Reactive oxygen species regulate angiogenesis and tumor growth through vascular endothelial growth factor.pdf

Reactive Oxygen Species Regulate Angiogenesis and Tumor Growththrough Vascular Endothelial Growth FactorChang Xia,1Qiao Meng,1Ling-Zhi Liu,1Yongyut Rojanasakul,2Xin-Ru Wang,3and Bing-Hua Jiang11Mary Babb Randolph Cancer Center,and Departments of Microbiology,Immunology and Cell Biology and2PharmaceuticalSciences,West Virginia University,Morgantown,West Virginia;and3Lab of Reproductive Medicine,Cancer Center,Nanjing Medical University,Nanjing,Jiangsu,ChinaAbstractReactive oxygen species(ROS)are associated with multiplecellular functions such as cell proliferation,differentiation,and apoptosis.However,the direct roles of endogenous ROSproduction still remain to be elucidated.In this study,wefound that high levels of ROS were spontaneously produced byovarian and prostate cancer cells.This elevated ROS produc-tion was inhibited by NADPH oxidase inhibitor diphenyleneiodonium(DPI)and mitochondria electron chain inhibitorrotenone in the cells.To further analyze the source of ROSproduction,we found that ovarian cancer cells have muchhigher expression of NOX4 NADPH oxidase,and that specificinhibition of NADPH oxidase subunit p47phoxdiminished ROSproduction.To analyze the functional relevance of ROSproduction,we showed that ROS regulated hypoxia-induciblefactor 1(HIF-1)and vascular endothelial growth factor(VEGF)expression in ovarian cancer cells.Elevated levels ofendogenous ROS were required for inducing angiogenesis andtumor growth.NOX4 knockdown in ovarian cancer cellsdecreased the levels of VEGF and HIF-1A and tumorangiogenesis.This study suggests a new mechanism of higherROS production in ovarian cancer cells and provides strongevidence that endogenous ROS play an important role forcancer cells to induce angiogenesis and tumor growth.Thisinformation may be useful to understand the new mechanismof cancer cells in inducing tumorigenesis and to develop newtherapeutic strategy by targeting ROS signaling in humancancer in the future.Cancer Res 2007;67(22):1082330IntroductionReactive oxygen species(ROS)are naturally produced by cellsthrough aerobic metabolism,and high levels of ROS in the cellsare associated with many diseases including cancer(1,2).Severallines of evidence indicate that ROS may be involved in humancarcinogenesis:(a)some growth factors such as epidermal growthfactor(EGF),insulin,and angiopoietin-1 increase ROS productionin the cells for regulating cell migration and proliferation(36);(b)natural antioxidants can inhibit cancer cell proliferation andtumor growth(710);(c)high levels of ROS are observed in somecancer cells,which may induce DNA damage leading to genomicinstability and tumor initiation(11,12);and(d)ROS induce theactivation of mitogen-activated protein(MAP)kinase,nuclearfactor nB(NF-nB),and activator protein 1,which are known to beassociated with cancer development(1315).However,undercertain conditions,ROS increase proapoptotic molecules such asp53 and p38 MAP kinase and induce cellular apoptosis(16,17).The direct role of ROS in tumor growth and angiogenesis remainsto be defined.Angiogenesis is important for tumor developmentand growth(18).The vascular endothelial growth factor(VEGF)isa major angiogenesis inducer and is regulated at transcriptionallevel by hypoxia-inducible factor 1(HIF-1)in response to hypoxia(19,20).HIF-1 is composed of HIF-1a and HIF-1h subunits(20,21).In this study,we found that ovarian cancer cells hadelevated levels of ROS production;thus,we further analyzed(a)what the source and mechanism of endogenous ROS productionin ovarian cancer cells were;(b)whether endogenous ROSproduction regulated HIF-1 and VEGF expression;(c)whetherROS generation in the cells was required for inducing angiogen-esis and tumor growth in vivo;and(d)whether ROS regulatedangiogenesis and tumor growth through HIF-1a and VEGFexpression.This work would provide the direct evidence of theendogenous ROS in regulating tumor growth and angiogenesisin vivo and a new sight in the underlying mechanisms.Materials and MethodsReagents and cell culture.The human ovarian cancer cells OVCAR-3and A2780 and immortalized ovarian surface epithelial cells IOSE 397 andIOSE 386 were maintained in RPMI 1640(Invitrogen)supplemented with10%fetal bovine serum(FBS),2 mmol/LL-glutamine,100 units/mLpenicillin,and 100 Ag/mL streptomycin and cultured at 37jC in a 5%CO2incubator.Trypsin(0.25%)/EDTA solution was used to detach the cells fromthe culture flask.Antibodies against HIF-1a and HIF-1h were from BDBiosciences.Antibodies against VEGF for ELISA and immunohistochemis-try were purchased from R&D System and Santa Cruz Biotechnology,respectively.Diphenylene iodonium(DPI)and rotenone were from Sigma.2,7-Dichlorofluorescein diacetate(CM2-DCFHDA)was from MolecularProbes.Intracellular H2O2staining.Ovarian cancer cells or immortalizedovarian surface epithelial cells were seeded in six-well plate at density of1?105cells per well on a coverslip overnight.The cells were stained withCM2-DCFHDA(5 Amol/L)for 15 min at 37jC,then washed with 1?PBSthrice,and fixed with 10%formaldehyde.Images were captured with a ZeissAxiovert 100 TV microscope with a 40?1.4 objective lens with a laserscanning confocal attachment(LSM 510;Zeiss).Quantification ofimmunofluorescence intensity was done using the confocal microscopewith 480-nm excitation and 540-nm emission settings.Immunoblotting analysis.The total cellular protein extracts wereprepared in radioimmunoprecipitation assay buffer(RIPA)and separated by7%SDS-PAGE.Membranes were blocked with 5%nonfat dry milk for 2 h andincubated with primary antibodies.Protein bands were detected byincubation with horseradish peroxidaseconjugated antibodies(Perkin-Elmer Life Sciences)and visualized with enhanced chemiluminescencereagent.Note:Supplementary data for this article are available at Cancer Research Online(http:/cancerres.aacrjournals.org/).C.Xia,Q.Meng,and L.Z.Liu contributed equally to this work.Requests for reprints:Bing-Hua Jiang,Mary Babb Randolph Cancer Center,andDepartment of Microbiology,Immunology and Cell Biology,West Virginia University,1801 Health Sciences South,Morgantown,WV 26506.Phone:304-293-5949;Fax:304-293-4667;E-mail:bhjianghsc.wvu.edu.I2007 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-07-0783www.aacrjournals.org10823Cancer Res 2007;67:(22).November 15,2007Research Article American Association for Cancer Research Copyright 2007 on December 6,2012cancerres.aacrjournals.orgDownloaded from DOI:10.1158/0008-5472.CAN-07-0783Transient transfection.Small interfering RNA(siRNA)duplex oligonu-cleotides targeting human p47phoxand NOX4 were purchased fromDharmacon.OVCAR-3 cells were cultured to 60%to 70%confluency in35-mm dishes and transfected with p47phoxsiRNA or NOX4 siRNA using X-tremeGENE siRNA transfecting reagent(Roche Applied Science)in serum-free OPTIMEM according to the manufacturers instruction.The cells wereswitched to fresh medium(1 mL)containing 10%FBS 3 h after thetransfection and cultured for 24 to 72 h.The p47phoxprotein expression inthe cells was analyzed by immunoblotting,and ROS levels in the cells wereanalyzed by DCFHDA staining.The cells transfected with NOX4 siRNA wereused for analyzing VEGF and HIF-1 expression and angiogenesis response.ELISA.Capture ELISA was done using a human VEGF ELISA kitaccording to the manufacturers instruction(R&D Systems).Opticaldensities were read at 405 nm,and the rate of VEGF secretion wascalculated as we previously described(22).Semiquantitative reverse transcription-PCR.Total RNAs were isolat-ed with TRIzol reagent(Life Technologies).First-strand cDNAs weresynthesized using total RNAs,avian myeloblastosis virus(AMV)reversetranscriptase,and an oligo(dT)primer(Promega).Primers used for PCRamplification were as follows:VEGF sense primer,5-TCGGGCCTCCG-AAACCATGA-3;VEGF antisense primer,5-CCTGGTGAGAGATCTGGTTC-3;glyceraldehyde-3-phosphate dehydrogenase(GAPDH)sense:5-CCACC-CATGGCAAATTCCATGGCA-3;GAPDH antisense:5-TCTAGACGGCAGGT-CAGGTCCACC-3;NOX4 sense:5-CTCAGCGGAATCAATCAGCTGTG-3;NOX4 antisense:5-AGAGGAACACGACAATCAGCCTTAG-3.Reverse tran-scription-PCR(RT-PCR)reaction was done for 30 cycles with each cyclefor 1 min at 94jC,1 min at 55jC,and 1 min at 72jC.Quantification of PCRproduct was done by electrophoresis.Luciferase assay.OVCAR-3 cells were cultured in 12-well platesand transiently transfected with human VEGF reporter(1 Ag)and h-galactosidase(0.2 Ag)plasmids.The cells were cultured for 48 h afterthe transfection,and relative luciferase activities were analyzed as wedescribed(22).Tumor-induced angiogenesis on chicken chorioallantoic mem-brane.White Leghorn fertilized chicken eggs were incubated at 37jCunder constant humidity.To investigate the effect of ROS inhibitors ontumor-induced angiogenesis,OVCAR-3 cells(1?106)with or without DPI(500 nmol/L)or rotenone(200 nmol/L)treatment were mixed at 1:1 ratiowith Matrigel and implanted onto the chorioallantoic membranes(CAM)atday 9.Tumor angiogenesis was analyzed 4 days after the implantation,andtumor growth was analyzed 9 days after the implantation.Similarly,OVCAR-3 cells were infected with 10 multiplicity of infection(MOI)adenovirus carrying green fluorescent protein,catalase,or GPx.the cellswere used to perform angiogenesis and tumor growth assay.The bloodvessel branches on the CAM were counted by two observers in a double-blind manner.The representative tumors were photographed.Immunohistochemistry staining.Tissues harvested from the CAMwere fixed in 10%formaldehyde overnight and then embedded in paraffin.Figure 1.Ovarian cancer cell lines generate higher levels of endogenous ROS.A,ovarian cancer cells OVCAR-3 and A2780 and immortalized ovarian surfaceepithelial cells IOSE 386 and IOSE 397 were seeded onto a glass coverslip in the six-well plate at 1?105cells per well for 24 h.CM2-DCFHDA(5 Amol/L)was addedinto the cell culture medium and incubated for 30 min.For the catalase treatment,catalase(750 units/mL)was added to A2780 cells 30 min before the additionof CM2-DCFHDA.The cells were washed thrice with 1?PBS and fixed with 10%buffered formalin.The representative images were captured with a confocalfluorescence microscope(at excitation wavelength,485 nm;emission wavelength,530 nm).Bar,50 Am.B,the mean value of DCF fluorescence intensity was obtainedfrom 10,000 cells at 485 nm excitation and 540 nm emission settings using a flow cytometer(Becton Dickinson FACSort).C,IOSE 386,A2780/CP70,SKOV-3,PC-3,and DU145 were seeded onto a glass coverslip in the six-well plate at 1?105cells per well for 24 h.CM2-DCFHDA(5 Amol/L)was added into the cell culturemedium and incubated for 30 min.The cells were washed thrice with 1?PBS and fixed with 10%buffered formalin.The representative fluorescence(top)andphase contrast(bottom)images were captured with a confocal fluorescence microscope.Bar,200 Am.D,OVCAR-3 cells were treated with solvent,5 Amol/L DPI,5 Amol/L rotenone,10 or 20 Amol/L LY294002 for 30 min and then stained with 5 Amol/L CM2-DCFHDA for 15 min.The relative fluorescence intensity was analyzed byflow cytometry and normalized to that of IOSE 397 cells.*,P 0.01,significant difference when the value of treatment was compared with that of the control.Cancer ResearchCancer Res 2007;67:(22).November 15,200710824www.aacrjournals.org American Association for Cancer Research Copyright 2007 on December 6,2012cancerres.aacrjournals.orgDownloaded from DOI:10.1158/0008-5472.CAN-07-0783Sections(5 Am)were collected on positively charged slides and deparaffi-nized in the following order:in xyline for 5 min,100%alcohol for 5 min,95%alcohol for 3 min,70%alcohol for 3 min,and then rinsed thrice in deionizedwater.Theslideswerethensteamedwithantigenretrievalbuffer 10mmol/Lcitrate(pH 8.0)for 10 min,cooled to room temperature for 20 min,andthen rinsed thrice in deionized water.The slides were incubated with 5%goat serum at room temperature for 1 h and then stained with antibodiesagainst VEGF or HIF-1a overnight at 4jC,followed by incubation withhorseradish peroxidaseconjugated secondary antibodies for 1 h at roomtemperature.Between each step,the sections were washed thrice with PBSbuffer.Statistical analysis.The data were analyzed using ANOVA by SPSSstatistics software package.All the results are expressed as mean F SE,andthe difference was considered significant at P 0.05.ResultsSpontaneous ROS production in ovarian and prostatecancer cells.We used intracellular DCFHDA staining method tomeasure the endogenous ROS levels in the cells and found that theROS levels in both OVCAR-3 and A2780 cells were 5-and 6-foldhigher,respectively,than those in immortalized ovarian epithelialcells IOSE 397 and IOSE 386(Fig.1A and B).The fluorescent signalwas completely inhibited by the addition of catalase to indicate thespecificity of ROS staining.To further test the ROS levels in othercancer cells,ovarian cancer cell lines A2780/CP70,SKOV-3,andprostate cancer cells PC-3 and DU145 were used.As shown inFig.1C,the ROS levels in these four cell lines were much higherthan that in IOSE 386 cells,indicating that ROS were also spon-taneously produced in other ovarian and prostate cancer cell lines.The endogenous ROS production was inhibited by DPI,NADPH-dependent oxidase inhibitor,and rotenone,the mitochondriacomplex I inhibitor(Fig.1D).Phosphoinositide-3-kinase(PI3K)inhibitor LY294002 did not inhibit the ROS generation.This resultsuggests that NADPH oxidase and the mitochondria respiratorychain are required for inducing ROS production in the cells.Role of NADPH oxidase in regulating ROS production inovarian cancer cells.The NADPH oxidase complex includes acatalytic subunit gp91phoxand regulatory subunits such as p47phox,p67phox,and Rac.The homologues of gp91phox(also known asNOX2)are called NOX family members.To test the expression ofNOX isoforms in ovarian cancer cells,the mRNA levels of NADPHoxidase isoforms were analyzed in OVCAR-3 and A2780 cells.Asshown in Fig.2A,NOX4 mRNA levels were much higher in ovariancancer cells than those in immortalized normal ovarian epithelialcells.NOX1,NOX3,and NOX5 mRNA levels were not detectable inthe ovarian cancer cells;and NOX2 mRNA level was not increasedin the cells(data not shown).This result indicates that NOX4overexpression may be responsible for higher NADPH activity toincrease ROS production in the cells.The expression of NOX4 wassignificantly inhibited by the treatment of SB431542,the inhibitorof transforming growth factor-h1(TGF-h1),and pyrrolidinedithiocarbamate(PDTC),a chemical inhibitor of NF-nB,indicatingthat the activation of TGF-h1 and NF-nB was involved in regulatingNOX4 expression(Fig.2B).To further test whether NADPH activitywas required for increasing ROS levels in the ovarian cancer cells,we inhibited p47phoxexpression,a regulatory subunit of NADPHoxidase,which is required for NADPH oxidase activity(2325).Theprotein levels of p47phoxwere markedly reduced by p47phoxsiRNAas compared with the cells with mock transfection(Fig.2C),indicating that p47phoxsiRNA was sufficient to inhibit end