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

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Evaluating the Effects of Fasting Conditions in Combination with Temozolomide for the Treatment of Glioblastoma Multiforme Northern Michigan University NMU Commons All NMU Master's Theses Student Works 7-2020 EVALUATING THE EFFECTS OF FASTING CONDITIONS IN COMBINATION WITH TEMOZOLOMIDE FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME Joseph Duffy [email protected] Follow this and additional works at: https://commons.nmu.edu/theses Part of the Cancer Biology Commons Recommended Citation Duffy, Joseph, "EVALUATING THE EFFECTS OF FASTING CONDITIONS IN COMBINATION WITH TEMOZOLOMIDE FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME" (2020). All NMU Master's Theses. 638. https://commons.nmu.edu/theses/638 This Open Access is brought to you for free and open access by the Student Works at NMU Commons. It has been accepted for inclusion in All NMU Master's Theses by an authorized administrator of NMU Commons. For more information, please contact [email protected],[email protected]. EVALUATING THE EFFECTS OF FASTING CONDITIONS IN COMBINATION WITH TEMOZOLOMIDE FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME By 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 i 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 By 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. iii Copyright by Joseph T. Duffy 2020 iv 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. v 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. vi 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 vii 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 viii 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
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