芹菜素通过组成性激活JNK敏化TRAIL诱导HeLa细胞凋亡

Sensitization of human cervical cancer HeLa cells to TRAIL-induced apoptosis by apigenin via constitutively activating JNK

目的研究芹菜素(API)与重组人可溶性肿瘤坏死因子相关凋亡诱导配体(TR AIL)合用对人宫颈癌H eLa细胞凋亡的影响,并探讨其作用机制是否涉及组成性激活JN K。方法体外培养人宫颈癌H eLa细胞。碘化丙啶(PI)染色流式细胞术(FC M)定量分析亚二倍体细胞(sub-G 1)百分率。比色法测定细胞C aspase-3活性。D N A琼脂糖凝胶电泳观察细胞D N A梯形条带。W estern Bloting检测细胞蛋白的表达。结果 API(20μm ol/L)和TR AIL(20ng/m L)以及两者合用作用48 h的人宫颈癌H eLa细胞系sub-G 1百分率分别是(3.56±0.20)%、(6.69±0.40)%和(59.87±4.20)%;H eLa细胞C aspase-3的活性分别是培养基对照组的1.4、1.5和39倍。D N A琼脂糖凝胶电泳显示:20μm ol/LAPI与20 ng/m L的TR AIL联合处理,展示出典型D N A梯形条带图谱,C aspase-3抑制剂zD EVD-fm k能够消除API和TR AIL合用促细胞凋亡作用。API以浓度依耐方式增高H eLa细胞JN K蛋白磷酸化水平。JN K特异性抑制剂SP600125能阻断API激活JN K,并抑制API增强TR AIL促细胞凋亡作用。结论芹菜素敏化TR AIL诱导细胞凋亡作用与其组成性激活JN K有关。

【Objective】 To investigate whether apigenin（API） enhance apoptosis induced by recombinant human soluble TNF-related apoptosis-inducing ligand（TRAIL） through constitutively activating JNK in human cervical cancer HeLa cell line.【Methods】 Human cervical cancer HeLa cells were treated with API and /or TRAIL in vitro.The characteristic features of cell apoptosis were examined by PI fluorescence staining combined with flow cytometry,assay of caspase-3 activity using colorimetric method,and DNA agarose gel electrophoresis.The expressions of protein were analyzed by Western blot.【Results】 Flow cytometry（FCM） analysis with PI staining indicated that the percentage of sub-G1 cells in human cervical cancer HeLa cells treated with API（20 μmol/L） or TRAIL（20 ng/mL） alone,and with combination for 48h were（3.5±0.20）%,（6.69±0.40）%,and（59.87±4.20）% respectively.Caspase-3 activity in HeLa cells was 1.4,1.5,39 fold in comparison with medium group.Co-treatment with API[0] and TRAIL increased the apoptosis of Hela cells as shown in apoptotic DNA ladder experience,which was ameliorated by zDEVD-fmk,the inhibitor of caspase-3.Western blot analysis indicated that API elevated the phosphorylation level of JNK in a time-dependent manner,which was blocked by the specific inhibitor of JNK,SP600125.Furthermore,SP600125 resulted in decrease the percentage of sub-G1 cells from（58.50±6.10）% to（15.70±2.46）%（P 0.01） in combination of TRAIL（20 ng/mL） and API（20 μmol/L）.【Conclusion】 The sensitization of HeLa cells to TRAIL induced apoptosis by API is associated with constitutively activating JNK.