Targeting Cell Cycle Checkpoint Related Protein Chk1 In Gastric Cancer Treatment

Objective:to investigate the role of Chk1 in gastric cancer cells,and evaluate the anticancer effect of Chk1 inhibitor LY2606368 in gastric cancer cells.Method:small inferring RNA(siRNA)targeting Chk1 was designed,MTS assay was used to determine the potential effect of Chk1 ablation by siRNA on the cell proliferation in p53 wild type gastric cancer cell line AGS and p53 mutant cell line MKN1,and also we checked the impact of Chk1 knockdown on the sensitivity to IR treatments.Then,colon formation assay and MTS assay were performed to determine the anticancer effect of Chk1 inhibitor LY2606368,molecule changes after LY2606368 were investigated by Western blot.Results:the results of MTS assay indicated that Chk1 ablation by siRNA could significantly inhibit the cell proliferation of AGS and MKN1 cells,also Chkl knockdown could sensitive the effect of IR treatment in these two gastric cancer cell lines.Besides,Chk1 inhibitor LY2606368 showed potent anticancer effect in AGS and MKN1 cells,the results of Western blot showed that LY2606368 treatment inhibited the expression of Chkl and increased the level of p-Chk1(Ser345),also it increased the expression of DNA damage related marker y-H2AX and apoptosis related protein cleaved caspase3 in AGS and MKN1 cells.Conclusion:Chkl ablation could not only significantly suppress the proliferation but also sensitize the effect of IR treatment in gastric cancer cells with p53 independent manner,and Chkl inhibitor LY2606368 also showed marked anticancer effect in gastric cancer cells.Objective:To explore the potential candidate which can combine with Chk1 inhibitor LY2606368 in gastric cancer treatment.Method:HR repair reporter assay was applied to check the effect of LY2606368 on cell HR repair ability,and hydroxyurea was used to synchronize the cell cycle,the potential synergistic anticancer effect of LY2606368 and PARP inhibitor BMN673 were examined by MTS assay and clonogenic assay,also we detected the apoptosis induction by LY2606368,BMN673 or combination treatment using Annexin V/PI staining,cell cycle analysis were performed to detect the change induced by drug treatment,western blot were performed to investigate molecular change in each group.Besides,we successfully established the gastric cancer patient derived xenograft mouse model,then we used this model to examine the anticancer effect of LY2606368,BMN673 and combination treatment.Results.HR repair reported assay indicated that LY2606368 significantly decreased the HR repair ability in U20S reporter cell.And we found LY2606368 had potent synergistic anticancer effect with PARP inhibitor BMN673 in gastric cancer cells AGS and MKN1,less colony formation in combination treatment groups also proved this synergistic effect.From the results of Annexin V/P1 staining,we detected more apoptosis induction by combination treatment in AGS and MKN1 cells,the results of western blot showed more expression of apoptosis related protein cleaved caspase3 contrasted to monotherapy grousp,we also found LY2606368 decreased the G2M arrest induced by BMN673,western blot showed decreased G2 phase marker Cylcin B1 and increased mitosis marker p-H3 in combination treatment groups.At last,LY2606368 and BMN673 combination treatment showed more potent anticancer effect in gastric cancer PDX nude mice model,lower tumor volumn and weight were observed in combination group.Conclusion:LY2606368 significantly impaired the HR repair ability,and showed marked synergistic anticancer effect with PARP inhibitor BMN673 in gastric cancer both in vitro and vivo.