药物诱导肺癌细胞衰老的体外研究

Drugs induce senescence of lung cancer cells in vitro

目的探讨药物诱导肿瘤细胞衰老的可能机制和对肿瘤治疗的潜在价值。方法用不同浓度的抗肿瘤药物顺铂(DDP)、Nutlin-3的活性异构体(Nutlin-3a)、4-异硫脲基丁腈盐酸盐(Kevetrin)分别处理人肺癌A549、H460、H2228细胞,采用β-半乳糖苷酶染色检测衰老细胞阳性率,EdU染色检测增殖细胞阳性率,CCK-8法检测细胞存活率,Real-time qPCR法检测肺癌细胞衰老相关分子p21 mRNA表达水平,Western blot法检测药物处理肺癌细胞衰老相关分子p53和p21蛋白表达情况。结果DDP、Nutlin-3a和Kevetrin可诱导A549、H460和H2228细胞胞体增大、蓝染衰老细胞增多,阳性细胞率与各自对照组比较差异均有统计学意义(均P<0.05)。DDP和Nutlin-3a处理的A549细胞,荧光显微镜检测发现EdU红色荧光增殖细胞阳性率与对照组相比明显减少(均P<0.05);此外,药物的细胞毒性实验也发现,DDP、Nutlin-3a和Kevetrin均可使肺癌细胞存活率明显降低(均P<0.05)。与各自对照组相比,6.25μmol/L DDP、8μmol/L Nutlin-3及100μmol/L Kevetrin处理组A549肺癌细胞p21 mRNA表达上调,差异均有统计学意义(均P<0.05);蛋白水平上,DDP、Nutlin-3a及Kevetrin作用肺癌细胞,其p53、p21蛋白表达上调,Kevetrin处理A549细胞p53蛋白表达变化不明显。结论DDP、Nutlin-3a和Kevetrin 3种抗肿瘤药物不仅能够抑制肺癌细胞增殖,而且能够诱导肺癌细胞衰老。

Objective To explore the mechanism of drug-induced senescence of lung cancer cells.Methods Human lung cancer cell lines A549,H460,and H2228 were treated with cisplatin(DDP),Nutlin-3a,and Kevetrin at different concentrations.The cell senescence was determined byβ-galactosidase staining and the cell proliferation was determined by EdU staining.Meanwhile,the morphology of senescence cells was observed under the microscope;CCK-8 test was applied to detect the proliferation inhibition of lung cancer cells;Real-time qPCR method was used to detect the expression of mRNA;Western blot was used to detect the expression of p53 and p21 protein.Results The treatment of A549,H460 and H2228 cells with DDP,Nutlin-3a and Kevetrin increased the cell senescence,compared with the corresponding control groups(all P<0.05).After treated with DDP and Nutlin-3a,the number of proliferating and dividing A549 cells was significantly reduced as shown by EdU staining under fluorescence microscope compared with the control group(all P<0.05).CCK-8 test showed that drugs inhibited cell proliferation significantly(all P<0.05).The mRNA expression levels of p21 in lung cancer cells were increased after treatment with 6.25μmol/L DDP,100μmol/L Nutlin-3a or 8μmol/L Kevetrin,while the protein expression levels of p53 and p21 were increased(all P<0.05).Conclusion Anti-tumor drugs DDP,Nutlin-3a and Kevetrin not only can inhibit cell proliferation in vitro,but also induce the senescence of lung cancer cells.