Author: Mostafa Khairy

Publisher:

Published: 2022

Total Pages: 0

ISBN-13:

Glioblastoma has a devastating prognosis, and a remarkably low survival rate. Current therapeutic strategies including surgery, radiation and temozolomide are only useful as a palliative therapy, with no significant increase in overall survival. In this study, we investigated multiple therapeutic approaches, both as a new main treatment and as an adjuvant therapy to enhance the sensitivity of glioblastoma cells to radiation. Histone acetylation and deacetylation is an important epigenetic mechanism for controlling gene expression and DNA repair. Histone acetylation and deacetylation are controlled by two classes of enzymes: Histone acetyltransferases (HATs) and Histone deacetylases (HDACs). Histone acetylation occurs on the lysine residues of histones, which neutralizes the lysine positive charge, leading to weakening of the electrostatic attraction between those residues and DNA. This induces a less condensed chromatin condition, which enhances the accessibility of DNA binding proteins to DNA. We postulated that the inhibition of histone acetylation will increase the sensitivity of glioblastoma cells to radiation. In the first part of my thesis, I used immunofluorescence microscopy to examine the effect of HAT inhibition on chromatin condensation and DNA double strand breaks after radiation, with no significant effect observed. HATs use acetyl CoA as the substrate for histone acetylation. Acetyl CoA is produced in the cell through various metabolic pathways, which include glucose, fatty acids, and glutamine metabolism. It has been reported that the acetyl CoA that is used for histone acetylation comes mainly from glucose metabolism in a panel of colorectal, prostate, glioblastoma and breast cancer cell lines. In the second part of my thesis, I investigated the effect of glucose concentrations in culture media on histone H3 and histone H4 acetylation, with the goal of controlling histone acetylation with glucose concentration and determining the impact on DNA repair. However, glucose concentration in the media had no effect histone acetylation. Finally, in the third part of my thesis, I examined whether tumor selective energy deprivation using 'ketogenic diet' culture media might affect the growth of glioblastoma cells. Research from different labs showed that glioblastoma cells have impaired mitochondrial function, and less active OXCT1 3-oxoacid-CoA transferase 1, leaving them with a disrupted ability to metabolize a ketogenic diet. We saw this as a therapeutic window opportunity, with a ketogenic diet affecting energy production in glioblastoma cells but not affecting the normal cells in the brain. Our results indicated that both healthy tissue and glioblastoma cells were unable to use the ketogenic diet for energy production. Therefore, we did not pursue this approach.Finally, in the last part of my thesis, I evaluated sulforaphane, a natural product that occurs in broccoli sprouts, as a new therapeutic approach for glioblastoma, for its anti-cancer properties. I found that sulforaphane exerted a cytotoxic effect on both U251 and U87 cells in a dose responsive manner. In addition, sulforaphane showed anti-invasion in U87 cells and cell cycle inhibition effects in U251 glioblastoma cells. Sulforaphane, however, did not enhance radiation cytotoxicity using the alamarBlue cytotoxicity assay. In conclusion, we believe that sulforaphane is a good candidate for in vivo studies and further research for glioblastoma therapy. In conclusion, in this thesis we investigated different strategies to increase the efficacy of radiation treatment in glioblastoma therapy. The methods we used included HAT inhibition, changing the glucose concentration to affect histone acetylation, ketogenic diet media and sulforaphane. Among these, only sulforaphane showed cytotoxic, anti-invasive and cell cycle inhibition effects on glioblastoma cells. For future directions, we plan to test sulforaphane synergy with radiation.