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The impact of oxidative stress and potential antioxidant therapy onfunction and survival of cultured pancreatic -islet cells
Kanase, Nilesh
DOCTOR OF PHILOSOPHY (AWARDED BY OU/ABERDEEN)
Award date:2011
Awarding institution:The University of Edinburgh
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Download date: 03. Jul. 2020
The impact of oxidative stress and potential
antioxidant therapy on function and survival of
cultured pancreatic β-islet cells
A thesis presented to
The University of Aberdeen
For the degree of
Doctor of Philosophy
By
Nilesh Kanase
(B. Pharmacy, MBA, M.Sc.)
School of Medical Sciences
University of Aberdeen
2010
i
Declaration
I hereby declare that the data published in this thesis are the result of my own work carried
out under the supervision of Professors Ian Megson, Sandra MacRury, Irfan Rahman and
Garry Duthie, at UHI Millennium Institute. This thesis has been completed entirely by myself
and has not previously been submitted for any other degree or qualification.
Nilesh S. Kanase
ii
Acknowledgements
I want to express my sincere thanks to Professors Ian Megson, Sandra MacRury, Irfan
Rahman and Garry Duthie. Ian gave me the opportunity to do PhD work in his lab in UHI,
and guided me throughout the thesis work. This dissertation could not have been written
without him, who not only served as my supervisor but also encouraged and challenged me
throughout the project work, never accepting less than my best efforts.
Other members of the lab, Sushama, Janet (lab managers), Ilene and Ivor (technical
assistants), Tim and Mathew (Post docs) encouraged me during the work and kept me
motivated. My fellow PhD students Michelle, Sherie, Matilda and Aditi were always there to
assist me and give me inputs on the work. I am also thankful to Dr Jun Wei for giving
valuable guidance in PCR work. I also want to extend my gratitude to Sandra MacRury for
her guidance and Irfan Rahman for managing time for meetings in UK all the way from USA
and for being always there to answer my queries through email at other times. Garry Duthie
for giving valuable suggestion and comments in the final thesis writing. This thesis could not
have been made possible without the dean Ian Leslie and the staff of UHI.
Not only my PhD but every walk of life is lightened by the blessings of my parents and this
thesis is dedicated to them. Last but not the least I want to thanks my family, friends and my
beloved wife for their never-ending support to me during this period.
iii
Abstract
An unfortunate consequence of aerobic life is the structural damage to organic compounds
such as DNA, proteins, carbohydrates and lipids that occurs as an effect of oxidative
reactions. Oxidative damage inflicted by reactive oxygen species is termed “oxidative stress”.
Biological systems contain powerful enzymatic and non-enzymatic antioxidant systems, and
oxidative stress denotes a shift in the pro-oxidant/antioxidant balance in favour of the former.
Diverse biological processes such as inflammation, carcinogenesis and ageing all appear to
involve reactive oxygen species. There is also considerable evidence that oxidative stress
resulting from increased production of reactive oxygen species (or their inadequate removal)
plays a key role in the pathogenesis of diabetic complications.
Dietary antioxidants have been suggested to decrease the risk of many chronic diseases.
Curcumin is a natural antioxidant derived from turmeric (Curcuma longa) and has been
recognised to possess therapeutic properties since ancient times. Much of the existing data for
curcumin stem from experiments performed at supra-physiological concentrations (µM-mM)
that are impossible to attain through oral ingestion. A consistent finding is that curcumin
provides direct protection against ROS-mediated damage.
Glutathione is a crucial endogenous intracellular antioxidant that is tightly controlled by
enzymes involved in its synthesis, utilization and breakdown. There is strong evidence of
changes in glutathione peroxidase or total GSH levels, but little is known about changes in the
rate-limiting enzyme for glutathione synthesis (glutamate-cysteine ligase; GCL) in pancreatic
β–islet cells in diabetes. It was therefore hypothesized that curcumin at low concentration
(those attainable in plasma after oral ingestion), though itself not acting as an antioxidant
might trigger to cause a shift in the GSH/GSSG ratio in favour of former by upregulating the
recycling and thus help combat oxidative stress and protect pancreatic β-islet cells.
iv
The results indicated that curcumin, DMC and BDMC were able to scavenge hydroxyl
radicals generated by menadione, but showed little scavenging ability against superoxide
radicals. None of the curcuminoids were able to scavenge the NO generated using DETA
NONOate. Plasma achievable nanomolar concentrations of curcuminoids are easily capable of
preventing the deleterious effects of oxidative stress in pancreatic -islet RINm5F cells, at
least in H2O2 model. The results point to an indirect method of protection, and one that
favours protection against mitochondrial dysfunction over loss of membrane integrity. None
of the curcuminoids showed a detrimental effect on insulin secretion, but the model did not
allow assessment of any potential positive effect on insulin secretion. Further the findings
confirmed that plasma attainable nanomolar concentrations of curcumin offered protection in
pancreatic -islet RINm5F cells against H2O2-induced damage by modulating the proportion
of GSSG:GSH in the favour of GSH and increasing the activity of SOD. This increase in
GSH and SOD levels was, at least in part, on account of an increase in GR and SOD-2 gene
expression. The intracellular mechanism driving this modulation of antioxidant gene was, by
virtue of blocking the H2O2-induced NF-κB activation.
v
Table of contents
Chapter 1: Introduction .......................................................................................................... 1
1.1 General Introduction ......................................................................................................... 1
1.2 Diabetes Mellitus .............................................................................................................. 3
1.2.1 Type 1 diabetes .......................................................................................................... 4
1.2.2 Type 2 diabetes .......................................................................................................... 5
1.2.3 Clinical manifestation of diabetes .............................................................................. 6
1.3 Oxidative stress and diabetes mellitus .............................................................................. 6
1.3.1 Reactive oxygen species (ROS) ................................................................................. 6
1.3.2 Sources of ROS .......................................................................................................... 9
1.3.3 Hyperglycemia induced oxidative stress ................................................................. 11
1.3.3.1 Advanced glycosylation end products (AGE) .................................................. 11
1.3.3.2 Polyol pathway ................................................................................................. 12
1.3.3.3 Activation of protein kinase C .......................................................................... 13
1.3.3.4 Enzymatic superoxide production .................................................................... 13
1.4 Cellular antioxidant defence ........................................................................................... 15
1.4.1 Enzymatic antioxidants ............................................................................................ 15
1.4.1.1 Superoxide dismutase (SOD) ............................................................................ 16
1.4.1.2 Catalase ............................................................................................................. 16
1.4.1.3 Thioredoxin system ........................................................................................... 16
1.4.1.4 Glutathione redox cycle .................................................................................... 17
1.5 Turmeric .......................................................................................................................... 21
1.5.1 Metabolism and bioavailability ............................................................................... 22
1.5.2 Antioxidant activity of curcumin ............................................................................. 23
1.5.3 Anticarcinogenic and antidiabetic activity of curcumin .......................................... 25
vi
1.5.4 Other bioactivities of curcumin ............................................................................... 26
1.6 Aim of the study ............................................................................................................. 28
Chapter 2: Materials and Methods ...................................................................................... 30
2.1 Materials ......................................................................................................................... 30
2.1.1 Chemicals ................................................................................................................. 30
2.1.2 Assay Kits ................................................................................................................ 32
2.1.3 Instruments ............................................................................................................... 32
2.1.4 Software ................................................................................................................... 33
2.1.5 Consumables ............................................................................................................ 33
2.2 Methods .......................................................................................................................... 34
2.2.1 Reverse phase – High performance liquid chromatography (RP-HPLC) ................ 34
2.2.2 Electron paramagnetic resonance (EPR) ................................................................. 35
2.2.3 Nitric oxide scavenging by NOATM
......................................................................... 36
2.2.4 MTT assay ............................................................................................................... 38
2.2.5 Lactate dehydrogenase (LDH) assay ....................................................................... 39
2.2.6 Insulin assay ............................................................................................................. 41
2.2.7 Glutathione (GSH) assay ......................................................................................... 42
2.2.8 SOD assay ................................................................................................................ 44
2.2.9 Quantitative real time polymerase chain reaction (RTQ-PCR) ............................... 44
2.2.10 NF-κB activation assay .......................................................................................... 45
2.2.11 Statistics ................................................................................................................. 46
2.2.12 Cell culture ............................................................................................................. 47
Chapter 3: Characterisation of commercially available curcuminoids: purity and free
radical scavenging capabilities ............................................................................................. 48
3.1 Introduction ..................................................................................................................... 48
vii
3.2 Results ............................................................................................................................. 49
3.2.1 Purity of curcuminoids ............................................................................................. 49
3.2.2 Effect of co-incubation with menadione on stability of curcuminoids .................... 54
3.2.3 EPR analysis ............................................................................................................ 55
3.2.4 NOATM
analysis ....................................................................................................... 60
3.3 Discussion ....................................................................................................................... 61
3.3.1 Purity and stability of curcuminoids ........................................................................ 61
3.3.2 Direct radical scavenging activity of curcuminoids ................................................ 63
3.4 Conclusions ..................................................................................................................... 67
Chapter 4: Effect of bioavailable concentrations of curcuminoids on pancreatic β-islet
cell viability and function ...................................................................................................... 69
4.1 Introduction ..................................................................................................................... 69
4.2 Results ............................................................................................................................. 72
4.2.1 Cell mitochondrial function and membrane integrity by MTT and LDH assay ...... 72
4.2.2 Effect of curcuminoids on Insulin secretion by RINm5F cells ............................... 80
4.3 Discussion ....................................................................................................................... 84
4.4 Conclusions ..................................................................................................................... 87
Chapter 5: Impact of curcuminoids on regulation of intracellular antioxidant defence
mechanisms ............................................................................................................................. 88
5.1 Introduction ..................................................................................................................... 88
5.2 Results ............................................................................................................................. 90
5.2.1 Effect of curcuminoids ± H2O2 on total-glutathione and GSSG level ..................... 90
5.2.2 Effect of curcuminoids ± H2O2 on SOD activity ..................................................... 92
5.2.3 Comparison of effect of curcuminoids ± H2O2 on calculated GSH and total SOD
levels ................................................................................................................................. 95
viii
5.2.4 Effect on curcuminoids ± H2O2 on gene expression by RTQ-PCR ......................... 95
5.3 Discussion ....................................................................................................................... 98
5.4 Conclusion .................................................................................................................... 103
Chapter 6: Effect of curcuminoids on hydrogen peroxide and tumor necrosis factor-α
induced NF-κB activation in pancreatic β-islet RINm5F cells ........................................ 105
6.1 Introduction ................................................................................................................... 105
6.2 Results ........................................................................................................................... 107
6.2.1 NF-κB activation assay in independent curcuminoid treated RINm5F cells ......... 107
6.2.2 NF-κB activation assay in curcumin + H2O2 or TNF-α cotreated RINm5F cells . 108
6.3 Discussion ..................................................................................................................... 112
6.4 Conclusion .................................................................................................................... 115
Chapter 7: General Discussion ........................................................................................... 117
7.1 Radical scavenging effects ............................................................................................ 118
7.2 Protective effects against H2O2-mediated cell death in β-islet cells ............................. 121
7.3 Up-regulation of intracellular antioxidant systems by curcuminoids ........................... 123
7.4 Conclusion .................................................................................................................... 127
7.5 Future work ................................................................................................................... 128
Chapter 8: References ......................................................................................................... 136
1
Chapter 1: Introduction
1.1 General Introduction
Food is an essential prerequisite for the body to perform vital tasks including growth, repair,
voluntary physical activity, involuntary activity and maintenance of body temperature. These
processes are ultimately driven by an enormous array of chemical reactions broadly
encompassed in the term „metabolism‟. Glycolysis is a central part of the metabolic processes
of all mammalian cells and involves the conversion of glucose to pyruvate producing ATP
(energy) in absence of oxygen. A family of transmembrane glucose transporters (GLUT)
facilitate the diffusion of glucose into cells. GLUT4 is the transporter responsible for glucose
uptake in skeletal, cardiac and adipose tissues and the hormone insulin stimulates this uptake.
Insulin is a peptide secreted by the β-islet cells of Langerhans in the pancreas. It is
synthesized as proinsulin in the rough endoplasmic reticulum, which consists of an amino-
terminal B chain, a carboxyl-terminal A chain and a connecting peptide known as the C-
peptide. Mature, active form of insulin is generated within the endoplasmic reticulum where
proinsulin is exposed to several specific peptidases that remove the connecting peptide, C-
peptide. Insulin and free C-peptide are packaged into secretory granules in the Golgi
apparatus, which accumulate in the cytoplasm of the pancreatic β-islet cells [1].
Entry of glucose into the pancreatic β-islet cells triggers the exocytosis of the granules (fig
1.1). The sequence of events involved in insulin secretion induced by glucose is as follows
[2]:
1. Glucose is transported into the pancreatic β-islet cells through facilitated diffusion by
GLUT2 glucose transporters.
2. Intracellular glucose is metabolized to ATP.
2
3. Elevation in the ATP/ADP ratio results in the closure of cell-surface ATP-sensitive K+
(KATP) channels, leading to cell membrane depolarization.
4. Extracellular Ca2+
influx into the pancreatic β-islet cells is facilitated by opening of the
cell-surface voltage-dependent Ca2+
channels (VDCC).
5. Exocytosis of insulin is triggered by a rise in free cytosolic Ca2+
.
Figure 1.1 Insulin release by pancreatic β-islet cells.
The secretion of insulin has a broad impact on metabolism. Along with its well known
hypoglycemic effect (by increasing glucose uptake into pancreatic β-islet cells) insulin can
also facilitate cellular K+ uptake and stimulate both protein synthesis and lipogenesis. Insulin
regulates metabolism by inducing post-translational modifications of preexisting molecules or
by altering the concentration of critical proteins. Post-translational modification of preexisting
insulin molecules is a well-recognized action of insulin, and has been extensively studied for
many years [3, 4 and 5]. It is also known that insulin can have positive and negative effects on
3
the transcription of specific genes like amylase, glucagon, growth hormone and many more
within the same cell [6, 7]. Insulin deficiency can therefore have serious physiological
consequences, resulting in excess of glucose in the extracellular environment (hyperglycemia)
and intracellular glucose depletion, a situation that has been referred to as „starvation in the
midst of plenty‟. The extensive consequence of insulin deficiency is demonstrated in humans
where this is associated with a common and serious pathological condition referred to as
„diabetes mellitus‟.
1.2 Diabetes Mellitus
The World Health Organization (WHO) November 2009, Fact sheet No 312 [8] estimates that
more than 220 million people worldwide suffer from diabetes mellitus. An estimated 1.1
million people died from diabetes-related illnesses in 2005 (the actual number is likely to be
much larger, because although people may live for years with diabetes, their cause of death is
often recorded as heart disease or kidney failure). WHO reports that both the number of
people suffering from the disease and the mortality is likely to double within the next 20
years. Diabetes UK reports [9] that there are 2.6 million people who have been diagnosed
with diabetes in the UK as of 2009, with up to half a million more people who have not been
diagnosed but have diabetes. The economic and human cost of this disease is devastating. It is
currently estimated that 10 % of the NHS budget in the UK [10] (around £9 billion a year;
based on 2007/2008 budget) is spent on diabetes and related illnesses [11].
In 2009, the prevalence of diabetes in the adult population across the UK is shown in table 1.1
[12]:
Country Prevalence Number of people
England 5.1 % 2,213,138
Northern Ireland 4.5 % 65,066
Wales 4.6 % 146,173
Scotland 3.9 % 209,886
Table 1.1 Prevalence of Diabetes in the adult population in countries that comprise UK
4
There is an increased risk of cardiovascular disease, kidney disease, eye disease and non
traumatic amputations in people with diabetes. However, despite the importance of the
disease in terms of population welfare and health economics, there are still wide gaps in our
knowledge about the aetiology and progression of the disease.
There are two main forms of diabetes:
1.2.1 Type 1 diabetes
The term, „Type 1 diabetes‟, has replaced former terms, including insulin-dependent diabetes
mellitus (IDDM), juvenile onset diabetes and childhood onset diabetes. Although the onset of
the disease can occur at any age, this form of diabetes usually strikes children and young
adults. In UK, it accounts for about 15 % of all people with diabetes [8]. Sufferers of type 1
diabetes are unable to produce sufficient insulin due to autoimmune-mediated destruction of
β-islet cells of Langerhans of the pancreas resulting in hyperglycemia due to insufficient
insulin production required to regulate blood glucose level. The exact mechanisms triggering
the autoimmune response are not known, but environmental factors have a particularly
dominant influence on the appearance of type I diabetes. Asian populations show a very low
rate of type 1 diabetes, whereas Finland shows the world‟s highest incidence [13]. However
the prevalence in Finland differ from other Baltic States, notably Estonia whose population is
linguistically and ethnically very similar to that of Finland, but suffers only a third the
incidence. Interestingly, Myers et al. have reported that macrolides produced by Streptomyces
species may function as a trigger in genetically susceptible people. These species are
ubiquitously present in soil and some can infest tuberous vegetables and thus may be taken up
through the diet [14]. Interestingly, Streptomyces species are the source of streptozotocin, an
agent used to produce experimental diabetes in rodents. Prevention and reversal of type 1
diabetes is not yet possible. Patients diagnosed with type 1 diabetes are dependent on
5
exogenous insulin for life. The frequency of this disease is relatively low compared to type 2
diabetes.
1.2.2 Type 2 diabetes
The term, „Type 2 diabetes‟, has replaced former terms, including non-insulin-dependent
diabetes mellitus (NIDDM), adult onset diabetes and obesity-related diabetes. In UK it
accounts for about 85 % of people with diabetes [8]. The pathogenesis of type 2 diabetes is
complex and, in the later stages of the disease, involves defects in both insulin sensitivity and
pancreatic β-islet cell function. Insulin sensitivity is an important factor arising due to cells
not being able to use insulin properly [15]. Diabetes is a progressive disorder and gradually
the pancreas becomes overwhelmed, along with the increase in the level of oxidative stress
and inflammation, loses its ability to produce insulin. Impaired glucose tolerance and
impaired fasting glucose are the related disorders. When these disorders are present along
with risk factors such as obesity, insulin resistance/hyperinsulinemia, hypertension and
dyslipedemia the term metabolic syndrome is used. Although patients with type 2 diabetes are
not dependent on exogenous insulin, they may sometimes require it in advanced stages of the
disease for control of blood glucose along with following a careful diet and exercise program
and taking oral medication. Some of the types of oral medicines used in the management of
type 2 diabetes are sulfonylureas (e.g. gliclazide, glimepiride, glibenclamide, glipizide),
biguanides (e.g. metformin), thiazolidinediones (also called glitazones: e.g. rosiglitazone,
pioglitazone). Type 2 diabetes is associated with obesity, lack of exercise (physical inactivity),
high-fat diets, aging and family history of diabetes (race/ethnicity also play role). Though
Type 2 diabetes is more frequent in the elderly, there is an alarming increase in type 2
diabetes in children as a result of changing lifestyles. It is most pronounced in non-European
populations (Native Americans, Pacific Islanders, and Australian aboriginals), while Asia
shows the highest potential for increases in people with type 2 diabetes [13].
6
1.2.3 Clinical manifestation of diabetes
A characterized essential clinical symptom of diabetes is abnormally high blood glucose
levels. The ideal values of glucose in the blood are,
4 to 7 mmol/l before meals.
less than 8 mmol/l after a meal
The most common symptoms of type 1 diabetes are polyuria, polydipsia, and polyphagia,
along with lassitude, nausea, and blurred vision. On the other hand, the symptoms of type 2
diabetes are often more nonspecific and include, increased tiredness and increased infections;
often these symptoms serve as early warning signs for disease onset. About 75 % of all deaths
among people with diabetes are attributed to cardiovascular disease; risk factors include high
blood pressure, high serum cholesterol, obesity and smoking [16].
1.3 Oxidative stress and diabetes mellitus
1.3.1 Reactive oxygen species (ROS)
There is a growing wealth of evidence to support a role of oxygen-centred free radicals in a
wide range of diseases. A considerable amount of data is published showing the involvement
of free radicals in the occurrence of diabetic complications. Oxidative stress is well known to
lead to destabilization of the cellular environment and free radical-induced damage. It causes
DNA, protein, carbohydrate and lipid damage, membrane dysfunction, protein inactivation
and accelerated aging [17-20]. Almost all of the pathways studied (fig 1.2) to understand the
mechanism of occurrence of diabetic complications show involvement of free radicals (and
thus oxidative stress). Free radicals though do not lead to diabetic complications by
themselves, they are shown to be involved in most of the aspects associated with diabetes
making their study particularly important [21, 22].
7
Figure 1.2 Pathways studied to understand the mechanism of occurrence of diabetic complications.
Oxidative stress is defined as a state characterized by imbalance between the antioxidants and
pro-oxidants (fig 1.3) on account of exposure to an increased number of reactive oxygen
species (ROS) and/or reactive nitrogen species (RNS), leading to a potentially unstable
cellular environment and damage [23, 24]. Oxidative stress represents a potentially toxic
insult to the intracellular environment. [25]. Oxidative stress as a result of increased
production of reactive oxygen species (ROS), or their inadequate removal by antioxidant
defence systems, plays a key role in the pathogenesis of late diabetic complications.
Hypotaurine
GSHSOD
α-TocopherolCatalase
LactatePyruvate
Taurine
Antioxidants
oxidants
Hydroxyl radical/ion
Superoxide anion
Hydrogen Peroxide
ROS
ROS Physiological levels
ROS Lacking
Oxidative Stress
Uric AcidHypotaurine
GSHSOD
α-TocopherolCatalase
LactatePyruvate
Taurine
Antioxidants
oxidants
Hydroxyl radical/ion
Superoxide anion
Hydrogen Peroxide
ROS
ROS Physiological levels
ROS Lacking
Oxidative Stress
Uric Acid
Figure 1.3 The crucial antioxidant and oxidant balance which has impact on the oxidative status.
AGEs
Damage to
antioxidant
enzymes
Glycated proteins
Elevated glucose Diabetic Complications
OXIDATIVE STRESS
ROS
Oxidized
glucose
Oxidized fatty
acids DNA damage
Other Sources
8
Superoxide (O2•-) is formed by one electron reduction of molecular oxygen (i.e. gain of a
single electron) by a variety of oxidases that take reducing equivalents from NAD(P)H
(nicotinamide adenine dinucleotide phosphate-oxidase) or NADP+ (or from
xanthine/hypoxanthine in the case of xanthine oxidase). Because it is an anion, it has limited
permeability through cell membranes [26]. Furthermore, superoxide is much more reactive
than nitric oxide (NO) and superoxide dismutase (SOD) catalyses it‟s rapid and spontaneous
disproportionation into molecular oxygen and H2O2. Its intracellular target range is therefore
limited to close proximity of its source. Under physiological conditions, its lifespan and range
of action is further limited by SOD. This enzyme exists in three isoforms, and accelerates the
spontaneous decay of O2•- to form H2O2 and molecular oxygen (O2). However, under
pathological conditions, O2•- can react with NO (with a reaction rate that is about three times
faster than the dismutation reaction with Cu/Zn SOD) [27] to form the potent oxidant
peroxynitrite (ONOO-). ONOO
- initiates both DNA single strand breakage and lipid
peroxidation and it also nitrates amino acid residues in proteins such as tyrosine and cysteine,
which affects many signal transduction pathways [28].
H2O2 is not a free radical. However, it is an oxidant that can serve as a second messenger. It
has a longer half-life than O2•- and can diffuse longer distances and is thus able to influence
signaling events at more distant sites. It is thought to propagate its signal by oxidizing active
site cysteine residues. If not degraded by catalase or glutathione peroxidase, it can, in the
presence of metals such as copper (Cu+) or iron (Fe
2+), lead to the production of the hydroxyl
radical (•OH) (Fenton reaction). This is the most reactive oxygen free radical and
unspecifically oxidizes all target molecules that it encounters. Therefore, •OH is not regarded
as a signaling molecule.
9
Figure 1.4 Scheme showing the formation of reactive oxygen species (ROS).
Some ROS are ideal signaling molecules since they are rapidly generated, highly diffusible,
and easily degradable and ubiquitously present in all cell types. They are important for the
normal functioning of the cell. However, excessively high levels of ROS cause damage to
cellular proteins, membrane lipids, and nucleic acids. Figure 1.4 illustrates the reactions by
virtue of which ROS are generated. This generated ROS may lead to oxidative stress followed
by controlled (apoptosis) or uncontrolled (necrotic) cell death.
1.3.2 Sources of ROS
Oxidoreductases are enzymes that catalyze redox reactions and are the major source of O2•- in
the cellular environment. Enzymes that play an important role in ROS production in the
vasculature include the following:
Xanthine oxidase (XO), which is a biochemically modified form of xanthine dehydrogenase
and catalyzes the oxidation of hypoxanthine and xanthine to uric acid with the concomitant
formation of O2•- and H2O2. The dehydrogenase form uses NAD
+ rather than oxygen as
electron acceptor and therefore does not produce ROS. XO activity is highest in liver and
intestine but is also found in endothelial cells [29].
10
Cytochrome P450 (CYP) monooxygenases are membrane bound, haem-containing oxidases,
found primarily in the liver. Apart from the group of CYP that metabolizes arachidonic acid,
these enzymes oxidise, peroxidise and/or reduce cholesterol, vitamins, steroids and
xenobiotics in an oxygen and NADPH-dependent manner. However, during their reaction
cycle, CYP enzymes can generate O2•-, H2O2 and
•OH by transferring electrons to the active
bound oxygen instead of the central haem iron [30]. Because these enzymes have been shown
to exist in endothelial and smooth muscle cells, their ROS production may play a significant
role in increasing oxidative stress within the vascular wall. Indeed, Fleming et al. [31] could
show that the isozyme CYP 2C9 was a functionally significant source of ROS in coronary
arteries.
The mitochondrial respiratory chain (in particular complex III and IV) can be another
source of ROS. This can occur through electron leak from the electron transport chain and
reduce oxygen to form O2•-. Although the percentage of electrons that leak from the normal
pathway, is quite low (1-2 %), mitochondria can, because of their high overall activity,
produce considerable amounts of ROS. Studies have also shown that mitochondria produce
ROS under physiological and pathological levels of glycemia via electron transport system
[32, 33]. However, due to the high concentrations of mitochondrial superoxide dismutase
(MnSOD) within the mitochondria, the O2•-
levels are kept low and only H2O2 permeates the
membrane to enter the cytosol.
The NAD(P)H oxidases are a group of plasma membrane-associated enzymes that have best
been studied in neutrophils. They catalyze the production of O2•-
by the one-electron reduction
of oxygen, using NADPH as the electron donor:
2O2 + NADPH 2O2•- + NADP+ + H+
11
Neutrophil NADPH oxidase activation results in the release of large amounts of O2•-
in the so-
called „oxidative burst‟, which plays an important part in host defence against microbial
infection. However, recent findings have established the existence of a family of
nonphagocytotic oxidases that share many characteristics with that of the NADPH oxidase
found in neutrophils. These oxidases have been identified in many cell types including
endothelial cells [34], smooth muscle cells [35] and adventitial fibroblasts [36]. To date, a
non-phagocyte NAD(P)H-oxidase is thought to be the major source of ROS production in the
vascular wall.
1.3.3 Hyperglycemia induced oxidative stress
ROS seem to play a crucial role in the pathogenesis of diabetes as has been shown by studies
in humans [37, 38]. The mechanisms by which diabetes can lead to hyperglycemia-induced
oxidative stress are multifactorial [39] and include the following sources of ROS generation:
1.3.3.1 Advanced glycosylation end products (AGE)
ROS in diabetes may be a result of the non-enzymatic interaction of glucose with the amino
side chains on proteins or lipoproteins (in particular lysine) leading through a series of
oxidative and non-oxidative reactions to form Schiff-bases called advanced glycosylation end
products (AGE), which spontaneously rearranges into an Amadori or Maillard products.
Some of the individual AGEs are formed in reactions of proteins with glucose only under
oxidative conditions and these are termed glycoxidation products. AGEs can accumulate in
tissue over time and can either on their own or via receptors for advanced glycated end
products (RAGE) promote ROS formation by inactivating enzymes by altering their structure
and functions. AGE–RAGE interaction modulates the expression of adhesion molecules and
the expression of proinflammatory/prothrombotic molecules such as vascular cell adhesion
molecule-1 (VCAM-1) in endothelial cells; in fibroblasts, it modulates the production of
collagen; in smooth muscle cells, it modulates the migration, proliferation, and expression of
12
matrix modifying molecules; and in lymphocytes, stimulates the proliferation and generation
of interleukin-2 (IL-2) [392-395]. A large body of evidence suggests that one consequence of
AGE–RAGE interaction is the generation of ROS, at least in part via the activation of
NADPH oxidase [396, 397 and 398]. Also, AGEs by themself may be linked to increased
generation of ROS by multiple mechanisms, such as by decreasing activities of superoxide
dismutase (SOD) and catalase, diminishing glutathione stores, and activation of protein kinase
C (PKC) [399, 400 and 401]. In particular, the glycosylation of low-density lipoproteins
(LDL) can decrease its receptor-mediated clearance and increase the susceptibility of LDL to
oxidative modification. This increases its uptake by macrophages resulting in foam cells, an
early step in the development of atherosclerosis. AGEs have also been shown to quench
endothelial derived NO [40, 41 and 42]. However, the role of AGE in endothelial dysfunction
is controversial and several authors found no beneficial effect of the AGE inhibitor
aminoguanidine on endothelium dependent vasodilatation [43, 44], suggesting that AGEs are
unlikely to interfere with vascular smooth muscle cell reactivity.
1.3.3.2 Polyol pathway
In tissues which do not require insulin for cellular glucose uptake such as blood vessels,
hyperglycemia activates the polyol pathway, in which aldose reductase reduces the aldehyde
form of glucose to form sorbitol. Because this reaction requires NADPH, an increase in the
polyol pathway flux may result in the depletion of cellular NADPH, which is a cofactor for
many enzymes including NOS and the antioxidant enzyme, glutathione reductase. Sorbitol is
then oxidized to fructose by the enzyme sorbitol dehydrogenase, while NAD+ is reduced to
NADH. It was proposed that the most likely mechanism by which this pathway induces
oxidative stress is due to decreased regeneration of reduced glutathione, as a result of the
depletion of the NADPH stores [402]. However, blocking the sorbitol pathway with aldose
reductase inhibitors improved peripheral nerve conduction but had little effect on diabetic
13
retinopathy, indicating that this may not be an important pathway in diabetic
microvasculopathy [45].
1.3.3.3 Activation of protein kinase C
Another glucose-induced alteration in cellular metabolism that may play a role in diabetic
complications is the activation of PKC [46]. Activation of PKC has now been demonstrated in
all vascular tissue involved in diabetic complications [47]. In vascular tissue, hyperglycemia
causes de novo synthesis of diacylglyerol (DAG), the physiological activator of PKC, mainly
through the glycolytic pathway. There was an increase in intracellular DAG levels, leading to
PKC activation found in endothelial cells and smooth muscle cells incubated with high
glucose concentrations [48]. Beckman et al. demonstrated that in humans, the PKC inhibitor
LY333531 prevented the hyperglycemia induced reduction in endothelium-dependent
vasodilation [49]. The activation of PKC can regulate many cellular functions, including
vascular permeability, contractility, cellular proliferation and signal transduction mechanisms.
A common denominator for abnormalities in the cellular functions may be the formation of
ROS by activation of PKC or phosphoinositide 3-kinases (PI3Ks) [50]. The activity of the
superoxide producing enzyme, NAD(P)H-oxidase, was shown to be increased in a PKC-
dependent manner in response to glucose [51], while in vessels from diabetic patients the
NAD(P)H-oxidase derived O2•-
production was abrogated by the PKC-inhibitor chelerythrine
[52]. Finally, in vitro studies have shown that in addition to PKC-mediated activation of O2•-
producing-enzymes, O2•-
per se is an activator of PKC, leading to increased oxidative stress in
a positive feedback manner [53].
1.3.3.4 Enzymatic superoxide production
Other sources of excess ROS generation in the setting of hyperglycemia may be ascribed to
the increased activity of several O2•- producing enzymes:
14
The enzyme NO synthase (NOS) can become uncoupled under certain pathophysiological
conditions. This happens when the cofactor tetrahydrobiopterin (BH4) is oxidized to
dihydrobiopterin (BH2) so that electrons flowing from the reductase domain to the oxygenase
domain are diverted to molecular oxygen rather than to L-arginine, with the resulting
formation of O2•-. Furthermore, ONOO
- may, at low concentrations, disrupt the zinc-thiolate
complex within the catalytic site of NOS, thereby uncoupling the enzyme [54]. The
uncoupled NOS thus turns into an O2•- producing enzyme, with a concomitant decrease in NO
production. Evidence for the existence of dysfunctional, uncoupled NOS in diabetes is shown
in studies on animal models [55] and diabetes in humans [52, 56].
Desco et al. [57] showed that in a rat model of type 1 diabetes, there was an increased release
of XO from the liver into the plasma. The binding of XO to the glucosaminoglycans on
endothelial cells can result in increased local O2•- formation, as observed in aortic rings of
these animals. Administration of heparin causes release of XO from the endothelial surface.
In addition, endothelial dysfunction in hypertensive diabetic patients was improved by acute
inhibition of XO by oxypurinol [58]. Moreover, increased levels of XO were found in the
plasma of diabetic mice, concomitant with increased O2•- levels, which were restored with
oxypurinol [59].
A link between mitochondria and oxidative stress in the setting of diabetes was found by
Nishikawa et al, who showed that hyperglycemia-induced ROS production in bovine aortic
endothelial cells was abrogated by inhibitors of the mitochondrial respiratory chain or by over
expression of either uncoupling protein-1 (UCP-1: an uncoupler of oxidative phosphorylation)
or the mitochondria specific manganese superoxide dismutase (MnSOD). Furthermore, this
group showed that normalizing mitochondrial ROS production using rotenone, an inhibitor of
electron transport chain complex I, thenoyltrifluoroacetone, an inhibitor of electron transport
chain complex II, or carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative
15
phosphorylation that abolishes the mitochondrial membrane proton gradient, or
overexpression of uncoupling protein-1, a specific protein uncoupler of oxidative
phosphorylation capable of collapsing the proton electrochemical gradient, blocked three
pathways of hyperglycemic damage: it prevented the glucose induced activation of PKC,
formation of AGE and sorbitol accumulation [33].
NAD(P)H-oxidase is known to be the major source of ROS in the vascular wall and any
dysfunction could play an important role in maintaining vascular tone. Kim et al. found an
increase in rac-1 membrane translocation associated with an increased NAD(P)H-oxidase
activity in aortic smooth muscle cells cultured in high glucose medium [60]. This group also
found an upregulation of p22phox mRNA in the aorta of OLETF rats, a model of type 2
diabetes [61]. Furthermore, in the saphenous veins and mammary arteries of diabetic patients,
the expression of the NAD(P)H-oxidase subunits p22phox, p67phox and p47phox was
increased, again associated with an increase in the activity of the enzyme [52].
1.4 Cellular antioxidant defence
Although production of ROS is an inevitable consequence of oxygen metabolism, safe
disposal or deactivation of these metabolites is essential to aerobic life. Thus, there is complex
cellular machinery devoted to detoxification of free radicals [62]. Clearly, the diversity of
cellular antioxidants matches that of pro-oxidants. Biological systems contain powerful
enzymatic and non-enzymatic antioxidants, the key elements of which are described below:
1.4.1 Enzymatic antioxidants
Three of the most important enzymes known to protect cells against oxidative damage are
superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). In addition to
directly acting enzymatic antioxidants, there are a number of indirectly acting ancillary
16
antioxidant enzymes like glutathione reductase (GR) [63], glutathione-S-transferases (GST)
[64], NAD(P)H:quinone oxidoreductase [65].
1.4.1.1 Superoxide dismutase (SOD)
SOD is a key antioxidant enzyme in the human body because of its great importance for the
regulation of free radical-mediated processes in biological systems [66]. SOD enzyme exists
in three isoforms, occurring either extracellularly (FeSOD: iron SOD), in the cytoplasm
(Cu/ZnSOD: copper and zinc SOD) or in the mitochondria (MnSOD: manganese SOD). It
spontaneously accelerates the dismutation of O2•- to form H2O2 and O2 in two steps (reaction 1
and 2). This reaction is catalyzed by iron or copper/zinc or manganese active centres present
in the SOD enzymes.
1.4.1.2 Catalase
For many years, the haem-containing [67] enzyme, catalase, has been considered to be one of
the most important antioxidant enzymes. Its predominant subcellular localization in
mammalian cells is in peroxisomes and it catalyzes the decomposition of hydrogen peroxide
to water and oxygen (reaction 3) [68].
Thus, catalase lowers the risk of hydroxyl radical formation from H2O2 via the Fenton-
reaction catalyzed by Cu or Fe ions [69]. Binding of NAD(P)H to catalase protects it from
inactivation and increases its efficiency [70, 71].
1.4.1.3 Thioredoxin system
Like glutathione peroxidase, thioredoxin reductase (TrxR) is a selenium-containing enzyme
whose activity can be influenced by dietary selenium. There are two forms of thioredoxin
(TRX): TRX1 (104 amino acids, found in the cytoplasm) and TRX2 (found in mitochondria).
2H2O2 O2 +2H2O (Reaction 3)
O2 + Cu(I)ZnSOD (Reaction 1) O2•- + Cu(II)ZnSOD
O2•- + Cu(I)ZnSOD + 2H+ H2O2 + Cu(II)ZnSOD (Reaction 2)
Catalase
17
The thioredoxin/thioredoxin reductase (TRX/TrxR) system functions to maintain the
cellular environment in a reduced state thus offering protection against free radicals (reaction
4a and 4b). Thioredoxins are small proteins with two cysteine residues at its active site, which
in the oxidized protein form a disulfide bridge located in a protrusion from the three-
dimensional structure of the protein [72]. The flavoprotein thioredoxin
reductase catalyzes the
nicotinamide adenine dinucleotide (NADPH)-dependent
reduction of the redox-active
disulfide in thioredoxin (Trx), which serves a wide range of functions in cellular proliferation
and redox control (reaction 4a and 4b) [73, 74 and 75]. Small increases in TRX can cause
profound changes in thiol-disulfide redox status in proteins [76]. TRX1 has also been reported
to induce transcription of superoxide dismutase [77]. Elimination of protein disulfides by the
thioredoxin system can be summarized as follows:
1.4.1.4 Glutathione redox cycle
Reduced glutathione (GSH) is an abundant, water-soluble, tripeptide made up of the amino
acids L-glutamine, L-cysteine, and L-glycine (γ-L-glutamyl-L-cysteinyl-glycine) (fig 1.5) [78,
79]. Its IUPAC name is 2-amino-5-{[2-[(carboxymethyl)amino]-1-(mercaptomethyl)-2-
oxoethyl]amino}-5-oxopentanoic. The importance of GSH is evident by its widespread utility
in humans, plants, mammals, fungi and some prokaryotic organisms [80].
NH2
COOH C CH2
H
CH2 CO NH C
CH2
SH
CO NH CH2 COOH
H
Glutamate Cysteine
Glycine
Figure 1.5 Structure of Glutathione (Reduced form)
TRX−(SH)2 + Protein−S2
TRX−S2 +NADPH + H+ TRX−(SH)2 + NADP+ (Reaction 4b) TrxR
TRX−S2 + Protein−(SH)2 (Reaction 4a)
18
GSH shows an unusual gamma (γ) peptide linkage between the side chain carboxyl group of
glutamate and the amine group of cysteine. This γ-linkage between the first two amino acids
(instead of the typical alpha linkage), helps GSH to resist degradation by intracellular
peptidases. This linkage is subjected to hydrolysis only by γ-glutamyltranspeptidase (GGT),
which is present on the cell surface of all cells, with particularly high concentrations in the
liver, bile ducts, and kidney [81, 82]. The thiol group (-SH; also known as sulphydryl) of
cysteine is the most active group, forming the important reaction centre of GSH providing its
potent reducing properties. Due to the presence of this highly reactive thiol group, GSH reacts
spontaneously with some electrophiles [83]. Most of the reactions of GSH take place in the
presence of catalysis from an extended family of enzymes known as GSH S-transferases [84].
For example, the reaction of GSH with hydrogen peroxide (H2O2) is catalyzed by glutathione
peroxidase (GPx), which reduces H2O2 to water (H2O) (reaction 5). Two such reduced
glutathione molecules undergo thiol-disulphide exchange and dimerise (fig 1.6), yielding
oxidized glutathione known as glutathione disulphide (GSSG) [85-89].
NH2
COOH C CH2
H
CH2 CO NH C
CH2
S
CO NH CH2 COOH
H
Glutamate Cysteine
Glycine
S
CH2
CO C
H
NHNHCH2COOH CO CH2 CH2
H
C COOH
H
Glycine
Cysteine Glutamate
GSSG
Figure 1.6 Structure of Glutathione disulfide (Oxidized form of GSH)
19
The enzyme, glutathione disulphide reductase (GR), rapidly reduces GSSG formed in the cell
back to GSH using NAD(P)H as a cofactor (reaction 6). Thus, there is a recycling loop
between oxidation of GSH and subsequent reduction of GSSG, helping to maintain a
predominant GSH-rich cell environment. Tight regulation of the GSH:GSSG loop is
imperative as maintaining an optimal ratio in the cell is critical for their survival [88, 90]. In
healthy cells, GSSG rarely exceed 10 % of total GSH. Typically, the GSH concentration in
cells ranges from 0.1-10 mM while GSSG is 5-15 μM, depending on the redox environment
of the cell. GSH in human tissues is most concentrated in the liver (up to 10 mM) and also in
the spleen, kidney, lens, erythrocytes, and leukocytes [91]. The plasma concentration of GSH
is in the low micromolar range (4.5 μM) [92]. 10-15 % of total intracellular GSH is in the
mitochondria [87, 90, 93, 94, and 95].
GSH has profound importance in maintenance of cellular homeostasis and diverse cellular
functions. A number of physiological roles are played by GSH such as amino acid transport,
protein and nucleic acid synthesis, active form maintenance of enzymes (thus also in enzyme
catalyzed various reactions), cell membrane maintenance (transmembrane transport), receptor
action, cell maturation, sepsis amelioration and cardiogenic shock (Fig. 1.7) [96, 97].
GSSG + NAD(P)H + H+ 2GSH + NADP+ (Reaction 6)
H2O2 + 2GSH GSSG +2H2O (Reaction 5)
GR
GPx
20
Figure 1.7 Various cellular functions in which GSH is involved. (Adapted and modified from 87)
The most important function of GSH is its antioxidant potential. GSH repairs free radical sites
(R•) that are generated on account of various cellular functions thus acting as a radical
scavenger, where R• represents
•OH and GS
• represents oxidized GSH (GSSG) (reaction 7). It
also scavenges various other reactive radicals, or radical sites in proteins and DNA
(quenching of radical centres on proteins and DNA) that are generated by various intracellular
reactions (reaction 8). Besides this, GSH also helps to maintain the sulphydryl (-SH) group of
proteins in the reduced state (reaction 9) and recycles vitamin C from its oxidized radical
(reaction 10) [98, 99]. Also, in the presence of glutathione peroxidase (GPx), GSH is involved
in transforming the hydroperoxide moiety of lipid hydroperoxide (LOOH) to generate alcohol
(LOH), yielding primary stable end-products of lipid peroxidation (reaction 11) [100, 101].
GSH serves as substrate and cofactor in numerous drug-metabolism reactions, primarily in
those catalysed by GSH-dependent oxidases and reductases [102]. It can also function as an
Synthesis of proteins, nucleic acid, leukotriene, prostaglandin
-SH enzyme regulation
Antioxidant defence
UV resistance, Redox related signal transduction
Transport and storage of cysteine
Cell growth, division, proliferation protection
Detoxification of xenobiotics in liver
Role of GSH
GSSG + LOH + H2O (Reaction 11) GPx
2GSH + LOOH
2GSH + 2Asc• GSSG + 2Asc (Reaction 10)
2GSH + PSSX GSSG + P (SH)2 X (Reaction 9)
GS• + DNA (Reaction 8)
GS• + RH (Reaction 7)
GSH + DNA• GSH + R•
21
antioxidant in an enzyme-independent manner as seen while it repairs DNA by donating
hydrogen [103]. GSH is also found to have anti-viral effects by virtue of attacking viruses
having different replicative mechanisms (HIV and other retroviruses, influenza and
parainfluenza viruses, rhinovirus and HSV-1) and inhibiting viral replication at different
stages [reviewed in 104]. Loss of GSH has been demonstrated and correlated to increased
cardiac oxidative stress in both human and experimental models of diabetes, [105, 106, and
107].
1.5 Turmeric
Since 1900 BC Asian medicine has been using the dried ground rhizome of the perennial herb
Curcuma longa Linn. Family: Zingiberaceae, called turmeric in English, haldi in Hindi and
ukon in Japanese [108]. It is referred to in the ancient Hindu scripture, the Ayurveda as
having numerous therapeutic activities for a wide variety of diseases and conditions [109].
Turmeric contains a wide variety of phytochemicals, including curcumin,
demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), zingiberene, curcumenol,
curcumol, eugenol, tetrahydrocurcumin, triethylcurcumin, turmerin, turmerones, and
turmeronols [110]. Extensive research within the last half century has identified curcumin,
DMC and BDMC as responsible for most of the biological activity of turmeric. Curcumin,
DMC and BDMC (fig 1.8) have been isolated from Curcuma mangga [111], Curcuma
zedoaria [112], Costus speciosus, Curcuma xanthorrhiza [113], Curcuma aromatica,
Cucruma phaeocaulis [114], Etlingera elation [115], and Zingiber cassumunar [116]. Most
currently available preparations of curcumin contain approximately 77 % curcumin, 18 %
demethoxycurcumin, and 5 % bisdemethoxycurcumin. Comparative chemical properties of
curcuminoids isolated from C. longa are given in table 1.2.
22
O OH
OCH3
OH
H3CO
HO Curcumin
O OH
OCH3
OHHO Demethoxycurcumin
O OH
OHHO Bisdemethoxycurcumin
Figure 1.8 Chemical structure (keto-enol form) of curcumin and its analogues.
Chemical properties Curcumin DMC BDMC
1. Molecular
formula
C21H20O6 C20H18O5 C19H16O4
2. Molecular
weight
368.38 338.39 308.33
3. Melting point
(oC)
183 168 224
4. Crystalline
nature
Orange prism Orange-yellow Yellow plates
5. Number of
methoxy groups
2 1 0
6. Solubility
- Insoluble
- Moderately
soluble
- Very
soluble
water, hexane
benzene, ether,
chloroform
ethanol, acetone,
DMSO
water, hexane
benzene, ether,
chloroform
ethanol, acetone,
DMSO
water, hexane
benzene, ether,
chloroform
ethanol, acetone,
DMSO
Table 1.2 Chemical properties of curcuminoids.
1.5.1 Metabolism and bioavailability
Clinical trials studies have reported that the systemic bioavailability of orally administered
curcumin in humans is relatively low [422-424]. Following oral consumption mostly
curcumin metabolites rather than curcumin itself are detected in plasma or serum [425, 426].
Curcumin is reduced to hexahydrocurcumin in the intestine and liver or alternately readily
conjugated to form curcumin glucuronides and curcumin sulfates [427]. Conjugated or
reduced metabolites of curcumin are reported to be less effective inhibitors of inflammatory
enzyme expression in cultured human colon cells than curcumin itself [428]. Serum
concentration of curcumin usually peaked 1-2 hours after oral intake of curcumin and
23
gradually declined within 12 hours as reported in a clinical trial conducted in Taiwan. The
average peak serum concentrations after taking 4000, 6000, and 8000 mg of curcumin were
0.51 +/- 0.11, 0.63 +/- 0.06 and 1.77 +/- 1.87 μM respectively [301]. Most recent clinical trial
conducted in the UK reported plasma concentrations of curcumin, curcumin sulfate, and
curcumin glucuronide were in the range of 10 nM one hour after a 3600 mg oral dose of
curcumin [429]. At a dose lower than 4000 and 3600 mg curcumin could not be detected in
serum in the respective studies. Poor absorption, rapid metabolism, and rapid systemic
elimination appear to be the major reasons contributing to the low plasma and tissue levels of
curcumin.
1.5.2 Antioxidant activity of curcumin
The antioxidant activity of curcumin was found to be equivalent to that of butylated
hydroxylanisole (BHA) and butylated hydroxyltoluene (BHT) [117]. Like curcumin,
demethoxycurcumin and bisdemethoxycurcumin also exhibited antioxidant activity [118].
Antioxidant capacities were found to be in the order of curcumin > DMC > BDMC in an in
vitro model systems, such as the phosphomolybdenum and linoleic acid peroxidation [119].
Curcumin can act as a scavenger of oxygen free radicals [120, 121, and 122] and it can protect
haemoglobin from oxidation [123]. In an in vitro test, curcumin significantly inhibited the
generation of ROS, such as superoxide anions and H2O2, and reactive nitrogen species (RNS),
which play an important role in inflammation [124]. Also, curcumin exerted a powerful
inhibitory effect against H2O2-induced damage in human keratinocytes and fibroblasts [125].
Oral administration of hydroalcoholic extract of C. longa to rabbits decreased the
susceptibility of LDL to lipid peroxidation in a dose-dependent manner [126]. Curcumin can
reduce the inflammatory response of ethanol by decreasing prostaglandin synthesis [127].
Curcumin helps maintain the membrane structural integrity and function and it also protects
against lead- and cadmium-induced lipid peroxidation in rat brain homogenates and against
24
lead-induced tissue damage in rat brain through metal binding mechanism [128].
Administering diabetic rats with curcumin reduced the blood sugar and glycosylated
haemoglobin (HbA1C) levels significantly. Curcumin supplementation has also been shown to
reduce oxidative stress in diabetic rats [129]. Dietary supplementation of curcumin (2 %, w/v)
to male ddY mice for 30 days significantly increased the activities of glutathione peroxidase,
glutathione reductase, glucose-6-phosphate dehydrogenase and catalase, as compared with the
same type mice fed normal diet. In several animal tumour bioassay systems, this may be one
of the possible mechanisms of cancer chemopreventive effects associated with curcumin
[130]. Since ROS have been implicated in the development of various pathological conditions
[131], curcumin has the potential to control these diseases through potent antioxidant activity.
The antioxidant capacity of curcumin is attributed to its unique conjugated structure, which
exists in equilibrium between the diketo and keto-enol forms that are strongly favoured by
intramolecular H-bonding [132]. Since demethoxycurcumin and bisdemethoxycurcumin have
similar structures to curcumin, they are thought to have similar bioactivities. Their respective
amounts needed for 50 % inhibition of lipid peroxidation were 20, 14, and 11 μg/mL; for 50
% inhibition of superoxides the respective concentrations were 6.25, 4.25, and 1.9 μg/mL, and
those for hydroxyl radical were 2.3, 1.8 and 1.8 μg/mL [120].
Typical radical-trapping ability as a chain-breaking antioxidant is shown by curcumin. The
nonenzymatic antioxidant process of the phenolic material is generally thought to be mediated
through the following two stages:
Where AH is the phenolic antioxidant, S is the substance oxidized, A•
is the antioxidant
radical and X• is another radical species or the same species as A
•. A
• and X
• dimerize to form
the non-radical product [110]. Linoleate as an oxidizable polyunsaturated lipid was used by
S-OO• + AH
A• + X• Nonradical materials (Step 2)
SOOH + A• (Step 1)
25
Masuda et al. to explore the antioxidant mechanism of curcumin. It was proposed that the
mechanism involved oxidative coupling reaction at the 3‟ position of the curcumin with the
lipid and a subsequent intramolecular Diels-Alder reaction by Masuda et al. [133]. Curcumin
was also confirmed to have metal binding ability. FT-IR spectrometric analysis showed that
both the hydroxyl groups and the β-diketone moiety of curcumin were involved in a metal-
ligand complexation, either directly bonding to the metal, or in intermolecular hydrogen
bonding [128].
Though numerous reports indicate that curcumin mediates antioxidant activity, there are also
some reports about its pro-oxidant role. Firstly, curcumin can induce the production of ROS
[134-137], which plays an important role in the anti-proliferative effects of this molecule
[138]. Secondly, curcumin binds thioredoxin reductase and this curcumin modified TrxR
enzyme strongly induces NAD(P)H oxidase activity, thus leading to the production of ROS
[139]. Recently it has been shown that curcumin, demethoxycurcumin,
bisdemothoxycurcumin, tetrahydrocurcumin, and turmerones differentially regulate anti-
inflammatory and anti-proliferative responses through a ROS-independent mechanism [140].
1.5.3 Anticarcinogenic and antidiabetic activity of curcumin
Curcumin acts as a potent anticarcinogenic compound. Among various mechanisms,
induction of apoptosis plays an important role in its anticarcinogenic effect. The cell
precipitates its own death in an orchestrated series of events through apoptosis. Cell
shrinkage, chromatin condensation, nuclear segmentation and internucleosomal fragmentation
of DNA, resulting in the generation of apoptotic bodies are the various stages of apoptosis
[141]. The antiproliferative effect of curcumin is mediated partly through inhibition of protein
tyrosine kinase, c-myc mRNA expression and bcl-2 mRNA expression [142]. Cellular
proliferation and apoptosis is known to be controlled by nuclear factor (NF)-κB. Curcumin
can also inhibit cell proliferation and induce apoptosis in human malignant astrocytoma cell
26
lines and head and neck squamous cell carcinoma (HNSCC) by inhibition of NF-κB activity
[143, 144]. For HNSCC, curcumin can induce cell apoptosis both in vitro and in vivo.
Curcumin caused lung cancer cell death by induction of apoptosis, which was found to be
independent of p53 status of the cell lines [145]. Other research showed curcumin-induced
apoptosis in melanoma cell lines in a manner that was also independent of p53 and the bcl-2
family [146]. Moreover, recent research found that curcumin had potent antiproliferative and
proapoptotic effects in melanoma cells by suppression of NF-κB and IKK activities but were
independent of the B-Raf/MEK (mitogen-activated/ERK, extracellular signal-regulated
protein kinase) and Akt (protein kinase B) pathway [147].
Both NF–κB and TNF-α have been linked with the induction of resistance to insulin in
diabetes mellitus [148-153]. Curcumin can be exploited in patients, as it has shown potential
to down regulate the activation of NF–κB and TNF-α expression and signaling [154-157].
Curcumin has been shown to overcome insulin resistance in several animal studies [158, 159].
1.5.4 Other bioactivities of curcumin
Extensive research within the past half century has proven that most of the beneficial
activities associated with turmeric are mediated by curcumin. Curcumin has been shown to
exhibit inhibition of NO-mediated effects [160, 161] as well as anti-inflammatory [162], anti-
allergy [163], anti-dementia [164], and anticancer [165] action and thus has a potential
activity against various malignant diseases such as diabetes, allergies, arthritis, Alzheimer‟s
disease, and other chronic illnesses. Although in most systems curcumin was found to be the
most potent analogue, [134, 166] bisdemethoxycurcumin was also found to exhibit high
activity [167]. There are also reports suggesting the mixture of all three to be more potent
than either one alone [168, 169]. Thus, there is an increasing literature to suggest that the
other curcuminoids also, to some extent, play a part in the exhibition of these therapeutic
activities.
27
Recent studies in lung cells have reported that curcumin (10 μM) inhibited NF-κB
expression/activation, IL-8 release, and neutrophil recruitment [403]. Curcumin inhibited NF-
κB transactivation by inhibiting the nuclear translocation from cytoplasm of the p65 subunit
of NF-κB, in association with the sequential suppression of IκB kinase (IKK)
phosphorylation, IκB-α degradation, p65 phosphorylation, and p65 acetylation [403, 404].
Curcumin is an interesting prospect for controlling chronic inflammatory diseases involving
the NF-κB signaling pathway as NF-κB regulates expression of a wide variety of genes that
are involved in the inflammatory processes. Curcumin is also shown as a downregulator of
the expression of iNOS, MMP-9, TNF-α, chemokines, cell surface adhesion molecules, and
growth factor receptors (such as epidermal growth factor receptor). Curcumin has also been
reported to modulate a number of key kinase signaling pathways such as mitogen-activated
protein kinases (MAPK) and protein kinase C (PKC) in a wide variety of different cell types.
In a recent study conducted by Koremu et al., [405] it was shown that in
monocytes/macrophages (U937 and MonoMac6 cells) curcumin at nanomolar concentrations
specifically restored cigarette smoke extract (CSE) or oxidative stress impaired
histone
deacetylase-2 (HDAC2) activity (reduction) and corticosteroid efficacy (lost under oxidative
stress) in vitro with an EC50 of approximately 30 nM and 200 nM, respectively. A common
feature of HDACs is ability to remove acetyl moieties from the ε-acetoamido group on lysine
residues of acetylated proteins, such as histones [406]. In general, these results in
condensation of the chromatin structure through tighter winding of the DNA around the core
histones thus dislodging the transcriptional machinery and occluding further transcription
factor binding, thereby resulting in gene silencing.
Histone acetylation by histone acetyltransferases (HATs) in contrast, disrupts the attractive
electrostatic interaction between the DNA and histones. This leads to the DNA unwrapping
28
from the core histones and allowing access for the transcriptional machinery thus resulting in
gene transcription [407, 408]. The anti-inflammatory actions of curcumin at 50 μM are
suggested to be propagated through inhibition of HAT activity, preventing NF-κB-mediated
chromatin acetylation [409]. Hence, it might be reasonable to hypothesize that in addition to
its role as an anti-inflammatory/antioxidant agent, curcumin may also assist in increasing the
efficacy of steroids via modulation of HDAC and HAT activity.
1.6 Aim of the study
Diabetes is associated with enhanced oxidative stress and decreased GSH (a natural
antioxidant) levels. This PhD research project was designed to test the following hypothesis:
A: that physiologically relevant concentrations of curcuminoids have significant scavenging
effects on ROS and RNS in vitro.
B: that curcuminoids inhibit oxidant-induced cell death in insulin-secreting pancreatic β-islet
cells.
C: that plasma achievable nanomolar concentrations of curcuminoids provide antioxidant
defence by influencing the GSH level in pancreatic β-islet RINm5F cells through modulation
of glutathione-related enzymes.
D: that plasma achievable nanomolar concentration of curcuminoids in presence of H2O2 and
TNF-α have any influence on the activation of transcription factor NF-κB in pancreatic β-islet
RINm5F cells.
In the present study various analogues of curcumin (pure curcumin, pure
demethoxycurcumin, pure bisdemethoxycurcumin and curcumin from Sigma-Aldrich, which
is a mixture of above mentioned pure curcuminoids) were examined. The over-arching driver
for the research was to establish the pathways by virtue of which curcuminoids could inhibit
29
oxidative stress in pancreatic β-islet cells at concentrations that could be achieved through
dietary intake, and to determine whether there was any structure-activity relationship between
the various analogues of curcumin tested (fig 1.9).
Figure 1.9 The pathway studied to understand the occurrence of diabetic complications.
Other Sources
OXIDATIVE STRESS
DNA damage
Elevated Glucose
Cell Death
CURCUMIN
AGEs
?
β-islet cell
Inflammation
Lipid damage
Protein damage
ROS
ROS
ROS
30
Chapter 2: Materials and Methods
2.1 Materials
2.1.1 Chemicals
2-vinylpyridine Sigma-Aldrich
Acetonitrile Sigma-Aldrich
Curcumin mixture Sigma-Aldrich
DETA NONOate Axxora
Dimethyl sulfoxide Sigma-Aldrich
di-Potassium hydrogen orthophosphate Sigma-Aldrich
DL-Dithiothreitol solution Sigma-Aldrich
DTNB Sigma-Aldrich
Dulbecco's phosphate buffered saline: without Ca & Mg PAA
Ethylene diamine tetraacetic acid (EDTA) Sigma-Aldrich
Ethylene glycol tetraacetic acid (EGTA) Sigma-Aldrich
Foetal bovine serum PAA
Glutathione Sigma-Aldrich
Glutathione disulphide Sigma-Aldrich
Glutathione reductase Sigma-Aldrich
Glibenclamide Sigma-Aldrich
Glycerol Sigma-Aldrich
HEPES Sigma-Aldrich
Hydrochloric acid Sigma-Aldrich
Hydrogen peroxide Sigma-Aldrich
L-arginine Sigma-Aldrich
31
L-cysteine Sigma-Aldrich
Leupeptin hemisulfate salt Sigma-Aldrich
Magnesium chloride Sigma-Aldrich
Mannitol Sigma-Aldrich
Menadione Sigma-Aldrich
Penicillin/Streptomycin PAA
Phenymethylsulfonyl fluoride Sigma-Aldrich
Potassium chloride Sigma-Aldrich
Potassium dihydrogen orthophosphate Sigma-Aldrich
Pure bisdemethoxycurcumin Sami Labs
Pure curcumin Sami Labs
Pure demethoxycurcumin Sami Labs
Pyrogallol Sigma-Aldrich
RINm5F cells ATCC
RPMI 1640 PAA
Sodium fluoride Sigma-Aldrich
Sodium orthovanadate Sigma-Aldrich
Sucrose Sigma-Aldrich
Sulfuric acid Sigma-Aldrich
Sulfosalicylic acid Sigma-Aldrich
Tempone-H•HCl Alexis Biochemicals
Tergitol® Solution, Type NP-40, 70 % in H2O Sigma-Aldrich
TNF-α R & D Systems
Triethanolamine Sigma-Aldrich
Trifluroacetic acid Sigma-Aldrich
Triton X-100 Sigma-Aldrich
32
Trypsin-EDTA (GIBCO) Invitrogen
β-NADPH Sigma-Aldrich
All other chemicals were of highest purity grade purchased from either Sigma-Aldrich or
Fisher. β-actin, GCL (both catalytic and modifier), GPx1, GR, SOD1 and SOD2 primers for
RTQ-PCR were purchased from Tebu-bio.
2.1.2 Assay Kits
Coomassie Plus (Bradford) protein assay kit Pierce
High capacity cDNA reverse transcription kit with RNase Inhibitor Applied Biosystems
Illustra Quick PrepTM
Micro mRNA purification kit GE healthcare
LDH assay kit Sigma-Aldrich
MTT assay kit Sigma-Aldrich
Superoxide dismutase assay kit Cayman Chemicals
SYBR® Green PCR Master Mix Applied Biosystems
TransAM NF-κB p65 Chemi Transcription Factor Assay Kit Active Motif
Ultra Sensitive Rat Insulin ELISA Kit Crystal Chem
2.1.3 Instruments
EPR Spectrometer (MiniScope 100) Magnettech
HPLC (1200 Series) equipped with Agilent
Quaternary Pump
Thermostatted auto-sampler injector
Diode array detector
Eclipse XDB-C18 column
Microplate reader (Varioskan Flash) Thermo Scientific
NanoDrop® ND-1000 Spectrophotometer Thermo Fisher
Nitric Oxide Analyzer (NOATM
model 280i) Analytix
33
pH meter (Hydrus 300) Fisherbrand
Plate centrifuge (BR.3.11) Jouan
Plate shaker/rocker (Shaker SI 100) Pharmacia
Quantica Real-Time Nucleic Acid Detection System Techne
Microson ultrasonic cell disruptor Microsonix
Thermocycler (2720) Applied Biosystems
2.1.4 Software
Chemstation software for LC 3D systems (B.02.01 SR1) Agilent
Miniscope32 (Version 1.0.0.1) Magnettech
Prism Software (Version 5.00) GraphPad
Quansoft (Version 1.1.0.21) Techne
SkanIt® Software for Varioskan
® Flash (Version 2.4) Thermo Scientific
2.1.5 Consumables
48 and 96 well plates Fisher Scientific
Corning® Costar
® cell culture plates, 6 well, flat and round bottom Sigma-Aldrich
Cryotube™ vials (2 ml) Nunc
Disposable glass capillaries 50 μl Magnettech
Eppendorf® epT.I.P.S. box Sigma-Aldrich
Eppendorfs tubes (1.5 and 2 ml) Sigma-Aldrich
T-25, T-75 and T-162 cm2cell culture flasks Sigma-Aldrich
34
2.2 Methods
2.2.1 Reverse phase – High performance liquid chromatography (RP-
HPLC)
Estimation of curcuminoids has been reported by high performance thin layer
chromatography (HPTLC) [170-172], HPLC [173-179], capillary electrophoresis [180, 181],
pH-zone-refining counter current chromatography [182], and supercritical fluid
chromatography [183]. However, none of the reports describe methods for simultaneous
determination of curcuminoids. To date, use of the modified stationary phases such as amino
bonded [175] and styrene divinylbenzene copolymer [176] and gradient elution [177] has not
produced clear separation of curcuminoids. The RP-HPLC method reported in this thesis was
developed for simultaneous determination of pure curcuminoids obtained from Sami labs,
India and from commercially available curcumin obtained from Sigma-Aldrich in a single
run.
a) Instrumentation
An HPLC (Agilent 1200 Series, Agilent Technologies, South Queensferry, UK) equipped
with the following was used for the purity experiments: quaternary pump (G1311A), injector,
thermostatted auto-sampler, (G1329A), and diode array detector (G1315B). Data acquisition
and analysis was carried out using Agilent Chemstation software for LC 3D systems (B.02.01
SR1).
b) Chromatographic Conditions
A new method was developed to measure curcuminoids. An Eclipse XDB-C18 column (5
μm, 4.6 mm x 150 mm, 993967-902, Agilent) was used at ambient temperature. The
optimized mobile phase consisted of acetonitrile:water (50:50 v:v), together with 0.1 %
trifluroacetic acid (TFA), (final pH of mobile phase adjusted to 3.0 with ammonia).The flow
35
rate for the mobile phase was adjusted at 1.5 ml/min. The eluent was monitored on the diode
array detector set at =420 nm. The injection volume was optimised to 10 l.
c) Stock solution and sample preparation
Fresh stock solutions (1 mM) of curcumin, DMC, BDMC and commercially available
curcumin (Sigma-Aldrich, Poole, Dorset, UK) were prepared in DMSO:PBS (final
concentration of DMSO was 0.01 %) every week and stored at -20 oC until used. Samples
were diluted to a final concentration of 200 μM. Each sample was analysed individually and
following co-incubation with 200 μM menadione at 37 oC for 30 minutes with an HPLC run-
time of 10 minutes.
2.2.2 Electron paramagnetic resonance (EPR)
EPR spin trapping is process in which short-lived radical species are intercepted by a so-
called spin trap, resulting in radical stabilization for sufficient time to be detected by EPR
spectroscopy. Information on the radicals generated both in biological and chemical situations
can be obtained from EPR spin trapping. EPR spectroscopy is a method that exposes a sample
to an external magnetic field simultaneously with microwave irradiation. Free radicals can be
unequivocally characterized by EPR because they contain unpaired electrons, which behave
like small magnets and thereby interact with the nucleus and electrons of neighbouring atoms.
The reaction principle of spin trapping is as follows:
R* + ST R-SA
*
where R* is a radical intermediate, ST is the spin trap and RSA
* is the radical spin adduct.
The possible direct scavenging effect of curcuminoids was determined using the three-line
spectrum of the 4-oxo tempo radical generated on account of reaction of spin trap, Tempone-
H.HCl (1-Hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine. HCl) with free radicals using EPR
spectrometry (Magnettech® Miniscope MS100 spectrometer). Spin-trapping experiments
36
were performed in PBS containing either a superoxide generator [pyrogallol (150 μM)] or
hydroxyl radical generator [menadione (150 μM)] along with a recognised spin trap for
oxygen-centred radicals [1-hydroxy- 2, 2, 6, 6-tetramethyl-4-oxo-piperidine.HCl (Tempone-
H.HCl) 50 μM] prepared in water containing 10 mM EDTA [184] and in the presence or
absence of curcuminoids. The reagents were added in the following order to eppendorf tubes
placed in a heating block set to 37 oC, PBS: curcuminoid (curcumin, DMC or BDMC)
followed by menadione (150 μM) or pyrogallol (150 μM) and finally Tempone-H.HCl (spin
trap; final concentration - 50 μM). The EPR signal intensity corresponding to the stable
radical adduct 4-oxo-Tempo formation (triplet centred at 3344G) was measured (arbitrary
units) immediately after addition of the last reagent (nominally time zero) using a 50 μl
disposable glass capillary tubes and then at 15 minutes intervals over 60 minutes with the
following parameter settings, Sweep: 49.1, Sweep time: 30 seconds, Smooth: 0.6s, Steps:
4096, Number (pass): 1, Modulation amplitude: 1500 (milli Gauss), microwave power: 7
(equivalent to 20 mW) and Gain: 1x101.
In control experiments in the absence of radical generators, small increases in the EPR signal
corresponding to the auto-oxidation of Tempone-H to 4-Oxo-Tempo were subtracted from the
corresponding experimental signals. Experiments with each test substances were performed
on six separate occasions (n=6).
2.2.3 Nitric oxide scavenging by NOATM
The chemiluminescent Nitric Oxide Analyzer (Sievers Model 280i) uses a high-sensitivity
detector for measuring nitric oxide based on a gas-phase chemiluminescent reaction between
nitric oxide (NO) and ozone (O3) as follows:
NO + O3 NO2* + O2
NO2* NO2 + hv
37
The emission from NO2* is in the red and near-infrared region and is detected by a
thermoelectrically cooled, red-sensitive photomultiplier tube. 0.5 ppb by volume is the
approximate detection limit for measurement of gas-phase NO by NOA Analyzer.
The potential of curcumin to scavenge NO was studied using an NO donor, DETA NONOate,
(Axxora, Nottingham, UK) in a Nitric Oxide Analyzer (Sievers NOATM
model 280i, Analytix,
Ltd., Peterlee, UK). The principle of the instrument is that NO generated in a purge vessel
(Fig 1) flushed with oxygen-free N2 gas is carried into the analyzer, where it reacts with
ozone, forming excited-state NO2*, which emits detectable light. The magnitude of NO
produced was calibrated as integral of the signal over time. The capacity of curcumin to
scavenge NO in PBS was analyzed by injecting the sample into the reaction chamber through
a rubber septum using a gas-tight syringe (fig 3.1). The reaction chamber has a teflon
stopcock valve and a needle valve, which was used to control the amount of carrier gas (N2)
entering the system. The cell pressure was set to 7.2 torr. N2 gas transfers the NO released in
purge vessel to Sievers NOA analyzer via a condenser equipped with cold water circulation
which dries the gas effluent from the purge vessel and thus protects the detector. The analysis
was performed by addition of DETA NONOate (100 μM) followed by the relevant
curcuminoid (1 nM - 5 μM) to the purge vessel containing 1 ml PBS. The purge system was
connected to a circulating water bath with controlled temperature (37 °C) permitting heating
of the reagents. Nitrogen gas was bubbled through a glass frit to mix the samples in the purge
vessel. Experiments with each of the test substances were performed on three separate
occasions (n=3).
38
Figure 3.1 Purge Vessel
2.2.4 MTT assay
The MTT system is a means of measuring the activity of living cells via mitochondrial
dehydrogenases. Thus MTT assay serves as a marker of mitochondrial functionality. The key
component of MTT assay is (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)
or MTT. The tetrazolium ring of MTT reagent is cleaved by the mitochondrial
dehydrogenases of viable cells, yielding purple formazan crystals which are insoluble in
aqueous solutions. The crystals are dissolved in acidified isopropanol and the resulting purple
solution is measured spectrophotometrically. There is a concomitant change in the amount of
39
formazan crystals formed depending upon an increase or decrease in cell number indicating
the degree of cytotoxicity caused by the test material.
The MTT assay was used to establish activity of living RINm5F pancreatic β-islet cells via
mitochondrial dehydrogenases. To ascertain if curcuminoids offered any protection against
the cytotoxic effect of H2O2, in separate experiments cells were cotreated with 50 μM H2O2 in
presence of curcuminoids (0.1 nM - 100 nM) for 24 hours. After incubation, MTT assays
were performed using the MTT in vitro toxicology assay kit as per the supplier‟s protocol
(Sigma-Aldrich Inc, UK). Briefly, at the end of incubation period, cell culture medium was
replaced with fresh medium supplemented with 20 μl of MTT solution and further incubated
for 2 hours, followed by adding MTT solubilisation solution to dissolve the formazan crystals
formed using a plate shaker. The resulting solution was transferred to 96-well plate and
absorbance was measured at 570 nm using a microplate reader (Varioskan Flash from Thermo
Scientific) and background absorbance was measured at 690 nm. Untreated cells were used as
control. Each sample was analysed in duplicate Experiments with each test substances were
performed on six separate occasions (n=6).
The percent of viable cells was determined according to the formula,
% of viable cells = (λ570 - λ690) treated cells X 100
(λ570 - λ690) untreated cells
where λ570 is the absorbance of dissolved formazan crystals at 570 nm and λ690 is background
absorbance at 690 nm.
2.2.5 Lactate dehydrogenase (LDH) assay
The amount of cytoplasmic LDH released into the medium represents the membrane integrity
of cells and it can be measured using the lactate dehydrogenase assay. The LDH assay is
based on the reduction of NAD by LDH. The resulting reduced NAD (NADH) causes a
stoichiometric conversion of a tetrazolium dye yielding a coloured compound which is
40
measured spectrophotometrically at 490 and 690 nm. Lysis of cells prior to assaying the
medium results in an increase or decrease in cell numbers with concomitant change in the
amount of substrate converted; this indicates the degree of inhibition of cell growth
(cytotoxicity) caused by the test material. When cell-free aliquots of the medium from
cultures with different treatments are assayed, then the amount of LDH activity can be used as
an indicator of relative cell viability as well as a function of membrane integrity.
Membrane integrity of RINm5F pancreatic β-islet cell was assessed by measuring
extracellular lactate dehydrogenase (LDH) using TOX-7 kit as per the supplier‟s protocol
(Sigma-Aldrich Inc, UK). Briefly the cells were cultured and treated for 24 hour with 0.1 -
100 nM curcuminoids in presence of 50 μM H2O2. 45 minutes before the end of 24 hours
incubation LDH assay lysis solution was added to the control untreated cells. Cells were
pelleted at 250 x g for 4 minutes using a plate centrifuge and 50 μl supernatant was
transferred to a new flat-bottom 96-well plate. 100 μl of lactate dehyrogenase assay mixture
was added and the plates were incubated at room temperature for 30 minutes covered with
aluminium foil in dark. 1.5 μl of 1 N HCl was added to each well to terminate the reaction and
absorbance of the resulting solution was measured at 490 and 690 nm using a microplate
reader (Varioskan Flash from Thermo Scientific). The amount of LDH leakage into media is
reversely correlated with cell viability. Each sample was measured in duplicate and
experiments with each test substances were performed on six separate occasions (n=6).
The percent of viable cells was determined according to the formula,
% of total LDH leakage = (λ490 - λ690) treated cells X 100
(λ490 - λ690) untreated lysed cells
where λ490 is the absorbance of tetrazolium dye at 490 nm and λ690 is background absorbance
at 690 nm.
41
2.2.6 Insulin assay
The insulin assay was performed as a marker of cell functionality after treatment with the
curcuminoids and H2O2. Insulin concentrations in the supernatant were measured using Ultra
Sensitive Rat Insulin ELISA Kit (Crystal Chem Inc., Downers Grove, IL, USA) according to
the manufacturer's instruction. Pilot experiments were performed to evaluate the effect of
various insulin secretagogues and inhibitors on insulin production in these cells with a view to
establishing the limitations of the model, given the high glucose concentration already present
in the medium (11.1 mM). RINm5F pancreatic β-islet cells were cultured in RPMI1640
phenol-red free medium containing 11.1 mM glucose. In pilot experiments RINm5F
pancreatic β-islet cells were treated with 10 mM L-arginine and 400 nM glibenclamide as
insulin secretagogues and with 10 mM L-cysteine as an insulin inhibitor for 1 hour. In
separate experiments, RINm5F cells were treated with 0.1 - 100 nM curcuminoids both in the
presence and absence of 50 μM H2O2 for 24 hours at 37 °C in 5 % CO2 in RPMI 1640 phenol-
red free medium (supplemented with 11.1 mM D-glucose). Insulin concentration in the
medium was measured according to the supplier‟s protocol. Briefly, 1:50 diluted sample
medium was placed in antibody-coated 96-well microplates and incubated at 4 °C for 2 hours
followed by five-times washing and adding anti-rat insulin enzyme conjugate antibody and
incubating for a further 30 minutes at room temperature. At the end of incubation time, wells
were washed seven times, followed by addition of enzyme substrate solution and incubation
in dark at room temperature for further 45 minutes. 1 N sulphuric acid was added to each well
to terminate the reaction and absorbance of the resulting solution was measured at 450 and
630 nm using a microplate reader (Varioskan Flash from Thermo Scientific). A standard
curve was constructed using the provided insulin standards. The concentration of insulin in
samples was derived from the standard curve. Experiments with each test substances were
performed on four separate occasions (n=4).
42
2.2.7 Glutathione (GSH) assay
Total-GSH (GSH + GSSG, in GSH equivalents) and GSSG levels were measured in RINm5F
cells (n=6, independent experiments) treated with 0.1 - 1 μM curcuminoids in presence of 50
μM H2O2 by means of the enzyme recycling assay according to the assay protocol of Rahman
et. al., [185].
A) Preparation of cytosolic extract: RINm5F cells were cultured in six-well plate till they
reached about 70-80 % confluence, followed by 24 hours treatment with 0.1 - 1 μM curcumin
and analogues independently or in presence of 50 μM H2O2. The cells were harvested by
treating with trypsin-EDTA, transferred to eppendorf tubes and washed two times with PBS
by centrifuging at 1,000 rpm for 5 minutes at 4 oC. The supernatant was discarded leaving ~
30 μl covering the pellet. 1 ml of ice-cold extraction buffer [0.1 % triton + 0.6 %
sulfosalicylic acid in 0.1 M phosphate buffer with 5 mM EDTA, pH 7.5 (prepared in PBS
instead of dH2O)] was added followed by vortexing 20 seconds and then sonication for 30
seconds, (2 times). To ensure complete lysis of cells an additional freezing at –70 oC for 10
minutes, followed by defrosting on ice for 2 times was also performed. After the second
freeze–thawing cycle, the cells were centrifuged at 5000 rpm for 5 minutes and immediately
the supernatant was transferred to pre-chilled eppendorf tubes which were used for protein,
total-GSH and GSSG measurement.
B) Detection of total-glutathione: GSH standards [26.4 - 0.41 μg/ml] were prepared in 0.1 M
phosphate buffer with 5 mM EDTA, pH 7.5 (prepared in PBS instead of d-H2O) and used to
construct a calibration curve. All other reagents a) 2 mg of 5,5‟-dithio-bis(2-nitrobenzoic
acid) (DTNB) also known as Ellman‟s reagent b) 2 mg of β-NADPH c) 40 μl glutathione
reductase (250 units/ml) were prepared in 3 ml of 0.1 M phosphate buffer with 5 mM EDTA,
pH 7.5 (prepared in PBS instead of d-H2O). Total-GSH was measured by adding 20 μl of PBS
to well A (blank) and 20 μl of each standard to wells B-H in column 1, followed by addition
of 20 μl of sample to wells A-H in columns 2 and 3. Equal volumes of DTNB and GSH
43
reductase solutions were mixed together and 120 μl of it was added to each well. 30 seconds
were allowed for conversion of GSSG to GSH, followed by addition of 60 μl of β-NADPH.
Immediately the absorbance was read at 412 nm in a microplate reader (Varioskan Flash
spectral scanning multimode reader from Thermo Electron Corporation) and measurements
were taken every 30 seconds for 2 minutes (5 readings in total). The rate of 2-nitro-5-
thiobenzoic acid formation (change in absorbance per min) was calculated and the actual total
glutathione concentration in the samples was determined by comparing with the standard
curve.
C) Detection of GSSG: Similarly, GSSG standards [26.4 - 0.41 μg/ml] were prepared in 0.1
M phosphate buffer with 5 mM EDTA, pH 7.5 (prepared in PBS instead of d H2O) and used
to construct a calibration curve. 100 μl of sulfosalicylic acid cell extract was transferred to 1.5
ml eppendorf tube followed by addition of 2 μl 2-vinylpyridine [diluted 1:10 in 0.1 M
phosphate buffer with 5 mM EDTA, pH 7.5 (prepared in PBS instead of d H2O)] to derivatize
GSH. The derivatization was carried out for 1 hour in a fume hood, followed by addition of 6
μl of trietanolamine [diluted 1:6 in 0.1 M phosphate buffer with 5 mM EDTA, pH 7.5
(prepared in PBS instead of d H2O)] to neutralize the derivatization reaction. The derivatized
samples were further assayed as per the method mentioned in step B for the total glutathione
measurement. GSSG standards (containing 2-vinylpyridine and triethanolamine) and sample
blanks containing only 2-vinylpyridine and triethanolamine were also assayed.
The protein concentration of the sulfosalicylic acid cell extracts was calculated as per the
supplier‟s protocol using Coomassie Plus (Bradford) protein assay kit obtained from Pierce,
Leicestershire, UK. Total-GSH, GSSG and calculated GSH concentration (by subtracting the
measured GSSG values from total-GSH) was expressed as nmoles/mg protein. Experiments
with each test substances were performed on six separate occasions (n=6).
44
2.2.8 SOD assay
To ascertain whether plasma achievable nanomolar concentration of curcuminoids had any
influence on intracellular SOD activity in RINm5F pancreatic β-islet cells, a SOD activity
assay was performed using a superoxide dismutase assay kit (Cayman Chemical, Ann Arbor,
MI). SOD assay makes use of tetrazolium salt for detection of superoxide radicals generated
by xanthine oxidase and hypoxanthine. In brief, the RINm5F pancreatic β-islet cells treated
for 24 hours at 37 °C in 5 % CO2 with curcuminoids alone and cotreated with 50 μM H2O2
were collected. The cells were resuspended in an ice-cold buffer containing 20 mM HEPES,
(pH 7.2), 1 mM EGTA, 210 mM mannitol and 70 mM sucrose and sonicated followed by
centrifugation at 4 °C for 15 minutes at 1500 x g. 10 μl of the resulting supernatant containing
both cytosolic SOD-1 (CuZnSOD) and mitochondrial SOD-2 (MnSOD) was added to a 96-
well plate already containing 200 μl radical detector (i.e. tetrazolium salt). 20 μl of xanthine
oxidase was added and the plate subjected to shaking on a plate shaker for 20 minutes.
Absorbance of the plate was measured at 450 nm using a microplate reader (Varioskan Flash
from Thermo Scientific). A standard curve was constructed using the provided SOD
standards. The concentration of SOD in samples was derived from the standard curve.
Experiments with each test substances were performed on five separate occasions (n=5).
2.2.9 Quantitative real time polymerase chain reaction (RTQ-PCR)
6.0 ~ 7.0 x 107 RINm5F pancreatic β-islet cells seeded in T-25 cm
2 flasks were treated with
0.1nM curcuminoids in the presence or absence of 50 μM H2O2 for 24 hours at 37 °C in 5 %
CO2. The cells were collected by trypsinization and subjected to RNA extraction using Illustra
Quick PrepTM
Micro mRNA purification kit (GE healthcare life sciences, Buckinghamshire,
UK) as per supplier‟s protocol. NanoDrop®
ND-1000 Spectrophotometer (Thermo Fisher
Scientific Inc.) was used to quantify the extracted RNA. Briefly, 1 µl sample was placed on a
clean measurement pedestal and measured at 260 and 280 nm. If the RNA yield was in the
45
range 20-40 ng/μl, cDNA was synthesised using a high capacity cDNA reverse transcription
kit (Applied Biosystems, Warrington, UK) as per manufacturer‟s protocol. SYBR® Green
PCR Master Mix (Applied Biosystems, Warrington, UK) was used to perform RTQ-PCR on
cDNA. GCL (both catalytic and modifier), GPx1, GR, SOD1 and SOD2 primers (tebu-bio,
Peterborough, UK) were used for RTQ-PCR. Experiments with each test substances were
performed on six separate occasions (n=6).
2.2.10 NF-κB activation assay
RINm5F pancreatic β-islet cells seeded in 6-well plates were treated with 10, 100 and 1000
nM curcuminoids independently or in the presence of either 50 μM H2O2 or 10 μg/ml TNF-α
for 30, 60 and 240 minutes at 37 °C in 5 % CO2. At the end of this incubation, the cells were
washed two times with ice cold PBS and collected by scraping and stored in cold PBS on ice.
Supernatant was removed by centrifuging the cells at 4 °C for 5 minutes at 1,000 rpm and the
pellet resuspended in 0.4 ml buffer A containing 10 mM HEPES, 10 mM potassium chloride
(KCl), 2 mM magnesium chloride (MgCl2), 1 mM dithiothreitol, 0.1 mM EDTA, 0.2 mM
sodium fluoride (NaF), 0.2 mM sodium orthovanadate (Na3VO4) along with 1 μg/ml
leupeptin* and 0.4 mM phenymethylsulfonyl fluoride (PMSF)*, * added to buffer A just
before the use and incubated for further 15 minutes on ice. 25 l of buffer B containing 10 %
Nonidet P40 in ddH20 was added and the tubes subjected to hard vortexing for 15 seconds,
followed by centrifugation at 4 C for 30 seconds at 13,000 rpm. The supernatant containing
cytosolic fraction was transferred to another tube while the pelleted nuclei fraction was
resuspended in buffer C containing 50 mM HEPES, 50 mM KCl, 300 mM NaCl, 0.1 mM
EDTA, 1 mM DTT, 10 % glycerol, 0.2 mM NaF, 0.2 mM Na3VO4 along with 0.1 mM PMSF
added just before the use. The pellet was resuspended by placing on a shaker in ice for 20
minutes. The suspension was subjected to centrifugation at 4 C for 5 minutes at 13,000 rpm.
Nuclear extract fraction present in the supernatant was transferred to a new cold Eppendorf®
46
tube and stored at -80 C. Protein concentration in cellular nuclear extracts was measured
using Coomassie Plus (Bradford) protein assay kit (Pierce, Leicestershire, UK) as per the
supplier‟s protocol.
Cellular nuclear extracts were used to measure the activity of NF-κB (p65 subunit) by
TransAM NF-κB p65 Chemi Transcription Factor Assay Kit (Active Motif, Belgium), in
accordance with the protocol provided with the kit. Briefly, based on the protein
concentration in the samples they were diluted such that 2 μg of protein would be present in
20 μl of sample. Samples were added to a microtiter plate coated with an oligonucleotide
containing the κB site of the immunoglobulin light chain gene promoter (5'-GGGACTTTCC-
3') along with 30 μl of binding buffer and incubated at room temperature for 1 hour with mild
agitation on a plate shaker. Wells were washed three times with 200 μl of washing buffer
followed by addition of 50 μl NF-κB antibody and a further incubation for 1 hour without
agitation. The wells were washed again three times with 200 μl of washing buffer followed by
a further incubation for 1 hour with 50 μl horseradish peroxidase-conjugated secondary
antibody without agitation. At the end of incubation, wells were washed a further four times
with 200 μl washing buffer. 50 μl chemiluminescent substrate (brought to room-temperature)
was added taking care so as to minimize the exposure to light. A microplate reader (Varioskan
Flash from Thermo Scientific) was used to measure the chemiluminescence. Experiments
with each test substances were performed on four separate occasions (n=4).
2.2.11 Statistics
If not mentioned otherwise, data from six independent experiments are shown ± SEM.
Statistical significance among multiple groups was tested with the One-way ANOVA and
Dunnett post-test using Prism version 5.00 software (GraphPad Software, San Diego, CA).
47
2.2.12 Cell culture
Pancreatic β-islet RINm5F cells, with insulin secreting properties, were obtained from ATCC
and grown in RPMI1640 cell culture medium. The medium was supplemented with 10 %
(v/v) fetal bovine serum and 1 % (v/v) penicillin-streptomycin. For experiments, 106 - 10
7
cells were treated with test compounds and harvested after 24 hour incubation with tested
compounds for the various assays. To maintain the exponential growth conditions, all cells
were routinely split twice a week and were cultured for no more than 40 passages after
thawing from stock.
48
Chapter 3: Characterisation of commercially available
curcuminoids: purity and free radical scavenging
capabilities
3.1 Introduction
Differences in the chemical characteristics of common curcuminoids curcumin, demethoxy-
curcumin (DMC) and bisdemethoxy-curcumin (BDMC) may influence their biological
activity. At the outset of this project, it was important to characterize curcuminoids from
different sources for their purity, susceptibility to oxidation in the presence of reactive oxygen
species (ROS) and their oxygen-centred radical and NO scavenging potential.
There are a number of methods reported for the quantitative analysis of curcuminoids.
Spectrophotometry is popular for assessment of high concentrations of curcuminoids [186],
but is not very sensitive and would not be capable of discriminating between different
curcuminoids in a mixture. As previously mentioned in general methods, high performance
thin layer chromatography (HPTLC), high performance liquid chromatography (HPLC),
capillary electrophoresis, pH-zone-refining counter-current chromatography, supercritical
fluid chromatography and mass spectrometry [187, 188, and 189] have also been reported for
analysis of curcuminoids. However, a method for simultaneous determination of
curcuminoids in a mixture has not been reported in any of these studies. Use of modifications
to the stationary phase, such as amino bonded and styrene divinylbenzene copolymer, and
gradient elution, have failed to yield acceptable curcuminoid separation. Despite the apparent
lack of convincing data, most, if not all, studies have used curcuminoids of unknown purity as
reference standards, relying simply on suppliers‟ recommendations as to the purity of their
compounds.
49
A number of studies in cell culture, tissues and in vivo [190-194] suggest that curcumin has
antioxidant properties. Equally, the free radical scavenging capability of curcumin,
particularly with respect to superoxide and hydroxyl ions [120, 195], is often attributed to its
H-donating phenolic groups [133, 196, 197, and 198]. However, the low plasma and tissue
levels of curcuminoids due to poor absorption, rapid metabolism and rapid systemic
elimination questions whether sufficient amounts of these agents can truly make a significant
contribution to the antioxidant pool. Thus, there is a need to determine the impact of direct
antioxidant scavenging potential of curcuminoids and to assess whether different analogues
have different activity in terms of free radical scavenging. Also, previous studies report that
curcumin has an indirect impact on nitric oxide through inhibition of inducible NO synthase
(iNOS) in activated macrophages [199, 200, and 201] but it is not clear whether curcuminoids
can scavenge NO directly.
The aims of the experiments described in this chapter were, firstly, to establish the purity of
the various preparations of curcuminoids that had been obtained from Sami Labs, India and
Sigma-Aldrich Inc., UK, as well as their stability in the presence of free radicals. Secondly,
experiments were devised to investigate the direct antioxidant and NO scavenging potential of
curcumin, DMC, BDMC and a commercially available mixture of curcuminoids. The results
would inform the studies described in subsequent chapters.
3.2 Results
3.2.1 Purity of curcuminoids
In the present study, a simple HPLC method was developed for the determination of purity of
curcuminoids obtained from various sources. To optimize the HPLC parameters, several
mobile phase compositions were evaluated: for example, increasing the water content of the
mobile phase was accompanied by increased peak separation, but at the same time it
prolonged the run time (fig 3.1: A). On the other hand, when the amount of acetonitrile was
50
increased in the mobile phase, there was a decrease in the run time, though peaks had poor
resolution (fig 3.1: B). It was found that with 50:50 v:v concentrations of water:acetonitrile,
optimal peak separation and resolution were obtained. Flow rates of mobile phase were
varied between 0.5 - 2 ml/min with no any noticeable difference in chromatogram peaks.
Mobile phase flow rate of 1.5 ml/min was used during the subsequent experiments. There was
a linear relationship between injection volume (10 l - 40 l) and the observed peak height on
the HPLC chromatogram (fig 3.2: A and B). An injection volume of 10 l was used in the
subsequent experiment. Curcumin, DMC and BDMC procured from Sami labs, India, each
generated a single peak with retention time at 3.1, 2.9 and 2.6 minutes respectively (fig 3.3:
A, B and C). On the other hand, commercially obtained “pure” curcumin from Sigma-
Aldrich, UK, generated 3 peaks at 3.1, 2.9 and 2.6 minutes, which coincide with those for
pure curcuminoids (fig 3.4: D). Based on the observed peak heights from the chromatogram
of commercial curcumin the approximate concentrations of curcumin, DMC and BDMC in
this mixture were found to be ~ 160 %, 55 % and 19 % respectively.
AB
51
Figure 3.1 HPLC chromatograms after 30 minutes incubation at 37 oC for 200 μM curcumin using
mobile phase consisting of A) acetonitrile: water (20:80 v:v); B) acetonitrile:water (80:20 v:v) monitored
at =420nm.
BB
AB
52
Figure 3.2 HPLC chromatograms after 30 minutes incubation at 37 oC for 200 μM curcumin using
mobile phase consisting of acetonitrile: water (50:50 v:v) and injection volume of A) 20 μl and B) 40 μl
monitored at =420nm.
AB
BB
53
BB
CB
54
Figure 3.3 HPLC chromatograms after 30 minutes incubation at 37 oC and a run time of 10 minutes
for A) 200 μM curcumin B) 200 μM DMC; C) 200 μM BDMC; D) 200 μM curcumin from Sigma-Aldrich
Inc., monitored at =420nm
3.2.2 Effect of co-incubation with menadione on stability of curcuminoids
Curcuminoid samples co-incubated with menadione at 37 oC for 30 minutes failed to affect
either the amplitude or retention time of the peaks generated for the curcuminoids in the
absence of oxidants.
Figure 3.4 HPLC chromatogram after 30 minutes incubation at 37 oC for and a run time of 10 minutes
for 200 μM commercial curcumin with 200 μM menadione at 37 oC.
D
55
3.2.3 EPR analysis
Preliminary experiments were performed to confirm radical generation by pyrogallol,
menadione, and to establish the minimum concentration of tempone-H to be used as spin-trap
to generate measurable spin adduct in a reasonable time-frame. The results from these
experiments indicated that menadione as well as pyrogallol caused a concentration-dependent
oxidation of tempone-H. The optimal respective concentrations used during subsequent
experiments were: menadione, 150 M; pyrogallol, 150 M; tempone-H, 50 M.
Co-treatment of curcumin, DMC, BDMC and commercial curcumin with menadione caused a
concentration-dependent depression of EPR signal (fig 3.5: A, B, C and D); the concentration
of each curcuminoid necessary to cause 50 % reduction in maximal response were found to be
16.0 ± 0.5, 16.0 ± 0.1, 16.0 ± 0.2 and 15.1 ± 0.2 M respectively.
On the other hand, none of the curcuminoid preparations successfully depressed the EPR
signal associated with pyrogallol, even at concentrations as high as 1 mM (fig 3.6: A, B, C
and D). By way of comparison, results obtained by another individual in the laboratory for
ascorbic acid under the same oxidising conditions with menadione or pyrogallol yielded
results of 79 M and 199 M required to cause a 50 % reduction in maximal response.
56
0 15 30 45 600
50
100
150
200150M Mena
1M Cur + 150M Mena
5M Cur + 150M Mena
10M Cur + 150M Mena
20M Cur + 150M Mena
Time (min)
EP
R S
ign
al In
ten
sit
y (A
U)
A
0 15 30 45 600
50
100
150
200150M Mena
1M DMC + 150M Mena
5M DMC + 150M Mena
10M DMC + 150M Mena
20M DMC + 150M Mena
Time (min)
EP
R S
ign
al In
ten
sit
y (A
U)
B
57
Figure 3.5 EPR spectrum intensity measured by conversion of spin trap, Tempone-H.HCl to 4-oxo tempo due to hydroxyl radicals generated by menadione in presence of: A) Curcumin B) Demethoxycurcumin; C) Bisdemethoxycurcumin; D) Sigma curcumin. Results expressed are mean of 6 independent experiments, ± SEM.
0 15 30 45 600
50
100
150
200150M Mena
1M BDMC + 150M Mena
5M BDMC + 150M Mena
10M BDMC + 150M Mena
20M BDMC + 150M Mena
Time (min)
EP
R S
ign
al In
ten
sit
y (A
U)
0 15 30 45 600
50
100
150
200150M Mena
1M Sig Cur + 150M Mena
5M Sig Cur + 150M Mena
10M Sig Cur + 150M Mena
20M Sig Cur + 150M Mena
Time (min)
EP
R S
ign
al In
ten
sit
y (A
U)
C
D
C
58
0 15 30 45 600
20
40
60
80
100
120
150M Pyro
100M DMC + 150M Pyro
200M DMC + 150M Pyro
500M DMC + 150M Pyro
1000M DMC + 150M Pyro
Time (min)
EP
R S
ign
al In
ten
sit
y (
AU
)
B
0 15 30 45 600
20
40
60
80
100
120
150M Pyro
100M Cur + 150M Pyro
200M Cur + 150M Pyro
500M Cur + 150M Pyro
1000M Cur + 150M Pyro
Time (min)
EP
R S
ign
al In
ten
sit
y (A
U)
A
59
Figure 3.6 EPR spectrum intensity measured by conversion of spin trap, Tempone-H.HCl to 4-oxo tempo due to superoxide radicals generated by pyrogallol in presence of: A) Curcumin B) Demethoxycurcumin; C) Bisdemethoxycurcumin; D) Sigma curcumin. Results expressed are mean of six independent experiments, ± SEM.
0 15 30 45 600
20
40
60
80
100
120
150M Pyro
100M BDMC + 150M Pyro
200M BDMC + 150M Pyro
500M BDMC + 150M Pyro
1000M BDMC + 150M Pyro
Time (min)
EP
R S
ign
al In
ten
sit
y (
AU
)
0 15 30 45 600
20
40
60
80
100
120
150M Pyro
100M Sig Cur + 150M Pyro
200M Sig Cur + 150M Pyro
500M Sig Cur + 150M Pyro
1000M Sig Cur + 150M Pyro
Time (min)
EP
R S
ign
al In
ten
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U)
C
D
60
3.2.4 NOATM
analysis
There was a rapid increase in NO signal detected by the NOA on addition of 100 μM DETA
NONOate. The signal reached a plateau at ~50 mV within ~ 5 minutes, at which point
increasing concentrations (1 nM to 5 μM) of a curcuminoid was injected into the purge vessel
in a cumulative manner (fig 3.7). None of the tested curcuminoids had any significant effect
on NO generation in this setting.
Figure 3.7 NO analyser plot showing a rise in the measured signal intensity on addition of DETA NONOate at 20 minutes followed by addition of 1nM-5µM curcumin at 32, 35, 37, 39 and 41 minutes respectively.
61
3.3 Discussion
3.3.1 Purity and stability of curcuminoids
In recent years, curcumin has attracted considerable attention due to its potentially beneficial
properties, giving rise to the suggestion that it might be an important drug candidate to
prevent and/or treat a number of diseases. It is a natural product isolated in abundance from
Curcuma species, although it can be readily synthesized [202]. It has been isolated from the
natural sources using recrystallization techniques [203, 204], but this carries challenges, most
notably a low yield, primarily on account of the difficult and complicated procedures required
for isolation and purification of the product. Curcumin is readily prepared to about 70 %
purity, with DMC, BDMC and other minor curcuminoids constituting the balance. Large
scale preparation of at least 99 % pure curcumin using conventional chromatographic or
recrystallization technique has proved quite difficult because of the similar physical properties
of these curcuminoids.
For quantitative curcumin analysis, several HPLC techniques have been developed during the
past 3 decades. Curcuminoid separation using an C18 column with a gradient solvent system
comprising methanol, acetonitrile, and 2 % acetic acid has been recently reported to determine
individual curcuminoids [177], where the. Another report on separation of curcuminoids
[178] used CLC-NH2 5 μm column with isocratic solvent system (ethanol:water 85:15, v:v).
However, since ethanol is an azeotrope, its use in the mobile phase with different water
mixtures can lead to compositional variations and variable results. Another HPLC method for
the separation of curcuminoids using fluorescence detection has also been reported [205,
206], but in all these cases, reproducible results with respect to optimal resolution and
baseline separation of peaks tends to be difficult to achieve, particularly when mixtures of
curcuminoids were analyzed.
62
The literature suggests that an amino-bonded stationary phase gives poor resolution of the
peaks and also has a catalytic effect upon curcumin degradation [170], so silica-bonded
(referred as sol-gel silica by the manufacturer); Eclipse XDB-C18 column was preferred as
the stationary phase for this study. In comparison to the results from various published articles
making use of citric acid, acetic acid and orthophosphoric acid as ionization suppressants,
TFA produced superior peak symmetry perhaps due to partial deactivation of the unmasked
silica sites on the stationary phase [207]. On the basis of these various optimisation
experiments, a mobile phase consisting of acetonitrile:water (50:50 v:v), and 0.1 % TFA
(adjusted to pH 3.0 with ammonia), at a flow rate of 1.5 ml/min and injection volume of 10
μl was used for analysis of curcuminoids.
Curcumin, DMC and BDMC provided as a gift by Sami Labs, India were found to be
relatively clean, on account of a single, clear peak with retention times of 3.1, 2.9 and 2.6
minutes respectively (fig 3.3: A, B, and C). On the other hand, commercially available
(Sigma-Aldrich) “pure” curcumin yielded three peaks on the chromatogram (fig 3.3: D), with
retention times that corresponded to the pure curcuminoids above, thus indicating that it was a
mixture of all the three (relative proportions based on peak heights ~ 160 %, 55 % and 19 %
respectively).
Previous reports suggest that curcumin is liable to degradation under varying pH conditions,
and also is not stable in different physiological matrices [208]. Representative experiments
performed with a mixture of curcumin and hydroxyl radical generator, menadione (fig 3.4),
after 30 minutes incubation at 37 oC showed peaks coinciding with individual sample
chromatograms, suggesting that there was no oxidative modification of any of the various
curcuminoids tested. Given that there was neither a decrease in the peak height or retention
time, nor formation of any new additional peaks, it is reasonable to conclude that any
oxidation of the samples under these conditions did not cause sufficient changes to the
63
characteristics of the curcuminoids to alter their column retention.Further experiments to
determine the effects of pyrogallol and DETA NONOate on curcuminoids are needed to be
carried out to ascertain the impact of these radical generators.
3.3.2 Direct radical scavenging activity of curcuminoids
Free radicals, owing to their unpaired electron, possess the property of paramagnetic
resonance. Unpaired electrons are magnetic on account of their ability to rotate. The spin trap,
tempone-H.HCl, on reaction with a free radical, is oxidized to the more stable 4-oxo-tempo
radical, which can be detected by EPR. The EPR signal amplitude thus measured is
proportional to the amount of 4-oxo-tempo radical generated [184, 209 and 210]. Thus, if a
free radical scavenger is present it would compete with the spin trap for the free radical,
causing a corresponding depression in the EPR signal. Use of menadione and pyrogallol as
hydroxyl and superoxide radical generators helped to ascertain the specificity of the free
radical scavenger towards these specific radicals. Optimisation of reactants was important
because concentrations of menadione and pyrogallol if very high would cause the spin trap to
saturate too quickly. Also as this is a competitive assay between spin trap and antioxidants, an
increase in the concentration of tempone-H.HCl would cause the concentration of antioxidant
to be increased to give the same depression as would have been observed employing lower
concentration of spin trap.
Initial experiments were performed to establish the radical generation by these compounds.
The result from these experiments indicated that menadione and pyrogallol caused a
concentration and time-dependent generation of oxidising radicals. The EPR study showed
that all of the curcuminoids tested were capable of scavenging hydroxyl radicals generated by
menadione (fig 3.5: A, B, C and D). The concentration of each curcuminoid necessary to
cause a 50 % reduction in maximal response were calculated to be 16.0 ± 0.5, 16.0 ± 0.1, 16.0
± 0.2 and 15.1 ± 0.2 M for curcumin, DMC, BDMC and commercially available (Sigma-
64
Aldrich) curcumin respectively, indicating that there was negligible difference in scavenging
activity between the various curcuminoids. It can thus be concluded that the methoxy group(s)
(2, 1 and 0 methoxy groups present in curcumin, DMC and BDMC respectively) are not
important in determining direct hydroxyl radical scavenging. However, the current study is
not able to distinguish the relative importance of the β-diketo group, the central methylenic
group or the phenolic groups in determining the reactivity towards the hydroxyl radical.
Further experiments using curcuminoid analogues synthesised with or without β-diketo group,
the central methylenic group or the phenolic groups would give more insight about their
relative importance in direct radical scavenging activity.
In contrast to the results with hydroxyl radicals, none of the curcuminoids showed any
antioxidant activity when challenged with pyrogallol-derived superoxide (fig 3.6: A, B, C and
D), even at concentrations of 1 mM (20 x that of tempone H). These results indicate a number
of things: 1. the radicals generated by pyrogallol are different from those generated by
menadione 2. curcuminoids are unable to effectively compete with tempone-H for these
radicals; ascorbate tested by another individual in the laboratory indicated that it was able to
scavenge radicals generated from both menadione and pyrogallol.
Earlier studies report contradictory interpretations in relation to the antioxidant activity of
curcumin. The central argument that arises from these studies surrounds whether the phenolic
ring or the β-diketo group or the central methylenic group in the heptadienone moiety is
responsible for the antioxidant activity of curcuminoids. According to Tonnesen et al. [211],
the diketone moiety of curcumin appears to be the part of the molecule involved in the
scavenging of oxygen-centred radicals, although the mechanism of action is not fully
understood. Sreejayan and Rao [166], on the other hand, have claimed that the phenolic group
is essential for the free radical scavenging activity and that the presence of the methoxy group
further increased the activity. Priyadarsini et al. [212] have also claimed that the phenolic
65
group is essential for the free-radical-scavenging activity and that the presence of the methoxy
group further increased the activity. Khopde et al. [213] followed the transient absorption at
=490–500 nm, which they took to be the evidence of phenoxyl radicals, thus supporting the
previous authors. However, Tonnesen and Greenhill [214] found that a curcumin analogue
lacking both a phenolic and methoxy group was just as active as curcumin in scavenging
hydroxyl radicals. Jovanovic et al. [215] proposed that curcumin is a superb H-atom donor
through transfer of H-atom from the central methylenic group rather than from the phenolic
group. Barclay et al. [196] showed that synthetic, non-phenolic, curcuminoids exhibited no
antioxidant activity in styrene/chlorobenzene solution, thus contradicting the findings of
previous authors.
It has become clear in recent years that NO is a vital biological messenger involved in the
cardiovascular and immune systems. Numerous studies report both the beneficial and harmful
effects of NO in the cardiovascular system. NO, via formation of peroxynitrite, has been
shown to cause harmful membrane lipid peroxidation leading to myocardial dysfunction [216,
217]. Many studies have also shown that enhanced formation of peroxynitrite
in the
myocardium contributes to ischemia/reperfusion injury both in vitro and in vivo, the
spontaneous loss of cardiac function, as well as cytokine-induced myocardial contractile
failure in isolated rat hearts and in dogs in vivo [218, 219 and 220]. In contrast, NO is
cardioprotective through coronary vasodilatation, and reduction of myocardial oxygen
consumption via upregulation of cGMP [221]. Both antiarrhythmic and anti-infarction [222]
effects of the NO donors have also been well documented. During infection and inflammation
there is an increased amount of NO generated through immunological stimulation and shows
cytotoxic or cytostatic activity against viruses and invasive organisms [223]. However, such
an increase in NO may also have adverse effects on host cells, thus causing tissue injury
[224]. Over expression of iNOS and thus its product nitric oxide [225], as well as ROS [226,
66
227] and peroxynitrite [228] is considered to be a major player in death and dysfunction of
isolated -islet cells.
Sreejayan and Rao [169] have reported that curcumin, along with DMC and BDMC, reduced
the amount of nitrite formed by the reaction between oxygen and NO generated from sodium
nitroprusside. There are various schemes proposed for release of NO from sodium
nitroprusside (SNP) in a review article by Butler and Megson [229]. They suggest that release
of NO from SNP is a complex process and it was therefore not found to be a very suitable
model in which to investigate the NO scavenging activity of curcumin, DMC and BDMC.
However, direct NO scavenging by curcuminoids using NOATM
has not yet been studied.
The results obtained in the studies described in this chapter indicate that none of the
curcuminoids tested showed any NO-scavenging activity. One of the reasons for such an
anomaly may be that the effects of curcumin in tissues might be mediated via its ability to
inhibit NO synthases [160] rather than direct NO scavenging by curcumin. Given that it is
known that some synthetic iNOS inhibitors prevent β-cell death in vivo [230], such a
possibility cannot be ruled out. Although the experimental set-up and results from this study
do not highlight the effect on NO synthase, it confirms that curcuminoids do not directly
scavenge NO. There is also reported evidence that curcumin is not a NO scavenger, rather it
shows ability to sequester nitrogen dioxide (NO2) that is formed as an intermediate in the
oxidation of NO to nitrite [231]. Also, given the fact that under pathological conditions, O2•-
reacts with NO to form a potent oxidant peroxynitrite it is reasonable to speculate from EPR
and NO analyzer results that curcuminoids do not have any impact on peroxynitrite
generation. Furthermore, literature reports that peroxynitrite decomposes to produce hydroxyl
radicals [417, 418 and 419].
NO• + O2•- ONOO-
ONOO- + H+ ONOOH
ONOOH •OH + NO2•
67
It is thus reasonable to hypothesize that curcuminoids may be able to affect the oxidative
process associated with generation of hydroxyl radicals from peroxynitrite based on the
hydroxyl scavenging activity evident from EPR studies.
3.4 Conclusions
HPLC analysis indicated that, curcumin, DMC and BDMC obtained from Sami Labs, India
are relatively pure, while the “curcumin” obtained from Sigma-Aldrich Inc.,UK was found to
be a mixture of curcumin, DMC and BDMC. Curcumin, DMC and BDMC were able to
scavenge hydroxyl radicals generated by menadione, but showed little scavenging ability
against superoxide radicals when tested by competitive EPR assay using tempone-H.HCl as
spin trap. The apparent selectivity for hydroxyl radicals is greater than with any other
antioxidants so far tested in our laboratory by another individual (including vitamin C,
glutathione and other thiols). None of the curcuminoids were able to scavenge the NO
generated using DETA NONOate. The fig 3.8 represents the conclusions that are derived
from this chapter.
68
Figure 3.8 Purity of curcuminoids and commercial curcumin analysed by HPLC and effect of curcuminoids and commercially obtained curcumin on hydroxyl and superoxide radical scavenging by EPR,; and on NO radical scavenging by NOA
TM analyser.
69
Chapter 4: Effect of bioavailable concentrations of
curcuminoids on pancreatic β-islet cell viability and
function
4.1 Introduction
The contribution of a depression in functional pancreatic β-islet cell mass to the development
of type 2 diabetes has been heavily debated for many years. Recently, a number of studies
have provided convincing evidence to support this hypothesis [232, 233, and 234]. Pancreatic
β-islet cell destruction in vitro may be effected through exposure to pro-inflammatory
cytokines such as TNF-α and IL-1β [235] or by extensive generation of reactive oxygen
species [236]. It is well established fact that pancreatic β-islet cells are more susceptible to
reactive oxygen species-induced toxicity compared to other cell types on account of the very
low level of antioxidant enzyme expression and activity in this particular cell type [237-240].
Thus, approaches towards prevention of pancreatic β-islet cell death while maintaining insulin
secretory function are of importance in moderating the progress of type 2 diabetes towards
insulin dependency.
As ROS-induced generation of oxidative stress is one of the important causes of pancreatic β-
islet cell death, utilization of antioxidants for protection of cells against oxidative stress is an
interesting therapeutic possibility. Thus, antioxidants, including vitamins C and E have been
used with some success for reduction of oxidative stress and protection of pancreatic -islet
cells against such an insult in vivo [22, 241, and 242]. Other antioxidants, including N-acetyl-
L-cysteine (NAC) have been shown to protect against nitrosative stress and associated
secondary complications in experimental diabetes [228]. Over expression of antiapoptotic or
antioxidant proteins such as thioredoxin [243], catalase [244], and metallothionine [245] have
also offered protection to pancreatic -islet cells from increased oxidative stress in vivo.
70
Additionally, treatment with antioxidants also salvages pancreatic -islets from oxidative
stress insult in vitro. For example rat pancreatic -islet cells were partially rescued from
streptozotocin induced oxidative stress damage in vitro by nicorandil, an antianginal and
antioxidant agent, by virtue of its radical scavenging effect [245]. Streptozotocin-induced
inhibition of insulin release and glucose metabolism in cultured rat pancreatic -islet cells and
cultured Min6 cells (mouse pancreatic β-islet cell line) is counteracted by melatonin and
polyenoylphosphatidylcholine (PPC) [246] [247].
Several studies have made use of hydrogen peroxide (H2O2) in an in vitro system, to generate
oxidative stress thus leading to oxidation of lipids, inactivation of enzymes, introduction of
DNA mutations, destruction of cell membranes and, ultimately, induction of pancreatic β-islet
cell death [248, 249 and 250]. The efficacy of curcumin has been widely observed in
recovering various diabetic secondary complications such as diabetic nephropathy/renal
lesions (in vivo) [251], retinopathy (in vivo) [252], wound healing (in vitro) [253, 254], and
levels of AGEs (in vivo) [150]. Curcumin treatment has been reported to increase the plasma
insulin level in streptozotocin-induced diabetic rats [255, 256] while contradictory results
reporting it as a hypoglycaemic agent in humans [257] have also been published. Curcumin
on oral administration through diet in streptozocin treated rats has been reported to reduce the
degree of systemic oxidative stress as well as retinal inflammation. In the same animal model
for diabetes, injection with a suspension of curcumin in carboxy methyl cellulose also reduced
diabetic nephropathy [251, 252 and 258]. Many studies have reported the in vitro efficacy of
curcumin in cytoprotection of various cell lines against toxicity [125, 259, 260, and 261]. The
importance of curcumin as a prophylactic on oral administration in vivo has been reported for
reduction of secondary complications in streptozotocin-induced diabetic animals [148, 158,
and 262] and in vitro as an islet protective agent against streptozotocin-induced death and
dysfunction by retarding and scavenging the generation of islet ROS [191]. It has also been
71
reported that inclusion of curcumin (10 μM) in islet cryopreservation medium enhances islet
viability after thawing and maintains islet functionality in culture [263]. Very recently, it has
also been reported that dietary curcumin (10 μM) scavenges the cellular ROS generated on
account of in vitro pro-inflammatory cytokines and also prevents the progression of multiple
low dose streptozocin induced diabetes in pancreatic β-islets isolated from C57/BL6J mice in
vivo [264].
Furthermore, although many aspects of curcumin-induced cytoprotection have been studied,
its efficacy in protecting RINm5F pancreatic β-islet cells against H2O2-induced free radical-
mediated damage has not been demonstrated so far. It was hypothesized that curcumin may
protect cultured pancreatic β-islet cells against H2O2-induced oxidative stress and resulting
cellular damage and dysfunction. Logically, some protection against oxidative damage to
pancreatic β-islet cells would be imparted by curcumin, but it would be of interest to
investigate the effect of such a protection on the functionality of cells in relation to insulin
secretion. To this end, implication of pathways regulating pancreatic β-islet cells turnover in
insulin secretory function and thus whether they act in concert or against will also become
clear. Depending on the prevailing curcumin and H2O2 concentrations and the intracellular
pathways activated, the findings will give an insight into whether A) H2O2 is deleterious to
pancreatic β-islet cell mass (and no significant protection is offered by curcuminoids) and
causes an inhibition or enhancement or no effect on insulin secretory function, B)
curcuminoids are protective to pancreatic β-islet cells (against H2O2) and inhibit or enhance or
have no effect on insulin secretory function.
As the fate of cells manifests via a variety of characteristics including varied cell population
[265], mitochondria and plasma membrane dysfunction [266], and fluctuating intracellular
reduction capacity as the result of oxidant-induced thiol oxidation and/or declining oxidant
defences [267], the cytotoxicity assay requires a selection of methods based on the prevailing
72
cellular situation. Measurement of lactate dehydrogenase (LDH) and glyceraldehyde-3-
phosphate dehydrogenase, which are expressed constitutively in mammalian cells and
released from damaged cells, is appropriate for the assessment of damaged membranes, such
as apoptosis-mediated membrane blebbing and necrosis-derived membrane disruption and
thus the cellular cytotoxicity associated with it [267, 268]. On the other hand, MTT enters the
cells via endocytosis and the reduction of MTT to a coloured insoluble formazan crystals
occur in the active mitochondria in viable cells, as well as outside the mitochondria in certain
cases [269, 270] and is used to verify mitochondrial reduction capacity and viability.
However, a tendency exists to randomly utilize various assay methods without taking the
cellular circumstances into consideration.
In the previous chapter it was shown that curcumin, DMC and BDMC obtained from Sami
Labs, India were pure, whilst curcumin obtained from Sigma-Aldrich, UK, was a mixture of
DMC, BDMC and curcumin. Curcumin, DMC and BDMC were able to scavenge hydroxyl
radicals generated by menadione, but showed little scavenging ability against superoxide
radicals and nitric oxide (NO). In this study in vitro effect of curcuminoids on H2O2-induced
oxidative stress in RINm5F pancreatic β-islet cells were studiedby using: a) LDH assay as a
measure of cytotoxicity, b) colorimetric MTT assay as a measure of mitochondrial function,
c) by quantifying insulin secretion as a measure of functionality.
4.2 Results
4.2.1 Cell mitochondrial function and membrane integrity by MTT and
LDH assay
Preliminary experiments were performed to test whether curcuminoids and H2O2 were (by
themselves) cytotoxic to RINm5F pancreatic β-islet cells. Cell mitochondrial functionality
and membrane integrity was assessed by the MTT assay and by the release of LDH into the
73
culture supernatant for the cells treated with increasing concentrations of curcuminoids
(ranging from 1 nM to 10 µM) and H2O2 (ranging from 5µM - 100 µM) for 24 hours. In
comparison to untreated RINm5F cells (100 % alive), those treated with 5, 25, 50, 75 and 100
µM H2O2 were found to have the % viability values 91.6 ± 1.2, 75.2 ± 1.7, 28.2 ± 1.9, 10.6 ±
1.4, 9.5 ± 0.7 (fig 4.1, A) respectively as calculated from MTT assay (Mean ± SEM, n=6).
The values obtained from the LDH assay for membrane integrity for cells treated with 5, 25,
50, 75 and 100 µM H2O2 in comparison to positive controls (100 % lysed) were 22.6 ± 0.9,
30.2 ± 1.4, 67.9 ± 2.2, 86.1 ± 2.4, 106.1 ± 4.9 respectively (fig 4.1, B) (Mean ± SEM, n=6).
Thus both the assays indicated that 50 µM H2O2 causes approximately 70 % cell death and
this is the concentration that was employed during the subsequent experimentation. Table 4.1:
A represents the mitochondrial functionality of 1 nM, 10 nM, 100 nM, 1 μM and 10 μM
curcuminoids treated cells (Mean ± SEM, n=6) in comparison to untreated cell controls
obtained by the MTT. Similarly the % membrane integrity for cells treated with curcuminoids
in comparison to untreated cell controls obtained by the LDH assay is given in table 4.1: B.
The values indicated that converse to H2O2 treatment; none of the curcuminoids up to the
highest concentration of 10 µM affected the cell mitochondrial and membrane integrity as
shown by both MTT and LDH assays.
A) MTT assay 1 nM 10 nM 100 nM 1 μM 10 μM
Curcumin 106.8 ± 1.4 99.0 ± 3.3 94.3 ± 2.1 95.0 ± 4.9 92.5 ± 2.3
DMC 107.3 ± 5.2 98.1 ± 3.7 104.3 ± 4.3 98.3 ± 3.6 101.0 ± 4.7
BDMC 109.5 ± 5.4 97.8 ± 3.0 105.5 ± 6.2 98.8 ± 5.7 98.6 ± 4.8
Curcumin from Sigma 106.8 ± 1.8 110.5 ± 2.4 98.5 ± 6.9 99.9 ± 6.3 93.2 ± 3.5
B) LDH assay 1 nM 10 nM 100 nM 1 μM 10 μM
Curcumin 17.7 ± 1.5 20.3 ± 2.6 21.2 ± 5.5 24.7 ± 3.9 25.3 ± 2.5
DMC 18.8 ± 3.8 21.4 ± 2.7 24.3 ± 3.6 26.8 ± 4.3 27.0 ± 6.9
BDMC 20.8 ± 3.8 18.3 ± 4.8 21.5 ± 3.6 23.9 ± 5.8 25.2 ± 6.2
Curcumin from Sigma 22.2 ± 3.3 25.7 ± 5.1 21.5 ± 3.2 23.5 ± 7.5 25.2 ± 7.2
Table 4.1 A) % cell alive in comparison to untreated cell controls on treatment with 1 nM-10 μM curcuminoids by MTT assay; B) % cytotoxicity in comparison to maximum lysis on treatment with 1nM-10 μM curcuminoids by LDH assay. Data is represented as Mean ± SEM for six independent experiments (n=6).
74
0
20
40
60
80
100
H2O2 5 M H2O2 25 M H2O2 50 M H2O2 75 M H2O2 100 M
MTT assay for RINm5F cells in presence of H2O2 (n=6)
Samples
Mit
oc
ho
nd
ria
l fu
nc
tio
n
(% o
f u
ntr
ea
ted
co
ntr
ol)
0
20
40
60
80
100
120
H2O2 5 M H2O2 25 M H2O2 50 M H2O2 75 M H2O2 100 M
LDH assay for RINm5F cells in presence of H2O2 (n=6)
Samples
LD
H l
ea
ka
ge
in
to m
ed
ia
(% o
f m
ax
imu
m l
ys
is)
Figure 4.1 A) MTT assay for mitochondrial function measurement in RINm5F cells treated with 5 - 100 µM H2O2 in comparison to untreated cell control (Mean ± SEM, n=6). B) LDH assay for membrane inegrity measurement in RINm5F cells treated with 5 - 100 µM H2O2 in comparison to maximum lysed cell control (Mean ± SEM, n=6).
During subsequent experiments, the effect of various concentrations of curcuminoids (ranging
from 0.1 nM to 100 nM) on the RINm5F cell death induced by 50 μM H2O2 were studied.
The results showed a concentration dependent prevention of the toxic effect of 50 μM H2O2 by
MTT assay. All the curcuminoids improved cell mitochondrial functionality and thus the
AB
B
75
viability in comparison to 50 μM H2O2 treated cells, as revealed by the MTT assay. There was
a concentration-dependent protective effect against H2O2-induced MTT reduction (fig 4.2, A
to D). 100 nM of curcumin, DMC, BDMC and sigma curcumin offered 68.9 ± 1.3, 72.9 ± 7.7,
83 ± 1.5 and 84.8 ± 5.4 % protection against H2O2-induced cell death respectively.
0
20
40
60
80
100
H2O2 50 M H2O2 50 M
+ Cur 0.1 nM
H2O2 50 M
+ Cur 1 nM
H2O2 50 M
+ Cur 10 nM
H2O2 50 M
+ Cur 100 nM
*****
***
*
MTT assay for curcumin in presence of H2O2 (n=6)
Samples
Mit
oc
ho
nd
ria
l fu
nc
tio
n
(% o
f u
ntr
ea
ted
co
ntr
ol)
0
20
40
60
80
100
H2O2 50 M H2O2 50 M
+ DMC 1 nM
H2O2 50 M
+ DMC 10 nM
H2O2 50 M
+ DMC 100 nM
MTT assay for DMC in presence of H2O2 (n=6)
***
***
***
*
Samples
Mit
oc
ho
nd
ria
l fu
nc
tio
n
(% o
f u
ntr
ea
ted
co
ntr
ol)
H2O2 50 M
+ DMC 0.1 nM
AB
BB
76
0
20
40
60
80
100
H2O2 50 M H2O2 50 M
+ BDMC 0.1 nM
H2O2 50 M
+ BDMC 1 nM
H2O2 50 M
+ BDMC 10 nM
H2O2 50 M
+ BDMC 100 nM
******
***
*
MTT assay for BDMC in presence of H2O2 (n=6)
Samples
Mit
oc
ho
nd
ria
l fu
nc
tio
n
(% o
f u
ntr
ea
ted
co
ntr
ol)
0
20
40
60
80
100
H2O2 50 M H2O2 50 M
+ Sigma 0.1 nM
H2O2 50 M
+ Sigma 1 nM
H2O2 50 M
+ Sigma 10 nM
H2O2 50 M
+ Sigma 100 nM
***
***
***
**
MTT assay for Sigma in presence of H2O2 (n=6)
Samples
Mit
oc
ho
nd
ria
l fu
nc
tio
n
(% o
f u
ntr
ea
ted
co
ntr
ol)
Figure 4.2 MTT assay (A to D) for viability measurement in RINm5F cells co-treated with 50 µM H2O2 in presence of A) Curcumin, B) DMC, C) BDMC, D) Sigma curcumin. Statistical significance was determined by ANOVA and Dunnett's multiple comparison post-test with 50 µM H2O2 as control using Prism version 5.00 software (GraphPad Software, San Diego, CA). (*** p<0.0001; ** p<0.001; * p<0.01 Mean ± SEM, n=6).
The concentrations of curcuminoids required to give a 50 % reduction in cell death were
derived from concentration–response relationships between the concentrations and %
inhibition of 50 μM H2O2-induced cell death. The calculated values were 1.9 ± 0.2, 1.1 ± 0.2,
0.6 ± 0.1, 0.8 ± 0.2 nM for curcumin, DMC, BDMC and sigma curcumin respectively (fig
4.3).
DB
CB
77
-11 -10 -9 -8 -7 -60
20
40
60
80
100
Cur
DMC
BDMC
Sigma curcumin
Log concentration (M)
Mit
och
on
dri
al
fun
cti
on
(% o
f u
ntr
eate
d c
on
tro
l)
Figure 4.3 Calculated values for protective effect of 0.1 - 100 nM curcuminoids in RINm5F cells co-treated with 50 µM H2O2, measured as mitochondrial functionality (MTT). Results are expressed as concentration required to give a 50 % reduction in cell death with SEM for six separate experiments. The experimental curves were fitted using a nonlinear regression model with a normalized response using Prism version 5.00 software (GraphPad Software, San Diego, CA).
In addition to the MTT assay, results from treatment of RINm5F cells with 50 μM H2O2
measured using the LDH assay indicated that there was disruption in almost ~70 % of cell
membranes causing release of lactate dehydrogenase (LDH) as compared to untreated cell
controls to which lysis buffer was added. Measurement of LDH released from the cytosol of
damaged RINm5F cells indicated that a concentration-dependent protection was offered by all
the curcuminoids against the toxic effect of 50 μM H2O2 (fig 4.4, A to D). 100 nM of
curcumin, DMC, BDMC and sigma curcumin treated cells showed 33.5 ± 3.2 %, 34.1 ± 3.7
%, 35.4 ± 3.3 % and 36.5 ± 6 % reduction in the LDH released as compared to 50 μM H2O2
treated cells respectively.
78
0
20
40
60
80
100
H2O2 50M H2O2 50M
+ Cur 0.1nM
H2O2 50M
+ Cur 1nM
H2O2 50M
+ Cur 10nM
H2O2 50M
+ Cur 100nM
******
***
LDH assay for curcumin in presence of H2O2 (n=6)
Samples
LD
H l
ea
ka
ge
in
to m
ed
ia
(% o
f m
ax
imu
m l
ys
is)
0
20
40
60
80
100
H2O2 50M H2O2 50M
+ DMC 0.1nM
H2O2 50M
+ DMC 1nM
H2O2 50M
+ DMC 10nM
H2O2 50M
+ DMC 100nM
LDH assay for DMC in presence of H2O2 (n=6)
*
******
Samples
LD
H le
ak
ag
e i
nto
me
dia
(% o
f m
ax
imu
m l
ys
is)
BB
AB
79
0
20
40
60
80
100
H2O2 50M H2O2 50M
+ BDMC 0.1nM
H2O2 50M
+ BDMC 1nM
H2O2 50M
+ BDMC 10nM
H2O2 50M
+ BDMC 100nM
**
******
LDH assay for BDMC in presence of H2O2 (n=6)
Samples
LD
H leak
ag
e i
nto
med
ia
(% o
f m
axim
um
lysis
)
0
20
40
60
80
100
H2O2 50 M H2O2 50 M
+ Sigma 0.1 nM
H2O2 50 M
+ Sigma 1 nM
H2O2 50 M
+ Sigma 10 nM
H2O2 50 M
+ Sigma 100 nM
******
LDH assay for Sigma in presence of H2O2 (n=6)
Samples
LD
H l
ea
ka
ge
in
to m
ed
ia
(% o
f m
ax
imu
m l
ys
is)
Figure 4.4 LDH assay (A to D) for cell membrane integrity measurement in RINm5F cells co-treated with 50 µM H2O2 in presence of A) Curcumin, B) DMC, C) BDMC, D) Sigma curcumin. Statistical significance was determined by ANOVA and Dunnett's multiple comparison post-test with 50 µM H2O2 as control using Prism version 5.00 software (GraphPad Software, San Diego, CA). (*** p<0.0001; ** p<0.001; * p<0.01 Mean ± SEM, n=6).
The calculated concentration required to give a 50 % reduction in maximum response (LDH
assay) were 125.8 ± 0.4, 123.3 ± 0.5, 114.6 ± 0.6, 113.8 ± 0.6 nM for curcumin, DMC,
BDMC and sigma curcumin respectively (fig 4.5).
CB
DB
80
-11 -10 -9 -8 -7 -60
20
40
60
80
100
Cur
DMC
BDMC
Sigma curcumin
Log concentration (M)
LD
H le
akag
e in
to m
edia
(% o
f m
axim
um
lysi
s)
Figure 4.5 Calculated values for protective effect of 0.1 - 100 nM curcuminoids in RINm5F cells co-treated with 50 µM H2O2, measured as membrane integrity (LDH). Results are expressed as SEM for six separate experiments. The experimental curves were fitted using a nonlinear regression model with a normalized response using Prism version 5.00 software (GraphPad Software, San Diego, CA).
Results from both MTT and LDH assays indicated that there was a increase in both the
mitochondrial functionality and membrane integrity in cells co-treated with 100 nM curcumin
and 50 μM H2O2. BDMC was found to be the most potent among all the tested curcuminoids
as evident from the MTT assay.
4.2.2 Effect of curcuminoids on Insulin secretion by RINm5F cells
Pilot experiments were performed to evaluate the effect of various insulin secretagogues and
inhibitors on insulin production in these cells with a view to establishing the limitations of the
model, given the high glucose concentration already present in the medium. 10 mM L-
arginine and 400 nM glibenclamide failed to show any significant impact on insulin secretion
compared to untreated control cells (6.4 ± 0.3; 8.4 ± 0.3 and 7.2 ± 0.7 ng/ml respectively).
However, a recognised inhibitor, L-cysteine (10 mM) caused a significant decrease in the
level of insulin secreted by the cells (2.4 ± 0.1 ng/ml; fig 4.6).
81
0
2
4
6
8
10
Untreated
cells
10 mM
L-arginine
10 mM
L-cysteine
400 nM
Glibenclamide
***
Insulin concentration for RINm5F cells treated with
L-arginine, L-cysteine and Glibenclamide (n=6)
Samples
Measu
red
In
su
lin
(ng
/ml)
Figure 4.6 Insulin secretion by RINm5F pancreatic β-islet cells in presence of 10 mM L-arginine, 10 mM L-Cysteine and 400 nM Glibenclamide. L-cysteine showed significant reduction in insulin secretion in comparison to untreated cells, while L-arginine and glibenclamide had no significant effect. Statistical significance was determined by one-way ANOVA and Dunnett’s post-test multiple comparisons with untreated control cells as control using Prism version 5.00 software (GraphPad Software, San Diego, CA). (Mean ± SEM, n=6).
Subsequent experiments were performed using curcuminoids (ranging from 0.1 - 100 nM)
independently or co-treated with 50 μM H2O2. None of the curcuminoid treatments by
themselves or in the presence of H2O2 caused any significant stimulation or inhibition of
insulin secretion as compared to untreated control cells (fig 4.7, A to D).
82
0
10
20
30
40
H2O2
50 M
Untreated
cells
Cur
0.1 nM
Cur
1 nM
Cur
10 nM
Cur
100 nM
Cur
0.1 nM
Cur
1 nM
Cur
10 nM
Cur
100 nM
+ H2O2
50 M
Insulin concentration for RINm5F cells treated
with curcumin and H2O2 (n=4)
Samples
Measu
red
In
su
lin
(n
g/m
l)
0
10
20
30
40
H2O2
50 M
Untreated
cells
DMC
0.1 nMDMC
1 nM
DMC
10 nM
DMC
100 nM
DMC
0.1 nMDMC
1 nM
DMC
10 nM
DMC
100 nM
+ H2O2
50 M
Insulin concentration for RINm5F cells treated
with DMC and H2O2 (n=4)
Samples
Measu
red
In
su
lin
(n
g/m
l)
AB
BB
83
0
10
20
30
40 Insulin concentration for RINm5F cells treated
with BDMC and H2O2 (n=4)
H2O2
50 M
Untreated
cells
BDMC
0.1 nM
BDMC
1 nM
BDMC
10 nM
BDMC
100 nM
BDMC
0.1 nM
BDMC
1 nM
BDMC
10 nM
BDMC
100 nM
+ H2O2
50 M
Samples
Measu
red
In
su
lin
(n
g/m
l)
0
10
20
30
40
H2O2
50 M
Untreated
cells
Sigma
0.1 nM
Sigma
1 nM
Sigma
10 nM
Sigma
100 nM
Sigma
0.1 nM
Sigma
1 nM
Sigma
10 nM
Sigma
100 nM
+ H2O2
50 M
Insulin concentration for RINm5F cells treated
with Sigma and H2O2 (n=4)
Samples
Measu
red
In
su
lin
(n
g/m
l)
Figure 4.7 Insulin secretion by RINm5F pancreatic β-islet cells treated with curcuminoids in comparison to untreated cells or co-treated with H2O2 in presence of all the four curcuminoids compared to 50 μM H2O2 showed no any significant change in insulin secretion. Statistical significance was determined by one-way ANOVA using Prism version 5.00 software (GraphPad Software, San Diego, CA). (p-values were 0.12, 0.11, 0.27, 0.41 for curcumin, DMC, BDMC and Sigma curcumin respectively. Mean ± SEM, n=4).
CB
DB
84
4.3 Discussion
It is well recognised that ROS produced as a result of chronic oxidative stress are not only
associated with development of type 2 diabetes but also play an important role in the long-
term development of associated complications [271-275]. Pancreatic β-islet cells continually
experience oxidative stress on account of AGE-formation, mitochondrial respiration as well
as elevated glucose concentrations leading to increased levels of reactive oxygen species in
type 2 diabetes [33, 273, 276, and 277]. The impact is exacerbated in this specific cell type
because it has intrinsically low antioxidant enzyme defences [237, 278]. In Type 2 diabetes,
excessive ROS impairs insulin synthesis [279-282], activates β-cell apoptotic pathways [283],
an orchestrated cellular event that occurs following oxidative and/or inflammatory stress and
thus leads to cell death [284, 285, and 286]. Previous studies have shown that treatment with
antioxidants improve pancreatic β-islet cell survival [287, 288].
H2O2 has been proposed to play a role in signal transduction for glucose stimulated insulin
secretion in pancreatic β-islet cells [289]. It has been used as a chemical inducer of oxidative
stress resulting in apoptosis in many different cell types [290, 291], and can be utilised in
models of oxidative stress for antioxidant testing [292]. In the present study, H2O2 was chosen
to induce oxidative stress in RINm5F pancreatic β-islet cells for the following reasons: (1)
oxidative stress is believed to be an important mediator of pancreatic β-islet cell death [17,
278, 293, and 294]; (2) H2O2 is a precursor via metal mediated reaction (Fenton) of highly
oxidizing, tissue-damaging radicals such as hydroxyl radicals, and is known to be toxic to
many systems [295-298]; (3) H2O2 plays a pivotal-role in oxidative stress because it is
generated from a wide range of sources and can readily diffuse in and out of tissues [299];
and (4) exogenous H2O2 can enter the cells and induce cytotoxicity due to its high membrane
permeability [300].
85
Though there are many studies reporting beneficial preventive activity of curcumin against
ROS induced pancreatic β-islet cell damage most, if not all, of previous experiments were
performed at concentrations (˃ μM) that are too high to be achieved in plasma after oral
ingestion. Preliminary concentration-response studies revealed that 50 μM H2O2 treatment for
24 hours caused, ~ 70 % reduction in mitochondrial function and membrane integrity as
measured by MTT and LDH assay respectively. Results from subsequent experiments
measuring the effect of nanomolar concentration of curcuminoids on H2O2-induced reduction
in mitochondrial function revealed a remarkably powerful protective effect of curcuminoids
by MTT assay, with the concentration required to cause a 50 % reduction in response to be <
10 nM for all the curcuminoid preparations tested. A Phase I clinical trial study reported that
the serum concentration of curcumin usually peaked at 1 to 2 hours after oral intake of
curcumin and gradually declined within 12 hours. The average peak serum concentrations
were 0.51 +/- 0.11, 0.63 +/- 0.06 and 1.77 +/- 1.87 μM after taking 4, 6, and 8 g of curcumin
respectively [301]. The calculated concentration required to cause a 50 % reduction in
response shows that this protective effect is in the nanomolar range for all the tested
curcuminoids and thus can be easily achieved in plasma. Interestingly, there were clear
differences in activity between the different curcuminoids with respect to efficacy: calculated
concentration required to cause a 50 % reduction in response showed that pure curcumin
(8.99 nM) was significantly less potent than DMC (1.15 nM), BDMC (0.41 nM) and Sigma‟s
curcuminoid mixture (0.69 nM). It is also reported that curcumin pre-treatment offers
protection to brain mitochondria against peroxynitrite in vitro by direct detoxification and in
vivo by the elevation of total cellular glutathione levels [380].
There are also some striking differences to the protective effects of curcuminoids when LDH
assays were used instead of MTT: firstly, the concentration required to afford 50 % protection
against H2O2-induced death was considerably higher in the LDH test group (calculated
86
concentration required to cause a 50 % reduction in response > 100 nM). Secondly, the
differential effects of the curcuminoid preparations as seen with the calculated concentration
required to cause a 50 % reduction in response for MTT assay are lost with the calculated
LDH assay values. The calculated concentration required to cause a 50 % reduction in
response for curcumin, DMC, BDM and Sigma‟s curcuminoid mixture were 125.8, 123.3,
114.6 and 113.8 nM respectively. These results perhaps say as much about the methods used
for detecting cell death and the chosen model of oxidative stress as they do about the
curcuminoids. The data hint at the fact that the mechanisms underlying H2O2-mediated effects
in terms of mitochondrial function and membrane integrity are, at least in part, different, and
that the protective effects of curcuminoids are biased in favour of those measured by MTT.
Furthermore, BDMC and DMC have a competitive advantage over curcumin in this respect.
To check whether the cytoprotective effect of curcuminoids against H2O2 also had any effect
on the functioning of the cells, insulin levels were measured. Pilot experiments revealed that
when RINm5F cells were treated with insulin secretagogues, 10 mM L-arginine and 400 nM
glibenclamide failed to cause any increase in the insulin secretion. The likely reason for such
a failure to stimulate insulin secretion may be attributed to the fact that 11.1 mM L-glucose
was already present in the cell culture medium; a concentration that is at the high end of the
physiological spectrum that might already be sufficient to fully activate insulin production
and secretion. On the other hand when treated with 10 mM L-cysteine as insulin inhibitor,
there was a significant reduction in the insulin level. In light of these findings, it is accepted
that the insulin assay in these experiments would only be able to detect a depression in insulin
secretion and would not be able to determine whether curcuminoids could actively stimulate
insulin secretion.
87
4.4 Conclusions
The results from experiments (fig 4.8) conclude that achievable nanomolar concentrations of
curcuminoids are easily capable of preventing the deleterious effects of oxidative stress in
pancreatic -islet cells, at least in this H2O2 model. The sensitivity of the cells to curcuminoid
effects is truly remarkable (low nM range) and all but excludes the possibility that there is a
direct antioxidant effect of these agents because they only represent a drop in the antioxidant
ocean. Instead, the results point to an indirect method of protection, and one that favours
protection against mitochondrial dysfunction over loss of membrane integrity. Neither
curcuminoids nor H2O2 (alone or in combination) have a detrimental effect on insulin
secretion, but the model did not allow assessment of any potential positive effect on insulin
secretion. There is also a suggestion that DMC and, particularly, BDMC are more effective
protective agents than curcumin, especially with respect to mitochondrial function.
Figure 4.8 Purity of curcuminoids and commercial curcumin analysed by HPLC and effect of curcuminoids and commercially obtained curcumin on hydroxyl and superoxide radical scavenging by EPR,; and on NO radical scavenging by NOA
TM analyser.
88
Chapter 5: Impact of curcuminoids on regulation of
intracellular antioxidant defence mechanisms
5.1 Introduction
Many cellular processes involve oxidation and reduction reactions, not only in the context of
signaling and regulation, but also as a part of metabolism and inflammatory host defence.
These redox processes take place throughout the cell and occur in many facets, from simple
electron transfer reactions to radical processes, hydride transfers and thiol/disulfide exchanges
[302]. Given the potential for free radical mediated tissue damage, it is perhaps not surprising
that the body has evolved the need to monitor, control and maintain an adequate antioxidant
defence mechanisms to protect itself. Clearly, the diversity of intracellular antioxidants
matches that of pro-oxidants.
Intriguingly, the levels of transcription, expression and activity of antioxidants enzymes (e.g.
catalase, superoxide dismutase, SOD; and glutathione peroxidise, GPx) have been reported to
be substantially lower in pancreatic β-islet cells as compared to other rat tissues [17, 237, and
240]. This lower level of antioxidant enzymes is likely to increase the susceptibility of these
cells in particular to oxidative damage [303]. Therefore, pharmacological or genetic strategies
to increase antioxidant enzyme expression in pancreatic β-islet cells have been extensively
studied.
Curcumin has been shown to exert a neuroprotective effect in rats with ischaemia/reperfusion
injury and this effect has been related to a direct scavenging effect of curcumin, as well as to a
curcumin-induced increase in antioxidant molecules, like reduced glutathione (GSH) and
enzymes (catalase, superoxide dismutase) [304, 305, and 306]. Also, previous cell culture
studies suggest that curcumin (concentrations between 5-30 μM) causes an increase in the
cellular GSH levels by enhancing the transcription of genes that encode glutamate cysteine
89
ligase; GCL (earlier called as γ-glutamyl-cysteinyl synthetase; γ-GCS) [307, 308]. Significant
enhancement in activity of antioxidant enzymes (GPx, glutathione reductase; GR (also called
as GRase or GSR), glucose-6-phosphate dehydrogenase, catalase) and phase II enzymes (e.g.
glutathione S-transferase and quinone reductase) in liver and kidney of mice fed with
curcumin, also suggest that it contributes to antioxidant defence in vivo [117, 309, and 310].
These results are in agreement with the earlier reported studies of Piper et al. [311], showing
that feeding curcumin to rats resulted in the induction of glutathione S-tranferase (GST), GPx,
and GCL
Considerable evidence suggests a linkage between diabetes and oxidative stress, leading to
the important question of the impact of curcumin on the antioxidant enzyme status of
hydrogen peroxide (H2O2) treated RINm5F pancreatic β-islet cells. The results from the
previous chapters indicate that curcuminoids are weak, direct scavengers of hydroxyl radicals
in vitro. Although not a powerful direct antioxidant scavenger, they offered protection at
achievable plasma nanomolar concentrations against deleterious effects of H2O2 in RINm5F
pancreatic β-islet cells. So, it was hypothesised that curcuminoids may be offering such a
protection indirectly by virtue of causing an upregulation in the activity and transcription of
intracellular antioxidant enzyme genes. This study aimed at measuring the effect of plasma
achievable nanomolar concentration of curcumin in presence of H2O2-induced oxidative stress
treatment on glutathione and SOD activity as well as GCL (both catalytic and modifier),
GPx1, GR, SOD1 and SOD2 gene expression in RINm5F pancreatic β-islet cells.
90
5.2 Results
5.2.1 Effect of curcuminoids ± H2O2 on total-glutathione and GSSG level
RINm5F cells treated with H2O2 (50 μM) did not show any significant impact on the
measured intracellular total GSH (oxidised + reduced) as compared to untreated control cells.
However, it caused a significant increase in the measured oxidised GSH (GSSG) in
comparison to untreated cells and a corresponding decrease in the calculated reduced GSH
(GSH). Co treatment of RINm5F cells for 24 hours with H2O2 and curcumin also did not show
any significant impact on the measured total GSH (oxidised + reduced) as compared to that in
H2O2 treated cells. However, it showed a concentration-dependent reversal of the H2O2-
mediated decline in GSH levels and also caused a concentration-dependent significant
decrease in the measured oxidised GSH (GSSG) level accompanied by a simultaneous
significant increase in the calculated reduced GSH (GSH) (fig 5.1, A). The same trend was
shown by the other curcuminoid preparation as well (fig 5.1, B to D).
91
92
Figure 5.1 Total GSH and GSSG assay in RINm5F cells co-treated with 50 µM H2O2 in presence of A) curcumin, B) DMC, C) BDMC, and D) sigma curcumin for 24 hours. Statistical significance was determined by ANOVA and Dunnett's multiple comparison post-test with untreated cells as control using Prism version 5.00 software (GraphPad Software, San Diego, CA). (*** p<0.0001; ** p<0.001; * p<0.01, mean ± SEM, n=6).
5.2.2 Effect of curcuminoids ± H2O2 on SOD activity
RINm5F cells treated with 50 μM H2O2 alone for 24 hours, resulted in a significant increase
in the total SOD activity in comparison to untreated cell controls. On the other hand none of
the 0.1, 1, 10 and 100 nM concentrations of curcumin had any significant effect on the total
SOD activity by themselves, but, co treatment of RINm5F cells with H2O2 and curcumin for
24 hours caused a significant increase in the SOD activity compared to untreated cell controls
(fig 5.2, A). The same trend was shown by the other curcuminoid preparation as well (fig 5.2,
B to D).
93
SOD activity in RINm5F cells treated
with curcumin and H2O2 (n=5)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
* * **
***
***
H2O2
50M
Untreated Cur
1nM
Cur
10nM
Cur
100nM
Cur
0.1nM
Cur
1nM
Cur
10nM
Cur
100nM
Cur
0.1nM
+ H2O2
50M
A
To
tal
SO
D A
cti
vit
y (
U/m
l)
SOD activity in RINm5F cells treated
with DMC and H2O2 (n=5)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
H2O2
50 M
Untreated DMC
1 nM
DMC
10 nM
DMC
100 nM
DMC
0.1 nM
DMC
1 nM
DMC
10 nM
DMC
100 nM
DMC
0.1 nM
+ H2O2
50 M
***
***
B
To
tal
SO
D A
cti
vit
y (
U/m
l)
94
SOD activity in RINm5F cells treated
with BDMC and H2O2 (n=5)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
H2O2
50 M
Untreated BDMC
1 nM
BDMC
10 nM
BDMC
100 nM
BDMC
0.1 nM
BDMC
1 nM
BDMC
10 nM
BDMC
100 nM
BDMC
0.1 nM
+ H2O2
50 M
***
******
C
To
tal
SO
D A
cti
vit
y (
U/m
l)
SOD activity in RINm5F cells treated
with sigma curcumin and H2O2 (n=5)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
H2O2
50 M
Untreated Sigma
1 nM
Sigma
10 nM
Sigma
100 nM
Sigma
0.1 nM
Sigma
1 nM
Sigma
10 nM
Sigma
100 nM
Sigma
0.1 nM
+ H2O2
50 M
****
******
D
To
tal
SO
D A
cti
vit
y (
U/m
l)
Figure 5.2 SOD activity in RINm5F cells treated for 24 hours with A) curcumin, B) DMC, C) BDMC, and D) sigma curcumin both individually and in the presence of H2O2. Statistical significance was determined by ANOVA and Dunnett's multiple comparison post-test with untreated cells as control using Prism version 5.00 software (GraphPad Software, San Diego, CA). (*** p<0.0001; ** p<0.001; * p<0.01, mean ± SEM, n=5).
95
5.2.3 Comparison of effect of curcuminoids ± H2O2 on calculated GSH and
total SOD levels
Comparison between the effect of curcuminoids on the measured SOD and calculated GSH
levels indicates that curcuminoids when present along with H2O2 has a more profound effect
on GSH than on SOD in comparison to H2O2 treated cells. Also the magnitude of effect on
GSH level was maximum with curcumin (Cur > DMC > BDMC > Sigma curcumin).
Figure 5.3 Comparative influence of curcuminoids on the measured SOD level (U/ml) and the calculated GSH level (nMoles/mg protein) in comparison to the 50 μM H2O2 treated RINm5F cells.
5.2.4 Effect on curcuminoids ± H2O2 on gene expression by RTQ-PCR
RINm5F cells treated for 24 hours with 50 μM H2O2, 1 nM curcuminoids and co treated with
1 nM curcuminoids and 50 μM H2O2 did not show any significant effect for GCLc and GCLm
gene expression in comparison to untreated cell control. On the other hand, 50 μM H2O2 by
itself caused an upregulation in GPx1 gene expression while it did not have any significant
effect on GR gene expression. All four curcuminoids did not have any significant effect by
96
themselves on GPx1 and GR gene expression. Also, cotreatment of cells with curcuminoids
and 50 μM H2O2 showed no significant effect on GPx1 gene expression but there was an
upregulation in GR gene expression observed.
RINm5F cells treated with 50 μM H2O2 alone caused an upregulation in SOD1 gene
expression compared to untreated cell control, though no significant effect eas observed on
the SOD2 gene expression. Both SOD1 and SOD2 gene expression was not significantly
changed with any of the 1 nM curcuminoids treatment. Conversely, cotreatment with
curcuminoids and 50 μM H2O2 indicated a significant upregulation in SOD2 gene expression
in comparison to untreated cell control (fig 5.4).
RTQ-PCR for RINm5F cells treated
with curcumin and H2O2 (n=6)
0
20
40
60
80
100
120
140
160GCLcGCLmGPx1GRSOD1
SOD2
H2O2
50 M
H2O2
50 M+
Cur1 nM
Cur1 nM
* **
**
**
H2O2
50 M
H2O2
50 M+
Cur1 nM
Cur1 nM
H2O2
50 M
H2O2
50 M+
Cur1 nM
Cur1 nM
H2O2
50 M
H2O2
50 M+
Cur1 nM
Cur1 nM
H2O2
50 M
H2O2
50 M+
Cur1 nM
Cur1 nM
H2O2
50 M
H2O2
50 M+
Cur1 nM
Cur1 nM
*
A
Ge
ne
Tra
ns
cri
pti
on
(% u
ntr
ea
ted
co
ntr
ol)
97
RTQ-PCR for RINm5F cells treated
with DMC and H2O2 (n=6)
0
20
40
60
80
100
120
140
160
GCLcGCLmGPx1GRSOD1
SOD2
H2O2
50 M
H2O2
50 M+
DMC1 nM
DMC1 nM
******
**
*
H2O2
50 M
H2O2
50 M+
DMC1 nM
DMC1 nM
H2O2
50 M
H2O2
50 M+
DMC1 nM
DMC1 nM
H2O2
50 M
H2O2
50 M+
DMC1 nM
DMC1 nM
H2O2
50 M
H2O2
50 M+
DMC1 nM
DMC1 nM
H2O2
50 M
H2O2
50 M+
DMC1 nM
DMC1 nM
B
Ge
ne
Tra
ns
cri
pti
on
(% u
ntr
ea
ted
co
ntr
ol)
RTQ-PCR for RINm5F cells treated
with BDMC and H2O2 (n=6)
0
20
40
60
80
100
120
140
160 GCLcGCLmGPx1GRSOD1
SOD2
H2O2
50 M
H2O2
50 M+
BDMC1 nM
BDMC1 nM
H2O2
50 M
H2O2
50 M+
BDMC1 nM
BDMC1 nM
H2O2
50 M
H2O2
50 M+
BDMC1 nM
BDMC1 nM
H2O2
50 M
H2O2
50 M+
BDMC1 nM
BDMC1 nM
H2O2
50 M
H2O2
50 M+
BDMC1 nM
BDMC1 nM
H2O2
50 M
H2O2
50 M+
BDMC1 nM
BDMC1 nM
** ****
**
C
Ge
ne
Tra
ns
cri
pti
on
(% u
ntr
ea
ted
co
ntr
ol)
98
RTQ-PCR for RINm5F cells treated
with sigma curcumin and H2O2 (n=6)
0
20
40
60
80
100
120
140
160 GCLcGCLmGPx1GRSOD1
SOD2
H2O2
50M
H2O2
50M+
Sigma1nM
Sigma1nM
** *** *
H2O2
50M
H2O2
50M+
Sig.1nM
Sigma1nM
H2O2
50M
H2O2
50M+
Sigma1nM
Sigma1nM
H2O2
50M
H2O2
50M+
Sigma1nM
Sigma1nM
H2O2
50M
H2O2
50M+
Sigma1nM
Sigma1nM
H2O2
50M
H2O2
50M+
Sigma1nM
Sigma1nM
*
D
Fo
ld C
ha
ng
e
(% o
f u
ntr
ea
ted
co
ntr
ol)
Figure 5.4 % fold change in level of GCLc, GCLm, GPx1, GR, SOD1 and SOD2 gene expression for cells treated for 24hours with H2O2 alone or co-treated with H2O2 in presence of A) curcumin, B) DMC, C) BDMC, and D) sigma curcumin in comparison to untreated RINm5F cells. Statistical significance was determined by ANOVA and Dunnett's multiple comparison post-test with untreated cells as control using Prism version 5.00 software (GraphPad Software, San Diego, CA). (*** p<0.0001; ** p<0.001; * p<0.01, mean ± SEM, n=6).
5.3 Discussion
The initial biochemical experiments mentioned in chapters 3 and 4 were directed towards
exploring the potential of curcuminoids in directly scavenging the free radicals. Results from
EPR studies concluded that curcuminoids were required at micromolar concentration to be an
effective direct scavenger of hydroxyl radicals generated by menadione, but even at such a
high concentration failed to scavenge superoxide radicals generated by pyrogallol. Even
though EPR studies showed that curcuminoids were only weak scavengers of free radicals,
they were still able to provide cytoprotection at nanomolar concentrations against the
oxidative stress induced on account of hydroxyl radicals generated by H2O2 in RINm5F cells.
To ascertain whether this cytoprotective effect was on account of upregulation of intracellular
antioxidant enzymes related to GSH modulation or to SOD, glutathione and superoxide
activity assays coupled with RTQ-PCR was performed.
99
The hypothesis that polyphenols can have pro-oxidative properties is not new, but is often
forgotten. Many publications have reported antioxidative and beneficial effects of
polyphenols, but study articles are now steadily appearing about apparently contradictory
investigations of the same substances, which might be related to their pro-oxidative effects.
One example is a publication by Surayanarayana et al. showing that dietary supplementation
of 0.002 % curcumin reduced the onset and maturation of cataracts in diabetic rats, but higher
concentrations of curcumin (0.01 %) in the diet accelerated the maturation of cataract [312].
Moreover, Galati and O‟Brien [313] reviewed investigations about the pro-oxidative related
toxicity of dietary phenolics. Pro-oxidative potential was found in almost every well known
polyphenolic antioxidant, including resveratrol, green/black tea phenolics, quercetin,
capsaicin, curcumin and many more. The authors concluded that every phenol ring-containing
flavonoid yields cytotoxic phenoxyl radicals upon oxidation by peroxidases. It is also
interesting that in study by Strasser et al. [314] a concentration of 25 μM curcumin, which
caused the highest increase in intracellular GSH, also reduced human myelomonocytic U937
cell viability. In this respect, it is worth mentioning that most of the in vitro studies [315, 316]
dealing with curcumin as an antioxidant use concentrations as high as 100 μM. Although in
these studies decreased ROS formation is observed after 1–2 hours incubation with curcumin,
it can be assumed that these high concentrations most likely lead to oxidative cell damage on
prolongation of the incubation time.
The GSH status of a cell is critical for various biological events since it offers antioxidant
defence in the cell, acts as an important detoxifying agent and is also involved in modulation
of redox-sensitive signal transduction and thus proinflammatory processes [317]. In the
present study, the effect of plasma achievable nanomolar concentration of curcumin on H2O2 -
induced oxidative stress in RINm5F cells was measured by quantifying total-GSH and GSSG
levels. Many authors have already investigated the effect on GSH content by curcumin, but
100
most of them have not measured the oxidised form of this redox couple, GSSG or have used
supraphysiological concentrations of curcumin. In RINm5F cells co-treated with H2O2 and
curcuminoids for 24 hours, there was a concentration dependent change in the proportion of
oxidised (GSSG): reduced (GSH) in favour of the latter in comparison to H2O2 treated cell
controls, thus indicating that curcuminoids were capable of elevating the GSH-dependent
antioxidant functionality. On the other hand, there was no significant change in the measured
total-GSH in RINm5F cells co-treated with H2O2 and curcuminoids in comparison to H2O2
treated cell controls, thus indicating that all the curcuminoids were involved in causing a shift
of oxidised GSH (GSSG) in favour of reduced state GSH (modulating the GSH:GSSG ratio),
while having no impact on the measured total-GSH concentration in the cells.
SOD is another important endogenous antioxidant present in the cells. The biological function
of SOD is very important because it prevents oxidative damage and inflammation, due to
subsequent formation of oxygen intermediates derived from the superoxide. The main
function of SOD is to scavenge superoxide radicals generated in various physiological
processes, thus preventing the oxidation of biological molecules, either by the radicals
themselves, or by their derivatives [318, 319]. None of the plasma achievable nanomolar
concentrations of curcuminoids by themselves caused any significant impact on the measured
SOD activity as compared to untreated RINm5F cell controls. On the other hand, H2O2
treatment by itself showed an increase in SOD activity in RINm5F cells and this increase was
further enhanced when curcumin was present along with H2O2. These findings indicate that
curcuminoids by themselves are not having any impact on SOD regulated antioxidant defence
in RINm5F cells, but when cells are stressed by H2O2, SOD regulated antioxidant defence is
activated to combat the stress and curcuminoids when present along with the stressor, H2O2
further enhance the SOD regulated antioxidant defence.
101
When the effect of curcuminoids on the measured total SOD level was compared with
calculated GSH levels it was found that curcuminoids when present along with H2O2 had a
more profound effect on calculated GSH level than on total SOD level in comparison to H2O2
treated cells. Curcumin showed higher magnitude of effect on GSH level in comparison to
the total-SOD level (Cur > DMC > BDMC > Sigma curcumin). It is already stated in the
literature that the methoxy group on the phenyl ring of curcuminoids is one of the functional
groups important for this compound's antioxidant effects [166, 320]. Curcumin has two
methoxy groups, DMC has one methoxy group, while BDMC has no methoxy groups. This
could explain to some extent the SAR that why curcumin is more potent in increasing the
GSH when present along with H2O2 and this finding is similar to that stated by Dairam et al.
[321] where they reported such a difference based on the methoxy groups in the
neuroprotective activity by influencing the GSH levels. Thus curcuminoids may be protecting
the cells against the cytotoxic effects of H2O2 by comparatively increasing the GSH levels
than via SOD levels.
Gene expression studies showed that curcuminoids, H2O2 and curcuminoids + H2O2 did not
have any significant impact on GCLc and GCLm gene expression in comparison to untreated
cell controls. These findings in keeping with the GSH data confirm that there was no impact
on GSH synthesis in RINm5F cells via either of GCLc or GCLm (rate-limiting enzyme for
glutathione synthesis). One of the reasons for failure in RINm5F pancreatic β-islet cells to up-
regulate GSH levels may be attributed to their lack of ability to respond to H2O2-induced
oxidative stress by up-regulating GCL subunit expression.
There was an increase in the GPx1 gene expression in RINm5F cells treated with H2O2 alone.
This increase may be attributed to the involvement of GPx1 gene expression in detoxification
of H2O2 with concomitant oxidation of glutathione [322]. GPx1 has also been shown to
metabolize other hydroperoxides including lipid hydroperoxides [323]. The accumulation of
102
H2O2 might have induced the activity of GPx leading to its up-regulation. These findings are
also in keeping with the GSH data for RINm5F cells treated with 50 μM H2O2. Thus,
RINm5F cells may be protecting themselves by up-regulation of GPx1 gene expression when
faced with oxidative stress generated on account of H2O2 alone. On the other hand neither of
the curcuminoids by themselves or when present along with H2O2 showed any significant
impact on GPx1 gene expression. This difference in GPx gene expression when curcuminoids
are present along with H2O2 might be an indication of more feasible machinery as opposed to
the compensatory adaptive mechanism in RINm5F cells to defend the gradual onset of
oxidative stress generated by H2O2.
Also, curcuminoids and H2O2 by themselves did not show any significant impact on GR gene
expression, but for RINm5F cells co-treated with curcumin and H2O2, there was an increase in
GR gene expression. This implies that curcuminoids up-regulated GR gene expression, but
only in the presence of H2O2, thus causing an increase in the recycling of oxidised GSH
(GSSG) to reduced GSH and favouring a healthy predominant GSH environment in the cells.
RINm5F cells when treated with H2O2 alone showed protection against the cytotoxic effects
by virtue of up-regulation of the GPx gene (a compensatory adaptive mechanism) but not up-
regulating the GR gene and thus getting rid of the accumulated H2O2. On the other hand when
H2O2 is present along with curcuminoids there was no increase in the measured total-GSH
level or GPx1 gene activity, but there was increase in the GR gene expression further
confirming this to be a more feasible mechanism of cell protection and keeping the level of
H2O2 so low that there was no up-regulation of GPx. Also, the impact of curcuminoids when
present along with H2O2 on the activities of GPx and GR may be correlated.
While curcuminoids by themselves failed to cause any significant impact on SOD-1 and
SOD-2 gene expression, when H2O2 is present alone there was a significant up-regulation in
SOD-1 gene expression observed indicating this to be a natural first line of antioxidant
103
defence by the cells. On the other hand when curcuminoids were present along with H2O2,
there was a significant up-regulation of SOD-2 genes. Thus independent activation of SOD-1
by H2O2 alone and of SOD2 in presence of H2O2 and curcuminoids indicates a symbiotic,
boosted antioxidant defence. Thus it is confirmed in the present study, that the natural
antioxidant defence of cells is not by the virtue of increasing the total GSH level but by
increasing the detoxification of H2O2 by up-regulating GPx1 and also by up-regulating SOD-1
genes. On the other hand curcuminoids offered protection against H2O2 by increasing the
recycling of GSH from GSSH by up-regulating GR and also by up-regulating SOD-2 gene
expression.
5.4 Conclusion
The findings from these experiments (fig 5.5) confirm that plasma attainable nanomolar
concentrations of curcumin offer protection in RINm5F cells against H2O2-indicted damage
by modulating the proportion of GSH in its reduced state and the activity of SOD. This
increase in GSH and SOD levels was, at least in part, on account of an increase in GR and
SOD-2 gene expression. Thus, cells must have devised a multitude of step by step
mechanisms by virtue of which they combat oxidative stress and also a wide range of cellular
antioxidants must be having specificity towards particular types of pathway to combat
particular free radicals. The intracellular mechanism driving this modulation of antioxidant
gene expression is the focus of the next chapter.
104
Figure 5.5 Effect of curcuminoids and commercial curcumin on GSH and SOD activity measured using GSH and SOD assay, as well as effect on GCLc, GCLm, GPx1, GR, SOD1 and SOD2 gene expression measured using RTQ-PCR.
105
Chapter 6: Effect of curcuminoids on hydrogen peroxide
and tumor necrosis factor-α induced NF-κB activation in
pancreatic β-islet RINm5F cells
6.1 Introduction
In the previous chapter it was observed that, plasma attainable nanomolar concentrations of
curcuminoids offer protection in pancreatic β-islet RINm5F cells against H2O2 induced
damage by modulating the proportion of GSH in its reduced state and the expression and
activity of SOD. This increase in GSH and SOD levels was, at least in part, on account of an
increase in GR and SOD-2 gene expression. Understanding how curcuminoids modulate the
intracellular mechanisms thus impacting the antioxidant gene expression via transcription of
nuclear factor kappa B (NF-κB) was therefore the next main focus.
Members of the inducible transcription factor NF-κB family play a pivotal role in various
responses leading to host defence, activating a rapid progression of gene expression, and
thereby regulating numerous immediate and long-lived cellular responses [324, 325]. These
active DNA-binding transcription factors are dimeric complexes composed of various
combinations of members of the Rel/NF-κB family of polypeptides [326]. NF-κB is a
transcription factor that serves as a key responder to changes in the environment. It plays a
critical role in many biological processes and thus it may be an evolutionarily conserved
signaling molecule [327]. Previous studies suggest that cytokines, such as tumour necrosis
factor-α (TNF-α), interleukin-1β (IL-1β) and interferon-γ (IFNγ) under in vitro conditions,
give rise to free radicals that cause translocation of NF-κB, from the cytoplasm into the
nucleus, eventually leading to cell death [328].
106
Blockade of the islet cell death pathway has been a primary approach employed for the
protection of islet cells from cytokine-induced death and dysfunction. This has been achieved
with some success by pre-treatment with various antioxidants and other compounds.
Inhibitors of NF-κB have also been employed to prevent in vitro as well as in vivo islet cell
death [329, 330 and 331]. NF-κB activation has also reported to be blocked by vitamin
metabolites such as [1,25-(OH) 2D3] and regulatory cytokine treatments such as IL-11, thus
offering protection to islets and RINm5F cells against cytokine-induced cell death [332, 333].
Induction and over expression of various enzymes scavenging reactive oxygen species (ROS)
in islets, as these cells have inherently low levels of these enzymes has also been tested by
many groups with a limited degree of success [243, 334, and 335]. A more comprehensive
approach is embraced by employing a compound that is capable of performing a range of
these functions, including free radical scavenging, induction of cellular defence responses,
cytoprotection and inhibition of important transcription factors, such as NF-κB, which
contribute to initiation of cell death cascade.
In previous chapters, it has been shown that curcuminoids are weak direct scavengers of free
radicals, but that they induce a multitude of intracellular defence responses by modulating the
proportion of GSH in its reduced state and the activity of SOD on account of an increase in
GR and SOD-2 gene expression. Also, curcuminoids offered protection against H2O2-induced
RINm5F cell death. Involvement of oxidative stress in activation of NF-κB DNA-binding
activity has been reported [359]. Matsuda et al. [336] have tried such a comprehensive
approach using silymarin, a polyphenolic flavonoid with a multitude of functions including
antioxidant, anticarcinogenic, anti-inflammatory and cytoprotective activities. Human islets
and RINm5F cells were protected against IL-1β- and IFNγ-mediated toxicity by suppression
of the c-Jun NH2-terminal kinase and JAK/STAT pathways by Silymarin. However, very few
of the studied compounds have, on their own, been able to confer complete protection against
107
cytokine-induced islet cell death along with maintenance of cellular homoeostasis and
function, both in vitro and in vivo. Following this lead, the efficacy of various curcuminoids,
for the protection of pancreatic β-islet RINm5F cells against the H2O2 and TNF-α induced
activation of NF-κB transcription factor was tested.
Previous studies have shown curcumin to offer protection against oxidative stress and cause
up-regulation of cellular defence proteins such as haem oxygenase-1 along with inhibition of
NF-κB [337, 338, and 339]. It has also been recently demonstrated that 10 μM curcumin
protects islets against streptozotocin (STZ)-induced death and dysfunction [191]. There is still
very little known about curcumins ability to protect the islets against H2O2-induced death
and/or dysfunction via activation/inhibition of NF-κB. Furthermore, very limited information
is available regarding in vivo ability of curcumin to prevent diabetogenesis. Thus to fill this
gap in our knowledge and to better understand the potential of curcumin, in this study in vitro
effect of plasma achievable nanomolar concentration of curcuminoids on H2O2 and TNF-α
induced activation of transcription factor NF-κB in pancreatic β-islet RINm5F cells was
investigated.
6.2 Results
6.2.1 NF-κB activation assay in independent curcuminoid treated RINm5F
cells
In the preliminary pilot study experiments (fig 6.1), levels of NF-κB (p65 subunit) in
pancreatic β-islet RINm5F cells were studied without any treatment over 30, 60, 120 and 240
minutes incubation time. NF-κB (p65 subunit) activation assay indicated that all the
pancreatic β-islet RINm5F cells examined after various incubation times, constitutively
expressed active NF-κB. In comparison to untreated cell controls, 50 μM H2O2 and 10 μg/ml
TNF-α treatment for 30, 60, 120 and 240 minutes caused an increase in the level of NF-κB
(p65 subunit) activation. There was though no noteworthy change in the H2O2 or TNF-α
108
activated NF-κB (p65 subunit) over all the measured treatment times. Similarly, pancreatic β-
islet RINm5F cells treated with 10, 100 and 1000 nM curcumin for 30, 60, 120 and 240
minutes caused no noticeable change in NF-κB (p65 subunit) activation in comparison to
untreated cell controls over time. Also, other curcuminoids did not show significant changes
in NF-κB (p65 subunit) activation in comparison to untreated cell controls (results not
shown). Based on pilot studies, subsequent experiments were performed using curcuminoids
(ranging from 10 to 1000 nM) independently or co-treated with 50 μM H2O2 or 10 μg/ml
TNF-α for 30, 60 and 240 minutes.
Figure 6.1 NF-κB activation in RINm5F cells treated with 50 µM H2O2, 10 µg/ml TNF-α, 10, 100 and 1000 nM curcumin for 30, 60, 120 and 240 minutes.
6.2.2 NF-κB activation assay in curcumin + H2O2 or TNF-α cotreated
RINm5F cells
RINm5F cells treated with 50 μM H2O2 alone for 30, 60 and 240 minutes (fig 6.2), resulted in
a significant increase in the NF-κB (p65 subunit) activation in comparison to untreated cell
controls. On the other hand, 10, 100 and 1000 nM concentrations of curcumin caused a
concentration-dependent reversal in the level of NF-κB (p65 subunit) activation with 1000
nM restoring it to normal level as seen in untreated cells after all the time points (fig 6.3).
109
Similarly 10 μg/ml TNF-α also caused a significant increase in the NF-κB (p65 subunit)
activation in comparison to untreated cell controls at all the measured time-points. Co-
treatment of RINm5F cells with 10 μg/ml TNF-α in presence of 10,100 and 1000 nM
curcumin caused a significant concentration-dependent decrease in the NF-κB (p65 subunit)
activation at all the time points, with 1000 nM curcumin effectively restoring the activation to
the basal untreated cellular level. The same trend was shown by the other curcuminoid
preparations (fig 6.3). Table 6.1 gives the calculated concentration required to give a 50 %
inhibition of the maximal response for various curcuminoids in presence of either 50 μM
H2O2 or 10 μg/ml TNF-α.
110
Figure 6.2 NF-κB activation in RINm5F cells treated with 10, 100, 1000 nM curcumin alone and with either 50 µM H2O2 or 10 µg/ml TNF-α for A) 30 min, B) 60 min, C) 240 min. Statistical significance was determined by ANOVA and Dunnett's multiple comparison post-test with untreated cell samples as control using Prism version 5.00 software (GraphPad Software, San Diego, CA). (*** p<0.0001; ** p<0.001; * p<0.01 Mean ± SEM, n=4).
AB
BB
CB
111
Figure 6.3 NF-κB activation in RINm5F cells treated with 10, 100, 1000 nM curcumin along with either 50 µM H2O2 or 10 µg/ml TNF-α for 30, 60 and 240 minutes. The experimental curves were fitted using a nonlinear regression model with a normalized response using Prism version 5.00 software (GraphPad Software, San Diego, CA). (mean ± SEM, n = 4).
Calculated
concentration
required to cause a
50 % inhibition of
maximal response
(nM)
30 min
H2O2 TNF-α
60 min
H2O2 TNF-α
240 min
H2O2 TNF-α
Curcumin 33.0 51.7 53.0 79.8 100.5 203.6
DMC 4.2 5.9 105.1 156.1 13.2 5.9
BDMC 13.9 102.0 15.0 60.5 6.2 6.9
Sigma curcumin 88.2 156.3 13.9 253.5 14.7 3.0
Table 6.1 Calculated concentration required to show a 50 % inhibition of maximal response in RINm5F cells treated with curcuminoids along with either 50 µM H2O2 or 10 μg/ml TNF-α for 30, 60 and 240 minutes. Numbers in bold are the lowest concentrations required to cause a 50 % inhibition of maximal response.
112
6.3 Discussion
The inducible transcription factor NF-κB, has been shown to be present in the cytoplasm of
every cell type in its inactive state and is conserved in animals all the way from Drosophila to
man. NF-κB 1 (p50/p105), NF-κB 2 (p52/p100), RelA (p65), RelB, and c-Rel are the five
different mammalian NF-κB family members that been identified and cloned. A highly
conserved Rel homology domain (RHD; ~300 aminoacids) responsible for DNA binding, a
dimerization domain, and the ability to interact with IκBs, the intracellular inhibitor for NF-
κB is shared by all the family members. The expression of a number of genes involved in
inflammation, proliferation and apoptosis is regulated by NF-κB. It can be activated by many
diverse stimuli via phosphorylation of the two major forms of IκB kinases (I kappa B kinases:
IκB), IκBα and IκBβ at their N-terminal serines, followed by their degradation and NF-κB
activation [340, 341, and 342]. These stimuli include bacterial and fungal products, viruses
and viral proteins, inflammatory cytokines, physiological stress, physical stress, oxidative
stress, environmental and occupational particles, heavy metals, intracellular stresses, viral or
bacterial products, UV light, X-rays, gamma radiation, chemotherapeutic agents, carcinogens,
cigarette smoke, hydrogen peroxide, chemotherapeutic agents, endotoxins, and tumour
promoters [343].
Reactive oxygen species (ROS) may either act directly causing induction of NF-κB activation
in a cell-specific manner [344, 345, and 346] or act indirectly as secondary messengers for
NF-κB activation [347, 348, and 349]. Previous studies have reported that NF-κB activation
can induce insulin resistance [350], and antidiabetic drugs can suppress NF-κB activation
through up-regulation of inhibitors of NF-κB [351-354]. Recently, it has been demonstrated
that H2O2 activates both alpha and beta IκB kinases through phosphorylation of
serine/threonine residues, which are components of the NF-κB activation signalsome [355].
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Also, it is well documented that TNF-α is one of the most potent activators of NF-κB, and the
expression of TNF-α itself is regulated by NF-κB [356, 357].
Singh and Aggarwal have demonstrated that 50 μM curcumin inhibits the activation of NF-κB
in different cancer cell lines [358]. It has been shown that curcumin inhibited, suppressed both
constitutive and inducible NF-κB activation and potentiated tumor necrosis factor (TNF)-
induced apoptosis. The amount of phosphorylated IKK was reduced on curcumin treatment,
which ultimately prevented the translocation of NF-κB to the nucleus. Curcumin has also
been shown as a regulator of genes expressed via activation of activator protein (AP1) and
NF-κB [360].
There are many studies reporting that curcumin is a potent blocker of NF-κB activation [358,
361-363]. One of the study reports that curcumin administration inhibits the activation of NF-
κB in the retina in streptozocin induced diabetes in rats [252]. Also, there is evidence that
curcumin inhibits TNF α-activated NF-κB signaling in adipocytes and thus significantly
reducing cytokine expression [364], but most of these studies were carried out in β-pancreatic
carcinoma cells and at high (μM) concentrations of curcumin which cannot be achieved in
plasma after typical oral ingestion. In the present study, the effect of plasma achievable
nanomolar concentration of curcumin on H2O2 and TNF-α induced NF-κB activation in
RINm5F cells was measured using NF-κB activation assay.
The result from the preliminary pilot study (fig. 6.1) indicated that NF-κB is constitutively
active in pancreatic β-islet RINm5F cells and that none of the studied 10, 100 and 1000 nM
concentration of curcumin, DMC, BDMC and curcumin (Sigma sourced) activated NF-κB,
whilst H2O2 and TNF-α were effective positive controls. The maximal NF-κB activation was
found after 30 min with H2O2 as well as with TNF-α; activation at later time points declined
with both agents. Based on the results from preliminary study, 30, 60 and 240 minutes time-
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points were selected for the full experiment. In further experiments it was clear that
curcuminoids caused a concentration-dependent depression of NF-κB activation, with almost
a complete prevention of the H2O2 or TNF-α induced component of NF-κB activation
observed in cells treated with 1000 nM curcuminoids (fig. 6.2). The same trend was also
observed after 60 and 240 minutes of treatment time. It has been shown with other inhibitor
studies that, blockade of NF-κB activation is on account of modification of NF-κB proteins
but in the case of curcuminoids such a blockade is not due to chemical modification [365, 366
and 367].
It is therefore important to explore the mechanism by which H2O2 and TNF-α activate NF-κB
so as to establish how curcuminoids may be blocking such activation. Both H2O2 and TNF-α
are known to produce reactive oxygen species/intermediates (ROS/ROI). It is possible,
therefore, that curcuminoids block the NF-κB activation by quenching the ROS but as the
concentration of curcuminoids required to cause direct scavenging of ROS as shown with the
EPR study is in micromolar range, this is not a possibility. It has also been shown that TNF-α-
induced activation of NF-κB is impaired by the inhibitors of mitochondrial electron transport
[368]. There are several indirect additional pieces of evidence to suggest a role for ROS as a
common and vital denominator [369, 370]. Many earlier studies also suggest an increase in
the cellular levels of reactive oxygen intermediates (ROIs) as an effective activator of NF-κB
on exposure to TNF-α, H2O2, interleukin-1, PMA, lipopolysaccharide, UV light, and γ-
irradiation [371]. Inhibition of a protein kinase has also been proposed as one of the
mechanisms by which curcumin may block NF-κB activation. In one of the in vitro studies,
curcumin has been shown to inhibit both serine/threonine protein kinase and protein tyrosine
kinase [372].
Both the NF-κB inducers used in this study, TNF-α and H2O2 have been shown to activate
both protein kinase C and protein tyrosine kinase. Protein kinase C and protein tyrosine
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kinase inhibitors have both been shown to block the TNF and H2O2-induced NF-κB activation
[373, 374]. Protein tyrosine kinase has also been shown to play a role in NF-κB activation by
ultraviolet light [375], lipopolysaccharide [376], hypoxia [377], and viral-sarcoma (v-src)
[378]. Schievien et al. have shown that protein tyrosine kinase inhibitors block NF-κB
activation induced by γ-irradiation, a stimulant thought to work through the immediate
generation of reactive oxygen intermediates (ROI), which thus further suggests involvement
of ROI generation-dependent NF-κB activation [379]. These studies thus suggest
involvement of a number of early events in NF-κB activation, all of them converging in IκBα
phosphorylation followed by its degradation and subsequent translocation of p65 into the
nucleus. NF-κB activation may also be blocked at another level due to a down-modulation of
the cytoplasmic pool of p65 subunit that could prevent formation of p50/p65 heterodimer.
Previous studies, however, reveal that p65 subunit was not down-regulated by curcumin but
its translocation to the nucleus was inhibited, most likely through inhibition of degradation of
IκBα [358].
6.4 Conclusion
The results from the current study indicate that the signal transduction pathway of NF-κB
activation inhibited by curcumin is at, or before, the phosphorylation step of NF-κB. As NF-
κB activation is shown to be inhibited by diverse agents indicate that this step is after or at the
step where the assorted signals converge. Overall the findings from this study conclude that,
by virtue of blocking the TNF-α and H2O2 induced NF-κB activation, curcuminoids may be
influencing the NF-κB regulated genes expression (fig 6.4) thus further confirming the results
observed in the RTQ-PCR study in previous chapter.
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Figure 6.4 NF-κB activation in RINm5F cells treated with TNF-α, H2O2 and curcuminoids plus either TNF-α or H2O2.
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Chapter 7: General Discussion
Curcumin, a natural polyphenol has gained widespread attention in recent years on account of
numerous reports of beneficial properties without any apparent toxic side-effects in a range of
clinical indications [381]. It has broad biological and pharmacological properties including
anti-inflammatory, antiviral, anti-infective, anti-proliferative and antioxidant effects [382]. It
has been shown to suppress the activation of NF-κB induced by various inflammatory stimuli.
It has also been shown to suppress the expression of various NF-κB-regulated genes,
including COX-2, Bcl-2, TNF, MMP-9, cyclin D1, chemokines and cell surface adhesion
molecules. Curcumin can induce apoptosis through consecutive activation of caspase 8, beta-
interaction domain (BID) cleavage, cytochrome c release, caspase 9 and caspase 3 [383]; it
has potent anti-cancer activity in the context of various human malignancies but is non-toxic
towards normal cells [382]. All of these reported findings have suggested curcumin to be a
multi-target, natural compound with potential as a nutraceutical.
The starting point for the present studies was an assessment of the chemical characteristics of
commercially available curcuminoids as a precursor for in vitro testing in rat pancreatic β-
islet RINm5F cell survival in the face of oxidative challenge, with a view to its therapeutic
potential in management of type 2 diabetes. First, the purity of several commercially available
curcuminoids was assessed: the results obtained from RP-HPLC analysis revealed that the
chromatograms for curcumin, DMC and BDMC (provided as a gift by Sami Labs, India) had
a single, clear peak with no peak-tailing, confirming that they were relatively pure. However,
the chromatogram for Sigma-Aldrich “pure” curcumin yielded three peaks with retention
times corresponding to those obtained with the pure curcuminoids from Sami Labs,
confirming that the preparation from Sigma is a mixture of the three curcuminoids, curcumin,
DMC and BDMC (relative proportions based on peak heights ~19:14:1 respectively). A
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significant proportion of previous studies have employed curcumin from this supplier, raising
doubts as to whether many of the literature findings relate to pure curcumin or to a mixture of
curcuminoids. The importance of this issue is dependent on the relative activity of the various
curcuminoids: should there be a large discrepancy in activity, the lack of purity of the test
compound becomes critical and highlights a need for separate studies with each of the pure
curcuminoids to establish any structure-activity relationship.
7.1 Radical scavenging effects
The antioxidant properties of curcumin have been the focus of a number of cell culture, tissue
and in vivo studies [190-194]. Tonnesen et al. [211], Priyadarsini et al. [212], and Jovanovic
et al. [215] have suggested that the free radical scavenging capability of curcumin,
particularly with respect to superoxide and hydroxyl ions, is on account of the β-diketo group,
the central methylenic group and H-donating phenolic group respectively. These three groups
are common to all 3 curcuminoids tested in this study [120, 133, 195-198]; it is the number of
methoxy groups that varies between these curcuminoids (n=2, 1 and 0 methoxy groups in
curcumin, DMC and BDMC respectively). Also, much of the previous in vitro
pharmacological and mechanistic work has been conducted with curcuminoid concentrations
that are several orders of magnitude above that which is likely to be found in vivo after
realistic dietary intake. There remains a need, therefore, for a careful analysis of the radical
scavenging potential of curcuminoids to establish the antioxidant potential of curcuminoids as
a group, to determine the relative merits of curcumin, DMC and BDMC in this capacity and
to establish their relative selectivity for superoxide and hydroxyl radical scavenging. To that
end, the EPR results for direct radical scavenging potential revealed that all of the tested
curcuminoids effectively compete with the spin-trap, Tempone-H (50 µM) for hydroxyl ions,
at concentrations <50 µM. Surprisingly, however, all of the tested curcuminoids failed to
scavenge superoxide radicals from pyrogallol, even at concentrations of 20 times that of the
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spin-trap). These findings suggest that the tested curcuminoids are considerably more
effective towards hydroxyl radical scavenging than towards that of superoxide radicals.
Continuing work in this laboratory is compiling an activity catalogue for well-known
antioxidants (e.g. vitamin C, GSH, cysteine, N-acetylcysteine, flavonoids) under identical
conditions; to date, the curcuminoids have emerged amongst the most potent scavengers of
hydroxyl radicals and amongst the weakest scavengers of superoxide. The wide variation in
ability to scavenge different oxygen-centred radicals is something of a surprise in that one
might assume that the highly reactive nature of radicals might lead to a fairly indiscriminate
effect of antioxidants. The reason for the particularly wide disparity in scavenging potential of
the curcuminoids is not yet clear, but it is reasonable to speculate that it is an inherent in the
core chemistry of these agents. The lack of impact of presence or otherwise of the methoxy
group(s) suggests that this particular moiety is not important in determining either the potency
of hydroxyl scavenging or the disparity between hydroxyl and superoxide scavenging, rather
these effects are likely to reside within the β-diketo group, the central methylenic group
and/or the H-donating phenolic groups; specific work with chemical analogues lacking these
elements would be required to identify the effective component(s).
The suggestion from previous work with this assay in our laboratory is that “traditional”
antioxidants have more balanced ability to scavenge hydroxyl and superoxide radicals but that
the potential to scavenge hydroxyl is marginally higher with the curcuminoids than any of the
other antioxidants so far tested. Given that the concentration of curcuminoids in the plasma
and cellular compartments is likely to be several orders of magnitude lower than the pre-
existing total endogenous antioxidant capacity in both the plasma and intracellular
compartments, there is no indication from the results obtained in this study that curcuminoids
derived from dietary intake could have any significant direct antioxidant activity. This is a
crucial finding because it raises a contentious issue that is often ignored in the literature: how
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can curcumin (and other phenolic compounds with low bioavailability) confer the beneficial
effects that are reported in vivo, apparently on account of their antioxidant effects, without
having the necessary scavenging capacity on account of exceptionally low bioavailability? As
a result other pathways by which curcuminoids might confer antioxidant capabilities were
explored.
Given the radical scavenging effects of curcumin, it was imperative to also examine its
potential to scavenge another radical species, such as nitric oxide (NO), which has largely
protective effects, particularly when generated in low concentrations from the endothelium in
the cardiovascular system. Experiments were designed to establish the NO-scavenging ability
of curcuminoids in the face of a NO-generating drug (DETA-NONOate). The results clearly
indicated that none of the curcuminoids tested showed any NO-scavenging activity. These
results are broadly in agreement with earlier reports suggesting that curcumin does not
scavenge NO - a useful attribute, given that NO (especially that derived from endothelial NO
synthase (eNOS)) is protective. There is some evidence, however, that curcumin can
sequester nitrogen dioxide (NO2) that is formed as an intermediate in the oxidation of NO to
nitrite [231]; as yet, it is unclear what impact this might have on cardiovascular physiology,
especially now that nitrite itself has been implicated as a vasoactive oxide of nitrogen.
However, curcumin is reported to have an impact on NO bioavailability through inhibition of
inducible NO synthase (iNOS) in activated macrophages [199, 200, and 201] and, in the light
of the fact that iNOS inhibitors have been shown to prevent pancreatic β-islet cell death in
vivo [230], this might be considered an additional benefit of curcumin from a therapeutic
perspective in type 2 diabetes. Certainly, an agent that has the potential to scavenge ROS
(directly or otherwise), not to scavenge NO, but to selectively inhibit iNOS is an attaractive
proposition with respect to combating oxidative stress in disease.
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7.2 Protective effects against H2O2-mediated cell death in β-islet
cells
Pancreatic β-islet cells have low level of antioxidant enzyme expression and activity, making
them particularly susceptible to oxidative stress [17, 237-240, 303]. Thus, approaches that
might help prevent ROS-mediated pancreatic β-islet cell death, whilst maintaining insulin
secretory function, might have some bearing in moderating the progression of type 2 diabetes
from a problem relating primarily to insulin resistance to one that also involves loss of insulin
secretory function. In type 2 diabetes, excessive ROS impairs insulin synthesis [279-282] and
activates β-cell apoptotic pathways [283], predisposing to early cell death [284, 285, and
286]. Previous studies have shown that treatment with antioxidants improves pancreatic β-
islet cell survival [287, 288]. Though there are many studies reporting beneficial preventive
activity of curcumin against ROS-induced pancreatic β-islet cell damage, most studies have
used very high concentrations (> μM) and do not take into account the limited bioavailability
of curcuminoids. This issue is paramount for determining both the potential of curcuminoids
as realistic antioxidants in vivo and the mechanism of action by which they might have
benefit, given that the early results presented in this thesis cast doubt over the importance of
direct scavenging of oxygen-centred radicals. The remainder of the studies presented in this
thesis concentrated on the impact of nanomolar concentrations of curcuminoids on
antioxidant activity in pancreatic β-islet cells.
Results from two different cell survival assays were designed to establish the impact of low-
level curcuminoid treatment on cell resistance to the oxidant H2O2; the MTT assay measures
mitochondrial function, whilst the LDH assay is an indicator of membrane integrity. The
results obtained indicate that only nanomolar concentrations of curcuminoids are required to
protect against the toxic effects of H2O2 and also suggest that the protective effects are more
evident in the MTT assay than the LDH assay, with BDMC and DMC proving significantly
more protective than curcumin. The difference in the cellular protection offered by curcumin,
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DMC and BDMC in the MTT assay is likely to be associated with the presence or absence of
methoxy group(s). BDMC, having no methoxy group, was found to be the most potent
followed by DMC and curcumin, indicating that the methoxy groups confer a disadvantage
with respect to this protective effect. It is not yet clear what the nature of the disadvantage is:
it seems unlikely that the presence of these groups would hinder diffusion into cells, given the
apparent lipophilicity of all three analogues, but they could cofer a degree of steric hindrance
should the protective effects involve interaction with a receptor or other protein. In case of the
LDH assay, there was no significant difference among the tested curcuminoids. Also, the
concentration required to inhibit 50 % of H2O2-induced cell death was much higher than that
for the MTT assay, giving rise to a number of implications: first, H2O2 has a different impact
on the two assays, implying that it affects mitochondrial function and membrane integrity
through different pathways. Second, the curcuminoids are more effective at modulating the
pathway(s) related to mitochondrial function rather than membrane integrity. These results
might simply be an artefact of the contrived nature by adding exogenous H2O2 as an oxidant
to the culture medium- the impact on the cell membrane could be irrepressible, particularly
with the use of nanomolar concentrations of curcuminoids. In contrast, the impact of
exogenous H2O2 on mitochondrial function will be milder on account of the highly reducing
intracellular environment and might, therefore, be more sensitive to relatively small changes
in intracellular antioxidant capacity. Notwithstanding, it still seems unlikely that nanomolar
concentrations of curcuminoids could have sufficient antioxidant capacity in their own right
to account for the profound protective effects seen in the MTT assay.
There was no effect on insulin secretion by treatment of pancreatic β-islet cells with
curcuminoids in the presence of H2O2. The results from these experiments conclusively
demonstrated that curcuminoids did not suppress insulin secretion, but it is not yet clear
whether they might be able to increase insulin secretion because the high glucose
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concentration in the culture medium for these cells probably ensured near-maximal insulin
secretion under control conditions. Further experiments in the presence of much lower
glucose concentrations would be required to conclusively determine whether or not
curcuminoids can increase insulin secretion, but for now there is no evidence to suggest that
curcuminoids influence insulin secretion directly. They might, nevertheless, have an impact
on insulin secretion through protection of -islet cells against oxidative damage in the in vivo
setting.
7.3 Up-regulation of intracellular antioxidant systems by
curcuminoids
Cells have a number of antioxidants to combat oxidative stress but, as stated earlier, the levels
of transcription, expression and activity of antioxidant enzymes in pancreatic β-islet cells (e.g.
catalase, superoxide dismutase; SOD and glutathione peroxidise; GPx) is substantially lower
in this cell type compared to that in other tissues [17, 237, and 240] thus, making them
particularly susceptible to oxidative damage [303]. Earlier reported cell culture studies
suggest that curcumin (concentrations between 5-30 μM) causes an increase in intracellular
GSH levels by enhancing the transcription of genes that encode GCL [307, 308]. The aim of
the studies reported in this thesis was to take the previous findings a step further and to
establish the effect of achievable nanomolar concentration of curcumin in presence of H2O2
on oxidised (GSSG) and reduced (GSH) levels, as well as that of SOD isoforms. The results
indicated that curcuminoids caused a relative change in the GSSG:GSH ratio in the favour of
latter, thus confirming that all of the studied curcuminoids were capable of elevating GSH-
dependent antioxidant functionality, thus offering protection against H2O2-induced oxidative
stress. Also, the magnitude of the effect on GSSG:GSH ratio was optimal with pure curcumin
in comparison to the other curcuminoids (activity: curcumin > DMC > BDMC > Sigma
curcumin), suggesting that the methoxy groups have some importance in determining activity
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in this respect. It is already known that the methoxy group on the phenyl ring of curcuminoids
is one of the functional groups that is important for the antioxidant properties of curcuminoids
[166, 320]. Dairam et al., [321], have reported that differences in the number of methoxy
groups in curcumin, DMC and BDMC influence the neuroprotective activity by influencing
the GSH levels. However, it is also worth noting that the presence or absence of methoxy
groups has a fairly modest effect on efficacy and that the major impact is due to the common
structural features, as mentioned earlier present across all the studied curcuminoids.
With respect to the influence of curcuminoids on SOD activity, none of the nanomolar
concentrations of curcuminoids by themselves caused a significant impact on the measured
SOD activity. However, when RINm5F cells were stressed with H2O2, there was an increase
in SOD activity and this increase was further enhanced when curcuminoids were present at
the same time as H2O2. These findings indicate that curcuminoids by themselves show a
selective response, not having any impact on SOD regulated antioxidant defence in RINm5F
cells, but when stressed, SOD regulated antioxidant defence is accentuated to combat the
oxidants. This is an interesting finding, suggesting that curcuminoids selectively switch on
intracellular SOD dependent response only in presence of oxidative stress.
Having established that curcuminoids in the nanomolar range altered the GSSG:GSH ratio as
well as SOD activity, it was imperative to drill down on the mechanisms that might be
involved in driving the effect, starting with potential transcriptional modification of enzymes
involved in GSH homeostasis and activity (GR, GCL, GPx), together with other prominent
antioxidants (SOD-1 and SOD2) using quantitative RT-PCR. Curcuminoids by themselves
failed to cause any significant impact on GR, SOD-1 and SOD-2 gene expression but when
present along with H2O2 there was a significant up-regulation of these genes. The effect of
curcuminoids favouring the GSSG:GSH ratio was thus confirmed to be on account of up-
regulation of the GR gene that encodes for the enzyme responsible for recycling of GSSG to
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reduced GSH and favouring a predominance of GSH in the intracellular environment. There
was also an up-regulation in SOD1 gene expression when RINm5F cells were treated with
H2O2 alone, confirming the increase in the level of SOD activity observed earlier. Up-
regulation of GR, SOD1 and SOD2 is thus in agreement with the results from the activity
assays obtained earlier. Furthermore, the up-regulation in SOD2 gene by curcuminoids in the
presence of H2O2 may be thus activating the natural first line of antioxidant defence by the
cells. The protective effect of curcuminoids was thus achieved only when cells were stressed
with oxidative stress generated on account of H2O2. It was also found that none of the
curcuminoids by themselves or when present along with H2O2 had any impact on GCLc or
GCLm genes, which encode for the rate-limiting enzyme for GSH synthesis. Though an up-
regulation of GPx gene transcription when treated with H2O2 alone was observed, neither of
the curcuminoids by themselves, or when present along with H2O2, showed any significant
impact on GPx1 gene expression. This difference in GPx gene transcription which is
responsible for detoxification of H2O2 may be an indicator of the natural cellular antioxidant
machinery, as opposed to the compensatory adaptive mechanism through up-regulation of
GR, SOD1 and SOD2 achieved on account of curcuminoids in presence of H2O2 in RINm5F
cells to defend against the gradual onset of oxidative stress generated by H2O2. There was no
significant difference between the various tested curcuminoids with respect to gene
expression and activity studies suggesting that the common structural features are important
for the beneficial effect.
Nuclear factor-κB (NF-κB) plays a pivotal role in inducing a number of genes including those
responsible for antioxidant activity. It has also been reported that in some cell lines direct
addition of H2O2 to culture medium activates NF-κB [383, 384]. Furthermore, in some cell
types there is increase in ROS in response to agents that activate NF-κB [385, 386, and 387].
Testing of whether H2O2 and tumour necrosis factor-α (TNF-α) is responsible for activating
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NF-κB in RINm5F cells and whether curcuminoids modulate such an activation thereby
impacting the antioxidant gene transcription was undertaken to explore this possibility.
TNF-α under in vitro conditions, gives rise to free radicals via mitogen-activated protein
kinases (MAPKs), which is responsible for activation of activator protein 1 (AP-1)
transcription factors and IκB kinases (IKK), which in turn activate NF-κB [389, 390, and
391]. This activation of NF-κB causes its translocation from the cytoplasm into the nucleus,
eventually leading to cell death [328]. Studies reported in this thesis established that
curcuminoids caused a concentration-dependent depression of both H2O2 and TNF-α -induced
NF-κB activation. TNF-α and H2O2 have been shown to activate both protein kinase C and
protein tyrosine kinase, which have in turn been reported to be inhibited by curcumin. [381,
382] Thus, this may be a possible mechanism by which curcuminoids inhibit NF-κB
activation. Also, previous results in this study indicated that curcuminoids up-regulated GR
gene expression; this up-regulation may be under the influence of NF-κB. However, the
concentration of curcuminoids required to cause GR up-regulation was very low, as opposed
to that required to inhibit NF-κB activation, suggesting that curcuminoids may also have an
impact on other systems [e.g. Nrf2 and antioxidant-responsive element (ARE)]. It has been
reported earlier that NF-κB activation could be attenuated by diverse Nrf2 activators, such as
phenethyl isothiocyanate (PEITC) and curcumin [384]. Furthermore, the literature suggests
that curcumin activates Nrf2 expression, which in turn binds to, and stimulates, antioxidant-
responsive element (ARE). ARE is responsible for initiating the transcription of various genes
coding for detoxifying enzymes and cytoprotective proteins. Further studies are required to
explore the impact of curcuminoids on Nrf2, AP-1 and ARE to unravel the effect on various
signaling pathways.
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7.4 Conclusion
The results from studies in this thesis (fig 7.1) suggest that curcuminoids are poor scavengers
of superoxide and, although effective scavengers of hydroxyl, are not present in the in vivo
environment at high enough concentrations to have a significant direct impact on antioxidant
capacity. Nevertheless, nanomolar concentrations of curcuminoids are effective protectors
against oxidative stress in rat pancreatic β-islet RINm5F cells by means of up-regulating
endogenous antioxidant systems (GSH, SOD), in part via inhibition of NF-κB. As only
nanomolar concentrations of curcuminoids show this beneficial effect, so dietary
interventions might be able to deliver sufficient curcuminoid concentrations to have a real
antioxidant effect. There are small differences between the activities of curcumin, DMC and
BDMC in some, but not all aspects of their function, suggesting that the methoxy groups have
only a minor part to play in the activity of curcuminoids. Commercially available curcumin,
which is a mixture of the three tested curcuminoids, is as effective as the individual
curcuminoids therefore there is no specific requirement of pure curcuminoids to derive its
beneficial activity.
With specific reference to the potential of curcuminoids in protecting β-islet cells against
oxidative stress in type-2 diabetes, the low levels of antioxidant enzyme expression and
activity make pancreatic β-islet cells particularly susceptible to oxidative stress and
curcuminoids may be helpful in the management of type 2 diabetes. However, on a cautionary
note, curcumin, in the form of turmeric, is consumed as a part of the daily diet in the Asian
sub-continent: a part of the world where type-2 diabetes is particularly prevalent. Like many
nutritional supplements, curcumin should not be regarded as a cure for type-2 diabetes, rather
a supplement that might form part of the nutritional advice given to patients to help manage
the condition. To that end, nutritional studies are now merited to confirm the benefits of
curcuminoids in the clinical setting.
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Thus the pathway explored in the course of this thesis is as follows:
Figure 7.1 Pathway by which curcuminoids show there beneficial activity in pancreatic β-islet RINm5F cells.
7.5 Future work
The findings of this project demonstrate that curcuminoids obtained from Sigma-Aldrich®
is a
mixture of curcumin, DMC and BDMC, raising concerns about whether the previous
literature finding relates to pure curcumin or mixture of curcuminoids. It was also found that
curcuminoids differ in their selectivity towards direct scavenging of various free radicals with
the most promising effect on hydroxyl radical scavenging (concentration of curcuminoid
required < 50 µM). Surprisingly though, there was no effect on superoxide and nitric oxide
scavenging even at concentrations as high as 1 mM. These results indicate that curcuminoids
would not be able to have any direct antioxidant scavenging effect at the concentrations
physiologically relevant after oral ingestion (low nM levels). To ascertain indirect antioxidant
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activity of curcuminoids at plasma-achievable nanomolar concentrations, cell culture
experiments were performed which suggested that curcuminoids offered protection in
pancreatic β-islet RINm5F cells against H2O2-induced toxicity, perhaps primarily via an
impact on mitochondrial function than membrane integrity. This findings hint at the fact that
the mechanisms underlying H2O2-mediated effects in terms of mitochondrial function and
membrane integrity are, at least in part, different, and that the protective effects of
curcuminoids are biased in favour of those measured by MTT. Furthermore, curcuminoids
caused an increase in the SOD activity and reversed the H2O2-induced depression in GSH
levels in terms of the GSH:GSSG ratio, thus further confirming its indirect antioxidant
activity. To further explore the mechanisms that might be involved in driving this increase
RTQ-PCR study was performed, which showed that curcuminoids when present along with
H2O2 caused an upregulation in GR and SOD2 gene expression thus further supporting the
hypothesis that curcuminoids have an indirect antioxidant activity. Furthermore, there was
also a concentration-dependent decrease in the H2O2 and TNF-α induced NF-κB (p65)
activation by curcuminoids confirming the involvement of NF-κB (p65) in the pathway by
which curcuminoids showed its beneficial activity.
Further experiments are needed to support the initial RP-HPLC findings about the purity of
studied curcuminoid analogues using liquid chromatography mass spectrometry and to also
explore the tissue/plasma concentration of curcumin upon oral administration. The results
would also give an insight about the metabolites of curcuminoids produced after oral
ingestion in experimental animal models. Also, direct radical scavenging activity using other
radical generators may be performed to make a further comparative assessment of the relative
activity of curcuminoids towards various radicals. The results would further confirm the
specificity of curcuminoids towards directly scavenging hydroxyl radicals with no impact on
superoxide and nitric oxide radical scavenging activity. In addition to MTT assay used to
130
measure the impact of curcuminoids on H2O2-induced oxidative stress in pancreatic β-islet
RINm5F cells, mitochondrial reactive species can be measured in mitochondria using the
fluorogenic probe, dichlorodihydrofluorescein diacetate (DCFDA or H2DCF). DCFDA is a
useful method to measure the production of ROS by a variety of cell types, in both flow-
cytometric and spectrofluorometric systems. The findings from this experiement would
inform about the intracellular ROS generated in RINm5F cells on account of H2O2 treatment
and explore in greater detail the intracellular action of curcuminoids to scavenge these H2O2-
induced ROS. With respect to the impact of curcuminoids on insulin levels, experiments
using RPMI-1640 medium containing low level of D-glucose (lower than 11.1 mM) should be
performed. RINm5F cells can be treated with increasing concentration of D-glucose
containing RPMI-1640 medium (0, 1, 2, 3, 4 and 5mM) and measured for insulin levels using
an insulin assay. Thus a concentration-response curve can be obtained and the impact of
presence/absence of curcuminoids along with H2O2 on RINm5F cells analysed. This
experiment would also overcome the limitation of insulin assay findings in the present study
and thus further explore whether the cytoprotective effect of curcuminoids against H2O2-
induced cell death causes an increase in the insulin level secreted by the cells.
Induction of phase II detoxification and antioxidant enzymes by curcuminoids is a major
protective mechanism against oxidizing substances capable of damaging DNA integrity and
initiating oxidative stress induced cytotoxicity [9, 11, 12]. Protection against electrophilic and
reactive oxygen species damage by induction of phase II genes encoding enzymes such as
such as UDP-glucuronosyl transferases, glutathione S-transferases (GSTs),
NAD(P)H:quinone oxidoreductases (NQOs), thioredoxin, glutathione peroxidase (GPx) and
N-acetyltransferases is a good strategy in reducing the risk of oxidative stress-induced
damage of pancreatic β-islet cells. The two main lines of defence against ROS are: (1) GSH,
the most abundant cellular non-protein thiol (studied in this project), and (2) a family of phase
131
II enzymes capable of converting reactive electrophiles to less toxic and more readily
excretable products, thus offering protection to cells [11-14]. Furthermore, since GSH levels
and phase II genes encoding enzyme activities do not usually operate to their maximum
capacity, their ability to be transcriptionally induced by curcuminoids should be able to
promote efficient protection against oxidative stress induced damages in pancreatic β-islet
cells. The influence of curcuminoids on GSH levels seen in this project should be followed by
the study of their influence on a sensor system known as the cytoplasmic oxidative stress
system NF-E2-related factor 2 – Kelch like ECH-associated protein (Nrf2–Keap1) [13, 24,
50].
Under basal (reducing) conditions, Keap-1 anchors the Nrf2 transcription factor within the
cytoplasm, targeting it for ubiquitination and proteasome degradation, thus repressing its
ability to induce phase II genes. When cells are exposed to oxidative stress, however, a signal
involving phosphorylation and/or redox modification is transmitted to the Nrf2–Keap1
complex. This signal leads to dissociation of Nrf2 from the Nrf2–Keap1 complex and its
nuclear translocation [10, 11, 31, 50 and 52]. Once inside the nucleus, Nrf2 heterodimerically
partners with other transcription factors [e.g. small musculoaponeurotic fibrosarcoma (sMaf):
MafF, MafG and MafK; JunD; activation transcription factor 4 (ATF4); polyamine-
modulated factor-1 protein (PMF-1)], and binds to the antioxidant/electrophile response
elements (AREs/EpREs) present in the promoter region of phase II genes [10, 11]. This
binding in turn leads to initiation of the transcription of antioxidant and phase II defence
enzymes, including glutathione-S-transferase (GST), NADP(H):quinone oxidoreductase 1
(NQO1), thioredoxin, and glutathione peroxidase (GPx) and that of Nrf2 as well [421]. To
test the hypothesis that curcuminoids have an impact on Nrf2 transcription factor and
therefore act to upregulate Nrf2-ARE/EpRE antioxidant signalling and thereby induce phase
II genes, RTQ-PCR and electrophoresis mobility shift assays (EMSA) may be employed.
132
Nuclear samples for EMSA would be prepared after treating RINm5F cells with curcuminoids
both in presence and absence of 50 µM H2O2 as described in section 2.2.10 for nuclear extract
preparation for NF-κB (p65) activation assay. The EMSA can be conducted using a γ-32P-
ATP end-labelled synthetic double-strand oligonucleotide probe for the ARE (5′-TGG GGA
ACC TGT GCT GAG TCA CTG GAG-3′) obtained from commercial source. Binding
reactions will be established in 20 μl of binding buffer using 4 μg of nuclear extract protein
per reaction for the consensus probe. In the binding reaction, nuclear extracts will be
incubated for 20 min at room temperature with Nrf2 probe. Samples will be then loaded and
electrophoresed through a 6% polyacrylamide gel at a constant voltage of 180 V. The gels
will be dried and scanned by exposure to x-ray films for different periods to obtain
autoradiograms appropriate for subsequent quantification by a densitograph. Supershift
analysis will be carried out using the anti-Nrf2 antibody (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA). Curcuminoids, as shown in this project, acts indirectly to confer antioxidant
activity and so may be expected to upregulate the level/activity of nuclear Nrf2.
Similarly, the CBP (CREB (cAMP responsive element binding protein) and its close
homologue, p300, has been shown to play a key role as a nuclear coactivator for a wide
variety of transcription factors involved in various different pathways including CREB,
activator protein 1 (AP-1), p53, as well as NFκB [442, 446]. Studies have also shown CBP
binding directly or through another member of p160 protein family, ARE-binding protein-1 to
the Nrf2 transactivation domain [442, 443]. It would therefore be interesting to study the
impact of curcuminoids on CBP/p300/p160 induced co-activation of transcription factor,
Nrf2. CBP has also been shown to interact and recruit the proteins possessing histone
acetyltransferase (HAT) activity into NFκB transcription activation complex. Histone
acetylation by HATs disrupts the attractive
electrostatic interaction between the DNA and
histones thus leading to the unwrapping of the DNA from the core histones and allowing
133
access for the NFκB transcriptional machinery, resulting in phase II gene transcription [444,
445]. CBP/p300 has, in addition, intrinsic HAT activity that acetylates histone and nonhistone
nuclear proteins [446, 447]. Converse to HATs, HDAC2 is responsible for condensation of
the chromatin structure through tighter winding of the DNA around the core histones and thus
displacing the transcriptional machinery and occluding further transcription factor binding,
thereby resulting in gene silencing. Whilst chronic oxidative stress has been shown to cause
reduction in HDAC2 activity and expression in
the lungs of patients with COPD [448, 449],
curcumin has been shown to restore the HDAC2 activity through regulation of its protein
expression status in ROS-stressed cells
in vitro. It would thus be interesting to explore the
impact of curcuminoids on the HAT/HDAC imbalance that exists under oxidative stress and
to ascertain whether it is able to maintain/restore the HDAC2 activity in pancreatic β-islet
cells subjected to H2O2-induced oxidative stress by employing, Nrf2 activity assay, HDAC
activity assay, siRNA studies, immunoprecipitation, western blotting, microarray gene chip
analysis and RTQ-PCR experiments.
The literature also reports that the activation of Forkhead box O3 (FOXO3) in cultured
fibroblasts and neuronal cells results in resistance to oxidative stress [410]. Genes involved in
DNA repair (GADD45), apoptosis (BIM and Fas ligand), ROS scavenging (MnSOD,
catalase) and cell-cycle arrest (p27KIP1) are transcriptionally induced by FOXOs [411, 430,
431]. In humans, SIRT1 (Silent information regulator 1) has been shown to deacetylate
FOXO, p53 and NF-κB transcription factors [432, 433]. Sirtuins (SIRT, Sir2 family of
enzymes) belong to class III HDACs that regulate gene expression in a variety of organisms
by deacetylation of modified lysine residues on histones, transcription factors and other
proteins. Since phosphorylation and acetylation regulate the transcriptional activity of
FOXO3, SIRT1-mediated deacetylation of FOXO3 leads to its activation and induction of cell
cycle arrest. Thus, high SIRT1 activity would promote cell survival [434, 435]. SIRT1 has
134
also been shown to upregulate stress-positive pathways via deacetylation of FOXO3
transcription factor leading to increased transcription of GADD45 (DNA repair) and MnSOD
(reactive oxygen detoxification) [436, 437]. Interaction of SIRT1 with p53 causes
deacetylation of the C-terminal regulatory domain [438], interestingly whereas depression of
SIRT1 leads to increased acetylation of p53, thereby increasing its pro-apoptotic and cell
senescence function [438, 439]. Decrease in the function of SIRT1 by NAD+ depletion leads
to accumulation of acetylated p53 which in turn causes oxidative stress induced cellular
senescence [440]. SIRT1 has also been shown to be downregulated in cells that have high
insulin resistance and induction of SIRT1 increases insulin sensitivity, suggesting that the
molecule is associated with improved insulin sensitivity [441]. Amelioration of oxidative
stress in cells and organs suggests that SIRT1 activation could have a real therapeutic benefit
under conditions of excessive ROS production. Alhough the precise role of SIRT1 in
oxidative stress and the detailed mechanism by which it deacetylates FOXO3 remains
uncertain, it would be important to fully elucidate the effects of curcuminoids on the role of
SIRT1 in the pancreatic β-islet cells subjected to H2O2-induced oxidative stress by
quantifying NAD+ levels, SIRT1 activity assay, SIRT1 carbonylation assay and RTQ-PCR
studies as well as interaction between SIRT1 and FOXO3 by western blotting, Chromatin
immunoprecipitation (ChIP) assays and RTQ-PCR. Curcuminoids would be expected to
activate FOXO3 as well as cause an increase in SIRT1 activity.
In conclusion this project provides evidence that advances the current understanding of
antioxidant activity of curcuminoids and the mechanism by virtue of which they alter the
antioxidant status of pancreatic β-islet RINm5F cells subjected to H2O2-induced oxidative
stress. Additionally, the project has clearly shown that curcuminoids at realistic plasma
achievable concentration have indirect antioxidant activity by virtue of modulating
intracellular antioxidant gene expression, as well as having an impact on the transcriptional
135
activator, NF-κB. Further in-depth research is required however, both in vitro and in vivo, in
order to fully understand the potential mechanism influenced by curcuminoids to combat the
H2O2-induced oxidative stress in pancreatic β-islet RINm5F cells and to further explore the
therapeutic potential of curcuminoids in diabetes.
136
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