Detection of Nε-(hexanoyl)lysine in the tropomyosin 1 protein in N-methyl-N'-nitro-N-nitrosoguanidine-induced rat gastric cancer cells

Abstract

Nε-(Hexanoyl)lysine, formed by the reaction of lysine with n-6 lipid hydroperoxide, is a lipid peroxidation marker during the initial stage of oxidative stress. The aim of the present study is to indentify Nε-(hexanoyl)lysine-modified proteins in neoplastic transformed gastric mucosal cells by N-methyl-N'-nitro-N-nitrosoguanidine, and to compare the levels of these proteins between gastric mucosal cells and normal gastric cells. Much greater fluorescence of 2-[6-(4'-hydroxy)phenoxyl-3H-xanthen-3-on-9-yl]benzoic acid, an index of the intracellular levels of reactive oxygen species, was observed for gastric mucosal cells compared to normal gastric cells. Nε-(Hexanoyl)lysine-modified proteins were detected by SDS-PAGE or two-dimensional electrophoresis and Western blotting using anti-Nε-(hexanoyl)lysine polyclonal antibody, and a protein band of between 30–40 kDa was clearly increased in gastric mucosal cells compared to normal gastric cells. Two Nε-(hexanoyl)lysine-modified protein spots in gastric mucosal cells were identified as the tropomyosin 1 protein by mass spectrometry using a MASCOT search. The existence of Nε-(hexanoyl)lysine modification in tropomyosin 1 was confirmed by Western blotting of SDS-PAGE-separated or two-dimensional electrophoresis-separated proteins as well as by the immunoprecipitation with anti-tropomyosin 1 antibody. These data indicate that Nε-(hexanoyl)lysine modification of tropomyosin 1 may be related to neoplastic transformation by N-methyl-N'-nitro-N-nitrosoguanidine in gastric epithelial cells.

Mitochondrial reactive oxygen species accelerate gastric cancer cell invasion

“…The data suggested the participation of Tropomyosin 1 and NOX-derived superoxide anion for forming invadopodia, which show an obvious amount in RGK-1 but few in RGM-1. ( 6 , 7 , 32 – 34 ) These factors should be impaired by expressed MnSOD. We confirmed the activation of cytoskeletal-associated protein due to clarify the mechanism of cellular movements through a mitochondrial ROS.…”

Acetic acid induces cell death: An in vitro study using normal rat gastric mucosal cell line and rat and human gastric cancer and mesothelioma cell lines

“…Rat gastric epithelial cell line (RGM-1, Riken Kagaku, Kyoto, Japan), rat gastric carcinoma cell line (RGK-1), human gastric cancer cell line (KATO III, Riken Kagaku), and human mesothelioma cell lines (ACC-MESO1: RIKEN BioResource Center, Tsukuba, Japan, and MSTO-211H: ATCC, Manassas, VA, USA) were used. [9][10][11][12] Both RGM-1 and RGK-1 cells were maintained in DMEM/F-12 (Wako, Osaka, Japan) medium, whereas KATO III cells and mesothelioma cells were maintained in RPMI 1640 medium (Wako). All media were supplemented with heatinactivated 10% fetal bovine serum (Biowest, Nuaille, France), and 100 U/mL of penicillin and 100 U/mL of streptomycin at 37°C under 5% CO2 in air.…”

Reactive oxygen species involved cancer cellular specific 5-aminolevulinic acid uptake in gastric epithelial cells

“…Western blotting analysis was performed according to previous reports. ( 25 , 29 , 30 ) Briefly, 15 µl of cell lysed solution (10 µg) from each cells were prepared with NuPAGE LDS Sample buffer containing Sample Reducing Agent (Invitrogen Japan K.K., Tokyo, Japan) and boiled at 70°C for 10 min. For SDS-polyaclylamidegel electrophoresis (SDS-PAGE), the cell lysed solutions were added into well of NuPAGE ® Novex ® 12% Bis-Tris gels.…”