Enhanced The Killing Effect Of Radiotherapy On A549cells Through Regulating APE1and P53interaction By Genistein

IntroductionLung cancer as the most common malignancy leads the first cause of cancer-related death in the world. It is estimated that non-small cell lung cancer(NSCLC) covered approximately80-85%of lung cancer. Although giving priority to the comprehensive treatment with surgery,radiotherapy,chemotherapy, immunotherapy and molecular targeted therapy represent significant development nowadays,currentive effects are still less than satisfactory.lt is an urgent affair to further improve the theraputic effects of NSCLC in the present field of oncology.ROS exerts an crutial role in tumor development.An array of experiments present that Ape1/Ref-1(apurinic/apyrimidinic endonuclease) and p53as two critical molecules within oxidative stress signaling pathways are essential for cell growth and cell apoptosis.Ape1/Ref-1identified as an unique multifunctional protein involving in numerous metabolism activities,such as DNA repair function,redox regulation,RNA metabolism and cell cycle arrest.The increased expression of Ape1/Ref-1can enhance lung cancer cells radiosensitivity and cause cell death.Genisten(GEN) acting as the dominant ingredients of soy isoflavones can efficiently reduce Ape1/Ref-1protein induced by ROS whereas enhance wildtype p53protein expression.The wild type p53plays a negative regulation role in Ape1/Ref-1expression which can restrain Ape1/Ref-1protein expression.On the other way,Ape1/Ref-1regulated p53DNA binding activity through both the redox dependent and independent way,further improve downstream p21,BAX and Bcl-2transcriptional activity.Ape1/Ref-1and p53interaction by genistein may be a central issues to radiosensitivity enhancement.However,some research investigated that there were endogenous and exogenous interactions between Ape1/Ref-1and p53in A549and Hela cell.Our study investigated Ape1/Ref-1and p53endogenous and exogenous interactions and their cross talk regulated by genistein may enhance NSCLC radiosensitvity. Thus genistein enhances NSCLC radiosensitivity can offer a potential experiment approach for tumor chemo-/radio-resistance.Objectives:1．To investigate the effects of cell growth and cell apoptosis by genisten and discussthe mechanism of Ape1/Ref-1and p53cross interaction induced by genistein onradiotherapy of NSCLC,further providing experimental evidence for genisten as a targettherapy enhancing NSCLC radiosensitivity.2．To prove the endogenous and exogenous interaction between Ape1/Ref-1and p53and the molecule mechanism from it,thus further discuss the effects of transcriptionregulation between each other on tumor radio-/chemo resistance.Methods:1．Cell viability assay examined A549cell growth treated with genistein alone orcombined with radiotherapy. With different doses of genistein treated cells for differenttimes,observing the cell growth inhibition respectively.Trypan blue staining assayexamined cell viability treated with genistein alone or combined with radiotherapy.Flowcytometry further assessed A549cell apoptosis.2．Western blot assay examined Ape1/Ref-1and p53proteins expression by genisteinalone or concurred with radiotherapy treatment.3． A549cells were cotransfected with Ape1/Ref-1promoter luciferase reporterconstruct and increasing amounts of WTp53expression vectors.Western blot assay detectedApe1/Ref-1and p53proteins expression respectively.The luciferase reporter gene assayexamined the regulation of Ape1/Ref-1transcriptional activity by WTp53.Meanwhile,EMSA verified p53DNA binding activity by genistein alone or combined with radiotherapytreatment.4． Co-immunoprecipitation assay proved the endogenous interaction betweenApe1/Ref-1and p53in A549,Hela and APE1WTcells.GST pull down assay further verifiedthe exogenous interaction. Immunofluorescence and Confocal Laser-scanning Microscopyexamined Ape1/Ref-1and p53proteins subcellular localization.Results: 1．Cell viability assay showes genistein alone or concurred with radiotherapy inhibitedA549cells growth through dose and time dependent manners.Trypan blue exclusion assayand Flow cytometry analysis examined cell growth numbers treated with genisteincombined with radiotherapy were significantly lower than other groups. Flow cytometryanalysis showed the early stage apoptosis rate of A549cells treated with genistein andradiotherapy was obviously higher than other treatment groups.2．Genistein downregulated Ape1/Ref-1protein expression whereas upregulated p53expression through time dependent and dose dependent manner.In addition,genisteinrepressed Ape1/Ref-1expression induced by radiotherapy while further improved p53protein expression induced by radiotherapy.3． A549cells were cotransfected with Ape1/Ref-1promoter luciferase reporterconstruct and increasing amounts of WTp53expression vectors.Western blot assay showedthat WTp53repressed Ape1/Ref-1protein expression.The luciferase reporter gene assayexamined the regulation of Ape1/Ref-1transcriptional activity by WTp53and the resultshowed that WTp53inhibited Ape1/Ref-1transcriptional activity.Meanwhile,EMSAverified p53DNA binding activity by genistein alone or combined with radiotherapytreatment and result displayed genistein induced p53DNA binding activity,thus stimulatingdownstream gene expression. This effects were further enhanced when concurred withradiotherapy.4． Co-immunoprecipitation assay proved the endogenous interaction betweenApe1/Ref-1and p53in A549,Hela and APE1WTcells.GST pull down assay further verifiedthe exogenous interaction. Immunofluorescence and Confocal Laser-scanning Microscopyexamined Ape1/Ref-1and p53proteins subcellular localization were mainly in nuclear.Conclusions:1．Genistein alone or concurred with radiotherapy inhibited A549cells growth throughdose and time dependent manners. Genistein downregulated Ape1/Ref-1protein expressionwhereas upregulated p53expression were also through time dependent and dose dependentmanner. WTp53repressed Ape1/Ref-1protein expression may through repressingApe1/Ref-1transcriptional activity. Genistein induced p53DNA binding activity,thusstimulating downstream gene expression. This effects were further enhanced when concurred with radiotherapy.2．Co-immunoprecipitation assay and GST pull down assay demonstrated that theendogenous and exogenous cross interaction between Ape1/Ref-1and p53.