Tan Xiang-Wen, Xia Hong, Xu Jin-Hua, Cao Jian-Guo
Laboratory of Medicine Engineering, Medical College, Hunan Normal University, Changsha 410006, Hunan Province, China.
World J Gastroenterol. 2009 May 14;15(18):2234-9. doi: 10.3748/wjg.15.2234.
To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin (ADFMChR) on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved.
HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry (FCM) using propidium iodide (PI) fluorescence staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor gamma (PPARgamma), NF-kappaB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting.
MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dose-dependent manner, with little effect on growth of L-02 cells, and when IC(50) was measured as 8.45 micromol/L and 191.55 micromol/L respectively, the potency of ADFMChR to HepG2 cells, was found to be similar to 5-fluorouracil (5-FU, IC(50) was 9.27 micromol/L). The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 (191.55/8.45), higher than 5-FU (SI was 7.05 (65.37/9.27). FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 micromol/L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were significantly higher when treated with 30.0 micromol/L ADFMChR than when treated with 30.0 micromol/L ChR (16.0%) (P < 0.05) and were similar to those obtained with 30.0 micromol/L 5-FU (41.0%). DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 micromol/L ADFMChR for 48 h and 72 h resulted in typical DNA ladders which could be reversed by 10.00 micromol/L GW9662, a blocker of PPARgamma. Western blotting analysis revealed that after 24 h of treatment with 3.0, 10.0, 30.0 micromol/L ADFMChR, PPARgamma and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-kappaB expression decreased; however, pre-incubation with 10.0 micromol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 micromol/L ADFMChR on PPARgamma and NF-kappaB protein expression in HepG2 cells.
ADFMChR induces apoptosis of HepG2 cell lines by activating PPARgamma, inhibiting protein expression of Bcl-2 and NF-kappaB, and increasing Bax expression.
研究5-烯丙基-7-亚甲基二氟甲基白杨素（ADFMChR）对人肝癌HepG2细胞系凋亡的影响及其相关分子机制。
体外培养HepG2细胞和L-02细胞，采用MTT法检测ADFMChR对其增殖的抑制作用。采用碘化丙啶（PI）荧光染色，通过流式细胞术（FCM）检测HepG2细胞的凋亡情况。通过DNA琼脂糖凝胶电泳观察DNA梯状条带。采用蛋白质免疫印迹法分析ADFMChR对HepG2细胞中过氧化物酶体增殖物激活受体γ（PPARγ）、核因子κB（NF-κB）、Bcl-2和Bax蛋白表达的影响。
MTT法显示，ADFMChR能显著抑制HepG2细胞的增殖，呈剂量依赖性，对L-02细胞生长影响较小，测得其对HepG2细胞的半数抑制浓度（IC50）为8.45 μmol/L，对L-02细胞的IC50为191.55 μmol/L，ADFMChR对HepG2细胞的抑制效力与5-氟尿嘧啶（5-FU，IC50为9.27 μmol/L）相似。ADFMChR对HepG2细胞的细胞毒性选择性指数为22.67（191.55/8.45），高于5-FU（选择性指数为7.05（65.37/9.27））。PI染色的FCM结果显示，用3.0、10.0和30.0 μmol/L ADFMChR处理HepG2细胞48 h后的凋亡率分别为5.79%、9.29%和37.8%，30.0 μmol/L ADFMChR处理组的凋亡率显著高于30.0 μmol/L白杨素（ChR）处理组（16.0%）（P < 0.05），且与30.0 μmol/L 5-FU处理组（41.0%）相似。DNA琼脂糖凝胶电泳显示，用10.0 μmol/L ADFMChR处理HepG2细胞48 h和72 h后出现典型的DNA梯状条带，10.00 μmol/L GW9662（一种PPARγ阻滞剂）可使其逆转。蛋白质免疫印迹分析显示，用3.0、10.0、30.0 μmol/L ADFMChR处理24 h后，HepG2细胞中PPARγ和Bax蛋白表达增加，而Bcl-2和NF-κB表达降低；然而，预先用10.0 μmol/L GW9662孵育可有效拮抗并减弱3.0、30.0 μmol/L ADFMChR对HepG2细胞中PPARγ和NF-κB蛋白表达的调节作用。
ADFMChR通过激活PPARγ、抑制Bcl-2和NF-κB蛋白表达以及增加Bax表达诱导HepG2细胞系凋亡。
