Down-regulation of myeloid cell leukemia 1 by epigallocatechin-3-gallate sensitizes rheumatoid arthritis synovial fibroblasts to tumor necrosis factor alpha-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 alpha (TNFalpha)-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 TNFalpha-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-kappaB.


RESULTS
In RA synovial fibroblasts, EGCG (5-50 microM) inhibited constitutive and TNFalpha-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 TNFalpha-induced Akt and NF-kappaB pathways. Caspase 3 activation by EGCG also suppressed RA synovial fibroblast growth, and this effect was mimicked by Akt and NF-kappaB inhibitors. Interestingly, Mcl-1 degradation by EGCG sensitized RA synovial fibroblasts to TNFalpha-induced PARP cleavage and apoptotic cell death.


CONCLUSIONS
Our findings indicate that EGCG itself induces apoptosis and further sensitizes RA synovial fibroblasts to TNFalpha-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.
Ahmed
Salahuddin
S
Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109-2200, USA. salahmed@umich.edu
Silverman
Matthew D
MD
Marotte
Hubert
H
Kwan
Kevin
K
Matuszczak
Natalie
N
Koch
Alisa E
AE
D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000503:physiopathology; D015153:Blotting, Western; D053148:Caspase 3 / Q000032:analysis; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002470:Cell Survival; D002478:Cells, Cultured; D015536:Down-Regulation / Q000502:physiology; D005347:Fibroblasts / Q000502:physiology; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D010003:Osteoarthritis / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis*; D013583:Synovial Membrane / Q000166:cytology / Q000503:physiopathology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016588:Anticarcinogenic Agents; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D002392:Catechin; C045651:epigallocatechin gallate; D053148:Caspase 3