Nitric Oxide Synthetase Induced Epithelial-mesenchymal Transition Promote Ovarian Cancer Metastasis

BACKROUND&OBJECTIVEEpithelial ovarian cancer(EOC)is the leading cause of death among women with reproductive tract carcinomas in China.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).EOC is characterized by high rate of metastases and resistance to chemotherapy and radiotherapy.At present,its exact cause remains unknown.Several factors are related to ovarian cancer,such as genetically determined susceptibility,hormones,alcohol,etc.Base on pathological and genetic features,ovarian cancers can be classified into Type I and Type II.Type I EOC(mucinous,clear cell,endometrioid,low-grade serous carcinoma)is characterized by mutations and dysfunction of K-RAS,B-RAF,PTEN.Type II tumor(high-grade serous carcinoma)is very aggressive and characterized by mutations of TP53 and BRCA1.Because of its high rate of mortality,the functional and mechanistic study on ovarian cancer is always a hot topic in the area of cancer research.Nitric oxide(NO)and nitric oxide synthases are ubiquitous in malignant tumours and are known to exert both pro-and anti-tumour effects.NO is synthesized by nitric oxide synthases(NOS),which are ubiquitously expressed in malignant tumours.NO regulates several physiological processes through the soluble-guanylyl-cyclase-cGMP pathway and S-nitrosylation,and has cytotoxic and genotoxic effects at high concentrations..As a signalling molecule,NO regulates various physiological and pathophysiological processes,such as vascular functions,neurological functions(neurotransmission and development of the nervous system)and,at relatively high concentration,cytotoxic functions(cytostasis and cytolysis).Interestingly,various studies have shown that NO can both promote and inhibit tumour progression and metastasis.The effects of NO in tumours seem to depend on the activity and localization of NOS isoforms,concentration and duration of NO exposure,and cellular sensitivity to NO.Tumour-cell-derived NO promotes tumour progression by induction of tumour-cell invasion,proliferation and the expression of angiogenic factors.The inducible isoform of NOS(iNOS),which produces high concentrations of NO,mediates neoplastic transformation in oncogene-and chemical-induced tumorigenesis models,although conflicting results are reported in the literature.Conversely,the transfection of iNOS-expressing constructs into NO-sensitive tumour cells inhibits tumour growth and metastasis.Host stromal-cell-derived NO,which is synthesized by iNOS,inhibits growth of NO-sensitive tumours but promotes growth of NO-resistant tumours.NO that is predominantly synthesized by endothelial NOS(eNOS)in vascular endothelial cells promotes angiogenesis directly and functions both upstream and downstream of angiogenic stimuli.Moreover,NO mediates recruitment of perivascular cells and,therefore,remodelling and maturation of blood vessels.NO that is synthesized by eNOS promotes tumour progression through the maintenance of blood flow,induction of vascular hyperpermeability and reduction of leukocyte-endothelial interactions.Induction of NO signalling can induce direct tumour-cell cytotoxicity or sensitize tumour cells to other treatments such as radiation.Conversely,blockade of NO signalling can inhibit neoplastic transformation,tumour angiogenesis and blood flow.Expression,activity and localization of NOS isoforms,concentration and duration of NO exposure,and cellular sensitivity to NO are important determinants of NO function.Further in vivo tumour studies with high spatial and temporal resolution should resolve conflicting issues in NO biology and guide the manipulation of NO signalling for future clinical use.Metastasis is a complex multi-step process.Ovarian cancer is prone to invade and disseminate in abdominopelvic cavity,therefore,how to inhibit cancer cell invasion and metastasis become the central issue in clinical treatment and basic research of ovarian cancer.Epithelial-mesenchymal transitions(EMT),during which epithelial cells transform from the polary epithelial phenotype to a high degree of movement of the fibroblast-Iike or mesenchymal phenotype,play an important role in early embryogenesis.In vitro and in vivo studies show that,EMT play an important role in the infiltration and dissemination of epithelial tumours.EMT which are activated during the progress,infiltration and invasion of epithelial tumour,are key molecular events of invasiveness acquirement of epithelial tumor cells,and play a major pathological role in the development and metastasis of malignant tumors spread process.As to the Biochemistry,cell changes the expression of epithelial markers(such as E-cadherin)to the expression mesenchymal markers(such as N-cadherin).Originally found in the study of embryonic development of Drosophila shows that,the Twist gene could regulate cell migration and tissue remodeling,and induct the mesoderm development.The current studies have confirmed that,NOS gene is closely related to neoplasm metastasis,there is no intensive study in the relationship between NOS and EMT,invasion and metastasis of ovarian cancer.Several studies show that NO has a positive effect on tumor growth and metastasis.NO increase various murine tumor growths and metastases in vivo;and NOS inhibitors L-NAME and L-NNA inhibit thei metastasis.Furthermore,enhancement of metastasis by iNO and eNOS is through the soluble guanylate cyclase(sGC)and mitogen-activate protein kinase(MAPK)pathway.Transplant breast carcinoma and melanoma in iNOS-/-mice exhibit decreased incidence and size of metastatic nodules,as well as longer survival.It has been noted that moderate,but not high,expression of iNOS in murine pancreatic adenocarcinoma cell scorrelates with rapid tumor growth and high metastatic potential On the other hand,high eNOS expression is positively correlated with vascular invasion in human colon cancer and trophoblast cancer cells.Activation of the sGC/cGMP pathway by eNOS causes cancer cell migration.Furthermore,tissue inhibitors of metalloproteinase(TIMP)-2 and TIMP-3 are inhibited by eNOS,providing another possible mechanism for eNOS-associated cell invasiveness.However,there is evidence supporting that NO and NOS inhibit tumor metastasis.Some studies show that endogenous iNOS expression and NO production are inversely correlated with metastasis Inhibition of tumor growth and metas-tasis with the transduction of iNOS in tumor cells is identi-fied in several mouse tumor models.The NOS inhibitors L-NAME and L-NMMA and eNOS-/-mice block vasculature-derived NO and induce metastasis of murine melanoma and hepatoma cells to the liver and lung.iNOS-/-mice with breast cancer and melanoma transplant show increased incidence and size of metastases,with sensitivity to NO-dependent cytotoxicity.Furthermore,transfer-ring iNOS-deficient tumor cells to iNOS-/-mice and iNOS+/+mice results in iNOS-related decreased tumor growth and metastasis Platelets enhance metastasis.This is a result of their ability to store angiogenic factors and stimulate the growth of blood vessels,and trap metastasizing tumor cells in blood vessels.Platelet-tumor cell aggregates more easily adhere to endothe-lial cells and disseminate hematogenously.Thus,NO derived from tumors actually prevents platelet aggregation through cGMP.And,NO production has been shown to be inversely proportional to the ability of tumor cells to aggregate with plate-lets.NO seems to have a role in eliminating metastasizing tumor cells.NO produced by endothelial cells is cytotoxic to metastasiz-ing tumor cells.It has also been shown that the liver rapidly releases NO in response to intravascularly arrested tumor cells and causes tumor cell apoptosis.Evidence is accumulating that NO has an important role in the regulation of tumorigenesis,tumour angiogenesis,vascular functions,progression and metastasis.On the basis of the available literature,one might conclude that,overall,NO promotes de novo tumorigenesis when associated with chronic inflammation,angiogenesis and the growth of established solid tumours,whereas it mediates anti-tumour-cell activity against haematogeneously disseminating tumour cells.Unfortunately,there are no preclinical studies yet that show causal relationships between NO production,lymphatic function and metastasis in tumours.However,many compelling histopathological studies demonstrate a positive correlation between NOS expression and/or activity,and lymph-node metastasis The array of conflicting reports in almost all aspects of NO biology hint at the complexity of the NO signalling network and confound the simple translation of preclinical data from bench to bedside.Recent studies highlight the importance of NO distribution,dose and exposure duration in governing the effects of NO.Therefore,studies of in vivo tumours with high spatial and temporal resolution are required to fully understand the role of NO in tumour progression.Owing to experimental challenges,the field currently suffers from a paucity of such data.Development of and progress with in vivoexperimental techniques,including quantitative imaging of NO,NOS activity and other functional parameters,are beginning to fill these gaps.Modulation of NO signalling might be used for the treatment of tumours.The literature supports both increasing and decreasing NO signalling as a potential strategy.Type and stage of tumour,and the expression,activity,and spatial and temporal distribution of NOS isoforms should be taken into consideration in deciding what kind of approach to use.However,there is little known about the functions of NOS in pathogenesis of ovarian cancer,which encourages us to explore the roles of NOS in proliferation,EMT,invasion and metastasis of ovarian cancer.This study used RNA interference(RNAi)technology,recombinant plasmid vector NOS1-ShRNA was designed by genecopoeia company,using cationic lipids Lipofectaming 2000,the plasmid vector NOS1-ShRNA was transfected in ovarian cancer cell line OVCAR3.After resistance monoclonal selected with puromycin,the effect of NOS 1 gene silence on the biological characteristics of ovarian cancer was detected.The role of NOS 1 gene in the occurrence and development process of ovarian cancer was observed,and the feasibility of gene therapy for ovarian cancer was explored.METHODS1.Based on gene expression profile data of ovarian cancer tissue and normal ovarian tissue in public resources GEO database,Compared ovarian cancer tissue and normal ovarian tissue NOS gene expression level,thus analyzed the correlation between the expression level and the prognosis of ovarian cancer and verifying NOS may participate in ovarian cancer development.2.Using L-NAME supress NOS protein function in ovarian cance.Observed ovarian cancer cell biology function change,Especially the regulation function of proliferation and migration.we detected Changes of EMT related gene expression level after administering NOS inhibitor L-NAME,thus expliciting NOS regulation role of EMT.3.Effect of NOS1 gene silence on the biological characteristics of ovarian cancerNOS1 gene interference fragment,recombinat plasmid vector NOS1/ShRNA was designed by genecopoeia company,using cationic lipids Lipofectaming 2000,the plasmid vector NOS1/ShRNA was transfected in ovarian cancer cell line OVCAR3,and use of negative space vector as the control.After resistance monoclonal selected with puromycin,a strong fluorescent clones expanding culture is picked,and analyzed the interference efficiency of NOS 1 by qRT-PCR and Western Blot.Expression of NOS 1 of NOS 1 gene after interference was detected by CCK8 and flat colony rate.The invasive of silent NOS1 gene exprssion was detected by invasion and migaration ability in vitro.4.Through the whole genome expression profile chip technology,Detecting NOS1 silence ovarian cancer cell line and its control cell lines,and screening NOS1 regulation of genes,Screening of NOS1 regulation of genes by comparing.Using bioinformatics methods Explorede NOS1 gene regulatory mechanism of ovarian cancer metastasis and EMT(signaling pathways and molecular targets).And by using western blotting technique,Validation the key signaling molecules regulated and targeted by NOSI of bioinformatics screening.thus Cleared Possible mechanisms of NOS 1 regulation of ovarian cancer metastasis.5.STATISTICAL ANALYSISThese data were analzed by SPSS 13.0 software.The indepedent samples t-test was used to analyze the datas which refer to qRT-PCR,migaration experiment,ivasion experiment,colony formation rate experiment and Scratch test.Proliferation experiments by CCK8 in vitro were assessed by means of the repeated-measures analysis of variance.P<0.05 meant the difference statistically significant.RESULTl.Analysis NOS expression in human ovarian tissue1)NOS expression in ovarian cancer tissue was higher than normal ovarian tissue:we analyzed 12 of the ovarian cancer tissue and normal ovarian tissue microarray in GEO database,it showed NOS(NOS1,NOS2,NOS3)expression in ovarian cancer tissue were higher than normal ovarian tissue.which suggested NOS was high associated with ovarian cancer survival and may be involved in ovarian cancer development.2)NOS expression level is negatively related to the prognosis of patients with ovarian carcinoma250 cases of ovarian tissue microarray were analyzed by Meta analysis showed that.Patients withhigher NOS1 or NOS2 expression have shortersurvivaltime,which suggested NOS was high associated with ovarian cancer survival and may be involved in ovarian cancer development.2.The influence of L-NAME on ovarian cancer cell biology1)L-NAME supress ovarian cancer cell proliferationSKOV3 proliferation is significantly reduced by CCK8 reagentafter administering a NOS inhibitor L-NAME.L-NAME also supresson colony formation in Ovarian cancer cells(P<0.05).2)L-NAME supress ovarian cancer cell migration and invasionscratch healing of L-NAME group cellscells were slower than control cells after 24h cultured in DMEM medium containing 2%fetal calf serum.and Migratory capabilities of SKOV3 is also significantly reduced after administering NOS inhibitor L-NAME(p<0.05).3)L-NAMEsupress EMT functionof ovarian cancer cell SKOV3 EMT functionCompared with control group,L-NAME groupsovarian cancer cell can induce the expression of E-cadherin in SKOV3 cells,while the expression of N-cadherin,Vimentin and Fibronectin was downregulated.(p<0.05).3.The influence of NOS1 silence on ovarian cancer cell biology1)Role of RNAi of NOS1 gene in cells of OVCAR3Recombinant plasmid vector NOS1/ShRNA was designed by genecopoeia company in view of the target gene NOS 1.It was confirmed by sequencing results that NOS1/ShRNA transfected cell OVCAR3.Interference clips and negative control OVCAR3 cells was selected with puromycin for getting resistance clones.Quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and Western Blot results showed NOS1 has the most obvious reduction of NOS 1 in interference cloning(named OVCAR3/NOS1-),the interference efficiency of NOS1 amounted to 94.5%in OVCAR3/NOS1-cells.2)Effect of NOS 1 gene silence on the biological characteristics of ovarian cancerNOS1 cell proliferation in vitro was observed after gene silence,it is showed that compared with OVCAR3/CON and OVCAR3,OVCAR3/NOS1-proliferation is significantly reduced.Three groups were significantly different(F=171.875,p=0.000).Flat cloning experiment results also showed OVCAR3/NOS1-cells in a single cell proliferation significantly was lower than other groups.The difference was stastistically significant(P<0.05).These resluts revealed that tumor growth was significantly inhibited in vitro after reduction of NOS 1 gene expression.Scratch test showed that,scratch healing of OVCAR3/shNOSl group were slower than OVCAR3/CON group.The difference was statistically significant(P<0.05).Migaration experiment in vitro detected the changes in the ability of cell.After NOS1 gene silence,the migaration of OVCAR3/shNOS1 was significantly lower than the OVCAR3/CON(P<0.05).It suggested that NOS1 silence significantly inhibited the migaration of ovarian cells in vitro.Invasion chamber assay in vito detected the changes in the ablility of cell invasion.After NOS1 gene silence,the invasion of OVCAR3/NOS1 was significantly lower than the OVCAR3/CON(P<0.05).It suggested NOS1 silence significantly inhibited the invasive ability of ovarian cancer cells in vitro.4.Investigate the molecular mechanism of NOS 1 regulation of EMT by Bioinformatics analysisNOS1-shRNA Stable transfection ovarian cancer cell lines(shNOSl-OVCAR3)and Control cell lines(conNOS1-OVCAR3)was compared by Gene expression profile chip.GenCLiP software screened 122 genes related to the EMT functional in NOS1-genes.34 signaling pathways were involved in 122 EMT genes regulated by NOS1 by KEGG website Analysis,by which Built NOS1 EMT-genes network by string online software.CONCLSION1.NOS expression in ovarian cancer tissue was higher than normal ovarian tissue,NOS expression level is negatively related to the prognosis of patients with ovarian carcinoma.2.In vitro experiments show that Ovarian cancer cells express NOS,Regulating the function of EMT,and Promote cell proliferation and migration.3.NOS1 through PI3K/AKT pathway regulate the EMT function of ovarian cancer cells.