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CTP: Phosphocholine Cytidyltransferase Alpha (CCTα) siRNA Induce Cell Death of Lung Cancer Cells

Rukia Marijani and Barack O. Abonyo

CTP: Choline cytidylyltransferase (CCT) is a rate limiting enzyme required for the synthesis of phosphatidylcholine (PC) through the CDP-choline pathway, which is critical for cell proliferation. Cells conditionally lacking CCT? activity undergo death by apoptosis. Inhibition of CCT? expression or activity in cancer cells using current molecular techniques would provide an excellent non toxic mechanism of killing cancer cells. The purpose of this study was to investigate the possibility of using CCT? siRNA sequences as anticancer agents by studying their ability to induce death of lung cancer cells. p53 positive, A549 and negative, H1299 lung epithelial cell lines were transfected with various concentrations (0-800 nM) of CCT? siRNA sequences (A, B, and C) and scrambled sequence siRNA (siRNA S) as control for 24 hrs. Inhibition of CCT? expression by various siRNA was determined by Western blot analysis. Olympus microscope was used to observe the impact of CCT? knockdown on cell structure and morphology while the role of CCT? siRNA in cell toxicity and survival was analyzed by Cell Titer Blue (CTB) and Lactose dehydrogenase (LDH) assays. All the CCT? siRNA sequences significantly inhibited CCT? protein expression. Pictures captured by the microscope depicted that untreated and siRNA S treated cells were intact and had visible nuclei while CCT?- siRNA transfected cells were shrunken with nuclei containing dark granular structures similar to apoptotic bodies. At higher CCT?-siRNA concentrations the cell membranes were completely degraded showing signs of cell lyses. CTB assay revealed that untreated cells exhibited high fluorescence intensity indicating presence of more viable cells, which decreased as CCT?-siRNA concentration increased. LDH assay showed that CCT?-siRNA transfected cells exhibited higher absorbencies than the untreated reflective of dead cells. Both assays indicated that siRNA S treated cells exhibited results similar to untreated cells, suggesting that the cells death was specific for CCTasiRNA inhibition. Furthermore, addition of PC did not improve viability of CCT?-siRNA treated cells. These results demonstrate that CCT?-siRNAs knocked down CCT? protein resulting in cell death implying that the sequences could be useful as anticancer agents.