【作者】 王永忠；

【导师】 冯振卿； 曹友清；

【作者基本信息】 南京医科大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 背景胃癌是我国常见的消化道肿瘤，病死率高。研究证实胃癌细胞中内源性一氧化氮与胃癌细胞生存或凋亡有关。许多研究表明新发现的叉头转录因子FKHRL1(FOXO3a)在许多细胞凋亡过程中起着十分重要的作用。ROCK蛋白酶是细胞凋亡过程中的效应分子，有研究表明乳腺癌细胞中抑制内源性一氧化氮产生能诱导癌细胞凋亡，其信号转导机制中有FKHRL1及ROCK蛋白酶的参与。然而一氧化氮(nitric oxide，NO)与ROCK蛋白酶及叉头转录因子FKHRL1之间相互作用的机制还不完全清楚。本研究应用一氧化氮合酶抑制剂NG-甲基-L-精氨酸(L-NMMA)抑制SGC-7901胃癌细胞内源性一氧化氮产生观察胃癌细胞凋亡，并进一步探讨SGC-7901胃癌细胞凋亡的信号转导机制。目的探讨L-NMMA对SGC-7901胃癌细胞凋亡的影响，并进一步探讨其信号转导的可能机制。方法1．应用硝酸酶还原法检测不同时间(12h、24h、48h)、不同浓度(10μM、20μM、30μM、40μM、50μM)一氧化氮抑制剂NG-甲基-L-精氨酸(N~G-Monomethyl一L-arginine，L-NMMA)处理组及对照组中SGC-7901胃癌细胞培养液中NO浓度。2．应用流式细胞仪及缺口末端标记技术(TUNEL)方法检测不同时间及不同浓度L-NMMA处理组及对照组中SGC-7901胃癌细胞凋亡。3．应用免疫荧光方法检测不同时间及不同浓度L-NMMA处理组及对照组中SGC-7901胃癌细胞内FKHRL1蛋白、p-FKHRL1(thr-32，ser-253)蛋白及ROCK蛋白表达。4．应用Western blot方法检测不同时间及不同浓度L-NMMA处理组及对照组中SGC-7901胃癌细胞中FKHRL1、p-FKHRL1(thr-32，ser-253)及ROCK表达。5．应用RNA干扰技术抑制SGC-7901胃癌细胞中FKHRL1表达，检测L-NMMA处理组及对照组中SGC-7901胃癌细胞凋亡。6．应用转基因方法转染野生型FKHRL1-HA和突变型FKHRL1-HA T32A基因，检测SGC-7901胃癌细胞凋亡。结果1．一氧化氮合酶抑制剂L-NMMA处理组与对照组比较，SGC-7901胃癌细胞凋亡明显增多(P＜0.01)。而且SGC-7901胃癌细胞凋亡与L-NMMA浓度有显著相关性(P＜0.01)，与处理时间无相关性(P＞0.05)。半胱天冬蛋白酶(caspase)3、6、7、8、10抑制剂Z-DEVD-fmk不能阻断SGC-7901胃癌细胞凋亡。2．免疫荧光检测显示L-NMMA处理组SGC-7901胃癌细胞胞核中叉头转录因子FKHRL1呈强阳性表达，p-FKHRL1(thr-32)胞浆内也呈强阳性表达，而p-FKHRL1(ser-253)在胞浆内表达没有明显变化。p-FKHRL1(thr-32)强阳性组SGC-7901胃癌细胞胞浆内ROCK蛋白也呈强阳性表达，统计学分析两者有相关性(P＜0.01)。3．ROCK蛋白激酶特异性抑制剂Y-27632明显抑制L-NMMA诱导SGC-7901胃癌细胞凋亡(P＜0.01)。磷酸肌醇化激酶(PI3K)特异性抑制剂wortmannin不能阻断L-NMMA处理组FKHRL1(thr-32)苏氨酸磷酸化。4．用RNA干扰方法抑制SGC-7901胃癌细胞中叉头转录因子FKHRL1表达，L-NMMA诱导SGC-7901胃癌细胞凋亡明显减少(P＜0.01)。5．转染野生型FKHRL1基因(FKHRL1-HA)，SGC-7901胃癌细胞凋亡明显增加(P＜0.01)，而且内源性NO产生明显减少(P＜0.01)。结论1．内源性一氧化氮抑制剂L-NMMA能诱导胃癌细胞凋亡，可为今后临床应用L-NMMA治疗胃癌提供实验依据。2．L-NMMA诱导胃癌细胞凋亡的可能机制是抑制内源性一氧化氮产生从而诱导叉头转录因子FKHRL1的表达及FKHRL1苏氨酸磷酸化(thr-32)，p-FKHRL1(thr-32)激活ROCK蛋白激酶引起胃癌细胞凋亡。在这凋亡信号转导过程中，没有半胱天冬蛋白酶(caspase一3)的参与以及非PI3K／Akt磷酸化途径。更多还原

【Abstract】 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.更多还原