Functional And Mechanistic Investigation Of PARP-1 And EMT In Ovarian Cancer

BackgruondOvarian cancer is one of the female reproductive system common malignant tumor,its incidence gynecologic malignant tumors in the third,after cervical cancer and endometrial cancer.However,patients with ovarian cancer with ascites or pelvic mass,most already is late clinical.Cytoreductive surgery and supplemented by platinum,paclitaxel combination chemotherapy is the important means for patients with ovarian cancer.And long plagued ovarian chemotherapy is the biggest obstacle to the development of drug resistance of tumor cells.Ovarian cancer patients of platinum drugs based on MDT first chemotherapy response rate was 85%,but most of the patients by chemotherapy meets the problem is chemotherapy drug resistance and failure finally.Ovarian cancer drug resistance,especially the occurrence of platinum resistance mechanisms and reverse way has become extremely urgent and important research subject.So the ovarian cancer drug resistance mechanisms,helps to take corresponding measures to reverse the resistance,improve patients sensitive to chemotherapy drugs,thus improve the survival rate and improve the after.In recent years,the research of cancer drug resistance mechanism has great progress,including the excessive expression of resistant protein,some protease’ changing in cells,which enhance cellular detoxification function,improve DNA damage repair ability,change the target molecules,etc.The enhancements of DNA damage repair ability is thought to be leading to important mechanism of drug resistance of tumor cells.Poly-ADP ribose polymerase 1(PARP-1)is a protein that exists in most eukaryotic cells posttranslational modification enzyme,and is a key molecular regulation of DNA damage repair pathways.In DNA damage repair,DNA replication,regulation of cell proliferation differentiation and apoptosis,plays an important role in the stability of the genome.When there is a gap or free end of the DNA,PARP 1 is activated,as a receptor molecules of DNA damage,recognition,binding to DNA breakup,activation,poly ADP ribose receptor proteins catalyzed base,participate in the repair of DNA.Research has shown that tumor cells after radiation therapy or cell.When DNA is widly damaged,PARP-1 can accelerate cell apoptosis.Research has shown that PARP-1 expresses higher in malignant tumour cells than normal cells,such as central nervous system tumors,malignant melanoma of skin,colorectal neoplasmas.Silencing PARP-1 or PARP-1 inhibitor can increase the cellular sensitivity of ovarian cancer cisplatin resistant cells to cisplatin effectively.Epithelial mesenchymal transitions(EMT)in the original play an important role in the invasion and metastasis of tumor cells.EMT is the polarity of epithelial cells into mesenchymal cells and invasion and migration ability of process,involved in embryonic development,organ formation and tissue fibrosis,malignant tumor progression of a variety of physiological and pathological process,is the key to tumor cell invasion and metastasis occurred.Studies have shown that EMT also play a key role in ovarian malignant progress,changes in epithelial ovarian cancer cells interstitial markers associated with tumor stage,prognostic factors.In recent years,more and more studies have found that EMT not only plays a significant role in tumor invasion and metastasis,and also there may be a correlation with tumor chemotherapy drug resistance.Mitogen activated protein kinase(MARK)pathway is one of important signal to the system of the organism,participating in regulating cell growth,proliferation,differentiation and malignant transformation and many other biological effect,is also important in the process of EMT regulation pathway.MARKs is a family that has many members,which play an important role in cell signaling has 3 kinds:JNK,p38,ERX.There are reports show PARP1/2 can activate with ERK1/2,JNK1 and other related MAPK signal pathways that can regulate gene transcription.The activation pathway of PARP-1 by pERK2 is different from the damaged DNA,we infer that ERK2 can regulate the expression of PARP-1.In the absence of DNA damage,PARP-1 can be activated by pERK1/2 directly without depending on the activity of kinase.We can infer,MAPKs participate in EMT with PARP-1,influence the drug resistance of tumor cell further.The molecular marker of EMT contains E-cadherin,vimentin,N-cadherin,fibronectin.Vimentin and E-cadherin are the key signature molecules.Vimentin is an intermediate filament protein in mesenchymal cell,with microtubule,microfilament form a cell support network and keep cells stability.The function of vimentin also contains:influence cell migrate,participate in cell signal transduction,regulate cell apoptosis,regulate gene expression.The higher expression of vimentin,the prognosis of tumor is worse.E-cadherin is a calcium-dependent single transmembrane glycoprotein in the distribution of epithelial,taking part in cell signal transduction,tissue morphogenesis,embryonic development.E-cadherin is regarded as epithelial cell adhesion agent,the reduced expression of E-cadherin is an important sign in EMT.We will make a further research whether PARP-1 take part in EMT affect ovarian cancer chemotherapy drug resistance,and effects of PARP-1 and EMT on epithelial mesenchymal transitions of the ovarian cancer.Part one Effects of PARP-1 inhibitor and ERK inhibitor on epithelial mesenchymal transitions of the ovarian cancer SKOV3 cellsObjective:To assess the effects of the poly(ADP-ribose)polymerase-1(PARP-1)inhibitor PJ34 and ERX1/2 inhibitor U0126 on the proliferation and epithelial mesenchymal transitions(EMT)of cisplatin resistant ovarian cancer SKOV-3 cells.Methods:Proliferation of SKOV-3 cells was evaluated using a 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide assay with PJ34 and U0126 treatment.Expression changes of E-cadherin and vimentin with PJ34 and U0126 treatment was examined using Western blot and quantitative PCR.In addition,invasion assay was performed in cells treated with PJ34 and U0126.Results:PJ34 and U0126 inhibited proliferation of SKOV-3 cells in a time dependent manner.PJ34 and U0126 suppressed the expression of vimentin and enhanced the expression of E-cadherin.PJ34 and U0126 reduced cell invasion.The inhibitory effects of PJ34 and U0126 were stronger than PJ34 alone.PJ34 inhibited the proliferation and invasion of SKOV-3 cells which can be enhanced by ERK1/2 inhibitor U0126.Conclusion:1.PARP-1、ERK1/2 affect ovarian cancer’s biological behavior through EMT.2.Combination of PARP inhibitor and ERK1/2 inhibitor can improve the clinical outcome of ovarian cancer patients3.Novel PARP-1 inhibitor and ERK1/2 inhibitor with low toxicity and high specificity need to be tested in the future.4.Targeted therapy using PARP inhibitor and ERK1/2 inhibitor may represent a novel therapeutic strategy in the prevention and intervention of ovarian tumors.Part two Expression of PARP-1、E-cadherin and vimentin in epithelial ovarian cancer SKOV3 cellsObjective:To investigate the expression of PARP-1,E-cadherin and vimentin in ovarian cancer SKOV-3 cells and cisplatin resistant ovarian cancer SKOV-3 cells,to investigation the relationship cisplatin resistant with PARP-1 and epithelial-mesenchymal transition(EMT).Methods:The expression of PARP-1,E-cadherin,vimentin was detected in the ovarian cancer SKOV-3 cells and cisplatin resistant ovarian cancer SKOV-3 cells by qRT-PCR and western blotting.Results:The positive expression rates of PARP-1,vimentin were significantly higher in the ovarian cancer SKOV-3 cells than that in the cisplatin resistant ovarian cancer SKOV-3 cells,whereas the positive expression rate of E-cadherin was the opposite.The differences were statistically significant(P<0.05).The relative mRNA,protein expression of PARP-1,vimentin in the ovarian cancer SKOV-3 cells was significantly higher than that in the cisplatin resistant ovarian cancer SKOV-3 cells(P<0.05),while the mRNA,protein expression of E-cadherin in the ovarian cancer SKOV-3 cells was remarkably lower than that in the cisplatin resistant ovarian cancer SKOV-3 cells(P<0.05).Conclusion:1.PARP-1,EMT can affect ovarian cancer drug resistance of platinum.2.PARP-1 can affect ovarian cancer drug resistance of platinum by vimentin andE-cadherin3.If there are other factors cooperate with PARP-1 influence the expression of vimentin and E-cadherin,remains to be further research.