Effect Of Pantoprazole On Proliferation And Apoptosis Of Hepatocellular Carcinoma Cells

Objective: Proton pump inhibitors(PPI),mainly used as treatment of gastric acid-related diseases,have been reported as cytotoxic drugs against several human tumor cells.However,its effect on hepatocellular carcinoma(HCC)are poorly investigated.The purpose of this study was to evaluate the effect of Pantoprazole(PPZ)on proliferation and apoptosis of HCC cells and seek for the possible mechaniam,hoping to expand the clinical use of PPZ in HCC treatment.Methods:(1)the effect of PPZ on the proliferation of Huh7,SK-Hep1,Hep G2 and PLC/PRF/5 cells was detected by CCK-8(Cell Counting Kit-8)assay;(2)the effect of PPZ on colony forming ability of SK-Hep-1 cell was detected by plate colony forming assay;(3)the effect of PPZ on the growth and proliferation of SK-Hep-1 cell was detected by the growth curve;(4)the effect of pantoprazole on the DNA synthesis of SK-Hep1 and Huh7 was detected by Edu assay;(5)the effect of PPZ on the apoptosis of SK-Hep-1 and Huh7 was detected by flow cytometry(Annexin V-FITC/PI);(6)the effect of PPZ on the late apoptosis of SK-Hep-1 and Huh7 was detected by TUNEL;(7)the effect of PPZ on the cell cycle of SK-Hep-1 and Huh7 was detected by flow cytometry cell cycle assay;(8)the effect of PPZ on the m RNA expressions of PNCA,MKI67,CDK1,CDK2,CDC25 C,CYCLIN A2,and CYCLIN E2 was detected by genechips and q PCR;(9)the effect of PPZ on the protein expression levels of CDK1,CDK2,CDK2,CDC25 C,CYCLIN A2,CYCLIN E2,Procaspase-9 and cleaved Caspase-9 was detected by Western-Blot.Results:(1)The results from CCK-8 showed that PPZ inhibited the proliferation of HCC cells dose-dependently and time-dependently after treated with different concentrations of PPZ(20,40,80,160,320,640?mol/L)for 24 h,48h,or 72 h,compared with the control group(p<0.05).(2)PPZ decreased significantly colony's number and size dose-dependently,compared with the control(p<0.05).(3)PPZ inhibited significantly the growth of SK-Hep-1 cells dose-dependently and time-dependently by growth curve analysis,compared with the control group(p<0.05).(4)The results from Edu showed that PPZ inhibited significantly DNA synthesizes,compared with the control group(p<0.05).(5)By flow cytometry(Annexin V-FITC/PI double staining),PPZ could induce significantly the early and late apoptosis at 160 and 320?mol/L,compared with the control group(p<0.05).(6)By TUNEL,PPZ could induce the late apoptosis at 160 and320?mol/L,compared with the control group.(7)The results from flow cytometry cell cycle analysis showed that cell cycle was blocked obviously in the G2/M phase and cells in S phase were decreased obviously by PPZ,compared with the control group(p<0.05).(8)The results from Genechips and q PCR showed that PPZ could downregulate significantly the m RNA expressions of PCNA,KI67,CDK1,CDK2,CDC25 C,CYCLIN A2,and CYCLIN E2,compared with the control group(p<0.05).(9)By Western-Blot,we found that PPZ could downregulate significantly the protein expressions of CDK1,CDK2,CDC25 C,CYCLIN A2,CYCLIN E2,and Procaspase-9 and increase the protein expression of cleaved caspase-9.Conclusion: PPZ could significantly inhibit proliferation and induce apoptosis of HCC cells,which may be related with G2/M block through downregulating PCNA,KI67,CDK1,CDK2,CDC25 C,CYCLIN A2,and CYCLIN E2.