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  • Title: Induction of apoptosis by L-NMMA, via FKHRL1/ROCK pathway in human gastric cancer cells.
    Author: Wang YZ, Feng ZQ.
    Journal: Biomed Environ Sci; 2006 Aug; 19(4):285-91. PubMed ID: 17044646.
    Abstract:
    OBJECTIVE: To investigate the apoptosis-inducing effect of endogenous nitric oxide (NO) suppression in gastric cancer cells and its mechanisms. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
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