Critical role of extracellular signal-regulated kinase phosphorylation on menadione (vitamin K3) induced growth inhibition.
BACKGROUND
Although it is widely known that the extracellular signal-regulated kinase (ERK) pathway stimulates cell growth and protects cells from death, recent findings have proposed a proapoptotic action of ERK phosphorylation. Because the authors found that vitamin K3 (VK3) was a potent growth inhibitor and an inducer for ERK phosphorylation through a specific pathway in the stomach cancer cell line, the critical role of ERK phosphorylation in VK(3)-mediated growth inhibitory effect was examined.


METHODS
The fluorochrome Hoechst 33258 assay (Hoechst AG [now Aventis] Frankfort, Germany) was used for counting cells (excitation at 360 nm; emission at 460 nm). For two-dimensional electrophoresis, cells were dissolved in urea standard buffer and applied first to isoelectronic focusing gels. Cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% polyacrylamide gels. To examine the phosphorylation of receptors, cell lysates were immunoprecipitated with receptor antibody.


RESULTS
VK3 induced phosphorylation of hepatocyte growth factor receptor (c-met), epidermal growth factor receptor (EGFR), or external signal-regulated kinase (ERK), which increased progressively to a maximum level at 30 minutes, in a dose-dependent manner and occurred at growth inhibitory concentrations. VK(3)-mediated growth inhibition and protein tyrosine phosphorylation were nullified completely by glutathione or L-cysteine but not by nonthiol antioxidants, thus suggesting that sulfhydryl arylation might have been involved in VK(3)-mediated action. The phosphorylation of EGFR and c-met by VK(3) appeared to be functional, because these were coimmunoprecipitated with growth factor receptor-bound protein 2 (Grb2) and SOS1 antibody. Epidermal growth factor (EGF) or hepatocyte growth factor (HGF) stimulated increase of cyclin D1 protein after 12 hours and increased DNA content after 3 days in culture. In addition, U0126, which is a potent inhibitor for ERK phosphorylation, antagonized increase of cyclin D1, thus suggesting that EGF- or HGF-mediated ERK phosphorylation might have played an essential role for cell growth. By contrast, ERK phosphorylation by VK3 was more prolonged and intense than the signal induced by the growth factors. U0126 reduced ERK phosphorylation and prevented growth inhibition by VK3. Two-dimensional gels showed VK(3)-induced additional phospho-ERK spots, compared with those obtained from growth factors. This extra spot was completely antagonized by U0126.


CONCLUSIONS
VK(3)-induced growth inhibition and protein tyrosine phosphorylation were mediated by the sulfhydryl arylation system. The tyrosine phosphorylation of EGFR or c-met by VK3 activated the Ras signaling pathway. The overexpressed ERK phosphorylation by VK3 seemed to originate from additional spots on two-dimensional gels, which played a critical role in VK(3)-induced growth inhibitory action despite the fact that ERK phosphorylation by growth factors had had an essential association with cell growth.
Second Department of Surgery, Gifu University School of Medicine, Gifu City, Japan.
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Osada
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Saji
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Osada
D017209:Apoptosis; D002455:Cell Division; D066246:ErbB Receptors / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000473:pathology*; D014407:Tumor Cells, Cultured; D014812:Vitamin K / Q000494:pharmacology*
D014812:Vitamin K; D066246:ErbB Receptors; D019859:Proto-Oncogene Proteins c-met; D020928:Mitogen-Activated Protein Kinases