Title: Effect of 5-aza-2'-deoxycytidine Induced CDX2 Gene Expression on Proliferation and Apoptosis of Human Gastric Cancer Cell Line MKN45
Abstract: Epigenetic gene silencing via DNA methylation is one of the important steps in the development of gastric cancer.CDX2,a regulator of intestinal phenotype,is shown to play a tumor-suppressive role in gastric cancer.Aims: To study the effect of 5-aza-2'-deoxycytidine(5-aza-CdR) on expression of CDX2 gene in human gastric cancer cell line MKN45 and its action on proliferation and apoptosis.Methods:Gastric cancer cell line MKN45 was treated with different concentrations of 5-aza-CdR(2.5,5,10(xmol/L).The methylation status of CDX2 gene promoter region was detected by methylation specific PCR(MSP).Real-time fluorescent quantitative RT-PCR and Western blotting assay were used to determine the expressions of CDX2 mRNA and protein,respectively.CCK8 assay and Annexin V-FITC/PI were used to examine the proliferative activity and apoptosis,respectively,and the morphology of apoptotic cells was identified by Hoechst 33258 staining.Caspase-3 activity was determined by colorimetric method.Results:Hypermethylation of CDX2 gene promoter region,extremely low expression of CDX2 mRNA and no expression of CDX2 protein were the characters found in MKN45 cells.After treatment with 5-aza-CdR for 72 hours,hypermethylated CDX2 gene promoter region was demethylated,and expressions of CDX2 mRNA and protein were up-regulated in MKN45 cells.Compared with control group,5-aza-CdR dose-dependently enhanced the inhibition of proliferation and apoptosis of MKN45 cells(P0.05).Hoechst 33258 staining displayed highly condensed chromatin of the nucleus as well as fragments of chromatin.Caspase-3 activity in MKN45 cells was increased in a does-dependently manner after exposure to 5-aza-CdR(P0.05).Conclusions:The CDX2 silencing in MKN45 cells may be related to methylation of 5' CpG island of CDX2 gene promoter region.5-aza-CdR may effectively reverse the aberrant methylation of CDX2 gene and induce re-expression of the gene,inhibit the proliferation and enhance apoptosis of MKN45 cells.This may be related to the activity of caspase-3.
Publication Year: 2011
Publication Date: 2011-01-01
Language: en
Type: article
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