Objective: To explore the molecular mechanism of sorafenib resistance in hepatoma cells and identify for new targets to reverse drug resistance.
Methods: THP-1 cells were induced into M2 tumor-associated macrophages (M2-TAMs) in vitro and identified by immunofluorescence. SMMC-7721 cells were co-cultured with M2-TAMs with or without sorafenib treatment. CCK-8 assay was used to observe the inhibitory effect of sorafenib on the cell proliferation. Annexin V/PI double staining and protein immunoblotting were used to assess the effect of sorafenib on the proliferation, apoptosis and the expressions of apoptosis-related proteins and autophagy-related protein in SMMC-7721 cells co-cultured with M2-TAMs in the presence or absence of the autophagy inhibitor chloroquine (CQ).
Results: The IC50 of sorafenib at 48 h was 2.25 μmol/L in SMMC-7721 cells cultured alone, and increased to 4.72 μmol/L in the cells co-cultured with M2-TAMs. Compared with the cells cultured alone, the co-cultured SMMC-7721 cells showed significantly reduced apoptosis rate in response to sorafenib (P < 0.01) and significantly increased expression of Bcl-2 and Bcl-2/Bax ratio (P < 0.05) with also increased LC3-II/LC3-I ratio (P < 0.001) and lowered expression of p62 (P < 0.05), suggesting a significantly enhanced level of autophagy. CQ treatment significantly inhibited the proliferation of the co-cultured SMMC-7721 cells (P < 0.05), increased the cell apoptosis (P < 0.05) and reduced the Bcl-2/Bax ratio (P < 0.01).
Conclusions: M2-TAMs can attenuate the inhibitory effect of sorafenib on the proliferation of hepatoma cells by increasing the level of autophagy, suggesting a new strategy for reversing sorafenib resistance induced by the tumor microenvironment by inhibiting autophagy.
目的: 探讨索拉菲尼治疗肝癌耐药的分子机制，寻找逆转耐药新靶点。
方法: 体外诱导THP-1细胞建立M2型肿瘤相关巨噬细胞（TAMs），免疫荧光鉴定M2型TAMs，将SMMC-7721细胞分为空白对照组、M2-TAMs共培养组、索拉菲尼组以及索拉菲尼与M2-TAMs共培养联合处理组，CCK-8法检测M2-TAMs对索拉菲尼抑制SMMC-7721增殖的影响；Annexin V/PI双染法、蛋白质免疫印迹法检测使用自噬抑制剂氯喹处理前后M2-TAMs对索拉菲尼促SMMC-7721细胞增殖、细胞凋亡的影响及细胞凋亡相关蛋白和自噬相关蛋白的表达量变化。
结果: 索拉菲尼对肝癌细胞SMMC-7721作用48 h的IC50为2.25 μmol/L，索拉菲尼对与M2-TAMs共培养（共培养给药组）48 h的SMMC-7721的IC50为4.72 μmol/L。共培养给药组与单独给药组相比细胞凋亡率明显下降（P < 0.01），Bcl-2表达明显增加，Bcl-2/Bax比值增加（P < 0.05），而自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ的比值升高（P < 0.001），p62蛋白表达下调（P < 0.05），自噬水平明显增强。氯喹处理后能明显抑制共培养给药组的细胞增殖（P < 0.05），促进细胞凋亡（P < 0.05），下调Bcl-2/Bax比值（P < 0.01）。
结论: M2-TAMs可通过促进SMMC-7721细胞自噬，降低索拉菲尼对肝癌细胞的增殖抑制作用，因此抑制自噬可能是逆转肿瘤微环境诱导索拉菲尼治疗耐药的新靶点。
Keywords: autophagy; drug resistance; sorafenib; tumor-associated macrophages.