Evaluating the Effects of Fasting Conditions in Combination with Temozolomide for the Treatment of Glioblastoma Multiforme

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7-2020

EVALUATING THE EFFECTS OF FASTING CONDITIONS IN COMBINATION WITH TEMOZOLOMIDE FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME

Joseph Duffy [email protected]

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EVALUATING THE EFFECTS OF FASTING CONDITIONS IN COMBINATION WITH TEMOZOLOMIDE FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME

Joseph T. Duffy

THESIS

Submitted to Northern Michigan University In partial fulfillment of the requirements For the degree of

MASTER OF SCIENCE

Office of Graduate Education and Research

June 2020

Dr. Robert Winn

Dr. Richard Rovin

Dr. Amber LaCrosse ABSTRACT

EVALUATING THE EFFECTS OF FASTING CONDITIONS IN COMBINATION WITH TEMOZOLOMIDE FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME

Joseph Duffy

Glioblastoma multiforme (GBM) is the most aggressive type of glioma and most lethal of adult brain tumors. The standard of care for GBM is maximal surgical resection followed by adjuvant treatment with the chemotherapeutic agent temozolomide (TMZ) and radiotherapy. The presence of TMZ-resistant cells in GBM that can reverse the TMZ induced cytotoxic lesions by expressing MGMT contribute to therapy resistance. A common trait of cancer is an altered carbohydrate metabolism. Known as the

Warburg effect, cancer cells will preferentially oxidize glucose to lactate in glycolysis. This less effective pathway of ATP production per mole of glucose means cancer cells must uptake significantly greater amounts of glucose to sustain physiological needs. One method that directly targets the bioavailability of glucose is fasting. Fasting lowers systemic glucose levels, lowers anabolic hormone levels (insulin and

IGF-1), and elevates ketone bodies like ß-hydroxybutyrate (ß-HB). This study sought to evaluate the effects of fasting in combination with TMZ on viability of GBM cell lines LN229 and T98G, and normal astroglial cell line SVG p12. The data from showed that glucose restricted media significantly enhanced the cytotoxic effects of TMZ in LN229 and T98G cells, while having no effect in SVG p12 cells. ß-HB in combination with TMZ potentiated the cytotoxicity of TMZ in LN229 and T98G cells, while reducing the cytotoxicity of TMZ in SVG p12 cells. Western blot analysis for MGMT determined T98G cells were the only cell line to express MGMT. Both glucose restricted media and ß-HB supplemented media in combination with TMZ significantly decreased the expression of MGMT levels in T98G cells. Together, this initial evidence suggests fasting may be a useful method to selectively increase cytotoxicity of TMZ in GBM cells, while protecting normal cells. Future research is needed to further assess the potential of fasting as an adjuvant therapy in the treatment of GBM.

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Joseph T. Duffy

2020

This thesis is dedicated to all those who have been affected by Cancer, especially those who are suffering

or have suffered from Glioblastoma Multiforme, and those who work tirelessly to advance the field of oncology. On a personal level, I would like to dedicate this thesis to Joe Duffy, Judy Duffy, Joyce Duffy,

Robert Duffy, and Tommy McGuire. You continue to motivate me on this quest to understand the nature

of cancer.

ACKNOWLEDGMENTS

The work presented in this thesis would not have been possible without the help of the Upper

Michigan Brain Tumor Center, graduate research assistantship, summer fellowship, and Excellence in

Education grant.

I would like to thank my research advisor, Dr. Robert Winn, and my committee members, Dr.

Richard Rovin and Dr. Amber LaCrosse, for their support and guidance throughout graduate school. I would also like to thank the other members of the Upper Michigan Brain Tumor Center for their various contributions, especially, Dr. Matt Jennings and Dr. Paul Mann for their guidance in cell and molecular work, and Dr. Sonia Geschwindt, Dr. Sheetal Acharya, John Paul Velasco, Dan Raymond, Allison Riehl,

Kyle Flickinger, and Nick Shortreed for their assistance throughout my research project.

Finally, I would like to thank Northern Michigan University for giving me a second chance at life, and for believing in me during a time when I did not believe in myself. For that I am forever in grateful.

TABLE OF CONTENTS

List of Figures.....…………………………………………………………………………………………(v) List of Abbreviations.……………………………………………………………………………………….…...(vi) Introduction…………………………………………………………………………………………………1 Chapter One: Literature Review.…………………………………………………………………………...5 Introduction………………………………………………………………………………………...5 Temozolomide: Mechanism of action, DNA repair mechanisms, and cellular resistance...... …………….5 The Warburg Effect in Cancer and Its Role in GBM…………………………………………...…8 Fasting: Systemic Changes and Role in Cancer Therapy……………………………………...…10 Conclusion……………………………………………………………………………………..…16 Chapter Two: Aims and Goals………………………………………………………………………….…17 Chapter Three: Culture and Maintenance of Cell Lines.………………………………………………….19 Chapter Four: Analyzing Cytotoxicity of Glucose Restricted Media in Combination with Temozolomide in GBM Cells……………………………………………………………………………..21 Introduction…...... 21 Methods and Materials…...... 21 Results...... …25 Discussion...... …31 Chapter Five: Analyzing Cytotoxicity of Glucose Restricted Media in Combination with Temozolomide in Administered Without DMSO in GBM and Human Astroglial Cells...... …………….33 Introduction……………………………………………………………………………………….33 Methods and Materials……………………………………………………………………………33 Results…………………………………………………………………………………………….37 Discussion………………………………………………………………………………………...42 Chapter Six: Analyzing Cytotoxicity of Glucose Restricted Media Supplemented with ß-Hydroxybutyrate in Combination with Temozolomide in GBM and Human Astroglial Cells…...... …47 Introduction……………………………………………………………………………………….47 Methods and Materials……………………………………………………………………………48

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Results…………………………………………………………………………………………….51 Discussion………………………………………………………………………………………...63 Chapter Seven: Analyzing the Effects of Glucose Restricted Media and ß-Hydroxybutyrate in Combination with Temozolomide on MGMT expression in GBM and Human Astroglial Cells..…….67 Introduction……………………………………………………………………………………….67 Methods and Materials……………………………………………………………………………68 Results…………………………………………………………………………………………….72 Discussion………………………………………………………………………………………...75 Chapter 8: Summary and Conclusions…………………………………………………………………….78 References…………………………………………………………………………………………………83

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LIST OF FIGURES

Figure 1. Temozolomide (TMZ) mechanism of action and cellular repair mechanism...……………….....7 Figure 2. Systemic changes induced by fasting...... 11 Figure 3. Morphology of adherent LN229, T98G, and SVG p12 cells…………………………………...20 Figure 4. Temozolomide (TMZ) dilution scheme...... 24 Figure 5. CellTiter-Glo 2.0 Assay principal...... 24 Figure 6. DMSO is cytotoxic to GBM cells...... 28 Figure 7. Glucose restriction sensitizes LN229 GBM cells to temozolomide treatment...... 29 Figure 8. Glucose restriction sensitizes T98G GBM cells to temozolomide treatment...... 30 Figure 9. Temozolomide (TMZ) dilution scheme...... 36 Figure 10. CellQuanti-Blue™ Assay principal...... 36 Figure 11. Glucose restriction sensitizes LN229 GBM cells to temozolomide treatment...... 39 Figure 12. Glucose restriction sensitizes T98G GBM cells to temozolomide treatment...... 40 Figure 13. Glucose restriction has no effect on SVG p12 normal astrocyte sensitivity to temozolomide treatment...... 45 Figure 14. Differential stress response (DSR) between normal cells and LN229 cells in response to fasting conditions...... 51 Figure 15. Glucose restricted media supplemented with ß-HB further sensitizes LN229 cells to temozolomide...... 54 Figure 16. Glucose restricted media supplemented with ß-HB further sensitizes T98G cells to temozolomide...... 55 Figure 17. Glucose restricted media supplemented with ß-HB has slight protective effects on SVG p12 normal astroglial cells sensitivity to temozolomide...... 56 Figure 18. ß-HB supplementation does not further sensitize LN229 cells cultured under glucose restricted media to temozolomide...... 57 Figure 19. ß-HB supplementation further sensitizes LN229 cells cultured under normal glucose media to high doses of temozolomide...... 58 Figure 20. ß-HB supplementation further sensitizes T98G cells cultured under glucose restricted media to temozolomide...... 59 Figure 21. ß-HB supplementation further sensitizes T98G cells cultured under normal glucose media to temozolomide...... 60

Figure 22. ß-HB supplementation protects SVG p12 cells cultured under glucose restricted media to temozolomide...... 61 Figure 23. ß-HB supplementation protects SVG p12 cells cultured under normal glucose media to temozolomide...... 62 Figure 24. Glucose restricted media and glucose restricted media plus ß-HB in combination with TMZ induces downregulates MGMT expression in T98G cells...... 74

LIST OF ABBREVIATIONS

GBM...... Glioblastoma Multiforme

TMZ...... Temozolomide

MTIC...... 5-(3-methyltriazen-1-yl) imidazole-4-carboximide

AIC...... 5-aminoimidazole-4-carboxamide

BER...... Base Excision Repair

MMR...... DNA Mismatch Repair

O6MeG...... O6-Methylguanine

T...... Thymine

DSB...... Double Stranded Breaks

MGMT...... O6-methyl-guanine-DNA methyltransferase

GLUT...... Glucose Transporter

RT-qPCR...... Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction

G6P...... Glucose 6-Phosphate

HK...... Hexokinase shRNA...... Short Hair-Pin RNA

LDHA...... Lactate Dehydrogenase A siRNA...... Small Interfering RNA

OS...... Overall Survival

IR...... Insulin Receptor

IGF1...... Insulin-Like Growth Factor Receptor 1

AKT...... RAC-Alpha Serine/Threonine-Protein Kinase mTORC1...... Mammalian Target of Rapamycin Complex 1

TCA...... Tricarboxylic Acid Cycle

KB...... Ketone Bodies

HMG-CoA....ß-Hydroxy-ß-Methylglutaryl-CoA

ß-HB...... ß-Hydroxybutyrate

BDH1...... 3-Hydroxybutyrate Dehydrogenase

OXCT1...... Succinyl-CoA by 3-Oxoacid-CoA Transferase

ACAT1...... Acetyl-CoA Acetyltransferase 1

HDAC...... Histone Deacetylase

IC50...... Half Maximal Inhibitory Concentration

STS...... Short-Term Starvation

ATCC...... American Type Culture Collection

DMEM...... Dulbecco's Modified Eagle Medium

DMSO...... Dimethyl Sulfoxide

PI3K...... Phosphoinositide 3-Kinase

AMPk...... AMP-Activated Protein Kinase

PTEN...... Phosphatase and Tensin Homolog

BCA...... Bicinchoninic Acid Assay

BSA...... Bovine Serum Albumin

GAPDH...... Glyceraldehyde 3-Phosphate Dehydrogenase

FFA...... Free Fatty Acid

SV40...... Simian Vacuolating Virus 40

KMT...... Ketogenic Metabolic Therapy

ERKD...... IRB-Approved Energy-Restricted Ketogenic Diet

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INTRODUCTION

Gliomas are the most common primary tumor of the brain.1 Gliomas arise from accumulation of mutations in glial cells of the brain. Glioblastoma multiforme (GBM) is the most aggressive type of glioma, classified by the World Health Organization as a grade IV glioma.2 The standard of care for GBM is maximal surgical resection of the tumor followed by adjuvant treatment with the chemotherapeutic agent temozolomide (TMZ) and radiotherapy. Despite these treatments, the rate of recurrence is ~90% with a median overall survival (OS), the length of time after resection of the disease the patient remains alive, of 15 months.3

TMZ is an orally administered prodrug that readily crosses the blood brain barrier and is the standard of care chemotherapeutic agent for GBM.3 TMZ is an alkylating agent that adds a methyl group on various bases of DNA, subsequently leading to base pair mismatch during DNA replication. These mismatches activate futile DNA repair mechanisms that ultimately leads to cell death. However, GBMs contain cells that are resistant to the TMZ induced cytotoxic lesions through expression of the DNA repair protein O6-methyl-guanine-DNA methyltransferase (MGMT).4–7 There is a direct correlation between

TMZ resistance and MGMT methylation status.4,6 Thus, further investigation into sensitization of TMZ- resistant cells is needed.

A common trait of cancer is the dysregulation of carbohydrate metabolism. First described in

1925 by Otto Warburg, cancer cells preferentially oxidize glucose to lactate in glycolysis, inhibiting mitochondrial respiration.8 Now known as the “Warburg effect”, this less effective pathway of ATP production demands that cancer cells must uptake and oxidize significantly more molecules of glucose to meet physiological needs. GBM oxidizes significantly more glucose than normal brain tissue.9 Glucose transporters (GLUT) 1 and 3 are significantly upregulated in GBM relative to lower grade tumors.10

Glycolysis and the Warburg effect also provides cells with intermediates needed for biosynthesis.

Glycolysis provides glucose 6-phosphate, glyceraldehyde 3-phosphate, and 3-phosphoglycerate, used to synthesize ribose sugars for nucleotides, glycerol in lipids, and nonessential amino acids respectively.11

Upregulation of key glycolytic enzymes, such as hexokinase (HK) and lactate dehydrogenase A (LDHA) is seen in GBM compared to normal brain tissue and lower grade gliomas.9,12,13 Knockdown of HK-214 and LDHA37 have both been shown to augment the efficiency of TMZ in human GBM cell lines.

Furthermore, multiple studies have identified hyperglycemic events during therapy as an unfavorable prognostic cofactor for OS in GBM patients.15,16,17–19 Thus, systemic restriction of carbohydrates could serve as a useful treatment for GBM.

One method to restrict available carbohydrates is fasting. Originally investigated for the treatment of epilepsy in 1921, fasting has since been investigated for its effects in many diseases, including cancer.20 Fasting increases the life span of rodents up to 60%, while also delaying the onset of many age related diseases.21,22 Short-term fasting (72 hours) leads to significant reduction in plasma glucose levels.

This fall in blood glucose is met with changes in hormonal signals, such as systemic reduction in insulin and insulin-like growth factor, increase in glucagon, and increase in epinephrine levels.23 Although the changes in hormonal signals appear to be involved in the cellular responses, the precise molecular mechanisms of fasting are not completely understood, but are undeniably multifactorial.

Energy allocation during fasting appears to be fundamental. That is, given a certain amount of energy at any given time, normal cells must reallocate energy between growth/replication and maintenance/repair/survival. Under fasting conditions, normal cells respond to the change in energy by reinvesting energy towards maintenance/repair/survival.24–27 Cancer cells on the other hand harbor oncogenic mutations that promotes self-sufficient growth and uncontrolled cell division independent of current energy availability. This differential stress response begins to explain why nutrient deprivation and fasting conditions have been shown to protect normal cells while sensitizing cancer cells to chemotherapy.28,29 Glucose restricted media has been shown to enhance the response of GBM cell lines to

TMZ, with no difference in cell death noted in a primary glia cell line subjected to restricted glucose media and TMZ treatments.30 Furthermore, fasting in combination with TMZ reduced tumor volume, and increased OS of GBM bearing rodents, with significant reduction in blood glucose and IGF-1 levels seen

2 in fasted subjects.30 However, changes in blood glucose and hormonal signals are not the only responses initiated by fasting, and glucose is not the only hepatically produced and systemically released metabolite during fasting conditions.

The brain preferentially uses glucose as fuel, using up to 60-70% of the total body glucose requirements. During fasting when glucose levels are decreased, the brain begins to utilize ketone bodies

(KB). KB are water soluble molecules synthesized by the liver and systemically released during fasting.

During fasting, Fatty acids (FAs) are transported to the liver where they are oxidized to acetyl-CoA.

Acetyl-CoA serves as the starting material for KB synthesis, where three acetyl-CoA molecules condense in a series of reaction to eventually form the KB β-hydroxybutyrate (β-HB). β-HB is released into circulation where it can be utilized as an energy source for the heart, skeletal muscle, kidney, and brain.31–35 Once in the cells of brain, β-HB is cleaved to form two molecules of acetyl-CoA in a series of reactions catalyzed by 3-hydroxybutyrate dehydrogenase (BDH1), 3-oxoacid-CoA transferase (OXCT1), and acetyl-CoA acetyltransferase (ACAT1). Unlike normal brain tissue, GBM tissue expresses low levels of OXCT1 and BDH1.36 Furthermore, β-HB has been previously shown to have inhibitory effects on histone deacetylase 1 (HDAC-1).37–39 HDACs are nuclear enzymes that function in epigenetic modifications through chromatin remodeling. Interestingly, HDAC-1 is found to be overexpressed in

GBM compared to normal brain tissue.40 Furthermore, HDAC inhibition by β-HB augmented the cytotoxic efficiency of TMZ in TMZ-resistant GBM cells.41 Thus, β-HB production during fasting could have additional anti-tumor effects that slows growth and sensitizes GBM to TMZ.

This study sought to explore the effects of fasting in combination with TMZ in human GBM Cell lines, we hypothesize that exposure to fasting conditions (glucose restricted and β-HB supplemented culture medium) will further potentiate cell death of GBM cells compared to either treatment alone.

Furthermore, this study sought to explore the effect of fasting conditioned media in combination with

TMZ in a normal glial cell line. A secondary goal of this study was to determine whether changes seen between fasting conditions and normal conditions were due to changes in the expression of MGMT, the

3 highly characterized gene involved in TMZ resistance. Should fasting effectively enhance the efficiency of TMZ in GBM cells while sparing normal brain cell cells, it may warrant further investigation in order to improve treatment efficiency in GBM.

CHAPTER 1: LITERATURE REVIEW

Introduction

Glioblastoma multiforme (GBM) is the most aggressive type of glioma and most lethal of adult brain tumors. The standard of care for GBM is maximal surgical resection of the tumor followed by adjuvant treatment with the chemotherapeutic agent temozolomide (TMZ) and radiotherapy. However,

GBM contains TMZ-resistant cells that contribute to repopulation of the tumor and recurrence of disease.

A hallmark of cancer is the presence of altered carbohydrate metabolism. The Warburg effect describes the process by which cancer cells preferentially oxidize glucose to lactate in glycolysis, even in the presence of oxygen. This less efficient method of ATP production per glucose molecule means cancer cells must uptake significantly more glucose to meet physiological needs. Fasting is a recently emerging practice that has been shown to have beneficial effects in cancer therapy, although the mechanism of action is not completely understood. This chapter highlights the mechanism of action of TMZ, the

Warburg effect in GBM, and the systemic changes that occur during fasting, with emphasis on the overlap of these different areas of research.

Temozolomide: Mechanism of action, DNA repair mechanisms, and cellular resistance.

Temozolomide (TMZ) is an orally administered prodrug that readily crosses the blood brain barrier and is the most commonly used chemotherapy agent for GBM.3 TMZ is a monofunctional DNA alkylating agent that is stable in an acidic environment, but is labile above pH 7. Following absorption,

TMZ is rapidly degraded under physiological pH to the active alkylating agent 5-(3-methyltriazen-1-yl) imidazole-4-carboximide (MTIC).42 MTIC reacts with water and breaks down to form 5-aminoimidazole-

4-carboxamide (AIC) and a methyldiazonium cation. The highly reactive methyldiazonium cation acts as a nonspecific alkylating agent that methylates DNA purinic bases at N7-guanine (70%), N1-adenine (12-

15%), N3-methyladenine (10%), and O6-guanine residues (6-8%).42,43 Methylation at N7-guanine, N1- adenine, and N3-adenine are accountable for the majority of methyl adducts. However, they are not

5 responsible for cytotoxic effects for they are readily repaired by the base excision repair (BER) system. It is the DNA mismatch repair (MMR) processing of the O6- methylguanine (O6MeG) lesions that is responsible for the cytotoxic effects of TMZ.

During DNA replication, TMZ induced O6MeG in the template strand is mispaired with thymine

(T) in the daughter strand, alerting MMR system (Figure 1).44,45 MMR is a highly conserved DNA repair system that repairs DNA base mismatches in the daughter strand created during DNA replication. In

MMR, base mismatches are recognized by the heterodimeric complex consisting of MSH2 and MSH6.

MSH2-MSH6 recruits another complex between MLH1 and PMS2 to initiate the MMR process. MMR excises T from O6MeG -T in the daughter strand. However, the O6MeG persists in the template strand, and subsequent mispairing of O6MeG with another thymine leads to repetitive rounds of MMR processing.46 These futile rounds of MMR processing and repair generates DNA double stranded breaks

45–48 (DSBs) during the late S/G2 phase of the cell cycle. Accumulation of DSBs triggers G2/M phase cell cycle arrest and late caspase activation.45 Thus, the cytotoxic effects of TMZ are due to the accumulation of double stranded breaks in the DNA that arrests the cell in the G2/M phase of the cell cycle, and ultimately induce apoptosis.43–46,49,50,50 Unfortunately, “Multiforme” describes the heterogeneous population of cells that are present within GBM, and cytotoxicity to TMZ is not equal in all cells lineages.

A population of TMZ-resistant cells has been identified in GBM that are able to reverse the TMZ induced cytotoxic lesions by expressing the DNA repair protein O6-methyl-guanine-DNA methyltransferase (MGMT) (Figure 1). MGMT functions by demethylating O6MeG, reversing TMZ mechanism of action, and contributing to chemoresistance.4–7 Furthermore, a direct correlation has been identified between TMZ resistance and the methylation status of MGMT.4,6 These cells contribute to repopulation of the tumor and recurrence of disease. Thus, further investigation into sensitization of

TMZ-resistant cells is needed.

Figure 1. Temozolomide (TMZ) mechanism of action and cellular repair mechanism. TMZ methylates DNA purinic bases at N7-guanine, N1-adenine, N3-methyladenine, and O6-guanine residues. N7MeG, N1MeG, N1MeA, and N3MeA are non-cytotoxic residues for they are readily repaired by the base excision repair (BER) system. O6- methyl-guanine-DNA methyltransferase (MGMT) expressed by cells readily removes the methyl group from O6MeG, contributing to cell survival and TMZ resistance. Only in MGMT negative cells does O6MeG persist in the DNA, where it is mispaired with thymine during DNA replication. This alerts the mismatch repair (MMR) system which excises thymine from the daughter strand of DNA. However, MMR fails to remove the methyl group so O6MeG persists in the template strand. This induces a futile MMR cycles that causes double-stranded breaks (DSBs) in the DNA, leading to late caspase activation and apoptosis.

The Warburg Effect in Cancer and Its Role in GBM A common trait of cancer is the presence of altered carbohydrate metabolism. The metabolic dysregulation of cancer cells was first discovered in 1925 by Otto Warburg.8 Warburg showed that even in the presence of oxygen, carcinoma cells preferentially oxidize glucose to lactate in glycolysis, inhibiting mitochondrial respiration. Now known as the “Warburg effect”, this less effective pathway of

ATP production per glucose molecule demands that cancer cells must take up and oxidize significantly more molecules of glucose to meet physiological needs. Glucose transporters (GLUT) are transmembrane proteins that facilitate the translocation of glucose across the cell membrane. In the brain, GLUT1 &

GLUT3 serve as the main neuronal glucose transporters.51–55 Through real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) analyses, one study found GLUT1 and GLUT3 to be upregulated in GBM relative to lower grade brain tumors, and extent of GLUT3 expression was found to be directly correlated to decreased OS.10 Another research group found that long term treatment of human

GBM cell lines T98G and U373 with TMZ increased the expression of GLUT3, suggesting a correlation between glucose uptake and acquired TMZ resistance.56 However, the Warburg effect is not exclusive to cancer cells, and similar method of aerobic glycolysis has been shown to occur in proliferating lymphocytes, macrophages, and pluripotent embryonic stem cells.57–60 Looking only at the rate of ATP production, the Warburg effect is much less energetically favorable than oxidative phosphorylation, so why do cancer, and proliferating cells, experience the Warburg effect?

One explanation for the use of the Warburg effect is it provides cells with intermediates needed for biosynthesis. Following glucose transport into the cell, glucose is converted to glucose 6-phosphate

(G6P) by hexokinase (HK) in the first step of glycolysis. G6P is important intermediate in the pentose phosphate pathway for the synthesis of ribose sugars, crucial components of nucleic acids. While HK-1 is the primary isoform found in the brain, gene set enrichment analysis of 25 GBM patients and 10 normal brains showed HK-1 to be significant downregulated and HK-2 significantly upregulated in GBM compared to normal brain tissue.12 A related study demonstrated that short hair-pin RNA (shRNA) knock

8 down of HK-2 decreased production of lactate and increased sensitivity to TMZ in U87 and U373 GBM cell lines.14 In addition to ribose, intermediates in the glycolytic pathway such as glucose 6-phosphate, glyceraldehyde 3-phosphate, and 3-phosphoglycerate can be used in the biosynthesis of glycogen, glycerol for lipids, and nonessential amino acids (serine, glycine, cysteine, etc.) respectively.11

The final step in the Warburg effect is the conversion of pyruvate to lactate catalyzed by lactate dehydrogenase A (LDHA). Although once thought of as an unusable byproduct of aerobic glycolysis, lactate has been shown to stimulate angiogenesis, promote migration, and contribute to immune evasion.61–64 The presence of the Warburg effect has been identified in GBM through evaluation of the fermentation of glucose to lactate. Analysis of pyruvate/lactate ratios revealed a 300% increase in glycolytic fermentation in GBM compared to non-neoplastic brain tissue.9 Immunohistochemistry (IHC) analysis of 9 grade IV (GBM), 7 grade III, and 10 grade II gliomas found LDHA expression to be significantly higher in grade IV (P<0.005) compared to the grade II samples.13 Additionally, small interfering RNA (siRNA) silencing of LDHA has been demonstrated to inhibit proliferation and induce apoptosis in a variety of GBM cell lines13,65. Furthermore, both siRNA knockdown of LDHA and LDHA inhibitors were able to augment the efficiency of TMZ in GBM cell lines compared to TMZ alone.13,65

These study allude to the importance of the Warburg effect in GBM growth and therapy resistance.

If glucose consumption promotes tumor growth and contributes to therapy resistance, then systemic glucose concentrations might be correlated to progression of disease and overall survival (OS).

A group of researchers from Mainz, Germany conducted a retrospective analysis on the effect of hyperglycemic events (blood glucose levels >10 mM) on OS during adjuvant therapy of 106 GBM patients. They concluded that patients who experience one or more hyperglycemic events had a 48.2% decrease in median OS (8.8 months) compared to patients who experience ≤ 1 such events

(16.6 months).15 A similar study of 191 GBM patients found that patients with mean blood glucose levels of >7.6 mM had a 37% decrease in median OS (9.1 months) compared to patients with mean glucose levels of <5.2 mM (14.5 months).16 Similar results supporting hyperglycemic events as an unfavorable

9 prognostic cofactor for OS has been shown in a multitude of other studies.17–19 Thus, systemic restriction of carbohydrates could serve as a useful treatment for GBM.

Fasting: Systemic Changes and Role in Cancer Therapy

The clinical relevance of fasting was first investigated for the treatment of epilepsy in 1911.66

They described seizures of lesser severity in patients who fasted compared to non-fasting epileptic patients. In the years since this observation, intermittent fasting has been the focus of many studies for its effects on cardiovascular disease, epilepsy, diabetes, depression, obesity, and cancer.24,31,67–70 In a fed state, insulin and insulin-like growth factor 1 (IGF1) are produced by the liver and systemically released where they bind to insulin receptor (IR) and insulin-like growth factor 1 receptors (IGFR-1) on the extrahepatic cells respectively. IGFR-1 is a receptor tyrosine kinase that regulates not only glucose metabolism but cell growth via the downstream RAC-alpha serine/threonine-protein kinase (AKT)/mammalian target of rapamycin complex 1 (mTORC1) pathway. Low blood glucose events due to fasting is detected by the pancreas, causing a reduction in the production of insulin, reduction in insulin-like growth factor (IGF), and an increase in the production of glucagon and epinephrine.71 A study on the effects of fasting and the concentration of glucose, insulin, and counter regulatory hormones in healthy individuals found that 72 hours of fasting decreased insulin levels from 421 pM to 127 pM, increased glucagon from 10 pM to 23 pM, and increased epinephrine levels from 8.4 ng/L to 20.9 ng/L.23 Plasma glucose levels fell from 93.7 mg/dL to 66.63 mg/dL after 2 days of fasting, and remained stable for the remainder of the fast.23 This observation can be explained by the liver’s response to changes in hormonal signals under nutrient deprived conditions. Under these conditions, glucagon and epinephrine signaling triggers glucose production in the liver via gluconeogenesis (figure 2). Glucagon signals the breakdown of glycogen to glucose 6-phosphate in the liver. Glucose 6-phosphate is hydrolyzed in the liver to form glucose that is systemically released to serve as fuel for the body.72 Once hepatic glycogen levels have been depleted, glucogenic amino acids are converted by the liver into citric acid cycle (TCA) intermediates and used to make glucose. Normal cells rely on these signals for reallocating energy between growth/reproduction

10 and maintenance/repair/survival. Under fasting conditions, cells respond to the change in the regulatory signals by reinvesting energy towards maintenance/repair/survival.24–27 Cancer cells on the other hand harbor oncogenic mutations that promotes self-sufficient growth and uncontrolled cell division independent of systemic growth hormone signaling.

Figure 2. Systemic changes induced by fasting. The body responds to fasting conditions by the systemic release of catabolic hormones glucagon, epinephrine, and cortisol. Drop in blood glucose levels is detected by the pancreas which by releasing glucagon. Cortisol signaling from the adrenal glands mobilizes fatty acids by activating hormone sensitive lipase (HSL) to breakdown triacylgltriaycerides (TAG) in adipose tissue. Epinephrine released by the adrenal gland stimulates proteolysis in skeletal muscle. Glucagon signaling in the liver stimulates gluconeogenesis and ketogenesis. Hepatic uptake of amino acids from skeletal muscle are used along with glycerol as the starting material for gluconeogenesis. ß-oxidation of fatty acids in the liver yields acetyl-CoA, which is used as the starting material for the synthesis of ketone bodies. Both glucose and ketone bodies are released systemically to act as energy sources for the rest of the body.73

Due to mutations in growth signaling genes in cancer, nutrient deprivation and fasting conditions have been shown to protect normal cells while sensitizing cancer cells to chemotherapy. A study investigating the response of normal rat glial cells cultured in glucose restricted media (0.5 g/L) and exposed to a high dose of the alkylating agent, cyclophosphamide (12 mg/mL), reported glucose restriction significantly decreased cyclophosphamide cytotoxicity (80% survival) compared to rat glial cells cultured in 1.0 g/L glucose (20% survival).28 Interestingly, the same dose of cyclophosphamide combined with glucose restricted media did not affect the cytotoxicity in two of three rat glioma cell lines

(C6 and A10-85), a human glioma cell line (LN229), and a human neuroblastoma cell line (SH-SY5Y ).

Cytotoxicity in the third rat glioma cell line (RG2) was significantly increased cyclophosphamide treatment under restricted glucose conditions.28 This same study also investigated the effect of high dose treatment of etoposide in fasted mice. Etoposide is a chemotherapy agent used for the treatment of testicular cancer, small cell lung cancer, and neuroblastoma.74 DNA topoisomerases II are essential enzymes to DNA replication that functions to relieve mechanical stress produced during DNA replication.

Etoposide forms a complex with topoisomerase II and DNA, leading to inhibition of topoisomerase II and the subsequent generation of double stranded breaks.75 The study reported that while treatment with a high dose of etoposide (100 mg/kg body weight) was lethal to 56% of ad-lib fed mice, no casualties were seen in mice who fasted 48 hours before treatment with the same dose of etoposide.28 Another extensive study by Changhan Lee et al., 2010 looking at the effects of fasting in combination with chemotherapy agents (doxorubicin or cisplatin) found that mice fasted for 48 hours (complete food withdrawal with water access) had a 50% reduction in tumor volume and an increased OS compared to ad-lib fed mice harboring murine melanoma, breast, neuroblastoma, and glioma tumors. To identify gene expression patterns associated with fasting conditions, they conducted genome-wide microarray analysis of non- neoplastic liver, heart, skeletal muscle, and subcutaneous 4T1 breast tumor tissue from mice previously fasted. They found that fasting differentially regulates proliferation-associated genes, such as insulin signaling adaptor, mitogen-activated protein kinase 6, homeobox protein TGIF1, eukaryotic translation initiation factor 4E-binding protein 1, cyclin-dependent kinase inhibitor 1, and p53-regulated protein

PA26, in which downregulation was seen in non-neoplastic tissue. Either up regulation or no change in expression was seen in the breast cancer tissue for the same genes. In order to determine a possible mechanism by which fasting augments chemotherapy (with cisplatin) and reduces tumor progression,

Changhan Lee et al., 2010 cultured melanoma (B16 cells) and breast (4T1 cells) in growth medium mimicking glucose concentrations in the ad lib-fed (2.0 g/L glucose) or fasting mice conditions (0.5 g/L glucose). Traditional cell culture medium is supplemented with 10% fetal bovine serum (FBS). In order to mimic the fasting induced reduction of growth factors like IGF-1 and insulin, all cell medium also contained 1% FBS. Following exposure to cisplatin (8 mM), a 20-fold increase in DNA damage was seen in both 4T1 and B16 cells exposed to fasting glucose concentrations compared to ad-lib glucose concentrations.29 This study demonstrated the differential stress response produced by fasting, protection of normal cells while sensitizing cancer cells to chemotherapy. However, changes in blood glucose and hormonal signals is not the only response initiated by fasting, and glucose is not the only hepatically produced and systemically released metabolite during fasting conditions.

The brain preferentially uses glucose as fuel, using up to 60-70% of the total body glucose requirements. When glucose levels are decreased, the brain begins to utilize ketone bodies (KB) as fuel.

KB are constitutively synthesized by the liver, detectable at micromolar levels (0.1-0.2 mM). However, in response to 48 hour fasting, KB serum concentrations can reach levels of 1-2 mM and upwards of 6-8 mM during prolonged starvation.34 KB are water soluble molecules synthesized by liver and systemically released to serve as fuel for the heart, skeletal muscle, kidney, and brain.31–35 Low insulin/IGF-1, and elevated glucagon, epinephrine, and cortisol signaling triggers the mobilization of fatty acids (FAs) from adipose tissue. Once systemically released, FAs are transported to the liver are subsequently oxidized to form acetyl-CoA, the starting material for ketone body synthesis (figure 2). In the liver, two acetyl-CoA condense to form acetoacetyl-CoA. Acetoacetyl-CoA is condensed with another acetyl-CoA molecule to form ß-hydroxy-ß-methylglutaryl-CoA (HMG-CoA). Subsequent cleavage of HMG-CoA by HMG-CoA lyase forms the first ketone body, acetoacetate. Acetoacetate is reduced by β-hydroxybutyrate

13 dehydrogenase to form the ketone body β-hydroxybutyrate (β-HB). The conversion of acetoacetate to β-

HB has two important functions. First, it stores a NADH energy equivalent that will be used by extrahepatic cells. Second, being that acetoacetate is a β-ketoacid, it is vulnerable to decarboxylation into the volatile waste product acetone. By converting acetoacetate to β-HB, a more stable molecule is produced that can be exported into circulation.73 Once in circulation, β-HB enters the brain through monocarboxylate transporter 1 and 2 (MCT1/2) expressed on the blood brain barrier.76 Once in the brain,

β-HB is oxidized back in the mitochondria to acetoacetate by 3-hydroxybutyrate dehydrogenase (BDH1), generating a molecule of NADH. Acetoacetate is activated by a transfer of CoA from succinyl-CoA by 3- oxoacid-CoA transferase (OXCT1), which is then cleaved by acetyl-CoA acetyltransferase 1 (ACAT1) to form two acetyl-CoA molecules to be used in energy production via TCA. However, β-HB is differentially utilized by normal and neoplastic brain tissue, and β-HB has additional inhibitory effects on class I histone deacetylase (HDAC) proteins.

GBM tissue has been shown to express low levels of enzymes involved in KB metabolism, OXCT1 and BDH1. IHC Analysis of 17 GBM specimens revealed low (5-20% of tumor cells) or very low (> 5% of tumor cells) expression of OXCT1 in 14 of 17 GBM samples. This same study found BDH1 expression to be either low or very low in all 17 GBM samples.36 β-HB has previously been shown to act as an inhibitor of HDAC-1.37–39 HDACs are nuclear enzymes that function in chromatin remodeling through the deacetylation of chromatin. Class I HDAC have previously been demonstrated to be overexpressed in GBM compared to normal brain tissue.40 Additionally, inhibition of class I HDAC by a variety of HDAC small molecule inhibitors significantly reduced proliferation of U87, LN18, and patient derived GSC cell lines.40,77 Furthermore, HDAC inhibition by β-HB was shown to sensitize TMZ- resistant GBM cells to TMZ.41 Thus, β-HB production during fasting could have additional anti-tumor effects that slows growth and sensitizes GBM to TMZ.

Fasting has been shown to enhance the response of GBM to TMZ both in vivo and in vitro. As proof of concept, one study found a three-fold lower TMZ half maximal inhibitory concentration (IC50)

14 value when A172, U251, and LN229 human GBM cell lines, and C6 and GL26 rat GBM cell lines were exposed to glucose restricted media (0.5 g/L glucose) compared to normal glucose media (2.0 g/L).30

Additionally, no difference in cell death was noted in the primary glia cell line when subjected to restricted glucose in combination with TMZ.30 This same study then monitored the response in vivo using luciferase firefly gene expressing GL26 (GLS6luc) murine cell line implanted intracranially in the right frontal lobe of C57BL/6 mice. Six days post implant, mice were subjected to 48 hours of short-term starvation (STS) with TMZ administration (15 mg/Kg) at 24 and 48 hours of STS. They found that STS in combination with TMZ significantly reduced tumor volume compared to the control and either treatment alone (p<0.05).30 Additionally, OS was slightly increased, with one subject alive 80 days after tumor implantation (p<0.05).30 In order to mimic the oncologic treatment conditions of human patients, they tested the effects of multiple chemotherapy and fasting cycles. Four days post GL2luc intracranial implantation, mice were subjected to STS for 48 hours with administration of TMZ at 24 and 48 hours.

Food was implemented days 6-9 before a second 24 hour fast followed by a second cycle of TMZ treatment. OS was significantly increased in STS + TMZ group compared to TMZ alone and control groups (p<0.01). Furthermore, OS was increased compared to mice who received a single dose of

TMZ/STS, suggesting that the additional second cycle of TMZ and STS can further delay morbidity.30

This study also monitored the effects of STS alone, TMZ alone, and STS + TMZ has on blood glucose and IGF-1 levels. Following 48 hours STS, a 70% reduction in blood glucose was seen in both STS and

STS + TMZ groups compared to control group, with no difference seen TMZ alone compared to control group. Furthermore, STS + TMZ showed a 60 % decrease in systemic IGF-1 levels compared to TMZ alone.30 The drop in both glucose and IGF-1 was thought to be a contributor to how STS further sensitizes

GBM cells to TMZ. Further investigation is needed to determine whether the multifactorial changes that occur during fasting can augment the efficiency of TMZ in GBM. In accordance with any new therapy, proper measures must also evaluate the effect of fasting in combination with TMZ on normal cells to prevent unintended effects and assure fasting as a safe practice.

Conclusion

TMZ is the standard chemotherapy agent used for the treatment of glioblastoma multiforme.

However, due to the presence of TMZ-resistant cells that can reverse the TMZ induced cytotoxic lesions by expressing MGMT, reoccurrence of disease is inevitable. A common trait of cancer is an altered carbohydrate metabolism. Known as the Warburg effect, cancer cells will preferentially oxidize glucose to lactate in glycolysis, inhibiting mitochondrial respiration. This less effective pathway of ATP production per molecule of glucose means cancer cells must uptake significantly greater amounts of glucose to sustain physiological needs. One method that directly targets the bioavailability of glucose is fasting. Short term fasting (72 hours) lowers systemic glucose levels, lowers anabolic hormone levels

(insulin and IGF-1), and elevates ketone bodies like ß-HB. Normal cells respond to this change in glucose bioavailability by reinvesting energy towards repair/maintenance/survival. However, cancer cells harbor oncogenic mutations that impair their ability to adjust to these changes. This differential response to nutrient deprivation and fasting conditions has been shown to protect normal cells while sensitizing cancer cells to chemotherapy. While the potential for the use of fasting in combination with chemotherapy for the treatment of cancer is supported by the literature, studies evaluating the effect of fasting in combination with TMZ for GBM is minimal. Studies designed to shed light on the effects fasting in combination with TMZ has on cell viability and expression of MGMT in GBM cells is needed to help evaluate its potential as a therapeutic agent for the treatment of GBM.

CHAPTER 2: HYPOTHESIS AND AIMS.

As suggested in the literature, the effects of fasting have been shown to lower systemic blood glucose and IGF-1/insulin production while increasing ketone body production. Previous in vitro analysis of glucose restriction, serum restriction, and ketone body supplementation in combination with TMZ has been shown to protect normal glial cells while increasing sensitivity of GBM cells. Thus, fasting induced systemic effects prior to treatment may assist in augmenting the efficiency of chemotherapy, while helping to protect normal cells against the cytotoxic effects of chemotherapy.

The purpose of this study is to evaluate the effects fasting conditions in combination with TMZ in human GBM cell lines. I hypothesize that exposure to physiological fasting conditions (glucose restricted and β-HB supplemented culture medium) prior to, and during TMZ treatment further potentiates cell death of GBM cells compared to either treatment alone or control groups. Additionally, I hypothesize that prior exposure to fasting conditions will protect normal glial cells against cytotoxic effects of TMZ.

I will use 3 specific aims to evaluate this hypothesis.

Aim 1: To test the cytotoxicity of LN229 GBM cells, T98G GBM cells, and SVG p12 astroglial cells exposed to glucose restricted media in combination with TMZ. Following a 24 hour exposure to either glucose restricted media (0.5 g/L glucose) or normal glucose media (2.0 g/L), I will treat cells with varying doses of TMZ for an additional 96 hours and perform cytotoxicity assay using CellTiter-Glo® 2.0

Cell Viability Assay and CellQuanti-Blue™ cell viability assay kit.

Aim 2: To test the cytotoxicity of LN229 GBM cells, T98G GBM cells, and SVG p12 astroglial cells exposed to ß-HB supplemented media in combination with TMZ. Following a 24 hour exposure to either glucose restricted media plus ß-HB (0.5 g/L glucose) or normal glucose media plus ß-HB (2.0 g/L,

2 mM β-HB), I will treat cells with varying doses of TMZ for an additional 96 hours and perform cytotoxicity assay using CellQuanti-Blue™ cell viability assay kit.

Aim 3: Determine if changes in cytotoxicity are due to a change in the expression of MGMT.

MGMT expression will be determined via immunoblotting using cells exposed to TMZ alone or in combination with glucose restricted media and fasting conditioned media.

CHAPTER 3: CULTURE AND MAINTENANCE OF CELL LINES.

Introduction

Human cell culture has long been used as significant tools in cancer research. The first cancer cell line was established in 1951 by Dr. George Gey, Mary Kubicek, and coworkers at Johns Hopkins

University. The cells obtained from a biopsy of a cervical tumor remained viable in a nutrient solution of chicken plasma.78,79 Although the medium and conditions for which human cells are grown has changed over the years, the nature of cancer cells to sustain replicative ability and achieve immortality has remained true. The experiments in the subsequent chapters of this thesis involve the use of established

GBM cell lines and SV40 transformed human astroglia cell line purchased from American Type Culture

Collection (ATCC). This chapter described the procedures used to culture and maintain these GBM cell lines.

Material and Methods

Established Cell Line Culture: Established GBM cell lines typically grow as adherent cultures that maintain rapid proliferative abilities and are insensitive to contact inhibition. Adherent human GBM cell lines LN229 and T98G as well as SVG p12 human astroglial cells (ATCC) were cultured in specially formulated Dulbecco's Modified Eagle Medium (DMEM) without glucose, L-glutamine, and sodium pyruvate (DMEM; Corning, Manassas, VA) supplemented with 4.5 g/L D-(+)-glucose (Sigma-Aldrich,

St. Louis, MO), 0.29 g/L L-glutamine (Gibco, Madison, WI), 0.11 g/L sodium pyruvate (Gibco), and 10% fetal bovine serum (FBS; Atlanta Biological, Atlanta, GA). Culture medium was sterile filtered using a

0.1 µm asymmetrical polyethersulfone membrane filter (ThermoFisher Scientific, Whaltham, MA). All cells were cultured under standard conditions (37 °C and 5% CO2).

Passaging of Cell Lines: Cells were passaged when confluence reached approximately 90%.

Cells were enzymatically disassociated from the flask using Trypsin/EDTA (Gibco), cells were

19 resuspended in DMEM, and centrifuged at 280 rcf for three minutes. Supernatant was removed and cell pellet was resuspended in DMEM. Cell count was determined using trypan blue exclusion method and cells were subsequently subcultured at the desired confluency in new flasks.

Figure 3. Morphology of adherent LN229, T98G, and SVG p12 cells. (a) Image of LN229 GBM cells. (b) Image of T98G GBM cells. (c) Image of SVG p12 human astroglial cells. All cells were culture under standard conditions in specialty DMEM supplemented with 4.5 g/L D-(+)-glucose, 10% FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate. Scale bar indicates a length of 250 µm. Images were obtained at 10X objective using EVOS M7000 imaging system.

CHAPTER 4: ANALYZING CYTOTOXICITY OF GLUCOSE RESTRICTED MEDIA IN

COMBINATION WITH TEMOZOLOMIDE IN GBM CELLS.

Introduction

Temozolomide (TMZ) is the standard chemotherapy agent for the treatment of GBM. Fasting induces systemic changes such as reducing anabolic hormones and systemic glucose concentration.

Glucose restriction has been shown to augment the efficiency of chemotherapy against cancer cells while having protective effects in normal cells. The experiments in this chapter were designed to analyze the effects of glucose restriction in combination with temozolomide in human GBM LN229 and T98G cell lines (ATCC) through CellTiter-Glo® 2.0 cell viability assay (Promega, Madison, WI). The data indicates that glucose restriction does augment the efficiency of TMZ in LN229 and T98G GBM cell lines.

However, the vehicle (DMSO) used to carry the drug also proved to be cytotoxic to both GBM cell lines.

Therefore, this initial evidence suggests fasting may be a useful method to increase the efficiency of TMZ for the treatment of GBM, but further investigation is warranted.

Methods and Materials

Preparation of Cell Culture Medium: Normal glucose media, DMEM containing 2.0 g/L D-(+)- glucose (Sigma-Aldrich), 0.29 g/L L-glutamine (Gibco), 0.11 g/L sodium pyruvate (Gibco), and 10%

FBS (Atlanta Biological), was prepared as stated previously. Glucose restricted media was prepared through a 1:3 dilution of the normal glucose media into glucose-free DMEM containing, 0.29 g/L L- glutamine (Gibco), 0.11 g/L sodium pyruvate (Gibco), and 10% FBS (Atlanta Biological). The final concentration glucose restricted media was 0.5 g/L g/L D-(+)-glucose.

Preparation of Temozolomide: Temozolomide (TMZ) was purchased from Sigma-Aldrich (St.

Louis, MO) and dissolved into 99.7% Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich) to a concentration of

50 mM. To ensure complete solubility of TMZ in DMSO, the solution was vortexed at maximum speed.

Serial dilution of the 50 mM TMZ stock was then performed 1:1 in DMSO to achieve the following

TMZ-DMSO concentrations: 50 mM, 25 mM, 12.500 mM, 6.250 mM, 3.125 mM, 1.562 mM, 0.781 mM,

0.391 mM, and 0.195 mM. The stock concentrations was subsequently used to create working concentrations in glucose restricted media (DMEM, 0.5 g/L glucose, 0.29 g/L L-glutamine, 0.11 g/L sodium pyruvate, 10% FBS) and normal media (DMEM (2.0 g/L glucose, 0.29 g/L L-glutamine, 0.11 g/L sodium pyruvate, 10% FBS) that all contained equal amounts of DMSO (2% DMSO v/v DMEM). A control solution of 2 % DMSO in both glucose restricted DMEM and normal DMEM was also prepared

(Figure 4B).

Cell Line Culture: Cells were cultured in DMEM as previously discussed. Briefly, cells were detached from flasks using trypsin-EDTA, pelleted by centrifugal force, and resuspended into one mL of

DMEM. Cell count of cell suspension was subsequently done using trypan-blue exclusion method. In order to minimize pipetting errors, appropriate amount of cell suspension was added to a conical tube containing DMEM to achieve a final concentration of 50,000 cells/mL, seeded 5,000 cells/well (100 µL) into 96-well tissue culture treated microplates (Corning) using a multi-channel pipettor, briefly centrifuged at 32 rcf to expedite attachment, and incubated for 2 hours under standard conditions.

At 2 hours after plating, the original complete medium was carefully removed using a multichannel pipettor and replaced with either glucose restricted media (DMEM, 0.5 g/L glucose, 0.29 g/L L-glutamine, 0.11 g/L sodium pyruvate, 10% FBS) or normal complete media DMEM, 2.0 g/L glucose, 0.29 g/L L-glutamine, 0.11 g/L sodium pyruvate, 10% FBS) for 24 hours. After 24 hours, media was removed and replaced with varying concentration of TMZ or the DMSO control using a multichannel pipettor. The final volume of media was 100 µL per well and each treatment group was prepared in triplicate. Plates were sustained in standard conditions. TMZ treatment lasted a total of 96 hours.

Cell viability assay: Following 96-hour exposure to TMZ, cytotoxicity was assessed by luminescence using CellTiter-Glo 2.0 cell viability assay (Promega) per manufacture’s protocol. Briefly,

CellTiter-Glo 2.0 reagent was removed from -20 °C and allowed to equilibrate to room temperature (RT).

Following this, 100 µL of CellTiter-Glo 2.0 reagent was added at a 1:1 ratio of cell culture medium to each well. Cell lysing was stimulated through agitation by placing plates on an orbital shaker for two minutes. Luminescent signal was stabilized through a ten-minute incubation at RT in the absence of light.

Finally, luminescence was recorded using a Modulus II Microplate Reader (Turner Biosystems,

Sunnyvale, CA). CellTiter-Glo is an extremely sensitive luciferin-based assay that determines the number of viable cells by quantifying intracellular ATP levels (Figure 5).

Statistical Analysis: Reported values indicate the mean luminescence values of three replicates from CellTiter-Glo 2.0 assay, adjusted to the blank controls, and converted to percent viability relative to the adjusted, average vehicle control luminescence values (equation 1). IC50 of TMZ for each cell line was generated by plotting the average cell viability for each dose against the log function of their respective TMZ dose (µM), fitted to a dose response curve in Prism 5 (GraphPad Software, San Diego,

CA). Significance was denoted by one asterisk (*) where p<0.05, two (**) where p<0.01, and three (***) where p<0.001. All data presented show the standard deviation of the mean (SD).

To determine the effects of glucose restriction and DMSO on cell viability, mean luminescence values of three replicates were converted to cell viability as previously described. The graph was generated by potting the average cell viability under each condition fit to a group column analysis in

Prism 5 (GraphPad Software). Differences between groups was found using two-way ANOVA and

Significance was denoted by one asterisk (*) where p<0.05, two (**) where p<0.01, and three (***) where p<0.001. All data presented show the standard deviation of the mean (SD). Post Hoc analysis using tukey was performed to look for differences between groups (not shown).

푅퐿푈푠푎푚푝푙푒 − 푅퐿푈푎푣𝑔. 푏푙푎푛푘 % 푉푖푎푏푖푙푖푡푦 = ( ) × 100% 푅퐿푈푎푣𝑔. 푣푒ℎ𝑖푐푙푒 − 푅퐿푈푎푣𝑔. 푏푙푎푛푘

Equation 1. Determining cytotoxicity from CellTiter-Glo 2.0. The experimental value from each replicate (RLUsample) was subtracted by the average blank (RLUavg. blank). This value was divided by the average vehicle value (RLUavg. vehicle) that was also subtracted by the RLUavg. blank. Finally, the value was multiplied by one hundred to convert it to percent viability.

Figure 4. Temozolomide (TMZ) dilution scheme. Stock 50 mM solution of TMZ in DMSO was prepared. Subsequent 1:1 serial dilution was prepared in DMSO ranging from 50 mM-0.2 mM TMZ. Equal parts of these dilutions were used to make the TMZ working concentrations ranging from 1000 µM to 3.9 µM in culture medium. A vehicle control (DMSO) was also prepared.

Figure 5. CellTiter-Glo 2.0 Assay principal. Mono-oxygenation of luciferin catalyzed by Ultra-Glo Luciferase enzyme in the presence of Mg2+, ATP and O2 to produce a stable “glow-type” luminescent.80

Results

The effects of glucose restriction and DMSO on viability of GBM cell lines.

DMSO is an organic, aprotic solvent that is used to solubilize an abundant of pharmaceutical compounds.81 Due to its amphipathic nature, DMSO is commonly used as the solvent of choice for drug delivery during in vitro studies.82,83 However, cytotoxicity to DMSO has been demonstrated at relatively low concentrations.84 Therefore, DMSO cytotoxicity was evaluated in GBM cells LN229 and T98G incubated in both normal and glucose restricted media. Glucose restriction alone significantly inhibited proliferation of LN229 (p <0.01) (figure 6A). Furthermore, glucose restriction significantly inhibited growth of T98G cells (p <0.001) (figure 6B) to a greater extent compared to LN229 (figure 6A).

Regardless of glucose restriction, 2% DMSO (v/v) was found to be cytotoxic in both LN229 (p <0.001) and T98G cells (p <0.001) (Figure 6).

Figure 6. DMSO is cytotoxic to GBM cells. DMSO cytotoxicity was tested in GBM cell lines (A) LN229 and (B) T98G. Cells were plated at 5,000 cells per well and incubated in low glucose (0.5 g/L glucose) or normal glucose (2.0 g/L glucose) DMEM supplemented with 10% FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate for 24 hours. Percent viability was determined through CellTiter-Glo® 2.0 cell viability assay after 96-hour treatment with 2% DMSO (3.55 M) v/v DMEM (white). Reported values indicate the mean of three replicates converted to percent of untreated cells cultured in 2.0 g/L glucose in Prism 5 (GraphPad Software). All data presented as mean (SD); * p<0.05, ** p<0.01, *** p<0.001, two-way ANOVA.

Cytotoxic effects of glucose restriction in combination with temozolomide in GBM Cell lines.

We sought to determine the effects glucose restriction on the efficiency of TMZ in both LN229 and T98G cell lines. As described earlier, cells were incubated in low glucose media (0.5 g/L) or normal glucose (2.0 g/L) DMEM for 24 hours prior to, and during treatment with TMZ. Following 96 hours of

TMZ treatment, cell viability was assessed using the CellTiter-Glo® 2.0 cell viability assay. Regardless of glucose concentration, both LN229 (Figure 7) and T98G (Figure 8) cells responded to increasing concentrations of TMZ in a dose-dependent manner. The results from a 96-hour treatment with TMZ in

LN229 cells indicate that glucose restriction augments the efficiency of TMZ (Figure 7). Under normal glucose media, a dose of 632.2 µM was necessary to cause 50% cell death (IC50) in LN229 cells (Table

1). Under glucose restricted media, the IC50 value of TMZ decreased to 429.5 µM in LN229 cells.

Similar results were obtained following 96 hours treatment of TMZ in T98G cells (Figure 8). The IC50 value decreased from 469.2 µM to 301.4 µM when T98G cells were cultured in glucose restricted media.

There were no outliers found in either T98G or LN229 cytotoxicity assays.

LN229

100

l 50

% 25 0.5 g/L glucose 2.0 g/L glucose 0 0 1 2 3 Log[Temozolomide]

Figure 7. Glucose restriction sensitizes LN229 GBM cells to temozolomide treatment. GBM cell line LN229 were tested for glucose restriction induced sensitization to TMZ. Cells were plated at 5,000 cells per well and incubated in low glucose (Red, 0.5 g/L glucose) or normal glucose (Black, 2.0 g/L glucose) DMEM supplemented with 10% FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate for 24 hours. Percent viability was determined through CellTiter-Glo® 2.0 cell viability assay after 96-hour treatment with 0-1000 µM TMZ. Reported values indicate the mean of three replicates converted to percent of vehicle control (DMSO) group in Prism software. All data presented as mean (SD).

T98G

100

l i

b 50

i V

25 0.5 g/L glucose 2.0 g/L glucose 0 0 1 2 3 Log[Temozolomide]

Figure 8. Glucose restriction sensitizes T98G GBM cells to temozolomide treatment. GBM cell line T98G were tested for glucose restriction induced sensitization to TMZ. Cells were plated at 5,000 cells per well and incubated in low glucose (Red, 0.5 g/L glucose) or normal glucose (Black, 2.0 g/L glucose) DMEM supplemented with 10% FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate for 24 hours. Percent viability was determined through CellTiter-Glo® 2.0 cell viability assay after 96-hour treatment with 0-1000 µM TMZ. Reported values indicate the mean of three replicates converted to percent of vehicle control (DMSO) group in Prism software. All data presented as mean (SD).

Chapter 4. Effects of glucose restricted media in combination with TMZ (DMSO vehicle) IC value of temozolomide (µM) 50

Cell line 0.5 g/L glucose 2.0 g/L glucose P Value LN229 (GBM) 429.5 638.2 <0.001 (***) T98G (GBM) 301.4 469.2 0.026 (*) Chapter 5. Effects of glucose restricted media in combination with TMZ IC value of temozolomide (µM) 50

Cell line 0.5 g/L glucose 2.0 g/L glucose P Value LN229 (GBM) 85.29 273.5 <0.001 (***) T98G (GBM) 371.4 678.3 0.005 (**) SVG p12 Astroglial) 92.80 77.19 0.125 (NS) Chapter 6. Effects of ß-HB in combination with TMZ IC value of temozolomide (µM) 50

0.5 g/L glucose + 2.0 g/L glucose + Cell line P Value 2.0 mM ß-HB 2.0 mM ß-HB LN229 (GBM) 76.66 189.1 <0.001 (***) T98G (GBM) 177.1 442.3 <0.001 (***) SVG p12 (Astroglial) 246.1 187.8 <0.001 (***)

0.5 g/L glucose + Cell line 0.5 g/L glucose P Value 2.0 mM ß-HB LN229 (GBM) 85.29 76.66 0.508 (NS) T98G (GBM) 371.4 177.1 <0.001 (***) SVG p12 (Astroglial) 92.80 246.1 <0.001 (***)

2.0 g/L glucose + Cell line 2.0 g/L glucose P Value 2.0 mM ß-HB LN229 (GBM) 273.5 189.1 0.024 (*) T98G (GBM) 678.3 442.3 0.004 (**) SVG p12 (Astroglial) 77.19 187.8 <0.001 (***)

Table 1. Summary of Temozolomide half maximal inhibitory concentration (IC50) value in GBM and normal astrocyte cells. Cells were cultured in low glucose (0.5 g/L glucose) or normal glucose (2.0 g/L glucose), with or without ß-HB, DMEM supplemented with 10% FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate for 24 hours. Percent viability was determined through CellTiter-Glo® 2.0 and CellQuanti-Blue™ Cell Viability Assay after 96-hour treatment with 0-1000 µM and 0-1500 µM TMZ, respictively. IC50 of TMZ for each cell line was generated by plotting the average cell viability for each dose relative to their control group against the log function of their respective TMZ dose (µM), fitted to a dose response curve in Prism 5 (GraphPad Software). Summary of IC50 value is shown with respect to the chapter in which they appear.

Discussion Glucose restriction enhanced the efficiency of TMZ to different extents in both GBM cell lines

(LN229 and T98G) (Figure 7 and 8). Glucose restriction sensitized both LN229 and T98 cell lines to

TMZ treatment (Figure 7 and 8). In LN229, the IC50 value decreased from 638.2 µM (Table 1) to 429.5

µM, a 32.7 % decrease, when cultured under glucose restricted media (p <0.001) (Table 1). In T98G cells, the IC50 value decreased from 469.2 µM to 301.4 µM, a 35.8% decrease, when cultured under glucose restricted media (p <0.001) (Table 1). So, at first glance, it appears the hypothesis that glucose restriction prior to, and during TMZ treatment would further sensitize GBM cells to TMZ is supported.

When comparing the IC50 values of LN229 and T98G under both normal and glucose restricted media, this data suggests that T98G cells are more sensitive to TMZ than LN229 cells. This data is surprising based on published MGMT status of these two cell lines. As discussed earlier (Chapter 2),

MGMT’s primary function is the removal of the TMZ induced, cytotoxic O6MeG residues of DNA. It has been shown that MGMT expression is directly correlated to TMZ resistance. In general, methylation located at the gene promoter acts to repress the expression of said gene. LN229 and T98G cells were chosen for these experiments because of their published differences in MGMT promoter status. The literature suggests that LN229 cells are hyper-methylated at the MGMT promoter region, do not express

MGMT, and are thus TMZ-sensitive.85 T98G cells have an unmethylated MGMT promoter, express

MGMT and are TMZ resistant.86 In the data we obtained, IC50 values under glucose restricted media were 429.5 µM in LN229 cells and 301.4 µM in T98G cells. Under normal glucose media, the IC50 were

638.2 µM in LN229 cells and 469.2 µM in T98G cells. The higher IC50 values obtained for LN229 compared to T98G cells suggest LN229 cells are more resistant to TMZ than T98G cells, which contradicts the literature. It may be possible the cell lines were switched during analysis using the microplate reader. This could be determined by performing short tandem repeat analysis to confirm the identity of the cell lines used. It may also be possible that the vehicle control (DMSO) may have

31 differentially affected the two cell lines. Consequently, the cytotoxicity of both cell lines to the drug vehicle was evaluated.

Granular TMZ was solubilized into liquid with the use of DMSO as a vehicle carrier. The maximum solubility of TMZ in pure DMSO (14.1 M) is 10 mg/mL (50 mM). At this concentration, the amount of DMSO necessary to achieve the highest working concentration of TMZ is 2% DMSO (v/v).

The cytotoxic effect of DMSO at a concentration of 2% v/v, as well as glucose restriction, was evaluated in both LN229 and T98G cell lines (Figure 6). As expected, glucose restriction significantly inhibited growth of both LN229 (figure 6A) and T98G cells (figure 6B). However, regardless of glucose restriction

DMSO induced significant cytotoxicity of both LN229 (figure 6A) and T98G cells (figure 6B). Since

DMSO was held constant at 2% v/v during the TMZ dose-dependent experiments, we can attribute the differences in viability cultured under glucose restricted media to changes in TMZ sensitivity within each cell line. However, the results from the subsequent chapter of this thesis, to determine the effects of glucose restriction with TMZ by dissolving TMZ straight into culture medium, indicates that LN229 and

T98G cells were affected differently by the DMSO.

CHAPTER 5: ANALYZING CYTOTOXICITY OF GLUCOSE RESTRICTED MEDIA IN

COMBINATION WITH TEMOZOLOMIDE ADMINISTERED WITHOUT DMSO IN GBM AND

HUMAN ASTROGLIAL CELLS.

Introduction

Temozolomide (TMZ) is the standard chemotherapy agent for the treatment of GBM. Fasting induces systemic changes such as reducing anabolic hormones and systemic glucose concentration.

Glucose restriction has been shown to augment the efficiency of chemotherapy against cancer cells while having protective effects in normal cells. The previous chapter evaluated the effects of glucose restriction in combination with TMZ on GBM cell lines LN229 and T98G. However, the organic solvent dimethyl sulfoxide (DMSO) utilized as a vehicle proved itself to be significantly cytotoxic to both LN229 and

T98G cell lines. This initial evidence suggesting glucose restriction can augment the efficiency of TMZ against GBM cells remains somewhat unclear.

The hydrophobic side chains of TMZ make it only mildly soluble in water. Nonetheless, TMZ is soluble in water up to a concentration of 0.31 mg/mL (1.6 mM).87 The experiments in this chapter were designed to analyze the effects of glucose restriction in combination with TMZ in human GBM cell lines,

LN229 (ATCC) and T98G (ATCC), as well as a human astroglial cell line, SVG p12 without the confounding factor of DMSO as a vehicle. The data indicates that glucose restriction does augment the efficiency of TMZ in LN229 and T98G GBM cell lines. Furthermore, glucose restriction did not affect the efficiency of TMZ in SVG p12 astroglial cell line. Therefore, this initial evidence suggests fasting may be a useful method to increase the efficiency of TMZ for the treatment of GBM.

Materials and Methods

Preparation of Cell Culture Medium: Normal glucose media, DMEM containing 2.0 g/L D-(+)- glucose (Sigma-Aldrich), 0.29 g/L L-glutamine (Gibco), 0.11 g/L sodium pyruvate (Gibco), and 10%

FBS (Atlanta Biological), was prepared as stated previously. Glucose restricted media was prepared

33 through a 1:3 dilution of the normal glucose media into glucose-free DMEM containing 0.29 g/L L- glutamine (Gibco), 0.11 g/L sodium pyruvate (Gibco), and 10% FBS (Atlanta Biological). The final concentration glucose restricted media was 0.5 g/L g/L D-(+)-glucose.

Preparation of Temozolomide: Temozolomide (TMZ) was purchased from Sigma-Aldrich (St.

Louis, MO) and dissolved into glucose restricted media (DMEM, 0.5 g/L glucose, 0.29 g/L L-glutamine,

0.11 g/L sodium pyruvate, 10% FBS) and normal media (DMEM, 2.0 g/L glucose, 0.29 g/L L-glutamine,

0.11 g/L sodium pyruvate, 10% FBS) to a concentration of 1500 µM. To ensure complete solubility of

TMZ in DMEM, the solution was vortexed at maximum speed. Serial dilution of the 1.5 mM TMZ stock was then performed 1:1 in media to achieve the following working concentrations: 1500 µM, 750 µM,

375 mM, 187.5 mM, 93.75 µM, 46.88 µM, 23.44 µM, 11.72 µM, and 5.86 µM A control solution for both glucose restricted DMEM and normal DMEM was also prepared (figure 11).

Cell Line Culture: Cells were cultured in DMEM as previously discussed. Briefly, cells were detached from flasks using trypsin-EDTA, pelleted by centrifugal force, and resuspended into one mL of

At 2 hours after plating, the original complete medium was carefully removed using a multichannel pipettor and replaced with either glucose restricted media (DMEM, 0.5 g/L glucose, 0.29 g/L L-glutamine, 0.11 g/L sodium pyruvate, 10% FBS) or normal complete media DMEM, 2.0 g/L glucose, 0.29 g/L L-glutamine, 0.11 g/L sodium pyruvate, 10% FBS) for 24 hours. After 24 hours, media was removed and replaced with varying concentration of TMZ or control (media only) using a multichannel pipettor. The final volume of treatment media was 100 µL per well and each treatment

34 group was prepared in triplicate. Plates were sustained in standard incubation conditions. TMZ treatment lasted a total of 96 hours.

Cell viability assay: Following 96-hour exposure to TMZ, cytotoxicity was assessed by florescence using CellQuanti-Blue™ cell viability assay kit (BioAssay Systems, Hayward, CA) per manufacture’s protocol. Briefly, CellQuanti-Blue™ reagent was removed from 4 °C and allowed to equilibrate to RT. Media was carefully removed using a multichannel pipettor and replaced was 100 µL of DMEM-phenol red free media (corning) supplemented with 10% FBS. Following this, 10 µL of

CellQuanti-Blue™ reagent was added to each well. Cells were briefly centrifuged at 32 rcf to homogenize

CellQuanti-Blue™ reagent in the culture medium and incubated for 2 hours under standard conditions.

After two hours, measurement of CellQuanti-Blue™ reagent fluorescence intensity was measured

(excitation wavelength = 525 nm, emission wavelength = 590 - 620 nm) using a Modulus II Microplate

Reader (Turner Biosystems, Sunnyvale, CA). CellQuanti-Blue™ is a sensitive assay that utilizes resazurin-based compound (non-fluorescent) that upon reduction by metabolically active cells is converted into a resorufin (highly fluorescent). Viable cells can readily reduce resazurin, while non-viable

(metabolically inactive) cells cannot. CellQuanti-Blue™ thus determines the number of viable cells by quantifying fluorescent intensity, which is directly proportional to the number of viable cells (Figure 12).

Statistical Analysis: Reported values indicate the mean fluorescence values of three replicates from CellQuanti-Blue™ assay, adjusted to the blank controls, and converted to percent viability relative to the adjusted, average vehicle control fluorescence values (equation 2). IC50 of TMZ for each cell line was generated by plotting the average cell viability for each dose against the log function of their respective TMZ dose (µM), fitted to a dose response curve in Prism 5 (GraphPad Software, San Diego,

CA). Significance was denoted by one asterisk (*) where p<0.05, two (**) where p<0.01, and three (***) where p<0.001. All data presented show the standard deviation of the mean (SD).

푅퐹푈푠푎푚푝푙푒 − 푅퐹푈푎푣𝑔. 푏푙푎푛푘 % 푉푖푎푏푖푙푖푡푦 = ( ) × 100% 푅퐹푈푎푣𝑔. 푣푒ℎ𝑖푐푙푒 − 푅퐹푈푎푣𝑔. 푏푙푎푛푘

Equation 2. Determining cytotoxicity from CellQuanti-Blue™. The experimental value from each replicate (RFUsample) was subtracted by the average blank (RFUavg. blank). This value was divided by the average vehicle value (RFUavg. vehicle) that was also subtracted by the RFUavg. blank. Finally, the value was multiplied by one hundred to convert it to percent viability.

Figure 9. Temozolomide (TMZ) dilution scheme. Stock 1500 µM solution of TMZ in culture medium was prepared. Subsequent 1:1 serial dilution was used to make the TMZ working concentrations ranging from 1500 µM to 5.86 µM in culture medium. A vehicle control (Media only) was also prepared.

Figure 10. CellQuanti-Blue™ Assay principal. Chemical structures of resazurin and resorufin. Reductases of metabolically active, viable cells reduce non-fluorescent resazurin in the presence of NADH into the highly fluorescent product resorufin.

Results

Cytotoxic effects of glucose restriction in combination with temozolomide in GBM Cell lines

We sought to determine the effects of glucose restriction on the efficiency of TMZ in LN229 and

T98G GBM cell lines. As described earlier, cells were incubated in low glucose media (0.5 g/L) or normal glucose (2.0 g/L) DMEM for 24 hours prior to, and during treatment with TMZ. Following 96 hours of TMZ treatment, cell viability was assessed using the of CellQuanti-Blue™ cell viability assay.

The results from a 96-hour treatment with TMZ in LN229 cells indicate that glucose restriction augments the efficiency of TMZ (Figure 11). When cultured in normal glucose media and treated with TMZ, the

IC50 value was 273.5 µM in LN229 cells. When cultured in restricted glucose conditions, the IC50 value of TMZ decreased to 85.3 µM in LN229 cells (p <0.001) (Table 1). T98G cells responded in a similar fashion following a 96-hour treatment with TMZ, with glucose restriction augmenting the efficiency of

TMZ (Figure 12). The IC50 value decreased from 678.4 µM to 371.4 µM when T98G cells were cultured in restricted glucose conditions (p =0.005) (Table 1).

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Figure 11. Glucose restriction sensitizes LN229 GBM cells to temozolomide treatment. GBM cell line LN229 were tested for TMZ sensitivity under glucose restricted media. LN229 cells were plated at 5,000 cells per well and incubated in low glucose DMEM (Red, 0.5 g/L glucose) or in normal glucose DMEM (Black, 2.0 g/L glucose) for 24 hours. Percent viability was determined through CellQuanti-Blue™ cell viability assay after 96-hour treatment with 0-1500 µM TMZ. Reported values indicate the mean of three replicates converted to percent of control (media only) in Prism software. Reported values presented as dose-response curve using Prism software.

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Figure 12. Glucose restriction sensitizes T98G GBM cells to temozolomide treatment. GBM cell line T98G were tested for TMZ sensitivity under glucose restricted media. T98G cells were plated at 5,000 cells per well and incubated in low glucose DMEM (Red, 0.5 g/L glucose) or in normal glucose DMEM (Black, 2.0 g/L glucose) for 24 hours. Percent viability was determined through CellQuanti-Blue™ cell viability assay after 96-hour treatment with 0-1500 µM TMZ. Reported values indicate the mean of three replicates converted to percent of control (media only) in Prism software. Reported values presented as dose-response curve using Prism software.

Cytotoxic effects of glucose restriction in combination with temozolomide in human astroglial cell line

In contrast to GBM cell lines, glucose restriction decreased the cytotoxicity of TMZ in SVG p12 cells. The IC50 value slightly increased from 77.2 µM to 92.8 µM when SVG p12 cells were cultured in restricted glucose conditions (Table 1). This slight decrease in IC50 suggests a protective effect when

SVG p12 cells were cultured in glucose restricted media, however the change in IC50 was found to be not significant (NS) (p =0.125) (Table 1).

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Figure 13. Glucose restriction has no effect on SVG p12 normal astrocyte sensitivity to temozolomide treatment. Normal human astrocyte cell line SVG p12 were tested for TMZ sensitivity under glucose restricted media. Cells were plated at 5,000 cells per well and incubated in low glucose (Red, 0.5 g/L glucose) or normal glucose (Black, 2.0 g/L glucose) DMEM supplemented with 10% FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate for 24 hours. Percent viability was determined through CellQuanti-Blue™ cell viability assay after 96-hour treatment with 0-1500 µM TMZ. Reported values indicate the mean of three replicates converted to percent of control (media only) in Prism software. Reported values presented as dose-response curve using Prism software.

Discussion

Glucose restriction enhanced the efficiency of TMZ in both GBM cell lines, LN229 and T98G. In

LN229, the IC50 value decreased from 273.5 µM to 85.29 µM, a 68.8% decrease, when cultured under glucose restricted media (p <0.001) (Table 1) (Figure 11). In T98G cells, the IC50 value decreased from

678.3 µM to 371.4 µM, a 45.3% decrease, when cultured under glucose restricted media (p <0.01) (Table

1) (Figure 12). These changes in IC50 values suggest that glucose restriction augments the cytotoxic effects of TMZ in GBM cell lines. Furthermore, the observed IC50 values were larger in T98G compared to LN229 cells under both glucose restricted media and normal glucose media. Thus, our data suggest that

T98G cells are inherently more resistant to TMZ than LN229 cells, a notion that is supported by the literature.

In contrast to GBM cells, glucose restriction slightly reduced the efficiency of TMZ in normal human astroglial cell line SVG p12. In SVG p12 cells, glucose restriction increased the IC50 value of

TMZ from 77.19 µM to 92.80 µM, a 20.2% increase (NS) (Table 1) (Figure 13). However, this change in

IC50 value was found to be not significant. Although glucose restriction with TMZ showed no significant effect in SVG p12, glucose restriction did potentiate TMZ cell death in both of GBM cell lines tested.

This observation seems to indicate that glucose restriction differentially effects normal and cancer cells by selectively augmenting the cytotoxic effects of TMZ in GBM cells. Thus, these results support the potential use of fasting in combination with TMZ for the treatment of GBM.

One hypothesis by which fasting enhances TMZ in LN229 cells is through the modulation of nutrient sensing pathways that governs cell behavior. In a fed state, anabolic signaling promotes cell growth/replication largely through the phosphoinositide 3-kinase (PI3K) pathway and mTORC1. Insulin and IGF-1 activation of PI3K/Akt/mTORC1 pathway leads to increased uptake of glucose and amino acids, activation of glycolytic enzymes phosphofructokinase and hexokinase, increased protein translation, increased lipid synthesis, increased nucleotide synthesis, and cell growth.88 In addition to activation of PI3K/Akt/mTORC1 pathway, the inactivation of AMP-activated protein kinase (AMPk)

41 occurs. AMPk is a protein complex whose activity is controlled by intracellular ATP/AMP levels. In a fed state, when ATP/AMP levels are high and ATP is bound to the regulatory subunit of AMPk, AMPk is inactive. In a fasted state, when ATP/AMP levels are low and AMP is bound to the regulatory subunit of

AMPk, AMPk is active.89,90 Active AMPk promotes cell survival/repair/maintenance by inhibiting mTORC1 and activating p53.91 p53 is one of the most studied tumor suppressor genes whose activation by cellular stress induces many different antiproliferative signals, such as cell cycle arrest, apoptosis, and

DNA repair. Activation of p53 leads to activation of phosphatase and tensin homolog (PTEN), a tumor suppressor gene that inactivates Akt.92,93 This inverse relationship between AMPk/p53/PTEN and

PI3K/Akt/mTORC1 is essential for maintaining cellular homeostasis throughout a range of nutrient conditions (Figure 14A). Cancer cells have oncogenic mutations that govern self-sufficient growth and altered metabolism. A comprehensive genomic characterization of GBM found mutations to at least one genetic event in the PI3k pathway in 86% of the 206 GBM samples.94 Mutations in the

PI3k/mTORC1/Akt pathway help to sustain proliferative signals even when nutrient availability is inadequate for growth and division. T98G have a mutation in PTEN, while LN229 cells have mutation in p53.95,96 These mutation hinders the ability of T98G and LN229 to maintain homeostasis in a fasting state, making them more susceptible to TMZ (Figure 14B). Therefore, future studies evaluating the relative expression and activity of PI3K/Akt/mTORC1 pathway and the AMPk/p53/PTEN pathway in response to fasting conditions is warranted to further understand the DSR between normal brain and GBM cells.

As discussed previously, depletion of glucose is only one of many systemic alterations that occur during fasting. Therefore, simply restricting the concentration of glucose within culture medium does not accurately reflect the response fasting with TMZ may have in patients. To account for an additional fasting induced adaptation, the next chapter of this thesis investigates the effects of ketone bodies in combination with TMZ on GBM and normal astroglial cell lines.

Figure 14. Differential stress response (DSR) between normal cells and LN229 cells in response to fasting conditions. (A) In a fed state (left), normal cells respond to high nutrient availability and Insulin/IGF-1 signaling through the activation of PI3K/Akt pathway. Activation of the PI3K/Akt pathways leads increases glucose metabolism and mTORC1 activation to increase protein, nucleotide, and lipid synthesis. High glycolytic flux increases intracellular ATP/AMP ratio, inhibiting AMPk activity. In a fasted state (right), low nutrient availability leads to a drop in ATP/AMP concentrations and the subsequent activation of AMPk. AMPk directly inhibits mTORC1, and activates p53 and PTEN to inhibit PI3k/Akt signaling. This inverse relationship in response relative nutrient availability allows normal cells to maintain homeostasis. (B) LN229 cells have mutation in p53 and T98G cells have a mutation in PTEN that impairs their ability to respond to fasting conditions. In a fed state (left), high nutrient availability and insulin/IGF-2 signaling activates PI3K/Akt/mTORC1 pathway to promote cell growth/division. In a fasted state (right), mutant p53 and PTEN inhibit LN229 and T98G ability to respond to fasting signals and continue to promote growth/division, making them more susceptible to TMZ. Active pathways are shown in black, inactive pathways are shown in red, and mutated pathways are shown in green. Image was drawn using Adobe Illustrator 2020.

CHAPTER 6: ANALYZING CYTOTOXICITY OF GLUCOSE RESTRICTED MEDIA

SUPPLEMENTED WITH β-HYDROXYBUTYRATE IN COMBINATION TEMOZOLOMIDE IN

GBM AND HUMAN ASTROGLIAL CELLS.

Introduction

Temozolomide (TMZ) is the standard chemotherapy agent for the treatment of GBM. During fasting, the reduction of blood glucose induces a variety of systemic changes to occur. Catabolic hormones signal the mobilization of fatty acids from adipose tissue. In the liver, fatty acids are broken down into acetyl-CoA which serves as the starting material in the synthesis of ketone bodies (KB). KB are hepatically produced and systemically released molecules that serve as an energy source for the heart, skeletal muscle, kidney, and brain.31–35 During fasting, serum concentrations of the KB ß-hydroxybutyrate

(ß-HB) can reach levels of 2 mM, and upwards of 6-8 mM during prolonged starvation.34 ß-HB readily crosses the blood brain barrier where it is oxidized by cells of the CNS into acetyl-CoA to serve as an energy source in the citric acid cycle.

In addition to its role as an energy substrate, β-HB has been previously to have inhibitory effects on histone deacetylase 1 (HDAC-1).37–39 This in turn augments the efficiency of TMZ in a TMZ-resistant

GBM cell line.41 The experiments in this chapter were designed to analyze the effects ß-HB in combination with temozolomide on the viability of two human GBM cell lines, LN229 and T98G, and a normal human astroglial cell line, SVG p12. The data indicates that ß-HB potentiated cytotoxicity of

TMZ in LN229 cells under normal glucose media but had no effect under glucose restricted media. In

T98G cells, ß-HB potentiated cytotoxicity of TMZ under both normal glucose media and restricted glucose conditions. In contrast to GBM cells, ß-HB reduced the cytotoxicity of TMZ in SVG p12 normal astroglial cells under both normal glucose media restricted glucose conditions. Therefore, this initial evidence suggests fasting may be a useful method to selectively increase cytotoxicity of TMZ in GBM cells, while protecting normal cells.

Materials and Methods

Preparation of Cell Culture Medium: Normal glucose media, DMEM containing 2.0 g/L D-(+)- glucose (Sigma-Aldrich), 0.29 g/L L-glutamine (Gibco), 0.11 g/L sodium pyruvate (Gibco), and 10%

FBS (Atlanta Biological), was prepared as stated previously. Glucose restricted media was prepared through a 1:3 dilution of the normal glucose media into glucose-free DMEM containing 0.29 g/L L- glutamine (Gibco), 0.11 g/L sodium pyruvate (Gibco), and 10% FBS (Atlanta Biological). The final concentration glucose restricted media was 0.5 g/L g/L D-(+)-glucose. Sodium ß-HB (Millipore Sigma) was dissolved into normal glucose media and glucose restricted media to a concentration of 2.0 mM.

Preparation of Temozolomide: Temozolomide (TMZ) was purchased from Sigma-Aldrich (St.

Louis, MO) and dissolved into glucose restricted media plus ß-HB and normal glucose media plus ß-HB to a concentration of 1500 µM. To ensure complete solubility of TMZ in DMEM, the solution was vortexed at maximum speed. Serial dilution of the 1.5 mM TMZ stock was then performed 1:1 in media to achieve working concentrations of 1500 µM, 750 µM, 375 mM, 187.5 mM, 93.75 µM, 46.88 µM,

23.44 µM, 11.72 µM, and 5.86 µM. A control solution of glucose restricted media plus ß-HB and normal glucose media plus ß-HB was also prepared.

Cell Line Culture: Cells were cultured in DMEM as previously discussed. Briefly, cells were detached from flasks using trypsin-EDTA, pelleted by centrifugal force, and resuspended into one mL of

At 2 hours after plating, the original complete medium was carefully removed using a multichannel pipettor and replaced with either glucose restricted media plus 2.0 mM ß-HB, or normal

45 glucose media plus 2.0 mM ß-HB for 24 hours. After 24 hours, media was removed and replaced with varying concentration of TMZ or control (media only) using a multichannel pipettor. The final volume of treatment media was 100 µL per well and each treatment group was prepared in triplicate. Plates were sustained in standard incubation conditions. TMZ treatment lasted a total of 96 hours.

Cell viability assay: Following 96-hour exposure to TMZ, cytotoxicity was assessed by luminescence using CellQuanti-Blue™ cell viability assay kit (BioAssay Systems) per manufacture’s protocol. Briefly, CellQuanti-Blue™ reagent was removed from 4 °C and allowed to equilibrate to RT.

Media was carefully removed using a multichannel pipettor and replaced was 100 µL of DMEM-phenol red free media (corning) supplemented with 10% FBS. Following this, 10 µL of CellQuanti-Blue™ reagent was added to each well. Cells were briefly centrifuged at 32 rcf to homogenize CellQuanti-Blue™ reagent in the culture medium and incubated for 2 hours under standard conditions. After two hours, measurement of CellQuanti-Blue™ reagent fluorescence intensity (excitation wavelength = 525 nm, emission wavelength = 590 - 620 nm) using a Modulus II Microplate Reader (Turner Biosystems,

Sunnyvale, CA). CellQuanti-Blue™ is a sensitive assay that utilizes resazurin-based compound (not fluorescent) that upon reduction by metabolically active cells is converted into a resorufin (highly fluorescent). Viable cells can readily reduce resazurin, while non-viable (metabolically inactive) cells cannot. CellQuanti-Blue™ thus determines the number of viable cells by quantifying fluorescent intensity, which is directly proportional to the number of viable cells (Figure 12).

Statistical Analysis: Reported values indicate the mean fluorescence values of three replicates from CellQuanti-Blue™ assay, adjusted to the blank controls, and converted to percent viability relative to the adjusted, average vehicle control fluorescence values. IC50 of TMZ for each cell line was generated by plotting the average cell viability for each dose against the log function of their respective

TMZ dose (µM) fitted to a dose response curve in Prism 5 (GraphPad Software, San Diego, CA).

Significance was denoted to one asterisk (*) if p<0.05, two (**) if p<0.01, and three (***) if p<0.001. All data presented show the standard deviation of the mean (SD).

Additional analysis was done to compare the average cell viability of each cell line when cultured in glucose restricted media verses glucose restricted media supplemented with ß-HB. In order to do so, the data from the chapter 5, cell viability under glucose restriction and normal glucose media, were compared to the average cell viability under glucose restricted plus ß-HB and normal glucose plus ß-HB, respectively dose response curve in Prism 5 (GraphPad Software). Significance was denoted to one asterisk (*) where p<0.05, two (**) where p<0.01, and three (***) where p<0.001, and all data presented show the standard deviation of the mean (SD)

Results

Effects of glucose restriction conditions plus ß-HB in combination with temozolomide in GBM cell lines

We sought to determine the effects glucose restricted media plus ß-HB on the efficiency of TMZ in LN229 GBM cell line. As described earlier, cells were incubated in glucose restricted media plus ß-HB

(0.5 g/L + 2.0 mM ß-HB) or normal glucose media plus ß-HB (2.0 g/L+ 2.0 mM ß-HB) DMEM for 24 hours prior to, and during treatment with TMZ. Following 96 hours of TMZ treatment, cell viability was assessed using the of CellQuanti-Blue™ cell viability assay. The results from a 96-hour treatment with

TMZ in LN229 cells indicate that glucose restricted media plus ß-HB augments the efficiency of TMZ

(Figure 15). The IC50 value of TMZ decreased from 189.1 µM to 76.7 µM when LN229 cells were cultured in restricted glucose media plus ß-HB (p <0.001) (Table 1) (Figure 15). Using the data from the previous chapter, we compared the effects of TMZ under glucose restriction conditions (chapter 5) to the effects of TMZ under glucose restriction plus ß-HB, and the effects of TMZ under normal glucose media to the effects of TMZ under normal glucose plus ß-HB in LN229 cells. The IC50 value of TMZ decreased from 85.29 µM to 76.66 µM when LN229 were cultured under glucose restriction plus ß-HB (NS) (Table

1) (Figure 16). The IC50 value of TMZ decreased from 273.5µM to 189.1 µM when LN229 were cultured under normal glucose media plus ß-HB (p = 0.0243) (Table 1) (Figure 17).

T98G cells responded in a similar fashion following a 96-hour treatment with TMZ, with glucose restriction plus ß-HB augmenting the cytotoxicity of TMZ (Figure 16). The IC50 value decreased from

442.3 µM to 177.1 µM (p <0.001) when T98G cells were cultured in restricted glucose conditions plus ß-

HB (p <0.001) (Table 1) (Figure 18). Using the data from the previous chapter, we again compared the effects of TMZ under glucose restriction conditions and normal glucose media, to their respective counterparts containing ß-HB in T98G cells. The IC50 value of TMZ decreased from 371.4 µM to 177.1

µM when T98G were cultured under glucose restriction plus ß-HB (p <0.001) (Table 1) (Figure 19). The

IC50 value of TMZ decreased from 678.3 µM to 442.3 µM when T98G were cultured under normal glucose media plus ß-HB (p <0.01) (Table 1) (Figure 20).

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Figure 15. Glucose restricted media supplemented with ß-HB further sensitizes LN229 cells to temozolomide. Human GBM cell line LN229 were tested for TMZ sensitivity under glucose restricted media supplemented with ß- HB. Cells were plated at 5,000 cells per well and incubated in glucose restricted media plus ß-HB (Red, 0.5 g/L glucose + 2.0 mM ß-HB) or normal glucose media plus ß-HB (Black, 2.0 g/L glucose + 2.0 mM ß-HB) DMEM supplemented with 10% FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate for 24 hours. Percent viability was determined through CellQuanti-Blue™ cell viability assay after 96-hour treatment with 0-1500 µM TMZ. Reported values indicate the mean of three replicates converted to percent of control (media only) in Prism software. Reported values presented as dose-response curve using Prism software.

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Figure 16. ß-HB supplementation does not further sensitize LN229 cells cultured under glucose restricted media to temozolomide. Human GBM cell line LN229 were tested for TMZ sensitivity under glucose restricted media plus ß-HB. Cells were plated at 5,000 cells per well and incubated in glucose restricted media (Black, 0.5 g/L glucose) or glucose restricted media plus ß-HB (Red, 0.5 g/L glucose + 2.0 mM ß-HB) DMEM supplemented with 10% FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate for 24 hours. Percent viability was determined through CellQuanti-Blue™ cell viability assay after 96-hour treatment with 0-1500 µM TMZ. Reported values indicate the mean of three replicates converted to percent of control (media only) in Prism software. Reported values presented as dose-response curve using Prism software.

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Figure 17. ß-HB supplementation further sensitizes LN229 cells cultured under normal glucose media to high doses of temozolomide. Human GBM cell line LN229 were tested for TMZ sensitivity under normal glucose media plus ß-HB. Cells were plated at 5,000 cells per well and incubated in normal glucose media (Black, 2.0 g/L glucose) or normal glucose media plus ß-HB (Red, 2.0 g/L glucose + 2.0 mM ß-HB) DMEM supplemented with 10% FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate for 24 hours. Percent viability was determined through CellQuanti-Blue™ cell viability assay after 96-hour treatment with 0-1500 µM TMZ. Reported values indicate the mean of three replicates converted to percent of control (media only) in Prism software. Reported values presented as dose-response curve using Prism software.

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Figure 18. Glucose restricted media supplemented with ß-HB further sensitizes T98G cells to temozolomide. Human GBM cell line T98G were tested for TMZ sensitivity under glucose restricted media supplemented with ß- HB. Cells were plated at 5,000 cells per well and incubated in glucose restricted media plus ß-HB (Red, 0.5 g/L glucose + 2.0 mM ß-HB) or normal glucose media plus ß-HB (Black, 2.0 g/L glucose + 2.0 mM ß-HB) DMEM supplemented with 10% FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate for 24 hours. Percent viability was determined through CellQuanti-Blue™ cell viability assay after 96-hour treatment with 0-1500 µM TMZ. Reported values indicate the mean of three replicates converted to percent of control (media only) in Prism software. Reported values presented as dose-response curve using Prism software.

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Figure 19. ß-HB supplementation further sensitizes T98G cells cultured under glucose restricted media to temozolomide. Human GBM cell line T98G were tested for TMZ sensitivity under glucose restricted media plus ß- HB. Cells were plated at 5,000 cells per well and incubated in glucose restricted media (Black, 0.5 g/L glucose) or glucose restricted media plus ß-HB (Red, 0.5 g/L glucose + 2.0 mM ß-HB) DMEM supplemented with 10% FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate for 24 hours. Percent viability was determined through CellQuanti-Blue™ cell viability assay after 96-hour treatment with 0-1500 µM TMZ. Reported values indicate the mean of three replicates converted to percent of control (media only) in Prism software. Reported values presented as dose-response curve using Prism software.

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Figure 20. ß-HB supplementation further sensitizes T98G cells cultured under normal glucose media to temozolomide. Human GBM cell line T98G were tested for TMZ sensitivity under normal glucose media plus ß- HB. Cells were plated at 5,000 cells per well and incubated in normal glucose media (Black, 2.0 g/L glucose) or normal glucose media plus ß-HB (Red, 2.0 g/L glucose + 2.0 mM ß-HB) DMEM supplemented with 10% FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate for 24 hours. Percent viability was determined through CellQuanti- Blue™ cell viability assay after 96-hour treatment with 0-1500 µM TMZ. Reported values indicate the mean of three replicates converted to percent of control (media only) in Prism software. Reported values presented as dose- response curve using Prism software.

Effects of glucose restriction conditions plus ß-HB in combination with temozolomide in human astroglial cell line

In contrast to GBM cell lines, glucose restricted media plus ß-HB cytotoxicity to TMZ in SVG p12 cells. The IC50 value increased from 77.2 µM to 92.8 µM when SVG p12 cells were cultured in glucose restricted media plus ß-HB (p <0.001), suggesting a protective effect (Table 1) (Figure 21). Using the data from the previous chapter, we again compared the effects of TMZ under glucose restriction conditions and normal glucose media, to their respective counterparts containing ß-HB in SVG p12 cells.

The IC50 value of TMZ increased from 92.80 µM to 246.1 µM when SVG p12 were cultured under glucose restriction plus ß-HB (p <0.001) (Table 1) (Figure 22). The IC50 value of TMZ increased from

77.19 µM to 187.8 µM when T98G were cultured under normal glucose media plus ß-HB (p <0.001)

(Table 1) (Figure 23).

SVGp12

100

l i

b 50

i V

25 0.5 g/L glucose + 2.0 mM ß-HB 2.0 g/L glucose + 2.0 mM ß-HB 0 0 1 2 3 Log[Temozolomide]

Figure 21. Glucose restricted media supplemented with ß-HB has slight protective effects on SVG p12 normal astroglial cells sensitivity to temozolomide. Normal human astroglial cell line SVG p12 were tested for TMZ sensitivity under glucose restricted media supplemented with ß-HB. Cells were plated at 5,000 cells per well and incubated in glucose restricted media plus ß-HB (Red, 0.5 g/L glucose + 2.0 mM ß-HB) or normal glucose media plus ß-HB (Black, 2.0 g/L glucose + 2.0 mM ß-HB) DMEM supplemented with 10% FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate for 24 hours. Percent viability was determined through CellQuanti-Blue™ cell viability assay after 96-hour treatment with 0-1500 µM TMZ. Reported values indicate the mean of three replicates converted to percent of control (media only) in Prism software. Reported values presented as dose-response curve using Prism software.

SVGp12

100

l i

b 50

i V

25 0.5 g/L glucose + 2.0 mM ß-HB 0.5 g/L glucose 0 0 1 2 3 Log[Temozolomide]

Figure 22. ß-HB supplementation protects SVG p12 cells cultured under glucose restricted media to temozolomide. Human astroglial cell line SVG p12 were tested for TMZ sensitivity under normal glucose media (A) or restricted glucose conditions (B) with or without ß-HB supplementation. (A) SVG p12 cells were plated at 5,000 cells per well and incubated in low glucose DMEM (Black, 0.5 g/L glucose) or low glucose DMEM plus 2.0 mM ß-HB (Red, 0.5 g/L glucose + 2.0 mM ß-HB) for 24 hours. Percent viability was determined through CellQuanti-Blue™ cell viability assay after 96-hour treatment with 0-1500 µM TMZ. Reported values indicate the mean of three replicates converted to percent of control (media only) in Prism software. Reported values presented as dose-response curve using Prism software.

SVGp12

100

l i

b 50

i V

25 2.0 g/L glucose + 2.0 mM ß-HB 2.0 g/L glucose 0 0 1 2 3 Log[Temozolomide]

Figure 23. ß-HB supplementation protects SVG p12 cells cultured under normal glucose media to temozolomide. Human astroglial cell line SVG p12 were tested for TMZ sensitivity under normal glucose media plus ß-HB. Cells were plated at 5,000 cells per well and incubated in normal glucose media (Black, 2.0 g/L glucose) or normal glucose media plus ß-HB (Red, 2.0 g/L glucose + 2.0 mM ß-HB) DMEM supplemented with 10% FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate for 24 hours. Percent viability was determined through CellQuanti-Blue™ cell viability assay after 96-hour treatment with 0-1500 µM TMZ. Reported values indicate the mean of three replicates converted to percent of control (media only) in Prism software. Reported values presented as dose-response curve using Prism software.

Discussion Glucose restriction plus ß-HB enhanced the efficiency of TMZ in both GBM cell lines LN229 and T98G. In LN229, the IC50 value decreased from 189.1 µM to 76.66 µM, a 59.5% decrease, when cultured under glucose restricted media plus ß-HB (p <0.001) (Table 1) (Figure 15). In T98G cells, the

IC50 value decreased from 442.3 µM to 177.1 µM, a 60.0% decrease, when cultured under glucose restricted media plus ß-HB (p <0.001) (Table 1) (Figure 18). Therefore, the hypothesis that exposure to glucose restricted media plus ß-HB prior to and during TMZ treatment would further sensitize GBM cells to TMZ is supported.

In contrast to GBM cells, glucose restricted media plus ß-HB slightly reduced the efficiency of

TMZ in normal human astroglial cell line SVG p12. In SVG p12 cells, the IC50 value of TMZ increased from 187.8 µM to 246.1 µM, a 31.0% increase, when cultured under glucose restricted media plus ß-HB

(p <0.001) (Table 1) (Figure 21). The change in IC50 indicates that more drug is necessary to kill the same number of cells when SVGp12 cells are cultured under glucose restricted media plus ß-HB. Thus, the hypothesis that exposure to glucose restricted media plus ß-HB prior to and during treatment would protect normal astroglial cells from the cytotoxic effects of TMZ was supported.

The statistical analysis between TMZ induced cytotoxicity of LN229 cells cultured under glucose restricted media (chapter 5) verse glucose restricted media plus ß-HB produced conflicting results. The

IC50 value of TMZ decreased from 85.29 µM to 76.66 µM, a 10.1% decrease, when cultured under glucose restricted media plus ß-HB (NS) (Table 1) (Figure 16). However, this change in IC50 value was found to be not significant. Comparison of normal glucose media (chapter 5) to normal glucose media plus ß-HB suggest ß-HB enhances the cytotoxic effects of TMZ in LN229 cells. The IC50 value of TMZ decreased from 273.5 µM to 189.1 µM, a 30.9% decrease, when cultured under normal glucose media plus ß-HB (p =0.0243) (Table 1) (Figure 17). These results suggest under normal media conditions, ß-HB enhances the cytotoxic effects of TMZ in LN229 cells.

The statistical analysis between TMZ induced cytotoxicity of T98G cells under glucose restricted media (chapter 5) verse glucose restricted media plus ß-HB suggests ß-HB sensitizes T98G cells to TMZ.

The IC50 value of TMZ decreased from 371.4 µM to 177.1 µM, a 52.3% decrease, when cultured under glucose restricted media plus ß-HB (p <0.001) (Table 1) (Figure 19). This drop in IC50 value suggests that ß-HB enhances the cytotoxic effects of TMZ in T98G cells cultured under glucose restricted media.

The IC50 value of TMZ decreased from 678.3 µM to 442.3 µM, a 34.8% decrease, when cultured normal glucose media plus ß-HB (p =0.0043) (Table 1) (Figure 20). This drop in IC50 value suggests that ß-HB enhances the cytotoxic effects of TMZ in T98G cells cultured normal glucose media. Taken all together, these results suggest that ß-HB enhances the cytotoxic effects of TMZ in T98G cells.

In contrast to GBM cell lines, statistical analysis between TMZ induced cytotoxicity of normal astroglial cell line SVG p12 under glucose restricted media (chapter 5) verse glucose restricted media plus

ß-HB suggests ß-HB protects SVG p12 cells to TMZ. The IC50 value of TMZ increased from 187.8 µM to 246.1 µM, a 165.19% increase, when cultured under glucose restricted media plus ß-HB (p <0.001)

(Table 1) (Figure 22). This increase in IC50 suggests ß-HB decreases the cytotoxic effects of TMZ in

SVG p12 cells cultured under glucose restricted media. The IC50 value of TMZ decreased from 77.19

µM to 187.8 µM, a 143.3% increase, when cultured under normal glucose media plus ß-HB (p <0.001)

(Table 1) (Figure 23). This increase in IC50 suggests ß-HB decreases the cytotoxic effects of TMZ in

SVG p12 cells cultured under glucose restricted media. Taken all together, these results suggest that ß-HB protects SVG p12 cells from the cytotoxic effects of TMZ.

CHAPTER 7: ANALYZING THE EFFECTS OF GLUCOSE RESTRICTED MEDIA AND β-

HYDROXYBUTYRATE IN COMBINATION TEMOZOLOMIDE ON MGMT EXPRESSION IN GBM

AND HUMAN ASTROGLIAL CELLS.

Introduction

Temozolomide (TMZ) is the standard chemotherapy agent for the treatment of glioblastoma multiforme (GBM). GBM cells expressing the DNA repair protein O6-methyl-guanine-DNA methyltransferase (MGMT) are resistant to the TMZ induced cytotoxic lesions. Fasting induces systemic changes such as reducing anabolic hormones, reducing systemic glucose concentration, and the production of ketone bodies (KB). KB are hepatically produced and systemically released molecules that serve as an energy source for the heart, skeletal muscle, kidney, and brain.31–35 Fasting has been previously shown to protect normal cells while sensitizing cancer cells to chemotherapy, a phenomenon known as the differential stress response (DSR).

The previous chapters showed glucose restricted media enhanced the cytotoxicity of TMZ in

LN229 and T98G GBM cells, while having no effect in SVG p12 normal astroglial cells. Furthermore, glucose restricted media plus ß-hydroxybutyrate (ß-HB), a ketone body, further enhanced the cytotoxic effects of TMZ in LN229 and T98G cells, while having protective effects in SVG p12 cells. However, the mechanism by which glucose restricted media plus ß-HB can induce DSR to TMZ remains to be determined. The experiments in this chapter were designed to analyze the effects glucose restriction media, and glucose restricted media plus ß-HB, in combination with TMZ has on the expression of

MGMT in GBM cell lines, LN229 and T98G, and a normal astroglial cell line, SVG p12. Western analysis showed T98G were the only cell line that expressed MGMT. In T98G, the data indicates that glucose restricted media led to a decrease in MGMT levels. Furthermore, glucose restriction plus ß-HB further decreased MGMT levels in T98G cells. Thus, the mechanism by which glucose restricted media enhances the cytotoxic effects of TMZ in T98G cells could be by decreasing MGMT levels.

Materials and Methods

Preparation of Cell Culture Medium: Normal glucose media, DMEM containing 2.0 g/L D-(+)- glucose (Sigma-Aldrich), 0.29 g/L L-glutamine (Gibco), 0.11 g/L sodium pyruvate (Gibco), and 10%

FBS (Atlanta Biological), was prepared as stated previously. Glucose restricted media was prepared through a 1:3 dilution of the normal glucose media into glucose-free DMEM containing 0.29 g/L L- glutamine (Gibco), 0.11 g/L sodium pyruvate (Gibco), and 10% FBS (Atlanta Biological). The final concentration glucose restricted media was 0.5 g/L g/L D-(+)-glucose. For ß-HB conditions, sodium ß-

HB (Millipore Sigma) was dissolved into normal glucose media and glucose restricted media to a concentration of 2.0 mM.

Preparation of Temozolomide: Temozolomide (TMZ) was purchased from Sigma-Aldrich (St.

Louis, MO) and dissolved into glucose restricted media, glucose restricted media plus 2.0 mM ß-HB, normal glucose media, and normal glucose media plus 2.0 mM ß-HB to a concentration of 1000 µM. To ensure complete solubility of TMZ in DMEM, the solution was vortexed at maximum speed. Working concentrations were prepared equal to the IC50 value [µM] for each treatment condition, in each cell line.

Amount of stock TMZ in DMEM (V1) [µL]to add was determined by dividing the product of IC50 (C2)

[µM] and total volume of 20 mL [20,000 µL] by the concentration of the TMZ stock [1000 µM] (equation

3). Summary of IC50 values can be seen in table 1.

Cell Line Culture: Cells were cultured in DMEM as previously discussed. Briefly, cells were detached from flasks using trypsin-EDTA, pelleted by centrifugal force, and resuspended into one mL of

DMEM. Cell count of cell suspension was subsequently done using trypan-blue exclusion method.

Appropriate amount of cell suspension was added to a conical tube containing DMEM to achieve a final concentration of 200,000 cells/mL and seeded 2,000,000 cells/flask (10 mL) into T-75 tissue culture treated flask (ThermoFisher Scientific). Cells were incubated under standard conditions and media was changed every second day.

Once cells reached approximately 80% confluency, the original complete medium was carefully removed, washed with Dulbecco’s Phosphate Buffered Saline (DPBS) (Sigma-Aldrich), and replaced with either glucose restricted media, normal glucose media, glucose restricted media plus 2.0 mM ß-HB, normal glucose media plus 2.0 mM ß-HB, or positive control media (DMEM; 4.5 g/L D-(+)-glucose, 10%

FBS, 0.29 g/L L-glutamine, and 0.11 g/L sodium pyruvate) for 24 hours. After 24 hours, media was removed, washed with (DPBS), and replaced with respective media conditions containing the IC50 value for each cell line or DMEM. Plates were sustained in standard incubation conditions. After 48 hours of treatment, cells received an additional 5 mL of their respective TMZ media conditions. Treatment lasted a total of 96 hours.

Preparation and quantification of cell lysates. Following 96 hours of treatment, media was removed and collected in a 50 mL conical tube. Cells were washed with DPBS, enzymatically disassociated from the flask using Trypsin/EDTA (Gibco), added to their respective conical tube, and centrifuged at 280 rcf for three minutes. Supernatant was carefully removed, and pellet was resuspended in 1 mL of cold DPBS (4°C). Cell count of cell suspension was subsequently done using trypan-blue exclusion method. Cell suspensions were transferred into labeled 1.5 mL Eppendorf tubes and centrifuged at 280 rcf for 3 min at 4°C. Supernatant was carefully removed, and cells were resuspended in appropriate amounts of cold radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Danvers,

MA) to achieve a concentration of 20,000 cells/µL RIPA. RIPA buffer is a lysis buffer that is used to solubilize cellular proteins for subsequent analysis. Cells lysates were gentle agitated using a micropipettor and placed on ice for 10 min. Agitation of cell lysate was repeated every 10 min for a total of 30 min. After 30 min, insoluble material was pelleted by centrifuging samples at 15,000 rcf for 15 min at 4 °C. Supernatants were carefully removed using a micropipettor and placed into labeled 1.5 mL

Eppendorf tubes on ice.

Following removal of supernatant, protein concentration of lysates was immediately quantified using a PierceTM bicinchoninic acid (BCA) protein assay kit (Thermo Scientific) per manufactural

63 protocol. Briefly, cell lysates were diluted 1:4 and 1:8 in RIPA. Diluted samples (25 µL) were placed into a 96 well microplate in triplicate. Bovine serum albumin (BSA) standard samples (25 µL) ranging from

2000 µg/mL to 25 µg/mL were also added to the 96 well microplate. BCA solution (200 µL) was added to each well and incubated at 37 °C for 30 min. BCA solution contains copper (II) sulfate. Cu2+ ions are reduced by the peptide bods present in proteins. Thus, the amount of Cu2+ ions reduced is directly proportional to the concentration of protein in each sample. Subsequent chelation between two molecules of BCA and one reduced Cu2+ ion produces a purple complex that absorbs light at a wavelength of 562 nm. Following 30 min of incubation, absorbance of each well was read using Modulus II Microplate

Reader (Turner Biosystems) at a wavelength of 560 nm. The average absorbance of each known BSA standard was calculated and a standard curve of absorbance as a function of protein concentration (µg/µL) was generated fit to linear regression model in excel. The slope of this resulting graph was subsequently used to determine the concentration of protein (µg/mL) in the cell lysates.

Western blot analysis. Treated LN229, T98G, and SVG p12 cell lysates were diluted using of

Laemmli Sample Buffer (15.6 mM Tris-HCl, 355 mM ß-mercaptoethanol, pH 6.8, 10% glycerol, 1%

SDS, 0.005% bromophenol blue) so that 35 µg of protein was loaded into each well of a 4-20% Midi-

PROTEAN® TGX Stain-Free™ gel (Bio-Rad). Samples were heated at 90 °C for 5 min to denature proteins. Proteins were separated by size using SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Protein ladder containing 1:1 dilution of Precision Plus Protein Unstained (Bio-Rad Laboratories,

Hercules, CA) and All Blue Standard (Bio-Rad) was used as molecular weight indicator. Protein gels were subjected to electrophoresis at a constant 150 V with varying current for one hour at 4 °C.

Following electrophoresis, proteins were transferred onto 0.2 µm nitrocellulose membrane (Bio-

Rad) using the Trans-Blot® Turbo Transfer System™ (Bio-Rad) at a constant 1.3 A with varying voltage for 7 min. Blots were blocked with a 5% non-fat dry milk (NFDM) in tris-buffered saline plus 0.1%

Tween-20 (TBST) (20 mM Tris base, 150 mM NaCl, 0.1% v/v Tween-20) for 90 min at RT with gentle agitation, and subsequently washed with TBST. Anti-MGMT Monoclonal Antibody (Invitrogen,

Carlsbad, CA) (1:100) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Monoclonal

Antibody (Invitrogen) (1:40,000) in 2.5% NFDM-TBST was added overnight at 4 °C with gentle agitation. The membrane was washed again with TBST and incubated with Goat anti-Mouse IgG (H+L)

Secondary Antibody, HRP (Invitrogen) (1:10,000) in 2.5% NFDM-TBST for 40 min at RT. Blots were washed a final time with TBST, followed by detection with Clarity™ Western ECL Substrate (Bio-Rad).

Chemiluminescent image was acquired using ChemiDoc™ MP Imaging System (Bio-Rad).

Statistical Analysis: Acquired images of western blots were analyzed using ImageLabTM Software

(Bio-Rad). Relative expression of MGMT between three replicates was determined by normalizing the optical density (OD) of MGMT bands against GAPDH bands. Comparison of the effects of glucose restricted media, normal glucose media, glucose restricted media plus ß-HB, and normal glucose media plus ß-HB on expression of MGMT was found by plotting the average relative MGMT expression between three replicates against their respective treatment condition fit to a group column analysis in

Prism 5 (GraphPad Software). Statistical significance between each treatment group was analyzed through one-way ANOVA in Prism 5 (GraphPad Software). Significance was denoted to one asterisk (*) if p<0.05, two (**) if p<0.01, and three (***) if p<0.001. All data presented show the standard deviation of the mean (SD).

퐶 × 20,000 µ퐿) 푉 = 2 1 1000 µ푀

Equation 3. Determining the amount of stock TMZ in DMEM needed to achieve working concentration. Working concentration were made for each cell line under each media condition equal to their respective IC50 value. Equation 3 describes the amount of stock TMZ in DMEM (V1) [1000 µM] needed to achieve the respective IC50 value. V1 [µL] was determined by dividing the product of IC50 (C2) [µM] and total volume [20,000 µL] by the concentration of the TMZ stock [1000 µM].

Results

Effects of glucose restricted media in combination with TMZ on the expression of MGMT in GBM cell lines and normal astroglial cell line.

We sought to determine the effect of glucose restricted media in combination with TMZ on the expression of MGMT in GBM cell lines, LN229 and T98G, and a normal human astrocyte cell line, SVG p12. As described earlier, cells were incubated in low glucose media (0.5 g/L) or normal glucose (2.0 g/L)

DMEM supplemented with 2.0 mM ß-HB for 24 hours prior to, and during treatment with TMZ.

Following 96 hours of TMZ treatment, cell lysates were generated, proteins separated by SDS-PAGE, transferred to nitrocellulose blot, and MGMT and GAPDH expression was detected using antibodies. Of cell lines tested, T98G were the only one that expressed the MGMT protein (Figure 24). The results from a 96-hour treatment with TMZ in T98G cells indicate that glucose restricted media downregulates the expression of MGMT (Figure 24A). The relative expression of MGMT decreased from 0.4044 to 0.2924 when T98G cells were cultured in glucose restricted conditions (p <0.01) (Figure 24B).

Effects of glucose restricted media plus ß-HB in combination with TMZ on the expression of MGMT in

GBM cell lines and normal astroglial cell line.

We sought to determine the effect of glucose restricted media plus ß-HB in combination with

TMZ on the expression of MGMT in GBM cell lines, LN229 and T98G, and a normal human astrocyte cell line, SVG p12. As described earlier, cells were incubated in glucose restricted media plus ß-HB (0.5 g/L glucose + 2.0 mM ß-HB) or normal glucose media plus ß-HB (2.0 g/L glucose + 2.0 mM ß-HB) for

24 hours prior to, and during treatment with TMZ. Following 96 hours of TMZ treatment, cell lysates were generated, proteins separated by SDS-PAGE, transferred to nitrocellulose blot, and MGMT and

GAPDH expression was detected using antibodies. Of cell lines tested, T98G were the only one that expressed the MGMT protein (Figure 24). The results from a 96-hour treatment with TMZ in T98G cells indicate that glucose restricted media plus ß-HB downregulates the expression of MGMT (Figure 24).

The relative expression of MGMT decreased from 0.3365 to 0.2457 when T98G cells were cultured in glucose restricted media plus ß-HB (p <0.01) (Figure 24B).

We compared the effects of TMZ under glucose restriction conditions and normal glucose media, to their respective counterparts containing ß-HB on the expression of MGMT in T98G cells. The results from a 96-hour treatment with TMZ in T98G cells indicate that ß-HB downregulates the expression of

MGMT under normal glucose media (Figure 24). The relative expression of MGMT decreased from

0.4044 to 0.3365 when T98G cells were cultured in normal glucose media plus ß-HB (p <0.05) (Figure

24B). The ratio of MGMT:GAPDH also decreased from 0.2924 to 0.2457 when T98G cells were cultured in glucose restricted media plus ß-HB (NS) (Figure 24B). However, no significance was noted between glucose restricted media and glucose restricted media plus ß-HB with respect to MGMT:GAPDH ratio.

Further comparison was done between the effects of TMZ under glucose restricted media plus ß-

HB to the effects of TMZ under normal glucose media on expression of MGMT in T98G cells. The results from a 96-hour treatment with TMZ in T98G cells indicate that glucose restricted media plus ß-HB downregulates the expression of MGMT (Figure 24). The relative expression of MGMT decreased from

0.4044 to 0.2457 when T98G cells were cultured in normal glucose media plus ß-HB (p <0.001) (Figure

24B).

Figure 24. Glucose restricted media and glucose restricted media plus ß-HB in combination with TMZ downregulates MGMT expression in T98G cells. The effects of glucose restricted media and ß-HB in combination with TMZ on the expression of MGMT was analyzed in GBM cell lines, LN229 and T98G, and human astroglial cell line, SVG p12. Cells were exposed to glucose restricted media (0.5 g/L glucose), normal glucose media (2.0 g/L glucose), glucose restricted media plus ß-HB (0.5 g/L glucose + 2.0 mM ß-HB), or normal glucose media plus ß-HB (2.0 g/L glucose + 2.0 mM ß-HB) for 24 hours. Lysates were prepared after 96-hour treatment with respective TMZ IC50 value for each cell line as described in the Materials and Methods. Proteins were separated through SDS-PAGE on 4-20% Midi-PROTEAN® TGX Stain-Free™ gels, transferred onto 0.2 µm Nitrocellulose, incubated with anti-MGMT and anti-GAPDH primary antibodies, and incubated with goat anti- Mouse secondary antibody. (A) Chemiluminescent images of LN229, T98G, and SVGp12 lysates under the four different media conditions. (B) Reported values of the average ratio of MGMT (23 kDA) OD to GAPDH (37 kDa) OD between three replicates in T98G cells presented as group column analysis using Prism software. All data presented as mean (SD); * p<0.05, ** p<0.01, *** p<0.001, one-way ANOVA.

Discussion Of the two GBM cell lines, LN229 and T98G, and the normal astroglial cell line, SVG p12, T98G was the only cell line that expressed the MGMT protein. The blots containing T98G cell lysate were the only blots in which a band was produced at 23 kDa, indicating the presence of MGMT (Figure 24A).

Glucose restricted media in combination with TMZ decreases MGMT levels in T98G cells. In

T98G, the relative expression of MGMT decreased from 0.4044 to 0.2924, a 27.7% decrease, when cultured under glucose restricted media (p <0.01) (Figure 24B). Due to the function of MGMT in TMZ resistance, decreasing MGMT levels would lead to increased efficiency in cytotoxicity of TMZ. Thus, the enhanced cytotoxicity of TMZ in T98G cells cultured under glucose restricted media could be due to the decrease in MGMT levels.

Glucose restricted media plus ß-HB in combination with TMZ decreases MGMT levels in T98G cells. In T98G, the relative expression of MGMT decreased from 0.3365 to 0.2457, a 27.0% decrease, when cultured under glucose restricted media plus ß-HB (p <0.01) (Figure 24B). Thus, it is likely that the enhanced cytotoxicity of TMZ in T98G cells cultured under glucose restricted media plus ß-HB is due to the decrease in MGMT.

Comparison of MGMT expression under normal glucose media to normal glucose media plus ß-

HB suggests ß-HB alone leads to the decreases MGMT levels in T98G cells. In T98G, the relative expression of MGMT decreased from 0.4044 to 0.3365, a 16.8% decrease, when cultured under normal glucose media plus ß-HB (p <0.01) (Figure 24B). Comparison of the expression of MGMT in T98G cultured under glucose restricted media to MGMT expression under glucose restricted media plus ß-HB showed slight reduction in MGMT levels. In T98G, the relative expression of MGMT decreased from

0.2924 to 0.2457, a 16.0% decrease, when cultured under glucose restricted media plus ß-HB (NS)

(Figure 24B). However, this 16.0% decrease in MGMT expression under glucose restricted media plus ß-

HB was found to be not significant. Taken all together, these results suggest ß-HB leads to the decrease of

MGMT in T98G cells. Thus, it is likely that the enhanced cytotoxicity of TMZ in T98G cells by ß-HB is due to ß-HB a reduction in MGMT.

Comparison of MGMT expression under normal glucose media to glucose restricted media plus

ß-HB suggests glucose restriction media plus ß-HB leads to robust reduction of MGMT in T98G cells. In

T98G, the relative expression of MGMT decreased from 0.4044 to 0.2457, a 39.2% decrease, when cultured under glucose restricted media plus ß-HB (p <0.001) (Figure 24B). Thus, it is likely that the enhanced cytotoxicity of TMZ in T98G cells by glucose restricted media ß-HB is due to reduction in

MGMT levels.

As previously discussed, fasting induces systemic changes such as reducing anabolic hormones, reducing systemic glucose concentration, and the production of KB. Glucose restricted media plus ß-HB was used in these experiments to mimic the drop in blood glucose and the increase in KB that occurs during fasting. Normal glucose media was used in these experiments to mimic the conditions that occur during ad-lib feeding. The most profound statistical in MGMT expression difference between treatment groups was found when comparing the effects of normal glucose media and glucose restricted media plus

ß-HB (p <0.001) (Figure 24B). Thus, these results suggest that fasting may be a useful treatment fasting may be a useful increase cytotoxicity of TMZ in GBM cells expressing MGMT.

Due to the role of MGMT in TMZ resistance, the reduction of MGMT provides an explanation by which ß-HB enhanced the cytotoxicity of TMZ in T98G. However, the mechanism by which ß-HB reduces MGMT levels in T98G cells remains to be determined. One such hypothesis by ß-HB reduces

MGMT levels in T98G cells is through ß-HB effects on HDAC. As discussed previously, HDAC are nuclear enzymes that function in epigenetic modifications through chromatin remodeling. HDACs are divided into four classes based on their homology to proteins isolated from yeast. Class I HDAC, including HDAC1, HDAC2, HDAC3, and HDAC8 share homology to yeast RPD3. HDAC8 is ubiquitously expressed but is highly abundant in the prostate, kidney, and brain.96 HDAC8 interaction with the proteasomal ubiquitin receptor ADRM1 has been previously shown to regulate MGMT

70 expression in GBM cell line T98G. In this study, treatment with the selective HDAC8 inhibitor PCI34051 led to a decrease in MGMT levels, increased TMZ-induced DNA damage, induced cell cycle arrest, and decreased cell viability of T98G cells.98 n-Butyrate is an endogenous four carbon chain fatty acid produced by the gut microbiome during bacterial fermentation of carbohydrates. n-Butyrate is a well characterized inhibitor of class I HDACs.99 Inhibition occurs due to Class I HDACs binding affinity to carbonic acid side chains of butyrate. Treatment with n-butyrate has antiproliferative effects in leukemia, breast cancer, and colorectal cancer cells through its inhibition of HDAC8 activity.100–102 Unfortunately, the short half-life and high hepatic clearance dampens the antiproliferative effects in vivo.103–105 Differing from n-butyrate by a single hydroxy group, ß-HB has also been shown to have inhibitory effects on Class

I HDACs. It is possible that ß-HB inhibits HDAC8 to reduce MGMT levels in T98G cells. However, future studies on the effects of ß-HB on HDAC8 activity and MGMT regulation is needed to determine the mechanism by which ß-HB reduces MGMT in T98G cells.

CHAPTER 8: SUMMARY AND CONCLUSIONS

GBM is the most common and deadly primary brain tumor in adults. The standard of care for

GBM is maximal surgical resection of the tumor followed by adjuvant treatment with the chemotherapeutic agent temozolomide (TMZ) and radiotherapy. Despite advancements in surgical procedures, chemotherapeutics, and radiotherapy, the prognosis for GBM remains dismal. The Warburg effect describes a common trait of cancer in which cancer cells preferentially oxidize glucose to lactate in glycolysis, inhibiting mitochondrial respiration. To meet physiological needs, cancer cells must oxidize significantly greater amounts of glucose. One method that directly targets the bioavailability of glucose is fasting. Fasting leads to rapid reduction of plasma glucose, reduction of anabolic hormones, and production of KB. Normal cells respond to these changes by reinvesting energy towards repair/maintenance/survival. Cancer cells harbor oncogenic mutations that hinders their ability to adjust to such changes. The DSR induced by fasting has been shown to protect normal cells while sensitizing cancer cells to chemotherapy.26,28,29

Summary of the effects of fasting in combination with TMZ the viability and expression of MGMT in GBM and normal glial cell lines.

This thesis sought to evaluate the effects fasting in combination with TMZ in human GBM cell lines, LN229 and T98G, and a normal astroglial cell line, SVG p12. Exposure of LN229 and T98G to glucose restricted media prior to and during treatment enhanced the cytotoxic effects of TMZ, while showing no effect in SVGp12 cells. Furthermore, exposure of LN229 and T98G to glucose restricted media plus ß-HB prior to and during treatment led to greater enhancement of the cytotoxic effects of

TMZ, while having protective effects in SVG p12 cells. We then sought to determine the effect of glucose restricted media and ß-HB on the expression of MGMT, a gene that functions in the removal of TMZ induced O6MeG cytotoxic lesions. Of all three cell lines tested, T98G were the only cell line that expressed MGMT protein. Exposure of T98G cells to glucose restricted media in combination with TMZ

72 lead to the downregulation of MGMT. Moreover, exposure of T98G cells to glucose restricted media plus

ß-HB led to further downregulation of MGMT. Thus, this study provided evidence for the use of fasting as an adjuvant therapy in the treatment of GBM.

Overview of study limitations and future directions.

Several limitations existed within the described experiments. As discussed earlier, the reduction of nutrient availability during fasting leads to numerous systemic alterations. Fasting induces the mobilization of fatty acids from adipose tissue, leading to increased plasma free fatty acid (FFA) concentrations. Fasting also induces reduction of anabolic hormones, such as insulin and IGF-1, and the release of catabolic hormones, such as epinephrine, glucagon, and cortisol. The media conditions of this study only accounted for two fasting induced adaptations, the reduction of glucose and the increase of ß-

HB. Future studies that account for additional fasting induced adaptations are needed to fully evaluate the potential of fasting as a therapy for GBM.

The choice of cell lines and the matrix by which they were grown in these experiments imposed several study limitations. All cell lines used were previously established cell lines. Both LN229 and T98G

GBM cell lines were established in 1979.96,106 Over this period of 41 years, these cells have become acclimated to life in culture. This has led to genomic alterations, such as chromosomal deletions/amplifications and gene expression profiles, that are exclusively present in GBM established cell lines. 107 Furthermore, T98G cells are no longer tumorigenic, that is they will not propagate in model organisms. These changes suggest that established GBM cell lines poorly represent the nature of primary

GBM. Human diploid cells are restrained to a finite number of cellular divisions before entering replicative senescence.108,109 SVG p12 is an astroglial cell line established in 1985 from 8-12-week-old human fetal glial cells immortalized through transfection with mutant simian vacuolating virus 40

(SV40).110 Even though SVG p12 is not cancerous, SV-40 transformation allowing continuous replication has caused SVG p12 to lose much of their in vivo characteristics. There is also evidence that

SVG p12 is contains an infectious BK polyomavirus.111 All cell lines were grown adherently as a

73 monolayer in tissue culture treated vessels. This two-dimensional method of cell culture does not accurately model the three-dimensional microenvironment of a tumor. Recent advancements in cell culture techniques have led to the development of artificial three-dimensional cerebral organoids that better represent in vivo GBM microenvironment.112 Therefore, future studies evaluating the effects of fasting using three-dimensional models of patient derived GBM cell lines is warranted.

We used western blot analysis to evaluate the effects of glucose restriction and ß-HB in combination with TMZ on the expression of MGMT. In doing so, we were only able to determine the relative amount of MGMT present in each cell line at the protein level. MGMT becomes inactive following the transfer of the methyl adduct from O6MeG, and this inactivation leads to the subsequent proteasomal degradation of MGMT. Thus, MGMT is a suicide enzyme that requires de novo synthesis for enzymatic recovery.113 The mechanism by which the relative amount of MGMT decreased in response to both glucose restriction and ß-HB in T98G cells, whether it be through increased consumption or transcriptional regulation, remains unknown. One way to determine this mechanism could be through performing RT-qPCR. By quantifying the relative amount of MGMT mRNA transcripts, RT-qPCR would show if the downregulation of MGMT was due to increased consumption or transcriptional regulation.

Overview of the clinical use of fasting and ketogenic metabolic therapies as an adjuvant therapy for cancer and GBM .

The clinical use of fasting as an adjuvant therapy has been evaluated in a variety of cancers. A pilot trial of 20 subjects evaluating short-term fasting (72 hour) in combination with platinum-based chemotherapy found fasting to be safe and feasible. Over the 72-hour period, levels of blood glucose, insulin, IGF-1, and ß-HB were monitored. Although reduction in insulin, IGF-1, and glucose and increase in ß-HB were found over the first 24-48 hours, the lack of continuing changes in glucose and ß-HB levels may indicate some subjects did not successfully complete the full 72 hour fast.114 Other studies evaluating the effects of short-term fasting on quality of life and tolerance to chemotherapy in breast and ovarian cancer patients found short-term fasting improved quality of life and fatigue related symptoms during

74 chemotherapy.106,107 These studies support the safety and feasibility of fasting in combination with chemotherapy for the treatment of GBM.

Although no studies have evaluated the use of fasting for the treatment GBM, there are various clinical studies evaluating ketogenic metabolic therapies (KMT) as adjuvants to standard GBM treatment.

These studies implemented a diet designed to mimic some the effects of fasting called the ketogenic diet

(KD).Created and put into practice in 1921 for the treatment of epilepsy, the KD features nutrient intake divided into of no-to-low carbohydrate (0-5%), moderate protein (30-45%), and high amounts of fat (60-

65%).117–119 Similar to fasting, KD forces the body to rely on endogenous glucose production. However, the rate at which glucose is synthesized endogenously is not sufficient for the body’s physiological needs.

In response, the production of ketone bodies in the liver occurs through ketogenesis as an alternative source of energy for the body.11 A pilot study evaluating the effects of the KD during treatment of recurrent glioblastoma found it to be well tolerated, with 17 of 20 subjects (85%) following the diet on average of 6.8 days per week. Of those 17 subjects, regular urine KB and blood glucose analysis was available in 13 subjects. Stable ketosis (>0.5 mM) was detectable in 12 of the 13 subjects (92%), but the

KD failed to significantly lower glucose levels (99 ± 21.8 to 9 2± 9.1). The median progression free survival (PFS) was only 5 weeks and PFS at 6 months was 0%. There was no control group, randomization, or adequate number of subjects to accurately estimate the efficiency of the KD.120 The advantage of KD is patients are still able to intake food while still reaching a state of ketosis. The disadvantage of KD is adequate protein intake provides substrates for gluconeogenesis, inhibiting the reduction of blood glucose.

Other studies evaluating the effects of KMT as monotherapy for GBM have used a caloric restricted KD. A case report of two GBM patients treated with and IRB-approved energy-restricted KD

(ERKD) as monotherapy found the ERKD to be safe and without major side effects. Both patients presented with GBM, underwent standard therapies, and reached therapeutic failure (recurrence of disease). Initiation of the ERKD therapy began with a supervised 48-hour water fast to induce ketosis,

75 followed by ERKD protocol that was to be administered for 12 weeks. To evaluate diet adherence and efficiency, body weight, blood glucose, and blood KB concentration were monitored daily using AM and

PM measurements, in which a goal of maintaining blood glucose levels of 50-70 mg/dL and blood KB levels of 3-8 mM was defined. In Patient 1, an initial weight loss of 6% was seen followed by weight stabilization during the first week of ERKD. Patient 1 PM KBs rose to >3 mM an PM KBs to > 2 mM, where they remain for the duration of the study. However, Patient 1 AM and PM blood glucose levels remained >80 mg/dL. After 4 weeks on the ERKD, patient 1 withdrew from the study due to impairment of vision and cognition in which tumor growth was demonstrated within an MRI. In Patient 2, AM and

PM blood KBs rose to >3 mM where they remained for the duration of the study. However, Patient 2 blood glucose levels remained >100 mg/dl for the duration of the study. Six weeks after starting ERKD, stable disease was demonstrated within the clinical exam and PET scan of patient 2. Unfortunately, progression of disease was evident 12 weeks of after the start of ERKD. Unlike short term-fasting where glucose levels can reach as low as 50-65 mg/dL, the inability of the ERKD to reduce and maintain glucose levels between 50 to 70 mg/dL was cited as an explanation for the failure of ERKD to control progression of disease. In addition, IHC analysis of key ketolytic enzymes BDH-1 and OXCT-1 in the patients’ tumors showed diminished expression of BDH-1 and OXCT-1 in the original tumor of Patient 1.

However, subsequent biopsies showed positive expression of BDH-1 in Patient 1. In patient 2, positive expression BDH1 and OXCT-1 were seen in the original tumor. These results suggest that these tumors may possess the ability to metabolize KB for energy to be used during cellular growth.121

Future directions to evaluate the effects of fasting as an adjuvant therapy to the standard of care for

GBM in a clinical setting.

Our results suggest fasting can selectively enhance the cytotoxic effects of TMZ in GBM cells while protecting astroglial cells. However, clinical studies are needed to determine the effects of fasting in combination with TMZ for the treatment of GBM in patients. During concurrent chemotherapy and radiotherapy, GBM patients are given TMZ 75 mg/m2 daily for up to 6 weeks. One month after

76 completion of concurrent therapy, patients begin maintenance cycles lasting 6-12 months. The first cycle is 28 days long and consists of TMZ 150 mg/m2 daily during days 1-5. The remaining 28-day cycles consist of TMZ 200 mg/m2 daily during days 1-5. One potential study design could implement short-term fasting (72-hour) as an adjuvant therapy during days 1-3 of the cycle. Another potential study design could implement a short-term fast beginning 24-hours prior to day 1 and lasting through day 2 for the entirety of the 28-day cycle. To maximize therapeutic efficiency, it is critical to determine the range of blood glucose and KB concentration that has the strongest anti-tumor effect. Both studies should monitor the relative blood insulin/IGF-1, glucose, and KB levels. These studies are needed to further assess the potential of fasting as an adjuvant therapy in the treatment of GBM.

REFERENCES

1. Ostrom QT, Gittleman H, Farah P, et al. CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2006-2010. Neuro-Oncol. 2013;15(Suppl 2):ii1-ii56. doi:10.1093/neuonc/not151

2. Louis DN, Ohgaki H, Wiestler OD, et al. The 2007 WHO Classification of Tumours of the Central Nervous System. Acta Neuropathol (Berl). 2007;114(2):97-109. doi:10.1007/s00401-007-0243-4

3. Stupp R, Hegi ME, Mason WP, et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol Lond. 2009;10(5):459-466.

4. Qiu Z-K, Shen D, Chen Y-S, et al. Enhanced MGMT expression contributes to temozolomide resistance in glioma stem-like cells. Chin J Cancer. 2014;33(2):115-122. doi:10.5732/cjc.012.10236

5. Gaspar N, Marshall L, Perryman L, et al. MGMT-Independent Temozolomide Resistance in Pediatric Glioblastoma Cells Associated with a PI3-Kinase-Mediated HOX/Stem Cell Gene Signature. Cancer Res. 2010;70(22):9243-9252. doi:10.1158/0008-5472.CAN-10-1250

6. Hegi ME, Hamou M-F, de Tribolet N, et al. MGMT Gene Silencing and Benefit from Temozolomide in Glioblastoma. N Engl J Med. Published online 2005:7.

7. Gessler F, Bernstock JD, Braczynski A, et al. Surgery for Glioblastoma in Light of Molecular Markers: Impact of Resection and MGMT Promoter Methylation in Newly Diagnosed IDH-1 Wild-Type Glioblastomas. Neurosurgery. 2019;84(1):190-197. doi:10.1093/neuros/nyy049

8. Warburg O. The Metabolism of Carcinoma Cells. J Cancer Res. 1925;9(1):148-163. doi:10.1158/jcr.1925.148

9. Wicks RT, Azadi J, Mangraviti A, et al. Local delivery of cancer-cell glycolytic inhibitors in high-grade glioma. Neuro-Oncol. 2015;17(1):70-80. doi:10.1093/neuonc/nou143

10. Flavahan WA, Wu Q, Hitomi M, et al. Brain tumor initiating cells adapt to restricted nutrition through preferential glucose uptake. Nat Neurosci. 2013;16(10):1373-1382. doi:10.1038/nn.3510

11. Nelson DL (David L 1942-. Lehninger Principles of Biochemistry. Fourth edition. New York : W.H. Freeman, 2005.; 2005. https://search.library.wisc.edu/catalog/999964334502121

12. Wolf A, Agnihotri S, Munoz D, Guha A. Developmental profile and regulation of the glycolytic enzyme hexokinase 2 in normal brain and glioblastoma multiforme. Neurobiol Dis. 2011;44(1):84- 91. doi:10.1016/j.nbd.2011.06.007

13. Di H, Zhang X, Guo Y, et al. Silencing LDHA inhibits proliferation, induces apoptosis and increases chemosensitivity to temozolomide in glioma cells. Oncol Lett. 2018;15(4):5131-5136. doi:10.3892/ol.2018.7932

14. Wolf A, Agnihotri S, Micallef J, et al. Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor growth in human glioblastoma multiforme. J Exp Med. 2011;208(2):313-326. doi:10.1084/jem.20101470

15. Mayer A, Vaupel P, Struss H-G, Giese A, Stockinger M, Schmidberger H. Strong adverse prognostic impact of hyperglycemic episodes during adjuvant chemoradiotherapy of glioblastoma multiforme. Strahlenther Onkol Organ Dtsch Rontgengesellschaft Al. 2014;190(10):933-938. doi:10.1007/s00066-014-0696-z

16. Derr RL, Ye X, Islas MU, Desideri S, Saudek CD, Grossman SA. Association between hyperglycemia and survival in patients with newly diagnosed glioblastoma. J Clin Oncol Off J Am Soc Clin Oncol. 2009;27(7):1082-1086. doi:10.1200/JCO.2008.19.1098

17. McGirt MJ, Chaichana KL, Gathinji M, et al. PERSISTENT OUTPATIENT HYPERGLYCEMIA IS INDEPENDENTLY ASSOCIATED WITH DECREASED SURVIVAL AFTER PRIMARY RESECTION OF MALIGNANT BRAIN ASTROCYTOMAS. Neurosurgery. 2008;63(2):286-291. doi:10.1227/01.NEU.0000315282.61035.48

18. Hagan K, Bhavsar S, Arunkumar R, et al. Association Between Perioperative Hyperglycemia and Survival in Patients With Glioblastoma. doi:info:doi/10.1097/ANA.0000000000000339

19. Adeberg S, Bernhardt D, Foerster R, et al. The influence of hyperglycemia during radiotherapy on survival in patients with primary glioblastoma. Acta Oncol Stockh Swed. 2016;55(2):201-207. doi:10.3109/0284186X.2015.1043397

20. Lee C, Longo VD. Fasting vs dietary restriction in cellular protection and cancer treatment: from model organisms to patients. Oncogene. 2011;30(30):3305-3316. doi:10.1038/onc.2011.91

21. Anderson RM, Shanmuganayagam D, Weindruch R. Caloric Restriction and Aging: Studies in Mice and Monkeys. Toxicol Pathol. 2009;37(1):47-51. doi:10.1177/0192623308329476

22. Fontana L, Partridge L, Longo VD. Extending Healthy Life Span--From Yeast to Humans. Science. 2010;328(5976):321-326. doi:10.1126/science.1172539

23. Højlund K, Wildner-Christensen M, Eshøj O, et al. Reference intervals for glucose, β-cell polypeptides, and counterregulatory factors during prolonged fasting. Am J Physiol-Endocrinol Metab. 2001;280(1):E50-E58. doi:10.1152/ajpendo.2001.280.1.E50

24. Mattson MP, Wan R. Beneficial effects of intermittent fasting and caloric restriction on the cardiovascular and cerebrovascular systems. J Nutr Biochem. 2005;16(3):129-137. doi:10.1016/j.jnutbio.2004.12.007

25. Kirkwood TBL, Kapahi P, Shanley DP. Evolution, stress, and longevity. J Anat. 2000;197(4):587-590. doi:10.1046/j.1469-7580.2000.19740587.x

26. Lee C, Safdie FM, Raffaghello L, et al. Reduced IGF-I differentially protects normal and cancer cells and improves chemotherapeutic index in mice. Cancer Res. 2010;70(4):1564-1572. doi:10.1158/0008-5472.CAN-09-3228

27. Brandhorst S, Wei M, Hwang S, Morgan TE, Longo VD. Short-term calorie and protein restriction provide partial protection from chemotoxicity but do not delay glioma progression. Exp Gerontol. 2013;48(10):1120-1128. doi:10.1016/j.exger.2013.02.016

28. Raffaghello L, Lee C, Safdie FM, et al. Starvation-dependent differential stress resistance protects normal but not cancer cells against high-dose chemotherapy. Proc Natl Acad Sci U S A. 2008;105(24):8215-8220. doi:10.1073/pnas.0708100105

29. Lee C, Raffaghello L, Brandhorst S, et al. Fasting Cycles Retard Growth of Tumors and Sensitize a Range of Cancer Cell Types to Chemotherapy. Sci Transl Med. 2012;4(124):124ra27. doi:10.1126/scitranslmed.3003293

30. Safdie F, Brandhorst S, Wei M, et al. Fasting Enhances the Response of Glioma to Chemo- and Radiotherapy. PLOS ONE. 2012;7(9):e44603. doi:10.1371/journal.pone.0044603

31. Balasse EO, Féry F. Ketone body production and disposal: Effects of fasting, diabetes, and exercise. Diabetes Metab Rev. 1989;5(3):247-270. doi:10.1002/dmr.5610050304

32. Robinson AM, Williamson DH. Physiological roles of ketone bodies as substrates and signals in mammalian tissues. Physiol Rev. 1980;60(1):143-187. doi:10.1152/physrev.1980.60.1.143

33. Lifescience Promoting Association, Daikyo-cho 25-3-1004, Shinjuku-ku, Tokyo 160-0015, Japan, Watanabe S, Hirakaw A, et al. Ketone Body Production and Excretion During Wellness Fasting. Diabetes Res - Open J. 2017;3(1):1-8. doi:10.17140/DROJ-3-129

34. Cahill GF. Fuel Metabolism in Starvation. Annu Rev Nutr. 2006;26(1):1-22. doi:10.1146/annurev.nutr.26.061505.111258

35. Browning JD, Baxter J, Satapati S, Burgess SC. The effect of short-term fasting on liver and skeletal muscle lipid, glucose, and energy metabolism in healthy women and men. J Lipid Res. 2012;53(3):577-586. doi:10.1194/jlr.P020867

36. Chang HT, Olson LK, Schwartz KA. Ketolytic and glycolytic enzymatic expression profiles in malignant gliomas: implication for ketogenic diet therapy. Nutr Metab. 2013;10:47. doi:10.1186/1743-7075-10-47

37. Shimazu T, Hirschey MD, Newman J, et al. Suppression of Oxidative Stress by β-Hydroxybutyrate, an Endogenous Histone Deacetylase Inhibitor. Science. 2013;339(6116):211-214. doi:10.1126/science.1227166

38. Kong G, Huang Z, Ji W, et al. The Ketone Metabolite β-Hydroxybutyrate Attenuates Oxidative Stress in Spinal Cord Injury by Suppression of Class I Histone Deacetylases. J Neurotrauma. 2017;34(18):2645-2655. doi:10.1089/neu.2017.5192

39. Wang X, Wu X, Liu Q, et al. Ketogenic Metabolism Inhibits Histone Deacetylase (HDAC) and Reduces Oxidative Stress After Spinal Cord Injury in Rats. Neuroscience. 2017;366:36-43. doi:10.1016/j.neuroscience.2017.09.056

40. Was H, Krol SK, Rotili D, et al. Histone deacetylase inhibitors exert anti-tumor effects on human adherent and stem-like glioma cells. Clin Epigenetics. 2019;11(1):11. doi:10.1186/s13148-018- 0598-5

41. Woolf EC, Bhattacharya A, Rossi AP, Davidson MM, Syed N, Scheck AC. EXTH-48. THE KETONE BODY β-HYDROXYBUTYRATE CHEMO- AND RADIO-SENSITIZES MALIGNANT GLIOMA CELLS BY INHIBITING HISTONE DEACETYLASE ACTIVITY AND DOWNREGULATING EXPRESSION OF RAD51. Neuro-Oncol. 2017;19(suppl_6):vi83-vi83. doi:10.1093/neuonc/nox168.340

42. Friedman HS, Kerby T, Calvert H. Temozolomide and treatment of malignant glioma. Clin Cancer Res Off J Am Assoc Cancer Res. 2000;6(7):2585-2597.

43. Marchesi F, Turriziani M, Tortorelli G, Avvisati G, Torino F, De Vecchis L. Triazene compounds: Mechanism of action and related DNA repair systems. Pharmacol Res. 2007;56(4):275-287. doi:10.1016/j.phrs.2007.08.003

44. Duckett DR, Drummond JT, Murchie AI, et al. Human MutSalpha recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine, or the cisplatin-d(GpG) adduct. Proc Natl Acad Sci U S A. 1996;93(13):6443-6447.

45. Quiros S, Roos WP, Kaina B. Processing of O6-methylguanine into DNA double-strand breaks requires two rounds of replication whereas apoptosis is also induced in subsequent cell cycles. Cell Cycle. 2010;9(1):168-178. doi:10.4161/cc.9.1.10363

46. Mojas N, Lopes M, Jiricny J. Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA. Genes Dev. 2007;21(24):3342- 3355. doi:10.1101/gad.455407

47. Perazzoli G, Prados J, Ortiz R, et al. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression. PLOS ONE. 2015;10(10):e0140131. doi:10.1371/journal.pone.0140131

48. Stojic L, Mojas N, Cejka P, et al. Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase. Genes Dev. 2004;18(11):1331-1344. doi:10.1101/gad.294404

49. Cejka P, Stojic L, Mojas N, et al. Methylation-induced G(2)/M arrest requires a full complement of the mismatch repair protein hMLH1. EMBO J. 2003;22(9):2245-2254. doi:10.1093/emboj/cdg216

50. D’Atri S, Tentori L, Lacal PM, et al. Involvement of the mismatch repair system in temozolomide- induced apoptosis. Mol Pharmacol. 1998;54(2):334-341.

51. Leino RL, Gerhart DZ, Bueren AM van, McCall AL, Drewes LR. Ultrastructural localization of GLUT 1 and GLUT 3 glucose transporters in rat brain. J Neurosci Res. 1997;49(5):617-626. doi:10.1002/(SICI)1097-4547(19970901)49:5<617::AID-JNR12>3.0.CO;2-S

52. Vannucci SJ, Gibbs EM, Simpson IA. Glucose utilization and glucose transporter proteins GLUT-1 and GLUT-3 in brains of diabetic (db/db) mice. Am J Physiol-Endocrinol Metab. 1997;272(2):E267- E274. doi:10.1152/ajpendo.1997.272.2.E267

53. Duelli R, Kuschinsky W. Brain Glucose Transporters: Relationship to Local Energy Demand. Physiology. 2001;16(2):71-76. doi:10.1152/physiologyonline.2001.16.2.71

54. Thorens B, Mueckler M. Glucose transporters in the 21st Century. Am J Physiol - Endocrinol Metab. 2010;298(2):E141-E145. doi:10.1152/ajpendo.00712.2009

55. Mantych GJ, James DE, Chung HD, Devaskar SU. Cellular localization and characterization of Glut 3 glucose transporter isoform in human brain. Endocrinology. 1992;131(3):1270-1278. doi:10.1210/endo.131.3.1505464

56. Le Calvé B, Rynkowski M, Le Mercier M, et al. Long-term In Vitro Treatment of Human Glioblastoma Cells with Temozolomide Increases Resistance In Vivo through Up-regulation of GLUT Transporter and Aldo-Keto Reductase Enzyme AKR1C Expression. Neoplasia N Y N. 2010;12(9):727- 739.

57. MacIver NJ, Michalek RD, Rathmell JC. Metabolic Regulation of T Lymphocytes. Annu Rev Immunol. 2013;31(1):259-283. doi:10.1146/annurev-immunol-032712-095956

58. Lunt SY, Vander Heiden MG. Aerobic Glycolysis: Meeting the Metabolic Requirements of Cell Proliferation. Annu Rev Cell Dev Biol. 2011;27(1):441-464. doi:10.1146/annurev-cellbio-092910- 154237

59. Kouidhi S, Elgaaied AB, Chouaib S. Impact of Metabolism on T-Cell Differentiation and Function and Cross Talk with Tumor Microenvironment. Front Immunol. 2017;8. doi:10.3389/fimmu.2017.00270

60. Abdel-Haleem AM, Lewis NE, Jamshidi N, Mineta K, Gao X, Gojobori T. The Emerging Facets of Non-Cancerous Warburg Effect. Front Endocrinol. 2017;8. doi:10.3389/fendo.2017.00279

61. Baumann F, Leukel P, Doerfelt A, et al. Lactate promotes glioma migration by TGF-beta2- dependent regulation of matrix metalloproteinase-2. Neuro-Oncol. 2009;11(4):368-380. doi:10.1215/15228517-2008-106

62. Goetze K, Walenta S, Ksiazkiewicz M, Kunz-Schughart LA, Mueller-Klieser W. Lactate enhances motility of tumor cells and inhibits monocyte migration and cytokine release. Int J Oncol. 2011;39(2):453-463. doi:10.3892/ijo.2011.1055

63. Kumar VBS, Viji RI, Kiran MS, Sudhakaran PR. Endothelial cell response to lactate: implication of PAR modification of VEGF. J Cell Physiol. 2007;211(2):477-485. doi:10.1002/jcp.20955

64. San-Millán I, Brooks GA. Reexamining cancer metabolism: lactate production for carcinogenesis could be the purpose and explanation of the Warburg Effect. Carcinogenesis. 2017;38(2):119-133. doi:10.1093/carcin/bgw127

65. Koukourakis M, Tsolou A, Pouliliou S, et al. Blocking LDHA glycolytic pathway sensitizes glioblastoma cells to radiation and temozolomide. Biochem Biophys Res Commun. 2017;491(4):932-938. doi:10.1016/j.bbrc.2017.07.138

66. Guelpa G. La lutte contre l’epiepsie par la desintoxication et par la reeducation alimentaire. Rev Ther Med Chir. 1911;78:8.

67. Hartman AL, Rubenstein JE, Kossoff EH. Intermittent fasting: A “new” historical strategy for controlling seizures? Epilepsy Res. 2013;104(3):275-279. doi:10.1016/j.eplepsyres.2012.10.011

68. Hussin NM, Shahar S, Teng NIMF, Ngah WZW, Das SK. Efficacy of Fasting and Calorie Restriction (FCR) on mood and depression among ageing men. J Nutr Health Aging. 2013;17(8):674-680. doi:10.1007/s12603-013-0344-9

69. Lee C, Longo VD. Fasting vs dietary restriction in cellular protection and cancer treatment: from model organisms to patients. Oncogene. 2011;30(Copyright (C) 2019 American Chemical Society (ACS). All Rights Reserved.):3305-3316. doi:10.1038/onc.2011.91

70. Li G, Xie C, Lu S, et al. Intermittent Fasting Promotes White Adipose Browning and Decreases Obesity by Shaping the Gut Microbiota. Cell Metab. 2017;26(4):672-685.e4. doi:10.1016/j.cmet.2017.08.019

71. Kawamori D, Kulkarni RN. Insulin modulation of glucagon secretion: the role of insulin and other factors in the regulation of glucagon secretion. Islets. 2009;1(3):276-279. doi:10.4161/isl.1.3.9967

72. Berg JM, Tymoczko JL, Stryer L. Food Intake and Starvation Induce Metabolic Changes. Biochem 5th Ed. Published online 2002. Accessed April 18, 2019. http://www.ncbi.nlm.nih.gov/books/NBK22414/

73. Nelson DL, Cox MM. Lehninger Principles of Biochemistry. W. H. Freeman; 2017.

74. Etoposide. National Cancer Institute. Published August 12, 2008. Accessed August 23, 2019. https://www.cancer.gov/about-cancer/treatment/drugs/etoposide

75. Montecucco A, Zanetta F, Biamonti G. Molecular mechanisms of etoposide. EXCLI J. 2015;14:95- 108. doi:10.17179/excli2015-561

76. Pardridge W. Blood-Brain Barrier Transport of Glucose, Free Fatty Acids, and Ketone Bodies. Adv Exp Med Biol. 1991;291:43-53. doi:10.1007/978-1-4684-5931-9_5

77. Alvarez AA, Field M, Bushnev S, Longo MS, Sugaya K. The Effects of Histone Deacetylase Inhibitors on Glioblastoma-Derived Stem Cells. J Mol Neurosci. 2015;55(1):7-20. doi:10.1007/s12031-014- 0329-0

78. Yao T, Asayama Y. Animal-cell culture media: History, characteristics, and current issues. Reprod Med Biol. 2017;16(2):99-117. doi:10.1002/rmb2.12024

79. Lucey BP, Nelson-Rees WA, Hutchins GM. Henrietta Lacks, HeLa Cells, and Cell Culture Contamination. Arch Pathol Lab Med. 2009;133:5.

80. Valley MP, Unch J. CellTiter-Glo® 2.0: A Novel Luminescent Cell Viability Assay with Greatly Enhanced Storage Stability. :1.

81. Szmant HH. PHYSICAL PROPERTIES OF DIMETHYL SULFOXIDE AND ITS FUNCTION IN BIOLOGICAL SYSTEMS. Ann N Y Acad Sci. 1975;243(1):20-23. doi:10.1111/j.1749-6632.1975.tb25340.x

82. Sang D, Li R, Lan Q. Quercetin sensitizes human glioblastoma cells to temozolomide in vitro via inhibition of Hsp27. Acta Pharmacol Sin. 2014;35(6):832-838. doi:10.1038/aps.2014.22

83. Cortese K, Daga A, Monticone M, et al. Carnosic acid induces proteasomal degradation of Cyclin B1, RB and SOX2 along with cell growth arrest and apoptosis in GBM cells. Phytomedicine. 2016;23(7):679-685. doi:10.1016/j.phymed.2016.03.007

84. Galvao J, Davis B, Tilley M, Normando E, Duchen MR, Cordeiro MF. Unexpected low‐dose toxicity of the universal solvent DMSO. FASEB J. 2014;28(3):1317-1330. doi:10.1096/fj.13-235440

85. Perazzoli G, Prados J, Ortiz R, et al. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression. PLOS ONE. 2015;10(10):e0140131. doi:10.1371/journal.pone.0140131

86. Dunn J, Baborie A, Alam F, et al. Extent of MGMT promoter methylation correlates with outcome in glioblastomas given temozolomide and radiotherapy. Br J Cancer. 2009;101(1):124-131. doi:10.1038/sj.bjc.6605127

87. Di Martino A, Kucharczyk P, Capakova Z, Humpolicek P, Sedlarik V. Enhancement of temozolomide stability by loading in chitosan-carboxylated polylactide-based nanoparticles. J Nanoparticle Res. 2017;19(2):71. doi:10.1007/s11051-017-3756-3

88. Laplante M, Sabatini DM. mTOR Signaling in Growth Control and Disease. Cell. 2012;149(2):274- 293. doi:10.1016/j.cell.2012.03.017

89. Xiao B, Sanders MJ, Underwood E, et al. Structure of mammalian AMPK and its regulation by ADP. Nature. 2011;472(7342):230-233. doi:10.1038/nature09932

90. Hardie DG. AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nat Rev Mol Cell Biol. 2007;8(10):774-785. doi:10.1038/nrm2249

91. Jones RG, Plas DR, Kubek S, et al. AMP-activated protein kinase induces a p53-dependent metabolic checkpoint. Mol Cell. 2005;18(3):283-293. doi:10.1016/j.molcel.2005.03.027

92. Nakanishi A, Kitagishi Y, Ogura Y, Matsuda S. The tumor suppressor PTEN interacts with p53 in hereditary cancer (Review). Int J Oncol. 2014;44(6):1813-1819. doi:10.3892/ijo.2014.2377

93. Stambolic V, MacPherson D, Sas D, et al. Regulation of PTEN transcription by p53. Mol Cell. 2001;8(2):317-325. doi:10.1016/s1097-2765(01)00323-9

94. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008;455(7216):1061-1068. doi:10.1038/nature07385

95. Schlapbach R, Fontana A. Differential activity of bcl-2 and ICE enzyme family protease inhibitors on Fas and puromycin-induced apoptosis of glioma cells. Biochim Biophys Acta. 1997;1359(2):174- 180. doi:10.1016/s0167-4889(97)00096-7

96. Ishii N, Maier D, Merlo A, et al. Frequent co-alterations of TP53, p16/CDKN2A, p14ARF, PTEN tumor suppressor genes in human glioma cell lines. Brain Pathol Zurich Switz. 1999;9(3):469-479. doi:10.1111/j.1750-3639.1999.tb00536.x

97. Wyngaert IV den, Vries W de, Kremer A, et al. Cloning and characterization of human histone deacetylase 8. FEBS Lett. 2000;478(1-2):77-83. doi:10.1016/S0014-5793(00)01813-5

98. Santos-Barriopedro I, Li Y, Bahl S, Seto E. HDAC8 affects MGMT levels in glioblastoma cell lines via interaction with the proteasome receptor ADRM1. Genes Cancer. 2019;10(5-6):119-133. doi:10.18632/genesandcancer.197

99. Steliou K, Boosalis MS, Perrine SP, Sangerman J, Faller DV. Butyrate Histone Deacetylase Inhibitors. BioResearch Open Access. 2012;1(4):192-198. doi:10.1089/biores.2012.0223

100. Gao S, Chen C, Wang L, et al. Histone deacetylases inhibitor sodium butyrate inhibits JAK2/STAT signaling through upregulation of SOCS1 and SOCS3 mediated by HDAC8 inhibition in myeloproliferative neoplasms. Exp Hematol. 2013;41(3):261-270.e4. doi:10.1016/j.exphem.2012.10.012

101. Wang Z-T, Chen Z-J, Jiang G-M, et al. Histone deacetylase inhibitors suppress mutant p53 transcription via HDAC8/YY1 signals in triple negative breast cancer cells. Cell Signal. 2016;28(5):506-515. doi:10.1016/j.cellsig.2016.02.006

102. Ghiaghi M, Forouzesh F, Rahimi H. Effect of Sodium Butyrate on LHX1 mRNA Expression as a Transcription Factor of HDAC8 in Human Colorectal Cancer Cell Lines. Avicenna J Med Biotechnol. 2019;11(4):317-324.

103. Nudelman A, Gnizi E, Katz Y, et al. Prodrugs of butyric acid. Novel derivatives possessing increased aqueous solubility and potential for treating cancer and blood diseases. Eur J Med Chem. 2001;36(1):63-74. doi:10.1016/S0223-5234(00)01199-5

104. Kuroiwa‐Trzmielina J, Conti A de, Scolastici C, et al. Chemoprevention of rat hepatocarcinogenesis with histone deacetylase inhibitors: Efficacy of tributyrin, a butyric acid prodrug. Int J Cancer. 2009;124(11):2520-2527. doi:10.1002/ijc.24212

105. Rephaeli A, Entin-Meer M, Angel D, et al. The selectivty and anti-metastatic activity of oral bioavailable butyric acid prodrugs. Invest New Drugs. 2006;24(5):383-392. doi:10.1007/s10637- 006-6213-1

106. Kiseleva LN, Kartashev AV, Vartanyan NL, Pinevich AA, Samoilovich MP. A172 and T98G cell lines characteristics. Cell Tissue Biol. 2016;10(5):341-348. doi:10.1134/S1990519X16050072

107. Li A, Walling J, Kotliarov Y, et al. Genomic Changes and Gene Expression Profiles Reveal That Established Glioma Cell Lines Are Poorly Representative of Primary Human Gliomas. Mol Cancer Res. 2008;6(1):21-30. doi:10.1158/1541-7786.MCR-07-0280

108. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res. 1961;25(3):585-621. doi:10.1016/0014-4827(61)90192-6

109. Campisi J. The biology of replicative senescence. Eur J Cancer Oxf Engl 1990. 1997;33(5):703-709. doi:10.1016/S0959-8049(96)00058-5

110. Major EO, Miller AE, Mourrain P, Traub RG, de Widt E, Sever J. Establishment of a line of human fetal glial cells that supports JC virus multiplication. Proc Natl Acad Sci U S A. 1985;82(4):1257- 1261.

111. Henriksen S, Tylden GD, Dumoulin A, Sharma BN, Hirsch HH, Rinaldo CH. The Human Fetal Glial Cell Line SVG p12 Contains Infectious BK Polyomavirus. J Virol. 2014;88(13):7556-7568. doi:10.1128/JVI.00696-14

112. Linkous A, Balamatsias D, Snuderl M, et al. Modeling Patient-Derived Glioblastoma with Cerebral Organoids. Cell Rep. 2019;26(12):3203-3211.e5. doi:10.1016/j.celrep.2019.02.063

113. Pegg AE, Dolan ME, Moschel RC. Structure, Function, and Inhibition of O6-Alkylguanine-DNA Alkyltransferase. In: Cohn WE, Moldave K, eds. Progress in Nucleic Acid Research and Molecular Biology. Vol 51. Academic Press; 1995:167-223. doi:10.1016/S0079-6603(08)60879-X

114. Dorff TB, Groshen S, Garcia A, et al. Safety and feasibility of fasting in combination with platinum- based chemotherapy. BMC Cancer. 2016;16(1):360. doi:10.1186/s12885-016-2370-6

115. Bauersfeld SP, Kessler CS, Wischnewsky M, et al. The effects of short-term fasting on quality of life and tolerance to chemotherapy in patients with breast and ovarian cancer: a randomized cross- over pilot study. BMC Cancer. 2018;18(1):476. doi:10.1186/s12885-018-4353-2

116. de Groot S, Vreeswijk MP, Welters MJ, et al. The effects of short-term fasting on tolerance to (neo) adjuvant chemotherapy in HER2-negative breast cancer patients: a randomized pilot study. BMC Cancer. 2015;15(1):652. doi:10.1186/s12885-015-1663-5

117. LaFountain RA, Miller VJ, Barnhart EC, et al. Extended Ketogenic Diet and Physical Training Intervention in Military Personnel. Mil Med. Published online March 16, 2019. doi:10.1093/milmed/usz046

118. Martin-McGill KJ, Lambert B, Whiteley VJ, et al. Understanding the core principles of a “modified ketogenic diet”: a UK and Ireland perspective. J Hum Nutr Diet Off J Br Diet Assoc. 2019;32(3):385- 390. doi:10.1111/jhn.12637

119. Roehl K, Falco-Walter J, Ouyang B, Balabanov A. Modified ketogenic diets in adults with refractory epilepsy: Efficacious improvements in seizure frequency, seizure severity, and quality of life. Epilepsy Behav EB. Published online March 10, 2019. doi:10.1016/j.yebeh.2018.12.010

120. Rieger J, Bähr O, Maurer GD, et al. ERGO: A pilot study of ketogenic diet in recurrent glioblastoma Erratum in /ijo/45/6/2605. Int J Oncol. 2014;44(6):1843-1852. doi:10.3892/ijo.2014.2382

121. Schwartz K, Chang HT, Nikolai M, et al. Treatment of glioma patients with ketogenic diets: report of two cases treated with an IRB-approved energy-restricted ketogenic diet protocol and review of the literature. Cancer Metab. 2015;3(1):3. doi:10.1186/s40170-015-0129-1