[STE029 Overcomes EGFR-TKI Resistance in Human Lung Adenocarcinoma]

Acquired and primary resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is still the bottleneck of clinical treatment of advanced non-small cell lung cancer (NSCLC). STE029 is a novel anticancer drug which consists of 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGCR) inhibitor and novel cancer cell membrane targeting molecular. This study aimed to investigate the reversal mechanism of EGFR-TKI resistance by STE029 in lung adenocarcinoma.CCK8 test was used to test the cell viability and survival rate of EGFR mutated PC9 cell (Gefitinib sensitive), PC9/BB4 cell (acquired Gefitinib resistant), and EGFR wild type A549 cell after treatment of STE029, Gefitinib or combination of both. EdU test was applied to detect changes in cell cycle and Hoechst 33258 was applied to detect apoptosis rate in overcoming the EGFR-TKI resistance. The activity of EGFR/PI3K/Akt, cell cycle and apoptosis signal pathways were examined. In vivo, nude mice were exposed to STE029, Gefitinib and STE029+Gefitinib for 5 wk. And the the tumor volume was measured and tumor weight was obtained on the last day.(1) PC9 cells was highly sensitive to Gefitinib, while PC9/BB4 and A549 cell showed significant resistance to Gefitinib treatment; (2) STE029+Gefitinib treatment could significantly decrease the 50% inhibitory concentrarion (IC₅₀) of Gefitinib in PC9, PC9/BB4 and A549 cells (P0.05, respectively); (3) In PC9 and PC9/BB4 cells, STE029+Gefitinib can block cell cycle and inhibit cell proliferation (P0.001), while there was no significant difference in apoptosis rate among three drug intervention groups (P0.05); However, apoptosis rate was increased in STE029+Gefitinib group in A549 cell (P0.01), while no significance detected in cell proliferation (P0.05). (4) In PC9 and PC9/BB4 cells, the combination of STE029 and Gefitinib could downregulate p-EGFR, p-Akt, p-Cyclin D1 and Cyclin D1 (P0.001), and upregulate the expression of GSK-3β (P0.001), and the expression of cleaved caspase-8, caspase-8 cleaved caspase-9, caspase-9 showed no difference among groups (P0.05). In A549 cells, the combination of STE029 and Gefitinib could downregulate p-Akt (P0.001) and upregulate cleaved caspase-8 and cleaved caspase-9 (P0.001); (5)In vivo, the combination of STE029 and Gefitinib effectively inhibited tumor development and progression compared to STE029 alone or Gefitinib alone, with significant difference (P0.05) in PC9 and PC9/BB4 xenografted tumor.STE029 could sensitize Gefitinib by inhibiting EGFR/PI3K/Akt pathway, blocking the tumor cell cycle and proliferation and inducing apoptosis through caspase-8 and caspase-9 dependent pathway. STE029 deserves further investigations in overcoming EGFR-TKI resistance in lung cancer.【中文题目：STE029逆转肺腺癌EGFR-TKI耐药 及其机制研究】 【中文摘要：背景与目的 表皮生长因子受体酪氨酸激酶抑制剂（epidermal growth factor receptor-tyrosine kinase inhibitor, EGFR-TKI）获得性耐药和原发性耐药至今仍是临床治疗晚期非小细胞肺癌（non-small cell lung cancer, NSCLC）的瓶颈。STE029是一种同时具有羟甲基戊二酸单酰辅酶A还原酶（3-hydroxy-3-methylglutarylcoenzyme A reductase, HMGCR）抑制剂抗肿瘤作用和肿瘤特异性细胞膜靶向功能的新型抗肿瘤药物。本研究旨在探讨STE029逆转肺腺癌EGFR-TKI的耐药机制。方法 STE029、吉非替尼单药及联合用药分别处理PC9、PC9/BB4、A549细胞，检测各细胞株的活性变化、细胞增殖、细胞凋亡，鉴定EGFR/PI3K/Akt信号通路、细胞周期及凋亡相关蛋白的表达；同时观察STE029、吉非替尼单药及联合用药对PC9、PC9/BB4裸鼠皮下移植瘤生长的影响。结果 ①PC9细胞为EGFR突变的敏感细胞，PC9/BB4细胞为EGFR突变的获得性耐药细胞，A549细胞为非EGFR突变的耐药细胞；②STE029联合吉非替尼作用于PC9、PC9/BB4、A549细胞后，吉非替尼的半数抑制浓度（50% inhibitory concentration, IC₅₀）值较对照组均显著降低（P0.05）；③STE029联合吉非替尼可抑制PC9、PC9/BB4细胞的增殖（P0.001），诱导A549细胞凋亡增加（P0.01）；④在PC9、PC9/BB4细胞中，联合用药组较单药组p-EGFR、p-Akt的表达明显下调（P0.000,1），GSK-3β表达升高（P0.001），其下游p-Cyclin D1及Cyclin D1表达明显降低（P0.001），cleaved caspase-8、caspase-8、cleaved caspase-9、caspase-9的表达在各给药组间未见明显差异（P0.05）；在A549细胞中，联合用药组p-Akt表达明显下调（P0.001），cleaved caspase-8、cleaved caspase-9表达均升高（P0.001），而GSK-3β、p-Cyclin D1、Cyclin D1、caspase-8、caspase-9的蛋白表达在各给药组间则无明显差异（P0.05）；⑤裸鼠体内，联合用药组PC9皮下移植瘤的生长明显受到抑制，差异有明显统计学意义（P0.01）；联合用药组PC9/BB4皮下移植瘤的生长速率亦明显降低（P0.05）。结论 STE029可在体内外明显增加非EGFR-T790M突变耐药的人肺腺癌细胞对吉非替尼的敏感性，其机制可能与STE029通过EGFR/PI3K/Akt信号通路调节GSK-3β、Cyclin D1的表达、阻滞细胞增殖、诱导细胞凋亡有关。 】 【中文关键词：STE029；EGFR-TKI；耐药；细胞凋亡；细胞周期；肺腺癌】.