Cellular effects of novel small molecule AMPK activator YLD466D in human prostate cancer
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5' adenosine monophosphate-activated protein kinase or AMPK is a heterotrimer complex that regulates cellular metabolism along with integrating various signaling pathways to organize a diverse range of cellular processes. Recently AMPK is being studied in cancer to elucidate it mechanism in regulating cellular proliferation, cell cycle progression and apoptotic induction. Direct AMPK activators are being developed to study these AMPK-mediated cellular effects in various types of cancer to provide future therapeutic benefit. Prostate cancer is the most commonly diagnosed form of cancer among men in the United States. It is second only to lung cancer as the leading cause of cancer-related deaths in males. The lethality of prostate cancer can be attributed to the progression of the tumor in becoming androgen-independent. Drugs and compounds known to activate AMPK have gained research focus to investigate AMPK as a new target for cancer treatment and drug development. Current diabetic medications already known to indirectly activate AMPK have a wide range of adverse side effects. With novel compounds being just recently screened for direct AMPK activation, in vitro studies must be done to establish to basic cellular effects of these compounds to augment further use as useful research tools. The objective of the present thesis was to first, establish the anti-proliferative effects of AMPK activators comparing a known diabetic drug, rosiglitazone with a recently screened novel direct AMPK activator YLD466D and then investigate the novel compound's cellular effects involved in reducing cancer cell proliferation. Results from in vitro experimentation on human prostate cancer cell lines (C4-2, and PC-3) in comparison with non-tumorigenic epithelial cell line (RWPE-1) reveal AMPK activators rosiglitazone and novel compound YLF466D are selective toward tumorigenic prostate cells at specific dose ranges. In vitro exposures of YLF466D were conducted. Cell viability was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Protein expression levels were examined by Western blotting. Reduced glutathione and oxidized glutathione levels were measured using the DTNB-GSSG reductase recycling assay method. The mitochondrial membrane potential was measured by using the mitochondrial probe JC-1 [5,5',6,6'- tetrachloro-1,1',3,3' tetra-ethylbenzimidazolo-carbocyanine iodide]. Cell cycle was analyzed by fluorescence cytometry with propidium iodide staining.