ASC Methylation Status And Its Relationship With Apoptosis In Human Colon Cancer Cell

Objective: Apoptosis associated speck—like protein containing CARD (ASC), also named target of methylation-induced silencing (TMS1), is an antioncogene, containing a pyrin(PYD) and a caspase recruitment domain (CARD), which play an important role in regulating apoptosis and immunological reaction. Mutation of many proteins which have a pyrin and a caspase recruitment domain are associated with autoimmune diseases and carcinogenesis. However, the methylation of ASC and the mechanism of apoptosis in human colon cancer cells and other cancers have not been clearly defined. In this study, we examined the role of ASC in human colon cancer cells LS174T, LoVo and SW480. It was indicated that the expression of ASC can enhance tumor cell apoptosis, suggesting a direct effect in cancer. Our datas further support that ASC is an antioncogene.Methods: 1.To explore the role of ASC in human colon cancer cells, we firstly detected the methylation of ASC in human colon cancer cells by methylation specific PCR(MSP), using the method of Protease K/hydroxybenzene and CpGenome DNA Modification Kit to extract, purify and modify the genome DNA. 2.To analyze the expression of ASC in human colon cancer cells by RT-PCR and Western blotting. 3. To restore the of expression of ASC in human colon cancer cell LS174T, we treated 5-aza-2-deoxycytidine with the LS174T cell in which ASC was completely methylation. 4. To analyze the restoring expression of ASC in LS174T cells by RT-PCR. 5. Compared with the LS174T cell treated with 5-aza-2- deox- cytidine, we analyze the effect of apoptosis caused by ASC methy- lation by Flow Cytometry and immunofluorescence in vitro experiment. 6.Compared with the LS174T cell treated with 5-aza-2-deoxycytidine, we analyze the quality of apoptosis genes, such as caspase-3, fas, Bcl-2, c-myc, Bax and P53 by Real time PCR.Results: 1.MSP showed that ASC was completely methylated in LS174T cells, partly methylated in LoVo cells and SW480 cells. 2.Both RT-PCR and Western blotting demonstrated that SW480 and LoVo cells expressed ASC, but LS174T cells did not express ASC, gene silencing. 3. Treated the LS174T cell with 5-aza-2-deoxycytidine, we observed that ASC restored expression by RT-PCR. Together, ASC restored unmethylation by MSP. 4. Treated the LS174T cell with 5-aza-2-deoxycytidine, restoring ASC expression, we found that the ratio of apoptosis was higher than the control by Flow Cytometry in vitro expriment. Moreover, the immuno- fluorescence phenomenon also supported the same result. 5.But the quantity of apoptosis genes, such as caspase-3, fas, Bcl-2, c-myc, Bax and P53 have not changed in LS174T cells by statistics, which treated or not treated with 5-aza-2- deoxycytidine. The mechanism that demonstrated ASC enhance the tumor cell apoptosis needed to be further researched.Conclusion: It was indicated that ASC was partly methylated in LoVo cells and SW480 cells, but completely methylated in LS174T cells. Treated with the 5-aza-2-deoxycytidine, restoring the expression of ASC can enhance LS174T cells apoptosis, compared with the control, suggesting a direct effect on cancer. So our datas further support that ASC is an an- tioncogene, which can enhance apoptosis, and inhibit cancer development.