The Study Of The Role Of APE1in Chemoresistance Of Epithelial Ovarian Cancer

Epithelial ovarian cancer (EOC), is one of the most frequent malignancies in femalereproductive system and has the highest mortality rate of all gynecological cancersworldwide. This high mortality is attributed in part to the lack of any reliable earlydetection method resulting in the majority of patients being diagnosed with advancedstage III or stage IV disease. Currently, the standard of care for ovarian cancer isaggressive surgical debulking followed by platinum-based combination chemotherapy.Although ovarian cancer is among the most chemosensitive malignancies at the time ofinitial treatment(surgery and taxane/platinum-based chemotherapy), most patients willultimately develop tumor recurrence and succumb to chemoresistant disease. Primary orsecondary chemoresistance is an important factor affecting chemotherapy and causedtumor recurrence, metastasis and higher mortality, which bring huge difficulties to clinicaltreatment. Therefore, it’s very importment to clarify the mechanisms of ovarian cancerrecurrence and chemoresistance and to take some effective measures to prevention and treatment of ovarian cancer.APE1is a multifunctional protein in DNA base excision repair pathway. It not onlyhas the activity of endonuclease but also is an essential enzyme in base excision repairpathway. It also acts as a major redox-signaling factor that involve in transcription factorregulation, tumor progression, oxidative stress, cell cycle and apoptosis. APE1plays animportment role in DNA damage and repair and apoptosis pathway regulation. A growingbody of evidence has demonstrated that APE1is not only critical for tumorigenesis andprogression in several human tumors and cancer cell lines, but also closely related to thechemosensitivity and radiosensitivity. It is reasonable to postulate that APE1maycontribute to the molecular mechanism of resistance to cisplatin-based chemotherapy, as avery potential tumor therapeutic target.Our previous results find that:①The expression and subcellular localization ofAPE1are involved in tumorigenesis, progression, chemosensitivity and prognosis inovarian cancer.②The cisplatin-resistant A2780/cis cells demonstrated higher expressionprotein levels of APE1than the cisplatin-sensitive A2780cells.③The overexpression ofAPE1had significant enhancement in cisplatin sensitivity. Above all the data, weproposed and confirmed that APE1involved in this resistance may provide new treatmentmodalities for ovarian cancer.A growing body of evidence has demonstrated that repair functions of APE1plays animportment role in tumorigenesis, progression, chemosensitivity and prognosis of variouscancers. Selective targeting of this DNA repair enzyme using RNA interference or specificsmall-molecule inhibitors blocking APE1repair functions have been shown to be effectiveinsensitizing cancer cells to both adiation and chemotherapy in vivo and in vitro.Some results about APE1’s role in the chemoresistance process in ovarian cancerhave been clarified. However, a number of questions are unclear. Our understanding ofAPE1DNA repair functions have been fully, but little is known about its Redox function.What important role does the redox function of APE1have in the process ofcisplatin-based chemotherapy? Whether intervention APE1can effectively enhancechemosensitivity in ovarian cancer? On the basis of the results of preliminary studies, some works have been done：According to characteristics of APE1structure, we constructed Ad5-APE1-EGFP andAd5-Redox-APE1-EGFP recombinant adenovirus, which contains APE1gene and itsredox structure, respectively. To detect the effect of the overexpression of APE1and itsredox in ovarian cancer cells, The infection efficiency was confirmed byfluorescence microscope and qRT-PCR and Western blot were performed to evaluate theexpression of APE1mRNA and protein respectively. MTT assays, clone formation assaysand transwell assays were performed to evaluate the effects of Ad5-APE1-EGFP andAd5-Redox-APE1-EGFP recombinant adenovirus on cell proliferation and Invasion. Drugsensitivity assays were performed to determine the cisplatin sensitivity of ovarian cancercells. The effect of Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFP recom-binant adenovirus on cell cycle was observed by flow cytometry analysis in infectedovarian cancer cells. Hoechst33258staining were performed to showed the apoptosis ratein ovarian cancer cells infected with Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFPrecombinant adenovirus after treated with cisplatin.Immunofluorescence was used toobserves ubcellular localization of APE1in ovarian cancer cells infected with Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFP. We constructed subcutaneous tumor model in nudemice and further confirmed that APE1and its resox played an iomportment role inchemoresistance in ovarian cancer and in tumor proliferation in vivo. We also constructedRedox-APE1siRNA lentivirus to down regulation the expression of APE1, and observedthe impact on biological function in ovarian cancer cell lines.Some results have been obtained：According to characteristics of APE1structure, wesuccessfully constructed recombinant adenovirus Ad5-APE1-EGFP andAd5-Redox-APE1-EGFP, Which contains APE1gene and its redox structure, respectively.The95%of ovarian cancer cells were infected with Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFP recombinant adenovirus by fluorescence microscope and qRT-PCR andWestern blot results showed that the expression of APE1mRNA and protein weresignificantly increased in ovarian cancer cells infected with Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFP, respectively（P＜0.05）. MTT assays, clone formation assays and transwell assays indicated that ovarian cancer cells infected with Ad5-APE1-EGFP andAd5-Redox-APE1-EGFP can increase the cell proliferative activity and the invasionability. Drug sensitivity assays demonstrated that the chemosensitivity of cisplatin-basedchemotherapy was significantly reduced. Cell cycle analysis by flow cytometry showedthat ovarian cancer cells infected with Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFPresulted in accumulation in G2and S phase. While the proportion of G0/G1phase weredecreased. Hoechst33258staining showed that the apoptosis rate was weakly decreased inovarian cancer cells infected with Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFP aftertreated with cisplatin. Immunofluorescence detection showed that the subcellular locationof APE1in ovarian cancer cells were cytoplasmic. We further confirmed that APE1andits resox played an iomportment role in chemoresistance in ovarian cancer and in tumorproliferation in vivo. We also successfully constructed Redox-APE1siRNA lentivirus todown regulation the expression of Redox-APE1, and observed the impact onbiological function in ovarian cancer cell lines. These results provided strong evidencesfor highlighting the potential of APE1to serve as a target for cancer therapeutics.In conclusion, on the basis of preliminary work, we successfully constructedrecombinant adenovirus Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFP, Which containsAPE1gene and its redox structure, respectively. We also successfully constructed APE1siRNA lentivirus to down regulation the expression of APE1. We observed the impact onbiological function and the chemosensitivity to cisplatin in vivo and in vitro. These resultsfurther provided strong evidences for the role of APE1in chemoresistance of ovariancancer and also may provide new treatment modalities for ovarian cancer.