The molecular basis of the phenotype observed when normal cells recover from camptothecin and methyl pyruvate

A thesis submitted to the Faculty of Science under the School of Molecular and Cell Biology, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2017.Cancer has become a major cause of death in modern society. New cancer cases are estimated to increase to 22.2 million by 2030. Many chemotherapeutic drugs, such as irinotecan, target the p53 pathway in rapidly dividing cells. However, chemotherapeutic drugs possess little discrimination between normal and cancer cells since they target DNA replication, a normal physiological process. Many studies indicate that cancer and normal cells prefer different glucose metabolic pathways. Cancer cells rely mainly on glycolysis while normal cells prefer the oxidative phosphorylation (OXPHOS). We hypothesise that manipulating the known metabolic programme of cancer cells to follow the OXPHOS pathway experienced by normal cells may alleviate side effects experienced by normal cells and accelerate cancer cell death. In order to enhance OXPHOS optimal pyruvate levels were used. To investigate the effect of this on normal and cancer cell division, cell viability and cytotoxicity was measured in real-time using xCELLigence technology. Flow cytometry was then used to determine the mode of cell death and cell cycle changes induced by the various treatments. In order to characterise the molecular bases of this, differential gene expression upon treatment with the various drugs was measured by RT-PCR and Western blot analysis. Furthermore, we conducted microarray analyses using the Human Cancer PathwayFinder array to determine pathways affected by the drug combination in A549 lung fibroblast cancer and a normal lung fibroblast (MRC-5). Differentially expressed genes with a fold change ≥ 2 were analysed by PANTHER, GeneMania and Reactome. Overall, this study shows that the reversal of the metabolic program in A549 and MDA-MB 231 cancer cells with chemotherapeutic agents accelerates the death of cancer cells while promoting the survival of normal MRC-5 cells. However, in the breast cancer cell line, MDA-MB 231 with mutant p53, it is observed that inhibition of proliferation is by another cell death mechanism. RT-PCR and western blot analyses indicate that introduction of exogenous methyl pyruvate enhanced the p53/p21 axis of the apoptotic pathway resulting in cancer cell death. While promoting survival in MRC-5 normal lung fibroblast cells by turning off the p53/p21 axis of the apoptotic pathway. Furthermore, the introduction of exogenous methyl pyruvate promotes MRC-5 cell proliferation by modulating the expression of RBBP6 isoform 1 and isoform 3. Microarray analysis indicates alterations in specific genes promote the survival and growth of MRC-5 normal cells and death of A549 lung cancer cells. This knowledge may provide an opportunity to protect patients who undergo chemotherapy from the harsh side-effects and increase the success rate of chemotherapeutic drugs.LG201