Objective This study investigated the role of the STAT3/survivin signaling pathway in the EML4-ALK–positive lung adenocarcinoma cell line H2228 before and after crizotinib-induced resistance. The mechanism of resista...Objective This study investigated the role of the STAT3/survivin signaling pathway in the EML4-ALK–positive lung adenocarcinoma cell line H2228 before and after crizotinib-induced resistance. The mechanism of resistance was studied. Methods Cell viability was determined using the MTT assay. Crizotinib-induced apoptosis in H2228 and H2228 crizotinib-resistant cells treated with the indicated doses of crizotinib was measured at different times(24 h, 48 h, 72 h) using flow cytometry. The levels of p-ALK, ALK, p-STAT3, STAT3, and survivin after treatment of cells with 0, 0.3, and 1 μM crizotinib for 72 h were determined using Western blot analysis. DNA sequencing was used to identify mutations in H2228 crizotinib-resistant cells. Results The crizotinib IC50 values in H2228 and H2228 crizotinib-resistant cells at 72 h were 334.5 n M and 3418 n M, respectively. The resistance index of H2228 crizotinib-resistant cells was 10.20. Crizotinib induced apoptosis in H2228 cells and reduced the levels of p-ALK, p-STAT3, and survivin. In contrast, no changes in the levels of p-ALK, p-STAT3, and survivin were observed in H2228 crizotinib-resistant cells. The mutations 2067G→A and 2182G→C in EML4-ALK were present in the H2228 crizotinib-resistant cells. Conclusion Crizotinib decreased the viability of H2228 cells in a dose- and time-dependent manner. In the STAT3/survivin pathway, downregulation of p-ALK, p-STAT3, and survivin might contribute to crizotinib-induced apoptosis in H2228 cells. However, the STAT3/survivin pathway in H2228 crizotinib-resistant cells was unaffected by crizotinib treatment. Acquired resistance in H2228 cells might be related to ALK mutations.展开更多
目的观察体外环境下克唑替尼对NSCLC细胞的放射增敏效果及其潜在机制。方法以H2228及H3122细胞株为实验对象,MTT法检测不同浓度克唑替尼对NSCLC细胞的抑制作用;平板克隆形成实验分别测定NSCLC细胞在克唑替尼作用下照射以及单纯照射的SF...目的观察体外环境下克唑替尼对NSCLC细胞的放射增敏效果及其潜在机制。方法以H2228及H3122细胞株为实验对象,MTT法检测不同浓度克唑替尼对NSCLC细胞的抑制作用;平板克隆形成实验分别测定NSCLC细胞在克唑替尼作用下照射以及单纯照射的SF,拟合细胞生存曲线并计算放射增敏比;流式细胞仪检测各细胞株的接受不同剂量放射线后细胞凋亡率及细胞周期分布的变化;Western Blot检测各细胞株接受放射线照射后STAT3及p-STAT3蛋白表达水平变化。结果 MTT实验:抑制率与浓度变化呈正相关。IC50分别为330 n M和102 n M。克隆形成实验:附加药物照射的细胞SF低于单纯照射组。H2228/H3122细胞株组中,SER(D0):1.064/1.004;SER(Dq):1.079/1.047。流式细胞术:两细胞的凋亡率与照射剂量呈正比。在H2228组细胞中G0/G1期细胞阻滞现象明显,H3122细胞各周期细胞比率未见明显变化。Western Blot法:应用克唑替尼联合时STAT3的磷酸化过程受到阻滞。H2228细胞株阻滞现象更为明显。结论克唑替尼对NSCLC H2228及H3122细胞株均有放射增敏作用,对H2228细胞株的放射增敏效果更加明显。展开更多
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 ...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(m RNA) 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 m RNA 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.展开更多
基金Supported by grants from the Bureau of Science and Technology,Guangxi Zhuang Autonomous Zone,China(No.201017)National Natural Science Foundation of China(No.81060188 and 81260357)
文摘Objective This study investigated the role of the STAT3/survivin signaling pathway in the EML4-ALK–positive lung adenocarcinoma cell line H2228 before and after crizotinib-induced resistance. The mechanism of resistance was studied. Methods Cell viability was determined using the MTT assay. Crizotinib-induced apoptosis in H2228 and H2228 crizotinib-resistant cells treated with the indicated doses of crizotinib was measured at different times(24 h, 48 h, 72 h) using flow cytometry. The levels of p-ALK, ALK, p-STAT3, STAT3, and survivin after treatment of cells with 0, 0.3, and 1 μM crizotinib for 72 h were determined using Western blot analysis. DNA sequencing was used to identify mutations in H2228 crizotinib-resistant cells. Results The crizotinib IC50 values in H2228 and H2228 crizotinib-resistant cells at 72 h were 334.5 n M and 3418 n M, respectively. The resistance index of H2228 crizotinib-resistant cells was 10.20. Crizotinib induced apoptosis in H2228 cells and reduced the levels of p-ALK, p-STAT3, and survivin. In contrast, no changes in the levels of p-ALK, p-STAT3, and survivin were observed in H2228 crizotinib-resistant cells. The mutations 2067G→A and 2182G→C in EML4-ALK were present in the H2228 crizotinib-resistant cells. Conclusion Crizotinib decreased the viability of H2228 cells in a dose- and time-dependent manner. In the STAT3/survivin pathway, downregulation of p-ALK, p-STAT3, and survivin might contribute to crizotinib-induced apoptosis in H2228 cells. However, the STAT3/survivin pathway in H2228 crizotinib-resistant cells was unaffected by crizotinib treatment. Acquired resistance in H2228 cells might be related to ALK mutations.
文摘目的观察体外环境下克唑替尼对NSCLC细胞的放射增敏效果及其潜在机制。方法以H2228及H3122细胞株为实验对象,MTT法检测不同浓度克唑替尼对NSCLC细胞的抑制作用;平板克隆形成实验分别测定NSCLC细胞在克唑替尼作用下照射以及单纯照射的SF,拟合细胞生存曲线并计算放射增敏比;流式细胞仪检测各细胞株的接受不同剂量放射线后细胞凋亡率及细胞周期分布的变化;Western Blot检测各细胞株接受放射线照射后STAT3及p-STAT3蛋白表达水平变化。结果 MTT实验:抑制率与浓度变化呈正相关。IC50分别为330 n M和102 n M。克隆形成实验:附加药物照射的细胞SF低于单纯照射组。H2228/H3122细胞株组中,SER(D0):1.064/1.004;SER(Dq):1.079/1.047。流式细胞术:两细胞的凋亡率与照射剂量呈正比。在H2228组细胞中G0/G1期细胞阻滞现象明显,H3122细胞各周期细胞比率未见明显变化。Western Blot法:应用克唑替尼联合时STAT3的磷酸化过程受到阻滞。H2228细胞株阻滞现象更为明显。结论克唑替尼对NSCLC H2228及H3122细胞株均有放射增敏作用,对H2228细胞株的放射增敏效果更加明显。
基金Supported by grants from the National Natural Sciences Foundation of China(No.81060188 and No.81260351)Guangxi Sciense&Technology Development Funds(No.2015139 and No.201017)
文摘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(m RNA) 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 m RNA 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.
