| Research on abnormal methylation of several tumor suppressors in human gastric cancerEffects of 5-Aza-CdR on proliferation and abnormal methylation of tumor supressor genes of human gastric cancer cell lines Gastric cancer is a kind of malignant tumor which threatens human beings' health, and its occurance is a pathological process which is many different genes involved. The gene promoter CpG island methylation is a important reason in tumor growth. In the study, we detected the effects of 5-Aza-CdR on proliferation of human gastric cancer cell lines SGC7901 and BGC823, methylation and epression of Apaf-1, p27kipl, DAPK1 gene and discussed the relationship between the methylation and expression of Apaf-1, p27kip1, DAPK1. we detect the expression and methylation state of DAPK1, p27kip1, Apaf-1, and explore the role of these genes concurrent methylation in gastric cancer tissues. CDDP is one of the important clinical anticancer drugs, and 5-aza-CdR is a specific demethylating agents. Many studies have indicated that promoter hypermethylation is in relation to chemotherapy resistance of cisplatin in many tumors. The experiment is to study whether the 5-aza-CdR can increase the sensitivity of gastric cancer cells to low dose CDDP.Materials and methodsMaterials: 35 surgical specimens of gastric carcinoma and their adjacent nonmal gastic tissues resected at the First Affiliated Hospital of China Medical University and the second Affiliated Hospital of China Medical University. Gasric cancer lines: BGC823 cell line, SGC7901 cell line. TRIzol Reagent(GIBCOL BRL company), RNAout(TakaRaå…¬å¸), demethylate agent 5-aza-2'-deoxycitydine(5-Aza-CdR) is the product of Sigma; PI is the product of Sigma; RT-PCR kit is the product of TaKaRa Company; Wizard DNA Clean-up(Promega company), Wizard DNA Clean-up(Promega company); RPMI 1640 culture medium is the product of Hyclone company. Apaf-1, p27kip1 monoclone antibody is the prduct of Santa cruz.Methods: SGC7901 cells and BGC823 cells were cultured in RPMI1640 and was treated with different concentrations of 5-Aza-CdR. The proliferation of the cells was detected by MTT assay, OA stain, flow cytometry. The methylation of Apaf-1, DAPK1, P27kip1 in the two cell lines was detected by MSP, and the expression of Apaf-1, DAPK1, P27kip1 was detected by RT-PCR and westernblot. SGC7901 cells and BGC823 cells were cultured in RPMI1640 and was treated with different concentrations of 5-Aza-CdR and low dose CDDP. The proliferation of the cells was detected by MTT assay and flow cytometry. The expression of Apaf-1, DAPK1, P27kip1 was detected by RT-PCR and westernblot. Using the semi-quantitative RT-PCR and western-blot analyses the expression of the Apaf-1, p27kip1, DAPKlin gastric cancer. Using the Loss of heterozygosity(LOH) analyses whether there is the loss of Apaf-1 gene in the domain of 12q22-23. Using the methylation specific PCR(MSP) analysis the promoter methylation of Apaf-1, p27kip1, DAPK1 gene in gastric cancer. Statistics analysis: Adopting t test of Excel.RESULTSâ… . Supressor genes methylation detection in gastric cancer1. The expression of Apaf-1, DAPK1, p27kip1's mRNA: Apaf-1, p27kip1, DAPK1 gene low expression is 17/35, 17/35 and 16/35(P＜0.05).2. The expression of Apaf-1, DAPK1, p27kipl's protein: There was 5 pous expression of Apaf-1 in gastric cancer and 29 pous expression of Apaf-1 in cancer-adjacent tissues; There was 3 pou expression of Apaf-1 in gastric cancer and 30 pous expression of Apaf-1 in cancer-adjacent tissues.3. The LOH frequencies of D12S346, D12S1706, D12S327, D12S1657 and D12S393is 34.3%, 8.3%, 57.1%, 11.4%, 42.8%. There was 50% LOH in two sites and 17% LOH in three sites.4. Methylation of DAPK1 was present in both cancer-adjacent tissues and gastric cancer tissue. P27kip1 and Apaf-1 rate of methylation were 25.71% and 22.86% in cancer-adjacent tissues 51.42% and 48.57% in gastric cancer respectively(p＜0.05).â…¡. Effects of 5-Aza-CdR on proliferation of human gastric cancer cell lines and abnormal methylation ofApaf-1 gene1. After SGC7901 and BGC823 cells were exposed to 5-Aza-CdR (1×10-7mol/L, 5×10-7mol/L, 1×10-6mol/L, 5×10-6mol/L), 5-Aza-CdR displayed a growth inhibitory effect on SGC7901 and BGC823 cells in a dose-and time-dependent manner, FCM analysis showed that G0/G1 phrase rate increased with exposing to 5-Aza-CdR (1×10-6mol/L, 5×10-6mol/L) for 72h(P＜0.01), the apoptosis rate increased too(P＜0.01).2. The methylation and the loss of Apaf-1, P27kip1, DAPK1 mRNA and protein of Apaf-1,, P27kip1, DAPK1 in SGC7901 and BGC823 cells were changed with 5-Aza-CdR.â…¢. The influence of 5-aza-CdR and low dose CDDP on gastric cell line1. MTT assay: The proliferation of SGC7901 cell and BGC823 cell added 5-aza-CdR and low dose CDDP was more inactive than that only added low dose CDDP.2. Annexin-v-FITC detecting the apoptosis: The apoptosis of SGC7901 cell and BGC823 cell added 5-aza-CdR and low dose CDDP was more than that only added low dose CDDP.3. the expression of gene: Analysis of mRNA and protein indicated the DAPK is both increased more greatly in SGC7901 cell and BGC823 cell when they were added 5-aza-CdR and low dose CDDP than that only added low dose CDDP.CONCLUSION1. The mechanism of 5-Aza-CdR's growth inhibitory effect on SGC7901 and BGC823 cells is related with blocking cell cycles, inducing apoptosis, and changing the methylation ofApaf-1 gene.2. The loss expression in SGC7901 and BGC823 cells is because of methylation states. 3. 5-aza-CdR can increase the gastric cells' sensitivity to low dose CDDP.4. DAPK gene acts a important role in 5-aza-CdR increasing the gastric cells' sensitivity to low dose CDDP.5. multiple genes concurrent methylation plays an important role in some gastric cancers, They happened at early stage.6. The expression of Apaf-1, DAPK1, p27kip1 is low in gastric cancer.7. gene's promoter Methylation of Apaf-1 and LOH of the gene in the domain of 12q22-23 both are the main reasons gene's promoter Methylation of p27kip1 is related with the in gastric cancer.
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