Non-steroidal anti-inflammatory drugs suppress the ERK signaling pathway via block of Ras/c-Raf interaction and activation of MAP kinase phosphatases.
Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit cancer cell growth, induce apoptosis and decrease tumor metastasis. We have previously reported that a NSAID NS398 repressed the expression of matrix metalloproteinase-2 (MMP-2) via inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway. In this study, we investigate the underlying mechanism of this inhibition. In vitro kinase assay indicated that NS398 could not directly inhibit c-Raf, MEK1 and ERK enzymatic activity. We found that NS398 increased the inhibitory phosphorylation of Ser259 in c-Raf, attenuated membrane recruitment of c-Raf and inhibited Ras/c-Raf interaction to attenuate activation of this kinase. This is a general effect for NSAIDs because sulindac sulfide, aspirin and indomethacin also inhibited the binding of c-Raf to Ras. Immunofluorescent staining verified that NS398 reduced the serum-induced membrane recruitment of c-Raf in cells. However, overexpression of constitutively active c-Raf only partly reversed NS398-induced inhibition of MMP-2 expression. Interestingly, we found that NS398 up-regulated the expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) and MKP-3. Block of MKP activity by sodium orthovanadate also partly counteracted the inhibitory effect of NS398. Overexpression of constitutively active c-Raf and treatment of sodium orthovanadate together completely reversed the inhibition of MMP-2 by NS398. Taken together, we conclude that NS398 and other NSAIDs act via inhibition of Ras/c-Raf interaction and up-regulation of MKPs to suppress the ERK-mediated signaling.
Pan
Mei-Ren
MR
Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
Chang
Hui-Chiu
HC
Hung
Wen-Chun
WC
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology; D054638:Dual Specificity Phosphatase 1 / Q000378:metabolism; D054642:Dual Specificity Phosphatase 6 / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D020778:Matrix Metalloproteinase 2 / Q000235:genetics*; D054639:Mitogen-Activated Protein Kinase Phosphatases / Q000378:metabolism*; D009578:Nitrobenzenes / Q000494:pharmacology*; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology*; D014638:Vanadates / Q000494:pharmacology
D000894:Anti-Inflammatory Agents, Non-Steroidal; D000970:Antineoplastic Agents; D009578:Nitrobenzenes; D013449:Sulfonamides; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D014638:Vanadates; D012694:Serine; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D048369:MAP Kinase Kinase 1; D054639:Mitogen-Activated Protein Kinase Phosphatases; D054638:Dual Specificity Phosphatase 1; D054642:Dual Specificity Phosphatase 6; D020778:Matrix Metalloproteinase 2; D016283:Proto-Oncogene Proteins p21(ras)