Effects Of Gossypol Acetic Acid On Proliferation And Methylation Of HMLH1 And MGMT Gene In Human Tongue Carcinoma Cell Line Tca8113

Objective1,To investigate the effects on proliferation and apoptosis in human tongue carcinoma cell line Tca8113 treated by GAA in vitro;2,To detect the effects on methylation level of hMLH1,MGMT in Tca8113 cells treated by GAA in vitro in order to provide the theory and experimental base for prevention and therapy on carcinoma by changing the methylation level of Tumor Suppressor Gene.Methods1,To cultivate Tca8113 cells in vitro, its morphology changing before or after being treated by GAA were observed through Light Microscope (LM).2,To detect the effects on proliferation inhibition in Tca8113 cells treated by different concentration of GAA through MTT assay, and then calculate IC50 of GAA.3,To observe apoptosis status changing in Tca8113 cells before or after being treated by GAA through cell flow cytometry (FCM).4,To detect methylation level changing of hMLH1,MGMT in Tca8113 cells after being treated by GAA for 48h,72h through nested Methylation specific PCR (nMSP).Results1,Experimental GAA group could be observed on growth and proliferation inhibition of Tca8113 cells after being treated by GAA for 24h to 72h through MTT assay, and the inhibition effects has a dose-dependent and time-dependent fashion. IC50 of GAA.for 24h,48h,72h was 51.06μmol/L,31.9μmol/L,14.36μmol/L respectively.2,Compared with the control group, the result of FCM assay indicated that GAA (30μmol GAA for 48h and 15μmol/L for 72h) induced a higher apoptosis rate of Tca8113 cells (P<0.05) 3,The result of nMSP showed that hMLH1 and MGMT gene hypermethylation in Tca8113 cells. The methylation of hMLHl and MGMT gene was reversed after GAA treatment (30μmol GAA for 48h and 15μmol/L for 72h).Conclusion1,The CpG island in promoter region of hMLH1 and MGMT gene is hypermethylated in human tongue carcinoma cell line Tca8113.2,GAA can suppress proliferation and induce apoptosis of human tongue carcinoma cell line Tca8113 in a dose-dependent and time-dependent fashion.3,GAA has its demethylation effect on the CpG island in promoter region of hMLH1 and MGMT gene in human tongue carcinoma cell line Tca8113.