Genistein在调节肝癌HepG2细胞Fas基因表达及诱导细胞凋亡中的作用

目的: 探讨三磷酸肌醇( IP3) 和Fas基因表达变化在genistein诱导肝癌细胞凋亡中的作用。方法:以肝癌HepG2 细胞培养72h为对照,以20,40.60,80μmol/ L genistein作用于 HepG2 细胞72h和60μmol/L genistein 作用于 HepG2 细胞6h、12 h、24 h、48 h、72 h，应用同位素试剂盒IP3 - [3H] Birtrak检测细胞IP3 含量，RT-PCR分析Fas mRNA表达，Western blotting 分析细胞Fas蛋白表达，流式细胞仪检测细胞凋亡率。结果: 各浓度的genistein作用于肝癌HepG2 细胞72 h，IP3 含量显著高于对照组[（17.7±1.3）pmol/106cells、（11.2±0.9）pmol/106cells、（4.9±0.5）pmol/106cells、（4.8±0.3）pmol/106cells vs （29.4±0.5）pmol/106cells]，Fas mRNA表达显著高于对照组[RI（灰度与面积之积与β-actin的相对强度）0.25±0.002、0.30±0.01、0.28±0.04、0.30±0.03 vs 0.19±0.01]，Fas蛋白表达显著高于对照组[RI1.08±0.01、 1.11±0.02、1.05±0.06、1.03±0.01 vs 0.17±0.01]，细胞凋亡率显著高于对照组[（10.1±0.9）%、（18.7±1.6）%、（28.7±2.5）%、（27.9±2.0）% vs （2.6±0.1）%]；60 μmol/L genistein 作用于肝癌HepG2 细胞6h、12 h、24 h、48 h、72 h，6h后IP3 含量显著高于对照组[（22.6±0.9）pmol/ 106cells、 (12.0±1.4) pmol/ 106cells、(7.5 ±0.8) pmol/ 106cells、(5.6 ±0.5) pmol/ 106cells、(4.3 ±0.6) pmol/ 106cells vs (29.2 ±0.6) pmol/ 106cells。P < 0.01）；12h后Fas mRNA表达显著高于对照组[RI0.24±0.01、0.24±0.01、0.24±0.02、0.30±0.001 vs 0.20±0.01]，6h后Fas蛋白表达显著高于对照组[RI 0.55±0.08、 1.01±0.03、1.62±0.03、1.46±0.09 、1.56±0.04 vs 0.18±0.01]，24 h后各时相细胞凋亡率为显著高于对照组[(7.4 ±0.5) %、(20.5 ±2.0) %、(30.7 ±1.6) % vs (2.6 ±0.1) %。P < 0.01]。结论: Genistein能减少IP3 生成,上调Fas基因表达,诱导肝癌细胞凋亡。

Genistein regulates Fas gene expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To probe into the role of 1 , 4 , 5 - trisphosphate inositol ( IP3) and Fas gene expression in apoptosis of HepG2 cells induced by genistein. METHODS: HepG2 cells were treated with different concentrations of genistein including 20,40.60,80μmol/ L for 72h and treated with 60μmol/ L genistein for 6h,12h, 24h, 48h and 72h. IP3 , Fas mRNA, Fas protein and apoptosis rate were assayed by IP3-[3H] Birtrak assay ,RT-PCR, Western blotting and flow cytometry , respectively. RESULTS: HepG2 cells incubated with each concentration of genistein for 72 h，IP3 continent was lower than that of control[（17.7±1.3）pmol/106cells,（11.2±0.9）pmol/106cells,（4.9±0.5）pmol/106cells,（4.8±0.3）pmol/106cells vs （29.4±0.5）pmol/106cells]，Fas mRNA expression was higher than that of control[RI which was the gray degree multiply area of Fas / the gray degree multiply area of β- actin was 0.25±0.002,0.30±0.01,0.28±0.04,0.30±0.03 vs 0.19±0.01]，Fas protein expression was higher than that of control[RI 1.08±0.01, 1.11±0.02, 1.05±0.06, 1.03±0.01 vs 0.17±0.01]，the apoptosis rate was higher than that of congtrol[（11.2±1.1）%,（15.5±1.1）%,（26.8±2.5）%,（27.1±1.5）% vs （2.6±0.1）%]； HepG2 cellls were incubated with 60μmol/L genistein for 6h, 12h, 24h, 48h, 72 h， IP3 continent was lower than that of control[（22.6±0.9）pmol/ 106cells, (12.0±1.4) pmol/ 106cells, (7.5 ±0.8) pmol/ 106cells, (5.6 ±0.5) pmol/ 106cells, (4.3 ±0.6) pmol/ 106cells vs (29.2 ±0.6) pmol/ 106cells。P < 0.01]；Fas mRNA expression was higher than that of control incubated with 60 μmol/L genistein for above 12h [RI 0.24±0.01、0.24±0.01、0.24±0.02、0.30±0.001 vs 0.20±0.01]，Fas protein expression was higher than that of control incubated with 60 μmol/L genistein for above 6h[RI 0.55±0.08、 1.01±0.03、1.62±0.03、1.46±0.09 、1.56±0.04 vs 0.18±0.01]， the apoptosis rate was significantly higher than that in control incubated with 60 μmol/L genistein for 24 h , 48 h and 72 h ([(7.4 ±0.5) %、(20.5 ±2.0) %、(30.7 ±1.6) % vs (2.6 ±0.1) %。P <0.01]. CONCLUSION: Genistein can induce apoptosis in HepG2 cells by reducing IP3 production and Fas gene expression.