克唑替尼耐药细胞H2228/CR对紫杉醇及顺铂的敏感性变化及机制

Changes of chemosensitivity of crizotinib-resistant cells H2228 / CR to paclitaxel and cisplatin

目的观察肺腺癌细胞H2228克唑替尼耐药前后对化疗药物紫杉醇和顺铂敏感性变化,并探讨其可能机制。方法采用克唑替尼体外低浓度梯度递增联合大剂量间断冲击方法处理H2228细胞,培养克唑替尼耐药细胞株H2228/CR。MTT法检测克唑替尼、紫杉醇及顺铂对H2228及H2228/CR细胞的增殖抑制率;实时荧光定量PCR法和Western blotting法分别检测H2228及H2228/CR细胞波形蛋白、N-钙黏蛋白、E-钙黏蛋白的mRNA和蛋白表达。结果经过6个月的培养,H2228/CR对克唑替尼明显耐药,且对紫杉醇及顺铂的敏感性降低。H2228/CR细胞E-钙黏蛋白mRNA及蛋白表达水平较亲代H2228细胞明显降低,而N-钙黏蛋白、波形蛋白的mRNA及蛋白表达水平较H2228细胞明显增高。结论克唑替尼耐药细胞H2228/CR对常规化疗药物紫杉醇、顺铂敏感性降低,其耐药机制可能与H2228/CR细胞发生上皮间质转化有关。

Objective To observe the sensitivity changes of lung adenocarcinoma H 2228 cells to paclitaxel and cispla-tin before and after resistance to crizotinib , and to explore the molecular mechanism .Methods Crizotinib-resistant H2228/CR cells were induced by continuous stepwise exposure to crizotinib combined with pulse therapy in vitro .MTT as-say was used to measure the growth inhibition ratio of H 2228 and H2228/CR cells treated with different concentrations of crizotinib, paclitaxel and cis-platinum.Real-time fluorescence quantification PCR and Western blotting were used to meas-ure the mRNA and protein expression of Vimentin , N-cadherin and E-cadherin in H2228 and H2228/CR cells. Results After being cultured for 6 months, H2228/CR cell displayed significantly resistance to crizotinib and its resist-ance to paclitaxel and cisplatin was decreased .The mRNA and protein expression of E-cadherin was decreased in H 2228/CR cells as compared with that of the parental cell line H 2228, while the mRNA and protein expression of N-cadherin and Vimentin was increased in H2228/CR cells.Conclusion The crizotinib-resistant H2228/CR cells decrease sensitivity to standard chemotherapeutic drugs paclitaxel and cisplatin , which may be associated with the epithelial-mesenchymal transi-tion of H2228/CR cells.