Cytotoxicity Of5-Aza-2’-deoxycytidine Against Gastric Cancer

1. Backgroud and aimsIt has long been established that malfunction of related-cancer genes plays a necessary and indispensable role in carcinogenesis. Fate in human cancer has been attributable to gene silence arised from methylation of CpG promoter region in tumor suppressor other than activation of oncogenes from gene mutation and exterior stimulants. In the process of tumorigenesis, impinge of DNA methylation catalysed by DNA methyltransferases on cancer is analogous to coding-region mutation, abrogating the expression of a number of gene inhibitors. Unlike gene mutation, DNA hypermethylation could be reversed from silenced status to active ones with employing DNA methyltransferases inhibitor. To date, there are two compounds sanctioned by FDA being applied to clinic comprehensively embracing5’-azacytidine and5-Aza-2’-deoxycytidine (5-Aza-CdR).5-Aza-CdR, which is only incorporated into DNA rather than RNA and protein, is at least10-fold more cytotoxic5’-azacytidine, serving as an encouraging DNA methyltransferases inhibitor.As one of the leading cause of cancer death worldwide, gastric cancer remains threatening around the world and most patients in advanced stages need chemotherapy. Recently, aberrant promoter hypermethylation has accounted for mechanism of gastric cancer. Exploring a sort of promising anti-cancer candidate has drawn considerably attention in worldwide associated cancer dormain.5-Aza-CdR, a unique cytosine analog, could reactivate silenced genes through suppressing DNA methyltransferases and induce cellular apoptosis, representing dramatic therapeutic activity in patients with myelodysplastic syndrome (MDS) in clinical trials. Weather5-Aza-CdR has cytotoxic against solid cancer including gastric cancer or not and if so, what molecular mechanisms this agent executes are our crux of investigation. On one hand, we aimed to examine the effects of the inhibitor of DNA methyltransferases (5-Aza-CdR) on gastric cancer cell line, evaluating whether it could effectively act as anti-gastric cancer candidate. On the other hand, it was focus to further shed light on the underlying mechanisms of5-Aza-CdR associated with diverse signal pathways and the status of DNA methylation on gene promoters, providing meaningful insights into5-Aza-CdR’s clinical activity. 2. MethodsGastric cell line with wild-type P53(AGS) was treated at different concentrations for indicated hours. Cell viability and the ability of proliferation were determined by MTT assay and colonial formation assay; Flowcytometry was performed to assess DNA contents of cells exposed to5-Aza-CdR at indicated time. To further test the influence of5-Aza-CdR on gastric cancer AGS cells, a series of assay such as Annexin V, DNA ladder, Hoechst33258staining as well as the acitivity of caspases was performed according to standard protocols. DNA damage marked by DNA double strands break (DDB) induced through incorporation of5-Aza-CdR into cancer cells DNA has been shown in many researches, so comet assay which can detect DDBs was conducted in AGS cells. In addition, Western blotting and RT-PCR were utilized to analyse the alterations of the expressions of various proteins and genes as well in presence of5-Aza-CdR or absence in AGS cells. Last but not least, AGS cells were assessed the status change of DNA methylation by Methylation Specific PCR (MSP) assay.3. Results and discussionFrom the results of our first part, AGS cells with wild-type P53treated with5-Aza-CdR at different concentrations for different time showed significantly inhibiton of cell viability and proliferation. More importantly, the cytotoxicity caused by5-Aza-CdR in AGS cells presented a dose and time-dependent manner. Data from flowcytometry demonstrated that5-Aza-CdR treatment resulted in arrest of G2phase in AGS cells. Besides that, we observed that apoptosis occurred in AGS with5-Aza-CdR exposure.Regarding mechanistic exploration demonstrated in the second part, we concluded these following results:1) apoptosis triggered by5-Aza-CdR in AGS cells unveiled a time-dependent pattern, in which apoptosis signalling was correlated intimately to mitochondrial pathway modified by releasing BAX and Cyt C and activating caspase9.2) From the data of comet assay, AGS cells treated with5-Aza-CdR for12-48hours respectively were observed DNA damage characterized by DNA double strands break along with time was accumulated, indicating time-dependent trend. ATM (ataxia-telangiectasia mutated), a sensor to DNA damage, was activated through phosphorylation at ser1981which was accompanied by initiation of downstream effctor factors such as P53, P21and caspases activation as well.3) P53status affected dramatically sensitivity of cells to5-Aza-CdR due to the fact that in presence of P53inhibitor, AGS cells exposed to5-Aza-CdR showed abrogated cytotoxicity.4)5-Aza-CdR reactivated silencing genes such as P16inactivated through DNA hypermethylation, to some extent playing a role in mechanisms of anti-cancer.4. ConclusionTake together, this study comprehensively enhances our understanding of the mechanisms underlying5-Aza-CdR cytotoxicity and reveals novel function for ATM-dependent P53accumulation as a component of the cellular response to DNA damage, which may help optimize gastric cancer patient responses to this agent in the future.