| 陈燕琼,吴丕华,宋向群.IGF-1R旁路活化在EML4-ALK阳性非小细胞肺癌Alectinib继发耐药中的作用[J].肿瘤学杂志,2018,24(5):418-424. |
| IGF-1R旁路活化在EML4-ALK阳性非小细胞肺癌Alectinib继发耐药中的作用 |
| IGF-1R Signaling Pathway Activation in Acquired Resistance of EML4-ALK Positive Lung Cancer Cell Line H3122 to Alectinib |
| 投稿时间：2017-08-01 |
| DOI：10.11735/j.issn.1671-170X.2018.05.B002 |
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| 中文关键词: 非小细胞肺癌 EML4-ALK融合基因 alectinib 胰岛素样生长因子1 耐药性 |
| 英文关键词:non-small cell lung cancer EML4-ALK fusion gene alectinib IGF-1 drug resistance |
| 基金项目:国家自然科学基金资助项目（81260357）；广西自然科学基金资助项目（2015GXNSFAA139162） |
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| 中文摘要: |
| 摘 要：［目的］ 探讨胰岛素样生长因子-1（IGF-1）是否以旁路激活的方式诱导EML4-ALK融合基因阳性肺癌细胞株H3122对Alectinib的耐药，并进一步探讨旁路信号激活在 Alectinib耐药中的作用。［方法］ 通过外源性加入人胰岛素样生长因子1（hIGF-1）刺激H3122细胞株构建Alectinib耐药细胞株H3122-IGF-CR；通过CCK8法检测H3122细胞株及H3122-IGF-CR对不同浓度Alectinib的敏感性，并计算半数抑制浓度IC50和耐药指数；PE Annexin V/7-AAD双染法检测细胞凋亡情况；qRT-PCR检测亲本细胞中IGF-1R的表达情况；蛋白质印记法检测PI3K/AKT, Ras-Raf-MEK-ERK/MAPK信号通路的变化情况。［结果］ qRT-PCR结果显示IGF-1R在H3122细胞中ct值为20.90±0.06，内参ct值为12.48±0.12，ΔCt值＜12， 提示IGF-1R在H3122细胞株中高表达。H3122细胞的活力能被Alectinib抑制，且呈剂量依赖性，其IC50值为0.0173μmol/L；当加入50ng/ml或100ng/ml的hIGF-1刺激后，其对Alectinib敏感性降低，IC50为0.358μmol/L和0.4001μmol/L，耐药倍数为20.6倍和23.0倍。0.03μmol/L Alectinib单药处理48h后H3122细胞的凋亡率为（26.43±0.23）%，而Alectinib联合50、100、150ng/ml hIGF-1刺激48h后，凋亡率分别为（18.95±0.48）%、（15.90±0.16）%、（13.70±0.36）%，显著性低于Alectinib单药组（P<0.05）。外源性hIGF-1与IGF-1R结合后，明显增加细胞中p-IGF-1R及其下游p-AKT、p-mTOR、p-P70S6K、p-ERK的蛋白表达水平（P<0.05）。此外，联合应用IGF-1R抑制剂Linsitinib (OSI-906)可以抑制因hIGF-1配体导致的H3122耐药细胞的活力。［结论］ hIGF-1可通过旁路激活的方式诱导EML4-ALK阳性非小细胞肺癌H3122细胞对Alectinib耐药，其机制可能为激活了其下游信号通路PI3K/AKT,MAPK/ERK，初步证实 IGF-1R信号通路与Alectinib继发耐药相关。 |
| 英文摘要: |
| Abstract：［Objective］ To explore the role of insulin-like growth factor-1(IGF-1) in acquired resistance of EML4-ALK fusion gene positive lung cancer cell line H3122 to Alectinib and its relation to activation of the bypass signaling pathway. ［Methods］ EML4-ALK positive cell H3122 cells were treated with human insulin-like growth factor 1(hIGF-1) to establish the acquired resistant cell line H3122-IGF-CR. The cell viability was measured by CCK8 assay；the cell apoptosis was detected by flow cytometry，the expression of IGF 1R in parental cells was detected by qRT-PCR. The protein levels and phosphorylation status of PI3K/AKT，Ras-Raf-MEK-ERK/MAPK signaling pathway were detected by Western blot. ［Results］ The ct value of the IGF-1R in H3122 cells and internal reference was 20.90±0.06 and 12.48±0.12，respectively with the Δct value <12(P<0.05). The viability of H3122 cell was inhibited by Alectinib in a dose-dependent manner with the IC50 value of 0.0173μmol/L. When treating with 50ng/ml or 100ng/ml hIGF-1，the activity of Alectinib was decreased，and the IC50 of the H3122-IGF-CR to Alectinib was 0.358μmol/L and 0.4001μmol/L with a resistance index of 20.6 and 23.0，respectively. After treatment with Alectinib at 0.03μmol/L for 48h，the apoptosis rate of H3122 cells was(26.43±0.23)%，while combined with the 50ng/ml，100ng/ml or 150ng/ml hIGF-1 for 48h，the apoptosis rates were(18.95±0.48)%，(15.90±0.16)% and (13.70±0.36)%，respectively(P＜0.05). Exogenous hIGF-1 significantly up-regulated the levels of pIGF-1R protein and its downstream proteins p-AKT，p-mTOR，p-70s6k and p-ERK. Combined with IGF-1R inhibitor Linsitinib(OSI-906) successfully inhibited the viability of H3122 cells even in the presence of hIGF-1. ［Conclusion］ hIGF-1 can induce the acquiring resistance of EML4-ALK positive non-small cell lung cancer H3122 cells to Alectinib by means of activating the bypass signaling pathway，indicating that IGF-1R signaling pathways is associated with Alectinib resistance. |
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