Study on the effect and mechanism of kaempferol in reversing drug-resistant Bel-7402/5-Fu cells
Objective To investigate the effect of kaempferol(KAE)on the function of drug-resistant Bel-7402/5-Fu cells.Methods Bel-7402/5-Fu cells were treated with KAE,and cells were divided into the control group and the drug group(0.064,0.320,1.600,8,40,200 µmol/L KAE).Cells were divided into the si-NC group and the DNA-PKcs interference group,or the control group,the KAE group,the KAE+si-DNA-PKcs group or the KAE+DMSO group,the KAE+MG132 group and the KAE+CQ group based on interfering DNA dependent kinase catalytic subunits(DNA-PKcs)or addition of proteasome inhibitor MG132 or autophagy inhibitor CQ.Cell proliferation was detected using CCK-8.The expression level of histone H2AX phosphorylation(γ-H2AX),DNA-PKcs,DNA double strand break repair/V(D)J recombinant protein(Artemis)and drug pump gene(P-gp)were analyzed using real-time fluorescence quantitative PCR(RT-qPCR)and Western blot assay.Cell cycle and apoptosis were detected by flow cytometry.The stability of DNA-PKcs proteins was analyzed by protein stability experiments.Ubiquitination of DNA-PKcs protein was evaluated by immunoprecipitation assay.Results Compared to the control group,treating cells with 8 µmol/L KAE for 24 h inhibited about 50%of cell proliferation ability.Therefore,this time and concentration were chosen for subsequent research.Compared to the control group,the expression level of γ-H2AX mRNA and protein significantly increased,while expression levels of DNA-PKcs,Artemis and P-gp mRNA and proteins significantly decreased in the KAE group(P＜0.05).Compared to the control group,KAE promoted cell cycle arrest in the G2/M phase of Bel-7402/5-Fu cells and increased cell apoptosis.Compared to the si-NC group,siRNA-1664 significantly downregulated the mRNA and protein expression levels of DNA-PKcs(P＜0.05).Compared with the KAE group,the effect of KAE was further promoted in the KAE+si-DNA-PKcs group of Bel-7402/5-Fu cells.Compared with the control group,the protein expression level of DNA-PKcs decreased in the KAE+DMSO group(P＜0.05).Compared with the KAE+DMSO group,the protein expression level of DNA-PKcs increased in the KAE+MG132 group(P＜0.05),while there was no significant change in the protein expression level of DNA-PKcs in the KAE+CQ group(P＞0.05).Compared to the control group,there was promoted ubiquitination of DNA-PKcs in the KAE+DMSO group,and the inhibited ubiquitination in the KAE+MG132 group(P＜0.05).Conclusion KAE may induce cell apoptosis and cell cycle arrest in drug-resistant Bel-7402/5-Fu cells.
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