猫爪草总皂苷对EGFR-TKIs耐药肺癌细胞作用的研究

Effect of Ranunculi Ternati Radix Saponins on reversing the EGFR-TKIs resistant of lung carcinoma cells via autophagy

目的 探讨猫爪草总皂苷通过抑制自噬活性逆转肺癌细胞耐表皮生长因子受体-酪氨酸激酶抑制剂(EGFR-TKIs)的作用机制。方法 采用浓度梯度递增法体外构建肺腺癌耐药细胞株PC-9/ER,分为空白组、10μg/mL组、20μg/mL组和50μg/mL组,空白组仅加入厄洛替尼10μmol/L处理,各猫爪草总皂苷组在加入厄洛替尼10μmol/L后,同时分别加入10、20和50μg/mL猫爪草总皂苷,共同干预48 h。MTT法检测细胞增殖活性。透射电镜和共聚焦显微镜观察细胞形态和自噬体数量。流式细胞术检测细胞凋亡水平,蛋白质印迹法分析LC-3Ⅱ、Beclin1、Bcl-2、BAX、Cleaved Caspase-3和Caspase-3蛋白表达水平。结果 厄洛替尼抑制PC-9/ER细胞增殖的IC为(8.76±0.13)μmol/L,耐药指数(RI)为60.41±5.14,说明PC-9/ER耐药细胞株构建成功。50μg/mL猫爪草总皂苷协同作用后,PC-9/ER细胞IC_(50)为(1.32±0.12)μmol/L,RI降低为9.10±1.30。与空白组相比,10、20和50μg/mL组PC-9/ER细胞中的自噬泡数量和酸性囊泡(包括自噬体)数量逐渐减少;凋亡率逐渐增加[(4.75±2.03)%,(7.66±2.14)%,(28.79±5.65)%,F=159.683,P<0.001]。与空白组相比,20μg/mL和50μg/mL组Bcl-2、Beclin1和LC-3Ⅱ/LC-3Ⅰ蛋白表达降低,BAX和Cleaved Caspase-3蛋白表达量升高,P<0.05。结论 猫爪草总皂苷可体外逆转PC-9/ER细胞对EGFR-TKIs的耐药性,其可能的作用机制与抑制自噬和诱导凋亡有关。

Objective To investigate the mechanism of Ranunculi Ternati Radix Saponins on reversing EGFR-TKIs resistance of lung cancer cells by inhibiting autophagy.Methods The cell line PC-9/ER was established by gradient increasing erlotinib concentration and divided into blank group, Ranunculi Ternati Radix Saponins(10,20,50 μg/mL) group.PC-9/ER cells in blank group were treated with Erlotinib 10 μmol/L,while PC-9/ER cells in Ranunculi Ternati Radix Saponins(10,20,50 μg/mL) group were treated with Erlotinib 10 μmol/L and Ranunculi Ternati Radix Saponins for 48 h.MTT assay was used to detect the proliferation activity.Autophagic vesicles and autophagysomes number were observed by transmission electron microscope and confocal microscope.The expression levels of LC-3Ⅱ,Beclin1,Bcl-2,BAX,Cleaved Caspase-3,Caspase-3 proteins were detected by Western blot analysis.Results The IC_(50) and RI of PC-9/ER cells to erlotinib were(8.76±0.13) μmol/L and 60.41±5.14 respectively, indicating that PC-9/ER cells were established successfully.The ICof PC-9/ER cells to erlotinib was(1.32±0.12) μmol/L,and the drug resistance index was reduced to 9.10±1.30.Compared with the blank group, the number of autophagic vesicles and acidic vesicles(including autophagosomes) in PC-9/ER cells in Ranunculi Ternati Radix Saponins(10,20,50 μg/mL) groups decreased gradually, meanwhile, the apoptosis rate increased gradually [(4.75±2.03)%,(7.66±2.14)%,(28.79±5.65)%,F=159.683,P<0.001].Compared with the blank group, the expression of Bcl-2,Beclin1,LC-3Ⅱ/LC-3Ⅰ in Ranunculi Ternati Radix Saponins(20,50 μg/mL) groups decreased, while the expression of BAX and Cleaved caspase-3 increased(P<0.05).Conclusion Ranunculi Ternati Radix Saponins can reverse the resistance of PC-9/ER cells to EGFR-TKIs in vitro, and its possible mechanism is related to inhibition of autophagy and induction of apoptosis.