| Ovarian cancer accounts for 3%of all cancers in women,but is over-represented in terms of cancer deaths(5%),ovarian cancer is one of the common gynecological tumor for women with the leading cause of death.As the population ages and expands,the annual number of ovarian cancer cases is expected to rise.Because signs and symptoms of ovarian cancer are frequently subtle at its early stage,70%of patients are diagnosed at the advanced stage(stage Ⅲ or stage Ⅳ).In recent years,the incidence of ovarian cancer in our country is on the rise,and tend to be younger.The most common cause of death are malnutrition and dyscrasia in ovarian cancer.So far,the situation has not changed radically.Therefore,to explore the mechanism of ovarian cancer,and inhibit the glycometabolism of ovarian cancer cell are the urgent task of cancer research.In addition to the morphological changes and function deficiency of malignanttumor cells compared with normal cells,another significant difference is the energymetabolism.Abnormal glucose metabolism is an important feature of tumor cellmetabolism.During the 1920s,Otto Warburg and colleagues made the observationthat tumors were taking up enormous amounts of glucose compared with what wasseen in the surrounding tissue.Additionally,glucose was fermented to produce lactateeven in the presence of oxygen,hence term "aerobic glycolysis".In normal differentiated cells,most of the cells are proceeding aerobic oxidative phosphorylation with high efficiency,produce 36 molecules of ATP;Aerobic glycolysis as its main form of energy metabolism to produce two molecules of ATP and generate a large amount of lactic acid under the condition of anaerobic.However,in many malignant cells,glycolysis metabolism as its main form of energy metabolism instead of mitochondrial oxidative phosphorylation even in the case of oxygen supply,this is known as the warburg effect.The discovery of the warburg effect makes glycolysis metabolic once become a research hotspot in tumor cells.With the development of molecular biology technology,cancer genes play a key role in the tumor,the research of glycolysis in tumor gradually fall.In 1990,due to positron emission computed tomography imaging technology in the application of tumor,glucose metabolism specificity phenoty of tumor cell has drawn great attention of people again.In recent years,the exploration through targeted glycolysis metabolic pathways strategy is enjoying more popularity for the treatment of malignant tumor.Warburg effect exists in most malignant tumor cells,and glycolytic activity associated with cell types and growth.Warburg effect reflects the tumor cell energy metabolism characteristics,namely the tumor cells in the way of mitochondrial oxidative phosphorylation insufficient supply of ATP,in turn,will rely more on glycolytic energy to meet the needs of its rapid growth.Compared with the mitochondria aerobic metabolism,glycolysis metabolism is a relatively inefficient production process.A molecule of glucose by aerobic metabolic pathway net generation 36 ATP molecules,and one molecule of glucose after glycolytic pathway net generation 2 molecules of ATP.So even if the mild inhibition of mitochondrial oxidative phosphorylation,glycolysis metabolic is significantly enhanced to add enough energy.Fructose phosphate kinase(Phosphofructokinase,PFK)is the most important glycolysis flow speed rate-limiting enzyme,includes two types:PFK-1 and PFK-2,these two enzymes can be respectively fructose 6-phosphoric acid catalysis for 1,6-diphosphate fructose and 2,6-diphosphate fructose.PFK-1 has three subtypes:PFK-M(muscle),PFK-L(liver)and PFK-P(platelets).Each subtype exists in the form of tetramer in the body,but the composition of tetramers are differences in different organs.In normal cells,PFK-1 are inhibited by high levels of ATP,phosphoenolpyruvate and citrate,while are activated by AMP,2,6-diphosphate fructose.Recent studies have found that the most closely relationship between several subtypes of fructokinase phosphofructokinase with malignant tumor is PFK1.In many tumor tissues,PFK1 expression is obviously enhanced,and adjusts the strength of the glycolytic pathway by changing the activity of PFK1.Nitric oxide synthase is widely distributed in the body,NOS were divided into three types,neuronal nitric oxide synthase(nNOS or NOS1),inducible nitric oxide synthase(iNOS or NOS2)and endothelial nitric oxide synthase(eNOS or NOS3)respectively.NOS1 and NOS3 were belonged to calmodulin dependent type,mainly exist in neurons,endothelial cells,and platelets,and were adjusted by concentration of calcium ions,produces low concentrations of NO,promoting the tumor growth.NOS2 was non calmodulin dependent type,widely exist in mammalian cells,its activity could not rely on intracellular calcium ion concentration.Under normal physiological conditions,the NOS2 activity is very low,only activation induced by some cytokines(e.g.,interferons,tumor necrosis factor alpha,lipopolysaccharide,interleukin-1 and inflammatory factor cytokines),persistently produce high levels of NO,play an important role in the killer cells.Recent studies have found that the expression of NOS 1 was higher in tumor tissues than in normal tissues.For example,NOS1 appeared low expression in high differentiation renal clear cell carcinoma,while high expression in poorly differentiated tumor.Besides,high expression of NOS1 also had association with microvascular invasion and metastasis,and of course predict a poor prognosis.In addition,NOS1 was also association with tumor immunology,and a study about 133 melanoma samples showed that high expression of NOS 1 could suppress the antitumor immune function.NO is a multifunction gas molecules,in mammals synthesis by NOS catalytic of L-arginine,easily through the cell membrane,the half-life is very short in the body.Physiological function of low concentration(pmol～fmol)NO in the body is mainly mediated by soluble guanylate cyclase acid receptors in cells(sGC),sGC is a vision of the heterodimer enzyme containing ferroheme.After NO combine with ferroheme,made the guanosine triphosphate(GTP)converted into guanosine 3’,5’-cyclic adenosine monophosphate(cGMP),caused the downstream signaling molecules,such as cGMP dependent protein kinase(PKG)mediated neurotransmitter conduction and inhibition of apoptosis.High concentration of NO(more than 300nM)could displayed a function of inhibiting tumor growth by mediating macrophages kill tumor effect,and inducing tumor cell apoptosis by activation of p53.Research has shown that NOS2 under the condition of inflammatory factor and so on,producing high concentration of NO inhibiting tumor cell division,induced cell apoptosis.In sum,NO promote tumor growth at most time,but suppress tumor growth at some conditions.In recent years,the relationship between NOS and tumors of the warburg effect was paid attention.Lamkin DM found that the prognosis of ovarian cancer was negative correlation with high glucose.Caneba CA confirmed that NO can promote the warburg effect of ovarian cancer.However,the specific mechanism of NOS 1 promote tumor warburg effect has not been reported.To this end,we hope to further explore the mechanism of NOS 1 regulating the Warburg effect.To provide new molecular targets for ovarian cancer prevention and the feasibility of gene therapy for ovarian cancer.METHODS1.Expression of NOS1 in ovarian cancerWe download 12 cases of gene expression profile data of ovarian cancer tissue and normal ovarian tissue in public resource GEO database,compared ovarian cancer tissue and normal ovarian tissue NOS1 gene expression level,verifying NOS1 may participate in ovarian cancer development.2.NOS1 products NO in OVCAR3 cellUsing real-time quantitative PCR(RT-PCR)and Western Blotting technology to observe NOS 1 expression in ovarian cancer cell line,and using NOS inhibitor(L-NAME),nNOS specific inhibitor(N-PLA),iNOS inhibitor(1400W)and donor(DETA-NONO)to explore the effect of NOS 1 on NO secretion in ovarian cancer cell,detecting the concentration of NO in ovarian cancer cells.3.Whether NOS1 combine with PFK1 in ovarian cancer cells Using Co-immunoprecipitation to observe NOS 1 whether can bind with PFK1 in overexpression of NOS 1 ovarian cancer cell line.4.Whether NOS1 regulate PFK1 Using NOS inhibitor(L-NAME),nNOS specific inhibitor(N-PLA),iNOS inhibitor(1400W)and overexpression of NOS1 to observe the effect of NOS1 on expression and activity of PFK1 in the ovarian cancer cells,verifying NOS1 can promote PFK1 activity in ovarian cancer cells.5.Whether NOS1 promote glycolysis of ovarian cancer cells by regulating PFK1 activity1)Using NOS inhibitor(L-NAME),nNOS specific inhibitor(N-PLA),iNOS inhibitor(1400W)and overexpression of NOS1 to observe the effect of NOS 1 on glycolysis of ovarian cancer cells,verifying NOS1 can promote the glycolysis of ovarian cancer cells.2)Observing the effect of NOS1 on glycolysis of ovarian cancer cells after SiPFK1,confirming NOS1 promote glycolysis of ovarian cancer cells by regulating PFK1 activity.6.Whether NOS1 regulate S-nitrosylation of PFK1 Using recombinant technology to observe expression of NOS1 on S-nitrosylation of PFK1,verifying NOS 1 can regulate the S-nitrosylation of PFK1.7.Whether NOS1 promote chemo-resistance of ovarian cancer cells by regulating PFK1 activity.1)Using NOS inhibitor(L-NAME),nNOS specific inhibitor(N-PLA),iNOS inhibitor(1400W)and overexpression of NOS1 to observe the effect of NOS1 on chemo-reaistance of ovarian cancer cells,verifying NOS1 can promote the chemo-resistance of ovarian cancer cells.2)Observing the effect of NOS1 on chemo-reaistance of ovarian cancer cells after SiPFKl,confirming NOS1 promote chemo-reaistance of ovarian cancer cells by regulating PFK1 activity.Statistical analysis SPSS 13.0 statistical software was used to analysis all the datas,test level at P<0.05 or P<0.01 was considered significant difference.First all datas were done F-test,and then completely random single factor analysis of One-Way ANOVA,Chi-squared test or t test of homogeneity of variance were used to compare the different expression between these groups.Experiment results1.NOS1 is high expression in human ovarian tissueNOS1 expression in ovarian cancer tissue was higher than normal ovarian tissue:we analyzed 12 cases of the ovarian cancer tissue and normal ovarian tissue microarray in GEO database,it showed NOS1、NOS2、NOS3 expression in ovarian cancer tissue were higher than normal ovarian tissue,but only the alteration of NOS 1 expression was statistically significant(P<0.05),which suggested NOS1 may be involved in ovarian cancer development.2.NOS1 is high expression in human ovarian cancer cell and secrete NONOS1 is high expression in human ovarian cancer cell lines:Real time PCR showed that mRNA levels of three NOS were detectable in all tested ovarian cancer cell lines,which NOS2 expression are comparatively relative higher than NOS1 and NOS3.Western blotting showed that protein levels of three NOS were detectable in all tested ovarian cancer cell lines.Concentration of NO is low in ovarian cancer:Griess assay showed that L-NAME、N-PLA and 1400W groups showed inhibition effect in secretion of NO when comparing with control group,L-NAME groups had statistically difference(P<0.05),among them,L-NAME showed the strongest inhibition.By contrast,DETA-NONO showed promotion effect in secretion of NO when comparing with control group(P<0.05).The concentration of NO is 15 nM in ovarian cancer.3.NOS1 can combine with PFK1 in ovarian cancer cellCo-Immunoprecipitation results showed that in the overexpression of NOS1 OVCAR3 cells,NOS1 can combine with PFK1.4.NOS1 regulates activity of PFK1Western blotting:L-NAME、N-PLA and 1400W groups showed no inhibition effect in the expression of PFK1 and PFKFB3 when comparing with control group;overexpression of NOS 1 group showed no promotion effect in the expression of PFK1 and PFKFB3 when comparing with vector group as well.Which suggested NOS1 may not regulate the expression of PFK1.PFK1 activity assay:L-NAME、N-PLA and 1400W groups showed inhibition effect in the activity of PFK1 when comparing with control group;L-NAME、N-PLA and 1400W groups had statistically difference(P<0.05),among them,N-PLA showed the strongest inhibition.By contrast,overexpression of NOS1 group showed significantly promotion effect in the expression of PFK1 when comparing with vector group(P<0.05).Which suggested NOS1 can regulate the activity of PFK1.5.NOS1 promote glycolysis of ovarian cancer cells by regulating PFK1 activity 1)NOS1 promote glycolysis of ovarian cancer cellThe effect of NOS1 expression on cell glycolysis was verified by glucose uptake,lactate secretion,ATP and NADPH assay.Similar as above,cells were incubated with NOS inhibitors(L-NAME,N-PLA,1400W)and then experiments were done.Results were showed as follows:Glucose uptake:L-NAME,N-PLA and 1400W groups showed significantly inhibition effect in the glucose uptake of OVCAR3 cell when comparing with control group;L-NAME,N-PLA and 1400W groups had statistically difference(P<0.05),among them,L-NAME showed the strongest inhibition and N-PLA showed the second strongest inhibition.By contrast,overexpression of NOS1 group showed significantly promotion effect in the glucose uptake of OVCAR3 cell when comparing with vector group(P<0.05).Lactate secretion:L-NAME,N-PLA and 1400W groups showed significantly inhibition effect in the lactate secretion of OVCAR3 cell when comparing with control group;L-NAME,N-PLA and 1400W groups had statistically difference(P<0.05),among them,L-NAME showed the strongest inhibition and N-PLA showed the second strongest inhibition.By contrast,overexpression of NOS 1 group showed significantly promotion effect in lactate secretion of OVCAR3 cell when comparing with vector group(P<0.05).ATP assay:L-NAME,N-PLA and 1400W groups showed significantly inhibition effect in the ATP production of OVCAR3 cell when comparing with control group;L-NAME groups had statistically difference(P<0.05),among them,L-NAME showed the strongest inhibition and N-PLA showed the second strongest inhibition.By contrast,overexpression of NOS1 group showed significantly promotion effect in ATP production of OVCAR3 cell when comparing with vector group(P<0.05).NADPH assay:L-NAME,N-PLA and 1400W groups showed significantly inhibition effect in the NADPH production of OVCAR3 cell when comparing with control group;L-NAME,N-PLA and 1400W groups had statistically difference(P<0.05),among them,L-NAME showed the strongest inhibition and N-PLA showed the second strongest inhibition.By contrast,overexpression of NOS 1 group showed significantly promotion effect in NADPH production of OVCAR3 cell when comparing with vector group(P<0.05).6.NOS1 regulates S-nitrosylation of PFK1Biotin-switch assay results showed that the S-nitrosylation of PFK1 was unregulated in overexpression of NOS 1 group compared with control group.7.NOS1 promote chemo-resistance of ovarian cancer cells by regulating PFK1 activity.NOS1 inhibition increases sensitivity of OVCAR3 to DDP induced cell death:CCK8 assay results showed that L-NAME,N-PLA and 1400W groups showed significantly sensitivity of OVCAR3 to DDP induced cell death when comparing with control group,L-NAME,N-PLA and 1400W groups had statistically difference(P<0.05),among them,L-NAME showed the strongest inhibition.By contrast,overexpression of NOS 1 group showed significantly sensitivity of OVCAR3 to DDP induced cell death when comparing with vector group(P<0.05).Conclusion:1.NOSs expression in ovarian cancer tissue was higher than normal ovarian tissue,but only the alteration of NOS1 expression was statistically significant.2.In vitro experiments show that NOS1 is high expression in ovarian cancer cells,promotes glycolysis by regulating the activity of PFK1 and inhibits the sensitivity of OVCAR3 to DDP induced cell death. |
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