足叶苦素抑制胰岛素样生长因子I受体表达对肝癌细胞增殖与运动的影响

Effects of inhibited IGF-IR expression on proliferation and apoptosis of human hepatocellular carcinoma cell lines

目的观察足叶苦素（PPP）抑制胰岛素样生长因子I受体（IGF-IR）后对肝癌细胞增殖和运动的影响。方法体外培养肝细胞L02和肝癌细胞（Bel-7404、Bei-7402、HepG2和Huh_7），以Westemblot分析IGF-IR表达，以磺酰罗丹明B法分析细胞活性；在PPP抑制IGF-IR后，以流式细胞术分析细胞周期，以ArmexinV-FITC法分析细胞凋亡；在z-VAD-FMK抑制caspases后，以均质发光法检测caspase-3／7活性；划痕试验分析细胞运动能力。对数据进行析因设计方差分析，采用t检验或one-wayANOVA法进行组间比较。结果肝细胞L02中几乎检测不到IGF-IR，各肝癌细胞中IGF-IR呈不同程度表达。PPP抑制肝癌细胞增殖呈时间和剂量依赖性，经1．0μmol／LPPP处理HepG2细胞24h，G1、S期和G2／M期细胞比例分别为2．1％±0．4％、11．0％±0．7％和87．1％±0．6％，且划痕不愈合Icaspase-3／7活陛显著增加（t=11．83，P〈0．01）；HepG2细胞凋亡率为16．4％±0．4％，明显高于对照组的5．8％±0．2％（f=14．05，P〈0．01）；z-VAD-FMK抑制casgases表达，HepG2细胞凋亡率为11．3％±0．7％，明显高于对照组的5．8％±0．2％（f=11．83，P〈0．01）。结论IGF-IR表达与肝癌细胞增殖、运动和凋亡相关，可能是肝癌分子靶向治疗的有效靶点。

Objective To investigate the therapeutic value of inhibiting the expression of insulin- like growth factor-I receptor （IGF-IR） using picropodophyllin （PPP） by studying the effects on proliferative and metastatic potentials of human hepatocellular carcinoma （HCC） using an in vitro cultured cell system. Methods IGF-IR expression in human HCC cell lines （Bel-7404, Bel-7402, HepG2, and Huh-7） and human hepatocytes （L02） was assessed＇at baseline （pre-treatment） and after PPP treatment by western blotting. Changes in cell cycle were analyzed by flow cytometry and in cell viability by sulforhodamine B staining. Early apoptosis was detecte0/by annexin-V/FITC and propidium iodide double-staining assay. Caspase-3/7 / activity was suppressed by/z-VAD-FMK and analyzed by homogeneous luminescence assay. Effects on cell motility were tested by v/ound-scratch test. Between-group differences were assessed by t-test or one-way analysis of variance. Results IGF-IR was markedly up-regulated in all HCC cell lines （vs. non-hepatoma hepatocytes）. HCC cells with PPP-inhibited IGF-IR showed time-dependent decreases in cell motility and After treatment with PPP for 24 hours, the proportion of HCC cells in G1 phase was 2.1% -4- 0.4%, viability. in S phase was 11.0% - 0.7%, and ＇in G2/M phase was 87.1% - 0.6%, and no healing was observed in the wound-scratch assay. The PPP treatment induced cell apoptosis, as evidenced by enhanced caspase-3/7activity; the proportion of annexin-V＋/PI cells was significantly higher in the HepG2 cells than in the non- hepatoma hepatocytes （16.4%± 0.4% vs. 5.8% ±0.2%, t = 14.05,P 〈 0.01）. After z-VAD-FMK treatment, the apoptosis rate was significantly higher in the HepG2 cells than in the non-hepatoma hepatocytes （ 11.3% ±0.7% vs. 5.8%± 0.2%, t = 11.83, P 〈 0.01）. Conclusion IGF-IR is associated with proliferation, cell motility, and apoptosis of HCC ceils, and may be a promising molecular target for HCC.