Non-small cell lung cancer (NSCLC) patients with epithermal growth factor receptor (EGFR) mutations can be treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs), however, development of acquired resistance could significantly limit curative effects of EGFR-TKIs. Different mechanisms of acquired resistance to first-generation and second-generation EGFR TKIs have been widely reported, but there were few reports on the resistant mechanism of third-generation EGFR-TKI such as osimertinib (AZD9291). In the present study, significant upregulation of Bcl-2 was found in AZD9291-resistant H1975 cells (H1975AR) compared with H1975, which may constitute an important resistant mechanism of acquired resistance to AZD9291. More importantly, our study showed that synergism between AZD9291 and Bcl-2 inhibitor ABT263 (0.25 μM) or ABT199 (1 μM) could effectively overcome the acquired resistance of AZD9291 in H1975AR in vitro. Flow cytometry analyses demonstrated that AZD9291 + ABT263/ABT199 caused a significantly different cell cycle distribution and produced significantly more apoptosis compared with either AZD9291 or ABT263/ABT199 treatment alone. Further multiscreen/Western blot analyses revealed that NF-κB was significantly downregulated in AZD9291 + ABT263/ABT199 treatment groups compared with AZD9291 or ABT263/ABT199 treatment alone, with a more significant reduction of NF-κB in AZD9291 + ABT199 compared with AZD9291 + ABT263. It is also noticeable that AZD9291 + ABT263 specifically caused a significantly reduced expression of p21 compared with AZD9291 or ABT263 treatment alone while AZD9291 + ABT199 specifically caused significantly reduced expressions of SQSTM1 and survivin, but increased expression of autophagosome marker LC3-II compared with AZD9291 or ABT199 treatment alone. Furthermore, cytotoxicity of AZD9291 + ABT199 could be partially reversed by autophagy inhibitor chloroquine. These results suggest that ABT263 and ABT199 may work through different signaling pathways to achieve synergistic cytotoxicity with AZD9291 in H1975AR. These findings suggest that Bcl-2 inhibitor may provide an effective option in combination therapy with EGFR-TKIs to treat NSCLC with EGFR-TKI acquired resistance.

具有超热生长因子受体 (EGFR) 突变的非小细胞肺癌 (NSCLC) 患者可以用 EGFR-酪氨酸激酶抑制剂 (EGFR-TKI) 治疗，但是，获得性耐药的发展可能会显着限制 EGFR-TKI 的疗效。第一代和第二代EGFR TKI获得性耐药机制不同，已有广泛报道，但关于奥希替尼（AZD9291）等第三代EGFR-TKI耐药机制的报道较少。在本研究中，与 H1975 相比，在 AZD9291 抗性 H1975 细胞（H1975AR）中发现 Bcl-2 显着上调，这可能构成对 AZD9291 获得性抗性的重要抗性机制。更重要的是，我们的研究表明 AZD9291 和 Bcl-2 抑制剂 ABT263 (0. 25 μM) 或 ABT199 (1 μM) 可以有效克服 AZD9291 在体外 H1975AR 中的获得性耐药。流式细胞术分析表明，与单独的 AZD9291 或 ABT263/ABT199 处理相比，AZD9291 + ABT263/ABT199 引起显着不同的细胞周期分布并产生明显更多的细胞凋亡。进一步的多屏/蛋白质印迹分析显示，与单独的 AZD9291 或 ABT263/ABT199 治疗相比，AZD9291+ABT263/ABT199 治疗组中的 NF-κB 显着下调，与 AZD9291+ABT199 相比，AZD9291+ABT199 中 NF-κB 的降低更显着. 同样值得注意的是，与单独使用 AZD9291 或 ABT263 处理相比，AZD9291 + ABT263 特异性地导致 p21 的表达显着降低，而 AZD9291 + ABT199 特异性地导致 SQSTM1 和存活蛋白的表达显着降低，但与 AZD921 或 AZD92 相比，自噬体标志物 LC3-II 的表达增加ABT199 单独治疗。此外，自噬抑制剂氯喹可以部分逆转 AZD9291 + ABT199 的细胞毒性。这些结果表明 ABT263 和 ABT199 可能通过不同的信号通路在 H1975AR 中与 AZD9291 实现协同细胞毒性。这些发现表明 Bcl-2 抑制剂可能为 EGFR-TKI 联合治疗提供有效的选择，以治疗具有 EGFR-TKI 获得性耐药的 NSCLC。但与单独使用 AZD9291 或 ABT199 处理相比，自噬体标志物 LC3-II 的表达增加。此外，自噬抑制剂氯喹可以部分逆转 AZD9291 + ABT199 的细胞毒性。这些结果表明 ABT263 和 ABT199 可能通过不同的信号通路在 H1975AR 中与 AZD9291 实现协同细胞毒性。这些发现表明 Bcl-2 抑制剂可能为 EGFR-TKI 联合治疗提供有效的选择，以治疗具有 EGFR-TKI 获得性耐药的 NSCLC。但与单独使用 AZD9291 或 ABT199 处理相比，自噬体标志物 LC3-II 的表达增加。此外，自噬抑制剂氯喹可以部分逆转 AZD9291 + ABT199 的细胞毒性。这些结果表明 ABT263 和 ABT199 可能通过不同的信号通路在 H1975AR 中与 AZD9291 实现协同细胞毒性。这些发现表明 Bcl-2 抑制剂可能为 EGFR-TKI 联合治疗提供有效的选择，以治疗具有 EGFR-TKI 获得性耐药的 NSCLC。