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 (H(2)O(2)). 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 microM for VK3 and 2.2 +/- 1.2 microM for H(2)O(2). By Western blot analysis, VK3 or H(2)O(2) was shown to induce rapid phosphorylation of extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinases (JNKs). H(2)O(2)-induced phosphorylation of ERK and JNK was almost complete inhibited by more than 100-muM genistein. VK3-induced JNK phosphorylation was blocked by 100-microM genistein, but ERK phosphorylation was not inhibited completely even if 400-microM genistein was used. H(2)O(2)-induced inhibition of cell proliferation was completely blocked by 400-microM genistein, but the VK3 effect was reduced 72.8 +/- 5.4% by the same concentration of genistein. H(2)O(2)-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 H(2)O(2)-induced growth inhibition. Treatment with a non-thiol antioxidant agent, catalase, completely abrogated H(2)O(2)-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 H(2)O(2)-induced growth inhibition, and L: -cystein, but not catalase, blocked VK3-induced effects on cell proliferation completely.


CONCLUSIONS
VK3-induced ERK phosphorylation occurs by a different mechanism from oxidative stress, and it might have an important role to induce growth inhibition.
Osada
Shinji
S
Surgical Oncology, Gifu University School of Medicine, 1-1 Yanagido, Gifu city, 501-1194, Japan. sting@gifu-u.ac.jp
Sakashita
Fumio
F
Hosono
Yosiki
Y
Nonaka
Kenichi
K
Tokuyama
Yasuharu
Y
Tanaka
Hidenori
H
Sasaki
Yoshiyuki
Y
Tomita
Hiroyuki
H
Komori
Shuji
S
Matsui
Satoshi
S
Takahashi
Takao
T
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D010190:Pancreatic Neoplasms / Q000201:enzymology / Q000473:pathology; D010766:Phosphorylation; D051381:Rats; D024483:Vitamin K 3 / Q000494:pharmacology*
D000975:Antioxidants; D024483:Vitamin K 3; D006861:Hydrogen Peroxide; D048049:Extracellular Signal-Regulated MAP Kinases