氯化两面针碱对类泛素化修饰抑制剂MLN4924介导肺癌耐药的作用与机制研究  

Study on effect and mechanism of nitidine chloride on drug resistance of neddylation inhibitor MLN4924 in lung cancer

作　　者：李蒙 张荔 赵伟丽 李立辉 LI Meng;ZHANG Li;ZHAO Weili;LI Lihui(Cancer Institute,Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]上海中医药大学附属龙华医院肿瘤研究所,上海200032

出　　处：《上海中医药大学学报》2023年第4期22-27,101,共7页Academic Journal of Shanghai University of Traditional Chinese Medicine

基　　金：上海市“科技创新行动计划”自然科学基金原创探索项目(21ZR1482200);上海市“科技创新行动计划”启明星项目(21QA1408900)。

摘　　要：目的:探索中药药效物质氯化两面针碱对A549 MLN4924耐药肺癌细胞株的影响及作用机制。方法:利用MLN4924低剂量处理A549细胞6个月,并进行细胞半数抑制率(IC50)分析和Western blot检测以确保顺利诱导出MLN4924耐药细胞株(A549-MR);氯化两面针碱浓度梯度和时间梯度处理A549-MR,并进行ATPLite细胞增殖分析、细胞计数检测其对A549-MR生长的影响;同时收集A549-MR细胞总蛋白进行Western blot检测氯化两面针碱抑制A549-MR生长的分子机制。结果:IC50分析和Western blot实验显示,经低剂量长时间处理A549细胞后,MLN4924对A549细胞的IC50从0.355μmol/L提高至10.643μmol/L,且其不能显著抑制Cullins家族蛋白的类泛素化修饰(neddylation);细胞增殖分析显示,氯化两面针碱显著抑制MLN4924耐药肺癌细胞生长;Western blot结果显示,氯化两面针碱能下调A540-MR细胞内的Cullins蛋白表达并引起其抑癌底物蛋白p21、p27等积聚。结论:氯化两面针碱通过抑制泛素连接酶(CRLs)复合体的活性,导致其下游抑癌蛋白底物积聚,从而抑制neddylation抑制剂MLN4924引起的肺癌耐药细胞增殖。Objective:To explore the influence and mechanism of the effective traditional Chinese medcine nitidine chloride on A549 MLN4924 drug-resistant lung cancer cell lines.Methods:A549 cells were treated with low dose of MLN4924 for 6 months,and half maximal inhibitory concentration(IC50)analysis and Western blot analysis were performed to ensure the successful induction of MLN4924 drug-resistant cell lines(A549-MR).A549-MR was treated with the concentration gradient and time gradient of nitidine chloride,and the effect of nitidine chloride on A549-MR growth was detected by ATPLite cell proliferation analysis and cell counting.Meanwhile,the total protein of A549 cells was collected for Western blot analysis to detect the molecular mechanism of inhibition of A549-MR growth by nitidine chloride.Results:IC50 analysis and Western blot analysis showed that after low-dose and long-time treatment of A549 cells,the IC50 of MLN4924 on A549 cells was increased from 0.355μmol/L to 10.643μmol/L,and the neddylation modification of Cullins family proteins wasn't significantly inhibited.Cell proliferation analysis showed that the proliferation of MLN4924 drug-resistant lung cancer cells was significantly inhibited by nitidine chloride,Western blot results showed that the expression of Cullins protein in A549-MR cells was downregulated by nitidine chloride and its tumor suppressor substrate protein p21 and p27 were induced to accumulate.Conclusion:By inhibiting the activity of the CRLs(cullin-RING ligases)complex,nitidine chloride can induce the accumulation of downstream tumor suppressor protein substrates,which can inhibit the proliferation of the drug-resistant lung cancer cells induced by the neddylation inhibitor MLN4924.

关 键 词：氯化两面针碱 MLN4924 耐药 泛素连接酶复合体 

分 类 号：R285[医药卫生—中药学]