influence multidrug resistance transporters …infïuence of cytokines on multidrug resistance...
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INFLUENCE OF CYTOKINES ON MULTIDRUG RESISTANCE
TRANSPORTERS IN HUMAN HEPATOMA CELL LINES
GIGI LEE
A thesis submitted in conformity with the requirement for the degree of Master of Science
Graduate Department of Pharmaceutical Sciences University of Toronto
O Copyright by Gigi Lee (2001)
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Canada
Infïuence of Cytokines on Multidrug Resistance Transporters
in Human Hepatoma Ceil Lines
Gigi Lee (M. Sc.), 201
Faculty of Pharmacy, University of Toronto
(Abstract)
Overexpression of P-Glycoprotein (PGP) and Multidrug Resistance Proteins (MW) is a
principle mechanism of drug resistance. Previously our laboratory demonstrated a
down-regulation in hepatic PGP expression and activity in endotoxin and Interleukind
(IL-6) treated rodents. We therefoie examined the effect of IL-6, IL- lfl and TNF-a on
PGP and MRP expression and activity in human hepatoma ce11 lines, (Hui37 and
HepG2). Treatment of HuH7 cells with IL-6, IL4 or TNF-a decreased the expression
and activity of PGP while causing an induction of MRP3 at the level of mRNA. In
HepG2 cells, POP expression and activity was down-regulated upon IL-1 or TNF-a
treatments, but not with IL-6. HepG2 cells treated with IL6 or IL-le also expressed
higher MRPl mRNA levels while a decrease in MRPl and M W 3 expression was seen
with TNF-a. We conclude that these cytokines may play a role in the regulation of these
transporters.
First, 1 would like to extend my gratitude and appreciation to my supervisor. Dr.
Micheline, Piquette-Miller for her support, encouragement, guidance and particularly
patience throughout my graduate studies. The completion of this project would not have
been possible without her constant support and assistance. Dr. R. Macgregor who had
inspired me to fulfill my dreams and pmvide suggestions to broaden my horizon. 1 would
also like to thank my advisors, Dr. Reina Bendayan, Dr. Peter Pennefather, Dr. Peter
O'Brien and Dr. Mike Rauth for both their F a t advice and technical assistance. Dr.
Usoa Busto for hcr participation as my extemal appraiser.
1 would like to express my deepest thanks to al1 those 1 had honor to work with over these
years: Annie Cheung, Julie Kalistsky, Won-Sang Lee, Wendy Tang. In particular Georgy
Hartmann, Mahadco Suichai and Adriane Yong : .ho had stood by me through al1
hardships and had provided me with continuous and ready assistance, support,
encouragement and friendship. ,
Last but not the least, my heartfelt thanks go to my dearest husband. His love.
understanding, klieve in me and continuous support throughout the whole course of rny
studies have made my dream come tme. Words fail to describe how grateful I am to my
most respectful parents, parents-in-law and my brother who had provided us with
emmnous support which haô made rny studies possible.
At the end, 1 would like to acknowledge my appreciation to the financial support
provided by University of Toronto Opa Fellowship, Edward Barber Scholarship and
Merck Fross t Post-Graduate Researc h Fcllow s hip.
Table of Contents
Abstract
Achow lcdgments
Table of Contents
List of Tables
List of Figures
List of Abbreviations and Syrnbols used
1. Introduction
1 . 1 Multidrug Resistance
1.2 PGP Overview
1.3 Overview of MRP farnily
1.4 Inhibition of PGP
1.5 n i e Inflarnmatory Mediatom
1.6 Cytokines and the expression of PGP and M W family
2 Rationaie and Experimental Justification
2.1 Background
2.2 Hypothesis
2.3 Project Objectives
2.4 Justification of Experimental Methods
Page
i
i i
iii
v
iii
3 Materials and Methods 42
4 Rcsults
4.1 Viability Studies
4.2 Expression of MDRl and MRP gene farnily membea in
HuH 7 and HepG2 cclls.
4.3 Effect of L 6 on PGP Expnssion and Activity
4.4 Effect of L l P on POP Expression and Activity
4.5 Effect of TNF-a on PGP Expression and Activity
4.6 Effm of IL6 on MRP Expnssion and Activity
4.7 Effect of IL1 on MRP Expression and Activity
4.8 Effect of TNF-u on MRP Expnssion and Activity
5 Discussion
5.1 Overview of the Project
5.2 Effect of IL6, IL- 1 and TNF-a on PGP in HuH 7 and HepG2 cells.
5.3 Effect of IL6, It lP and TNF-bt on MRP in HuH 7 and HepGZ cells.
5.4 Clinicall Physiologicai Implications of our study
5.5 Summary
6 Conclusion
Conclusions and Future Studics
7 Rcfercnces
List of Tables
Table Page
1 Expression of MRP family genes in humans 9
II List of primer sequences. PCR product sizes and cycle numbers 50
used in RT-PCR assay.
m Viability nsults of contml and cytokine treated HuH 7 and 53
HepG2 cells.
IV Summary of the effects of cytokine treatment on PGP in HuH 7 1 14
and HepG2 ceIl lines.
V Summary of the effects of cytokine treatment on MRP in HuH 119
7 and HepG2 ce11 lines.
List of Figures:
Ti tle
Accumulation of 5-CF in HuH 7 cells with varying doses of 5-
CFDA
Accumulation of 5-CF in HuH 7 cells over 60 minutes
Accumulation of 5-CF in HepG2 cells with varying doses of 5-
CFDA
Accumulation of 5-CF in HepG2 cells over 60 minutes
Time course of 5-CF efflux in HuH 7 cells
Time course of 5-CF efflux in HepG2 cells
Time course of Rh123 emux in HuH 7 cells
Time course of Rh 123 efflux in HepG2 cells
RT-PCR gel depicting relative MRRl and MRP farnily gene
expression in HuH 7 cells
RT-PCR gel depicting relative MDRl and MRP farnily gene
expression in HepG2 cells
Influence of IL6 matment duration on POP transport activity in
HuH 7 cells
Influence of IL-6 concentrations on PGP transport activity in
HuH 7 cells
Influence of IL-6 matment duration on POP transport activity in
Hep02 cells
Influence of I L 4 concentrations on POP transport activity in
HepG2 cells
Effect of IL6 on MDRl mRNA expression in HuH 7 and HepG2
cells
Effect of IL6 on PGP protein expression on HuH 7 and HepG2
cells
Influence of L I S treatment duration on PGP transport activity in
Page
3 1
32
33
34
36
37
39
40
54
55
58
59
60
6 1
62
63
66
HuH 7 cells
influence of IL-le concentrations on POP transport activity in
HuH 7 cclls
Influence of IL- I trcatment duration on PGP transport activity in
HepG2 cells
Influence of ILI B concentrations on POP transport activity in
HepG2 cells
Effect of IL1 $ on MDRl rnRNA expression in HuH 7 and
Hep02 cclls
Effect of IL1 B on POP expression on HuH 7 and Hep02 ce1 1s
Influence of TNF-a tnatment duration on PGP transport act i vity
in HuH 7 cells
Influence of TNF-u concentrations on POP transport activity in
HuH 7 cells
influence of TNF-a treatmcnt duration on POP transport activity
in HepG2 cells
Influence of TNF-a concentrations on POP transport activity in
Hep02 cells
Effcct of TNF-a on MDRl mRNA expression in HuH 7 and
HepG2 cells
Effect of TNF-a on PGP expression on HuH 7 and HcpG2 cells
Influence of IL6 tnatment duration on MRP transport activity in
HuH 7 cclls
influence of IL6 concentrations on MRP transport activity in
HuH 7 cells
Eff- of IL6 on MRP3 and MW6 mRNA expression in HuH 7
cells
Influence of IL6 trcatment duration on MRP transport activity in
HepG2 cclls
influence of IL-6 concentrations on MRP transport activity in
vii
Hep02 cells
Effect of IL6 on MRP mRNA expression in HepG2 cells
Influence of IL- 1 B trcatment duration on MRP transport activity
in HuH 7 cells
Influence of ILla concentrations on MRP transport activity i n
HuH 7 cells
Effect of LI$ on MRP3 and MRP6 mRNA expression in HuH 7
cells
Influence of IL-le treatment duration on MRP transport activity
in HepG2 cells
Influence of IL- 1 P concentrations on MRP transport activity in
HepG2 cells
Effect of IL4 P on MRP mRNA expression in HepG2 cells
Influence of TNF-a tkatment duration on MRP transport activity
in HuH 7 cells
Influence of TNFa concentrations on MRP transport activity in
HuH 7 cells
Effect of TNF-a on MRP3 and MRP6 rnRNA expression in HuH
7 cells
Influence of TNPa treatment duration on MRP transport acti vity
in He@? cells
Influence of TNF-a concentrations on MRP transport acti vi ty in
HepG2 cells
Effect of TNF-a on MRP mRNA expression in HepG2 cells
List of Abbreviations:
5-CF
5-CFDA
a-MEM
AP- 1
APP
APR
APRF
CRI'
CYP
DMEM
FBS
GAPDH
m - y
IL4 1
IL-1R
IL-1s
IL-6
L 6 R
JAK
LIF
LPS
5-Carboxyfîuoiescein
5-Carboxyfluonscein diacetate
a-Minimum Essential Medium
Activator Protein- 1
Acute Phase protein
Acute Phase Response
Acute Phase nsponse factor
C-Reactive Protein
Cytochrome P450
Dulbecco's Modified Eagle's Medium
Fetal Bovine Semm
Glyceraldehyde 3 Phosphate Dehydrogenase
Interferon -y
Interleukin- 1 1
Interleukin- 1 receptor
intcrleukin 1s
Interleukin-6
Interleukin-6 rcceptor
Janus Kinase
Leukemia Inhibitory Factor
Li pop1 ysaccaride
LTC4
MAP
MDR
MRP
MTI'
NF-IL6
NF--
PBS
PGP
Rh 123
SAA
TGF-8
TNFR
Mitogen activated protein
Multidrug rcsistance
Multidmg nsistancc protein
Nuclear Factor- interleukin 6
Nuclear Factor- kappa B
Phosphate Buffered Saline
P-gl ycoprotein
Rhodamine 123
Semm Amyloid A
Transforming growth factor-p
Tumor Necrosis Factor receptor
Tumor Necrosis Factor-a
INTRODUCTION
lntroduc tion
1.1 Multidnig Resistance
Liver cancer is a leading cause of morbidity worldwide. Simultaneous resistance of
turnor cells to multiple cytotoxic dmgs, or multidrug resistance (MDR). is a major
obstacle to the successful chemotherapeutic treatment of cancer. (Germann, 1996,
Hooper et al, 2001) Mechanisms by which tumor cells can acquim MDR include the
failure of drug uptake or activation, alteration in the level of target enzymes such as
topoisomerase 11, mutations in these target enzymes, enhanced expression of detoxifying
enzymes such as glutathione S-transferases, increased anticancer dmg efflux, and
alterations in the tumor microenvironment (e.g., vascularization, hypoxia) (Germann,
1996). An incrcase in drug efflux seems to bc a major mechanism as i t reduces the
iniracellular accumulation of cytotoxic drugs through an overcxpression of several
members of the ATP-binding Cassette superfamily of transporters, namely, P-
glycopmtein (PGP) and the family of multidrug resistance-associated proteins (MW)
(Gcmnann 1996, Hopper et al 200 1). These active efflux transporters. which are highly
expressed in the healthy liver, arc of'ten induced in malignant cells, and thus impart a
MDR phenotypc to numerous anticancer drugs including vincristine, doxorubicin and
taxol. (Fardcl et al, 1996)
1.2 PGP Overview
PGP is a 170 kDa transporter which functions as an ATP-dependent efflux pump for a
variety of dnigs. (Farâel et al, 1996) Its protein structure consists of 2 membrane-bound
domains. each consisting of 6 transmembrane segments and a nucleotide binding domain
which will bind and hydrolyze ATP. (Sharom, 1997) The mechanism by which PGP
transports dmgs is still unknown, but it is thought that POP acts as a "flippase" in the
membrane of the cell. (Fardel et al, 1996, Sharom, 1997) It is a member of the MDR
gene family, which includes MDRI and MDR3 in humans and rndrla, indrlb and mdr2 in
rodents. Only Class 1 MDRI genes (MDRI, mdrla and mdrlb) encode for the dnag
efflux transport protein. (Germann, 1996) MDR3Mr2 encodes for a phospholipid
transporter with specific affinity to phosphatidylcholine. (Gemann, 1996) PGP/MDRl
actively secretes a broad spectnim of stnicturally unrdated diverse chemicals, including
many important organic cationic drugs. (Gemann 1996, Sharom 1997, Endicott et al,
1989) t
It has bccn demonstrated that the overexpression of PGP in cancer cells imparts
resistance to a variety of anticamer drup (e.g. vinblastine, vincristine, doxorubicin.
daunombicin, etoposide and palictaxol) as well as many othcr non-cytotoxic dmgs such
as dexamethasone . aldosterone and fexofenadine. (Farde1 et al, 1996, Cvetkovic et al,
1999) Severai substrates of PGP also compctitively inhibit the efflux activity of the
transporter (e.g. verapamil and cyclosporin A) through cornpetitive inhibition. (Gemann,
19%).
In humans, high levels of MDRl mRNA are exprcssed in the adrenal glands, kidney,
livet, jejunum, ileum. colon and bloodlbrain barrier. (Gennann, 1996, Endicott et al,
1989) MDRl gene proàucts am exprcssed on the apical surface of secretory epithelial
cells lining luminai spaces, e.g. the columnar epithelium of the small and large intestine,
bmsh border of proximal rend tubule and bile canaliculi of hepatocytes. (Fardel et al.
1996, Endicott et al, 1989)
Physiobgical implications of PGP
While the physiological role of POP has not been established, the presence of PGP on
the mucosal surface of the small and large intestines suggested that PGP may assume a
general protective role and is involvcd in the defense against xenobiotics ingested with
food. (Gennann, 1996, Endicott et al, 1989) The expression of PGP in the liver and
kidney suggests its role in the excretion of xenobiotics or possibly endogenous
metabolites. (Gcrmann, 1996, Endicott et al, 1989) The prcsence of PGP in the
bloodlbrain banier suggests a role for PGP in the protection of the organism from central
nervous system toxicity. This is supported by the phenotypes of the mdrl (4) knockout
mice, which an normal, but exhibit incrcased sensitivity tc vinblastine and showed
alterations in phamacokinetics and tissue distribution of several dmgs (incnase
accumulation in ihe brain and decrcase rate of climination). (Germann, 1 996) In mdr la
(4) knock out rnice, the phmacoicinetics and tissue distribution of several PGP
substrates such as dexarnethasone, digoxin and cyclosporin A have been shown to k
altercd. (Schinkel et al, 1995, Germann, 1996)
Class II MDR pnes ( M M and rndr2) are involved in hepatic phospholipid transport.
(Fardel et al. 1996) in mir2 (4) knockout mice. impaired phospholipid transport into
the bile was observed which cause progressive liver disease over time (Bradshaw D,
t 998).
In summary, PGP plays a significant role in dmg transport and protecting cells from toxic
substance. Thereforc by understanding the mcchanisms of physiological nplation of
PGP may lead to some insights into the control of the expression and aciivity of POP
which may lead to novel ways in overcoming multidrug resistance.
1.2 Overview of MRP Family
The human multidnrg resistance associated protein (MW) family of transporters consist
of at kast 7 membcrs: MRPl-7. (Hopper et al, 2001) They arc a farnily of 190 kDa
proteins which belong to the ATP-binding cassette transporter superfamil y (which
includes PGP and the cystic fibrosis transconductance replatory protein, among many
others acmss species from bacteria to man) (Borst et al, 1999). MRP family membea
mediate unidirectional ATP-dependent transport of anionic conjugates and amphiphilic
anions. (Kuwano et al. 1999). Within the MRP farnily, homology is highest between
MRPl , MRP2, MRP3 and MRP6. (Borst et al. 1999, Hooper et al, 200 1 )
MRPl. MRP2 and MRP3 have ken hinctionally characterizcd as conjugate export
pumps. (Borst et al, 1999) MRPl and MRPZ s h m similar substrate specificities. with
high aninities for the glutathione, glucuronide and sulfated conjugates while MRP3
demonstrates a prefenntial affinity for glucuronide conjugates. (Borst et al, 1999)
Transfection snidies of thesc transporters in human cancer cells have shown that they can
confer nsistance to cytotoxic drugs such as vincristine. vinblastine and methotrexate.
(Borst et al. 1999, Kuwano et al, 1999) MRPS vanspons cyclic nucleotides but not
glutathione or glucuronide conjugates and ha9 not ken shown to confer multidrug
&stance. (Schcffer et al, 2000, Hopper et al, 2001) MRP4 transports mainly
nucleoside analogues but can confer iesistancc to methotnxate. (Hopper et al, 2001).
Substrates for MW6 and M W 7 have not yet bcen identified. (Borst et al. 1999, Hooper
et al. 2001) However, in addition to dnigs, MRPI. MRP2 and MRP3 also transport
endogenous substrates. MRPl has a high affhity for transporthg Leukotriene C4
- . . (LTC4). (Kuwano et al, 1999) MRP2 has a major role in transporting bilirubin (Kuwano
et al, 1999) while MRP3 is a bile salt transporter for glycocolare and taurocholate.
(Takada et al, 2000)
Table 1 shows the expression pattern of MRP in humans. MRPI, MRP4 and MRPS an
widely distnbuted throughout the body whereas MRP2, MRP3 and MRP6 appear to be
mainly expressed in liver, kidney and gut. (Borst et al. 1999) in hepatocytes, MRP l and
MRP3 are located on the basolateral membrane while MRPî is located on the canalicular
membrane. (Bont et al, 1999) MRP6 has becn shown to exist on both lateral and
canalicufar membranes in rat hepatocytes. (Madon et al, 2000)
Physiologicol implications of the MRP farnily
In addition to transport of endogenous substances, defense against toxic compounds and
maintaining cellular homeostasis is a major role of the MRP transporters. For example,
MRPl is located at the basolateral membranes of epithelial cells in the choroid plexus,
which is essential for the exchange of metabolites ktween blood and cerebrospinal fluid.
(Borst et al, 1999) Hence, decrcase in expression of MRPI may result in accumulation
of drugs in the cercbrospinal fluid and increased CNS toxicity. (Borst et al. 1999)
Mutations in the MRPZ gene can cause hbin-Johnson Syndrome which is a hereditary
disease characterized by conjugated hypcrbilirubinemia. (Kuwano et al, 1999) MRP3
has a metabolic role in the removal of olpanic acids from bile and has been shown to be
upregulatcd under conditions of cholestasis. (Bont et al, 1999) Mutations in the MRP6
gcne can lcad to Pseudoxanthoma elasticum which is a heritable connective tissue
disorder with abnonnalities in the elastic structures in the body such as eyes,
cardiovascular systcm with consideraôle morbidity and mortality. (Ringpfeil et al, 200)
As a rcsult, it is equally as important to understand the ngulation of the MRP family
transporters since they share similar substrates as PGP and can also confer multidrug
resistance.
MRPS
MRP6
MRP7
Main location in the body
Ubiquitous: testis, skeletal muscle, lung
Liver, Kidney, p t
Liver, kidney, p t , pancreas, adrenal glands I Lung, prostate, muscle. testis. bladder (al1 I expnssed at low levels)
Liver, kidney
Colon, skin, tcstis 1
Table 1: Expression of M W family genes in humans. (Borst et al, 1999. Hopper et al,
200 1, and Kuwano et al, 1999)
1A Inhibition of PGP
With the discovery that these enlux transporters an often induced in malignant cells and
can impart a MDR phenotype on trcatment with numerous anticancer drugs, then have
been attcmpts to inhibit these transporters. However, these enorts have met with little
success because, for some of the inhibitors (e.g. verapamil), the inhibi tor concentration
that is requircd to block PGP is much higher than the normal therapeutic dose range.
These extnme doses can lead to toxic side effects (cg., cardiac effects). (Fardel et al,
1996, Walther 1994) Furthemore, studies have shown that PGP inhibitors such as
veraparnil, can cause an induction of MDRl pne expression in human cell lines.
(Walther 1994) However, this may be a cell dependent phenornenon as verapamil has
also been shown to downregulate PGP in leukernia cells. (Fardel et al, 1 996) Thereforc
the= is a need to find other means to rcgulate the expression and activi ty of these
transporters.
Numerous reports indicated that the hepatic expression of PGP and MRP2 are decnased
under conditions of experimental inflammation and cholestasis in rodents. (Hartmann et
al, 2001, Kool et al 1999, Sukhai et al in press, Trauner et al 1997) Bacterial or viral
infections as wcll as experimcntal modcls of -te inflammation evoke an acute phase
nsponse which is associated with alterations in the expression of many liver denved
proteins including the cytochromc P450 (CYP) mctabolizing enzymes such as CYP 1Al
and CYP 3A4 (Hasler et al, 1999, Muntanc-Rclat et al, 1999, which can in turn alter
dmg metabolism and disposition. (Wright, 199 1, Hasler et al, 1999) It is believed that
these changes arc brought about by the rclease of proinflammatory cytokines. It is
known that the CYP and PGP gene families s h m many similar substrates, inhibitors and
inducers such as nfarnpicin and dexamethasone, and these compounds can induce both
PGP and CYP3A expression in rats and primary human hepatocytes (Raynes, 2000.
Wacher et al. 1995. Salphati 1998. Schuetz et al, 1996) hence it was fel t that there might
exist common ngulatory pathways for the expression of CYP and PGP. Therefore. the
primary goal of this project is aimed at detennining whether inflammation and
proinflammatosy cytokines could elicit any changes in the expression and replation of
these transporters. Thus the effects of these pro-infiammatory cytokines on MDRl and
MRP activity and expression wen examined in human hepatoma cells.
1.5 The Infiammatory Mediators
Upon acute inflammation, a group of innammatory mediators tenned the
proinfiammatory cytokines an released from macrophages in a cosrdinated manner.
Thcre are at least 8 proinflammatory cytokines: Interleukin 6 (IL-6). Interleukin lp (IL-
I S), Tumor Necrosis Factor- u (TNF-u ), Interleukin 1 1 (IL 1 1 ), Oncostatin M.
Leukemia Inhibitory Factor (m. Interferon- y (1FN.y) and Transforming Grow th
Factor+ (TGF-B). (Richard et al, 199 1, Kushner 1.. and Mackiewicz A.. 1993) Among
them, a-6, IL-le, and TNFa, are the principal mediators of the acute phase response
(APR). (Kushner I., 1993, Fey O., 1990) Release of these cytokines in senim trigger an
APR in liver which in Nni aiters the expression of numerous proteins which are
collcctively t e m d the acute phase proteins (APP) (Richard et al, 199 1, Kushner 1.. 1993,
Fey, G., 1990). Some APP such as Serum Amyloid A ( S M ) and C-Reactive Protein
(CRP) an induced upon onset of the APR while some negative APP, such as dbumin,
are significantly down-regulated (Richard et al, 1991 and Fey, G., 1990) Although the
acute phase proteins an structurally and hinctiondly diverse, they are generally involved
in the defense of the host against tissue damage or infection.
IL6
IL6 is the principal mediator of hepatic changes in gene transcription and protein
synthesis rates. It is a cytokine with plciotropic activitks, which stimulates the
production of APP such as antiproteinases and fibnnogen in the liver. These APP are
specifically induced by IL6 only. IL6 also cause the secretion of immunoglobulins in B
cells. (Alcira 1992). and the proliferation of hematopoietic progmitor cells. (Akira et al,
1990) It is proâuced by a number of differcnt cc11 types such as fibroblasts,
macrophages, T and B lymphocytes but it is not produced under normal circumstances.
(Akira, 1990)
Signal transduction is initiated when IL6 is bound to the IL6 specific cell-surface
receptor (L6R). (Rose-John, 1993) This complex then activates the JAWSTAT
Tyrosine Kinase pathway, which induces activation of APRFlSTAT3. IL-6 will also
activates a mitogen activated protein (MM) Kinase Cascade which induces the
production of NF-M. (Akira, S 1997) NF-U's induction is the principal phway by
which IL6 rncdiatcs changes in APP expression during acutc inflammation. (Akira.
1990, Akira, 1992) Aside from NF-IL6, other transcription factors including AP-I
(Raingeaud et al 1995) an also reporteci to k induced through the activation of MAP
Kinase Cascades. Hcnce these 3 transcription factors are thought to be involved in the
IL6 signal transduction pathway.
I L l P
The proinflammatory cytokine, ILI is a key mediator of the host response to
inflammation. ILlp can stimulate T-cells, production of growth facton and induces the
biosynthesis of several APP during infection, tissue damage or stress. (Dinarello, 1996,
Jensen et al, 2000) IL4 afTccts the hcpatic APR through stimulation of IL6 s yndiesis in
fibmblast and other stroma1 cclls. (Richard et al, 1991) Together with IL-6, IL- 1
stimulates production of opsonins and transport proteins such as C-Reactive Protein,
SAA. and a 1 -acid glycoprotein. (Richard et al, 199 1)
Upon inflammation, IL 1 $ is pmduced by macrophages and binds to IL4 receptors (Il
IR) located on ceIl surface (Richards et al, 1991). which associate with the cytoplasmic
kinase IRAK. IRAK in tum activates TRAF6 Kinase to phosphorylate and activate NF-
KB. (Jensen et al, 2000) NF-KB can then translocate to the nucleus where it activates
transcription by binding to specific binding sites in the promoters of numerous genes.
(Jensen et al, 2000) Additionally, L l S can also activates the MAPK Cascade directly to
induce the production of transcription factor, NF-IL6 Transcription factor AP- 1 has also
been dcmonstrated to be induced by IL1 (Diiffado et al. 1994)
TNFIa
TNF-a also plays a significant role in host inflarnmatory and immune response. TNF-a is
predominantly produced by activated macrophages and can be induced by natural killer T
and tumor cells. (Walther et al, 1995) TNF-a acts on macrophages and endothelial cells
where it induces IL- 1 and IL-6. (Brouckaert, P. and Libert, C. 1993) TNF-a has
pleitmphic functions such as activation of T-cells, mutagenesis of fibroblasts and
activation of neutmphil functions. (Nitta et al, 1994) TNFix also stimulates a number of
APP genco, sirnilar to those regulatcd by IL4$ including fibrinogen, al -a&
glycoprotein and albumin but cffects of TNF-a have ken shown to be weaker than IL-
lp. (Brouckaert, P. and Likrt, C. 1993)
TNF-a binds with T N F a spcific rcceptors and activates the transcription factor NF-
KB, however this signaling pathway occurs through a different signal transduction
pathway than that of IL1 p. TNFa binds to its own TNF rcccptor (TNFR) to activate
the TRAFl and TRAFZ complcx which triggers the MAPK Cascade and phosphorylates
NF--. (Cleveland and Ihle, 1995, Liu et al, 1996, Wiegmann et al, 1994, Cao et al,
1 996, Malinin et al, 1 997)
ûther Pro-in@mnta!o~ cytoùines
Besides I L I & IL6 and TNF-a. then arc 5 other proinflammatory cytokines in which
some of them exhibit similar sipal transâuction and have similar physiological functions
as IL6 while othea have th& own unique hinctions and signal transduction pathways.
Oncostatin M. IL4 1 and LF, which al1 proâuce nsponses similar to IL-6, are tenned IL
6 likc farnily cytokines. They elicit direct acute phase alterations in hepatic plasma
protein synthesis through similar pathways to that of IL-6 and through interaction with
specific Rcepton. (Kushner, I, and Mackiewicz, A. 1993) However they have a much
weaker effect than IL-6. (Kushner, L, and Mackiewicz, A. 1993)
IFN-y induce the expression of numerous proteins which mediate antiproliferative and
immunornodulatory effects. (Melem et al, 2000) This cytokine interacts with I F N q
specific rcceptor and activates JAKl and JAK2 Tyrosine Kinases which phosphorylate
the STATl protein and in mm homodimerizes to fonn a GAP complex. GAF
tmnslocatcs into the nucleus and bind to the appropriate elemcnts in the promotet of IFN-
y inducible genes. (Melem et al, 2000)
TGF-0 is involved in the regulation of cell growth and function, particularly during
development of tepair. It has potent proliferative and antiprolifcrative effects depending
on the ce11 type. (Zhang et al. 1994) TGF-p has its own unique signal transduction
pathway which involves binding to the TGF-B nceptors which thcn activates the SMAD
transcription factor cascade. (Wrana 1998)
Overall. these proinflarnmatory cytokines have important roles in inflammatory reactions
and in rcsponse to infection. Besides their action on neutrophils r d macrophages, L6,
ILlp and TNF-a have the most pronounced effcct on the livcr during an APR evoking
substantial changes in the expression and production of the acute phase proteins. (Fey,
G., 1990).
1.6 Cytokines a d the expression of PGP and the MRP f d l y
Thcm have been numerous studies published in literature and fmm our laboratory which
demonstrate that acute inflammation and cytokines have an influence on PGP and MRP
expression. In Our laboratory, the induction of acute inflammatory response in rodents
using lipopolysaccaridc and turpentine administration (Piquette-Miller et al, 1998) have
shown a reduction in the hepatic expression and activity of PGP at the level of mRNA.
Thus it has been postulated that these changes are brought about by the release of
proinflarnmatory cytokines such as IL6, IL4 and TNF-a.
Prcvious rssults generated from our laboratory also demonstrated that IL-6 cause
significant reductions in the expression and activity of the mdrI genes in vivo in rnice
(Hartmann et al, 2000) and in vitro in cultured rat hepatocytes. (Sukhai et al, in press)
This reduction has been shown to occur through a suppression of mdrl gene transcription
in IL6 trcated rat hepatocytes. (Sukhai et al, 20ûûb) Thus, it has been postulated that
IL6 is likcly involved in the downreplation of POP expression and activity during acute
inflammation. In mice, IL- 1 P causes a substantial down-replation of mdrla gene
expression whereas TNF-a imposes a signifcant induction in rndrlb gem expression.
(Hartmann et al, 2001) IL-lfl also dccre~ses the expression and activity of POP in
culturcd rat hepatocytes, howevcr this is only evident at the levcl of protein. (Sukhai et
al, in press)
Numerous exarnples in the literaain have also &monstrateci effccts of cytokines on the
expression of PGP and MRP family members. In vitro studies in four human colon
-- - carcinoma ceIl lines have demonstrated that trcatment A with IL2, IFN-y or TNF-a elicit a
down-regulation on the expression of PGPlMDRI at the level of mRNA. (Stein et al,
19964 Stein et al. 1996b.Walther et al. 1994) However the degne of reduction and
effect on POP was seen to differ betwccn these ceIl lines thus suggesting ceIl lines
specific differcnces in cellular and molecular mechanisms by which cytokines modulate
expression of the PGP/MDRl pne. It has ken suggested that these influences on gene
transcription could involve activation of the transcriptions factors previously identified.
e.g. NF-* and AP-1 as in the case of TNF-a
However, contradictory results on the rcgulation of POP have also ken reported. in
endotoxemic rat liver, the expression of nzdrlb was induced with no change in the
expression of indrla. (Vos et al, 1998) Since neither quantitative nor statistical analysis
of nsults were presented, it is difficult to compare their study findings w ith those
obtained in our laboratory. On the other hand, it is possible that the degree of
endotoxemia in rats could play a rok in altenng the pattern and extent of cytokine
rcleasc. For instance, TNF-or, which is induced in endotoxemia, has been shown to both
incnase and decrease d l b expression. (Hartmann et al, 2001, Walther et al, 1994 )
Futhermore, our results demonstrating an induction of mdrlb expression upon TNF-a
trcatment suggests that di Hcrent in fiammatory models may impart dissimilar effec ts on
mdrlb expression. Studies utilising other modtls of inflammation such as chronic
adjuvant-induced arthritis did not detect changes in the hepatic expression of PGP
(Piquette-Miller. unpublished results) which indicates a dependency on the degree of
inflammation.
With regards to the MRP farnily, TNFa and IL4 have both k e n shown to decrease the
expression of MRPZ mRNA in isolatcd rat hepatocytes, with It 1 king a stronger
suppressor of hepatic MRP2 mRNA expression than TNF-a. (Nakamura et al, 1999) It
h a beni demonstrated that endotoxin administration in rats imparts a reduction of
M W 2 while imposing an induction in the Ievels of MRP 1. (Vos et al. 1998). Other rat
models of cholestasis, including bile duct ligation also cause a reduction in the expression
of MRP2 dong with a substantial induction of MRP3. (Hirohashi et al, 1999) Each of
these modcls arc associated with an increase in rclease of the proinflammatory cytokines.
Thus, studies in our laboratory have examined the impact of APR and individual
cytokines on MRP expnssion.
As a rcsult, it is evident that cytokines can have an influence on the expression of PGP
and MRP family transporters. Due to the overlapping and synergistic effects of these
cytokines, it is likely that these cytokines modulate MDRI gene expression in a unique
manncr which relies upon cytokine concentration, ce11 type and species. Hence we would
li ke to sec if similar changes on the expression of MDR transporters occur in human
hepatocytes and to understand whether changes in the expression of these transporters in
diseases which involve cytokine induction could be involved in d~g-disease interactions.
LI Background
As discussed in introduction. studies pcrfonned in our laboratory have demonstrated that
hepatic expression and activity of PGP arc reduced in rodent models of acute
inflammation. (Piquette-Miller et al, 1998, Hartmann et al, 2000) Acute inflammation
induced by LPS and turpentine administration am well characterizcd in vivo models of
inflammation. (Piquette-Miller et al, 1998) IL-6, IL4 and TNF-cx are major mediators
of acute inflammation with IL-6 king the principal mediator. Significant mdrl gene
suppression have been observed in IL6 treated rat hepatocytes. (Sukhai et al, in press)
More rcccntly it has k e n demonstrated that IL6 trcated mice exhibit significantly lower
levels of hepatic POP expression (Hartmann et al, 2 0 ) and a similar effect was also
obscmd in IL6 trcated cultured rat hepatocytes. (Sukhai et ai, in press) The IL-6
mediated rcduction of PGPImdrI expression in isolated rat hepatocytes has also been
recently shown to occur at the level of gene transcription. (Sukhai et al, 2ûûûb)
Downrcgulation of PGP protein but not mRNA expression has ben observed in IL4
txeated rat hepatocyics. (Sukhai et al, in press) A simiiar downrcgulation of mdrla gene
expression was obscrved in IL l trcated mice. (Hartmann et al, 200 1 ) On the other
hand. TNF-a was shown to induce expression of mdrlb in mice. (Hartmann et al, 2001)
Other groups of mearchers have also demonseaicd that cellular exposure to cytokines
such as TNF-a, IL2 and IFN-y can significantly downreplate POP at the rnRNA level
in human colon carcinoma ce11 lines. (Walther et al, 1994)
It has also been rcported in literature that MRP2 is significantly reduced in
experimentally induced cholestasis and inflammation in rats (Vos et al. 1998) while
levels of MRP3 (Hirohashi et al, 1999) and MRPl (Vos et al, 1998) are induced.
Expcriments in out laboratory have also show that expression of mrp2 was nduced
upon LPS-induced acute inflammation (Hartmann, unpu blished iesults) and these
changes appear to occur through activity of the proinflammatory cytokines. Indeed,
studics in cytokine-treated micc indicated a suppression of nrp2 mRNA in mice treated
with L 6 and Lie. (Hartmann, unpublished rcsults)
As cytokines appear to be highly involved in the regdation of PGP and MRP
transporten in rodents, we wanted to establish whether similar rcgulaiory pathways exist
in humans. Hence we examined the effect of these proinflammatory cytokines on the
expression and activity of POP and MRP in vitro using two human hepatoma cell lines.
Morcover, it will k helpful to sec if observable changes in POP, previously nported in
human colon carcinoma cell lines, is a general rcplatory response of PGP to cytokines or
whether this response is organ or cell specific.
2.2 Hypdhcsis
Pro-infammatory cytokines I L 4 IL- I f l and TNF-a will influence the expression
of the MDR eflw transporters, PGP anù the MRP family in human heputorna ce11
fines.
23 Project Objectives
1. To examine the influence of IL-6, LI and TNF-a on the functional
activities of PGP and MRP family
2. To establish whether cytokine mediated changes in the functional
activities of PGP occur through changes in protein or mRNA expression.
3. To ascertain the intluence of these cytokines on mRNA levels of M W
family.
1) Human Hepatoma ce11 lines as a suitable in vitro mode1
In theory, an ided way to study the impact of cytokims on tfflux transporter expression
and ectivity would be to perform in vivo human studies. However, there arc a large
number of confounding factors and technical challenges associated with these studies
which make in vivo studies less than an idea such as inability to obtain tissue sarnples and
confounding influences of genetics and dnigs. Therefom. we felt it was necessary to
initially pcrform these experiments in an in vitro system. Primary culture of human
hepaiocytes would k a good choice to examine changes in MDR transportes in response
to cytokines because they possess very similar overall characteristics and msponses to
cytokines as seen in whole human liver and these cells express PGP, MRP2 , and MRP3,
similar to normal human liver. However, as we have limited acccss to fresh human
livers, experiments involving pcimary human hepatocyte cultures would suffcr from the
lack of sample availability and quantity. Furthermore changes in gene expression with
culturing times has not been fully chanrterized in human primary cultures of
hepatocytes. Hence we then looked into the use of continuous ce11 lines. Human
hepatocyte ce11 lines available from the American type culture collection (ATCC)
catalogue (http:l/ ww w .atcc.org) includcd several hepatoma ce11 lines.
We chose to perfonn these studies in two human hepatoma ce11 lines, Hep02 and HuH 7.
These ce11 lines wem chosen as both ce11 lines werc developed from human liver
specimens (US Patent) and express these transporters. Othcr available ce11 lines, such as
Chang Liver and Hep38, have k e n shown to k contaminated with Hela cells and
Hepatitis B virus. (http://www.atcc.org) Another hepatoma cell line, SK-Hep 1 expresses
only low kvels of PGP and is poorly differentiated, therefon this ceIl line was not felt to
be a good model. (Chiu et al, 1989)
Hep02 celfs
A) Morphological characteristics of liver parenchyml cells.
Hep02 cells, onginally developed from a 15 year old caucasian male w ho had
hepatoblastoma biopsy sample. It is a well established rnodel for studying cytokine-
mediated effects. (US Patent) It is a very well-diffenntiated epithelial cell line that has
bcen shown to rctain phenotypic characteristics of liver cells. (Stockel et al, 2000) It has
been tested and ch-terized to rctain many parcnchymal hepatocyte&xific functions
such as albumin and bile acid synthesis. It has been widely used by researchers as in
vitro system to study hepatic protein synthesis and replation by the influence of
cytokines. (Gutiemz-Ruiz, 1999, Daffada et al, 1994, Stonans et al, 1998) Hence we felt
that this ceIl line would be a suitable model for these studies based on its morphological
characteristics of liver parenchyrnal cells and its ability to synthesis plasma proteins like
normal hepatocytes when triggered by cytokines.
B) Presence of cytokine-specijic receptors on ce11 surf4ce
1t has bcen shown that HepG2 cells express the receptors for many cytokines including
IL-4, ILS, L 7 , IL1 1 and M. (Stonans et al, 1998) Under normal conditions, IL6
rnRNA is not dctectable but is inducible upon exposurc to appmpnate treatment. Fcr
example, ILI and TNF-a are able to trigger the production of IL6 mRNA in HepG2
cclls. (Stonans et al, 1 998) Furthemore, studies have s hown that IL-6. IL 1 P and TNF-
a can bind to teceptors on the surface of HepG2 cells and stimulate protein synthesis in
thest cells. (Sonne et 41, 1990) The binding affhity of IL6 has aiso been compared
ktwecn isolated rat hepatocytes and HepG2 cells; with both cc11 types showing a high
affinity for this cytokine.
C) A bility ro elicit APR
HepG2 cells is a valid model for the human hepatic pannchymal cells as they can bind to
IL4 (Scheffer et al, 2000) and elicit the typical acute phase protein tesponse such as a-l-
antichymotrysin and cemloplasmin induction. However. the extent of induction of the
APR is less than that observed in primary culture of human hepatocytes. (Scheffer et al,
2000) Similarly, both IL4 a and TNF-a are able to stimulate the production of positive
acute phase proteins such as cenilosplasmin and suppnss the synthesis of the negative
acute phase protein, albumin, afker 20 houn of trcatment in HepG2 cells. (Kobsel. N and
Ramadori, 0.. 1994) Thenfore, with the pnsence of the I L 4 receptor on HepG2 cells
and their ability to produce APP after stimulation by IL-6, L l p and TNF-CX, HepG2
cclls is an appropriate in vitro model for studying regulatory mechanisms induced by
cytokines in hepatocytes. Other studies have also shown that upon treatment by L6, IL
1 B and TNF-a on HepG2 celh, transcription factors such as AP- 1, NF-KB. NF-IL6 are
induccd which suggests that the signal transduction pathways of these cytokines arc intact
and funciional in HepG2 cells. (Daffads et al, 1994)
D) Erpnssion of dlrerent MDR transpotters of interest
Furthcnnort, it has b e n shown that in a monolayer, certain fractions of HepG2 cells
fomed apical micro-villi lined vacuoles betwccn adjacent cells, which resemble bile
canaliculi. (Roelofsen el at, 1997) MDR transporters such as PGP, MRPl and MRP2 aie
al1 present on thcse cells. (Roelofsenet al, 1997) With the use of immunofluorescence
microscopy, PGP present on HepG2 cells have been shown to k located apically as seen
in normal hepatocytes. (Roelofsen et al, 1997) Whercas MRP 1 has been shown to be
localized mainly to the lateral membrane. (Bont et al, 1999)
As a result, HepG2 cells resemble an appropriate in vitro human liver mode1 for our
proposed study since this ceIl line can generate an acute phase nsponse just like normal
hepatocytes upon cytokine trcatment and expresses the MDR transporten that we are
interestcd in studying.
HuH 7 cells
HuH 7 is another well-differcntiated hepatoma cell line that possesses an enzyme pattern
suitable for studying cytokine-mediated induction of acute-phase proteins. (Malle et a,
1999) It has been shown that in nsponse to IL 1 and IL-6 treatments, HuH 7 cells are
also capable of synthezising APP including S M , u 1 -antichymotrypsin, ai -protease
inhibitors etc. (Fardel et al, 1996, Rayncs, 1993) In another study, HuH 7 cells
nspondcd to TNF-a tmtment. (Arndt et uf, 1992) HuH 7 cells also express efflux
transporters namely POP and MRPJ, hence with thcir ability to rcspond to these 3 pro-
inflammatory cytokine treatments, HuH 7 cells dso serves as an appropriate mode1 for
our studies.
Although both HepG2 and HuH 7 cc11 lines may differ phenotypically from normal
human hepatocytes, they.nevertheless have retained the qualitative and quantitative acute
phase nsponse of human livcr and may k interpnted to reflcct general mechanisms of
acute phase protein induction. (Malle et al, 1999, Rayms et al, 1993, Scheffer et al, 2000,
Stonans, 1998) Given these limitations, the rcsults presented may indicate the
importance of specific effects on these MDR efflux transporters upon individual cytokine
trcatmen t .
2) Justification of our choice of cytokines for our studies
iL-6, L I a and TNFs w e n chosen for our study. They are the 3 major pro-
inflammatory cytokines elicited during acute inflammation and based on pnvious work
conducted in Our laboratory, these 3 cytokines have k e n demonstrated to impart
significant changes in the expression of PGP and hlRP2 both in vivo and in vitro rodents
models. (Hartmann et al, 200 1, Sukhai et al, in press)
Cells wen treated with physiologie doses of each cytokine over W 2 houn: IL-6 (0-
25nghnl); L 1 fl(û-û.Sng/ml) and TNFa (O-O.bg/rnl). Maximum concentrations wen
chosen to rcflect those found in patient scrum during extreme septic shock. (Barrierc et
al, 1995) Since these cytokines arc elicited shortly afler inflammation we studied their
effects on MDR transporters over 0-72 houa. For example, IL6 is induced within 45 to
6û Mnutes upon inflammation and declines within 6 hours with the rnajority of IL6
mediated effects on APP (CRP) occumng within 20-24 hours, (Sehgaî, 1993)
3) Jwt~~cution of the em assays used in OUT study
Indomethacin-inhibitable 5-CF e m assay
MRP hinctional activity was assayed using the fluorescent MRP substrate, 5-CF as
prcviously used by othcr laboratories. (van der Kolk et al, 1998, Courtois et al, 1999) 5-
CFDA by itself is not fluorescent, but after it difhiscs into cells, intracellular esterases
convert 5-CFDA to a fluorescent anion 5-CF. (Van der Kolk et al, 1998) 5-CF has k e n
shown to k a specific substrate of the MRP family of transporters, but is not a substrate
of PGP. (Van der Kolk et 01, 1998) Efflux of 5-CF was exarnined in the presence and
absence of the MRP-specific inhibitor, Indomethacin. (Draper et al, 1997) Indomethacin
has k e n shown to be a specific inhibitor of MRP but does not inhibit PGP (Draper et al,
1997, Courtois et al, 1999). The functional activity of MRP could thus be detemined by
measuring the amount of 5-CF effiuxed from the cells in the pnsence or absence of
indomethacin.
4) Establishment and optirniration of the 5-CF assay
5-CF Accumulation Study
In order to detcnnine concentrations of 5-CFDA needed for adequate fluorescent
detcction. we incubated Hep02 and HuH 7 cells (3 million cells) into 6-well culture
clustem with 0-5 phd of 5-CFDA over 060 minutes. These concentration ranges and
incubation timcs have k e n commonly reportcd in literature. (van der Kolk et al, 1998.
Courtois et al, 1999) At the end of incubation priod, cells wen washed 3 times with
ice-cold Phosphate Buffered Saline (PBS), then lysed with 1% Triton and the
intracellular fluorescence intensity was measurcd using a spccaofluorimeter (Shimadzu).
at excitation wavelength 492nm and emission wavelengt h 5 1 8nm. (Haugland, R., 1996)
The upper limit of detection of the fluorimeter is at 1ûûû uni& hence any readings
bcyond this limit require further dilutions of the sample in order to get a reading.
Thenfore we wmted to be able to detect 5-CF over the linear concentration range
without htrther dilutions.
Our results demonstrated a proportional concentration-dependent increase in 5-CF
accumulation in both ce11 lincs. (Figure 1 and 3) Fluorescence readings increased
rapidly in both ce11 lines reaching a plateau after 30 minutes of incubation. (Figure 2 and
4) Based on these studies, we pre-incubated HepG2 and HuH 7 cells with 2pM and 4
pM of 5-CFDA ~pectively for 30 minutes for further studies.
Concentration of SCFDA (PM)
Figure 1. Accumulation of 5-CF in HuH 7 cells following incubation with varying doses
of 5-CFDA for 30 minutes. Values w c n reportcd as fluorescence intensity of 5-CF in
ceIl lysatc (mean f SEM. n=3/group)
lïme (minutes)
Figure 2: Accumulation of 4 pM of 5-CFDA in HuH 7 cells over 60 minutes. Values
werc nported as fluorescence intensity of intmcellularly accumulaied 5-CF (mean f
SEM, n=3/group).
O t 2 3 4 5 6
Concentration of 5-CFDA (PM)
Figure 3: Accumulation of 5-CF in HepG2 cells following incubation with varying doses
of 5-CFDA for 30 minutes. Values werc reported as fluorescence intensity of 5-CF in
ce11 lysate (mean f SEM, n=3/group)
O 10 20 30 40 50 60 70
Time (minutes)
Figure 4: Accumulation of 2 pM of 5-CFDA in HepG2 cells over 60 minutes. Values
wen reportcd as fluorescence intensity of intracellularly accumulated 5-CF (mean f
SEM, n=3/group).
5-CF e f l w assay
In order to establish the efflux time for the assay, cells were first loaded with 5-CFDA for
30 minutes and then medium was nmoved and nplaced with S-CFDA-free medium in
the pnsence and absence of 2ûQM of Inâomethacin.(Bakos et al, 2000) Xndornethacin is
a specific inhibitor of MRP and the concentrations have been shown to maxirnally inhibit
MRP mediated efflux. 5-CF was then allowed to efflux over 0-60 minutes. We then
detennined the time at which maximum differtnces in indomethacin-inhibitable efflux
could bc observtd.
As illustrated from figures 5 and 6. sipificant differcnces in efflux with or without the
MRP inhibitor, indomethacin were observed at 30 minutes. Hence a 30 minute efflux
tirne was utilized for dl subsequent cytokine studies.
+ lndomethacin
I
\- - indomethacin
time (minutes)
Fiprc 5: Time course of 5-CF efflux in HuH 7 cells. Cells wen preloaded with 5-
CFDA for 30 minutes and the cfflux of S-CF was measured over 60 minutes in the
pnsencc and abBerne of 2ûûpM i n d o d a & . Vahies w m reportcd as 46 of 5-CF
mmaining in the cells (mean f SEM. n=3/gmup). * @.OS.
+ Indomet hacin
- Indomethacin
Time (minutes)
Figure 6: Time course of 5-CF efflux in HepG2 cells. Cells were preloaded with 5-
CFDA for 30 minutes and the efflux of 5-CF was measured over 60 minutes in the
presence and absence of 200pM indomethacin. Values were reported as % of 5-CF
remaining in the cells (man f SEM, n=3/gmup), *p<O.OS
5) GG918-In hibitable Rh 123 Em Assay
PGP functional activity was assayed using the fluorescent PGP substrate, Rhodamine 123
(Rh 123) as previously described. (Piquette-Miller et al, 1998) Efflux of Rh 1 23 can be
inhibitcd by POP specifc inhibitor, GG9 18. GG9 18 has been tested to be a specific
inhibitor of PGP and does not significantly affect MRP efflux ovet those concentrations.
(Utsunomiya et al, 2000) Additional studies also included the use of indornethacin in
ordcr to inhibit MRP mediated efflux of Rh 123. The functional activity of PGP can be
determined by the amount of Rh 123 efflux with the presence and absence of GG9 18.
6) Establishment a d Optirnizution of Rh123 e f l u assay
Similar to the 5-CF efflux assay, WC monitond the efflux of Rh123 in HepG2 and HuH 7
cclls that were preincubated with 2.6pM Rh 123. In both ce11 lines, samples were taken
betwccn û-60 minutes in order to determine the time at which maximum difference in
efflux with and without PGP specific inhibitor, GG918. It was observed from figures 7
and 8, that significant and measurable diffennces in efflux was observed at 45 minutes.
Hcnce 45 minute efflux tirne was utilized for al1 efflux studies.
Time (minutes)
Figure 7: Time course of Rh123 efflux in HuH 7 cells. Cells w e n preloaded with Rh 123
for 15 minutes and the efflux of Rh 1 23 was measurcd over 60 minutes i n the presence
and absence of 1pM GG9 18. Values wcrc nported as I of Rh 123 remaining in the cells
(mean f SEM. n=3/group), *@.OS.
-- . . I . . O 10 20 30 40 50 60 70
Time (minutes)
Figure 8: Time course of Rh123 efflux in Hep02 cells. Cells werc preloaded with Rh 123
for 15 minutes and the efflux of Rh 123 was measurcd over 60 minutes in the presence
and absence of 1pM 00918. Values were rcported as I of Rh123 remaining in the cells
(mean f SEM. n=3/group), *pcû.05.
-- 7) RT-PCR used to establish gene-specifc effects
MRP 1, MRPZ and -3 are al1 transporters that share sirnilar substrate specificities and
the 5-CF efnux assay does not differcntiate between MRP farnily members. Hence we
also mn semiquantitative RT-PCR analysis with primers specific for MRPI, MRP2.
M W 3 and MRP6 which allowed us to examine cytokine mediated effects on individual
MRP family members.
MATERIALS AND METHODS
METHODS
Chernicals and nagents
Recombinant human L6, L l p and TNF-a; 5-carboxyfhorescein diacetate (5-CFDA);
Rhodarnine 123 (Rb 123); Indomethacin, Triton X- 100, MTT, Mannitol, Protease
Inhibitor Cocktail, Tris-HCI, Acrylamide, SDS, Glycine. and Methanol were purchased
from Sigma (ON). Al1 culture media, trypsin and fetd bovine serum (FBS) were
purchased from Canadian Life Technologies (Hamilton. ON). The monoclonal Antibody
C494, specific to human MDRI, was purchased from ID labs (London. ON). The PGP
inhibitor GG9 18 was kindly donated by Dr. A. M. Rauth. (Ontario Cancer Institute,
Mncess Margartt Hospital, University of Toronto).
Cell lines
The human hepatocellular carcinoma ce11 line, HepG2, obtained from the American Type
Culture Collection (Maryland, USA), passage #79, was maintained in a-Minimum
Essential Medium (a-MEM) supplemented with 10% FBS. HuH 7 cells. kindly donated
by Dr. C. Richardson (Ontario Cancer Institute, ON), passage # 3040 were maintained in
Dulbecco's Modificd Eagle's Medium (DMEM) with 10% FBS. Both cell lines were
gmwn at 37°C in a humidified incubator equilibrated with 5% CO2. Medium was
replaceci twicc a week and cells werc trypsiniscd and subculturcd every 7 &YS. Viability
of control and trcated cells werc asscsseâ by Trypan Blue exclusion and M'ïTassay.
=-- - Qtokine Tnatment
HepG2 and HuH 7 cells werc tnated with M. IL1 a and TNF-a for 0-72 hours
using doses within the physiologie range of each cytokine: IL6 (0-25ng/ml);
I L I B (0.0.5 nghnl) and TNF- a (0-0.5 nglml). The concentration and treatment time
at which maximal changes of the functional activity of M W and PGP wen determined
by 5-CF and Rh123 eMux assays, as described klow. Further experiments were
perfomd at these time points and cytokine concentrations, RNA and protein isolated
from the cells and RT-PCR and western blots are performed as described klow in order
to determine the expression of each transponer at the mRNA and protein level.
M W Fwictional Assay, Indomethmin-in hibitable 5-CF e m assay
Hem2 or HuH 7cells were seeded ont0 6-well ce11 culture clusten and incubated with
individual cytokines as described above. Control and cytokine utated cells were then
washed and pre-incubated with 2 or 4 pM 5-CFDA for 30 minutes, the medium was
nmoved and replaced with 5-CFDA-frtt medium in the presence and absence of 200
indomethacin and allowcd to efflux for 30 minutes. (These concentrations and times
were determined as discussed in rationale) At O and 30 minutes of incubation, cells wen
washed and the intracellular fluorescence intensities was measured on spectrofluorimeter
at excitation wavelengtb 492nm and emission wavelength of 5 18nm. Intracellular 5-
Carboxyfiuorcscin (5-CF) fluonscence intensity was conected on the basis of protein
content as assesscd by the Bio-Rad pmtein assay kit. The 30 minute efflux of 5-CF was
calculated as follows:
M W - mediated efflux was then estimated as a fraction of the 30 minute efflux which
was inhibited by indomethacin:
MRP-mediated efflux = %5-CF efflux (no indomethacin)-%S-CF efflux (+indomethacin)
Time- and Dose-dependency studies were conducted for each cytokine tnatment over its
physiological dose range and over 0-72 houn of trcatrnent. (n=6) ~reatrnents which
elicited maximum changes in MRP huictional activity wen further investigated for
changes in MRP mRNA levels using semi-quantitative RT-PCR.
Rh123 e#hu assay
Both HepG2 and HuH 7 cells were pmpared in the sarne manner as above and
preincubated with 2.6 ph4 Rh 123 (Piquette-Miller et al, 1998, Sukhai et al, 200 1 ) for 15
minutes and allowed to efflux in FBS-fm medium in the presence or absence of the POP
specific inhibitor. 1)rM GG918. At O and 45 minutes. cells were washed and lysed in the
same way as mcntioned kfon. Intracellular fluorescence intensity was measured at
excitation wavelength 5 18nm and emission wavelength at 532 nm. Intracellular Rh 123
fluorescence intensity was comcted on the bais of protein content as assessed by the
Bio-Rad Protein assay kit. PGP mediated efflux was analyzed as follows:
%Rh123 efflux = m123P" f ~ h 1 2 3 1 ~ ~ ~ " X 100
[Rh 1 231'
And:
P-gp mcdiated efflux = % Rh 123 efflux (no GG9 18) 0% Rh 123 eMux (-9 1 8)
Mï7' Assay
M'IT assay was perfonned to examine the effects of cytokines on the viability and
proliferation of Hep02 and HuH 7 cells with each cytokine tnatment. MTT is converted
from the tetnizolium salt into a fonnazan dye by mitrochondrial dehydrogenase which
measures the proliferation of cells. (Lobner, D. 2000) Cells w m seeded ont0 96 well
culture cluster and incubated with cytokines ( n d ) as descrikd above. 0.Smglml MTT
(3 - (4 ,5 -d imethy l -2 - th iazo ly l ) -2$ -d iph~o l ium bromide) was added to the
cells and incubated for 2 hours at 3 7 ' ~ . Cells wcrc then solubilised in a solution
containing 10% SDS. Formazan dyc concentration were then detected using a microplate
reader (Bio-Rad) at emission wavelcngth of 540nm. Absorbame of untreated controls
wcre taken as 100% controls. (Walther et ui, 1995) This assay allows us to detennine
the ctll proliferative ability of Hep02 and HuH 7 cells under the txtatment of these
cytokines.
Protein Isolation
Cells were seeded on 60mm petri-dishes, then truued with individual cytokines.
Cytokine concentrations and incubation times utilized were those that demonstrated
maximal effects on PGP and MRP. Crude membrane proteins were isolated from contml
and ücated HepG2 and HuH 7 cells as descnbed pnviously. (Sukhai et al, 200 1) Cells
harvested by trypsinisation wen homogmized in lysis bu ffer containi ng 300rnM
Mannitol, 1 % protcase inhibitor cocktail, lOrnM Tris -HCI. pH 7.1 with a Polytron tissue
homogeniser. The homogenatcs wen centrihiged at 800g for 10 minutes and the
supernatant was centrifuged 15000 g for 30 minutes. The pellet was reconstituted in
rcsuspcnsion bufler containing SOrnM Mannitol, 1% protease inhibitor cocktail. lOmM
Tris -HCL, pH 7.1.
Western Blot Andysis
POP protein expression was exarnined on Western blots. Briefly, 8 pg of protein sample
was separated on SDS-PAGE electrophorcsis gels (Stacking gel 4% acrylamide,
separating gel 6% aciylarnide. SDS ~nn ing buffer contains 12mM Tris-HCI pH 8.3,
1.7mM SDS and 96mM Glycine) and transfemd to nitrocellulose membranes
(Amersham Pharmacia Biotech) by electroblotting in transfer buffer containing 25rnM
Tris, 0.19M glycine and 20% Methanol for 2 hours at 250mV. The blots are blocked in
TBST containing 5% Mlk powder. M e r blocking, the blots wcrc washed 3 times in
TBST and incubated with the PGP spccific monoclonal antibody. C494 for 2 hours. This
antibody specifically rccognized human MDRl PGP and has k e n shown not to cross-
nact detectably with MDR3 PGP. Afar incubation, blots an washed again and
incubated for 1.5 houa w ith secondary antibody , horseradish pcroxidase. (Amenharn
Phmacia Biotech) The bound antibody was visualized using ECL reagent and optical
density was quantitated using Kodak Digital Science 1D Image Analysis software
(Easb~n Kodak).
We also attempted to perform western blots on MRP2 protein using the primary antibody,
M2IiI6, however no specific bands could be identified from the blots. This could be a
rrsult of insumcient specificity of the antibody. However, no antibody capable of
differentiating ktween the diffennt mcmbea of the MRP gene family is as yet available.
rendering Western blots of MRP protein impracticably difficult to interpnt.
RNA Isolation
Cells were seeded on 60mm petri dishcs, then incubated with each cytokine. Total RNA
was exüacted from both control and trcated cells using the Amersharn ~ u i c k ~ r e ~ ~ RNA
isolation kit according to the manufacturer's spccifications. RNA concentration was
quantitated ai wavelcngth 26ûnm on an UltraSpcc 2000 Spectrophotometcr (Amersham)
Semi-Quantitative RT- PCR
The mRNA levels of each MRP gene, as well as MDRl and GAPDH or P-Actin were
deteamincd by a semiquantitative RT-PCR assay as describcd prcviously, with minor
modifications. (Sukhai et al. in press) Briefly, cDNA was synthcsized from total RNA
(SM) using the Fint Strand cDNA Synthesis Kit (MBI Fermentas, Fiamborough, ON),
according to the manufacturer's protacol. PCR standard curves for each gene pmduct
(GAPDH or PActin, MRPI, 2,3,6 and MDR I ) werc generatcd h m serial dilutions of
RT product and optimal amounts of tanplate werc determined from the linear portions of
the nsulting PCR calibration curves. PCR was performed using 50 pmol sense and anti-
sense pnmea (Table 1, DNA Synthesis Center, University of Toronto. ON), 1-2.5 pl of
RT product (as determined above) and 2.5 units of Taq DNA polymerase (MBI
Fermentas) in a reaction volume of 10p1 . The tubes were incubated in a GeneAmp PCR
system (Perkin-Elmes, Nonvalk, Ci') at 9 4 ' ~ for 3 minutes to denature cDNA and
primcrs. The cycling program was 9 4 ' ~ for 45 seconds, SS'C or 6 0 ' ~ for 30 seconds
depending on difiennt genes and 7 2 ' ~ for 75 seconds for the defined numkr of cycles
as shown in Table 2. Samples were incubated at 7 2 ' ~ for an additional 7 minutes after
the final cycle. PCR products werc nin on a 2% agarose gel, stained with SYBR Gold
Stain (Molecular Probes, Eugene.OR) and quantitated using Kodak Digital Science 1 D
Image Analysis software. Optical intensities of the PCR products were normalized to the
intensities of the GAPDH or B-Actin PCR products generated.
Stutisticol Anulysis
ui our pnliminary accumulation and enlux establishment studies, sample size of 3 was
used. Whereas in cytokine treatmcnt studies, al1 trcated and conml sarnples were
obtained from 3-6 individuai culture plates per expriment which is a common sample
size used by other rcsearchers performing similar studies. Experiments were performed
on separate days with similar findings. The unpaircd Student's t-test was used to
caiculatc significance with a p4 .05 considend as statistically significant. Analysis was
pcrfonned using Micmsoft Exce19712000 and SigmaPlot 4.0/2000.
GAPDH 5'-ACC ACA GTC CAT GCC ATC AC-3' 5'-TCC CAC CAC CCT GTT GCT GTA-3'
$-ACTIN 5'- AGA GCT ACG AOC TOC CL% AC-3' S ' -AU GCC ATG CCA ATC TCA TC-3'
MDRI 5'-GTG CTG G'IT GCT GCT TAC AT-3' 5'-CCC AGT GAA A M TGT TGC CA-3'
MRPl 5'-ATG TCA CGT GGA ATA CCA GC-3' 5'-GAA GAC TGC ACT CCC TTC CI'-3'
MW2 5'-CTG CCT CIT CAG AAT C'IT AG-3' 5'-CCC AAG TTG CAG GCT GGC C-3'
MW3
# Cycles HepG2 Cells
5'-GAT ACG CTC GCC ACA GTC C-3' 5'-CAG ?TG CCG TGA TGT GCC TG-3'
M W 6
# C y c k Hua7 Cells
5'-GTG GTG 'ITI' GCT GTC CAC AC-3' 5'-ACG ACA CCA GGG TCA ACT TC-3'
NIA
Table II. List of primer sequenccs, PCR product sizes and cycle numbers used in RT-PCï
assay. NIA = not applicable
RESULTS
4.1 Viabuity Shidla
Trypan Blue studils
Aiterations in cet1 viability when thcsc cells wen subjected to different cytokine
treatments were cxarnined by the use of the Trypan Blue Exclusion Method. As shown
from Table 3, viability was maintaincd above 98% in both ce11 lines after treatment with
L i e (0.25nglm1.48 hours); IL6 (IOnglml, 24 hours) and TNP-a (Olng/rnl, 24 houn).
No statistical ciifference in the viability of control and cytokine trcated cells were seen.
This means that the concentration and trcatment time of cytokines that were used in out
studies did not cause significant ce11 death and hence it is unlikely that increased ce11
death contributes to our findings.
MIT Assay
The M ï T assay allows us to examine the proliferative ability of both HepG2 and HuH 7
cells under each cytokine treatment. In HuH 7 cells. we observed that IL-6 and TNF-a
matment did not affect the viability and proliferative ability of the cells but that IL4 P
imposcd a slight decrcase (10%) in proliferation of the cells. (Table III) However in
HepG2 cells, we did not dctect any significant rcductions in the proliferation of the cells
a k r tteatment with each of the cytokines. (Table m) Indeed, in Hep02 cclls, the
cytokines IL-6 and L 1 rcsulted in a small stimulation in the proliferation of cells. This
hm k e n pnviously nported to be effects of IL6 and IL4 in certain human cells such
as peripheral T cells. (Fey, G.. 1990, Roitt et al, 1998)
-.
Cells
HepG2
I
HuH 7
Trcatment L
IL6 (IOnglml. 24 hours)
Cell Viability and Proiiferation (8 controls)
L l p (0.2kglml,48 hours) TNF-a (0.2ng/ml, 24 hours) Contra1
Table ïik Viability rcsults of control and cytokine treated HuH7 and HepG2 cells. Values are rqmited as (mean i SEM, n=3/groap for Trypan Blue studies and n=6 for MTI' assay) *@.OS.
Trypan Blue Exclusion
Method
100.3 f 0.43 96
IL4 p (0.25nglml. 48 hours) TNF-u (O.Îngîml,24 hours)
MTl' Assay
121.8 I 7 .29 % *
100.5 f 0.46 %
100.0 f 0.58 %
100.0 f 1 66 %
-
127.5 f 5.07 % *
108.1 f 4.74 %
100,Of 3.22 %
99.3 f 1 .O3 %
98.2 f 1.27 %
89.6 f 3.45 % *
105.8 f 7.06 %
4.2 -sion of M . and the MRPgene famiiy members in HuH 7 a d HepG2
c e f i
RT-PCR analysis of RNA isolatcd h m HuH7 cells demonstrateci expression of MDRl,
MW3 and MRP6 (Figure 9). Results obtained from HepG2 cells indicaied that these
cclls express M M B MRPl, 2,3 and 6 (Figure 10).
Figure 9: Repnscntative RT-PCR gel depicting relative MDRl and MRP family gene expression in HuH 7
cells. As descrikd in methods, total RNA was isolatcd from HuH 7 cells. RT-PCR was performcâ, PCR
products were separateci on 2% agarose pls, stained with SYBR Gold stain and analyzed with Kodalc Digital
Science Image Analysis Software. Lem #l contains GAPDH. #Z MDRI. #3 MRPI, W4 MRPî, #5 M ' 3 , #6
MRP6. Lane M contains a 100 bp DNA ladder.
Figure 10: Representative RT-PCR gel depicting relative MDR 1 and MRP family gene expression in
Hem2 cells. As described in methods, total RNA was isolated from HepG2 cells, RT-PCR was
performed PCR products were separated on 2% agarose gels, stained with SYBR Gold stain and
analyzed with Kodak Digital Science Image Analysis Software. Lane #l contains GAPDH, #!2 MDRl,
#3 MRPl, #4 MRP2, #5 MRP3, #6 MRP6. Lane M contains a 100 bp DNA ladder.
4.3 Effbcts of IL-6 on PGP Expression and Activity
Funcüonal Aetivi@ of PGP
of ïL-6 on HuH 7 cell~
As compmd to controls, results from time dependent studies shows that a slight but
significant downregulation of PGP-mediated effîux of Rh 123 (16-20%) is evident at 24
hour trcatmcnt of IL-6. From dose studies, the maximal decrcase of PGP mediated efflux
was observed at IOng/ml of IL6 tnahmnt. (Figures 1 1 and 12).
gffect of IL6 on Heffi2 cellg
From both time and dose studies, treatment with 0-25nglml IL6 over the treatment
period of 0-48 hours failed to demonstrate any changes in the PGP-mediated efflux of
Rh123. (Figure 13 and 14). Then was difficulty in trcating both ce11 lines for 72 hours,
they exhibited a decnased growth pattern beyond 48 houn and started to die ai 72 hours.
Thenfore we wen unable to obtain rcsults on functional activity for 72 hours IL-6
treatment since the majority of cells detached during the washing steps of the efflux
assay.
niRNA Erpnssion of MDRl in HuH 7 and HepG2 cells
MDRl mRNA levels were significantly reduced (35% ) in IL6 trcated HuH 7 cells
(IOng/ml, 24 hours) which cornlates with the rtduction we observed in functional
activity of POP. This suggests that the ccduction in PGP activity is rcgulated at the
mRNA level. (Figure 15). However. no significant changes in MDR 1 mRNA level could
be observed in He@ cells.
Protein Ekpression of PGP in HuH 7 mid HepG2 cells
Protein levels of PGP were also significantly rcduccd (20%) in HuH 7 cells w hich
cornlates with the reduction scen ai functional and mRNA level while no significant
changes could be obwwed in HepG2 cells. (Figure 16).
12 24 48
Incubation period (hours)
Figure 1 1: Influence of IL6 tnatment duration on PGP transport activity in HuH 7
cells. PGP mediattd efflux of Rh 123 was measurcd in HuH 7 cells after û-48 hours of
incubation with IL6 (lûng/mi). POP transport activity was cxamined using Rh 123 as
describai in the methods. Values an rcporied as 96 controls (mean t SEM) of the 45
minutes GG9 1 8-inhibitabk Rh 123 efnux (n=Wgmup), *p<O.OS.
O 0.5 1 10
IL4 dose (ng(ml)
Figure 12: influence of I L 4 concentrations on POP transport activity in HuH 7 cells.
PGP mediatcd efflux of Rh123 was measund in HuH 7 cells after 24 hour incubation
with 0-25 nghnl of IL-6. PGP transport activity was examined using Rh 123 as descrikd
in methods. Values are reportcd as % controls (maan t SEM) of the 45 minute GG918-
inhibitable Rh 123 efflux (n=3-6lgroup). *@.OS.
Control O IL-6 tmated 1
12 24 48
Incubation period (hours)
Figure 13: Influence of IL6 treatment duration on POP transport activity in Hep02 cells.
POP rnediafcd efflux of Rh 123 was m e a s u d in HrpGZ cclls aftcr 0-48 hours of
incubation with IL6 ( lOng/ml). PGP transport activity was examined using Rh 1 23 as
descrikd in methods. Values are reportcd as 96 controls (mean t SEM) of the 45
minutes GG9 18-inhibitable Rh 123 cfflux (n=3-Wgroup), *pcû.O5.
P-GP mediated Effîux (GG918 Inhibible-Rh1 23 effîux, % controls)
control IL4 treated
Figure 15: Effect of L-6 on MDRl mRNA expression in HuH 7 and HepG2 cells. RT-
PCR was performed on total RNA extracted from 24 hou; IL6 treated ( 1 Onglml) or
control cells as described in methods. Values (mean t SEM, n=lgroup) are reported as
96 of controls. Raw intensitics of MDRl genes werc normalised with respect to GAPDH.
*p<0.05.
Figure 16: Effect of IL-6 on PGP protein expression on HuH 7 and HepG2 cells.
Western blots were performed on crude membrane fractions fiom control or cells treated
with 24 hour IL6 (IOng/ml) using the MDRIIPGP specific antibody. C494 described in
mcthods. Values (mean t SEM. n=4/group) an nported as % of controls. *pd.OS.
4.4 En& d IL-la O!? PGP e % m ana 8cthity
Functional activiv
As compared with controls, sipificant reduction (39%) of POP-mediated Rh 123 efflux
was observed after 48 to 72 houn of incubation (Figure 17) In cells incubated with IL-
i for 48 hours. significant changes of 27% werc seen only at the concentration of
0.25ng/ml IL- 1 (Figure 1 8). Concentrations of O. 1 ng/ml also imposed n substantial
rcduction. however this effect did not nach statistical significance.
Effect of IL-1 0 on HmG2 cells
The L l p mediated suppression of POP activity was more pronounced in HepG2 cells.
(Figure 19) A reduction in POP activity was seen in cells treatcd with IL- I after 12-72
hours of incubation. (Figure 19) This decrease was significant from 48 to 72 hours with
a 55% decrease of PGP mediated activity seen at 48 hours. A concentration dependent
reduction was seen over concentration range of O-O.Sng/ml (Figure 20). We did not
exceed concentrations above 0.5ng/ml as the physiological concentrations in human
rarety exceed 0.35ng/mI. (Barriere et al, 1995) Furthemon, concentrations above
O.Sng/mi are often associated with high mortality.
nrRNA expression of MDRl in HuH 7 and HepG2 cells
Our RT-PCR results did not detect any significant effects of IL1 on the mRNA Ievels
of MDRl in either HuH 7 and HepG2 cells. (Figure 21) This implicates that the
reduction we observed on functional activity does not likely occur at the
RNNtranscriptional level, hence western blots were performcd to determine if the
protein expression of PGP was a l t ed upon &le treatment.
Protein expression of PGP in HuH 7 und HepG2 cells
As RT-PCR did not detect changes in MDR 1 rnRNA levels upon treatment with IL 1 P,
we examined protein expression on western blots. The monoclonal antibody, C494 was
used to specifically detect the MDRl gene product, POP. This antibody docs not cross
nact with SPGP or MDR3 gene products. Our western blots dctected a significant 16
and 36% decrease in HepG2 and HuH 7 cells , nspcctively. (Figure 22) This implies that
the reduction in PGP protein expression is accounted for the observed reduction in POP
functional activity. Hence IL4 P regulates the activity of PGP through its protein
expression rather than gene transcription.
Control 0 l L-1@ treated
12 24 48 72
Incubation period (houn)
Figure 17: Influence of IL4 f! treatment duration on PGP transport activity in HuH 7
cells. PGP mediated efflux of Rh123 was measured in HuH 7 cells after 0-72 hours of
incubation with IL1 (0.25nglml). POP transport activity was examined using Rh 123
as describcd in methods. Values arc nported as 96 controls (mean + SEM) of the 45
minutes GG9 1 &inhibitable Rh 123 efflux (ndd/group), *@.OS.
IL4 P dose (ng/ml)
Figure 18: Influence of IL-I concentrations on PGP transport activity in HuH 7 cells.
PGP mediated efflux of Rh 123 was measured in HuH7 cells after 48 hour incubation with
0.0.5 ng/ml of L I P . PGP transport activity was exarnined using Rh 123 as descnbed in
methods. Values are reported as % controls (mean t SEM) of the 45 minute GG9 18-
inhibitable Rh 123 cfflux (n=6/group). pO.05.
= Control 0 IL-1p treated
T
12 24 48 72
Incubation period (hours)
Figure 19: influence of IL- 1 treatment duration on POP transport activity in HepG2
cells. POP mediated efflux of Rh 123 was measund in Hep02 cells after 0-72 houn of
incubation with IL 1 (0.25nglml). PGP transport activity was examined using Rh 123
as described in methods. Values arc rcported as % controls (mean t SEM) of the 45
minutes GG9 18-inhibitable Rh 123 efflux ( n d p u p ) , *pd.05.
0.05 O. 1 0.25
11-1 B dose (nglml)
Figure 20: Influence of IL-1P concentrations on POP transport activity in HepG2 cells.
PGP mediated efflux of Rh 123 was measund in HepG2 cells after 48 hour incubation
with û-û.5 ngfml of IL1 p. POP transport activity was exarnined using Rh 123 as
descrikd in methods. Values are rcported as % controls (mean + SEM) of the 45 minute
009 18-inhibitable Rh 123 efflux (n=o/group), *p<0.05.
= Control i IL4 treated 1
HuH 7
Figure 2 1 : Effect of IL1 on MDRl mRNA expression in HuH 7 and HepGZ cells. RT-
PCR was performed on total RNA extractcd from 48 hours LI B treated (0.25ng/ml) or
control cells as described in methods. Values (mean t SEM, n=S/group) are reported as
% of controls. Raw intensities of MDRl genes were normalised with respect to P-Actin.
'p4.05.
Control 0 IL-1 treated
Figure 22: Effect of IL-lp on PGP expression on HuH 7 and HepG2 cells. Western blots
werc perfomed on crude membrane fractions from control or cells treated with 48 hours
IL- 1 B (0.25ng/ml) descri bed in methods. Values (mean + SEM. n=4/group) are nported
as % of controls. *pcO.05.
4.5 Mect of TNFsron PCP expression a n à d v i t y
Functional A ctivity
ct of TNF-a on HuH 7 cells
As compared to controls. a signi ficant down-regdation (35%) in POP-mediated Rh 1 23
efflux was observed after 24 to 72 hours of treatment with O.Ong/ml TNF-a treatment.
(Figure 23) Similar to that seen with LlP, a concentration dependent reduction in POP
activity was seen with TNF-u (Figure 24). We did not exceed concentrations above
O.Sng/ml as plasma concentrations of TNF-a in surviving septic patients rarely exceed
O.kg/rnl and plasma concentrations of TNF-a which exceed O.Sng/ml is usually
associated with mortality. (Barrien et al, 1995)
Effect of TNF-a on H ~ D G ~ cells
in contrast to that seen in HuH 7 cells, the pattern of PGP suppression was sonewhat
different in HepG2 cells. As compared to controls, downregulation of POP-mediated
Rh 123 efflux was observed within 12 hours of TNF-a treatment but maximum
suppression (33%) was seen at 24 hours. (Figure 25) After that, the functional activity
returned to normd at 48 and 72 hours which suggests that the suppressive effect of TNF-
a in HepG2 cells was a rapid and transient response. A 30% suppression of POP activity
was seen at concentrations of 0.05 - 0.2 ng/ml. Increasing dose did not result in
substantial changes to this downregulation. (Figure 26) concentrations above O.Sng/ml
werc not conside~d with the same reason as above.
noRNA Expression of MDRI in HuH 7 and HepG2 cells
RT-PCR nsults indicated that MDRl mRNA levels were down-regulated in both ce11
lines after 12 hours and 24 houts of tnatments. (Figure 27) In HuH 7 cells, both 12 and
24 hour incubation periods showed significant decnase (25%) in MDRl mRNA level.
This implies that the reduction of MDRl functional activity in TNF-a treated HuH 7
cells arc likely replated at transcriptional level. It is not surprising to see that reduction
of MDRl mRNA starts earlier than 24 houn as transcription always occurs kfon
translation, hence it is logical to see a reduction in mRNA levels befon the ectual
reduction is evident in the function of the transporter since it takes time from
transcription to translation to proper protein phosphorylation and transportation to the
proper membrane site.
However in HepG2 cells, significant down-regdation was observed at 12 hours (2 1 %)
while at 24 hours. a 16% down-ngulation was observed but this was not significant.
This could be due to early and short-acting effect of TNF-a. 1 could suppress gene
transcription of MDRI only immediately after trcatment. This matches with what we
have seen with the functional activity elicited by TNF-a wherc fast reduction of PGP
mediated efflux was observed upon treatment. After 48 houa, this reduction effect
disappearcd and a retum to normal levels was evident. This implies that the effect of
TNF-a on MDRIPGP in HepG2 cells may k differcnt from its effects in HuH 7 cells as
the down-regdation of MDRl gene in HuH 7 cells lasted longer and was more sustained
than that observcd in HepG2 cells.
Protein Expression of PGP in HuH 7 and HepG2 cells
As cornparrd to contcols, siBDificantky lower levels of PGP pro- expression were
obsemd in both TNF-a treated HuH 7 and He62 cells (32% and 39 1 respectively).
(Figure 28) This coincides with what we observed at both functional and mRNA level of
PGP/MDRI expression.
= Contml 0 TNF-a treated
12 24 48 72
lncu bation peiiod (hours)
Figure 23: Influence of TNFu matment duration on POP transpoit activity in HuW 7
cclls. PGP mediated efflux of Rh123 was mcasurcd in HuH 7 cells after 0-72 hours of
incubation with TNF-a (0.2nglml). POP transport activity was exarnined using Rh123 as
described in methods. Values are ceportcd as % controls (mean t SEM) of the 45
minutes GG9 18-inhibitable Rh 123 efflux (n=6/group), *pcû.OS.
TNF-a dose (nglml)
Figure 24: Influence of TNF-a concentrations on POP transport activity in HuH 7 cells.
PGP mcdiated efflux of Rh 123 was measured in HuH 7 cells &ter 24 hour incubation
with 0-0.5 nghnl of TNF-a. POP transport activity was exarnined using Rh î 23 as
described in mcthods. Values arc reportcd as % controls (mean t SEM) of the 45 minute
GG9 1 &inhibitable Rh 123 efflux (n=6/gmup), *p<O.OS.
I Control 0 TNF-a treated
lncu bation period (hours)
Figure 25: influence of TNF-a treatment duration on PGP transport activity in HepG2
cells. PGP mediated efflux of Rh 123 was measured in HepG2 cells after 0-72 hours of
incubation with TNFa (0.2ngirnl). PGP transport activity was examined using Rh 123 as
descnbed in methods. Values arc reportcd as % controls (mean t SEM) of the 45
minutes GG9 1 binhibitable Rh 123 efflux (n=o/group), *p<O.OS.
Figure 26: Influence of TNF-a concentrations on PGP transport activity in HepG2 cells.
PGP mediated efflux of Rh 123 was measured in HepG2 cells after 24 hour incubation
with 0-0.5 nglml of TNF-a PGP transport activity was examined using Rh 123 as
descrikd in methods. Values an nported as % controls (mean t SEM) of the 45 minute
0 0 9 1 8-inhibitable Rh 1 23 efflux (n=6/pup), *pçO.OS.
Control I r 12 hour tieatment
24 hour treatrnent
Figure 27: Effect of TNF-a on MDRI rnRNA expression in HuH 7 and Hepû2 cells. RT-
PCR was performed on total RNA extracted from 12 and 24 houa TNF-a treated
(0.2ng/ml) or control cells as described in methods. Values (mean t SEM, n=S/group)
arc reported as % of controls. Raw intensities of MDRI genes were normalised with
respect to B-Actin. *p<O.OS.
= Control 0 TNF-a treated
HuH 7 HepG2
Figure 28: Effect of TNF-a on POP expression on HuH 7 and HepG2 cells. Western
blots were performed on cmde membrane fractions from control or cells trcated with 24
houa RJF-a (0.2nglml) as described in methods. Values (mean t SEM, n=4/group) are
reportcd as % of controls. *p<0.05.
4.6 Effcct of I L 4 on MRP Expression and Acüvîty
Functional Activity
Effcct of IL6 on HuH 7 cells
As comparai to controls. a significant upngulation of MRP mediated 5-CF efflux was
obscrved after 12 to 48 hours of 1Onglml IL6 treatment with a 1.65 fold increase seen at
24 houn. (Figure 29) This induction was found to k dose dependent as increasing
concentrations of IL6 resulted in an increased induction of MRP transport activity.
(Figure 30)
Effect of IL6 on HeDG2 cells
Similar to HuH 7 cells, a significant upregulation of MRP mediated 5-CF efflux was
obstrved after 12 to 48 houn of IL6 matment. (Figure 32) As compared to controls, a
2 fold increase in MRP transport activity was seen in cells treated with 24 hours. A
similar dose dependency in this induction was appannt in Hep02 cells. (Figure 33) in
both ce11 lines, IL6 concentrations of 25ng/ml or longer trcatment time (48 hours)
appcared to saturate MRP induction or even impose a slight dccrcase in MRP mediated 5-
efflux. This could occur due to the toxic effccts of IL6 which occur at these high
concentrations. Physiologic concentrations of IL6 in septic patients rarely exceed
lOng/ml. (Barriete et al, 1995)
mRNA expression of MRP family genes
HuH 7 cetls
RT-PCR nsuits indicpted that only M W 3 mRNA bels demonstrate signifiant
upregulation (50%) while mRNA levels of MW6 were not altend. (Figure 3 1 ) Since
HuH 7 cells did not express MRPl or MRP2, the induction in MRP mediated f'unctional
activity on IL6 tnatment is likely due to MRP3 induction.
HepG2
RT-PCR results showed that MRPl mRNA levels were 1.7 fold higher (p<0.05) in IL-6
treated cells as compared to control. (Figure 34) The mRNA levels of MRP2, MRP3 and
MRP6 did not show any significant changes in IL6 treated cells. Hence the upregulation
in MRP functional activity in IL6 treatment is likely due to MRPl induction.
e IL. C,
T
Figure 30: Influence of IL-6 concentrations on MRP transport activity in HuH 7 cells.
MRP mediated emux of 5-CF was measured in HuH 7 cells after 24 hour incubation with
0-25 ng/ml of IL-6. MRP transport activity was examined using 5-CF as described in
mehods. Values am rcported as % controls (mean t SEM) of the 30 minute
indomcthacin-inhibitable 5-CF cfflux (n=3-Wgmup), *p<0.05.
control 0 111-6 treated
Figure 31: Effect of IL6 on MRP3 and M W 6 mRNA expression in HuH 7 cells. RT-
PCR was performed on total RNA extracted from 24 hour IL6 treated (lûnglml) or
control HuH 7 cells as descrikd in methods. Values (mean t SEM. n=S/group) are
mponed as % of contmls. Raw intcnsities of M W 3 and MRP6 gcnes were normalised
with respect to GAPDH. *paû.OS.
Incubation period (hours)
Figure 32: Influence of iL-6 treatment duration on MRP transport activity in HepG2
cells. MRP mediated efflux of 5-CF was measad in HepG2 ceiis after 0-48 h o u of
incubation with IL6 (lûnglml). MRP transport activity was examined using 5-CF as
described in mtthods. Values an nported as 9b controls (mcan t SEM) of the 30
minute indomethach-inhibitable 5-CF efflux (n=36/group), *@.OS.
ri.
u" L.
Y
x .A h)
i control iI3 IL06 treated
L!: .-.:$ +"'
T . .. -2 .. ...
.- i > ' 7
-., ., .-,TM C
"M y5 :& 'a! -3 ."?
<j q Z 3 3 -:r : Y,
"r J F' .; 'f : -$ . .. ..
1
Figure 34: Effcct of IL6 on MRP mRNA expression in HepG2 cells. RT-PCR was
perfonned on total RNA extractcd from 24 hour IL-6 treated (10nglml) or control HepG2
cells as describal in methods. Valucs (mean t SEM. n=S/group) are reported as % of
controls. Raw intcnsities of different MRP genes werc nonnaliscd with respect to
GAPDH. *p<0.05.
A7 E f Y ' o f IL-lp aa MRP JhptCSSlOP anà ActMty
MRPfunctional activiîy
As compared to controls. a significantly higher MRP mediated 5-CF efflux was observed
after 24 to 72 houa of treatment with IL4 p. (Figure 35) A 1.5 to 1.6 fold increase was
sctn after 48 to 72 hours of IL 1 fl exposun. A maximal induction in MRP activity was
seen at concentrations of 0.25ng/ml. Higher concentrations of up to O.Jng/ml did not
substantially induce MRP activity f'urther. (Figure 36)
effi2 cells
In He*? cells, significant higher levels of MRP-mediated 5-CF was observed after
treatment times of 12 to 72 hours with IL 1 P. (Figure 38) ïncnases of 1.5 to 1.7 fold
were seen after 24 to 48 hours. This tffect is diminished after 72 hours of treatmcnt. The
induction of M W activity was seen at concentrations of O.lng/ml with peak effect
occumng at concentration of 0.25ng/mi. (Figure 39) Hence it appears that the effect of
IL 1 fl on MRP expression in HepGZ cells occurs aftcr shorter incubation periods than in
HuH 7 cells which indicates that regdatory diffetences may exist between these 2 ceIl
lines. However the general rcgulatory pattern cemains the same upon IL-1 fl treahnent.
niRNA expression of MRP fmiiy genes
uH 7 cclls
Similar to what was seen with IL16 treatments, a two fold induction of MRP3 mRNA was
seen in IL 1 treated cells while levels of MRPo mRNA were not affected. (Figure 37)
89
This indicates that the observable induction in MKP functioaal activi ty upon IL- 1
matment is likely due to the induction of MRP3.
eDG2 ctlls
Similar to that secn with IL6 treatments, RT-PCR results in LI t~ated cells showed
that mRNA levels of only MRPl was significantly induced from that of controls while
the mRNA levels of MRPZ. MRP3 and MRP6 wen not significantly affectai by IL- 1 a treatment. (Figure 40) This implies that MRPl is likely responsible for the induction of
MRP hinctional activity seen after IL 1 $ treatments.
Incubation period (hours)
Figure 35: Influence of IL- 1 P treatment duration on MRP transport act i vity in HuH 7
cells. MRP mediated efflux of 5-CF was measurtd in HuH 7 cells after 0-72 hours of
incubation with IL 1 P (0.25ng/ml). MRP vansport activity was examined using 5-CF as
descikd in methods. Values are reported as % controls (man t SEM) of the 30 minute
indomethacin-inhibitable 5-CF cfflux (n=6lgmup), *p<O.OS.
IL1 B Dose (ngml)
Figure 36: influence of IL- 1 concentrations on MRP transport activity in HuH 7 cells.
MRP mcdiated tfflux of 5-CF was measured in HuH 7 cells after 24 hour incubation with
0-0.5 ng/ml of IL 1 B. MRP tnuispon activity was examined using 5-CF as described in
methods. Values are reportcd as % controls (mean I SEM) of the 30 minute
indomethacin-inhibit able 5-CF efflux (n=6/pup), *p<O.OS.
Control 0 IL4 treated 1
Fipre 37: Effect of IL- 1 on MW3 and MRP6 rnRNA expression in HuH 7 cells. RT-
PCR was performed on total RNA extracted from 48 hour IL4 B treated (0.2Snglml) or
control HuH 7 cells as described in methods. Values (mean + SEM, n=5/group) are
reportcd as % of controls. Raw intensities of MRP3 and MRP6 genes were nonnalised
with respect to p-Actin. *pcû.05.
I Control i IL4 fl treated 1
12 24 48 72
Incubation period (hours)
Figure 38: Influence of IL18 treatxncnt duration on MRP transport activity in HepG2
cells. MRP mediated efflux of 5-CF was measuted in HepG2 cells after 0-72 hours of
incubation with IL4 (0.25nglml). MRP transport activity was examined using 5-CF as
descrîbeû in methods. Values arc reportcd as % controls (mean t SEM) of the 30 minute
indomethacin-inhibitable 5-CF efflux (n=6/group), *p<O.OS.
Figure 39: Influence of IL-IB concentrations on MRP transport activity in HepG2 cells.
M W mcdiated efflux of 5-CF was mcasured in HepG2 cells after 24 hour incubation
with 0-0.5 ng/ml of LI p. MRP transport activity was examincd using 5SF as
dcscrikd in methods. Values are reportcd as % controls (mean t SEM) of the 30 minute
indomethacin-inhibitable 5-CF efflux (n=6/group), *p<O.OS
= Control i-i IL-1 treated
F i p n 40: Effect of IL4 P on MRP mRNA expression in He@? cells. RT-PCR was
prformed on total RNA extractcd from 48 hour IL-1 treated (0.25nghl) or contrul
HepG2 cells as described in methods. Values (mean t SEM, n=S/group) are reporteci as
% of conbols. Raw intensities of different MRP genes were nonnalised with respect to
&Ach. *pd.OS.
4.8 Effect of TNF-a on MRP Expression and Activity
Functional uctivity
ct of TNF-a on HuH 7 cells
As compared to controls, TNF-a caused a significant upreplation of MRP-mediated 5-
CF efflur in HuH 7 cells from 12 to 24 hours. A 3 fold incnase in MRP activity was
seen after 24 hour tnatment times. (Figure 41) This induction was dose dependent with a
maximal incnase of 2.6 fold observed at 0.2nglml of TNF-a treatmeni. (Figure 42)
Higher concentration did not cause further induction.
Effect of TNF-a on HeG2 ctlls
As opposed to that seen in HuH 7 cells, TNFa imposed a significant down-regulation of
MRP-mediated 5-CF efflux in HepG2 cells after 12 to 24 hours. (Figure 44) This
suppressive effect cont inued for 72 hours. This down-ngulation of MRP-mediated 5-CF
efflux was dose-dependent with maximal reduction of 22% observed at O.Zng/ml.
(Figure 45) Higher concentrations did not cause furthet reduction.
inRNA upression of MRP family genes
IImhdls
M W 3 mRNA lcvel was significantly induced by 1.5 to 2.0 fold at 12 houa and 24 hours
of TNF-a trcatmcnt, rcspectivcly. (Fiprc 43) MW6 mRNA was not affccted by TNF-a
trcatment, as was the case with IL6 and IL-1 p. Hence it is likely that the increased MRP
activity seen after TNF-a tnatment occumd through an induction of MRP3
transcription.
RJF-a matments did not significantly affect mRNA levels of MRP2 and MW6 in
HepG2 cells. On the other hand, MRPI mRNA levels were significantly decreased at 12
hours and 24 hours with a maximal nduction s a n after 24 hours of treatment (45%).
btenstingly, MRP3 mRNA levels were also significantly duced at 12 hours (but to a
lesser extent, only about 16%) but not at 24 houn. (Figure 46) This implies that the
reduction we saw in MRP-mediated 5-CF eMux is likely due primarily to MRPI down-
regulation, however, changes in MRP3 likely also contribute to those changes.
It is interesting to see that TNF-a elicited totally different and opposite effects on the
MRP function and expression in these two ce11 lines. This could k possibly due to the
different regulatory pathways that exist betwecn the two ce11 lines.
Control i TNFu treated l
12 20 48
Incubation period (hours)
Figure 41: Influence of TNF-a treatment duration on MRP transport activity in HuH 7
cells. MRP mediatcâ efflux of 5-CF was measured in HuH 7 cells after 0-72 hours of
incubation with TNF-a (0.2ngmi). MRP transport activity was examined using 5-CF as
descrikd in methods. Values are reported as % controls (mean t SEM) of the 30 minute
indomethacin-inhibitable 5-CF efflux (n=6/group), *pd.OS.
TNF-a dose (ng/ml)
Figure 42: Influence of TNFa concentrations on MRP transport activity in HuH 7 cells.
MRP mediated tfflux of 5-CF was mcasured in HuH 7 cells aftet 24 hour incubation with
0-0.5 ng/ml of TNF-a MRP transport activity was examined using 5-CF as descrikd in
methods. Values an reported as % controls (mean I SEM) of the 30 minute
indomethacin-inhibitable 5-CF effîux (n-dgroup), *p<O.OS
Control EL= 12 hours treatment
24 hours treatment
Figure 43: Effect of TNF-a on MRP3 and MRP6 mRNA expression in HuH 7 cells. RT-
PCR was performed on total RNA extracted from 12 and 24 hour TNF-a treated
(O.tng/ml) or control HuH 7 cells described in methods. Values (mean t SEM.
n=5Igroup) are reported as % of contmls. Raw inruisities of MW3 and M W 6 genes
were normalised with respect to p-Actin. *@.OS.
24
Incubation peii
48
od (hours)
Figun 44: Influence of TNF-u treatment duration on MRP transport activity in HepG2
ah. MRP mediatcd eMux of 5-CF was measured hi HepGZceffs after 0-72 hours of
incubation with TNFa (0.2nglml). MRP transport activity was examined using 5-CF as
describeci in methods. Values are reportcd as % controls (mean SEM) of the 30 minute
indomethacin-inhibitable 5-CF cfflux (n=6/gmup), *pc0.05.
TNF-a dose (ng/ml)
Figure 45: Influence of TNF-a concentrations on MRP transport activity in Hep02 cells.
MRP mediated efflux of 5-CF was measured in HepG2 cells after 24 hour incubation
with 0-0.5 nglml of TNF-a. MRP transport activity was examined using 5-CF as
described in methods. Values arc reportcd as % controls (mean I SEM) of the 30 minute
indomethacin-in hi bi table 5-CF effiux (n=6/group), *p<0.05
= Control L 12 hurs treatrnent = 24 hours treatment
Figure 46: Effect of TNF-a on MRP mRNA expression in HepG2 cells. RT-PCR was
performed on total RNA extracted from 12 and 24 hour TNF-a treated (0.2ng/ml) or
control HepG2 cells as descrikd in methods. Values (mean t SEM, n=S/group) are
reported as % of controls. Raw intensities of different MRP genes were nomalised with
respect to PActin. *p<O.OS.
DISCUSSION
DISCUSSION
5.1 Ovenkw of the pmject
Major findings from these studies demonstrated the existence of differentiai regulatory
patterns on MDR efflux transporters on tnatment with the pro-inflammatory cytokines,
L 6 , L l p and TNFs in two human hepatoma ceIl lines. b general, we found that the
PGP and MRP farnily of transporters werc rcgulated in an opposing manner in both ce11
lines and after ail cytokine treatrnents. PGP was shown to be downregulated with MRP
uprcplated upon L6, IL-lp and TNF-a trcatment in most cases and was shown to be
regulated at the level of mRNA (and, perhaps, transcriptional) by IL6 and TNF-a. Most
changes occumng during hepatic rcsponse to inflammatory stimuli occur through
changes in gene regulation. These changes are thought to occur primarily through
activities of IL6 but have also k e n seen after exposure to IL 1p and TNF-a. For
exampk, treatments with IL6, IL-lp or RJF-a have been demonstrated to increase
levels of the positive acute phase proteins such as fibrinogen, ceruloplasmin, SAA as well
as suppress expression of the negative APP, aibumin, in HepG2 cells. (Kobsel, 1994,
Richards et al, 1991) Furthennon, Cytochrome P450 3A4, has ken shown to be down-
ngulated at mRNA level with L 6 , IL4 P and TNF-a treatments in Cacol
cells. (Bertilsson et al, 2001) Both hepatoma ce11 lines that WC used in our studies, HepG2
and HuH 7 have been shown to elicit partial acute phase response upon treatments with
the pproiflammatory cytokines, IL6, ILlB and TNF-a in that they maintain their ability
to ngulate a broad range of APP genes like normal iiver cells, hence they are an
appropriate in vitro mode1 for studying changes in MDR transporters upon cytokine
trcatment. (Won et al, 1993. Kobsel 1994, Koj et al, 1993) However, the pattern of
- A expression of APP genes and th& nphtion in culhind cells may not be identical to
primary hepatocytes and cytokines only elicit partial inflammatory nsponse in cultured
cells, thenfore the actual changes in PGP and MRP expression rnight be even mon
pmfound in vivo. (Won et al. 1993)
It appearcd that L l b altered PGP activity through a diffennt rcgulatory pathway, that
is, through alterations in protein expression of PGP. These nsults coincide with what we
have secn in IL6 and L l P treated isolated rat cultured hepatocytes where IL6
downrcgulates POP at mRNA level and IL- 1 rcduces the POP protein expression but
not at the kvel of mRNA. (Sukhai et al, in press) As IL-6, IL1 and TNFa are the
pnnciple mediators of acute inflammation, the down-regulation of hepatic POP function
and expression that our laboratory has prcviously observed in experimentally-induced
inflammation in rats (Piquette-Miller et al, 1998) are likely due to modulation by these
cytokines. Furthemore, IL-6 trcatments in cultured rat hepatocytes and experimentdly
induccd inflammation in rats has been shown to impose a suppression in gene
transcription rates of the mdrla and mdrlb genes. (Sukhai et al, 2000b) Thus, iL-6 likely
plays a major d e in this downngulation of PGP.(Sukhai et al, 20ûûb). Similar to the
rodent models, out studies conducted in this pmject indicatc that down-ngulation of
PGPlMDRl by IL6 and TNF-a also exists in human in vitro systems.
MRPl and MRP3 have been prcviously reported to k induccd in rodenis models of
hepatic cholestasis. (Borst et al, 1999, Hirohashi et al, Nakamura et al. 1999. Vos et al,
1998) Our studies indicated that MRPl and MRP3 are induced in human hepatoma cells
upon treatment with IL6 ami IL la, TNF-a was Plso fouDd ta induce levcls of MRP3 in
HuH 7 cells. Thenfore, these 3 principal proinflammatory mediators are likely involved
in the downregulation of POP and upregulation of MRPl and MW3 expression during
inflammation.
Overall, our results suggests that pro-infiammatory cytokines, L6. IL4 and TNF-a are
involved in POP and MRP family ngulation in human in vitro mdel. Concentration of
cytokines that we used were observed in patient serum during sepsis. Maximum
concentrations were chosen to reflect those found in patient semm during septic shock.
(Bhere and Lowry, 1995) This suggests that expression of POP and MRP family .
members may be altend clinically, in infiammatory disease statcs (e.g., inflammation
brought on by bacterial and viral infection). As many drugs have ken identified as
substrates of PGP and MRP family proteins and the functional activi ty of these
transporters were altered by cytokine exposure. it is plausible that the disposition,
elimination and plasma concentration of their substrates could be influenced in diseases
which arc associated with cytokine induction. Hence, the potential for dmg-disease
interactions with these transporters is large. For example, nduction in POP expression
during acute inflammation may decrcasc the elimination of W P drug substrates: digoxin
and fexofenadinc, hence increase the accumulation of these dnip inside the body which
may lead to systemic toxicity. This can k very sipificant especially for dnigs that haâ
narrow therapeutic window like digoxin and dosage adjustment will be necessary. As a
rssult, development of in vitro systems will allow us to predict potential discase effects
on the hinctional activity of these transportea. As these interactions can influence the in
vivo pharmacokinetics of numcmus phemiacological agents. this may allow us to predict
and acquire information on the in vivo dnig disposition, allowing dosage adjustment in
different disease States.
Clinically, these findings are important kcause they can be demonstrated in human cells
despite the limitations that ceIl lines have. These rcsults can help us to understand and
explain the differcnces in expression panmis of MDR efflux transporters in normal and
diseased tissues. Of particular interest is that when one farnily of efflux transportes, PGP
is dowmgulated, a simultaneous increase in the expression of another efflux transporters
(MRP family) occurs. Since these transporters share many common substrates, this may
help to explain why certain therapies aimed at suppressing one mechanism of drug
nsistance, often fails clinically. Therefore, elucidating the mechanisms by which PGP
and MRP are rcgulated may k useful in the development of therapeutic strategies that
may modulate the expression of these transporters and hence increase the accumulation
of chemotherapeutic agents inside tumor cells which might help to overcome multidnig
resistance.
5.2 ELlid IL-fiJL-lp anci T-on PGP in BuH 7 and HepG2 cek
in general, in cytokine-treated HuH 7 cells. significant reductions in functional activity,
immunodetectable levels and mRNA expression of PGP werc scen within 24 hours after
treatment with IL6 or TNF-a whenas a nduction in hinctional activity, protein
expression but not mRNA expression were seen after 48 hours of treatment with IL 1 p.
i) IL6
Reduction in the protein expression and activity of PGP after IL6 tnatment was
essociated with a cornsponding decrrase in MDRl rnRNA levels. Hence, the
mechanisms by which IL6 regulate POP at mRNA kvel could be a result of either
decrcase in mRNA stability or a decrase in gene transcription. Nuclear run-on and
rnRNA stability studies perfomd in our laboratory have found that IL-6 decreases gene
transcription of d r f a and mdrlb in cultured rat hepatocytes rather than through changes
in mRNA stability. (Sukhai et al, 2 0 b ) It is plausible that IL6 decrease PGP/MDRI
expression through changes in transcription. However, in ordcr to differentiate ktween
the two, further studies such as nuckar run-on assay can be used to measure gene
transcription rates. Alternative studies could compare mRNA expression under cytokine
trcatments by means of the transcriptional inhibitor, Actinomycin D.
ii)TNF- a
SiMlar to IL-6, iemiction in expression and activity of POP was also observed with a
deciease in MDRl mRNA level in TNFa trcatcd cclls. 1t is possible that both IL6 and
TNF-a mediate transcriptional changes in the protein and mRNA levels of PGP through
induction of the transcription factors C m P , AP-1 and NF-- similar to which occurs
with the hcpatic acutc phase proteinSc Iîhas ken rcported chat transcription factors such
as NF-ICB is involved in the induction of mddb in cultured rat hcpatocytes by TNF-a
through direct binding to the NF-KB binding site present on the mdrJb promoter
region.(Ros et al, 2001) bdeed regulatory regions on the untranslated promoter sequence
of the MDRl gene possess binding sites for these transcription factors. (Combates et al,
1994, Sundseth et al, 1997) However. fûrther studies such as gel-shift assays are nceded
to confimi the involvement of these binding sites.
iii) IL48
The functional activity of I L I mediated rcduction in PGP was associated with a
dccrcase in PGP protein expression as shown by the western blot analysis. These
westerns used the PGP specific antibody C494. Since MRP hinctional activity and
mRNA expression of MRP3 in HuH 7 cclls an induced in Lie, this indicatcs that these
cells possess the II.,- I nceptors and signaling systems. The prcsmce of JL- I response in
HuH 7 cells was also documented in literatun. (Raynes et al, 1993, Malle et al, 1999)
niercfon. it is possible that regulation of POP by IL4 P exists at the level of protein.
This regulation of MDR I at the kvel of proiein could be the result of al t e d translation
rates of the PGP or by altend protein stability.
HsQmzb
Similarly, TNF-a and IL1 in HepG2 cells wcrc found to impose significant reâuction
in the expression and activity of PGP afker 24 and 48 hours trcatmcnt. This likely occurs
thmgh mechanisms or pathways similar to HuH 7 cells. While PGP activity and
expression were somewhat lower in IL6 treated cells, this was not significant.
i ) IL-6
S ine Hem2 cells display a less pmnounced duction in PGP expression and activity
than that seen in HuH 7 cells upon IL6 trcatment, this suggests that IL6 mediated
pathways of POP downreplation may not be functional in al1 hepatoma ceIl line.
It is possible that HepG2 cells partially lack L 6 responsive signaling mechanisms that
lead to suppression of PGPIMDRl expression and function, thenfore. differences in
MDRI gene replation could result from differences in IL-6 nsponsiveness. This could
k due to an absence of various transcription factors which bind to regulatory elements on
the MDRl promotcr. Again, significant differences may exist between HepG2 and HuH7
cells rcgarding the spectrum and the interplay of transcription factors involved in the
cytokinc-mediated response. In addition, we cannot preflude the possibility that
regulatory elements may be mutated in one ce11 line while remaining non-mutated in the
other ce11 line, hence nsulting in differcnt rcsponses to IL-6. Furthemore, differences in
the expression of PGP ktween the two ce11 lines may explain why it is more difficult to
detect changes in HepG2 cells as POP expression and activity were somewhat lower than
that scen in HuH 7 cells.
ii) W a
TNFa imposed a significant duction in MDRI mRNA levels suggesting that mgulation
occurs through transcriptionai changes. In literature, contradictory results have been
npwted on the c f k t of MDRI expsion nwdistsd by TNFu. Some wlier siudies
done in 4 human colon carcinoma cell lines showed that TNF-01 can down-regulate
expression of POP at mRNA level which agrees with what we have seen in our studies.
(Walther et al, 1994) This may imply that TNFir downregulatory effect on PGP may not
bc an organ or tissue specific phenomenon. However, one ment report had shown that
MDRl mRNA expression was significantly induced in TNF-a Crtated Caco2 cells.
(Bertilsson et al, 2001) but the concentration of TNF-a in their experimental design is
lOng/ml which is 30 fold mon than the maximum tolerated dose for septic shock
patients, (Bariem et al, 1995), hence it is not Iikely that TM-a mediated induction of
POP would be seen in physiologic conditions. It also makes it hard to compare Our
rcsults with them since the cells might react differently under such extnme stress
conditions.
.i) IL- 1 fl
Similar to what was seen in HuH 7 cells. the hinctional activity was suppressed in IL-1 a trcatment with a significant dccrease in PGP protein expression only. Since both MRP
fûnctionai activity and mRNA expression of MRPl in HepG2 cells were induced by L
1s , and the pnsence of IL4 itsponse in HepG2 cells werc well documented. (Daffada
et al, 1994, Stonais et al, 1998) the regulation of PGP by Ll is mediated at the protein
level. The cesults of our study in both HuH 7 and HepG2 cells agee with what has k e n
obsewed in IL 1 trcated isolated rat hepatocytes where changes in hinctional activity
and protein expression of POP werc observai without any changes at mRNA level.
exist across species and possibly in human.
1 1 1 Hep02 cells 1 HuH 7 ctlls
Table IV: Summary of the effects of cytokine treatment on PGP in HuH 7 and HepG2
ce11 lines. (t, mcans no change)
Cytokine
IL6
TNF-a
Functional activity
Functional Activity
C)
Rotein mRNA
t)
Protein
C)
J.
4
mRNA
C)
++
5 3 Effe t of IL-6, IC1P and TNF9 oa MRP in HuH 7 and HepG2 ceüs
In general, cytokine treatments imposed an induction on MRP hinctional efflux activity,
suggesting similar rrgulatory pathways exist. Treatrncnt with L 6 , IL4 and TNF-
a resulted in an induction of MRP mediated efflux in HuH 7 cells. With the high
homology among al1 MRP family memkrs, they often share common substrates and it is
possible that 5-CF can be transportcd by different MRP family members. The induction
in MRP hinctional activity in HuH 7 cclls was associated with a corresponding incnase
in MW3 mRNA expnssion with no change in MRPo mRNA expression. Furthemore,
MRPl and M W 2 wcn not detectable in this ceIl line. This implies that MRP3 is likely
responsible for the induction of MRP functional activity in HuH 7 cells. As it is possible
that other, non-characterized efflux cransporten could contribute to S-CF efflux in
hepatocytes, we cannot exclude the possibility that changes in the expression of other
transporters could contribute to Our findings. However we found that changes in rnRNA
expression mirromci those secn in functional activity.
In HepG2 cells, IL6 and IL- 1 were also found to impose an increase in the hinctional
activity of MRP. However, this incnased enlux activity was associated with a
comsponding induction of MRPl mRNA expression whercas rnRNA levels of MRP2,
M W 3 and MRP6 were not altered during these treatments. This implies that induction in
expression of MRPl is likely responsibk for the observed upngulation of MRP function
instead of MRP3 as seen in HuH 7 cells. Differences in the induction pattern of MRP3
ktwccn the two ce11 lines could k the rcsult of altemate MRP expression patterns which
exist. Among the MRP family , MRPl and MRP3 share the gmatest homology; (Kool et
al. 1999) they sharc similar substnitc spccificity and are both located on the basolateral
membrane. (Borst, 1999) Hence it is plausible that upon treatment with cytokines, the
basolateral expression of MRP would k upmgulated in ordcr to remove toxic by
products or metabolites h m the cells. In Hem2 cells, which express both MRPl and
MRP3, MRPl appws to k prefemtidly induced, whereas in HuH 7 cells which does
not express MRP 1, MRP3 is induced to perform similar functions as MRP 1. The
induction of either MRPl or MRP3 has been documented in literature upon inflammation
in d e n t s (Borst et al, 1999, Kool et al. 1999, Vos et al, 1998). As both proteins cany
out similar functions, it is unlikely that both would be physiologically rrquired in a stress
response. Hence induction of one of these efflux transporters is likely sufficient.
It is intensting that TNF-a elicited an opposite effect on MRP functional and mRNA
expression between the two cell lines. In HuH 7 cells, TNF-a caused a sipificant
uprcgulation in MW-mediated efflux with a comsponding increase in expression of
MRP3 rnRNA. On the contrary, TNF-a caused a significant dom-replation of MRP-
mediated efflux in HepG2 cells with a comsponding reduction of 36% in MRPI mRNA
and a smallcr but significant reduction in MRP3 rnRNA level. Changes at the level of
mRNA imply that the replation of MRP functional activity likely occurs through
changes in gene transcription. nius differences obscrved ktween the two ceIl lines
suggests that different regulatory patterns might exist, or transcription factors involved
may k differcntially activated in these ceIl lines. As mention4 in the previous section,
opposite regdatory effects on MDR transporters w m reportai in literature in different
human colon carcinoma ceIl lines. Expression of MDRl was found to be upregulatcd in
Caco 2 cells upon TNF-a tnatment but down-ngulated in M o , HTI 15 colon
carcinoma ce11 lines. (Walther et al, 1994, Bertilsson et al 2001)
As antibodies specific to individual MRP proteins are not readily available, we felt it
would k much more efficient to examine mRNA expression of each gem. We primarily
utilizcd RT-PCR to ascertain effects of these cytokines on individual genes of the MRP
family. However, Our results cannot exclude the potential for alterations in the expression
and activity of other MRP proteins. Furthemiore, protein stability and/or translation may
also k changed.
It has been reported that hepatic MRP2 expression is significantly down-rcgulated in rats
during obstructive cholestasis. (Trauner, M et al, 1997) Reccnt studies conducted in Our
laboratory have demonstrated that MRP2 is significantly down-rcgulated at the level of
rnRNA upon both LPS and IL6 treatment in vivo in mice. (Hartmann, unpublished data)
Hence it was surprising that the expression and activity of MRP2 was not altered in
HepG2 cells after treatment with these 3 pro-inflammatory cytokines. This could k due
to mutated regdatory sequences on the M W 2 promoter during the conversion of cclls
into the continuous ce11 line. This could mike the cells unnsponsive to the binding of
transcription factors. similar to the inferences we dmw earlier. In addition, Denson es al,
reportcd that IL6 did not impose significant changes in M M 2 promoter activity in
Hep02 cells. (Denson et al, 2000) Thercfon it is not surprising that MRP2 was not
Pltercd by IL6 tnatment. On the other hand. LPS administration had been demonstnued
to impose a 90% and 85% nduction in M W 2 gene expression in vivo studies in rats and
in mice ttspectively. @enson et al, 2000, Hartmann, unpublished results). Such
diffmnces in the extent of effect implies that the proinfiammatory cytokines may not be
solely responsible for changes in MRP2 gene expression. ûther compounds, such as bile
acids which accumulate in rcsponse to endotoxin tnatment in vivo. and could also
contribute to the down regulation of MRPZ seen in vivo. (Denson et al, 2000)
As effects were seen at the level of mRNA, it is possible that these pro-inflammatory
cytokines mediate their effects on MRP3 through changes in gene transcription. It is
known that iL-6, I L I and TNFs mediate their effects primarily through induction of
the AP- 1, STAT3, NF-IL6 and NF-- transcription factors. (Manos, 200 1, Su khai et al,
2000a. Jensen et al, 2000) Furthemore, putative binding sites for AP- 1, NF-IL6 and NF-
XB have ken identified on the promoter region of MRP3. (Takada et al, 2000) Hence it
is possible that IL-6, IL 1 $ and TNF-a rcplate the MRP3 transcription through these
transcription factors. As the promoter region of MRPl has yet to k cloned (based on a
search of the GenBank database located at http://www .ncbi.nlm.nih.gov), it is difficult to
know whether putative binding sites for these transcription factors are pnsent on the
MRP I promoter.
I HuH7 cells I Hep02 cells
Table V: Summary of the effects of cytokine tnatment on MRP in HuH 7 and HepG2
ceIl lines.
Cytokine
IL6
Functiond activity
Functional Activity
mRNA
M R P ~ ~
tnRNA
MRP 1 T
5.4 Ciinical 1 Physiolgical Implications of our study
From these studies, we have clearly shown that the MDR efflux transporters namcly PGP
and MRP family can be regulatcd by pro-infiammatory cytokines. These rcgulatory
effects appear to occur relatively fast upon trcatment. usually within 24 and 48 hours.
Since the concentration that we used in the studics cuc within physiological ranges
observed in patients with sepsis (Barrien et al, 1995), it is likely that these changes could
occur clinically.
The MDR and MRP efflux transporters hinction (Kuwano et al, 1999, Borst et al, 1999,
Sharom 1997 and Gemann, 1996) to remove toxic drugs and toxins from cells and
thercforc act as protective mechanism for liver cells. (Germann, 1996) Hcnce, up or
downregulation of these transporters could affect the elimination of these dnigs or toxins
and may either dccrcase the thempcutic effect of these dmgs or incrcase the toxicity due
to an excessive accumulation of the dmg. In out studies with IL6 and IL- 1 in both cell
lines, upqulation of either MRPl or MRPJ which are localized on the basolateral
membrane of hepatocytes occumd concumntly to the downregulation of POP, which is
located on the canalicular membrane. Based on these changes, it is anticipated that the
biliary elimination of dmgs which an substrate for PGP and MRPl or MRP3 such as
vincristine would k rcduced duc to the decrcasc in POP. However. a decrcased cfflux
through POP Mght not lead to an incrase in accumulation of the drug inside the ceIl
simply k a u s e at the same time, the induction of MRPl and MRP3 also increases the
cfflux of the drug to the blood. Therefom, increased plasma concentration of the h g
could rcsult and may hirther increase dmg toxicitics. As intraccllulm dmg concentrations
arc ducai , this could furthet contributc to dt idrug resktancc. h temu of tumor cells
which overcxpress these transporters, the downregulation of MDR 1 is offset by the
induction of MRP. thus as the majority of antineoplastic agents WC substrates for both
POP and MW, MDR l down-regdation would not necessarily be associated with
incrcased intracellular drug accumulation. Efflux experiments using a common substrate
of PGP and MRP (cg. vinblastine) might k able to confirm Our hypothesis.
The effect of TNFs on MRP function and expression was diffennt in HepG2 from HuH
7 cells. The downregulatory effects on both PGP and MRPl which we observed in
HepG2 cells could be used therapeutically to increase chemonsponsiveness to dnigs that
are common substrates of both transporters. On the other hand, in HuH 7 cells, we saw
upnplation of MRP3 whilc MDRl rnRNA was downregulated. The different pattern of
rcgulation in MRPl expression and function shown between the 2 ce11 Iines suggests one
question: is this decrease in MRP 1 shown in HepG2 cells a cell-line specific phenornenon
or does TNF-a have a different regdatory effect on MRPl and MRP3? Hence future
studies will be needed to further elucidate the pathway of how TNF-a ngulates these
transporters.
It will be helpful if we can identify pathways by which these MDR transporters arc
ngulated. This may explain or identify changes in h g disposition or elimination upon
discase conditions such as sepsis or acute inflammation when L6, IL- 1 p or TNF-OC arc
at high levels. This Mght be particularly usefut for dmgs that have narrow therapeutic
windows, as too high or low doses c m lead io either increase toxicity or decrease efficacy
of the h g . Anochcr use could be tocbign mechanisms in regulating the expression and
activity of these transporters thus ultimately may help to decnase the effiux of dmgs out
of the cells and hence overcome situations of multidrug resistance.
Although the physiological roles of the downregulation of PGP and induction of
individual MRPs arc not clear, it is postulateci that the induction of MRP may increase the
elimination of intracellular glutathionc conjugatcd toxins which would othcmise
accumulate within the cell and hcnce lead to seven damage. Even though the
physiological substrates of PGP have not well characterized yet. steroids such as
corticosteroids. aldosterone and some peptides w e n found to be substrates of POP.
(Sharom, 1997) Therefore, the decrease in PGP during inflammation may help to retain
these substrates inside the ce11 which might have an effect in maintaining the cell's
integrity.
5.5 suaimsry
Based on our studies, we observed a ngulatory effect of the pro-inflammatory mediators,
L6. IL- 1 and TNF-a on POP and MRP hinctional activities and expression in HepG2
and HuH 7 ce11 lines. In general these cytokines imposed a down-replation of
PGP/MDRl expression and activity while causing an increase in MRP activity and
expression. The down-replation of POP and induction of MRPs may play an important
role in maintaining the cell's integrity under inflammatory stress. Furthemore, the
opposing rcgulatory mechanisms of these transporters would bc of particular importance
since suppression of one transporter was counteractcd by the uprcgulation of another
transporter. This could contribute to difficulty in overcoming multidnig rcsistance as
ôoth PGP and MRPs are capable of confering nsistance to anti-cancer drugs.
CONCLUSION
Conclusion and Future studies:
Overall, Our data indicates that the proinflarnarntory cytokines, L 6 , IL- 1 and TNF-a
impose changes in the expression and activity of the PGP and MRP families of ABC
transporters. These changes wcre scen both at the level of rnRNA with L 6 and TNF-a,
indicating changes in gcne transcription or at the level of protein, in the case of I L I p.
While in general a similar pattern of replation was seen in the two cell lines, there were
some discrepancies. For example: TNF-a imposeci an induction in MRP activity in HuH
7 cells while a suppressive effect was observed in HepG2 cells.
1t is likely that L6, IL-1 $ and TNFa cm regulate the expression of these transporters
through different mcchanisms when one examines the different effects of these cytokines
on the regulation and expression of POP and M W as well as in the time course. It will
k interesting to delineate the effects of these cytokines on these transporten in human or
in human primary hcpatocytes if possible since primary cultun generally retain
physiologically exprcssed transporters and intact sipaling pathways. Future studies to
exainine the signal transduction mechanisms by which these cytokines regulate the
expression of these transporters are important since it may find a means to regulate the
expression of these transporters in overcoming multidrug rcsistance.
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