Extracellular signal-regulated kinase phosphorylation due to menadione-induced arylation mediates growth inhibition of pancreas cancer cells

Background Cytotoxicity of Vitamin K3 (VK3) is indicated to have the same mechanism with oxidative stress (H2O2). In the present study, we analyzed the differences and/or similarities in the cellular responses to oxidative stress and VK3 to clarify the mechanism of growth inhibition. Methods Cell viability was determined by a test method with 3-[4, 5-dimethyl-thiazol]-2, 5-dephenyl tetrazolium bromide (MTT). Expressions of cellular proteins were evaluated by Western blot analysis. Results The IC50 was calculated to be 47.3 +/- 4.1 mu M for VK3 and 2.2 +/- 1.2 mu M for H2O2. By Western blot analysis, VK3 or H2O2 was shown to induce rapid phosphorylation of extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinases (JNKs). H2O2-induced phosphorylation of ERK and JNK was almost complete inhibited by more than 100-mu M genistein. VK3-induced JNK phosphorylation was blocked by 100-mu M genistein, but ERK phosphorylation was not inhibited completely even if 400-mu M genistein was used. H2O2-induced inhibition of cell proliferation was completely blocked by 400-mu M genistein, but the VK3 effect was reduced 72.8 +/- 5.4% by the same concentration of genistein. H2O2-induced JNK phosphorylation and ERK phosphorylation were inhibited by staurosporine, protein kinase C (PKC) inhibitor. VK3-induced JNK phosphorylation was also blocked, but ERK phosphorylation was not affected. Staurosporine had no effect on VK3- or H2O2-induced growth inhibition. Treatment with a non-thiol antioxidant agent, catalase, completely abrogated H2O2-induced JNK and ERK phosphorylation, but a thiol antioxidant, L-cystein, had no effect on phosphorylation of them. The VK3-induced JNK phosphorylation was inhibited by catalase, but not L-cystein. But ERK phosphorylation was not inhibited by catalase and was abrogated completely by the thiol antioxidant. Catalase, but not L-cystein, blocked H2O2-induced growth inhibition, and L-cystein, but not catalase, blocked VK3-induced effects on cell proliferation completely. Conclusion VK3-induced ERK phosphorylation occurs by a different mechanism from oxidative stress, and it might have an important role to induce growth inhibition.