脂氧合酶抑制剂对HepG2细胞增殖及凋亡的影响

Effect of nordihydroguaiaretic acid on the proliferation and apoptosis of HepG2 cell

目的探讨脂氧合酶抑制剂对 HeoG2细胞增殖及凋亡的影响及其相关机制。方法应用不同浓度脂氧合酶抑制剂去甲二氢愈创木酸(NDGA)处理 HepG2细胞,利用 MTT 比色法、生长曲线、细胞克隆形成观察 NDGA 对 HepG2细胞的增殖作用,H-E 染色、流式细胞术观察细胞凋亡并行细胞周期分析,细胞免疫化学或 Western 印迹技术测定 Caspase-3、增殖细胞核抗原(PCNA)、野生型(wt)P53、c-erbB-2蛋白表达水平变化。结果 NDGA 对 HepG2细胞具有明显生长抑制作用,不同浓度NDGA(50、100、150、200 μmol/L)处理 HepG2细胞72h 的抑制率分别为27.34%、41.76%、57.08%和69.17%,其 IC_(50)值为109.13μmol/L;细胞克隆形成率分别为21.63%、17.33%、12.53%、7.97%,明显低于对照组的31.70%。同时 NDGA 可诱导 HepG2细胞凋亡,与细胞周期被阻滞于 G_1/S 期有关。细胞免疫化学、Western 印迹分析显示 NDGA 对 HepG2细胞的 PCNA、c-erbB-2蛋白表达有抑制作用,并可促进 Caspase-3、wtP53蛋白表达。结论 NDGA 对 HepG2细胞有较强的细胞毒作用且以剂量、时间依赖方式抑制细胞增殖,并诱导凋亡,使细胞周期阻滞于 G_1/S 期。此作用与改变某些原癌基因、抑癌基因的蛋白表达水平有关。

Objective To investigate the effects of the lipoxygenase inhibitor, nordihydroguaiaretic acid （NDGA）, on the proliferation and apoptosis of HepG2 cell and its relevant mechanism. Methods The cells were treated with different concentrations of NDGA 50,100,150 or 200 μmaol/L for up to 72 hrs. The effects of NDGA on the growth and survival of human hepatoma cell line HepG2 were determined by MTT assay, growth curve and colonyforming assay, and the apoptosis and cell cycle were determined by flow cytometry. The protein expression of Caspase-3, wtP53 or c-erbB-2 was determined by Western blot, and proliferating cell nuclear antigen （PCNA） staining was detected by immunohistochemistry. Results NDGA had marked suppressive effect on the growth of HepG2 cell line. The inhibitory rates of the cells in concentration of 50, 100, 150 or 200 μmaol/L were 27.34%, 41.76%, 57.08% or 69.17%, respectively, in 72 hrs. NDGA had a remarkable cytotoxic effect to HepG2 with IC50 of 109.13μmaol/L. Colony-forming rate of HepG2 cells treated with NDGA in concentration of 50, 100, 150 or 200 μmaol/L were 21.63%, 17.33%, 12.53% and 7.97% respectively, which were much lower than that of control group（31.70%）. At the same time, NDGA could induce the apoptosis of HepG2 with the cell cycle arrested on G1/S phase. In immunohistochemistry and Western blot analysis, NDGA could inhibit the protein expression of PCNA, e-erbB-2 in human hepatoma HepG2 cells, but the protein expression of Caspase-3 and wtP53 in HepG2 cells were up-regulated. Conclusion NDGA could inhibit proliferation of HepG2 cells in a time- and dose-dependent manner, and induce the apoptosis with cell cycle arrested on G1/S phase, which may be associated with the levels of the protein expression of some oncogenes and tumor suppressor genes.