Study On The Blocking Effect Of Nitidine Chloride On DNA Damage Repair Induced By Top I Inhibitor And Its Mechanism

Objectives:1.To study the synergistic inhibition effect that NC and SN38 on HT29 cells,blocking DNA damage repair induced by SN38 and inducing DNA damage.2.To explore the molecular mechanism of NC on blocking DNA repair.3.To evaluate the effect of NC on proliferation and apoptosis in HT29 cells.Methods:1.HT29 cells were used as testing cells to evaluate proliferation and apoptosis in HT29 cells by the combination of NC with SN38 using MTT and flow cytometry.2.Comet assay was employed to determine the effect of NC and SN38 on DNA damage and repair,and immunofluorescence and western blotting test were used to detect the effect of NC and SN38 on the protein levels of p-H2 AX.3.Western blotting assay was performed to detect the effect of NC and SN38 on the protein levels of Eme1,p-Src(Tyr419),Stat3,p-Stat3(Tyr705),p-Stat3(Ser727),and further to explore the molecular mechanisms of DNA damage caused by NC.4.The Eme1-overexpressed HT29 cells which Eme1 was expressed stably wassuccessfully constructed by the recombinant lentivirus plasmid.Comet assay was employed to determine the effect of NC and SN38 on DNA damage and repair,and western blotting assay was performed to detect the effect of NC on the protein level of Eme1.5.CCK8 and flow cytometry was carried out to evaluate the effect of NC on cell viability and apoptosis.6.Western blotting assay was used to detect the effect of NC on the protein levels of Cyt-c and caspase-3,and further to explore the pathway of apoptosis induced by NC.Results:1.MTT and flow cytometry results presented that the combination of NC with SN38 inhibited the proliferation of HT29 cells significantly and the proportion of apoptotic cells significantly increased after being treated with NC and SN38,compared with the cells treated with single SN38(P<0.05).2.Comet assay showed that cells treated with SN38 led to only a slightly increase of the tail length and tail DNA% compared with control group(P>0.05).However,a significant increase in tail length was observed in the presence of NC and SN38 compared with the one of SN38 alone.Western blotting and immunofluorescence analysis showed an increasing protein level of p-H2 AX when treating HT29 cells with the combination of NC and SN38,compared with the treatment with single SN38(P<0.05).3.Western blotting showed that the protein expression of Eme1,p-Src(Tyr419),Stat3,p-Stat3(Tyr705),p-Stat3(Ser727)was significantly increased in response to SN38 alone compared with control groups,and this up-regulation was suppressed when the cells treated with combination of NC and SN38(P<0.05).4.HT29 cells obtained high transfection efficiency,and western blottingshowed that the protein expression of Eme1 was stably expressed in HT29 cells.Comet assay showed that cells treated with SN38 led to only a slightly increase of the tail length and tail DNA%.However,a significant increase in tail length was observed in the presence of NC compared with the Eme1-OV group.Western blotting showed that the protein expression of Eme1 was down-regulated by NC.5.Compared with the control group,the cell viability rates of the NC groups decreased obviously and the apoptosis rates increased prominently(P<0.05).6.The results of western blotting showed that the protein levels of Cyt-c and caspase-3 in the NC groups were notably up-regulated compared with the control group(P<0.05).Conclusions:1.NC and SN38 have synergistic inhibition effect on HT29 cells,and NC down-regulates the protein level of Eme1,blocking DNA damage repair and inducing DNA damage.2.NC blocks DNA damage repair and the related molecular mechanism might be associated with inhibiting Src-Stat3 signal pathway.3.NC significantly inhibits the proliferation of HT29 cells and induces apoptosis,and the related molecular mechanism might be associated with the Cyt-c/caspase-3-mediated mitochondrial apoptosis pathways.