Essential role of caspases in epigallocatechin-3-gallate-mediated inhibition of nuclear factor kappa B and induction of apoptosis.
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.
Gupta
Sanjay
S
Department of Urology, Case Western Reserve University, Cleveland, OH 44106, USA.
Hastak
Kedar
K
Afaq
Farrukh
F
Ahmad
Nihal
N
Mukhtar
Hasan
H
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002294:Carcinoma, Squamous Cell; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006801:Humans; D008297:Male; D008954:Models, Biological; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000187:drug effects; D011471:Prostatic Neoplasms; D017434:Protein Structure, Tertiary / Q000187:drug effects; D013997:Time Factors; D014158:Transcription, Genetic
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D016328:NF-kappa B; D009842:Oligopeptides; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D002392:Catechin; D004247:DNA; C045651:epigallocatechin gallate; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases