Critical role of extracellular signal-regulated kinase phosphorylation on menadione (Vitamin K-3) induced growth inhibition

BACKGROUND. Although it is widely known that the extracellular signal-regul ated kinase (ERK) pathway stimulates cell growth and protects cells from de ath, recent findings have proposed a proapoptotic action of ERK phosphoryla tion. Because the authors found that vitamin K-3 (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 VK3-mediated growth inhibitory effect was examined. METHODS. The fluorochrome Hoechst 33258 assay (Hoechst AG [now Aventis] Fra nkfort, Germany] was used for counting cells (excitation at 360 nm; emissio n 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 electr ophoresis (SDS-PAGE) using 10% polyacrylamide gels. To examine the phosphor ylation of receptors, cell lysates were immunoprecipitated with receptor an tibody. RESULTS. VK3 induced phosphorylation of hepatocyte growth factor receptor ( c-met), epidermal growth factor receptor (EGFR), or external signal-regulat ed kinase (ERK), which increased progressively to a maximum level at 30 min utes, in a dose-dependent manner and occurred at growth inhibitory concentr ations. VK3-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 in volved in VK3-mediated action. The phosphorylation of EGFR and c-met by VK3 appeared to be functional, because these were coimmunoprecipitated with gr owth factor receptor-bound protein 2 (Grb2) and SOS1 antibody. Epidermal gr owth 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 ERI; phosphory lation, 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 inte nse than the signal induced by the growth factors. U0126 reduced ERK phosph orylation and prevented growth inhibition by VK3. Two-dimensional gels show ed VK3-induced additional phospho-ERK spots, compared with those obtained f rom growth factors. This extra spot was completely antagonized by U0126. CONCLUSIONS, VK3-induced growth inhibition and protein tyrosine phosphoryla tion were mediated by the sulfhydryl arylation system. The tyrosine phospho rylation of EGFR or c-met by VK3, activated the Ras signaling pathway. The overexpressed ERK phosphorylation by VK3 seemed to originate from additiona l spots on two-dimensional gels, which played a critical role in VK3-induce d growth inhibitory action despite the fact that ERK phosphorylation by gro wth factors had had an essential association with cell growth. Cancer 2001; 91:1156-65. (C) 2001 American Cancer Society.