| Shaozhang Zhou,Zhixin Dong,Jinyi Lv,Aiping Zeng,Huilin Wang,Ruiling Ning,Xiangqun Song. The role of the HGF/c-Met signaling pathway in crizotinib-induced apoptosis in lung cancer with c-Met amplification. Oncol Transl Med, 2017, 3: 116-126. |
| HGF/c-Met信号通路在克唑替尼诱导c-Met基因扩增的肺癌细胞凋亡作用研究 |
| The role of the HGF/c-Met signaling pathway in crizotinib-induced apoptosis in lung cancer with c-Met amplification |
| Received:December 04, 2016 Revised:June 13, 2017 |
| DOI：10.1007/s10330-016-0210-0 |
| 中文关键词: HGF/c-Met信号通路，H1993细胞，H2228细胞，克唑替尼，肿瘤抑制 |
| 英文关键词: HGF/c-MET signaling pathway; H1993 cells; H2228 cells; crizotinib; apoptosis |
| 基金项目:国家自然科学基金项目（面上项目，重点项目，重大项目）广西科技厅资助项目（编号：201017 和2015139） |
| Author Name | Affiliation | E-mail | | Shaozhang Zhou | The Second Department of Chemotherapy, The Affiliated Tumor Hospital, Guangxi Medical University, Guangxi 530021, China | zhoushaozhang@qq.com | | Zhixin Dong | Guangxi Medical University Graduate School, Guangxi 530021, China | | | Jinyi Lv | Guangxi Medical University Graduate School, Guangxi 530021, China | | | Aiping Zeng | The Second Department of Chemotherapy, The Affiliated Tumor Hospital, Guangxi Medical University, Guangxi 530021, China | | | Huilin Wang | The Second Department of Chemotherapy, The Affiliated Tumor Hospital, Guangxi Medical University, Guangxi 530021, China | | | Ruiling Ning | The Second Department of Chemotherapy, The Affiliated Tumor Hospital, Guangxi Medical University, Guangxi 530021, China | | | Xiangqun Song* | Affiliated Tumor Hospital of Guangxi Medical University | xiangquns@163.com |
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| 中文摘要: |
| 目的：探讨HGF/c-Met信号通路在克唑替尼诱导不同的肺腺癌细胞系和肿瘤异种移植模型细胞凋亡的作用。
方法：细胞实验中，采用噻唑蓝（MTT）法检测克唑替尼作用于H2228，H1993及A549细胞的活力抑制情况。采用流式细胞术检测3种细胞在克唑替尼作用后的凋亡率；采用蛋白质印迹法检测HGF/c-Met信号通路关键蛋白的变化情况。动物实验中，将裸鼠随机分为5组。建立H1993及A549细胞裸鼠移植瘤模型。免疫组织化学染色法观察裸鼠移植瘤组织中HGF、c-MET及p-MET的表达情况。RT-qPCR法研究c-MET、AKT的mRNA表达量。蛋白质印迹法研究HGF/c-MET及其下游信号通路磷酸化水平的影响。
结果：细胞实验中，MTT结果表明克唑替尼作用72 h后，H1993、H2228和A549细胞的细胞活力抑制率均呈剂量依赖性升高。流式细胞术检测发现随着克唑替尼作用时间的延长，细胞凋亡率呈时间依赖性增加。蛋白质印迹法检测结果提示在H199和H2228细胞中，p-MET，p-AKT，p-ERK随着时间的延长蛋白水平呈现下降趋势。动物实验中，IHC检测结果可知，HGF和c-MET蛋白在H1993细胞移植瘤的表达亦均高于A549细胞移植瘤。p-MET蛋白在H1993细胞模型组的表达显著高于其余各组，而在H1993细胞克唑替尼组的表达显著低于其余各组。c-MET、AKT的mRNA于H1993细胞移植瘤的表达均高于A549细胞移植瘤。蛋白质印迹法结果可知，c-MET、AKT、ERK蛋白于H1993细胞移植瘤的表达量均显著高于A549细胞移植瘤；p-MET蛋白则H1993细胞模型组表达量高于其余各组；p-AKT蛋白于H1993细胞移植瘤的表达量均高于A549细胞移植瘤；p-ERK蛋白于H1993细胞克唑替尼组表达量最少。
结论：HGF/c-Met信号通路可能与克唑替尼抑制细胞增殖和凋亡诱导相关。 |
| 英文摘要: |
| Objective?This study aimed to study the role of the HGF/c-Met signaling pathway in crizotinib-induced apoptosis of various lung adenocarcinoma cell lines and xenograft tumor models.
Methods?In vitro, H2228, H1993, and A549 cells were treated with crizotinib. The inhibition of proliferation was quantitated by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was quantified by flow cytometry. Expression of key proteins of the HGF/c-Met signaling pathway was examined by western blotting. In vivo, H1993 and A549 tumor cell xenograft models were established. Immunohistochemical analysis was used to determine protein expression of HGF and c-MET and the amount of phospho-c-MET (p-c-Met). Real-time quantitative polymerase chain reaction (PCR) was applied to examine the messenger RNA (mRNA) expression of c-MET and serine/ threonine protein kinase (AKT). The expression and activation of the key proteins were evaluated by western blotting.
Results?In vitro, the growth of H1993, H2228, and A549 cells was inhibited after crizotinib treatment for 72 h. Apoptotic rates of H1993 and H2228 cells increased with the crizotinib concentration and exposure time. In vivo, the growth-inhibitory rate of crizotinib for H1993 xenografts was 72.3%. Positive expression rates of HGF and c-MET in H1993 xenografts were higher than those in A549 xenografts; the p-c-MET amount was the largest in H1993 xenograft control but the lowest in the H1993 xenograft with crizotinib treatment. The mRNA expression levels of c-MET and AKT in H1993 xenografts were higher than those of A549 xenografts. The protein levels of c-MET, AKT, and extracellular regulated protein kinases (ERK) in H1993 xenografts were higher than those in A549 xenografts; the p-AKT amount was higher in H1993 xenograft control than in A549 xenografts; the largest amount of p-c-MET was detected in H1993 xenograft control; the amount of p-ERK was the lowest in the H1993 xenograft with crizotinib treatment.
Conclusion?The HGF/c-Met signaling pathway may mediate crizotinib-induced apoptosis and inhibition of proliferation of lung adenocarcinoma cells.
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