FW-04-806对HER2阳性胃癌细胞的抗肿瘤活性及其机制研究

摘要

目的：研究大环双内酯类化合物FW-04-806对HER2阳性胃癌细胞的抗肿瘤活性及其机制，探讨其与拉帕替尼的联合作用。方法 MTT法检测FW-04-806对HER2阳性胃癌细胞增殖抑制作用；结晶紫染色法检测集落形成抑制能力；流式细胞术检测细胞蛋白表达、凋亡诱导和周期阻滞；免疫共沉淀法检测蛋白间相互作用；免疫组化法观察蛋白的表达变化；免疫印迹法检测细胞增殖和凋亡通路相关蛋白的表达；体内异种移植瘤模型检测抑瘤效果。结果 FW-04-806明显抑制HER2阳性胃癌细胞 NCI-N87、OE19的增殖和集落形成能力，半数抑制率( IC50)分别为(24．17±0．02)、(29．61±0．03)μmol·L-1；剂量依赖性诱导阻滞细胞于G2-M期，并增加凋亡比例；200 mg·kg-1实验组对OE19瘤块的抑瘤率为48．0%( P<0．01)；诱使 Hsp90/CDC37复合物解离；降解HER2、Akt蛋白；抑制HER2、Akt和ERK的磷酸化，增加cleaved caspase-3、cleaved parp的表达；FW-04-806与拉帕替尼体外联用对NCI-N87胃癌细胞具有协同作用，能抑制增殖，提高凋亡比例。结论 FW-04-806对HER2阳性胃癌细胞具有良好的体内、外抗肿瘤活性；与拉帕替尼联合具有协同作用。%Aim To investigate the efficacy and mech-anism of FW-04-806 against HER2-positive gastric cancer cell lines,and the combination effect of FW-04-806 with lapatinib. Methods MTT assay was used to assess cell proliferous inhibition of FW-04-806 . The in-hibitory effect of colony formation was tested by colony formation. The protein expression, apoptotic induction and cell cycle arrest were detected by flow cytometry. Co-immunoprecipitation was used to investigate protein-protein interactions. The expression change of proteins was showed by immunohistochemistry. Western blot was applied to reveal the protein expression of related pro-liferous and apoptotic signaling pathway. The tumor growth inhibition was evaluated in tumor xenograft model. Results FW-04-806 obviously inhibited cell proliferation and colony formation in HER2 positive gastric cancer cell lines NCI-N87, OE19, with IC50 of (24. 17 ± 0. 02 ) , ( 29. 61 ± 0. 03 ) μmol · L-1 , re-spectively;FW-04-806 induced G2-M arrest and apop-tosis in a dose-dependent manner;200 mg · kg-1 of FW-04-806 showed tumor growth inhibition of 48. 0%( P < 0. 01 ) . In addition, FW-04-806 dissociated Hsp90/CDC37 complex, followed by degradation of HER2 and Akt,inhibiting the phosporylation of HER2, Akt and ERK, and increasing expression of apoptotic proteins,such as cleaved caspase-3 and cleaved parp. Furthermore,the combination of FW-04-806 with lapa-tinib in vitro was synergistic in NCI-N87 , which en-hanced the inhibition of cell proliferation and increased apoptotic rates. Conclusions FW-04-806 shows po-tent efficacy against HER2-positive gastric cancer cell lines in vitro and in vivo;FW-04-806 is synergistic with lapatinib.