唑来膦酸与紫杉醇体外协同抗肺腺癌细胞增殖及诱导凋亡的分子机制研究

  目的   探讨唑来膦酸（zoledronic acid，ZOL）及联合紫杉醇（paclitaxel，PTX）体外对人肺腺癌A-549细胞增殖影响及潜在机制。   方法   实验组包括不同浓度ZOL组、2 nM PTX组与ZOL联合应用PTX加药顺序不同3个组。MTT方法观测不同浓度ZOL与联合PTX对A-549细胞增殖的影响；流式细胞仪检测各组细胞凋亡；RT-PCR检测各组ERK和AKT mRNA的变化；Western blot检测各组ERK、AKT蛋白表达及磷酸化水平和Bcl-2的表达。   结果   :ZOL能抑制A-549细胞生长，该作用随ZOL浓度增加而增强；ZOL与PTX具有协同抗肿瘤作用，作用大小与两药加药顺序有关，产生最大抑制作用的顺序为先PTX后ZOL；RT-PCR结果显示不同组ERK和AKT mRNA表达水平分别为空白对照组最高，单药ZOL组次之，再次为PTX组，先PTX后ZOL组最低；Western blot结果显示各实验组中ERK、AKT蛋白表达水平无差异，但ERK和AKT磷酸化水平有差异，分别为空白对照组最高，单药ZOL组次之，再次为PTX组，先PTX后ZOL组最低，Bcl-2的表达水平亦是如此。   结论   ZOL联合PTX可能通过下调RAF/MEK/ ERK信号通路中ERK基因mRNA的表达，降低ERK磷酸化水平，抑制ERK激酶活性，进而抑制A-549细胞生长和增殖；同时可能通过下调PI3K/AKT信号通路中AKT基因mRNA的表达，降低AKT磷酸化水平，进而减弱AKT活性，进一步降低抗凋亡蛋白Bcl-2水平，从而起到促凋亡作用。 

Molecular mechanism of synergistic antitumor activity and induced apoptosis of zoledronic acid and paclitaxel

  Objective   This study aimed to investigate the inhibitory effect of zoledronic acid (ZOL) alone or the combined treatment of ZOL and paclitaxel (PTX) on the cell growth of lung cancer cell line in vitro.   Methods   The effects of different concentrations of ZOL alone, 2 nM PTX, and combined treatment of ZOL and PTX on the growth of A-549 cell line were determined using methyl thiazolyl tetrazolium (MTT) method. The mechanism of the curative effect was analyzed by flow cytometry on the basis of apoptotic rate. AKT, phospho-AKT, ERK, phospho-ERK, and Bcl-2 expressions were determined by western blot analysis. AKT and ERK gene expressions were determined by RT-PCR.   Results   MTT results showed that ZOL alone could inhibit the growth of lung cancer cell line A-549 in a dose-dependent manner. The combined therapy of ZOL and PTX could inhibit cell growth. This combined treatment is more effective than the single treatment with either ZOL or PTX alone. The synergistic inhibition rate is dependent on drug sequencing. Furthermore, maximum inhibition was induced by sequence order, i.e., initial treatment with PTX and then with ZOL. RT-PCR results demonstrated that the mRNA of ERK and AKT of the group treated with PTX and then ZOL were lower than that in other treatment groups. Western blot analysis results demonstrated that the ERK and AKT levels of the treated groups were parallel in the cell line. However, the lowest phospho-ERK, phospho-AKT, and Bcl-2 levels were observed in the PTX then ZOL group. The cell lines treated with PTX alone and ZOL alone ranked second and third among the lowest results, respectively. The highest level was observed in the control group.   Conclusion   The combined ZOL and PTX treatment induced the downregulation of phospho-ERK, phospho-AKT, and Bcl-2 protein expressions in RAF/MEK/ERK and PI3K/AKT signaling pathway. This pathway could be one of the synergistic antitumor mechanisms of the two drugs.