丁酸钠与索拉非尼可能通过YAP诱导铁死亡协同抑制肝癌细胞增殖

doi:10.12122/j.issn.1673-4254.2024.07.23

摘要/Abstract

摘要：

目的探讨丁酸钠（NaB）联合索拉非尼（Sora）是否协同诱导铁死亡抑制肝癌细胞增殖及其机制。方法 CCK8、平板克隆实验和倒置光学显微镜观察探讨NaB或（和）Sora对HepG2肝癌细胞增殖活性的影响；GSH法和C11-BODIPY 581/591检测NaB或（和）Sora是否诱导HepG2细胞发生铁死亡；公共数据库TCGA分析肝癌组织与正常组织中YAP基因表达差异；Western blotting检测NaB或（和）Sora对HepG2细胞中YAP蛋白表达及其磷酸化水平的影响。结果 CCK8结果显示，2 mmol/L NaB联合Sora可显著降低HepG2肝癌细胞对Sora的IC₅₀（P<0.001），联合指数法证实Sora与NaB联合具有协同作用（CI<1），铁死亡抑制剂（Fer-1）和YAP激活剂（XMU）则可逆转NaB联合Sora对HepG2细胞的增殖抑制作用（P<0.001、P<0.001）；平板克隆实验结果显示，相较NaB或Sora单独处理，NaB联合Sora可显著抑制HepG2细胞克隆球形成（P<0.01，P<0.01）；形态学观察结果显示，NaB或Sora单独处理均可使HepG2细胞分散，形态皱缩且数量减少，而两者联合处理可加剧这一现象；GSH法和C11-BODIPY 581/591检测结果显示，较NaB或Sora单独处理，NaB联合Sora可进一步降低细胞内GSH水平（P<0.001，P<0.05）和脂质ROS水平（P<0.05，P<0.01），Fer-1和XMU可逆转NaB联合Sora对HepG2细胞内GSH水平的下调作用（P<0.001，P<0.01）和脂质ROS水平的上调作用（P<0.01，P<0.05）；TCGA分析结果显示，肝癌组织与正常组织相比YAP mRNA高表达（P<0.001）；Western blotting结果显示，较NaB或Sora单独处理，NaB联合Sora进一步下调HepG2细胞YAP蛋白表达水平（P<0.05，P<0.05），上调YAP蛋白磷酸化水平（P<0.05，P<0.01）。结论 NaB联合Sora可能通过抑制YAP诱导铁死亡，进而协同抑制肝癌细胞增殖。

关键词: 丁酸钠, 索拉非尼, 肝癌, 铁死亡, YAP, 细胞增殖

Abstract:

Objective To investigate whether sodium butyrate (NaB) and sorafenib synergistically induces ferroptosis to suppress proliferation of hepatocellular carcinoma cells and the possible underlying mechanisms. Methods CCK8 assay and colony formation assay were used to assess the effects of NaB and sorafenib, alone or in combination, on proliferation of HepG2 cells, and ferroptosis of the treated cells was detected with GSH assay and C11-BODIPY 581/591 fluorescent probe. TCGA database was used to analyze differential YAP gene expression between liver cancer and normal tissues. The effects of NaB and sorafenib on YAP and p-YAP expressions in HepG2 cells were invesitigated using Western blotting. Results NaB (2 mmol/L) significantly reduced the IC₅₀ of sorafenib in HepG2 cells, and combination index analysis confirmed the synergy between sorafenib and NaB. The ferroptosis inhibitor Fer-1 and the YAP activator (XMU) obviously reversed the growth-inhibitory effects of the combined treatment with NaB and sorafenib in HepG2 cells. The combined treatment with NaB and sorafenib, as compared with the two agents used alone, significantly inhibited colony formation of HepG2 cells, further enhanced cellular shrinkage and dispersion, and decreased intracellular GSH and lipid ROS levels, and these effects were reversed by Fer-1 and XMU. TCGA analysis revealed a higher YAP mRNA expression in liver cancer tissues than in normal liver tissues. NaB combined with sorafenib produced significantly stronger effects than the individual agents for downregulating YAP protein expression and upregulating YAP phosphorylation level in HepG2 cells. Conclusion NaB combined with sorafenib synergistically inhibit hepatocellular carcinoma cell proliferation possibly by inducing ferroptosis via inhibiting YAP expression.

Key words: sodium butyrate, sorafenib, hepatocellular carcinoma, ferroptosis, YAP, proliferation

何华星, 刘璐琳, 刘颖茵, 陈纳川, 孙素霞. 丁酸钠与索拉非尼可能通过YAP诱导铁死亡协同抑制肝癌细胞增殖[J]. 南方医科大学学报, 2024, 44(7): 1425-1430.

Huaxing HE, Lulin LIU, Yingyin LIU, Nachuan CHEN, Suxia SUN. Sodium butyrate and sorafenib synergistically inhibit hepatocellular carcinoma cells possibly by inducing ferroptosis through inhibiting YAP[J]. Journal of Southern Medical University, 2024, 44(7): 1425-1430.

图/表 3

图1 NaB联合Sora对HepG2肝癌细胞增殖活性的影响

Fig.1 Effects of NaB and sorafenib (Sora) on growth of HepG2 cells. A: CCK8 assay for assessing viability and Sora IC50 of HepG2 cells with combined treatment with NaB and Sora. B: Plate cloning experiment of the treated cells. C: Cell morphology of the treated cells (Original magnification: ×400). *P<0.05, **P<0.01 vs control group, ***P<0.001 vs control Sora; ##P<0.01.

图3 YAP基因在肝癌组织和正常组织中的表达

Fig.3 Expression of YAP gene in liver cancer tissues and normal liver tissues. ***P<0.001.

图4 NaB联合Sora对HepG2肝癌细胞中YAP、p-YAP蛋白表达水平的影响

Fig.4 Effect of NaB and Sora on expression levels of YAP and p-YAP in HepG2 cells. A, B: Expression levels of YAP and p-YAP detected using Western blotting (n=3). *P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.01.

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