Arie Sullivan

8/9/2015

ON THE ACTION

MECHANISM OF

CHLOROTOXIN:

APOPTOSIS MSc Research Project

Arie Sullivan

1

Contents

Abstract………………………………………………………………………………………………….2

Introduction…………………………………………………………………………………………….3

Materials and methods………………………………………………………………………….12

Results..…………………………………………………………………………………………………16

Discussion……………………………………………………………………………………………..25

Further investigation…………………………………………………………………………….33

Appendix……………………………………………………………………………………………….34

References…………………………………………………………………………………………….46

Arie Sullivan

2

Abstract.

Scorpion venom is a complex mixture of biologically active compounds, of which some are being

increasingly studied for their therapeutic properties. The family of chlorotoxin (CTX) -like peptides

exhibit insecticidal activity with a yet only little known of its mechanism of action. The primary

activity of CTX is in protection against invertebrate predators, however, CTX’s activity is not

restricted to invertebrates, with growing incoming research reporting CTX binding specificity to

cells of malignant brain tumors, namely glioma. Additionally, studies investigating CTX-based

tissue staining claim CTX-binding extends to tumors of neuroectodermal origin. Deciphering the

mode of action of CTX largely relies on understanding the interactions of CTX at a molecular level,

specifically, ascertaining the exact CTX receptors would provide the parameters in which CTX can

be used safely and therapeutically. Investigations on alternate forms of CTX (BmKCT) report

apoptosis induction in glioma with IC50 values of 0.28µM. It is shown herein that contrary to

expectation, CTX does not reach an IC50 value for any cell line, potentially demonstrating

alternative mechanisms for CTX in its action on tumorous/non-tumorous cell lines. This report

also discloses no apoptogenic properties for CTX as determined by a series of experiments

including statistical analyses of CTX effect on highly migratory cells of neuroectodermal origin,

specifically SHSY5Y; non-migratory breast cancer cell line MCF7; and migratory non-cancerous

human keratinocyte cell line HaCaT. Rather, a new mechanism of action, necrosis induction in

SHSY5Y, is demonstrated for CTX. Moreover, no significant effect on cell line HaCaT expressing

MMP-2, suggests that MMP-2 as a lone CTX target is questionable.

Arie Sullivan

3

Introduction.

Glioma and Neuroblastoma

The glioma family form 65% of primary brain tumors (Wang and Ji, 2005) and include Glioblastoma

multiforme (GBM) and anaplastic astrocytomas, the most aggressive of primary brain tumors

which at best, are accompanied by dismal prognoses (Holland, 2000). Ne uroblastoma (NB),

suspected of originating from neural crest-derived sympathoadrenal progenitor cells, has a high

metastatic potential, and is the most common extracranial tumor in children, accounting for 8-

10% of childhood cancers (Kim et al., 2014; McHugh, 2007).

Despite the progression made over the last decade, a monogenic Mendelian syndrome of

heritable NBs has not been established, moreover, the influences of both epigenetic factors and

of the presumed hereditary components remain to be determined. Genetic factors for a diversity

of human pathologies have been identified by whole exome sequencing yet only recently has it

been applied in ascertaining genetic factors linked to glioma and NB tumorigenesis (Kim et al.,

2015). Thus, efficient therapeutic interventions for both gliomas and NB remain scarce.

NB and glioma cells however, both show an unusual propensity to disperse from the tumor site

with a high metastatic rate, subsequently invading neighboring healthy tissue (Merzak et al.,

1994). As well as sharing metastatic potential, gliomas and NBs have been associated by

embryonic nature, cellular characteristics and tumorigenesis (Kriegstein and Alvarez-Buylla,

2009). Moreover, Notch signaling has been demonstrated to initiate irreversible differenti ation

from Neurogenesis to Gliogenesis by dominant inhibition of BMP-2 in neural crest stem cells

(Morrison et al., 2000). Thus, an investigation into the targeting capacity of CTX to NB would form

a continuation of the research characterizing CTX’s mechanism of action.

Malignant cell invasion

Invasive tumor cells escape surgical removal and geographically circumvent lethal radiation

exposure and chemotherapy (Nakada et al., 2007). This evading ability stems from a unique

capacity of glioma cells to actively migrate through two types of extracellular space in the brain,

the perivascular space present around all blood vessels, and the spaces in between the neurons

and glial cells making up the brain parenchyma and white mater fiber tracts (Paw et al., 2015).

The migration of glioma cells through these extracellular space necessitates particular changes in

cell morphology. Key signaling GTPases that regulate cell morphology and mediate receptor-

initiated signaling in the regulation of glioma invasion are Rho family GTPases including Rac, RhoA

and Cdc42 (Kwiakowska and Symons, 2013).

Arie Sullivan

4

The mechanism of action for NB brain metastasis is not as well -characterized as that of glioma,

but the invasive capacity of many tumor cells necessitates particular mechanisms of action which

can be summarized in three sequential steps. The first step is modification of cell adhesion

property by interaction with the extracellular matrix (ECM) via adhesion proteins such as

integrins, specifically αvβ3 and αvβ6 mediate cell adhesion (Deryugina and Bourdon, 1996). Since

NB is highly metastatic, upregulated expression of αvβ3 is common and has been recognized as a

prognostic indicator for NB (Ribatti et al., 2004). The second step is degradation of the

extracellular matrix (ECM) via proteolytic enzymes such as members of the matrix

metalloproteinase (MMP) family. Importantly, the extracellular space in anatomic arrangements

varies profoundly, such as in the basal lamina between myelinated axons or thin fibrous ECM of

the blood vessel basement membranes (Brown, 2011). This indicates the presence of more than

one matrix ligand and potentially separate mechanisms for invasion, further complicating the

mode of action in translocation of neoplastic cells through host ECM barriers. Finally, a change of

cell shape and volume, is necessitated for migration through the narrow spaces formed from

degradation of the ECM (Kim et al., 2004). This shape shifting ability is mediated via ion flow such

as Cl-, K+ and their respective volume-regulated ion channels (Kim et al., 2004). The ability to

perform all three steps sequentially allows invasive cells to penetrate areas that would otherwise

be impossible, specifically, allowing glioma cells to penetrate the blood-brain-barrier (BBB) (fig.1).

With such distinctive characteristics however, often comes in equal measures, distinctive

mechanisms of action.

Fig.1 The blood brain barrier (BBB) and the diversity of glial and neural cells implicated in

glioma /astrocytoma tumorigenesis. Image acquired from Slayden, 2005.

Arie Sullivan

5

Venoms as therapeutics

The first use of scorpion venom as a drug can be traced back to almost 2000 years in China, in

treating apoplexy, epilepsy, spasm, migraines, tetanus and pyocutaneous amongst more (Fan et

al., 2010). Interestingly, these diseases are nowadays categorized as channelopathies, implying

the active component is a key regulator of ion channels (Zhijian et al., 2006; Goudet et al., 2002).

Various other venoms isolated from a number of species have been hailed as possessing

antiproliferative, cytotoxic, apoptogenic, and immunosuppressive properties. They have been

recognized as a rich source for numerous bioactive compounds possessing therapeutic potentials

like enzyme and non-enzyme proteins, ions, free amino acids, and other organic and inorganic

substances.

Studies on the mode of action of cardiotoxin III, isolated from Naja naja atra snake venom, in

human colorectal cancer (colo205) revealed apoptosis induction, confirmed by DNA

fragmentation (Tsai et al., 2006). Spider venom isolated from Macrothele raven has been reported

to affect cell viability in a dose-dependent manner and induce apoptosis and necrosis in breast

cancer (MCF7) cells (Gao et al., 2007). A number of scorpion venom components have been

known to mediate cell proliferation, cell growth and cell cycle (Das Gupta et al., 2007). Specifically,

venom from the scorpion Odontobuthus doriae has been shown to decrease cell viability, induce

reactive nitrogen intermediates, depolarize mitochondria membranes and increase caspase-3

activity and thus, apoptosis in human neuroblastoma (SHSY5Y) cells (Zargan et al., 2011).

Additionally, a recombinant form of the scorpion venom component Chlorotoxin (CTX) Buthus

martensii Karsch Chlorotoxin-like Toxin (BmKCT), divergent by only 7 amino acids of the 36

characterized in wild-type CTX, (fig.2a) has been reported to induce glioma cell apoptosis (Wang

and Ji, 2005; Fu et al., 2007). Several other related scorpion venom peptides possess similar

amino acid sequences with matching cysteine residues and minimal divergence from the

consensus sequence (Arzamasov et al., 2014). This offers a rational hypothesis that wild-type CTX,

a natural 36 amino acid peptide derived from the venom of scorpion Leiurus quinquestriatus, may

possess similar apoptogenic properties.

2 a)

G

Arie Sullivan

6

Fig.2 a) Amino acid sequence alignment of BmKCT peptide with Chlorotoxin by matching cysteine

residues. Green pleated sheets indicate the sequences forming β-pleated sheets, the blue helices

represent sequences forming α-helices. This highlights the differences in amino acid sequence and thus,

structure despite both CTX and BmKCT having a ββαβ fold b) 3D structure of CTX c) 3D structure of BmKCT.

Image acquired from Dardevet et al., 2015.

Chlorotoxin (CTX)

Leiurus Quinqestriatus chlorotoxin (CTX hereafter), a small peptide compact in structure and able

to penetrate the BBB (fig.1), is known among other low molecular-mass and cysteine-rich

peptides, to inhibit recombinant small-conductance chloride channels (DeBin and Strichartz,

1991; DeBin et al., 1993). Cell membrane chloride channels have been implicated in cell

proliferation and invasive cell migration of primary brain tumor cells, namely glial and neural cells

(Olsen et al., 2003). Moreover, cell membrane channel inhibitors play an important role in cellular

mitogenesis (Ghallagher et al., 1996) and have been associated with the control of signal

transduction in the metastatic cascades (Laniado et al., 2001).

However, despite numerous incoming reports confirming the binding specificity of CTX for tumor

cells, there has been contradicting reports as to the exact mechanism of action of CTX, with three

potential receptors being recognized to date. Cl - channels, discovered in 1993, characterized the

name ‘chlorotoxin’ (DeBin et al., 1993), followed by matrix metalloproteinase -2 (MMP-2) a

decade later. Finally, the late annexin A2 was discovered as a potential receptor, reported as being

the target for a biotinylated recombinant derivative of CTX, TM601 (Kesavan et al., 2010). Annexin

A2 was confirmed as a molecular target of TM601 by reduced CTX binding as a direct result of

annexin A2 siRNA knockout (Dardevet et al., 2015).

All three receptors, Cl - channels, MMP-2 and annexin A2 are involved in cell migration (Mao et al.,

2007; Reunanen and Kahari, 2000; Tatenhorst et al., 2006). As such, the targeting capacity of CTX

b) c)

Arie Sullivan

7

has been coupled to migrating cancer cells, namely gliomas, melanomas, small cell lung

carcinomas, neuroblastomas, ganglioneuromas, adrenal pheochromocytomas,

medulloblastomas and Ewings’s sarcomas (Lyons et al., 2002). CTX has been demonstrated as a

highly specific marker for these selected tumors in biopsy tissues, frequently associating CTX with

the term ‘tumor paint’ (Butte et al., 2014). However, deciphering the exact mechanism of action

that allows such specific targeting to tumor cells has been met with difficulty since the potential

characterized CTX targets are associated with the migratory capacity of cells, primarily known to

inhibit cell migration. Although it is the constituents involved in the invasive facet of malignant

cells that are recognized as the principal target for CTX, expression of membrane proteins involved

in cell migration is not limited to malignant cells. Cell migration is a natural mechanism vital for

embryonic development and tissue repair, and the potential target receptors for CTX are not only

expressed in highly invasive tumor cells, but also in healthy migratory cell lines such as human

keratinocyte (HaCaT). Thus, despite receptors such as MMP-2, CL- ion channels and annexin A2

having altered expression in selected malignant invasive tumor cells, these do not form an

absolute marker for these cells, and non-specific CTX binding remains a potential concern

necessitating further investigation.

Chlorotoxin as ‘tumor paint’

Migratory neural stem cells and neoplastic cells of neuroectodermal origin, including sensory,

sympathoadrenal, enteric and parasympathetic neurons of the peripheral nervous system,

Schwann cells, melanocytes and endocrine cells all share a common embryonic origin. Similarly,

these share genetic and antigenic phenotypes with gliomas (Lyons et al., 2002). This suggests that

CTX’s specificity as a marker for gliomas may extend to other tumor cells of neuroectodermal

origin. Indeed, histochemical staining of human biopsy tissues demonstrated binding of CTX at a

rate of 90% CTX positive cells in each section, extending to peripheral neuroectodermal tumors

as well as gliomas (Lyons et al., 2002). This ‘tumor painting’ capacity of CTX has provided surgeons

with unprecedented, real-time biophotonic information clearly defining tumor margins and

associated cancer cells (Stroud et al., 2012). Beyond the ‘tumor painting’ ability of CTX via

membrane receptor binding, lies the consequential intracellular signaling partly defining the

‘mechanism of action’ of CTX.

Chloride ion channels

Plasma membrane anion chloride channels are implicated in a number of functions, including

control of excitability in neuron and muscle, cell volume regulation, transepithelial transport and

sensory transduction (Hartzell et al., 2005). Thus far, three classes of structures have been

identified, voltage-gated ion channels (VGIC), postsynaptic Cl - channels; the cystic fibrosis

transmembrane conductance regulator Cl - channels; and the CLC family of Cl - channels (Duran et

al., 2010). Since CTX has been reported to bind specifically to CLC-3 (Rao et al., 2015), the CLC

family of CL- Channels are of foremost interest.

Arie Sullivan

8

Fig.3 CLC chloride anion channel a) 3D structure of CLC channel b) mechanism of action of CLC channel.

Images acquired from OPM database and Lisal and Maduke, 2009 respectively.

Apoptotic normotonic shrinkage of cells is coupled to facilitation of regulatory volume decrease

(RVD) which is attained by parallel operation of Cl - and K+ channels under hypotonic conditions

(Maeno et al., 2000). Cl- channel blockers, such as 4,4’-diisothiocyanatostibene-2, have been

shown to inhibit cell shrinkage (Wei et al., 2004), indicating a necessity for Cl - channel mediated

fluid secretion for invasive migration (fig.4). Thus, the inhibitory effects of CTX on Cl - channels

hold promising prospects for halting the invasiveness of NBs and gliomas. However, the role of Cl-

channels have been highlighted in a number of cell types treated with apoptotic stimuli (Chen et

al., 2008). Specifically, the rapid outflow of Cl - ions, triggered by intrinsic or extrinsic apoptotic

stimuli has been recognized and confirmed by apoptosis inhibition in the presence of Cl - channel

blockers (Szabo et al., 1998; Nietsch et al., 2000). Additionally, RVD has also been prevented by

blocking regulatory Cl - or K+ channels, halting the succeeding biochemical and morphological

events leading to apoptosis (Maeno et al., 2000).

Fig.4 Cell volume regulatory mechanisms a) cell shrinkage via efflux of Cl - via Cl - channels along

with obligated water (Sontheimer, 2004) and b) outflow of Cl - during depolarization of the

membrane (Scott and Holmes, 2012)

a) b)

a) b)

Arie Sullivan

9

As well as depolarization of the mitochondrial membrane during apoptosis, depolarization of the

plasma membrane (PM) has been reported in a number of papers investigating apoptosis (Nolte

et al., 2004; Mann et al., 2001). Moreover, a correlation between modulation of the membrane

potential and protection against apoptosis has been demonstrated (Nuccitelli et al., 2006),

suggesting a protective mechanism from apoptosis by preventing depolarization of the PM.

Despite additional research being necessitated to establish the exact association between

apoptosis and depolarization of the PM, these reports, at least in part, confirm the implication of

ion channels in the apoptotic process. A further complication is the overexpression of CIC-3

chloride channels in glioma that facilitate outward rectifying currents overwhelm CIC-2 channels

that facilitate inward rectifying currents, causing a net outflow of Cl - and subsequently,

depolarization of the PM (Olsen et al., 2003). Since depolarization of the PM is a necessity to pass

the G2/M checkpoint in the cell cycle (Blackiston et al., 2009), overexpression of CIC-3 channels

strongly favors cell proliferation.

Since CTX has been reported to induce apoptosis (Cheng et al., 2014) and Cl- channels are a

recognized target for CTX, it is perhaps not surprising that there exists contradicting reports as to

the mode of action of CTX. With reports claiming the induction of apoptosis by recombinant CTX,

BmKCT; and Cl- channels being well-characterized receptors for CTX, the claim of apoptosis

induction by CTX when considered alongside reports of CTX Cl - channel inhibition, prompts a need

for further investigation. Further complicating matters, a study by Maertens et al. (2009) revealed

no detection of any change in whole-cell membrane currents by EPC-7 patch clamp amplifier post

CTX treatment, leading to a claim that CTX does not inhibit Cl- channels.

Matrix metalloproteinase -2 (MMP-2)

Matrix metalloproteinases (MMPs) form a family of multi -domain proteins implicated in the

physiological degradation of the extracellular matrix and connective tissue. MMPs bear a catalytic

site from which tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) can control MMP activity.

Specifically, via interaction between the hemopexin domain of MMP-2 and the C-terminal domain

of TIMP-2 (fig.5) (Morgunova et al., 2002). Additionally to TIMP-2, inhibition of MMP-2 enzymatic

activity is thought to occur via association with a complex of proteins composed of MMP-9, αvβ3

integrin and membrane-type matrix metalloproteinases (MTI-MMPs). Interestingly, CTX is

suspected of binding to more than one of these receptors resulting in internalization of the

complex by endocytosis (Deshane et al., 2002; McFerrin and Sontheimer, 2006). Endocytosis of

MMP-2/TIMP-2 complex has previously been associated with low density lipoprotein receptor-

related protein (LRP), confirmed by inhibition of endocytosis by exposure to natural LRP ligand

antagonist receptor-associated protein (RAP) (Emonard et al., 2004; Sternilight and Werb, 2009).

Receptor-mediated endocytosis of MMP-2/TIMP-2 has not been considered to a great extent as

a potential mechanism of action of CTX. However, one study reports that the maximum inhibitory

capacity of CTX on glioma invasion was reduced by 50% in the presence of filipin (Deshane et al.,

2003). Since filipin’s mechanism of action involves inhibition of the raft/caveolae endocytosis

pathway (Schnitzer et al., 1994), this is indicative that CTX induces endocytosis of MMP-2.

Arie Sullivan

10

a)

As MMP-2 has been characterized as a mediator for the degradation of the ECM, glioma’s capacity

for invasion and metastasis is owed, at least in part, to readily detectable expression of MMP -2,

-9 and TIMP-2 (Wang et al., 2003) Similarly, NB’s invasive malignancy stages have been positively

correlated with expression of MMP-2 and -9 (Zhu et al., 2010). Moreover, stable nucleic acid lipid

particle (SNALP) internalization in U87 glioblastoma cells (fig.5 b), HEK293T human embryonic

kidney cells and mouse primary astrocytes with CTX-coupled liposomes encapsulating FAM-

labeled anti-miR-21 oligonucleotides revealed intensive red (lipid) and moderate green

(oligonucleotide) fluorescence detectable throughout cellular cytoplasm (Costa et al., 2013).

These findings contribute to the growing body of research recognizing the internalization effect

of CTX on PM receptors. Thus, CTX-mediated endocytosis indeed offers a rational mechanism of

action for CTX in the inhibition of glioma metastatic capacity, and prompts a need for further

investigation.

Despite the binding specificity and associated ‘tumor painting’ capacity of CTX being associated

with MMP-2 expressing tumors, the growing field of genetic manipulation has allowed for CTX

tumor targeting to extend beyond these parameters. For example, using a chlorotoxin Cy5.5

bioconjugate in targeting breast cancer cells (MCF7), modified to express MMP-2 via MMP-2

encoding plasmid transfection (fig.5 b), facilitates CTX binding, strongly favoring MMP-2 as the

Fig.5 3D structure of MMP-2/TIMP-2 complex and method for MMP-2 transfection. a) The proteinase

(MMP-2) and inhibitor (TIMP-2) interact via the hemopexin domain and C-terminal domain respectively.

Catalytic and structural Zn2+ ions are red and Ca2+ ions are purple. The turquoise ell ipsoids (III and V)

indicate areas of interaction between the proteinase and inhibitor b) CTX triggers gene transfection for

cancer cell therapy. Images acquired from Morgunova et al., 2002 and Hmed et al., 2013 respectively

b)

Arie Sullivan

11

Fig.6 Annexin A2 bound to calcium. Calcium is represented by black spheres

bound to the α-helices. Image acquired from Schramel, 2014.

CTX target receptor (Veiseh et al., 2007). This confirms MMP-2 bearing tumor cell lines can be

readily detected by CTX.

Annexin A2

Annexin A2 (fig.6) mediates a number of biological processes and has been implicated in cell

migration and metastasis (Zhang et al., 2013). Importantly, annexin A2 silencing inhibits invasion,

migration and tumorigenic potential of cancer cells (Yao et al., 2013). Loss of annexin A2 has also

been reported to cause tumor cell apoptosis via proapoptotic p38 mitogen activated protein

kinase (p38MAPK), c-Jun N-terminal kinase (JNK) and Akt signaling (Madureira et al., 2011). The

annexin A2 tetramer has also been shown to localize on the surface of human breast carcinoma

and glioma cells where it is suspected that interaction with procathepsin-B facilitates tumor

invasion and metastasis (Mai et al., 2000). Taken together, these findings suggest an additional

potential mode of action for CTX in halting malignant invasion.

This paper constitutes an investigation into the hypothesis that CTX induces apoptosis in tumors

cells of neuroectodermal origin, specifically NB cell line SHSY5Y. In order to determine the

potential target receptors of CTX, the investigation will include a control non-cancerous human

keratinocyte (HaCaT) migratory cell line for consideration of MMP-2 receptors and Cl- channels,

as well as a non-invasive breast cancer cell line MCF7, for considering effects of CTX on tumors

lacking significant MMP-2 expression and consequentially, with lower degrees of overall invasive

capacity.

The study will test this hypothesis by means of a number of experimental procedures based on

determining apoptosis/necrosis levels post-exposure to CTX. Initially, a qualitative cell viability

assay based on ATP levels will be performed -/+CTX for all cell lines. Since ATP levels are exquisitely

regulated in cells, detectable loss/gain in ATP levels should reflect loss/gain in cell viability. All

three cell lines will be subsequently treated, stained and observed under inverted fluorescent

microscopy -/+CTX to provide quantitative data regarding apoptotic and necrotic effects of CTX.

A statistical analysis will be performed to determine the significance of the quantitative data

regarding apoptosis and necrosis. Finally, any apoptosis will be confirmed by detection of DNA

Arie Sullivan

12

fragmentation (Nagata, 2000) using ethidium bromide stained gels. On the detection of apoptosis,

a cytochrome-C assay will finally be performed to confirm depolarization of mitochondrial

membrane, caspase 3 activation and thus, true apoptosis (Fig.2).

A discussion will consider previous and current research with the aim of determining if the findings

regarding apoptosis induction by CTX remain consistent and whether these are comparable with

studies on other characterized venoms.

The selected cell lines for the current study will provide additional information regarding CTX

receptors since each cell line differs in MMP-2 expression and thus, in associated capacity for

malignant invasion. Certain predictions can be drawn in so far as that differing expression of each

of the three potential receptors would reflect CTX binding ability. As MMP-2 and annexin A2 are

highly expressed in both NB and HaCaT cells (Blanchard et al., 1996) but not MCF7 (Wang and Lin,

2014), an effect on NB and HaCaT but not MCF7 would favor MMP-2 or annexin A2 as the primary

CTX receptors. If effect is observed on CTX treatment of MCF7, receptors other than MMP-2 and

annexin A2 should be considered. Non-specific binding of CTX to non-tumor cells could also be

determined by investigating the binding capacity of CTX to non-tumor, migratory cell lines, known

to express the three potential CTX target receptors, such as human keratinocyte cells (HaCaT).

The concentrations of CTX proposed herein are based on minimum concentrations under which

an apoptotic effect has been observed in previous studies investigating apoptosis in glioma tumor

cells (Veiseh et al., 2009; Soroceanu et al., 1999) (appendix 2).

Materials and Methods.

Cell lines and cell cultures

Human breast cancer cell line MCF7, and neuroblastoma SHSY5Y (obtained from Sheffield Hallam

University) were maintained in Minimum Eagle’s medium (Gibco) supplemented with 2mM L-

glutamine and 10% heat inactivated fetal calf serum, Non-essential amino acids and

penicillin/streptomycin. Human keratinocyte cell line HaCaT (obtained from Sheffield Hallam

University) was maintained in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with

10% heat inactivated fetal calf serum and penicillin/streptomycin.

Fig.7. Flow diagram of the experimental procedure considered in determining apoptosis induction by CTX.

Arie Sullivan

13

Reagents

All reagents used herein are purchased from Sigma™ unless otherwise stated. CellTiterGlo™ kit

was purchased from Promega™. DNA Ladder Detection kit was purchased from Abcam™.

Chlorotoxin was supplied by the Peptide Institute, Inc.

Bicinchoninic acid (BCA) assay

2mL of bovine serum albumin (BSA) was prepared at a concentration of 2mg/mL and frozen in

100µL aliquots. 10mL of 4% CuSO4 solution was then prepared and stored at 4°C. 60µL of 4%

CuSO4 solution was added to 3mL of BCA stock solution to make a working BCA solution. Two

100µL BSA aliquots (2mg/mL) were thawed and used to prepare five BSA standards ranging from

125, 250, 500, 1000 to 2000mg/mL. 20µL of each BSA standard was sequentially transferred to a

96-well plate in triplicates, followed by the addition of 200µL of the working solution to each well

containing BSA standards. The plate was covered and incubated at room temperature for 45

minutes. The absorbance was subsequently read in a spectrophotometer set at a wavelength of

570nM.

Cell viability assay -/+CTX

A cell viability assay without CTX was performed using Breast cancer cells (MCF7) neuroblastoma

cells (SHSY5Y) and human keratinocyte cells (HaCaT) to generate standard curves and determine

optimum concentration of cells to be used for subsequent assays +CTX. The capacity range of the

CellTiterGlo™ kit is from 0-50,000 cells/well in a 96-wellplate format, this was subsequently

confirmed by the plateau phase which occurs at approximately 40,000 cells/well for both cell lines

MCF7 and SHSY5Y. HaCaT generated a linear standard curve without a plateau phase for up to

50,000 cells. A concentration within the linear luminescence phase for all cell lines was

determined (20,000 cells/well), this will provide the largest variation in luminescence from loss of

cell viability.

An ATP standard curve was also generated for quality control of the viability assay. 1µM ATP was

prepared in culture medium. Ten fold dilutions of ATP were then prepared (1µM to 10nM). A 96-

well plate was sequentially loaded with the varying concenttrations of ATP. A volume of

CelTiterGlo™ reagent equal to the volume of ATP standards in each well was added and the

platewas shaken gently on an orbital shaker for 2 minutes. The plate was then incubated at room

temperature for 10 mimnutes to stabilize the luminescence signal and luminescence was

recorded using a Victor™ multiplate reader. The standard curve for ATP was inset to the cell

viability curves for each cell line (appendix 2a), b) and c)).

A cell viability assay with CTX was performed for MCF7, SHSY5Y, and HaCaT. Cell concentrations

(20,000 cells/well) within the luminescence linear phase of the cell viability assay -CTX (fig.2) were

prepared for both cell lines for optimum detection of change in luminescence. All cell lines were

plated out at 250µL on a 96-well format, with the outer wells containing PBS (appendix 4).

Arie Sullivan

14

Cells in three wells were lysed using CellLytic™ according to protocol for cell lines MCF7, HaCaT

and SHSY5Y and a BCA assay was performed to determine MCF7 and SHSY5Y cell concentration

relative to HaCaT cell concentration (20,000 cells/well) by determining total protein content in

mg.

Two concentrations of CTX (0.25mM and 0.025mM) were prepared from a stock CTX

concentration of 10mg/mL. 2.5µL of 0.25mM CTX was added to wells containing 250µL of cells in

media (20,000 cells/well) producing a 2.5µM CTX treatment. 2.5µL of 25µM was added to wells

containing 250µL of cells in media (20,000 cells/well) producing a 0.25µM CTX treatment. All

treatments were plated out in triplicates on a 96-well plate format for all three cell lines. A –ve

control was prepared by plating 250µL (20,000 cells/well) in 3 wells in triplicate for each cell line.

Luminescence was recorded at time 1 hour and 24 hours, and the results imported into a

Graphpad™ spreadsheet.

Fluorescent microscopy -/+CTX

A series of cell concentrations (31,250 cells/mL - 200,000 cells/mL) were prepared by serial 2-fold

dilutions and plated out (250µL/well) in a 96-well format. The plate was incubated for 24 hours at

37°C to allow cells to adhere. 10µL of Hoechst 33342 stain and 10µL of Propidium Iodide (PI) was

added to 1mL media for detection of live and dead cells respectively. A ll wells containing various

cell concentrations were stained with 10 µL of the prepared dye and incubated in the dark at room

temperature for 30 minutes. All wells were subsequently observed under fluorescent microscopy

to determine the optimum cell concentration for fluorescent microscopy +CTX.

The optimum cell concentration (80,000 cells/mL) was plated out (250µL) in triplicates for each

CTX dilution (2.5µM and 0.25µM) and one triplicate untreated to generate a –ve control, for

observation at time 1 and 24 hours. An additional triplicate was plated out for each cell line for a

BCA assay. The plate was incubated at 37°C for 24 hours to allow cells to adhere before being

treated with CTX. Two concentrations of CTX (0.25mM and 25µM) were prepared from a stock

CTX concentration of 10mg/mL as per above. 2.5µL of 0.25mM CTX was added to wells containing

250µL of cells in media (20,000 cells/well) producing a 2.5µM CTX treatment. 2.5µL of 25µM was

added to wells containing 250µL of cells in media (20,000 cells/well) producing a 0.25µM CTX

treatment. All wells were treated including –ve control. The plate was placed on an orbital shaker

and shaken gently for 10 minutes before returning to the incubator. A Hoechst 33342 and

Propidium Iodide stain was prepared as per above. Following 30 minutes incubation, 10µL of the

stain was added to each well, the plate was incubated in the dark at room temperature for further

30 minutes and all wells were subsequently observed directly under inverted fluorescent

microscopy after a total of 1 hour of exposure to CTX. A BCA assay was performed to compare cell

concentration for each cell line. The plate was returned to 37°C incubation and the process

repeated at time 24 hours. A statistical analysis was subsequently performed to determine the

apoptotic index for each time interval.

Arie Sullivan

15

DNA fragmentation detection -/+CTX

7 X 105 cells for SHSY5Y, MCF7 and HaCaT were gently trypsinized and pelleted by centrifugation

at 1000rpm for 5 minutes. Cells were washed with PBS briefly and re-pelleted by centrifugation

at 1000rpm for 5 minutes, the supernatant was removed carefully using a pipette. Cells were then

lysed with 35µL TE (Tris-HCL/EDTA) buffer with gentle pipetting. 5µL Enzyme A solution was added

and the samples mixed by gentle vortex, all samples were then incubated at 37°C for 10 minutes.

5µL of Enzyme B solution was added to each sample and all samples were incubated at 50°C for

30 minutes. The salt concentration was raised to precipitate nucleic acids out of solution by adding

5µL of Ammonium Acetate Solution to each sample and vortexed to mix well. 50µL of Isopropanol

was added to each sample and vortexed to mix well and all samples were kept at -20°C for 10

minutes. All samples were then centrifuged for 10 minutes to precipitate DNA . All DNA sample’s

viscosity was reduced by passing samples through a 26.5G needle via an insulin syringe to facilitate

sample loading into the gel (Hagberg et al., 2000; Catalani et al., 2013). The electrophoresis tank

was filled with 1 X TAE buffer containing 0.5µg/mL ethidium bromide. The samples were then re-

suspended in DNA suspension buffer and loaded into wells of a 1.2% agarose gel containing

0.5µg/mL ethidium bromide (Fig.4). Electrophoresis was performed at 100V for 1 hour and the

gel visualized under UV and photographed.

7 X 105 cells for SHSY5Y, MCF7 and HaCaT were suspended in 1mL of media and loaded into 3 rear

wells of a 6-well plate to generate a –ve control. 7 X 105 cells for SHSY5Y, MCF7 and HaCaT were

suspended in 1mL of media in 1mL Eppendorf tubes and 1µL of 2.5mM CTX was added to each

tube, generating a final concentration of 2.5µM CTX treatment. All treated cells were placed in

the corresponding 3 front wells of the 6-well plate (appendix 5). The 6-well plate was incubate at

37°C for 24 hours.

Cells in three separate wells were lysed using CellLytic™ according to protocol for cell lines MCF7,

HaCaT and SHSY5Y and a BCA assay was performed to determine MCF7 and SHSY5Y cell

concentration relative to HaCaT cell concentration (700,000 cells/well) by determining total

protein content in mg.

Following incubation at 37°C for 24 hours, cells in all wells were washed in PBS, then gently

trypsinized, pelleted and re-suspended in media. All cells were placed in 1.5mL Eppendorf tubes.

All subsequent steps were followed as per the apoptosis DNA ladder detection protocol -CTX. All

samples were re-suspended in DNA suspension buffer and loaded into wells of a 1.2% agarose gel

containing 0.5µg/mL ethidium bromide. Each CTX-treated sample was loaded adjacent to the

corresponding control (Fig.6). Electrophoresis was performed at 100V for 1 hour and the gel

visualized under UV and photographed.

Arie Sullivan

16

Results.

BCA assay

A set of BSA dilutions (125, 250, 500, 1000 and 2000mg/mL) were prepared for a BCA assay to

generate a standard curve which will serve throughout this investigation to compare cell

concentrations plated out for various experiments by measuring differences in protein

concentration in mg/mL.

Cell viability assay -/+CTX

Initially, optimum cell concentrations to use for cell viability assays were determined by

measuring luminescence generated from serial dilutions of each cell line. This generated standard

curves (appendix 1) which reveal the linear phase of luminescence, thus, the optimum cell

concentration to use for subsequent assays +CTX was chosen within this linear phase, namely

20,000 cells. An ATP standard curve was also generated for quality control. It can be observed

that the highest relative light unit (RLU) value for HaCaT (416462) was significantly higher than

that of MCF7 (9127) and SHSY5Y (7175).

Following the calibration to determine optimum cell concentrations, a cell viability assay was

performed for all cell lines +CTX in which 20,000 cells were plated out per well in a 96-well format

(appendix 3). The cell concentrations plated out for each cell line were compared by BCA assay in

separate wells (appendix 5). The cell concentration was compared to that of the control HaCaT

and displayed in percentage difference from HaCaT (table.1).

Cell Line Absorbance

570nm

Y = Mx + C Protein mg/mL Total

Protein

Difference %

from HaCaT

SHSY5Y 1.798 X = 2,746 2,746mg/mL 687mg 0.29% MCF7 1.901 X = 2,916 2,916mg/mL 729mg 6.04% HaCaT 1.795 X = 2,741 2,741mg/mL 685mg --

Table 1. Comparison of total protein concentration for SHSY5Y and MCF7 compared to HaCaT as determined

by BCA assay. Measured in percentage di fference from HaCaT in cel l concentrations for both cel l l ines .

Arie Sullivan

17

a)

b)

c)

Fig.8 Effect of CTX on cellular

ATP metabolism for human

keratinocyte (HaCaT), breast

cancer (MCF7), and

neuroblastoma (SHSY5Y) cell

lines, presented as percentage

of cell proliferation. a) No loss

of HaCaT cell viability following

1 hour CTX exposure for both

CTX concentrations (0.25µM

and 2.5µM). A small decrease

in cell viability can be observed

following 24 hours exposure

for both CTX concentrations b)

A similar outcome can be

observed for cell l ine MCF7,

with no loss of cell viability

following 1 hour exposure but

a small decrease following 24

hours CTX exposure c) For cell

l ine SHSY5Y, there is no loss of

cell viability following one hour

exposure to CTX. In contrast,

following 24 hours CTX

exposure, SHSY5Y has a small

decrease in cell viability at a

CTX concentration of 0.25µM

CTX and a more evident loss of

cell viability on 24 hours

exposure to 2.5µM CTX. The

percentage of cell proliferation

in a), b) and c) was normalized

to control cells (untreated).

Values are presented as means

± SD (n=3).

Arie Sullivan

18

Fluorescent microscopy -/+CTX

An initial 500,000 cells/mL dilution was prepared from which serial 2-fold dilutions were

performed and plated out in a 96-well format. This will allow to determine optimum cell

concentration for observation of apoptosis and necrosis for all cell lines under inverted

fluorescence microscopy.

A level of necrosis can be observed beyond cell concentrations of 125,000 cells/mL (appendix 6),

thus, the optimum cell concentration for observing effects of CTX was selected at 80,000 cells/mL.

It should be noted that even in low concentrations, SHSY5Y shows a level of necrosis, this should

be taken into account when considering subsequent observations of CTX effects on SHSY5Y.

The optimum cell concentration (80,000 cell/mL) was plated out at 250µL/well (20,000 cells/well),

incubated for 24 hours then treated with 0.25µM and 2.5µM CTX. The plate was subsequently

observed under inverted fluorescent microscopy following 1 and 24 hours of exposure (Fig.9) .

A level of necrosis can be observed for HaCaT post CTX treatment at a concentration of 0.25µM

for 24 hours. More evident necrosis can be perceived at a concentration of 2.5µM for both 1 and

24 hours when compared to control which appears relatively unaffected following 24 hours +CTX

incubation time.

100µm

Control 0.25µM CTX 2.5µM CTX

1 Hour

24 Hours

20 X

a) b) c)

d) e) f)

Fig.9 HaCaT under inverted fluorescence microscopy a) HaCaT control after 1 hour incubation b) HaCaT after 1 hour

exposure to 0.25µM CTX, c) HaCaT after 1 hour exposure to 2.5µM CTX, d) Control after 24 hours incubation, e) HaCaT

after 24 hours exposure to 0.25µM CTX, f) HaCaT after 24 hours exposure to 2.5µM CTX. The arrows inset indicate

apoptosis by observation of nuclei fragmentation and membrane blebbing.

100µm

Arie Sullivan

19

1 Hour

a) b

)

c)

d) e) f)

b)

a) b) c)

d) e) f)

1 Hour

Despite a minimal level of necrosis that can be observed for 24 hour exposure to CTX at a

concentration of 2.5µM, no significant effect is demonstrated. Although MCF7 cells appear to

coagulate and form islands (Fig. 10 e and f), this is not the case, the lighter blue cells are in fact

protruding from the basement cell layer. This was confirmed by the ability to observe 90-100%

confluence of the cells on altering the focus slightly.

SHSY5Y appears to be undergoing a high level of necrosis following 24 hours CTX treatment at

both 0.25µM and 2.5µM concentrations when compared to control. A low level of necrosis can

be perceived throughout but remained relatively constant for the control, whereas there is an

observable increase in necrotic cells upon treatment with CTX. The CTX treated SHSY5Y appear to

200µm

Control 0.25µM CTX 2.5µM CTX

24 Hours

Fig.11 SHSY5Y under inverted fluorescence microscopy a) SHSY5Y control after 1 hour incubation, b) SHSY5Y after

1 hour exposure to 0.25µM CTX, c) SHSY5Y after 1 hour exposure to 2.5µM CTX, d) Control after 24 hours incubation,

e) SHSY5Y after 24 hours exposure to 0.25µM CTX, f) SHSY5Y after 24 hours exposure to 2.5µM CTX.

10 X

10 X

Fig.10 MCF7 under inverted fluorescence microscopy a) MCF7 control after 1 hour incubation, b) MCF7 after 1

hour exposure to 0.25µM CTX, c) MCF7 after 1 hour exposure to 2.5µM CTX, d) Control after 24 hours incubation,

e) MCF7 after 24 hours exposure to 0.25µM CTX and f) MCF7 after 24 hours exposure to 2.5µM CTX.

Control 0.25µM CTX 2.5µM CTX

1 Hour

24 Hours

200µm

1 Hour

a)

a)

b)

b) c)

c)

d) e) f)

d) e) f)

Arie Sullivan

20

a) b) c)

d) e)

f)

f)

c)

form clusters of both live and dead cells. The distinct effect CTX on SHSY5Y when compared to

HaCaT and MCF7 cells, prompted a repetition of the experiment, and include a 48 hour incubation

time with CTX.

A low level of necrosis can be observed throughout (fig.12) as in fig.11, at time 24 hours there is

a marked clustering of live and dead cells (fig.12, b and c). At time 48 hours, a high level of necrosis

occurs for SHSY5Y treated with both concentrations of CTX (fig.12, e and f). It is total necrosis for

SHSY5Y treated with 2.5µM CTX, demonstrating strong necrotic effects from CTX when

considering the control.

Apoptosis and Necrosis +CTX

Since apoptosis and necrosis can be directly visualized under inverted fluorescent microscopy

(fig.9-12), the acquired data from microscopy can be propagated to perform quantitative analyses

on necrotic and apoptotic cells in a given population. Three images of each cell line were taken at

time 1 hour and 24 hours post CTX treatment, all cells were counted and mean values were

generated from all three images then converted into percentage values, these were subsequently

plotted in column charts (fig. 14-16). Despite some apoptosis (fig.13) observable for HaCaT, this

occurred as an exception rather than a rule, with limited amounts of apoptosis taking place.

Control 0.25µM CTX 2.5µM CTX

24 Hours

48 Hours

Fig.12 SHSY5Y under fluorescence microscopy for 24 hours and 48 hours a) SHSY5Y control after 24 hours

incubation, b) SHSY5Y after 24 hours exposure to 0.25µM CTX, c) SHSY5Y after 24 hours exposure to 2.5µM CTX, d)

Control after 48 hours incubation, e) SHSY5Y after 48 hours exposure to 0.25µM CTX and f) SHSY5Y after 48 hours

exposure to 2.5µM CTX.

10 X

200µm a) b) c)

d) e) f)

Arie Sullivan

21

a) b)

Initially, a D’Agostino & Pearson Normality test was performed for each cell line at each time point

to confirm response variable residuals are normally distributed. A statistical analysis should

determine any significant effect from CTX concentration on cell type, thus the use of a one-way

analysis of variance (one-way ANOVA) statistical test is appropriate.

A D’Agostino & Pearson statistical test confirmed Gaussian distribution of the data from HaCaT

(appendix 7 a, and b). A one-way analysis of variance (one-way ANOVA) was performed for 1 hour

and 24 hours CTX exposure (appendix 8, tables a and b).

The one-way ANOVA revealed no significant effect from CTX concentration on cell type

(live/apoptotic/necrotic) (P value = 0.5572) following 1 hour exposure to CTX. For 24 hours CTX

exposure, there is no significant effect from CTX concentration on cell type (P value = 0.2761). The

statistical analysis therefore suggests there is no significant effect from CTX on HaCaT.

Fig.14 Live, apoptotic and necrotic cells as a percentage of cell population for HaCaT at a) time 1 hour (0.25µM and 2.5µM)

and b) time 24 hours (0.25µM and 2.5µM). For each condition (Control, 0.25µM and 2.5µM), results are presented as

percentage of viable, apoptotic and necrotic cells. Values are presented as means ± SD (n=3)

a) b)

Fig.13 HaCaT apoptosis under

20X inverted fluorescent

microscopy. Arrows indicate

nuclei fragmentation and

membrane blebbing. The inset

represents a 40X close up of a

fragmented nucleus.

Arie Sullivan

22

a) b)

A D’Agostino & Pearson statistical test confirmed Gaussian distribution of the data from MCF7

(appendix 7 c, and d). A one-way ANOVA statistical test was performed (appendix 8, tables c and

d). One-way ANOVA revealed no significant effect from CTX concentration on cell type after 1

hour exposure (P value = 0.0788), No significant effect from CTX concentration on cell type

following 24 hours exposure (P value = 0.8516) therefore it can be assumed that CTX does not

significantly affect MCF7 cells.

A D’Agostino & Pearson statistical test confirmed Gaussian distribution of the data from SHSY5Y

(appendix 7 e, and f). A two-way ANOVA statistical test was performed (appendix 8, table e and

f).

Again, there is no significant effect from CTX concentration on cell type following one hour

exposure (P value = 0.0993). The one-way ANOVA for SHSY5Y following 24 hours of exposure to

Fig.15 Live, apoptotic and necrotic cells as a percentage of cell population for MCF7 at a) time 1 hour (0.25µM and

2.5µM) and b) time 24 hours (0.25µM and 2.5µM). For each condition (Control, 0.25µM and 2.5µM), results are presented as percentage of viable, apoptotic and necrotic cells. Values are presented as means ± SD (n=3)

Fig.16 Live, apoptotic and necrotic cells as a percentage of cell population for SHSY5Y at a) time 1 hour (0.25µM

and 2.5µM) and b) time 24 hours (0.25µM and 2.5µM). For each condition (Control, 0.25µM and 2.5µM), results are

presented as percentage of viable, apoptotic and necrotic cells. Values are presented as means ± SD (n=3)

a) b)

a) b)

Arie Sullivan

23

CTX suggests a significant effect from CTX concentration on cell type (P value = 0.0010), thus

suggesting that CTX affects levels of apoptosis/necrosis in NB cell line SHSY5Y.

A D’Agostino & Pearson statistical test confirmed Gaussian distribution of the data from SHSY5Y

(appendix 7 g and h). A one-way ANOVA statistical test was performed for SHSY5Y following 48

hours CTX exposure (appendix 8, table g), and suggests a significant effect from CTX on SHSY5Y

(P value = < 0.0001).

DNA fragmentation detection -/+CTX

Genomic DNA was extracted from all cell lines and run on a 1.2% agarose gel containing 0.5µg/mL

ethidium bromide for 1 hour. Genomic DNA samples proved difficult to manipulate with pipettes,

the high viscosity owing to un-sheared long genomic DNA strands in the cell lysate (Boynton et

al., 1999; Yukl et al., 2014). The result of loading high viscosity samples into the wells of the

agarose gel can be observed (appendix 9 a), the samples have aggregated in the wells and despite

strong signals, the definition of the bands is poor. The reason for no signal on SHSY5Y (appendix

9 a) is unknown. In an effort to reduce the viscosity, the experiment was repeated using a 2-fold

dilution of cells, producing a stained gel that revealed tighter DNA bands for the sample, however,

the genomic DNA bands are faint and difficult to distinguish (appendix 9 b).

Viscosity of genomic DNA can be reduced mechanically, by enzyme digestion or sonication.

Sonication however, produces DNA fragmentation to a size of 300-500bp (Sambrook and Russel,

2006) whilst enzyme digestion such as endonuclease DNA digestion can produce randomly sized

DNA fragments (Miyazaki, 2002). Therefore both sonication and enzyme digestion of genomic

a) b)

Fig.17 Live, apoptotic and necrotic cells as a percentage of cell population for SHSY5Y at a) time 24 hours (0.25µM

and 2.5µM) and b) time 48 hours (0.25µM and 2.5µM). For each condition (Control, 0.25µM and 2.5µM), results are

presented as percentage of viable, apoptotic and necrotic cells. Values are presented as means ± SD (n=3)

Arie Sullivan

24

DNA are inappropriate for use in sample preparation when detecting apoptotic DNA

fragmentation. Thus, a reduction in viscosity was attempted mechanically, by passing samples

through a 26.5G needle via insulin syringe as outlined in methods. Passing Genomic DNA through

a 26.5G needle mechanically shears very long DNA strands, reducing viscosity and easing

manipulation of the samples. However, to maintain a distinction between apoptotic DNA

fragmentation and mechanical shearing, a single pass is recommended so as to avoid a false-

positive result. (Hagberg et al., 2000).

Despite some DNA fragmentation occurring, the technique revealed better defined genomic DNA

bands (appendix 10) and thus optimized the technique to differentiate genomic DNA from DNA

fragmentation.

Cells in three separate wells were lysed using CellLytic™ according to protocol for cell lines MCF7,

HaCaT and SHSY5Y and a BCA assay was performed to determine MCF7 and SHSY5Y cell

concentration relative to HaCaT cell concentration (700,000 cells/well) by determining total

protein content in mg (appendix 10).

All cell lines were treated with 2.5µM CTX and incubated for 24 hours, DNA was extracted

according to methods and run on a 1.2% agarose gel containing 0.5µg/mL ethidium bromide for

1 hour. The gel was subsequently visualized under UV (fig.15).

Genomic DNA bands produce a strong signal for MCF7 control and MCF7; SHSY5Y control and

SHSY5Y. A weaker signal was generated for HaCaT and no signal for HaCaT control, the reason for

this is unknown. The genomic bands are localized just below the well as in the case of genomic

DNA extraction. Although some DNA fragmentation occurs, fragments characteristic of apoptotic

DNA fragmentation are not apparent. Some smearing of DNA fragments can be observed for

MCF7 control and MCF7, but as in the case of SHSY5Y control and SHSY5Y and HaCaT control and

HaCaT, the fragments appear identical to those generated in the genomic DNA extraction. Thus,

it appears that CTX does not induce apoptotic DNA fragmentation in these cell lines.

Cell Line Absorbance 570nm

Y = Mx + C Protein mg/mL Total Protein

Difference % from HaCaT

SHSY5Y 1.132 X = 1,636 1,636g/mL 409mg 11.09% MCF7 1.235 X = 1,808 1,808mg/mL 452mg 1.74%

HaCaT 1.254 X = 1,839 1,839mg/mL 460mg --

Table 2. Comparison of total protein concentration for SHSY5Y and MCF7 compared to HaCaT as determined

by BCA assay. Measured in percentage di fference from HaCaT in cel l concentrations for both cel l l ines .

Arie Sullivan

25

Discussion

Despite CTX’s highly specific targeting capacity for migratory tumor cells, potential binding to

tumors of neuroectodermal origin remains to be investigated. Different methods have been

applied in exploiting the specificity of CTX binding, including induced expression of potential CTX

target receptors in alternate tumor cell lines; targeting CTX to define tumor margins facilitating

surgical removal of tumor mass; bioconjugation of CTX to therapeutic compounds; and

endocytosis of liposomes encapsulating modified oligonucleotides or siRNAs. The majority of the

research in characterizing these alternative methods for CTX use however, has been focused on

glioma.

In this work, the tumor-targeting capacity of CTX to a cell line of neuroectodermal origin, namely,

NB cell line SHSY5Y was investigated. Additionally, CTX’s biological activity on migratory cell line

HaCaT and non-migratory cancer cell line MCF7 was also evaluated. In accordance with previously

Fig.15 DNA extraction from 7 X 105 cells for MCF7, SHSY5Y, HaCaT, 100bp and BSTEII MW markers. All

DNA was passed through a 26.5G needle for ease of manipulation. DNA fragments can also be observed

from mechanical shearing. There is no observable difference between genomic DNA extraction (control)

and CTX treated samples.

Arie Sullivan

26

reported studies (Wang and Ji, 2005; Fu et al., 2007), the induction of apoptosis following

exposure to CTX delineates a mechanism of action for CTX. The suggested mechanism of action

was herein assessed to determine if it remains consistent across alternate migratory and non-

migratory cell lines.

Three experiments were designed to investigate CTX’s capacity for apoptosis induction, namely,

cell viability, fluorescent microscopy, and DNA fragmentation assays. Taken together, the results

from these experiments provide a reliable platform on which to assess the hypothesis that CTX

induces apoptosis in tumor cell lines of neuroectodermal origin.

Cell viability assay

Since intracellular ATP levels are exquisitely regulated, a correlation between the presence of ATP

and number of viable cells can be established. The homogenous automated high-throughput

screening (HTS) method is based on a ‘glow type’ signal produced at 560nm in the generation of

Oxyluciferin, AMP, PPi and CO2 from d-luciferin, O2 and ATP. The luciferase reaction can be used

directly to quantify the number of metabolically active cells in culture. The effects of CTX on cell

viability was measured by decrease/increase in signal strength, directly reflecting the amount of

ATP present and thus, viable cells. In the calibration of the cell viability assay to determine

optimum cell concentration for subsequent assays, the cancer cell lines MCF7 and SHSY5Y

(appendix a, and b) produced significantly lower signals (9127 RLU and 7175 RLU, respectively)

than the non-cancerous HaCaT cell line (416462 RLU) (appendix 1, c). This can be attributed to

‘the Warbug effect’, whereby the primary source of ATP in cancer cells is switched from

mitochondrial oxidative phosphorylation to aerobic glycolysis (Amoedo et al., 2013). Despite the

inefficiency of aerobic glycolysis in generating ATP, particular cancer-associated mutations allow

cancer cells to metabolize nutrients in a manner more conducive to proliferation than efficient

ATP production (Van der Heiden et al., 2009). Since ATP levels were measured for two cancerous

cell lines, the loss in sensitivity of the assay owing to ‘the Warbug effect’ should be considered.

Particularly, the mechanism favoring cell proliferation over ATP metabolism can lead to the

generation of misleading results if ATP levels are no longer proportional to the number of viable

cells.

Despite this shortcoming, it is possible to assess changes in cell proliferation proportional to loss

or gains in luminescence signal for each cell line independently, converting changes in

luminescence signal into percentage loss or gain to enable comparison. MCF7 and HaCaT did not

show any significant decrease in cell viability following 1 and 24 hours incubation with CTX

(0.25µM and 2.5µM) (fig.8 a, and b). SHSY5Y showed a slight decrease in cell viability following 24

hours exposure to 0.25µM CTX and a moderate decrease following 24 hours exposure to 2.5µM

CTX (fig.8 c), indicating a dose-dependent effect of CTX on SHSY5Y. However, a 50% inhibitory

concentration (IC50) was not reached by either CTX concentration on either cell lines, a contrast

to the IC50 of approximately 0.28µM for BmKCT reported on glioma cell line SHG-44 (Fu et al.,

2007). Comparable studies on scorpion venom component III (SVCIII) revealed cell viability IC 50’s

of 0.39µM and 0.53µM for SVCIII on human leukemia cell lines THP-1 and Jurkat respectively (Song

Arie Sullivan

27

et al., 2012). Taken together, the inhibitory concentrations of these scorpion venoms on cancer

cell lines suggests that either CTX has a lower binding affinity for cancer cells than alternate

scorpion venoms; that the target receptor is not expressed or expression is significantly reduced

in the cell lines used herein; or that CTX possesses a mechanism of action other than inhibition of

cell proliferation. Non-the-less, the data from figure 8-c indicates that CTX does impact SHSY5Y

cell viability when compared to control.

Fluorescent Microscopy

The effect of CTX on all cell lines was further assessed by visualizing live cells stained with Hoechst

33342 and dead cells counterstained with PI, 1 and 24 hours post-CTX treatment (fig.9-12).

Visualizing under inverted fluorescent microscopy provides the ability to directly observe the

effects of CTX on cells, thus generating data of a more sensitive nature. All data for each cell line

and each time interval was statistically analyzed by one-way ANOVA to generate P values

reflecting the probability of a significant effect of CTX concentration on cell type.

The data shown in figure 9 and 14 disclose the effect of CTX on HaCaT with little to no necrosis

observed following 1 hour exposure for both 0.25µM and 2.5µM CTX (P value = 0.5572). Following

24 hours exposure, a minor level of necrosis can be observed at a concentration of 0.25µM and a

small level of necrosis at a concentration of 2.5µM (P value = 0.2761). By one-way ANOVA analysis,

there is no significant effect of CTX on HaCaT.

Figure 10 and 15 disclose CTX effect on MCF7 with no significant apoptosis/necrosis throughout

all CTX treatments after 1 hour (P value = 0.0788). Following 24 hours exposure, the one-ANOVA

analysis suggests an interaction between CTX concentration and cell type (P value = 0.8516). One-

way ANOVA reveals no significant effect of CTX on MCF7.

Finally, the effects of CTX concentration on SHSY5Y (fig.11, 12, 16 and 17) following 1 hour

exposure disclose no effect from CTX (P value = 0.0993), suggesting that CTX has no significant

effect on apoptosis/necrosis after 1 hour exposure to CTX. After 24 hours CTX exposure however,

a significant effect from CTX on cell type can be observed (P value = 0.0010). The results for

SHSY5Y prompted an additional test using 0.25µM and 2.5µM CTX treatment for 24 and 48 hours

exposure. In confluence with previous experiments, a high level of necrosis can be observed for

0.25µM CTX, and total necrosis for 2.5µM CTX following 48 hours exposure (P value = < 0.0001).

The column charts generated for SHSY5Y reflect the one-way ANOVA test by an observable

increase in necrotic cells for SHSY5Y following 24 hour exposure to 0.25µM CTX and a marked

increase in both necrotic and apoptotic cells for SHSY5Y following 24 hour exposure to 2.5µM CTX.

It is worth noting the clustering of cells following CTX treatment for SHSY5Y when considered

alongside the control (fig.11 d, e and f). These clusters of inconsistent size and shape have been

observed with SHSY5Y when grown onto nanorough substrates (Brunetti et al., 2010). Similar

clustering has been observed on treating SHSY5Y with between 12.5 and 50mg/mL guarana

(Zeidan-Chulia et al., 2013). Despite the aggregation of SHSY5Y cells being previously attributed

Arie Sullivan

28

to the formation of neurospheres (Moors et al., 2009), this is not likely the case since these are

known to form during growth and are not present within the control group herein. Additionally,

there was no cell aggregation on observation of the cells following 24 and 48 hours CTX exposure,

indicating the previous clustering could have been an anomaly rather than an organized

formation.

Since CTX has shown to be significant in determining necrosis levels in SHSY5Y, and not HaCaT,

the role of MMP-2 alone as the target receptor for CTX can be brought into question since both

these cell lines express MMP-2. As no effect was observed for HaCaT, it can be deduced that it is

not likely that CTX acts alone on MMP-2 but rather, other receptors are implicated.

Despite CTX affecting SHSY5Y cell viability, investigation into apoptogenic capacity of CTX on

SHSY5Y revealed little to no apoptosis, rather, the different approaches into determining the

mechanism of action of CTX revealed a significantly higher capacity to induce necrosis than

apoptosis.

DNA fragmentation

To further test CTX apoptosis induction, a test for DNA fragmentation was performed on all cell

lines following 24 hour 2.5µM CTX exposure (fig. 15) which generated genomic DNA bands but no

apoptotic DNA fragmentation despite a number of repetitions of the experiment under different

conditions. Without the presence of DNA fragmentation, apoptosis is not likely implicated in the

mechanism of action of CTX on SHSY5Y.

Following reports of apoptosis induction on glioma cell line SHG-44 and breast cancer cell line

MCF7 by recombinant Buthus martensii Karsch chlorotoxin (BmKCT) (Fu et al., 2007; Li et al.,

2014), the mechanism of action of Leiurus quinqestriatus chlorotoxin (CTX) was speculated to also

implicate apoptosis, this was not the case. Despite NB having a neuroectodermal origin and

reports claiming 8 positive results of 9 tested for CTX tissue staining (Dardevet et al., 2015), the

report herein indicates CTX does not induce apoptosis, but rather, necrosis. This prompts a need

for further investigation into the differences in components and structure betwee n the two

molecules that result in the ability of one form to induce apoptosis and the other to induce

necrosis. From the amino acid sequence of BmKCT, it is possible to generate its molecular

structure (fig.16) for comparison with CTX.

Arie Sullivan

29

Fig.16 Molecular structures for

BmKCT and CTX a) Molecular

structure and amino acid sequence

of CTX including all four di-sulfide

bonds, containing a total 36 amino

acid residues (Chemblink database)

b) Molecular structure of

recombinant BmKCT including all

four di-sulfide bonds, containing a

total of 35 amino acids. All differing

or additional amino acids are

presented in blue in both molecular

structures and amino acid

sequences. All amino acids

presented in black are of the same

structure for both BmKCT and CTX.

Glycine residues at the C-terminal of

BmKCT are presented in red.

Altogether, a total of 9 amino

acids are substituted between

the two molecules, with

additional glycine residues

present at the C-terminal of

BmKCT. Interestingly, the glycine

residues at the C-terminal of

BmKCT are thought to play an

important role in analgesic

activity of the peptide (Zhang et

al., 2010; Zhao et al., 2013), thus

potentially causing BmKCT and

derivatives to possess altered

mechanisms of action. For

example in the capacity of BmKCT

to induce apoptosis, and CTX to

induce necrosis despite both

molecules possessing a βαββ

conformation and the same di-

sulfide bonds between cysteine

residues. Both molecules have

been reported to inhibit glioma

tumor growth by as of yet,

undefined mechanisms.

a)

b)

Arie Sullivan

30

Glycine receptor (GlyR) Cl- channels belong to a family of ligand-gated ion channel receptors best

known for mediating inhibitory neurotransmission in motor and sensory reflex circuits of the

spinal cord (fig.17) (Webb and Lynch, 2007). Disruption of GlyR expression causes reduced ability

to conduct chloride ions resulting in neurological disorder, hyperekplexia (Andrew and Owen,

1997; Xiong et al., 2014). Flatteringly, ion channels mediating neurological signaling are frequently

the target for potent venoms used to paralyze prey (Cannon, 2006).

Studies have demonstrated that glioma cell-GlyRs do not serve as typical neurotransmitter

receptors, with knockdown of GlyR α1 subunit expression resulting in impaired tumorigenicity

(Forstera et al., 2014). Another report suggests GlyRs could have an influence on radial migration

during late embryonic development (Nimmervoll et al., 2011). Moreover, application of glycine

was shown to impede radial migration in neuronal and non-neuronal cells (Avila et al., 2013;

Dender et al., 2010; Van den Eynden et al., 2009), suggesting GlyR activation causes cell migration

arrest. Since GlyRs are widely distributed throughout the whole cortex, it is thought that they

provide significant contributions in controlling cell migration. Studies on rodent models report

glycine-induced inhibition of cell proliferation, migration and tumor growth by 5% (Rosa et al.,

1999). GlyR activation causing downstream effects on cell migration potentially implicates GlyR in

the invasive capacity of cells, outlining a potential mode of action for BmKCT’s additional C-

terminal glycine residue. However, to date, there are no reports of GlyR channel inhibitors such

as strychnine or choline having the capacity to circumvent glycine-induced migration inhibition.

The presence of such GlyR antagonists would be expected to blunt the effects of glycine and

downstream processes.

Glycine also triggers calcium influx by activating GlyRs and glycine transporters (GlyTs),

depolarizing the plasma membrane. Calcium influx has the potential to trigger apoptosis through

plasma membrane channels (fig.18). Specifically, calcium influx into the mitochondrion induces

permeability transition in the membrane of an adjacent mitochondrion, forming a chain reaction

leading to a significant elevation in cytochrome-c levels initiating downstream caspases and

formation of the apoptosome (Mattson and Chan, 2003). Thus, it could be suggested that the

additional glycine residues at the C-terminal of BmKCT play a pivotal role in the apoptogenic

capacity of BmKCT, perhaps via GlyR Cl - channel activation.

Determining the function of additional glycine residues at the C-terminal of BmKCT may assist in

deciphering amino acid sequence/molecular structure relationship and could be use d to predict

the mechanism of action of related venom components since these post-translationally modified

peptides most often share similar sequence consensus, matching cysteine residues, di -sulfide

bonds and thus, structural conformation (Arzamasov et al., 2014).

Arie Sullivan

31

Fig.17 Glycine signaling in macroglial cells. Upon ligand binding, GlyR activation causes chloride efflux leading to cellular

depolarization. The depolarization causes activation of VGCC resulting in calcium influx inducing numerous downstream

effects (cell proliferation, migration and differentiation). Inactivation of the Gl yR may be caused by endocytosis of the

receptor by as of yet unknown mechanisms. Image acquired from Van den Eynden et al., 2009)

Fig.18 The role of GlyR, calcium and cytochrome-c as inter-organellar messengers in apoptosis. a) Upon ligand binding,

GlyR activation causes chloride efflux leading to cellular depolarization. Depolarization causes activation of VGCC

resulting in calcium influx which induces release of cytochrome-c, b) cytochrome-c then diffuses to adjacent

endoplasmic reticulum and binds IP3R receptors c) enhancing calcium release from the endoplasmic reticulum, d)

released calcium causes overall increase in cytoplasmic calcium concentration, e) resulting in calcium uptake by

mitochondria throughout the cell that triggers release of cytochrome-c from all mitochondria. f) cytochrome-c induces

formation of the apoptosome in which caspases are activated, g) caspases and nuclease are the final step in the

apoptotic process, cleaving protein substrates and DNA respectively. Image adapted from Mattson and Chan, 2003

Arie Sullivan

32

Despite indications of different mechanism of action for CTX and BmKCT, studies investigating

glioma tumor growth inhibition report near identical inhibition rates (fig.19) in female SD rats

bearing allografted tumors by subcutaneous injection of C6 glioma cell suspension (Fan et al.,

2010).

Although the study demonstrates GST-CTX and GST-BmKCT have identical tumor growth

inhibition rates, the exact mechanism by which inhibition is achieved remains to be conclusive.

The data drawn from the report herein suggests necrosis induction for CTX whilst other studies

indicate apoptosis induction for BmKCT, both necrosis and apoptosis having the capacity for

tumor growth inhibition.

The internalization of membrane expressed receptors has also been recognized as a potential

mechanism in halting metastasis of migrating tumor cells but only limited research has focused

on the implicated mechanism of action. A number of nanoparticles have been demonstrated to

internalize successfully when bioconjugated with CTX (Stroud et al., 2012; Akcan et al., 2011;

Cheng et al., 2014). Specifically, monomeric and dimeric forms of CTX were demonstrated to

induce internalization in glioma A172 cells, halting migratory capacity (Kasai et al., 2012). MT1-

MMP has been shown to internalize from cell surface by clathrin-mediated and independent

pathways involving caveolae in HT1080 fibrosarcoma cells, downregulating invasive capacity

(Remacle et al., 2003). Moreover, studies investigating mutations affecting MT1 internalization,

found a subsequent disruption of invasion-promoting activity whereas those mutations not

affecting internalization promoted invasion (Uekita et al., 2001). Since CTX has been reported to

bind to membrane receptors Cl - channels, MMP-2 and annexin A2 leading to internalization of the

complex, these findings suggest a strong association with endocytosis of specific membrane

receptors and subsequent inhibition of migratory capacity of malignant invasive cells.

Fig.19 Tumor growth inhibitory effect of GST-CTX and GST-BmKCT a) Results from statistical analysis of average tumor

weight among groups, with NS and GST treatment as controls. Despite a significant inhibition of tumor growth

demonstrated when compared to control (**), there was no significant difference between GST-CTX and GST-BmKCT

inhibition rates b) Data of tumor weight and inhibition rate between GST-CTX and GST-BmK demonstrated significant

inhibition of tumor growth for both GST-CTX and GST-BmKCT. Results acquired from Fan et al., 2010.

Abbreviations: NS, Normal Saline; GST, Glutathione

transferase; CTX, Leiurus Quinqestriatus chlorotoxin; BmKCT,

Buthus martensii Karsch chlorotoxin.

a) b)

Arie Sullivan

33

Further investigation

Despite increasing reports narrowing the search for potential CTX target receptors, a number of

possibilities remain feasible for the action mechanism of CTX. It is possible to characterize the CTX

target receptor by western blot. Western blot analysis will enable to probe for the target receptor

using an iodinated form of CTX by substitution of tyrosine for iodine at residue 29. The tyrosine

residue at position 29 has been demonstrated as not critical for the function of CTX by intact

activity following tyr29 iodination (Dardevet et al., 2015). The labeling of CTX and binding to protein

of interest on the nitrocellulose membrane should allow to visualize and subsequently determine

the molecular weight of the protein of interest. Alternately circumventing the iodination step,

antibodies can be raised against bound CTX on the nitrocellulose membrane and visualized by

labelled secondary antibody to primary antibody.

However, both CLC-2 and GlyR as target Cl- channels for CTX are indistinguishable by western blot

owing to similar molecular weights of 97kDa and 106kDa (Britton et al., 2000). Internalization of

GlyR causes ubiquitin molecules to induce proteolytic cleavage of the GlyR α1-subunit into a

glycosylated 35kDa N-terminal fragment and a 17kDA COOH-terminal fragment (Lynch, 2004).

This allows for western blot analysis to be used in combination with CTX-bioconjugation mediated

endocytosis to determine which of the two receptors is the true CTX target, as well as ascertaining

whether endocytosis of target membrane proteins is occurring.

CTX-bioconjugated particles showing successful internalization followed by western blot analysis

revealing bands at 35kDa and 17kDa which would indicate receptor-mediated endocytosis of CTX-

target membrane protein GlyR. CTX-bioconjugated particles showing successful internalization

and western blot analysis revealing bands at approximately 100kDa would indicate receptor-

mediated endocytosis of CTX-target membrane protein ClC-2. Finally, CTX-bioconjugated particles

showing failed internalization and western blot analysis revealing bands at approximately 100kDa

would suggest either GlyR or ClC-2 as target membrane proteins and no receptor-mediated

endocytosis.

A further hypothesis to investigate is the ability of CTX as a Cl - channel blocker to prevent

apoptosis. This could be achieved using glioma cell line SHG-44, replicating the conditions under

which apoptosis was observed on BmKCT treatment. The experiment could be repeated in the

presence of CTX, with a speculation that CTX will inhibit apoptosis by Cl - channel inhibition. This

will assist in determining whether CTX is preventing apoptosis.

Recent advances have been made in categorizing and organizing data regarding scorpion toxins.

The construction of molecular databases for scorpion toxins (Srinivasan et al., 2002) forms an

integral part in allowing confluent research to adjoin. Such collaborative databases offer

promising future prospects in deciphering the therapeutic value these numerous compounds

possess.

Arie Sullivan

34

Appendix

1) Standard curves demonstrating the linear phase for a) MCF7, b) SHSY5Y and c) HaCaT to

determine cell concentration for optimum detection of luminescence variance (20,000

cells) d) ATP standard curve for QC.

R² = 0.9992

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

0 10000 20000 30000 40000 50000 60000

Lum

ines

cen

ce (

RLU

)

Cells/well

HaCat

a) b)

)

c)

R² = 1

0

100

200

300

400

500

600

0 0.5 1

RLU

Concentration (µg/mL)

ATP Standard d)

Arie Sullivan

35

2) Preparation of two CTX concentrations from 10mg/mL stock

1 Mole = 3995g/L

10mg/mL = 10g/L

10 / 3995 = 2.5 X 10-3 M

X 1000 = 2.5mM

a) 10µL (2.5mM) was diluted in 90µL de-ionized water to produce a 0.25mM.

b) 2.5µL (0.25mM) diluted in 250µL media produces a 2.5µM CTX concentration.

c) 10µL (0.25mM) was diluted in 90µL de-ionized water to produce a 0.025mM.

d) 2.5µL (0.025mM) diluted im 250µL media produces a 0.25µM CTX concentration.

3) 96-well plate set up for cell viability assay +CTX.

Arie Sullivan

36

4) 6-well plate set up, row A) MCF7, SHSY5Y and HaCaT plated out at 7 X 10 5 cells. Row B) MCF7,

SHSY5Y and HaCaT plated out at 7 X 10 5 cells with 2.5µM CTX treatment.

5) a) Absorbances at 570nm for both cell lines MCF7 (1.901) and SHSY5Y (1.798) determine

protein concentration in mg/mL by BCA assay for cell viability assay. Values from the

multiscan are incorporated into the BCA standard curve to determine difference in

protein concentration in mg/mL.

Arie Sullivan

37

b) Absorbances at 570nm from the multiscan are incorporated into the BCA standard curve

to determine difference in protein concentration in mg/mL for SHSY5Y ( ), MCF7 ( ) and

HaCaT ( )

6) Optimization of cell concentration for fluorescent microscopy a) Cell concentration/mL for

SHS Y5Y, b) Cell concentration/mL for MCF7 c) Cell concentration/mL for HaCaT. The optimum cell

concentration was selected at 80,000cells/mL.

y = 0.0006x + 0.1502

R² = 0.9956

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 500 1000 1500 2000 2500 3000

Ab

sro

ba

nce

57

0n

M

Concentration µg/mL

BCA Assay

Absorbance Mean

SHSY5Y

MCF7

HaCaT

Linear (Absorbance Mean)

b) MCF7

10X

a) SHSY5Y

10X

c) HaCaT

10X

200µm

200µm

200µm

31,250 cells/mL 62,500 cells/mL 125,000 cells/mL 250,000 cells/mL

Arie Sullivan

38

7 a) D’Agostino & Pearson test to determine Gaussian distribution of the data for HaCaT at

time 1 hour.

b) D’Agostino & Pearson test to determine Gaussian distribution of the data for HaCaT at time

24 hours.

D’Agostino & Pearson test Live cells Apoptotic cells Necrotic cells

Number of values 9 9 9 Minimum 75.59 0.0 0.9100 25% Percentile 85.61 1.355 2.805 Median 93.33 2.990 4.420 75% Percentile 95.06 3.945 10.43 Maximum 95.45 4.950 24.41 Mean 90.24 2.668 7.094 Std. Deviation 6.704 1.631 7.343 Std. Error of Mean 2.235 0.5436 2.448 Lower 95% Cl of mean 85.08 1.414 1.450 Upper 95% Cl of mean 95.39 3.921 12.74 D’Agnostino & Pearson test K2 6.336 0.6282 11.62 P-value 0.0421 0.7304 0.0030 Passed normality test (alpha=0.05) No Yes No P-value summary * ns * Sum 812.1 24.01 63.85

D’Agostino & Pearson test Live cells Apoptotic cells Necrotic cells

Number of values 9 9 9 Minimum 89.80 0.0 0.0 25% Percentile 91.97 0.4100 1.055 Median 94.11 1.690 2.080 75% Percentile 97.72 3.925 4.815 Maximum 98.85 5.620 10.20 Mean 94.75 2.160 3.086 Std. Deviation 3.188 1.940 3.386 Std. Error of Mean 1.063 0.6467 1.129 Lower 95% Cl of mean 92.30 0.6687 0.4830 Upper 95% Cl of mean 97.20 3.651 5.688 D’Agnostino & Pearson test

K2 0.9373 0.9200 6.247 P-value 0.5539 0.3925 0.0440 Passed normality test (alpha=0.05)

Yes Yes No

P-value summary Ns Ns * Sum 852.8 19.44 27.77

Arie Sullivan

39

c) D’Agostino & Pearson test to determine Gaussian distribution of the data for MCF7 at time 1

hour.

d) D’Agostino & Pearson test to determine Gaussian distribution of the data for MCF7 at time 24

hours.

D’Agnostino & Pearson test Live cells Apoptotic cells Necrotic cells

Number of values 9 9 9 Minimum 73.91 0.0 0.0 25% Percentile 82.07 1.695 0.6350 Median 89.08 10.05 1.340 75% Percentile 96.08 17.26 1.915 Maximum 97.95 25.69 2.670 Mean 88.53 10.15 1.322 Std. Deviation 8.338 9.081 0.8240 Std. Error of Mean 2.779 3.027 0.2747 Lower 95% Cl of mean 82.12 3.165 0.6889 Upper 95% Cl of mean 94.94 17.13 1.956 D’Agnostino & Pearson test K2 1.310 1.198 0.01015 P-value 0.5196 0.5495 0.9949 Passed normality test (alpha=0.05) Yes Yes Yes P-value summary ns ns ns Sum 796.8 91.31 11.90

D’Agostino & Pearson test Live cells Apoptotic cells Necrotic cells

Number of values 9 9 9 Minimum 79.93 0.0 0.0 25% Percentile 91.06 1.485 0.0 Median 93.28 3.460 0.8600 75% Percentile 98.27 7.000 3.455 Maximum 100.0 19.75 4.440 Mean 93.17 5.333 1.494 Std. Deviation 6.022 5.996 1.804 Std. Error of Mean 2.007 1.999 0.6013 Lower 95% Cl of mean 88.54 0.7246 0.1078 Upper 95% Cl of mean 97.80 9.942 2.881 D’Agnostino & Pearson test K2 5.651 13.01 2.134 P-value 0.0593 0.0015 0.3441 Passed normality test (alpha=0.05) Yes No Yes P-value summary Ns * Ns Sum 838.6 48.00 13.45

D’Agostino & Pearson test Live cells Apoptotic cells Necrotic cells

Number of values 9 9 9 Minimum 73.91 0.0 0.0 25% Percentile 82.07 1.695 0.6350 Median 89.08 10.05 1.340 75% Percentile 96.08 17.26 1.915 Maximum 97.95 25.69 2.670 Mean 88.53 10.15 1.322 Std. Deviation 8.338 9.081 0.8240 Std. Error of Mean 2.779 3.027 0.2747 Lower 95% Cl of mean 82.12 3.165 0.6889 Upper 95% Cl of mean 94.94 17.13 1.956 D’Agnostino & Pearson test K2 1.310 1.198 0.01015 P-value 0.5196 0.5495 0.9949 Passed normality test (alpha=0.05) Yes Yes Yes P-value summary ns ns ns Sum 796.8 91.31 11.90

Arie Sullivan

40

e) D’Agostino & Pearson test to determine Gaussian distribution of the data for SHSY5Y at time

1 hour

f) D’Agostino & Pearson test to determine Gaussian distribution of the data for SHSY5Y at time

24 hours

D’Agostino & Pearson test Live cells Apoptotic cells Necrotic cells

Number of values 9 9 9 Minimum 78.65 0.0 3.950 25% Percentile 83.32 0.3950 5.955 Median 91.16 0.8800 6.610 75% Percentile 93.16 10.12 7.740 Maximum 96.05 14.61 9.220 Mean 88.58 4.700 6.723 Std. Deviation 5.936 5.572 1.481 Std. Error of Mean 1.979 1.857 0.4935 Lower 95% Cl of mean 84.01 0.4172 5.585 Upper 95% Cl of mean 93.14 8.983 7.861 D’Agnostino & Pearson test K2 1.262 1.832 0.9792 P-value 0.5319 0.4000 0.6129 Passed normality test (alpha=0.05) Yes Yes Yes P-value summary ns ns ns Sum 797.2 42.30 60.51

D’Agostino & Pearson test Live cells Apoptotic cells Necrotic cells

Number of values 9 9 9 Minimum 30.44 0.0 6.610 25% Percentile 34.85 0.0 14.33 Median 67.81 9.700 24.16 75% Percentile 75.42 43.23 28.15 Maximum 81.27 50.30 45.71 Mean 58.45 18.08 23.47 Std. Deviation 20.27 21.23 11.22 Std. Error of Mean 6.757 7.076 3.739 Lower 95% Cl of mean 42.87 1.767 14.84 Upper 95% Cl of mean 74.03 34.40 32.09 D’Agnostino & Pearson test K2 3.256 2.786 1.576 P-value 0.1963 0.2483 0.4549 Passed normality test (alpha=0.05) Yes Yes Yes P-value summary ns ns ns Sum 526.1 162.8 211.2

Arie Sullivan

41

g) D’Agostino & Pearson test to determine Gaussian distribution of the data for SHSY5Y at time

24 hours

h) D’Agostino & Pearson test to determine Gaussian distribution of the data for SHSY5Y at time

48 hours

D’Agostino & Pearson test Live cells Apoptotic cells Necrotic cells

Number of values 9 9 9 Minimum 70.17 0.0 1.040 25% Percentile 78.13 0.0 4.305 Median 84.5 0.0 15.49 75% Percentile 95.68 0.0 21.87 Maximum 98.96 0.0 29.83 Mean 85.81 0.0 14.18 Std. Deviation 9.617 0.0 9.619 Std. Error of Mean 3.206 0.0 3.206 Lower 95% Cl of mean 78.42 0.0 6.787 Upper 95% Cl of mean 93.2 0.0 21.58 D’Agnostino & Pearson test 0.3617 0.3611 K2 P-value 0.8346 0.8348 Passed normality test (alpha=0.05) Yes Yes P-value summary Ns Ns Sum 772.3 0.0 127.6

D’Agostino & Pearson test Live cells Apoptotic cells Necrotic cells

Number of values 9 9 9 Minimum 0.0 0.0 1.29 25% Percentile 1.665 0.0 2.26 Median 27.27 0.0 72.72 75% Percentile 97.48 0.0 98.97 Maximum 98.7 0.0 100 Mean 41.42 0.0 58.66 Std. Deviation 43.69 0.0 43.92 Std. Error of Mean 14.56 0.0 14.64 Lower 95% Cl of mean 7.832 0.0 24.9 Upper 95% Cl of mean 75 0.0 92.43 D’Agnostino & Pearson test 3.231 3.221 K2 P-value 0.1987 0.1998 Passed normality test (alpha=0.05) Yes Yes P-value summary Ns Ns Sum 372.8 0.0 528

Arie Sullivan

42

8 a) One-way ANOVA test for HaCaT following 1 hour exposure to CTX

b) One-way ANOVA test for HaCat following 24 hours exposure to CTX

c) One-way ANOVA test for MCF7 following 1 hour exposure to CTX

d) One-way ANOVA test for MCF7 following 24 hours exposure to CTX

ANOVA table SS DF MS F DFn, DFd) P-Value Significant?

CTX Treatment 14.40 3 7.199 F (2, 6) = 0.6457 P = 0.5572 No Residuals 68.69 6 11.15 Total 81.29 8

ANOVA table SS DF MS F DFn, DFd) P-Value Significant?

CTX Treatment 125.4 2 62.72 F (2, 6) = 1.607 P=0.2761 No Residuals 234.2 6 39.03 Total 359.6 8

ANOVA table SS DF MS F DFn, DFd) P-Value Significant?

CTX Treatment 209.5 2 104.8 F (2, 6) = 3.996 P = 0.0788 No Residuals 157.3 6 26.21 Total 366.8 8

ANOVA table SS DF MS F DFn, DFd) P-Value Significant?

CTX Treatment 15.12 2 7.560 F (2, 6) = 0.1650 P = 0.8516 No Residuals 275 6 45.83 Total 290.1 8

Arie Sullivan

43

e) One-way ANOVA test for SHSY5Y following 1 hour exposure to CTX

f) One-way ANOVA test for SHSY5Y following 24 hours exposure to CTX

g) One-way ANOVA test for SHSY5Y following 48 hours exposure to CTX

ANOVA table SS DF MS F DFn, DFd) P-Value Significant?

CTX Treatment 151.3 2 76.67 F (2, 6) = 3.478 P = 0.0993 No Residuals 130.5 6 21.76 Total 281.9 8

ANOVA table SS DF MS F DFn, DFd) P-Value Significant?

CTX Treatment 2954 2 1477 F (2, 6) = 26.55 P = 0.0010 Yes Residuals 333.8 6 55.63 Total 3288 8

ANOVA table SS DF MS F DFn, DFd) P-Value Significant?

CTX Treatment 15224 2 7612 F (2, 6) = 929.6 P < 0.0001 Yes Residuals 49.13 6 8.189 Total 15273 8

Arie Sullivan

44

9 a) Genomic DNA extraction from 10 X 105 cells for MCF7, SHSY5Y, HaCaT, and a 100bp molecular weight

marker (MW). b) Genomic DNA extraction from 5 X 105 cells for MCF7, SHSY5Y, HaCaT, and a 100bp MW

marker.

10) Genomic DNA extraction from 7 X 105 cells for MCF7, SHSY5Y, HaCaT, 100bp and BSTEII MW markers.

The wells were included in the figure so as to distinguish the bands from the wells. Some DNA fragments

can also be observed from mechanical shearing.

a) b)

Arie Sullivan

45

11 a) Absorbances at 570nm for cell lines MCF7 (1.235), SHSY5Y (1.132) and HaCaT (1.254)

determine protein concentration in mg/mL by BCA assay for DNA fragmentation assay. Values

from the multiscan are incorporated into the BCA standard curve to determine difference in

protein concentration in mg/mL.

b) Absorbance at 570nm from the multiscan are incorporated into the BCA standard curve

to determine difference in protein concentration in mg/mL for SHSY5Y ( ),MCF7 ( ) and HaCaT

( ).

y = 0.0006x + 0.1502R² = 0.9956

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 500 1000 1500 2000 2500 3000

Ab

sro

ba

nce

57

0n

M

Concentration µg/mL

BCA Assay

Absorbance Mean

SHSY5Y

MCF7

HaCaT

Linear (Absorbance Mean)

Arie Sullivan

46

References

AKCAN, M., STROUD, M. R., HANSEN, S. J., CLARK, R. J., DALY, N. L., CRAIK, D. J. and OLSON, J.,

M. (2011) Chemical Re-engineering of Chlorotoxin Improves Bioconjugation Properties for Tumor

Imaging and Targeted Therapy. J. Med. Chem. 54(3), 782-787 DOI: 10.1012/jm101018r

AMOEDO, N. D., VALENCIA, J. P., RODRIGUES, M. F., GALINA, A. and RUMJANEK, F. D. (2013)

How Does the Metabolism of Tumor Cells Differ from that of Normal Cells. Biosci. Rep. 33(6)

e00080 DOI: 10.1042/BSR20130066

ANDREW, M. and OWEN, M. J. (1997) Hyperekplexia: Abnormal Startle Response due to Glycine

Receptor Mutations. Br. J. Psychiatry. 170, 106-108

ARZAMASOV, A. A., VASSILEVSKI, A. A. and GRISHIN, E. V. (2014) Chlorotoxin and Related

Peptides: Short Insect Toxins from Scorpion venom. Russian Journal of Bioorganic Chemistry.

40(4), 359-369 DOI: 10.1134/S1068162014040013

AVILA, A., VIDAL, P.M., DEAR, T. N., HARVEY, R. J., RIGO, J. M. and NGUYEN, L. (2013) Glycine

Receptor α2 Subunit Activation Promotes Cortical Interneuron migration . Cell. Rep. 4(4), 738-750

DOI: 10.1016/j.cellrep.2013.07.016

BLACKISTON, D. J., McLAUGHLIN, K. A. and LEVIN, M. (2009) Bioelectric Controls of Cell

Proliferation: Ion Channels, Membrane Voltage and the Cell Cycle. Cell. Cycle. 8(21), 3527-3536

BLANCHARD, S., BARWISE, J. L., GERKE, V. GOODALL, A., VAUGHAN, P. F. and WALKER, J. H.

(1996) Annexins in the Human Neuroblastoma SH-SY5Y: Demonstration of Relocation of

Annexins II and V to Membranes in Response to Elevation of Intracellular Calcium by Membrane

Depolarization and by the Calcium Ionophore A23187. J. Neurochem. 67(2), 805-813

BOBEK-BILLEWICZ, HEBDA, A., STASIK-PRES, G., MAJCHRZAK, K., ZMUDA, E. and TROJANOWSKA,

A. (2010) Measurement of Glycine in a Brain and Brain Tumors by Means of 1H MRS. Folia.

Neuropathol. 48(3), 130-139

BOYNTOPN, Z. L., KOON, J. J., BRENNAN, E. M., C<LOUART, J. D., HOROWITZ, D. M., GERNGROSS,

T. U. and HUISMAN, G. W. (1999) Reduction of Cell Lysate Viscosity during Processing of Poly(3-

Hydroxyalkanoates) by Chromosomal Integration of the Staphylococcal Nuclease Gene in

Pseudomonas Putida. Appl. Enviorn. Mircobiol. 65(4), 1524-1529

BRITTON, F. C., HATTON, W. J., ROSSOW, C> F., DUAN, D., HUME, J., R. and HORROWITZ, B.

(2000) Molecular Distribution of Volume-regulated Chloride Channels (ClC-2 and ClC-3) in Cardiac

Tissues. Am. J. Physiol. Heart. Circ. Physiol. 279(5), H2225-33

Arie Sullivan

47

BROWN, N. H. (2011) Extracellular Matrix in Development: Insights from Mechanisms Conserved

between Invertebrates and Vertebrates. Cold Spring Harb. Perspect. Biol. 3(12), p: 1005082 DOI:

10.1101/cshperspect.a005082

BRUNETTI, V., MAIORANO, G., RIZELLO, L., SORCE, B., SABELLA, S., CINGOLANI, R. and POMPA, P.

P. (2010) Neurons Sense Nanoscale Roughness with Nanometer Sensitivity . Proc. Natl. Acad.

USA. 107(14), 6264-6269 DOI: 10.1073/pnas.0914456107

BUTTE, P. V., MAMELAK, A., PARRISH-NOVAK, J., DRAZIN, D., SHWEIKEH, F., GANGALUM, P. R.,

CHESNOKOVA, A., LJUBIMOVA, J. Y. and BLACK, K. (2014) Near-Infrared Imaging of Brain Tumors

Using the Tumor Paint BLZ-100 to Achieve Near-Complete Resection of Brain Tumors. Neurosurg.

Focus. 36(2), p: E1 DOI: 10.3171.2013.11.FOCUS13497

CANNON, C. S. (2006) Channelopathies of the Nervous System. Prin. Mol. Med. Pp-1088-1096

CATALANI, S., CARBONARO, V., PALMA, F., ARSHAKYAN, M., GALATI, R., NUVOLI, B., BATTISTELLI,

S., CANESTRARI, F. and BENEDETTI, S. (2013) Metabolism Modifications and Apoptosis Induction

after Cellfood™ Administration to Leukemia Cell Lines. J. Exp. Clin. Cancer. Res. 32(1), p: 63 DOI:

10.1186/1756-9966-32-63

CHEN, M. J., SEPRAMANIAM, S., ARMUGAM, A., CHOY, M. S., MANIKANDAN, J., MELENDEZ, A. J.,

JEYASEELAN, K. and CHEUNG, N. S. (2008) Water Ion Channels: Crucial in the Initiation and

Progression of Apoptosis in Central Nervous System. Curr. Neuropharmacol. 6(2), 102-116 DOI:

10.2174/157015908784533879

CHENG, Y., ZHAO, J., QIAO, W. and CHEN, K. (2014) Recent Advances in Diagnosis and Treatment

of Gliomas Using Chlorotoxin-based Bioconjugates. Am. J. Nucl. Mol. Imaging. 4(5), 384-405

COSTA, P.M., CASDOSO, A. L., MENDOCA, L. S., SERANI, A., CUSTODIA, C., CONCEICAO, M.,

SIMOES, S., MOREIRA, J. N., PEREIRA de ALMEIDA, L. and PEDROSO de LIMA, M. (2013) Tumor-

targeted Chlorotoxin-coupled Nanoparticles for Nucleic Acid Delivery to Glioblastoma Cells: A

Promising System for Glioblastoma Treatment. Mol. Ther. Nucleic Acids. 2, p: e100 DOI:

10.1038/mtna.2013.30

DARDEVET, L., RANI, D., AZIZ, T. A. E., BAZIN, I., SABATIER, J.M., FADI, M., BRAMBILLA, E. and

WAARD, M. D. (2015) Chlorotoxin: A Helpful Natural Scorpion Peptide to Diagnose Glioma and

Fight Tumor Invasion. Toxins. 7(4), 1079-1101 DOI: 10.3390/toxins7041079

DAS GUPTA, S., DEBNATH, A., SAHA, A., GIRI, B., TRIPATHI, G., VEDASIROMONI, J. R. and GOMES,

A. (2007) Indian Black Scorpion (Heterometrus bengalensis Koch) Venom Induced

Antiproliferative and apoptogenic Activity Against Human Leukemic Cell Lines U937 and K562.

Leuk. Res. 31(6), 817-825

DEBIN, J. A. and STRICHARTZ G. R. (1991) Chloride Channel Inhibition by the Venom of the

Scorpion Leiurus Quinquestriatus. Toxicon. 29(11), 1403-1408

Arie Sullivan

48

DEBIN, J. A., MAGGIO, J. E. and STRICHARTZ, G. R. (1993) Purification and Characterization of

Chlorotoxin, a Chloride Channel Ligand from the Venom of the Scorpion . Am. J. Physiol. 264(2 Pt

10:C361-9

DENDER, D. G., HECK, N., RIEDEMANN, T., WHITE, R., KILB, W. and LUHMANN, H. J. (2010)

GABAC Receptors are Functionally Expressed in the Intermediate Zone and Regulate Radial

Migration in the Embryonic Mouse Neocortex . Neuroscience. 167, 124-134 DOI: 10.1016/j.

neuroscience.2010.01.049

DERYUGINA, E.I. and BOURDON, M. A. (1996) Tenascin Mediates Human Glioma Cell Migration

and Modulates Cell Migration on Fibronectin. J. Cell. Sci. 109 (Pt3), 643-652

DESHANE, J., GARNER, C. C. and SONTHEIMER, H. (2002) Chlorotoxin Inhibits Glioma Cell

Invasion via Matrix Metalloproteinase-2. J. Biol. Chem. 278, 4235-4144 DOI:

10.1074/jbc.M205662200

DURAN, C., THOMPSON, C. H., XIAO, Q. and HARTZELL, H. C. (2010) Chloride Channels: Often

Enigmatic, Rarely Predictable. Annu. Rev. Physiol. 72, 95-121.

EMONARD, H., BELLON, G., TROEBERG, L., BERTON, A., ROBINET, A., HENRIET, P., MARBAIX, E.,

KIRKEGAARD, K., PATTHY, L., EECKHOUT, Y., NAGASE, H., HORNEBECK, W., COURTOY, P. J. (2004)

Low Density Lipoprotein Receptor-related Protein Mediates Endocytic Clearance of pro-MMP-

2.TIMP-2 Complex Through a Thrombospondin-independent Mechanism. J. Biol. Chem. 279(52),

54944-54951

FAN, S., SUN, Z., JIANG, D., DAI, C., MA, Y., ZHAO, Z., LIU, H., WU, Y., CAO, Z. and LI, W. (2010)

BmKCT Toxin Inhibits Glioma Proliferation and Tumor Metastasis. Cancer Letters. 291(2), 158-

166 DOI: 10.1016/j.canlet.2009.10.011

FORSTERA, B., DZAYE, O. D., WINKELMANN, A., SEMTNER, M., BENEDETTI, B., MARKOVIC, D. S.,

SYNOWITZ, M., WEND, P., FAHLING, M., JUNIER, M. P., GLASS, R., KETTENMANN, H. and MEIER,

J. C. (2014) Intracellular Glycine Receptor Function Facilitates Glioma Formation in Vivo. J. Cell.

Science. DOI: 10.1242/jcs.146662

GALAGHER, J. D., FAY, M. J., NORTH, W. G., and McCANN, F. V. (1996) Ionic Signals in T47D

Human Breast Cancer Cells. Cell Signal. 8(4), 279-284

GAO, L., YU, S., WU, Y. and SHAN, B. (2007) Effect of Spider Venom on Cell Apoptosis and

Necrosis Rates in MCF-7 Cells. DNA Cell Biol. 26(7), 485-489.

GOUDET, C., CHI, C. W. and TYTGAT, J. (2002) An Overview of Toxins and Genes from the Venom

of the Asian Scorpion Buthus martensii Karsch. Toxicon. 40, 1239-1258

Arie Sullivan

49

HAGBERG, A., BARBANY, G., KROOK, H., SAMIOTAKI, M. and LANDERGREN, U. (2000) Expression

Profiling Accross Many Samples via Manifold-assisted mRNA Processing. Nucleic acids Res.

28(11): e54

HARTZELL, C., PUTZIER, I. and ARREOLA, J. (2005) Calcium-activated Chloride Channels. Annu.

Rev. Physiol. 67, 719-758

HMED, B., SERRIA, H. T. and MOUNIR, Z., K. (2013) Scorpion Peptides: Potential Use for New

Drug Development. J. Toxicol. 2013, 958797 DOI: 10.1155/2013/958797

HONG, S., BI, M., WANG, L., KANG, Z., LING, L. and ZHAO, C. (2014) CLC-3 Channels in Cancer.

Onc. Report.507-514 DOI: 10.3892/or.2014.3615

KASAI, T., NAKAMURA, K., VAIDYANATH, A., CHEN, L., SEKHAR, S., EL-GHLBAN, S., OKADA, M.,

MIZUTANI, A., KUDOH, T., MRAKAMI, H. and SENO, M. (2012) Chlorotoxin Fused to IgG-Fc

Inhibits Glioblastoma Cell Motility via Receptor-mediated Endocytosis. J. Drug. Deliv. 975763

DOI: 10.1155/2012/975763

KESAVAN, K., RATLIFF, J., JOHNSON, E. W., DAHLBERG, W., ASARA, J. M., MISRA, P., FRANGIONI,

J. V. and JACOBY, D. B. (2010) Annexin A2 is a Molecular Target for TM601, a Peptide with

Tumor0targeting and Anti-angiogenic Effects. J. Biol. Chem. 285(7), 4366-4374 DOI:

10.1074/jbc.M109.066092

KIM, H., ZHENG, S., AMINI, S. S., VIRK, S. M., MIKKELSEN, T., BRAT, D. J., GRIMSBY, J., SOUGNEZ,

C., MULLER, F., HU, J., SLOAN, A. E., COHEN, M. L., VAN MEIR, E. G., SCARPACE, L., LAIRD, P. W.,

WEINSTEIN, J. N., LANDER, E. S., GABRIEL, S., GETZ, G., MEYERSON, M., CHIN, L., BARNHOLTZ-

SLOAN, J. S. and VERHAAK, R. G. (2015) Whole-Genome and Multisector Exome Sequencing of

Primary and Post-Treatment Glioblastoma Reveals Patterns of Tumour Evolution. Genome. Res.

25(3), 316-327 doi: 10.1101/gr.180612.114

KIM, M. J., CHENG, G. and AGRAWAL D. K. (2004) Cl- Channels are Expressed in Human Normal

Monocytes: A Functional Role in Migration, Adhesion and Volume Change. Clin. Exp. Immunol.

138(3), 453-459

KIM, Y. S., LEE, H. A., LIM, J. Y., KIM, Y., JUNG, C. H., YOO, S. H. and KIM, Y. (2014) β-Carotene

Inhibits Neuroblastoma Cell Invasion and Metastasis in vitro and in vivo by Decreasing Level of

Hypoxia-inducible Factor-1α. J. Nutr. Biochem. 25(6), 655-664 DOI:

10.1016/j.jnutbio.2014.02.006

KRIEGSTEIN A. and ALVAREZ-BUYLLA, A. (2009) The Glial Nature of Embryonic and Adult Neural

Stem Cells. Annual Review if Neuroscience. 32, 149-184 DOI:

10.1146/annurev.neuro.051508.135600

Arie Sullivan

50

KWIATKOWSKA, A. and SYMONS, M. (2013) Signaling Determinants of Glioma Cell Invasion. Adv.

Exp. Biol. 986, 121-141 DOI: 10.1007/978-94-007-4719-7_7

LANIADO, M. E., FRASER, S. P. and DJAMGOZ, M. B. (2001) Voltage-gated K(+) Channel Activity in

Human Prostate Cancer Cell Lines of Markedly Different Metastatic Potential: Distinguishing

Characteristics of PC-3 and LNcaP cells. Prostate. 46(4), 262-274

LI, W., LI, Y., ZHAO, Y., YUAN, J. and MAO, W. (2014) Inhibition Effects of Scorpion Venom

Extracts (Buthus martensii Krasch) on the Growth of Human Breast Cancer MCF-7 Cells. Afr. J.

Tradit. Complement. Alter. Med. 11(5), 105-110

LISAL, J. and MADUKE, M. (2009) Proton-coupled Gating in Chloride Channels. Roy. Soc. Pub.

364(1514) DOI: 10.1098/rstb.2008.0123

LYNCH, W. J. (2004) Molecular Structure and Function of the Glycine Receptor Chloride Channel.

Physiol. Rev. 84(4), 1051-1095 DOI: 10.1152/physrev.00042.2003

LYONS, S. A., O’NEAL, J. and SONTHEIMER, H. (2002) Chlorotoxin, a Scorpion-Derived Peptide,

Specifically Binds to Gliomas and Tumors of Neuroectodermal Origin . Glia. 39(2), 162-173

MADUREIRA, P. A., HILL, R., MILLER, V. A., GIACOMANTONIO, C., LEE, P. W. and WAISMAN, D. M.

(2011) Annexin A2 is a Novel Cellular Redox Regulatory Protein Involved in Tumorigenesis .

Oncotarget. 2(12), 1075-1093

MAENO, E., ISHIZAKI, Y., KANASEKI, T., HAZAMA, A. and OKADA, Y. (2000) Normotonic Cell

Shrinkage Because Disordered Volume Regulation is an Early Prerequisite to Apoptosis . Proc.

Natl. Acad. Sci. pp: 9487-9492

MAERTENS, C., WEI, L., TYTGAT, J., DROOGMANS, G. and NILIUS, B. (2009) Chlorotoxin does not

Inhibit Volume-regulated, calcium-activated and cyclic AMP-activated chloride channels. B. J.

Pharm. 129(4), 791-801

MAI, J., FINLEY, R. L., WAISMAN, D. M. and SLOANE, B. F. (2000) Human Procathepsin B interacts

with the Annexin II Tetramer on the Surface of Tumor Cells. J. Biol. CHem. 275, 12806-12812

MANN, C. L., BORTHER, C. D., JEWELL, C. M. and CIDLOWSKI, J. A. (2001) Glucocorticoid-induced

Plasma Membrane Depolarizing During Thymocyte Apoptosis: Association with Cell Shrinkage

and Degradation of the Na(+)/K(+) adenosine Triphosphate. Endocrinology. 142(12), 5059-5068.

MAO, J., WANG, L., FAN, A., WANG, J., XU, B., JACOB, T. J. and CHEN, L. (2007) Blockage of

Volume-activated Chloride Channels Inhibits Migration of Nasopharyngeal Carcinoma Cells . Cell.

Physiol. Biochem. 19(5), 249-258

MATTSON, M. P. and CHAN, L. (2003) Calcium Orchestrates Apoptosis. Nature. Cell Biology. 5,

1041-1043 DOI: 10.1038/ncb1203-1041

Arie Sullivan

51

McFERRIN, M. B. and SONTHEIMER, H. (2006) A Role for Ion Channels in Glioma Cell Invasion.

Neuron. Glia. Biol. 2(1), 39-49 DOI: 10.1017/S17440925X06000044

McHUGH, K. (2007) Renal and Adrenal Tumors in Children. Cancer Imaging. 7(1), 41-51 DOI:

10.1102/1470-7330.2007.0007

MERZAK, A., McCREA, S., KOOCHEKPOUR, S. and PILKINGTON, G. J. (1994) Control of Human

Glioma Cell Growth, Migration and Invasion in Vitro by Transforming Growth Factor Beta 1. BR.

J. Cancer. 70(2), 199-203

MIYASAKI, K. (2002) Random DNA Fragmentation with Endonuclease V: Application to DNA

shuffling. Nucleic Acids Res. 30(24), p: e139

MORGUNOVA, E., TUUTILA, A., BERGMANN, U. and TRYGGVASON, K. (2002) Structural Insight

into the Complex Formation of Latent Matrix Metalloproteinase-2 with Tissue Inhibitor of

Metalloproteinase -2. Proc. Natl. Acad. USA. 99(11), 7414-7419

MORRISON, S. J., PEREZ, S. E., QIAO, Z., VERDI, J. M., HICKS, C., WEINMASTER, G. and

ANDERSON, D. J. (2000) Transient Notch activation initiates an Irreversible Switch from

Neurogenesis to Gliogenesis by Neural Crest Stem Cells. Cell. 101, 499-510

MUNSON, J., BONNER, M., FRIED, L., HOFMEKLER, J., ARBISER, J. and BELLAMKONDA, R. (2013)

Identifying New Small Molecule Anti-invasive Compounds for Glioma Treatment. Cell Cycle.

12(14), 2200-2209 DOI: 10.4161/cc.25334

NAGATA, S. (2000) Apoptotic DNA Fragmentation. Exp. Cell. Res. 256(1), 12-18

NAKADA, M., NAKADA, S., DEMUTH, T., RAN, N. L., HOELZINGER, D. B. and BERENS, M. E. (2007)

Molecular Targets of Glioma Invasion. Cell. Mol. Life. Sci. 64(4), 458-478

NIETSCH, H. H. ROE, M. W., FIEKERS, J. F., MOORE, A. L. and LIDOFSKY, S. D. (2000) Activation of

Potassium and Chloride Channels by Tumor Necrosis Alpha. Role in Liver Cell Death. J. Biol.

Chem. 275(27), 20556-20561

NIMMERVOLL, B., DENTER, D. G. SAVA, I., KILB, W. and LUHMANN, H., J. (2011) Glycine

Receptors Influence Radial Migration in the Embryonic Mouse Neocortex . Neuroreport. 22, 509-

513 DOI: 10.1097/WNR.obo13e32834Saafe

NOLTE, F., FRIEDRICH, O., ROJEWSKI, M., FINK, R. H., SCHREZENMEIER, H. and KORPER, S. (2004)

Depolarization of the Plasma Membrane in the Arsenic Trioxide (As2O3)- and anti-CD95-induced

Apoptosis in myeloid cells. FEBS. Lett. 578(1), 85-89

NUCCITELLI, S., CERELLA, C., CORDISO, S., ALBERTINI, M. C., ACCORSI, A., DeNICOLA, M.,

D’ALESSION, M., RADOGNA, F., MAGRINI, A., BERGAMASCHI, A. and GHIBELLI, L. (2006)

Arie Sullivan

52

Hyperpolarization of Plasma Membrane of Tumor Cells Sensitive to Anti-apoptotic Effects of

Magnetic Fields. Ann. N. Y. Acad. Sci. 1090, 217-225

OLSEN, M. L., SCHADE, S., LYONS, S. A., AMARAL, M. D. and SONTHEIMER, H. (2003) Expression

of Voltage-Gated Chloride Channels in Human Glioma Cells. J. Neurosci. 23(13), 5572-5582

PAULUS, W., BAUR, I., BEUTLER, A. S. and REEVES, S. A. (1996) Diffuse Brain Invasion of Glioma

Cells Requires beta 1 Integrins. Lab. Invest. 75(6), pp: 819-826

PAW, I., CARPENTER, R. C., WATABE, K., DEBINSKI, W. and LO, H. W. (2015) Mechanisms

Regulating Glioma Invasion. Cancer Letters. 362(1), 1-7 DOI: 10.1016/j.canlet.2015.03.015

PETRAT, F., BOENGLER, K., SCHULZ, R. and GROOT, H. (2012) Glycine, a Simple Physiological

Compound Protecting by yet Puzzling Mechanism(s) Against Ischemia-Reperfusion Injury: Current

Knowledge. Br. J. Pharmacol. 165(7), 2059-2071 DOI: 10.1111/j.1476-5381.2011.01711.x

RAO, V. R., PEREZ-NEUT, M., KAJA, S. and GENTILE, S. (2015) Voltage-Gated Ion Channels in

Cancer Cell Proliferation. Cancer. 7(2), 849-875 DOI: 10.3390/cancers7020813

REMACLE, A., MURPHY, G. and ROGHI, C. (2003) Membrane type-1matrix Metalloproteinase

(MT1-MMP) is Internalized by two Different Pathways and is Recycled to the Cell Surface. J. Cell.

Sci. 3905-3916

REUNANEN, N. and KAHARI, V. (2000) Matrix Metalloproteinases in Cancer Cell Invasion. Landes.

Bioscience

RIBATTI, D., MARIMPIETRI, D., PASTORINO, F., BRIGNOLE, C., NICO, B., VACCA, A. and PONZONI,

M. (2004) Angiogenesis in Neuroblastoma. Ann. N.Y. Acad. Sci. 1028, 133-142 DOI:

10.1196/annals.1322-014

ROSE, M. L., CATTLEY, R. C., DUNN, C., WONG, V., LI, X. and THHURMAN, R. G. (1999) Dietary

Glycine Prevents the Development of Liver Tumors Caused by the Peroxisome Proliferator WY-

14,643. Carcinogenesis. 20(11), 2075-2081

SAMBROOK, J. and RUSSEL, D. W. (2000) Fragmentation of DNA by Sonication. CSH. Protoc.

2006(4), pii.pdb.prot4538 DOI: 10.1101/pdb.prot4538

SCABO, I., LEPPLE-WIENHUES, A., KABA, K. N., ZORATTI, M., GUBLINS, E. and LANG, F (1998)

Tyrosine Kinase-dependent Apoptosis in T-lymphocytes. Proc. Natl. Acad. Sci. 95(11), 6169-74

SCHNITZER, J. E., OH, P., PINNEY, E. and ALLARD, J. (1994) Filipin-sensitive Caveolae-mediated

Transport in Endothelium: Reduced Transcytosis, Scavenger Endocytosis, and Capillary

Permeability of Select Macromolecules. J. Cell. Biol. 127(5), 1217-1232

SCHRAMEL, S. (2014) Structure – Function of Annexin A2 & A5. Duluth. J. Underg. Res. 108-111

Arie Sullivan

53

SCOTT, R. C. and HOLMES, G. L. (2012) Before Epilepsy Unfolds: Opening up the potassium Door

in Neonatal Seizures. Nature. Med. 18, 1624-1625 DOI: 10.1038/nm.2987

SONG, X., ZHANG, G., SUN, A., GUO, J., TIAN, Z., WANG, H. and LIU, Y. (2012) Scorpion Venom

Component III Inhibits Cell Proliferation by Modulating NF-κB Activation in Human Leukemia

Cells. Exp. Ther. Med.4, 146-150 DOI: 10.3892/etm.2012.548

SONHEIMER, H. (2009) An Unexpected Role for Ion Channels in Brain Tumor Metastasis . Exop.

Biol. Med. 233(7), 779-791 DOI: 10.3181/0711-MR-308

SONTHERIMER, H. (2004) Ion Channels and Amino Acid Transporters Support the Growth and

Invasion of Primary Brain Tumors. Mol. Neurobiol. 29(1), 61-71 DOI: 10.1385/MN:29:1:61

SOROCEANU, L., MANNING, T. J. Jr. and SONTHEIMER, H. Modulation of Glioma Cell Migration

and Invasion Using Cl(-) and K(+) Ion Channel Blockers. J. Neurosci. 19(14), 5942-5954

SRINIVASAN, K.N., GOPALAKRISHNAKONE, P., TAN, P. T., CHEW, K. C., CHENG, B., KINI, R. M.,

KOH, J. L., SEAH, S. H. and BRUSIC, V. (2002) SCORPION, a Molecular Database of Scorpion

Toxins. Toxicon. 40(1), 23-31

STERNLIGHT, M. D. and WERB, Z. (2009) How Matrix Metalloproteinases Regulate Cell Behavior.

Annu. Rev. Cell. Dev. Biol. 17, 463-516 DOI: 10.1146/annurev.cellbio.17.1.463

STROUD, M. R., HANSEN, S. J. and OLSON, J. M. (2012) In Vivo Bio-imaging Using Chlorotoxin-

based Conjugates. Curr. Pharm. Des. 17(38), 4362-4371

SZABO, I., LEPPLE-WIENHUES, A., KABA, K. N., ZORATTI, M., GULBINS, E. and LANG, F. (1998)

Tyrosine Kinase-dependent activation of a chloride channel in CD95-induced Apoptosis in T

Lymphocytes. Proc. Natl. Acad. 95, pp: 6169-6174

TATENHORST, L., RESCHER, U., GERKE, V. and PAULUS, W. (2006) Knockdown of Annexin 2

Decreases Migration of Human Glioma Cells in Vitro. Neuropathol. Appl. Neurobiol. 32(3), 271-

277

TSAI, C. H., YANG, S. H., CHIEN, C. M., LU, M. C., LO, C. S., LIN, Y. H., HU, X. W. and LIN, S. R.

(2006) Mechanism of Cardiotoxin III-induced Apoptosis in Human Colorectal Cancer COLO205

Cells. Clinical and Experimental Pharmacology and Physiology. 33(3), 177-182.

DOI:10.1111/j.1440-1681.2006.04334.x

TYSNES, B. B., LARSEN, L F., NESS, G. O., MAHESPARAN, R., EDVARDSEN, K., GARCIA-CABRERA, I.

and BJERKVIG, R. (1996) Stimulation of Glioma-cell Migration by Laminin and Inhibition by Anti-

alpha3 and anti-beta1 Integrin Antibodies. Int. J. Cancer. 67(6), 777-784

Arie Sullivan

54

UEKITA, T., ITOH, Y., YANA, I., OHNO, H and SEIKI, M. (2001) Cytoplasmic Tail-independent

Internalization of Membrane type 1 Matrix Metalloproteinase is Important for its Invasive-

promoting activity. J. Cell. Biol. 155(7), 1345-1356 DOI: 10.1083/jcb.200108112

VAN den EYNDN, J., ALI, S. S., HORWOOD, N., CARMANS, S., BRONE, B., HELLINGS, N., STEELS, P.,

HARVEY, R. J. and RIGO, J. M. (2009) Glycine and Glycine Receptor Signaling in Non-Neuronal

Cells. Front. Mol. Neurosci. 2, 9 DOI: 10.3389/neuro.02.009.2009

VAN der HEIDEN, M. G., CANTLEY, L. C. and THOMSPON, C. B. (2009) Understanding the

Warburg Effect: The Metabolic Requirements of Cell Proliferation. Science. 324(5930), 1029-

1033 DOI: 10.1126/science.1160809

VEISEH, M., GABIKIAN, P., BAHRAMI< S. B., VEISEH, O., ZHANG, M., HACKMAN< R. C.,

RAVANPAY, A. C., STROUD, M. R., KUSUMA, Y., HANSEN, S. J., KWOK, D., MUNOZ, N. M., SZE, R.

W., GRADY, W. M., GREENBERG, N. M., ELLENBOGEN, R. G. and OLSON, J. M. (2007) Tumor

Paint: A Chlorotoxin:Cy5.5 Bioconjugate for Intraoperative Visualization of Cancer Foci. Cancer.

Res. 67(14), 6882-6888

VEISEH, O., GUNN, J. W., KIEVIT, F., SUN, C. and FANG, C. (2009) Inhibition of Tumor Cell Invasion

with Chlorotoxin-Bound Superparamagnetic Nanoparticles. HHS. Small. 5(2), 256-264 DOI:

10.1002/smll.200800646

VEISEH, O., GUNN, J. W., KIEVIT, F. M., SUN, C., FANG, C., LEE, J. S. and ZHANG, M. (2009)

Inhibition of Tumor-cell Invasion with Chlorotoxin-bound Superparamagnetic Nanoparticles.

Small. 5(2), 256-264 DOI: 10.1002/smll.200800646

WANG, C. Y. and LIN, C. F. (2014) Annexin A2: Its Molecular Regulation and Cellular Expression in

Cancer Development. Disease Markers. Vol 2014, Article ID: 308976

WANG, M., WANG, T., LIU, S., YOSHIDA, D. and Teramoto, A. (2003) The Expression of Matrix

Metalloproteinase-2 and -9 in Human Gliomas of Different Pathological Grades. Brain . Tumor.

PAthol. 20(2), 65-72

WEBB, T. I. and LYNCH, J. W. (2007) Molecular Pharmacology of the Glycine Receptor Chloride

Channel. Curr. Pharm. Des. 13(23), 2350-2367

WEI, L., XIAO, A. Y., JIN, C., YANG, A. LU, Z. Y. and YU, S. P. (2004) Effects of Chloride and

Potassium Channel Blockers on Apoptotic Cell Shrinkage and Apoptosis in cortical Neurons .

Plugers. Arch. 448(3), 325-334

XIONG, W., CHEN, S. R., HE, L., CHENG, K., ZHAO, Y. L., CHEN, H., LI, D. P., HOMANIACS, G. E.,

PEEVER, J., RICE, K. C., WU, L. G., PAN., H. L. and ZHANG, L. (2014) Presynaptic Glycine receptors

as a Potential Therapeutic Target for Hyperekplexia Disease. Nat. Neurosci. 17(2), 232-239 DOI:

10.1038/nn.3615

Arie Sullivan

55

YAO, D. F., ZHANG, H. J., YAO, M., HUANG, H., WANG, L., YAN, M. J., YAN, X. D., GU, X., WU, W.

and LU, S. L. (2013) Annexin A2 Silencing Inhibits invasion, migration, and tumorigenic Potential

of Hepatoma Cells. World. J. Gastroenterol. 19(24), 3792-3801 DOI: 10.3748/wjg.v19.i24.3792

YUKL, S. A., KAISER, P., KIM, P., LI, P. and WONG, J. K. (2014) Advantages of Using the

QIAshredder instead of Restriction digestion to prepare DNA for droplet digital PCR.

Biotechniques. 56(4), 194-196 DOI: 10.2144/000114159

ZARGAN, J., SAJAD, M., UMAR, S., NAIME, M., ALI, S. and KHAN, H. A. (2011) Scorpion

(Odontobuthus doriae) Venom Induces Apoptosis and Inhibits DNA Synthesis in Human

Neuroblastoma Cells. Mol. Cell. Biochem. 348(1-2), 173-181. DOI: 10.1007/s11010-010-0652-x

ZEIDAN-CHULIA, F., GELAIN, D. P., KOLLING, E. A., RYBARCZYK-Filho, J.L., AMBROSI, P., TERRA, S.

R., PIRES, A., S., TEIXEIRAdaROCHA, J. B., BEHR, G. A. and MOREIRA,J. C. F. (2013) Major

Components of Energy Drinks (Caffeine, Taurine, and Guarana) Exert Cytotoxic Effects on Human

Neuronal SH-SY5Y Cells by Decreasing Reactive Oxygen Species Production. Oxid. Med. Cell.

Longev.2013, p: 791795

ZHANG, R., CUI, Y., ZHANG, X., YANG, Z., ZHAO, Y., SONG, Y., WU, C and ZHANG, J. (2010) Soluble

Expression, Purification and the Role of C-terminal Glycine Residues in Scorpion Toxin BmK AGP-

SYPU2. BMB. Rep. 43(12). 801-806 DOI: 10.5483/BMBRep.2010.43.12.801

ZHANG, W., ZHAO, P., XU, X. L., CAI, L., SONG, Z. S., CAO, D. Y., TAO, K. S., ZHOU, W. P., CHEN, Z.

N. and DOU, K. F. (2013) Annexin A2 Promotes the Migration and Invasion of Human

Hepatocellular Carcinoma Cells In Vitro by Regulating the Shedding of CD147-Harboring

Microvesicles from Tumor Cells. PLoS One. 8(8), p: e67268 DOI: 10.1371/journal.pone.0067268

ZHAO, Y. S., ZHANG, R., XU, Y., CUI, Y., LIU, Y.F., SONG, Y. B., ZHANG, H. X. and ZHANG, J. H.

(2013) The Role of Glycine Residues at the C-terminal Peptide Segment in Antinociceptive

Activity: a Molecular Dynamics Simulation. J. Mol. Model. 19)3), 1295-1299 DOI: 10.007/s00894-

012-1666-y

ZHIJIAN, C., FENG, L., YINGLIANG, W., XIN, M. and WENXIN, L. (2006) Genetic Mechanisms of

Scorpion Venom Peptide Diversification. Toxicon. 47, 348-355

ZHU, H., ZHENG, J., XIAO,X., ZHENG, S., DONG, K., LIU, J. and WANG, Y. (2010) Environmental

Endocrine Disruptors Promote Invasion and Metastasis of SK-N-SH Neuroblastoma Cells. Oncol.

Rep. 23(1), 129-139