Gupta Sanjay, Hastak Kedar, Afaq Farrukh, Ahmad Nihal, Mukhtar Hasan
Department of Urology, Case Western Reserve University, Cleveland, OH 44106, USA.
Oncogene. 2004 Apr 1;23(14):2507-22. doi: 10.1038/sj.onc.1207353.
Green tea constituent (-) epigallocatechin-3-gallate (EGCG) has shown remarkable cancer-preventive and some cancer-therapeutic effects. This is partially because of its ability to induce apoptosis in cancer cells without affecting normal cells. Previous studies from our laboratory have shown the involvement of NF-kappa B pathway in EGCG-mediated cell-cycle deregulation and apoptosis of human epidermoid carcinoma A431 cells. Here we show the essential role of caspases in EGCG-mediated inhibition of NF-kappa B and its subsequent apoptosis. Treatment of A431 cells with EGCG (10-40 microg/ml) resulted in dose-dependent inhibition of NF-kappa B/p65, induction of DNA breaks, cleavage of poly(ADP-ribose) polymerase (PARP) and morphological changes consistent with apoptosis. EGCG treatment of cells also resulted in significant activation of caspases, as shown by the dose- and time-dependent increase in DEVDase activity, and protein expression of caspase-3, -8 and -9. EGCG-mediated caspase activation induces proteolytic cleavage of NF-kappa B/p65 subunit, leading to the loss of transactivation domains, and driving the cells towards apoptosis. EGCG-mediated induction of apoptosis was significantly blocked by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VAD-FMK), and moderately blocked by the specific caspase-3 inhibitor Z-DEVD-FMK. Further, pretreatment of cells with Z-VAD-FMK was found to suppress the cleavage of NF-kappa B/p65 subunit, thereby increasing nuclear translocation, DNA binding and transcriptional activity, thus protecting the cells from EGCG-induced apoptosis. Taken together, these studies for the first time demonstrate that EGCG-mediated activation of caspases is critical, at least in part, for inhibition of NF-kappa B and subsequent apoptosis.
绿茶成分(-)表没食子儿茶素-3-没食子酸酯(EGCG)已显示出显著的防癌和某些抗癌治疗作用。部分原因在于它能够诱导癌细胞凋亡而不影响正常细胞。我们实验室之前的研究表明,NF-κB通路参与了EGCG介导的人表皮样癌A431细胞的细胞周期失调和凋亡过程。在此,我们展示了半胱天冬酶在EGCG介导的NF-κB抑制及其随后的凋亡过程中的关键作用。用EGCG(10 - 40微克/毫升)处理A431细胞导致NF-κB/p65的剂量依赖性抑制、DNA断裂的诱导、聚(ADP-核糖)聚合酶(PARP)的裂解以及与凋亡一致的形态学变化。EGCG处理细胞还导致半胱天冬酶的显著激活,表现为DEVD酶活性以及半胱天冬酶-3、-8和-9的蛋白表达呈剂量和时间依赖性增加。EGCG介导的半胱天冬酶激活诱导NF-κB/p65亚基的蛋白水解裂解,导致反式激活结构域的丧失,并促使细胞走向凋亡。EGCG介导的凋亡诱导被半胱天冬酶抑制剂N-苄氧羰基-Val-Ala-Asp(OMe)-氟甲基酮(Z-VAD-FMK)显著阻断,并被特异性半胱天冬酶-3抑制剂Z-DEVD-FMK适度阻断。此外,发现用Z-VAD-FMK对细胞进行预处理可抑制NF-κB/p65亚基的裂解,从而增加核转位、DNA结合和转录活性,从而保护细胞免受EGCG诱导的凋亡。综上所述,这些研究首次证明EGCG介导的半胱天冬酶激活至少部分对于抑制NF-κB和随后的凋亡至关重要。