Agarwal Chapla, Singh Rana P, Agarwal Rajesh
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Carcinogenesis. 2002 Nov;23(11):1869-76. doi: 10.1093/carcin/23.11.1869.
Grape seed extract (GSE), rich in the bioflavonoids commonly known as procyanidins, is one of the most commonly consumed dietary supplements in the United States because of its several health benefits. Epidemiological studies show that many prostate cancer (PCA) patients use herbal extracts as dietary supplements in addition to their prescription drugs. Accordingly, in recent years, we have focused our attention on assessing the efficacy of GSE against PCA. Our studies showed that GSE inhibits growth and induces apoptotic death of human PCA cells in culture and in nude mice. Here, we performed detailed studies to define the molecular mechanism of GSE-induced apoptosis in advanced human PCA DU145 cells. GSE treatment of cells at various doses (50-200 micro g/ml) for 12-72 h resulted in a moderate to strong apoptotic death in a dose- and time-dependent manner. In the studies assessing the apoptotic-signaling pathway induced by GSE, we observed an increase in cleaved fragments of caspases 3, 7 and 9 as well as PARP in GSE-treated cells after 48 and 72 h of treatment. Pre-treatment of cells with general caspases inhibitor, z-Val-Ala-Asp(OMe)-FMK or caspase 3-like proteases inhibitor [z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-FMK], almost completely (approximately 90%) inhibited the GSE-induced apoptotic cell death. In a later case, GSE-induced caspase-3 activity was completely inhibited. Selective caspase 9 inhibitor [z-Leu-Glu(OMe)-His-Asp(OMe)-FMK] showed only partial inhibition of GSE-induced apoptosis whereas GSE-induced protease activity of caspase 9 was completely inhibited. Upstream of caspase cascade, GSE showed disappearance of mitochondrial membrane potential and an increase in cytochrome c release in cytosol. Together, these results suggest that GSE possibly causes mitochondrial damage leading to cytochrome c release in cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic death of human PCA DU145 cells. Furthermore, GSE-caused caspase 3-mediated apoptosis also involves other pathway(s) including caspase 9 activation.
葡萄籽提取物（GSE）富含通常被称为原花青素的生物类黄酮，由于其多种健康益处，它是美国最常用的膳食补充剂之一。流行病学研究表明，许多前列腺癌（PCA）患者除了服用处方药外，还将草药提取物用作膳食补充剂。因此，近年来，我们将注意力集中在评估GSE对PCA的疗效上。我们的研究表明，GSE在培养的人PCA细胞和裸鼠中均能抑制生长并诱导凋亡性死亡。在此，我们进行了详细研究以确定GSE诱导晚期人PCA DU145细胞凋亡的分子机制。用不同剂量（50 - 200μg/ml）的GSE处理细胞12 - 72小时，以剂量和时间依赖性方式导致中度至强烈的凋亡性死亡。在评估GSE诱导的凋亡信号通路的研究中，我们观察到在处理48小时和72小时后，GSE处理的细胞中半胱天冬酶3、7和9以及PARP的裂解片段增加。用通用的半胱天冬酶抑制剂z-Val-Ala-Asp(OMe)-FMK或半胱天冬酶3样蛋白酶抑制剂[z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-FMK]对细胞进行预处理，几乎完全（约90%）抑制了GSE诱导的凋亡细胞死亡。在后一种情况下，GSE诱导的半胱天冬酶-3活性被完全抑制。选择性半胱天冬酶9抑制剂[z-Leu-Glu(OMe)-His-Asp(OMe)-FMK]仅部分抑制GSE诱导的凋亡，而GSE诱导的半胱天冬酶9的蛋白酶活性被完全抑制。在半胱天冬酶级联反应的上游，GSE显示线粒体膜电位消失，细胞溶质中细胞色素c释放增加。总之，这些结果表明，GSE可能导致线粒体损伤，导致细胞溶质中细胞色素c释放，并激活半胱天冬酶，导致PARP裂解，从而使人PCA DU145细胞发生凋亡性死亡。此外，GSE引起的半胱天冬酶3介导的凋亡还涉及其他途径，包括半胱天冬酶9的激活。
