Investigating the effects of the inhibition of citrate transport protein on breast cancer cells

Cellular metabolic alterations in cancer cells are among the hallmarks of the cancer which is one of the most important medical problems of our age. Increased rate of glycolysis and synthesis pathways along with decreased rate of degradation pathways are mmong these alterations. Anabolic pathways required for cells to create novel cells are protein, nucleotide and fatty acid synthesis pathways. Novel studies suggest that proteins playing a role in these pathways such as ATP-citrate lyase (ACLY), acetyl-CoA carboxilase and fatty acid synthease have high activity in cancer cells. However the role of citrate transport protein (CTP) which enables transportation of citrate from mitochondria into the cytoplasm where it plays a critical role for fatty acid synthesis as a source for cytoplasmic acetyl-CoA, is rather unknown. In this study the importance of citrate transport protein for cellular processes is examined by its inhibition in breast cancer cell lines via siRNA or chemical inhibition. Among the cell lines; MCF-10A represents normal epithelial cells, MCF-7 represents less-aggressive breast cancer and MDA-MB-231 represents metastatic breast cancer. After siRNA treatment the efficiency of the inhibition was demonstrated via western blotting and the alterations in cytoplasmic citrate levels were detected via spectrophotometry. Cell viability was assessed by spectrophotometric crystal violet assay while the alterations in apoptosis, necrosis and cell cycle regulation were evaluated via flow cytometry. Autophagy was assessed both by fluorescence microscopy and flow cytometry. Lastly the effects of the inhibition on acetylation of histones, an epigenetic regulation process which requires cytopalsmic acetyl-CoA, were detected spectrophotometrically. During the experiments ACLY was also inhibited along with CTP and the results were evaluated comparatively Obtained results suggest the proteins of interest are expressed in all cell lines and siRNA treatment can effectively silence the proteins. An association between basal cytoplasmic citrate levels and the aggressiveness of the cancer was determined and these levels were reduced partially by siRNA treatment and effectively by chemical inhibition. According to the data obtained from viability experiments inhibition of CTP and ACLY inhibit cancer cell viability without affecting normal cells. This inhibition is more evident in cells treated with chemical inhibitor. The reason of the inhibition of cell viability was also studied, however no alteration in apoptosis, necrosis, cell cycle or autophagy strong enough for to cause such inhibition was detected. Lastly inhibition was also detected to inhibit histone acetylation which was more prominent in cells treated with chemical inhibitor accordance with previous experiments. Data obtained throughout this thesis demonstrates the importance of CTP and ACLY for cell viability and histone acetylation, and there processes can be interfered with by inhibiting those proteins. High potency of chemical inhibition compared to RNA interference suggest the importance of extracellular citrate as a source of cytoplasmic citrate and that this pathway is effected by chemical inhibition. However effectiveness of the inhibition of both protein on cancer cells without affecting normal cells demonstrates that targeting these proteins is advantageous for cancer therapy strategies.