木犀草素联合顺铂诱导人类肝癌HepG2细胞凋亡及对C-jun氨基末端激酶信号通路的影响

The apoptosis of HepG2 cells and effect of C-jun N-terminal kinase signaling pathway induced by luteolin combined with cisplatin

目的 探讨木犀草素作为凋亡增敏剂增强顺铂诱导人类肝癌HepG2细胞凋亡的作用.方法 将木犀草素与顺铂单独或联合处理HepG2细胞.采用Hoeehst染色试剂盒检测细胞凋亡,分光光度法检测Caspause-3活性,乳酸脱氢酶（LDH）释放法检测细胞死亡情况,蛋白质印迹法检测磷酸化C-jun氨基末端激酶（JNK）及总JNK蛋白的表达情况.结果 木犀草素能显著增强顺铂诱导的HepG2细胞Caspase-3活性以及细胞凋亡.不同浓度的木犀草素（0～ 20 μmol/L）与顺铂（10 μg/ml）联用后,联合组的细胞死亡率较各浓度单药处理组增加更为明显,最高达47.66％,各浓度单药处理和联合应用之间的细胞死亡率差异有统计学意义（F=535.48,P＜ 0.01）,联合处理的疗效优于单药应用.顺铂（10 μg/ml）组、木犀草素（20 μmol/L）单药处理组以及顺铂（10 μg/ml）与木犀草素（20μmol/L）联合处理组的Caspase-3活性分别是阴性对照组的1.94倍、1.74倍和8.12倍,且联合处理后HepG2细胞内JNK信号通路得到持续活化.结论 木犀草素进一步增强了顺铂介导的HepG2细胞内JNK信号通路的活化,从而增强顺铂诱导的人类肝癌HepG2细胞的凋亡.木犀草素有望作为凋亡增敏剂与抗肿瘤药物联合应用.

Objective To investigate the increased cytotoxicity induced by luteolin and cisplatin co- treatment and validate its mechanism. Methods HepG2 cells were pretreated with luteolin for 1 hour or remained untreated and followed by exposure to cisplatin. Cells were stained with Hoechst, and Caspase-3 activity was detected by spectrophotometry. Cell death was detected by LDH release assay. Phosphated C-jun N-terminal kinase （JNK） and total JNK1 were detected by Western blot. Results Luteolin significantly enhanced Caspase-3 activity and HepG2 cells apoptosis by cisplatin. The cell death rates by co-treatment with different concentration of luteolin （0-20 μmol/L） and cisplatin （10 p,g/ml） were much higher than cisplatin or luteolin alone treatment. Combined treatment of luteolin （20/xmol/L） with cisplatin （10 p^g/ml） killed 47,66 % of HepG2 cells. There were significant differences on cell death between combination group and single reagent group （F = 535.48, P 〈 0.01）. Combination group showed better efficacy than single reagent group. The Caspase-3 activity of HepG2 cells treated by luteolin （20 p^mol/L）, cisplatin （10 p^g/ml） or luteolin （20 p^mol/L） plus cisplatin （10 p,g/ml） were 1.94, 1.74 and 8.12 times of negative control group respectively. Combination treatment induced sustained activation of JNK signaling pathway. Conclusions Enhancing cisplatin-induced JNK activation by luteolin leads to increased cytotoxicity in HepG2 cells. The combination of luteolin and cisplatin is an effect apporch for improving the anticancer value of cisplatin, which has imphcations in cancer prevention and therapy.