Effects Of Endogenous Nitric Oxide On Apoptosis Of Gastric Cancer Cells And Its Singal Transduction Pathways

BACGROUND Gastric carcinoma is a digestive canal cancer of highdeath rate in our country. It was reported that the apoptosis of gastriccarcinoma cells was relation to endogenous nitric oxide and nitric oxideplayed an important role in the survival of disseminating gastriccarcinoma cells. Recently, there have been numerous studies regardingthe forkhead transcriptional factor FKHRL1 (FOXO3a) playing a criticalrole in the regulation of apoptosis in a wide variety of cells. ROCK kinaseis an effector molecule in cell apoptosis, it was reported that the breastcarcinoma cell apoptosis was triggered by endogenous nitric oxidesuppression, which initiates signaling of FKHRL 1 to ROCK kinase as aneffector molecule. However, this signaling mechanism of that observationhave not been identified. The aim of this study is to investigate effects ofendogenous nitric oxide on apoptosis of gastric cancer cells and thesignaling mechanism of FKHRL1-induced apoptosis by nitric oxidesuppression.OBJECTIVEThis study is to investigate the apoptosis-inducing effects and signalingof transduction pathways during endogenous NO suppression in gastriccancer SGC-7901 cell line.METHODS1. The concentration of NO measured by nitrate reductase in culturemedium of SGC-7901 cells incubated (12h,24h,48h) in the presence of L-NMMA (10-50μM) and in the absence(control).2. The gastric cancer cell apoptosis was determined with terminaldeoxynucleotidyl trnasferase-mediated dUTP-fluoresce in nick endlabeling(TUNEL) method. Apoptotic index of gastric cancer cells wasdetected by flow cytometry.3. Immunofluorescence assay applied to localize the intracellularphosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1 protein.4. Expression of phosphorylated FKHRL1 (thr-32, ser-253)protein,FKHRL1 protein in gastric cancer cells was analyzed using Westernblotting.5. Two human FKHRL1 siRNA template DNA sequence weredesigned and synthesized. The annealed siRNA template was insertedinto pSliencer 3.1 plasmid. The recombinant plasmid (pSliencer 3.1FKHRLI) was transformed into DH5αBacillus coli strain and identifiedby restrictive enzyme digestion and sequence analysis. The effect ofpSliencer FKHRL1 on the FKHRL1 expression of human gastriccarcinoma SGC-7901 cells was detected by Western blotting.6. Transfection of FKHRL1-HA wild type and mutant FKHRL1-HAT32A constructs was performed by lipofectamine plus reagent.RESULTS1. Gastric cancer cells were significantly apoptotic after treatment withNG-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor) compared with the control (P＜0.01). The apoptosis of gastric cancer cellsinduced by L-NMMA was dose-dependent and time-independent. TheZ-DEVD-fmk, a caspase-3,6,7,8,10 inhibitor, did not prevent theapoptosis.2. The results of immunofluorescence assays showed that FKHRL1protein strongly expressed in the nucleus and p-FKHRL1 thr-32 proteinstrongly expressed in the cytoplasm of SGC-7901 cells when endogenousnitric oxide generation was blocked by L-NMMA, but no any change inFKHRL1 ser-253 phosphorylation. Nevertheless, ROCK protein stronglyexpressed in p-FKHRL1 thr-32-positive SGC-7901 cells.3. The wortmannin, an inhibitor of phosphoinositol-3-OH kinase(PI3K), didn't block the phosphorylation FKHRL1 thr-32 protein inducedby L-NMMA. However, Y-27632, a specific inhibitor of the proteinkinase ROCK, significantly blocked apoptosis induced by phosphorylatedFKHRL1 thr-32 (P＜0.01), which was mediated by L-NMMA.4. A significant decrease in apoptosis (P＜0.01) were observed whenHuman FKHRL1 shRNA were transfected into gastric carcinomaSGC-7901 cells, which inhibited FKHRL1 mRNA expression.5. A significant decrease in NO generation (P＜0.01) and a significantincrease in apoptosis (P＜0.01) were observed when FKHRL1-HAwild-type cells were transfected, which caused increased FKHRL1 thr-32phosphorylation. CONCLUSIONS1. Endogenous NO suppression by L-NMMA induced gastriccarcinoma cells apoptosis, and provided the supporting data for thetreatment of gastric cancer in the future.2. The mechanism of gastric carcinoma cell apoptosis triggered byL-NMMA maybe by promoting FKHRL1 expression and FKHRL1 thr-32phosphorylation and initiating signal of FKHRL1 to ROCK kinase. Thisapoptotic signaling process was PI3K/Akt as well as caspase-3independent.