MAP kinase subtypes and Akt regulate diosgenin-induced apoptosis of rheumatoid synovial cells in association with COX-2 expression and prostanoid production

Abstract

Abstract. In the present study, we investigated the signalling pathways involved in diosgenin-induced apoptosis in human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) in vitro with particular interest on Akt and MAPKs activation in relation to arachidonic acid metabolism via COX-2 pathway. MAPK activation was measured by ELISA quantification in diosgenin-treated human RA FLS. Expression of Akt and phospho-Akt was analyzed by Western blot analysis. Nuclear factor-κB (NF-κB) translocation was evaluated by electromobility shift assay. The prostanoid production (COX-2 activity) was measured by quantitative ELISA. Diosgenininduced apoptosis in the presence of MAPK or Akt inhibitors was detected by a quantitative determination of DNA fragmentation. Treatment of human RA FLS with 40 μM diosgenin caused an activation of p38 and JNK and an inhibition of ERK phosphorylation. Akt and NF-κB are potentially required for diosgenin-induced apoptosis in human RA FLS because 40 μM diosgenin abrogated Akt phosphorylation which correlated with an inhibition of NF-κB nuclear translocation. SB203580 and SP600125 (p38 and JNK inhibitors) reduced diosgenininduced DNA fragmentation whereas U0126 and LY294002 (MEK and PI3 kinase/Akt inhibitors) caused an amplification of proapoptotic effect of diosgenin. Diosgenin increased COX-2 activity resulting in PGE 2 and 6-keto-PGF 1· overproduction in human RA FLS. All MAPK inhibitors markedly reduced diosgenin-induced PGE 2 and 6-keto-PGF 1· synthesis except for SP600125 on 6-keto-PGF 1· production. These results provide, for the first time, strong evidence that a combined association implicating a MEK inhibitor (U0126) and diosgenin is the most effective in inducing very strong apoptosis with down-regulation of COX-2 expression and activity in human RA FLS.

Inhibition of human rheumatoid arthritis synovial cell survival by hecogenin and tigogenin is associated with increased apoptosis, p38 mitogen-activated protein kinase activity and upregulation of cyclooxygenase-2

“…At this stage of our study, we showed that three structurally similar saponins caused apoptosis of synovial cells but with different signalling pathways compared with results previously described for diosgenin (28) where three MAPK pathways were implicated; hecogenin or tigogenin induced apoptosis only by the p38 pathway. To confirm the implication of the p38 kinase signalling pathway in hecogenin-or tigogenininduced apoptosis in human RA FLS, we used a specific inhibitor for pretreatment before addition of saponins.…”

“…Diosgenin induced apoptosis in human RA FLS by inhibition of ERK activation but also by activation of p38 and JNK (28). However, in the absence of the 5,6-double bond in their structures, we demonstrated that only the p38 pathway was implicated in hecogenin-and tigogenin-induced apoptosis.…”

“…Recently, Yen et al (41) reported that diosgenin promoted angiogenesis in preosteoblast-like cells by a hypoxiainducible factor-1α-dependent mechanism involving the activation of p38 MAPK. Our recent study showed that, diosgenin (40 μM) inhibited ERK activation but activated p38 and JNK in human erythroleukemia (HEL) cells (22) but also in human RA FLS (28).…”

“…Recently, we described, for the first time, that diosgenin induced apoptosis in human RA FLS with COX-2 expression (16) but also inhibited the activation of survival transducing factors such as ERK while activating apoptotic transducers such as p38 and JNK (28). The aim of our study was to compare hecogenin and tigogenin [lacking the 5,6-double bond compared to diosgenin (18)] with our previous results concerning diosgenin (16,28). Hecogenin and tigogenin only differ in the presence or absence respectively of a keto group at C-12 (Fig.…”

A review on pharmacological and analytical aspects of diosgenin: a concise report

“…52 Combined association implicating a MEK inhibitor (U0126) and diosgenin is the most effective in inducing very strong apoptosis with down-regulation of COX-2 expression and activity in human RA FLS. 53 Diosgenin induced megakaryocytic differentiation of HEL cells through a combined activation of ERK and inhibition of the p38 MAPK pathways. Inhibition of ERK activation by a MEK inhibitor abrogated diosgenin-induced differentiation.…”