榄香烯通过c-Met信号通路逆转由肝细胞生长因子诱导的吉非替尼耐药作用

β-elemene reverses hepatocyte growth factor-induced resistance to gefitinib in PC-9 lung cancer cells via c-Met pathway

目的:研究肝细胞生长因子(hepatocyte growth factor,HGF)诱导肺癌PC-9细胞对吉非替尼耐药的作用,并探讨榄香烯逆转此耐药作用的可能机制。方法:应用不同浓度的吉非替尼和榄香烯单独或联合作用于HGF诱导的PC-9细胞株。MTT法检测细胞活力,并计算吉非替尼的半数抑制浓度(half maximal inhibitory concentration,IC50);Transwell小室实验检测榄香烯对HGF诱导的吉非替尼耐药PC-9细胞侵袭能力的影响;Western blot检测PC-9细胞中c-Met、AKT及其磷酸化蛋白的水平。结果:榄香烯可以显著抑制肺癌PC-9细胞的活力(P<0.05),随着药物剂量的增加,榄香烯对PC-9细胞的生长抑制率显著上升,在给药24 h后,其IC50为169.31 mg/L。肺癌PC-9细胞对吉非替尼敏感,随着吉非替尼浓度的增加,其对PC-9细胞生长的抑制作用不断增强,IC50为0.30μmol/L;外源性HGF(50μg/L)对肺癌PC-9细胞的生长有明显的促进作用,并诱导吉非替尼耐药。同时,使用cMet抑制剂SU11274联合吉非替尼作用于HGF诱导的PC-9细胞时,其存活率比吉非替尼单独作用于HGF诱导的PC-9细胞明显降低(P<0.05)。Transwell小室结果显示,与对照组相比,HGF能显著提高肺癌PC-9细胞的侵袭能力;与HGF联合吉非替尼组相比,榄香烯、HGF联合吉非替尼作用能明显抑制肺癌PC-9细胞的侵袭能力。Western blot结果显示,与对照组相比,HGF可显著上调p-Met和p-AKT的蛋白水平;与HGF联合吉非替尼组相比,榄香烯、HGF联合吉非替尼作用能明显下调p-Met和p-AKT的蛋白水平(P<0.01)。结论:榄香烯可逆转HGF诱导的肺癌PC-9细胞对吉非替尼耐药,其机制可能与抑制HGF活化的c-Met及其下游信号通路有关。

AIM:To investigate the effect ofβ-elemene on reversing hepatocyte growth factor(HGF)-induced resistance to gefitinib in PC-9cells,and to explore its possible mechanisms.METHODS:The gefitinib-resistant PC-9cells induced by HGF were treated withβ-elemene or/and gefitinib.The cell activity was measured by MTT assay.The effect ofβ-elemene on the invasion ability in HGF-induced resistance to gefitinib in PC-9cells was detected by Transwell migration assay.The protein levels of p-Met,c-Met,p-AKT and AKT in PC-9cells of each group were determined by Western blot.RESULTS:The results of MTT assay showed that the cell activity of PC-9cells was significantly inhibited byβ-elemene(P<0.05).IC50ofβ-elemene for PC-9cells was169.31mg/L.IC50of gefitinib for PC-9cells was0.30μmol/L.Exogenously adding recombinant HGF induced significantly resistance to gefitinib in PC-9cells.Moreover,SU11274(an inhibitor of c-Met)significantly decreased the viability of the cells exposed to HGF and gefitinib(P<0.05).Combined treatment withβ-elemene and gefitinib in the presence of HGF(50μg/L)significantly decreased the viability of PC-9cells as compared with the PC-9cells treated with gefitinib alone in the presence of HGF(P<0.05),so did the result of the cell migration.The protein levels of p-Met and p-AKT were significantly up-regulated by HGF,while the protein levels of p-Met and p-AKT were markedly down-regulated in the cells treated withβ-elemene and gefitinib compared with gefitinib alone in the presence of HGF.CONCLUSION:β-elemene reverses HGF-induced resistance to gefitinib in lung cancer PC-9cells,likely due to the inhibition of HGF-induced activation of c-Met and its down streams signaling pathways(P<0.01).