| 王诗忆,刘铁鑫,何陈佳,涂力英,朱 亮,吕倩明,雷绘敏.靶向Bcl-2克服非小细胞肺癌EGFR-TKIs获得性耐药[J].,2023,(15):2801-2808 |
| 靶向Bcl-2克服非小细胞肺癌EGFR-TKIs获得性耐药 |
| Targeting Bcl-2 Overcomes Acquired Resistance to Multiple Generations of EGFR-TKIs in Non-Small Cell Lung Cancer |
| 投稿时间：2023-03-07 修订日期：2023-03-28 |
| DOI：10.13241/j.cnki.pmb.2023.15.001 |
| 中文关键词: 非小细胞肺癌 表皮生长因子受体-受体酪氨酸激酶抑制剂（EGFR-TKIs）耐药 B细胞淋巴瘤/白血病-2基因（Bcl-2） 核红细胞2相关因子2（NRF2） |
| 英文关键词: Non-small cell lung cancer (NSCLC) Epidermal growth factor receptor-tyrosine kinase receptor inhibitors（EGFR-TKIs） resistance B-cell leukemia/lymphoma 2 (Bcl-2) Nuclear factor erythroid 2-related factor 2 (NRF2) |
| 基金项目:国家自然科学基金项目（82273950；82003772；82103050） |
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| 中文摘要: |
| 摘要 目的：探讨靶向抗凋亡蛋白Bcl-2克服非小细胞肺癌EGFR-TKIs耐药的作用及重定位Bcl-2靶向抑制剂用于克服耐药的可行性。方法：通过药物浓度梯度递增法构建非小细胞肺癌多代EGFR-TKIs耐药株，根据亲本细胞和多代EGFR-TKIs耐药的非小细胞肺癌细胞的RNA-seq数据筛选出潜在耐药相关基因Bcl-2，通过Western blot 检测其在耐药细胞中的蛋白水平。为了探讨Bcl-2诱导耐药的作用，采用siRNA干扰和使用Bcl-2的抑制剂ABT199抑制Bcl-2，通过CCK8法、IncuCyte实时监测和Western blot等方法检测其对亲本和耐药细胞的细胞活力、药物敏感性、增殖和凋亡的影响。随后使用奥希替尼分别处理亲本及耐药细胞，通过Western Blot检测NRF2受到药物作用后及在耐药细胞中的蛋白水平，并以临床公共数据库分析辅助验证。使用siRNA干扰或NRF2抑制剂ML385敲低或抑制NRF2功能，借助Western Blot和CCK8法检测其对Bcl-2表达水平及对EGFR-TKI敏感性的影响；通过加入NRF2的激动剂Ki696探究其对Bcl-2的诱导作用、对EGFR-TKI敏感性的影响及取消靶向Bcl-2逆转耐药的作用。结果：Bcl-2在EGFR-TKIs耐药细胞中上调；敲低或抑制Bcl-2后，可选择性抑制耐药细胞的生长和活力，并诱导凋亡，且能逆转包括第三代药物奥希替尼在内的多代EGFR-TKIs耐药；EGFR-TKI可在敏感细胞中诱导NRF2的上调，且耐药细胞中上调的Bcl-2受NRF2调控。结论：EGFR-TKIs耐药细胞通过上调抗凋亡分子Bcl-2获得耐药，该分子的上调受NRF2的调控，靶向Bcl-2则可以逆转耐药。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the effect of anti-apoptotic protein Bcl-2 on the resistance of non-small cell lung cancer（NSCLC）to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Methods: Lung adenocarcinoma cells resistant to multiple generations of EGFR-TKIs were constructed by gradient drug escalation method. The potential EGFR-TKIs resistance-related gene Bcl-2 was screened out according to the RNA-seq data of multi-generation EGFR-TKI resistant NSCLC cells versus parental cells. Consequently, Bcl-2 expression in drug-resistant cells was then detected by Western blot. To verify the effect of Bcl-2 protein on drug resistance, negative intervention methods such as siRNA interference and BCl-2 inhibitor ABT-199 were used to knock down and inhibit the protein, respectively. The effects of Bcl-2 on cell viability, drug sensitivity, proliferation and apoptosis in parental and drug-resistant cells were confirmed via CCK8, IncuCyte monitoring and Western blot. NRF2 expression was examined via western blot after exposure to osimertinib in parental and drug-resistant cells. The upstream mechanism was further demonstrated and validated by phenotype experiments combined with public clinical databases analysis as supplementation. The effects of knockdown or inhibition of NRF2 by siRNA or NRF2 inhibitor ML385 on expression of Bcl-2 and sensitivity to EGFR-TKI was confirmed via western blot and CCK8 method. The induction of Bcl-2 expression, the sensitivity to EGFR-TKI and reversal of effects induced by targeting Bcl-2 were demonstrated by adding NRF2 agonist Ki696. Results: Bcl-2 was found overexpressed in EGFR-TKIs resistant cells. The resistance to multi-generation EGFR-TKIs including the third-generation - Osimertinib - can be reversed by interventions on Bcl-2 protein through genetically targeted means or Bcl-2 selective inhibitors. In the meantime, cell growth and cell viability were inhibited while cell apoptosis was induced selectively in drug-resistant cells after targeting Bcl-2. The upstream transcription factor NRF2 was induced by EGFR-TKI in sensitive cells, and was proven to be in charge of the upregulation of Bcl-2. Conclusion: Anti-apoptotic molecule Bcl-2 is up-regulated in EGFR-TKIs resistant non-small cell lung cancer cells by the antioxidant element NRF2 and it endows cancer cells with drug resistance, while retargeting Bcl-2 can reverse this resistance. |
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