The Effect Of APE1/Ref-1on The Biology Behavior Of Ovarian Cancer And The Related Molecular Mechanism

Background Ovarian cancer is a common malignancy in female genitaltract which has low incidence and high mortality. Owing to absence ofsymptoms in early stage and effective methods for early diagnosis, manypatients were diagnosed in advanced stage. Ovarian cancer is a disease which isapt to resist to chemotherapy and metastasis. Although comprehensivetreatments including surgery and chemiotherapy are widely used, thefive-surivival rate is still low and the prognosis remains poor. The study onmolecular mechanism of ovarian cancer progression and drug resistanceincluding searching its related target gene is meaningful for early diagnosis andis effective therapy of ovarian cancer.APE1/Ref-1(apurinic/apyrimidinic endonuclease/redox effector factor-1) isan attractive gene in tumor study during the past few years. It is a perfect paradigm of multifunctional protein. It is not only a rate-limiting enzyme inBER pathway, but also an important factor which can regulate transcription byactivating many transcription factors. In addition, it is involved in someimportant cell movements, such as apoptosis, cell cycle control and oxidateaignal transduction. Some studies showed that APE1/Ref-1expressed widely innormal tissue. However, in several malignancy tumors including cervical cancer,colonic cancer, glioma, and small cell lung cancer, its expression was increasedor cell localization was changed. Since it expresses differently between normaltissue and cancer tissue, APE1/Ref-1may be a potential oncogene which isrelated with tumorigenesis and progression. Furthermore, in view of theimportant role of APE1/Ref-1in DNA repair and cell apoptosis, there aregrounds for believing that it has close relationship with tumor chemotherapyresistance and may be a potential target point of oncotherapy.Purpose1. To study the relationship between APE1/Ref-1expression and clinicalpathology, angiogenesis and cell proliferation of ovarian cancer; toreveal the role ofAPE1/Ref-1on tumorigenesis and progression.2. To investigate the effect of APE1/Ref-1on biological behaviour ofovarian cancer cell including proliferation, apoptosis and invasion.Further understanding the molecular mechanism of APE1/Ref-1effecton tumorigenesis and progression; to provide theoretical basis for genetherapy with APE1/Ref-1as target point；3. To investigate the effect of APE1/Ref-1on cisplatin resistance ofovarian cancer in molecular level; to reveal the molecular mechanismand lie the foundation for study on reversal of drug resistance withAPE1/Ref-1as target point. Contents1. The relationship between APE1/Ref-1expression and clinicalpathology, angiogenesis and cell proliferation of ovarian cancer:a) Immunohistochemistry was employed to investigate the expression ofAPE1/Ref-1protein in65formalin-fixed paraffin-embeded ovariancancer tissues. Western Blot and realtime PCR were employed toinvestigate the expression of APE1/Ref-1protein in36samples ofovarian cancer tissues.b) Immunohistochemistry was employed to investigate the expression ofAPE1/Ref-1, PCNA and CD34protein in65formalin-fixedparaffin-embedded ovarian cancer tissues. Analyzing the relationshipbetween the expression of APE1/Ref-1with proliferation andangiogenesis of ovarian cancer.2. The effect of APE1/Ref-1on biological behaviour of ovariancancer cell including proliferation, apoptosis and invasion:a) Using Western blot and RT-PCR detect the expression of APE1/Ref-1protein and mRNA in five ovarian cancer cell lines.Immunofluorescence was used to detect the APE1/Ref-1subcellularlocalization of HO-8910cells.b) HO-8910cells were transfected with APE1-pEGFP-N1plasmid or thecontrol vector by liposome2000. Fluoroscope was used to detect thetransient transfection efficiency.c) Using G418selection to obtain the stable expression cell colonies ofHO-8910. Fluoroscope, Western Blot and realtime PCR wereemployed to detect the expression of target gene.d) Using MTT assay method，clone formation test and flow cytometry analysis to investigate the effect of APE1/Ref-1on HO-8910cellproliferation. Using cell adhesion experiment, cell scratch experimentand transwell chamber experiment to analyze the effect ofAPE1/Ref-1on HO-8910cell adhesion, immigration and invasion.e) Using Western blot to investigate the expression ofAKt、p-AKt、CyclinD1、MMP2and E-cadherin.3. The effect of APE1/Ref-1on cisplatin resistance of ovarian cancerin molecular level:a) MTT assay and flow cytometry analysis were used to analyze theeffect of APE1/Ref-1on HO-8910cell proliferation and apoptosis bycisplatin induced.b) Western blot was used to investigate the activated expression ofCaspase-4、-8、-9proteins in cisplatin-induced apoptosis process.c) Flow cytometry analysis and Western blot were used to analyze theeffect of APE1/Ref-1on mitochondria apoptotic pathway. Special kitswere used to detect the change of mitochondrial transmembranepotential and intra-cellular ATP level. Western blot was used to detectthe expression of cytochrome C, Bcl-2, Bax and cleaved caspase-3which were involved in mitochondria apoptotic pathway.Results1. The relationship between APE1/Ref-1expression and clinicalpathology, angiogenesis and cell proliferation of ovarian cancer:a) By immunohistochemical analysis, in65ovarian cancer specimens,the expression of APE1protein was positive in58samples (89.23%),and highly expressed in45ovarian cancer patients (69.23%). Theexpression of APE1protein was negative in7samples (10.77%), and lowly expressed (including negative and low expression) in20samples (30.77%). The subcellular localization of APE1wascytoplasm only, nucleus only or cytoplasm and nucleus both. Thenucleal localization only was rare in65ovarian cancer samples.b) By immunohistochemical analysis, the expression of APE1proteinwas related with FIGO stage, differentiation and lymphadenmetachoresis (P<0.05) rather than age, family history, pathologysubtypes, tumor size and ascites(P>0.05); the expression of APE1protein was decreased from poor to well differentiated ovarian cancersamples and increased from stage Ⅰ to Ⅳ samples. APE1proteinhighly expressed in lymphaden metachoresis samples.c) By immunohistochemical analysis, PCNA localized in the nuclei ofcells and expressed highly in60%(39/65) ovarian cancer samples.The positive immunostaining rage was from6.9-95.6%. The PCNAindex was53.5±2.3in ovarian cancer samples with high APE1expression (grade III+IV), and the data was decreased to18.6±3.0inovarian cancer samples with low APE1expression (grade I+II)(P<0.05).The PCNA index was40.3±4.1in ovarian cancer samples with positiveAPE1expression, and the data was decreased to12.1±2.6in ovariancancer samples with negativeAPE1expression (P<0.05);d) By immunohistochemical analysis, in65ovarian cancer samples theMVD was50.7±6.5in samples with high APE1expression (gradeIII+IV), and the data was decreased to23.6±3.0in samples with lowAPE1expression (grade I+II)(P<0.05). The PCNA index was39.3±4.1in samples with positive APE1expression, and the data was decreased to20.1±3.5in samples with negativeAPE1expression (P<0.05); e) By Western Blot analysis, in36fresh ovarian cancer samples, theexpression of APE1protein increased along with the decreased levelsof differentiation and increased FIGO stages. The expression of APE1was higher in lymphaden metachoresis samples than in no lymphadenmetachoresis samples. These results were in accordance withimmunohistochemical results.f) By realtime PCR analysis, the expression of APE1mRNAwas relatedwith FIGO stage, differentiation and lymphaden metachoresis (P<0.05)rather than age, family history, pathology subtypes, tumor size andascites(P>0.05); These results were in accordance withimmunohistochemical and Western Blot results.2. The effect of APE1/Ref-1on biological behaviour of ovariancancer cell including proliferation, apoptosis and invasion:a) By Western blot and realtime PCR analysis, there were expressions ofAPE1protein and mRNA in all five ovarian cancer cell lines. Theexpression was decreased progressively in SKOV3、CP70、A2780、8910PM、HO-8910cell lines.b) By immunofluorescence analysis, the subcellular localization of APE1protein in HO-8910cell was both cytoplasm and nucleus.c) HO-8910cells were transfected with APE1-pEGFP-N1plasmid for48hours. By fluorescence microscope, it was found that most cellsbecame round and the transient transfection efficiency wasapproximate50-60%.Using G418selection we obtained the stableexpression cell colonies of HO-8910; Using fluorescence microscope,it was found the stable transfection efficiency was about95%.d) By Western blot and RT-PCR analysis, the expression of APE1 mRNA and protein were notable increased in HO-8910cells withAPE1-pEGFP-N1plasmid transfected.e) By curve of growth analysis, the HO-8910cells withAPE1-pEGFP-N1plasmid transfected proliferated more quickly. Byclone-forming assays analysis, the clone-forming rate was increasedin the HO-8910cells with APE1-pEGFP-N1plasmid transfected. Byflow cytometry analysis, the ratio of G1phase of HO-8910cells withAPE1-pEGFP-N1plasmid transfected was decreased and the ratio ofG2+S phase was increased.f) The bioadhesion to matrigel, invasion ability of HO-8910cells withAPE1-pEGFP-N1plasmid transfected was increased. There was nosignificant difference in immigration ability.g) By Western blot analysis, pAKt, Cyclin D1, MMP-2and E-cadherinprotein expression increased in HO-8910cells with APE1-pEGFP-N1plasmid transfected. There was no significant difference of AKtprotein expression.3. The effect of APE1/Ref-1on cisplatin resistance of ovarian cancerin molecular level:a) By MTT analysis, treated with cisplatin at the same dose, theinhibiting rate and IC50to DDP was not significantly differentbetween HO-8910cells transfected with the control vector and normalHO-8910cells (P>0.05). The inhibiting rate of HO-8910cellstransfected with APE1-pEGFP-N1plasmid was significantly lowerthan that of transfected with the control vector (P>0.05), and the IC50to DDP was increased1.35fold (from19.84μM to29.86μM).b) Apoptosis analysis by flow cytometry showed that treated with cisplatin, the apoptosis rate of normal HO-8910cells enhanced from4.5±0.6%to74.6±8.1%. The rate was not significantly differentbetween HO-8910cells transfected with the control vector and normalHO-8910cells (P>0.05). The apoptosis rate of HO-8910cellstransfected with APE1-pEGFP-N1plasmid was significantlydecreased13.1%than that of transfected with the control vector.c) By Western blot analysis, the normal HO-8910cells were treated withcisplatin for12hours, there were significantly expression of cleavedcaspase-4and cleaved caspase-9proteins. There was no significantlyexpression of cleaved caspase-8all the time. DDP induced caspase-4and caspase-9activation but not caspase-8.d) Mitochondrial transmembrane potential analysis by flow cytometryshowed that treated with cisplatin for24hours, JC-1%of HO-8910cells transfected with APE1-pEGFP-N1plasmid was enhanced15%than that of transfected with the control vector (65.69±6.53vs.50.87±9.61); Intracellular ATP level analysis: Treated with cisplatinfor24hours, ATP level of HO-8910cells transfected withAPE1-pEGFP-N1plasmid was significantly enhanced than that oftransfected with the control vector (8.56±1.73vs4.64±1.03).e) By Western blot analysis, the expression of Cyt c protein in cytoplasmand mitochondria of normal HO-8910was coincident with HO-8910cells transfected with the control vector, but the expression in cytoplasmof HO-8910cells transfected with APE1-pEGFP-N1plasmid was lowerand in mitochondria was higher than the other two groups.f) The expression of Bcl-2、Bax and cleaved caspase3protein in normalHO-8910was coincident with HO-8910cells transfected with the control vector, but the expression of Bcl-2in HO-8910cellstransfected with APE1-pEGFP-N1plasmid was increased and Baxand cleaved caspase3were decreased than the other two groups.Conclusion1. In ovarian cancer, the expression of APE1was relevant with celldifferentiation, FIGO stages, and lymphaden metachoresis. Theexpression of APE1was related with PCNA index and MVD. APE1plays an important role in on tumorigenesis and progression.2. Up-regulating the expression of APE1can enhance the proliferation ofHO-8910cells in vitro. The effect was exerted by activation ofPI3K/AKt pathway and APE1maybe the potential regulatory factor.3. Up-regulating the expression of APE1can enhance the adhesion andinvasion ability of HO-8910cells in vitro. The effect was exerted byup-regulated MMP-2and E-cadherin expression.4. Up-regulating the expression of APE1can increase the sensitivity toDDP and inhibit apoptosis by DDP induced of HO-8910cells in vitro.APE1can enhance the tolerance of DDP in HO-8910cells.5. The effect that DDP induced apoptosis of HO-8910cells was exertedby activation of mitochondria and endoplasmic reticulum apoptosispathway, rather than by Fas/FasL apoptosis signal pathway.6. The effect of APE1on DDP resistance of HO-8910cells in vitromight be attributed to APE1’s function on regulation mitochondriaapoptosis pathway.