| Haiyan Peng,Wenhua Zhao,Cuiyun Su,Xiangqun Song,Aiping Zeng,Huilin Wang,Ruiling Ning,Shaozhang Zhou. Role of the STAT3/survivin signaling pathway in the EML4-ALK-positive lung adenocarcinoma cell line H2228 before and after crizotinib-induced resistance. Oncol Transl Med, 2015, 1: 73-77. |
| EML4-ALK融合基因阳性人肺腺癌H2228细胞株对克唑替尼耐药前后stat3/survivin信号通路变化的检测 |
| Role of the STAT3/survivin signaling pathway in the EML4-ALK-positive lung adenocarcinoma cell line H2228 before and after crizotinib-induced resistance |
| Received:December 16, 2014 Revised:April 03, 2015 |
| DOI：10.1007/s10330-014-0039-7 |
| 中文关键词: EML4-ALK融合基因，H2228细胞，克唑替尼，细胞凋亡，stat3/survivin信号通路 |
| 英文关键词: EML4-ALK fusion gene; H2228 cell line; crizotinib; apoptosis; STAT3/survivin signaling pathway |
| 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） |
| Author Name | Affiliation | E-mail | | Haiyan Peng | Guangxi Medical University | 784951431@qq.com | | Wenhua Zhao | Affiliated Cancer Hospital, Guangxi Medical University, Guangxi, China | 315333096@qq.com | | Cuiyun Su | Affiliated Cancer Hospital, Guangxi Medical University, Guangxi, China | | | Xiangqun Song | Affiliated Cancer Hospital, Guangxi Medical University, Guangxi, China | | | Aiping Zeng | Affiliated Cancer Hospital, Guangxi Medical University, Guangxi, China | | | Huilin Wang | Affiliated Cancer Hospital, Guangxi Medical University, Guangxi, China | | | Ruiling Ning | Affiliated Cancer Hospital, Guangxi Medical University, Guangxi, China | | | Shaozhang Zhou* | Affiliated Cancer Hospital, Guangxi Medical University, Guangxi, China | zhoushaozhang@qq.com |
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| 中文摘要: |
| 【摘要】 目的：探讨stat3/survivin信号通路在克唑替尼（crizotinib）诱导的EML4-ALK融合基因阳性的人肺腺癌细胞H2228细胞株凋亡中的作用。方法：采用噻唑蓝（MTT）法检测克唑替尼对H2228细胞增殖抑制作用，计算IC50；采用浓度梯度递增方法诱导H2228耐药细胞株，并测定其耐药指数；采用流式细胞术检测300nmol/L、1000nmol/L克唑替尼分别作用于H2228亲本细胞和耐药细胞（H2228 crizotinib resistance，H2228 CR）24h、48h、72h后的凋亡率，用蛋白质免疫印迹（Western Blot，WB）的方法检测0nmol/L、300nmol/L、1000nmol/L克唑替尼分别作用于H2228亲本细胞和耐药细胞72h后p-ALK、ALK和stat3/survivin信号通路中p-stat3、stat3和survivin各蛋白表达量及活化水平的变化。结果：克唑替尼以剂量依赖性方式诱导H2228增殖抑制。克唑替尼作用H2228亲本细胞和H2228耐药细胞72小时的IC50值分别为334.5nmol/L和3418nmol/L。H2228耐药细胞的耐药指数为10.20。克唑替尼对H2228细胞有促凋亡作用(P<0.05)，且能使H2228细胞中的融合蛋白EML4-ALK V3的活化形式p-ALK明显受到抑制，其下游的p-stat3及存活蛋白survivin的表达呈下降趋势，但克唑替尼对H2228耐药细胞株不具有促凋亡作用，且不能使p-ALK的表达受到抑制，下游通路stat3的活化水平及存活蛋白survivin的表达无下降趋势。结论：克唑替尼以剂量和时间依赖的方式抑制人肺腺癌细胞株H2228的增殖。克唑替尼诱导的H2228细胞凋亡可能与stat3/survivin通路有关，并能下调p-ALK、p-stat3和survivin的表达，但对H2228耐药细胞没有影响。 |
| 英文摘要: |
| 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 nM and 3418 nM, 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. |
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