Down-Regulation of Myeloid Cell Leukemia 1 by Epigallocatechin-3-Gallate Sensitizes Rheumatoid Arthritis Synovial Fibroblasts to Tumor Necrosis Factor α-Induced Apoptosis

Objective. Overexpression of the antiapoptotic protein myeloid cell leukemia 1 (Mcl-1) in rheumatoid arthritis (RA) synovial fibroblasts is a major cause of their resistance to tumor necrosis factor a (TNF alpha)-induced apoptosis. This study was undertaken to evaluate the efficacy of epigallocatechin-3-gallate (EGCG) in down-regulating Mcl-1 expression and its mechanism of RA synovial fibroblast sensitization to TNF alpha-induced apoptosis. Methods. EGCG effects on cultured RA synovial fibroblast cell morphology, proliferation, and viability over 72 hours were determined by microscopy and a fluorescent cell enumeration assay. Caspase 3 activity was determined by a colorimetric assay. Western blotting was used to evaluate the apoptosis mediators poly(ADP-ribose) polymerase (PARP), Mcl-1, Bcl-2, Akt, and nuclear translocation of NF-kappa B. Results. In RA synovial fibroblasts, EGCG (5-50 mu M) inhibited constitutive and TNF alpha-induced Mcl-1 protein expression in a concentration- and time-dependent manner (P < 0.05). Importantly, EGCG specifically abrogated Mcl-1 expression in RA synovial fibroblasts and affected Mcl-1 expression to a lesser extent in osteoarthritis and normal synovial fibroblasts or endothelial cells. Inhibition of Mcl-1 by EGCG triggered caspase 3 activity in RA synovial fibroblasts, which was mediated via down-regulation of the TNF alpha-induced Akt and NF-kappa B pathways. Caspase 3 activation by EGCG also suppressed RA synovial fibroblast growth, and this effect was mimicked by Akt and NF-kappa B inhibitors. Interestingly, Mcl-1 degradation by EGCG sensitized RA synovial fibroblasts to TNF alpha-induced PARP cleavage and apoptotic cell death. Conclusion. Our findings indicate that EGCG itself induces apoptosis and further sensitizes RA synovial fibroblasts to TNF alpha-induced apoptosis by specifically blocking Mcl-1 expression and, hence, may be of promising adjunct therapeutic value in regulating the invasive growth of synovial fibroblasts in RA.