活性氧在顺铂致HepG_2凋亡中的作用

Roles of reactive oxygen species in HepG_2 cell apoptosis induced by CDDP

目的 利用Acivicin阻断GGT的功能 ,观察其单独对HepG2 凋亡及联用顺铂后对HepG2 的凋亡、细胞内ROS产生的影响。方法 细胞毒性测定采用MTT法。Acivicin和顺铂分别或联合处理HepG2 细胞。采用DCFH DA能被ROS作用发光的特性。通过流式细胞仪测定细胞内ROS的变化。凋亡检测采用细胞凋亡原位检测法 (TUNEL法 )。结果 MTT法测定HepG2 IC50 值Acivicin为 1 4mmol L ,顺铂为 67μmol L。Acivicin 1 4mmol L和顺铂 67μmol L以及Acivicin( 1 4mmol L) +顺铂 ( 67μmol L)HepG2 2 4h细胞内的ROS显著升高 ,尤其是在当与顺铂联用时HepG2 细胞内的ROS升高就更加明显。TUNEL法测定Acivicin( 1 4mmol L)在 2 4、48、72h致HepG2 的凋亡率分别分 ( 16 3± 3 5 ) %、( 2 7 9± 4 3 ) %、( 4 7 2±3 0 ) %。与对照组相比差异显著 (P <0 0 5 )。顺铂 ( 67μmol L)HepG2 2 4h的凋亡率为 ( 73 4± 1 5 ) %,Acivicin( 1 4mmol L)+顺铂 ( 67μmol L)HepG2 2 4h的凋亡率为 ( 94 7± 0 5 ) %,较单用显著增加 (P <0 0 1)。结论 顺铂致HepG2 凋亡的机制之一是增加细胞内ROS的产生。Acivicin抑制GGT ,干扰GSH代谢 ,可致HepG2 细胞内的ROS升高 ,使细胞凋亡 ,并增强顺铂的细胞毒性。

Objective To investigate the effects of acivicin on HepG 2 cell apoptosis and the effects of acivcin with cis diaminedichloroplatinum (CDDP) on HepG 2 cell apoptosis and on production of intracellular ROS. Methods HepG 2 cells were treated with acivcin, CDDP, and acivicin combined with CDDP. The cytotoxicity was measured by MTT assay. Flow cytometry was used to detect the intracellular ROS, and the rate of apoptosis was determined by TUNEL assay. Results The concentration of acivicin to cause HepG 2 IC 50 was 1.4 mmol/L, and CDDP was 67 μmol/L. Significant increases in intracellular ROS content were observed in HepG 2 cells 24 h after treatment with acivicin (1.4 mmol/L), CDDP (67 μmol/L), or acivicin (1 4 mmol/L) plus CDDP (67 μmol/L), especially with acivicin (1 4 mmol/L) plus CDDP (67 μmol/L). HepG 2 cell apoptotic rates induced by acivicin (1 4 mmol/L) at 24, 48, and 72 h were (16 3±3 5), (27 9±4 3), (47 2±3 0), respectively, and a significant difference was observed compared to that of control group ( P <0.01). HepG 2 apoptotic rate induced by CDDP (67 μmol/L) at 24 h was (73 4±1 5), while that by acivicin (1.4 mmol/L) plus CDDP (67 μmol/L) at 24 h was (94.7±0.5). The increase caused by combination of acivcin and CDDP was more significant than that by either of them ( P <0.01). Conclusions One of the mechanisms of HepG 2 apoptosis induced by CDDP is to increase intracellular ROS. Acivicin inhibits GGT and interferes GSH metabolism, which may lead to an increase in intracellular ROS level of HepG 2 cell, resulting in apoptosis and enhanced CDDP cytotoxicity.