Wogonin and fisetin induction of apoptosis through activation of caspase 3 cascade and alternative expression of p21 protein in hepatocellular carcinoma cells SK-HEP-1
Wogonin and fisetin induction of apoptosis through activation of caspase 3 cascade and alternative expression of p21 protein in hepatocellular carcinoma cells SK-HEP-1
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DOI：
10.1007/s00204-002-0346-6
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发表时间：
2002-06-01
影响因子：
6.1
通讯作者：
Yang, LL
中科院分区：
文献类型：
--
作者：
Chen, YC;Shen, SC;Yang, LL
汉黄芩素和非瑟酮均为黄酮类化合物，广泛分布于植物中。我们最近的研究表明，在七种结构相关的黄酮类化合物中，汉黄芩素和非瑟酮对人早幼粒白血病细胞HL-60显示出最强的凋亡诱导活性。在本研究中，我们进行了分子研究，以评估汉黄芩素和非瑟酮对肝癌细胞SK-HEP-1的凋亡作用。汉黄芩素和非瑟酮对SK-HEP-1细胞的细胞毒作用呈剂量依赖性，并伴有DNA断裂。Giemsa染色显微镜观察显示，汉黄芩素和非瑟酮在80 μ M的剂量下诱导SK-HEP-1细胞肿胀和凋亡小体的出现，这是凋亡的特征。此外，流式细胞术分析显示在汉黄芩素和非瑟酮处理的SK-HEP-1细胞中亚二倍体细胞增加。这些数据表明汉黄芩素和非瑟酮是SK-HEP-1细胞凋亡的有效诱导剂。用诱导凋亡浓度的汉黄芩素或非瑟酮处理引起半胱天冬酶3/CPP 32活性的诱导，但不诱导半胱天冬酶1活性。此外，半胱天冬酶3抑制剂Ac-DEVD-CHO，而不是半胱天冬酶I抑制剂Ac-YVAD-CHO，逆转汉黄芩素和非瑟酮对SK-HEP-1细胞的细胞毒性作用。此外，在汉黄芩素和非瑟酮处理的SK-HEP-1细胞中检测到包括聚（ADPribose）聚合酶（PARP）和D4-GDI蛋白的半胱天冬酶3底物的切割，以及半胱天冬酶3原蛋白的减少。p53蛋白的增加与汉黄芩素和非瑟酮诱导的细胞凋亡有关;然而，p53控制的基因p21（Waf/Cip-1）仅在汉黄芩素（非瑟酮）处理的SK-HEP-1细胞中诱导。血清饥饿可提高SK-HEP-1细胞p21（Waf/Cip-1）蛋白表达，增强汉黄芩素（非fiseitn）诱导SK-HEP-1细胞凋亡的活性。本研究从分子水平证明汉黄芩素和非瑟酮通过激活caspase 3级联反应、诱导p53蛋白和p21（Waf/Cip-1）蛋白的交替表达诱导肝癌细胞SK-HEP-1凋亡，从而发挥细胞毒作用。
Wogonin and fisetin are flavonoids, which are widely distributed in plants. Our recent study demonstrated that, among seven structurally related flavonoids, wogonin and fisetin showed the most potent apoptosis-inducing activities in human promyeloleukemic cells HL-60. In the present investigation, we performed molecular studies to assess the apoptotic effects of wogonin and fisetin on hepatocellular carcinoma cells SK-HEP-1. Both wogonin and fisetin showed dose-dependent cytotoxic effects on SK-HEP-1 cells, accompanied by DNA fragmentation. Microscopic observation tinder Giemsa staining showed that wogonin and fisetin, at the dose of 80 muM, induced cellular swelling and the appearance of apoptotic bodies, characteristics of apoptosis, in SK-HEP-1 cells. Furthermore, flow cytometry analysis showed an increase of hypodiploid cells in wogonin- and fisetin-treated SK-HEP-1 cells. These data demonstrated that wogonin and fisetin were effective inducers of apoptosis in SK-HEP-1 cells. Treatment with an apoptosis-inducing concentration of wogonin or fisetin caused induction of caspase 3/CPP32 activity, but not of caspase 1 activity. In addition, a caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase I inhibitor Ac-YVAD-CHO, reversed the cytotoxic effects of wogonin and fisetin on SK-HEP-1 cells. Further, cleavage of caspase 3 substrates including poly(ADPribose) polymerase (PARP) and D4-GDI protein, and decrease of pro-caspase 3 protein were detected in wogonin- and fisetin-treated SK-HEP-1 cells. Increase of p53 protein was associated with wogonin- and fisetin-induced apoptosis; however, a p53-controlled gene, p21(Waf/Cip-1), was only induced in wogonin- (not fisetin-) treated SK-HEP-1 cells. Serum starvation elevated p21(Waf/Cip-1) protein expression, and enhanced the apoptotic induction activity of wogonin (not fiseitn) in SK-HEP-1 cells. Our study has provided molecular evidence to demonstrate that wogonin and fisetin had effective cytotoxic effects through apoptosis induction in hepatocellular carcinoma cells SK-HEP-1; activation of caspase 3 cascade, induction of p53 protein and alternative expression of p21(Waf/Cip-1) protein were involved.