Abstract: Objective To investigate the effects of methionine （Met） deficiency on human hepatocellular carcinoma （HCC） cell lines HepG2 and Huh7 growth, and whether Met deficiency synergizes with regorafenib in tumor growth. Methods IC50 of regorafenib in normal complete medium and methionine-deficient complete medium were detected by CCK8 assay. Apoptosis and cycle flow experiment were employed to identify the apoptosis induction and cell cycle effect of regorafenib on these two cell lines under different medium conditions. Clone formation assay was conducted to explore HCC cell proliferation. Changes of mitochondrial membrane potential were detected by JC-1 fluorescence probe. Liposome peroxidation assay was used to measure lipid peroxidation levels. Results This research revealed that methionine deficiency decreased the IC50 of regorafenib in human hepatocellular carcinoma （HCC） cell lines HepG2 and Huh7. Methionine deficiency synergized with regorafenib in inhibiting tumor proliferation,inducing apoptosis and decreasing mitochondrial membrane potential; while HepG2 cell cycle had no significant effect. Both of them increased but had no synergistic effect on the lipid peroxidation level in HepG2. Conclusion Methionine restriction and regorafenib played a synergistic role in inhibiting cell proliferation, which provided a theoretical basis for clinical treatment on reducing the dose of regorafenib, increasing drug sensitivity and improving patients' quality of life in clinic practice.

Key words: hepatocellular carcinoma, methionine, regorafenib, drug susceptibility

摘要： 目的 探索甲硫氨酸（Met）缺乏对人肝癌（HCC）细胞系HepG2和Huh7的生长影响和甲硫氨酸限制作用是否协同瑞戈非尼抑制肿瘤生长。方法 CCK8实验检测瑞戈非尼分别在正常培养基和甲硫氨酸缺乏的培养基的IC50;凋亡实验、周期实验检测瑞戈非尼在不同培养基条件下对两种肝癌细胞株的凋亡诱导情况和对细胞周期影响;克隆形成实验评估细胞增殖情况;JC-1荧光探针实验检测细胞线粒体膜电位变化;脂质体过氧化实验检测胞内脂质过氧化反应。结果 甲硫氨酸缺乏可降低瑞戈非尼在人肝癌细胞株HepG2和Huh7的IC50值;甲硫氨酸缺乏协同瑞戈非尼抑制肿瘤增殖、诱导凋亡、降低线粒体膜电位;而在细胞周期方面对HepG2影响无显著差异;瑞戈非尼和甲硫氨酸限制均可增加HepG2细胞膜脂质过氧化水平,但并无协同作用。结论 甲硫氨酸限制和瑞戈非尼在抑制细胞增殖、诱导凋亡等方面起到协同作用;为临床减少瑞戈非尼用药剂量和增加药物敏感性、改善患者生活质量提供体外实验的理论依据。

关键词: 肝癌, 甲硫氨酸, 瑞戈非尼, 药物敏感性