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Title: Pyruvate Dehydrogenase Kinase (PDK): An attractive target for anticancer therapy
Author: Madhok, Brijesh Madanmohan
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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Cancer cells are highly dependent on glycolysis. which provides protection against the hypoxic tumour microenvironment. Colorectal cancer cells have been reported to undergo increased glycolysis, the so-called 'Warburg's effect'. Previous research in our laboratory revealed increased expression of hypoxic and glycolytic markers to be associated with aggressive colorectal cancer phenotype. Pyruvate dehydrogenase kinase (PDK) is a crucial mitochondrial enzyme, which plays an important role linking glycolysis to oxidative phosphorylation. Inhibiting PDK with its pharmacological inhibitor, Dichloroacetate, or with RNA interference has been reported to have anti-cancer effects in other solid cancers such as head and neck squamous cell cancer. Since colorectal cancer cells are also known to be reliant on glycolysis, my aim was to determine if switching metabolism from glycolysis towards mitochondrial respiration, by inhibiting PDK, would reduce growth preferentially in colorectal cancer cells over normal cetis, and to examine the underlying mechanisms. Representative colorectal cancer (HT29, SW4BO and LaVa) and non-cancerous cell lines (HB2 and 293) were treated with Dichloroacetate (DCA), an inhibitor of PDK. 20mM DCA did not reduce growth of non-cancerous cells but caused significant decrease in cancer cell proliferation, which was associated with apoptosis and G2 phase cell cycle arrest. DCA reduced lactate levels in growth media and induced dephosphorylation of E10 sub-unit of pyruvate dehydrogenase complex in all cell lines, but the intrinsic mitochondrial membrane potential was reduced in only cancer cells. PDK exists in four isoenzymes (PDK 1-4). Next, I aimed to examine expression levels of the individual PDK isoenzymes in the panel of cell lines used, downregulate the individual PDK isoenzymes using short interierence RNA (siRNA), and • - 6 - investigate whether a subset of the PDK isoenzymes represents the actual target of DCA. Expression levels of individual PDK isoenzymes varied amongst the cell lines in a manner that did not suggest which was the main target of DCA. Down regulation 01 individual PDK isoenzymes failed to induce apoptosis in the cancer cells and, interestingly there was a compensatory rise in the mRNA levels of the other PDK isoenzymes. Treatment with siRNAs directed against various combinations of PDK isoenzymes did not induce apoptosis in cancer cells. Treatment of cells with DCA led to an up regulation of PDK isoenzymes, particularly PDK4. These results indicate that it is difficult to isolate a subset of PDK isoenzymes as potential therapeutic targets since a combination of functional redundancy and compensatory up-regulation among the isoenzymes means that sufficient overall PDK activity is maintained. Cancer cells are most sensitive to radiation in the G2-M phase of the cell cycle. Since DCA induced G2 arrest in the colorectal cancer cells, my hypothesis was that it sensitises these cells to radiation. HT29, SW480, LoVo, and 293 cells were treated with 10 and 20mM DCA, and irradiated. DCA did not induce any change in the sensitivity of 293 or HT29 cells to radiation. In contrast, SW480 and LoVo cells were significantly sensitised to radiation after pre-treatment with 10mM, but not 20mM DCA. There were minimal effects of 10 and 20mM DCA on the cell cycle profiles of all the cell lines studied. The cell lines derived from the poorly differentiated or metastatic cancers were sensitised to radiation following pretreatment with DCA, and cell lines derived from well-differentiated colorectal cancer or non-cancerous epithelial cells were resistant to any such sensitisation. However I am unable to provide any evidence to explain this radiosensitisation effect. This warrants further experiments to confirm the underlying molecular basis of these effects of DCA .
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available