Effect Of Zebularine On Apoptosis Of Esophageal Cancer Cells And Its Mechanism

Objectives To investigate the effect and mechanism of zebularine on apoptosis of esophageal cancer cells.Methods The optimal concentration and action time of zebularine on cultured esophageal cancer cells ECA109 and KYSE170 were established by CCK-8 method.The concentration and time with inhibition rate of 50%were 100μmol·L^-1 and 48 h.Under this condition,the apoptosis rates of zebularine-treated cells were detected by flow cytometry,and the expression of apoptosis-related proteins and Wnt signaling pathway molecules were determined by Western blot.The expression levels of upstream negative regulatory genes of Wnt pathway was detected by RT-PCR,and the methylation statuses of human secretory frizzled related protein 2 and Dickkopf 3 were detected by methylation specific PCR.The mechanism of zebularine on apoptosis of esophageal cancer cells was observed in further by knocking down SFRP2 and Dkk3.Results Zebularine could inhibit the viability of ECA109 and KYSE170 cells in a dose and time-dependent manner.When the inhibition rate of zebularine on cells was 50%,the concentration and action time of zebularine were 100μmol·L^-1 and 48 h respectively.Zebularine could induce apoptosis,and the apoptosis rates of ECA109 and KYSE170cells were 29.68%and 23.41%respectively,which were significantly higher than those of the control group（10.64%and 12.13%）（P<0.05）.Zebularine could down-regulate Bcl-2 apoptotic protein,up-regulate Bax,cleaved-caspase-3 and cleaved PARP apoptotic protein,and could also decrease the expression ofβ-catenin,cyclin D1 and c-myc protein.The m RNA expression levels of SFRP2 and Dkk3 were increased,while the methylation levels of SFRP2 and Dkk3 promoters were decreased significantly after zebularine treatment.Knocking down of SFRP2 and Dkk3 could reduce zebularine-induced apoptosis,and increase the expression ofβ-catenin,cyclin D1 and c-myc protein.Conclusion Zebularine could induce apoptosis of esophageal cancer cells by reducing the methylation of SFRP2 and Dkk3 promoters and then inhibiting Wnt pathway.Figure 10;Table2;Reference 107...