Induction of apoptosis by L-NMMA, via FKHRL1/ROCK pathway in human gastric cancer cells / 生物医学与环境科学(英文)
Biomedical and Environmental Sciences
;
(12): 285-291, 2006.
Article
in English
| WPRIM
| ID: wpr-229687
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the apoptosis-inducing effect of endogenous nitric oxide (NO) suppression in gastric cancer cells and its mechanisms.</p><p><b>METHODS</b>Apoptosis 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.</p><p><b>RESULTS</b>Gastric 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.</p><p><b>CONCLUSIONS</b>L-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.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Pathology
/
Phosphorylation
/
Stomach Neoplasms
/
Transfection
/
Signal Transduction
/
Protein Serine-Threonine Kinases
/
Apoptosis
/
Omega-N-Methylarginine
/
Phosphatidylinositol 3-Kinases
/
Cell Line, Tumor
Limits:
Humans
Language:
English
Journal:
Biomedical and Environmental Sciences
Year:
2006
Type:
Article
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