Essential role of caspases in epigallocatechin-3-gallate-mediated inhibition of nuclear factor kappa B and induction of apoptosis.. Sanjay Gupta; Kedar Hastak; Farrukh Afaq; Nihal Ahmad; Hasan Mukhtar (2004) Oncogene display abstract
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.