欧前胡素通过下调c-met的表达提高肺癌CD133~＋细胞亚群对吉非替尼的敏感性

Imperatorin increased sensitivity of lung cancer CD133^+ cell subsets to gefitinib by down-regulating c-met expression

目的:研究PC9 CD133^+细胞亚群对吉非替尼的耐药性并探讨欧前胡素提高吉非替尼抗肺癌活性的机制。方法:MTT法检测PC9细胞在吉非替尼和欧前胡素处理下的细胞活力。Western blot实验检测吉非替尼和欧前胡素对PC9细胞c-met表达水平、caspases活化水平及表皮生长因子受体(EGFR)、PI3K、AKT磷酸化水平的影响。流式细胞术检测欧前胡素和吉非替尼对PC9细胞系的CD133^+细胞亚群种群比例的影响及PC9细胞在二者处理下的凋亡率。结果:PC9 CD133^+细胞亚群对吉非替尼的敏感性显著低于PC9 CD133^-细胞亚群。吉非替尼能显著抑制PC9 CD133^-细胞亚群EGFR/PI3K/AKT的活化,但对PC9 CD133^+细胞亚群该通路的影响不大。吉非替尼单独处理能提高PC9细胞系中CD133^+细胞亚群的比例,然而联用欧前胡素后PC9 CD133^+细胞亚群的种群比例显著下降。Western blot实验表明欧前胡素能显著降低PC9 CD133^+细胞亚群的c-met蛋白表达水平表明c-met是欧前胡素的治疗靶点。MTT、Western blot、流式细胞术实验结果表明在PC9 CD133^+细胞亚群中,欧前胡素通过抑制c-met的表达提高吉非替尼对PI3K/AKT的抑制作用,从而诱导PC9 CD133^+细胞亚群发生caspases活化和凋亡。结论:欧前胡素通过下调c-met的表达提高肺癌CD133^+细胞亚群对吉非替尼的敏感性,两者存在协同抗肿瘤效应。

AIM：To investigate the role of imperatorin in reversing the resistance of the PC9 CD133 cell subsets to gefitinib.METHODS：MTT assay was performed to evaluate the viability of PC9 cells treated with imperatorin and gefitinib.The expression of c-met,activation of caspases and phosphorylation of epidermal growth factor receptor（EGFR）,PI3 K and AKT in the PC9 cells treated with imperatorin and gefitinib were determined by Western blot.The percentage of CD133~＋ cell subsets population and the apoptotic rate of the PC9 cells treated with imperatorin and gefitinib were analyzed by flow cytometry.RESULTS：The sensitivity of the PC9 CD133~＋ cell subsets to gefitinib was significantly lower than that of the PC9 CD133^- cell subsets.Treatment with gefitinib alone significantly inhibited the protein levels of EGFR/PI3K/AKT in the PC9 CD133~ cell subsets but not the PC9 CD133~＋ cell subsets.Treatment with gefitinib alone increased the percentage of CD133~＋ cell subsets population in the PC9 cells.However,combination of gefitinib with imperatorin significantly inhibited the enrichment of CD133~＋ cell subsets population.Imperatorin down-regulated c-met expression,suggesting the c-met was the target of imperatorin in the PC9 CD133~＋ cell subsets.The results of MTT assay,Western blot analysis and flow cytometry indicated that imperatorin increased the gefitinib induced inhibition of PI3K/AKT protein levels by down-regulating the expression of c-met,which subsequently induced the cleavage of caspases and apoptosis in the PC9CD133~＋ cell subsets.CONCLUSION：Imperatorin increases the sensitivity of lung cancer CD133~＋ cell subsets to gefitinib by down-regulating the expression of c-met,and the synergistic anti-tumor effect exists between imperatorin and gefitinib.