山萘酚逆转肝癌耐药细胞Bel-7402/5-Fu的作用机制研究.

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 DNAPKcs (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. [ABSTRACT FROM AUTHOR]