Induction of apoptosis by L-NMMA, via FKHRL1/ROCK pathway in human gastric cancer cells.
- Author:
Yong-Zhong WANG
1
;
Zhen-Qing FENG
Author Information
- Publication Type:Journal Article
- MeSH: Antineoplastic Agents; toxicity; Apoptosis; Caspase 3; metabolism; Cell Line, Tumor; Enzyme Inhibitors; toxicity; Forkhead Box Protein O3; Forkhead Transcription Factors; analysis; metabolism; Humans; Intracellular Signaling Peptides and Proteins; metabolism; Nitric Oxide; antagonists & inhibitors; Phosphatidylinositol 3-Kinases; metabolism; Phosphorylation; Protein-Serine-Threonine Kinases; metabolism; Proto-Oncogene Proteins c-akt; metabolism; Signal Transduction; Stomach Neoplasms; enzymology; metabolism; pathology; Transfection; omega-N-Methylarginine; toxicity; rho-Associated Kinases
- From: Biomedical and Environmental Sciences 2006;19(4):285-291
- CountryChina
- Language:English
-
Abstract:
OBJECTIVETo investigate the apoptosis-inducing effect of endogenous nitric oxide (NO) suppression in gastric cancer cells and its mechanisms.
METHODSApoptosis of gastric cancer cells was detected by flow cytometry. Expression of phosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1 in gastric cancer cells was analyzed using Western blotting. Immunofluorescence assay was performed to localize the intracellular phosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1. Transfection of FKHRL1-HA wild type and mutant FKHRL1-HA T32A constructs was performed by lipofectamine plus reagent. NO generation was determined by Griess reaction.
RESULTSGastric cancer cells were significantly apoptotic after treatment with N(G)-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), compared with the control (P<0.01). The apoptosis of gastric cancer cells induced by L-NMMA was dose-dependent and time-independent. However, the Z-DEVD-fmk, a caspase-3, 6, 7, 8, 10 inhibitor, did not prevent the apoptosis. The immunofluorescence assays showed that FKHRL1 protein was strongly expressed in the nucleu and p-FKHRL1 thr-32 protein was strongly expressed in the cytoplasm of SGC-7901 cells when endogenous nitric oxide generation was blocked by L-NMMA, but no change in FKHRL1 ser-253 phosphorylation. Nevertheless, ROCK protein was strongly expressed in p-FKHRL1 thr-32-positive SGC-7901 cells. The wortmannin, an inhibitor of phosphoinositol-3-OH kinase (PI3K), did not block the phosphorylated FKHRL1 thr-32 protein induced by L-NMMA. However, Y-27632, a specific inhibitor of the protein kinase ROCK, significantly blocked apoptosis induced by phosphorylated FKHRL1 thr-32 (P < 0.01), which was mediated by L-NMMA. A significant decrease in NO generation (P < 0.01) and a significant increase in apoptosis (P < 0.01) were observed when FKHRL1-HA wild-type cells were transfected, which caused increased FKHRL1 thr-32 phosphorylation.
CONCLUSIONSL-NMMA triggers gastric carcinoma cell apoptosis, possibly by promoting FKHRL1 thr-32 phosphorylation and initiating signal of FKHRL1 to ROCK kinase. This apoptotic signaling process is PI3K/Akt as well as caspase-3 independent.
