Open Access Research Article Marijani and Abonyo Pharm Anal Acta 2011, 2:3 10.4172/2153-2435.1000121 Volume 2 • Issue 3 • 1000121 Pharm Anal Acta ISSN: 2153-2435 PAA, an open access journal Keywords: CCTa; siRNA; Phosphatidylcholine (PC); apoptosis Introduction e synthesis of phosphatidylcholine (PC) through the CDP- choline pathway is a crucial process in mammalian cells development, as PC has a critical role in membrane structure and cellular signaling [1]. Certain cancers are associated with changes in expression of genes and proteins involved in the replenishing the membranes through synthesis of the phosphatidylcholine (PC). Consistently elevated PC levels have been observed in several types of cancers including human breast cancer cells in culture [2,3]. Indeed, progression of cells from G0 to G1 is accompanied by an increased rate of PC synthesis [4]. van der Sanden et al. [5] reported that inhibition of PC synthesis induced expression of the endoplasmic reticulum stress and apoptosis-related protein CCAAT/enhancer-binding protein-homologous protein (CHOP/GADD153) leading to programmed cell death. Because PC biosynthesis is very much connected to cancer development, several drugs have been designed to inhibit its biosynthetic pathway. For instance, the inhibition of PC synthesis both in vivo and in vitro by the antitumor phospholipid analogs, ET-18-OCH3 (or edelfosine) and hexadecylphosphocholine (or miltefosine) correlates with their cytotoxic properties [6]. e observations that choline deficiency induces apoptosis and that PC supplementation does not rescue cells from apoptosis in the presence of antineoplastic phospholipids [7] strongly supports the hypothesis that sufficient amount of PC is required for cell proliferation including that of cancerous cells. e limiting enzyme in the PC synthesis is CTP: Phosphocholine Cytidylyltransferase (CCT), which is derived from two genes on distinct chromosomes in human and mouse. Phosphate cytidylyltransferase 1 alpha (Pcyt1a) gene encodes the CCTα isoform and Phosphate cytidylyltransferase 1 beta (Pcyt1b) gene encodes the CCTβ1 (in human only), β2, and β3 isoforms [8]. e CCTα and CCTβ proteins are similar in several ways, for instance they have nearly identical amino acid sequences in the catalytic domain (residues 72–233 for both), which is highly conserved among all CCTs from yeast to mammals and contains two motifs, NTP binding motif (HXGH) and RTEGISTS, which are involved in the binding of CTP to membranes [9-11]. Additionally, they have an almost identical membrane interaction domain (residues 256–288), which consists of three consecutive 11-residue repeats that form an amphipathic α-helix that regulates CCT activity [12,13]. On the contrary, the amino terminus of CCTβ completely differs that of CCTα and does not include a nuclear localization sequence as CCTα. Two CCTβ transcripts are expressed in human tissues, CCΤβ1 is a smaller protein that lacks the carboxyl terminal domain while CCTβ2 has carboxyl terminus [14]. e carboxyl terminal domain of CCTα and β2 are distinct, opening the possibility that the proteins are differentially regulated by anionic lipids and/or phosphorylation. CCTα *Corresponding author: Barack O. Abonyo, Florida A & M University, College of Arts and Sciences, Department of Biological Sciences, Tallahassee, FL 32307, Telephone: 850-561-2553; FAX: 850-599-3347; E-mail: abonyo.barack@famu. edu, luo31@hotmail.com Received March 10, 2011; Accepted March 30, 2011; Published May 21, 2011 Citation: Marijani R, Abonyo BO (2011) CTP: Phosphocholine Cytidyltransferase Alpha (CCTα) siRNA Induce Cell Death of Lung Cancer Cells. Pharm Anal Acta 2:121. doi:10.4172/2153-2435.1000121 Copyright: © 2011 Marijani R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract 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 CCTα activity undergo death by apoptosis. Inhibition of CCTα 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 CCTα 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 CCTα siRNA sequences (A, B, and C) and scrambled sequence siRNA (siRNA S) as control for 24 hrs. Inhibition of CCTα expression by various siRNA was determined by Western blot analysis. Olympus microscope was used to observe the impact of CCTα knockdown on cell structure and morphology while the role of CCTα siRNA in cell toxicity and survival was analyzed by Cell Titer Blue (CTB) and Lactose dehydrogenase (LDH) assays. All the CCTα siRNA sequences significantly inhibited CCTα protein expression. Pictures captured by the microscope depicted that untreated and siRNA S treated cells were intact and had visible nuclei while CCTα- siRNA transfected cells were shrunken with nuclei containing dark granular structures similar to apoptotic bodies. At higher CCTα-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 CCTα-siRNA concentration increased. LDH assay showed that CCTα-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 CCTa- siRNA inhibition. Furthermore, addition of PC did not improve viability of CCTα-siRNA treated cells. These results demonstrate that CCTα-siRNAs knocked down CCTα protein resulting in cell death implying that the sequences could be useful as anticancer agents. CTP: Phosphocholine Cytidyltransferase Alpha (CCTa) siRNA Induce Cell Death of Lung Cancer Cells Rukia Marijani and Barack O. Abonyo* Florida A & M University, College of Pharmacy and Pharmaceutical Sciences Tallahassee, FL 32307 P h a r m a c e u t i c a A n a l y t i c a A c t a ISSN: 2153-2435 Pharmaceutica Analytica Acta