Studies Of The Mechanisms Of ROS/HO-1 Pathway In The Epithelial-mesenchymal Transition Induced By Autophagy Defect In Ovarian Cancer Cells And The Clinical Significance Of HO-1 In Ovarian Cancer

BackgroundOvarian cancer is the leading cause of death among all gynecological malignancies,which accounting for 3%gynecological malignancies,while,5%of all the new deaths.Though both cytoreductive surgery and cisplatin-based chemotherapies have contributed to ovarian cancer treatment,the prognosis of ovarian cancer patients remained gloomy.The high rate of distant metastasis is still considered to be the leading cause of recurrence and death in ovarian cancer patients.Based on data from Surveillance,Epidemiology,and End Results Program(SEER)18 2006-2012,the five-year survival rate was up to 92.1%of patients diagnosed with Stage I.However,over 75%of ovarian cancer patients were diagnosed with metastasis.As a result,the five-year survival rate declined sharply to 17%(stage IV).Hence,it’s of great importance for identifying the molecules or signaling pathways involved in metastasis to improve treatment efficiency and prognosis in ovarian cancer.Epithelial-mesenchymal transition(EMT)process was firstly identified in the context of embryogenesis,where it made differentiated epithelial cells transform to invasive mesenchymal cells,participating in embryonic development,tissue damage regeneration,organ fibrosis and cancer metastasis.EMT plays a critical role in tumor spreading and dissemination,which tumor cells lose their epithelial morphology and detach from the primary site and invade surrounding tissues and blood vessels.Emerging evidence has been confirmed that EMT process participates in the onset of metastasis in epithelial ovarian cancer(EOC).Moreover,EMT provides tumor cells properties of cancer stem cells,apoptosis resistance and immune privilege,resulting in the distant metastasis and treatment obstacle.Therefore,the targeted treatment that can reverse EMT may inhibit the metastasis of EOC and benefit EOC patients.Autophagy has reported to be vital in the maintenance of cellular homeostasis by cellular cleaning.Autophagosome degrades cell contents such as proteins,lipids,carbohydrates,nucleates,even damaged or old organelles and pathogens through combining with lysosomes.Emerging evidence has reported that autophagy played important roles in cancer metastasis.It has been clarified that autophagy could reverse EMT by degrading transcript factors of EMT such as Snail,Slug and Twist.Autophagy can promote EMT and cancer metastasis in cholangiocarcinoma and hepatocellular carcinoma,while,autophagy can reverse EMT and inhibit metastasis in glioma.However,it’s still unknown whether autophagy can impair the migration and invasion in ovarian cancer by EMT.Heme oxygenase-1(HO-1)is one of the heat shock proteins,weight of 32Kda.HO-1 was firstly identified as the rate-limiting enzyme,which catalyzes the degradation of cellular heme to liberate free iron,carbon monoxide(CO)and biliverdin in mammalian cells.Meanwhile,HO-1 can be induced by different oxidative stress such as hypoxia,ROS,heavy metals,UV and so on,participating in the maintenance of cellular homeostasis.Emerging evidence has proved that HO-1 overexpression is found in many cancers and associated with poor prognosis and tumor stage,including prostate cancer,renal carcinoma,rectal cancer,thyroid carcinoma and glioblastoma.Moreover,HO-1 was reported to be able to promote tumor cells proliferation,angiogenesis,chemotherapy resistance and distant metastasis.However,the roles of HO-1 played in ovarian cancer need to be elucidated.Our work provides the first study to investigate the relationship between autophagy and aggressivity of ovarian cancer and we further investigate the mechanisms of ROS/HO-1 pathway in EMT induced by autophagy defect in ovarian cancer cells and clinical significance of HO-1 in ovarian cancer patients.PartⅠ The study of effect and mechanism of autophagy on themigration and invasion abilities of ovarian cancer cells Objective:1.To test the autophagy level,migration and invasion abilities of A2780 and Skov-3 ovarian cancer cells.2.To investigate the effects and mechanisms of autophagy on the migration and invasion abilities of A2780 and Skov-3 ovarian cancer cells.Methods:Western blotting was used to examine the expressions of autophagy related proteins(Beclin-1,P62,LC3)and EMT associated proteins(Keratin,Vimentin,N-cadherin,Zeb1)of ovarian cancer cells.Transwell chamber was used to test the migration and invasion abilities of ovarian cancer cells.Immunofuresence was performed to examine the immunoreactivity of epithelial marker(Keratin)and mesenchymal marker(Vimentin)of ovarian cancer cells.EBSS and Rapamycin was used to induce autophagy,while,chloroquine was used to inhibit autophagy.CCK-8 was used to detect the cell viability of ovarian cancer cells after autophagy induction.Results:1.Identification of morphology,autophagy level,migration and invasion abilities of A2780 and Skov-3 cells.Inverted microscope was used to identify the morphology of A2780 and Skov-3 cells:A2780 cell exhibited a round like phenotype,while Skov-3 cell exhibited a fibroblastoid like phenotype.Transwell chamber was used to test the migration and invasion abilites of A2780 and Skov-3 cells:Skov-3 exhibited higher invasive ability than A2780 cell(P<0.01).Western blotting was used to examine the expressions of autophagy related proteins and EMT associated proteins of A2780 and Skov-3 cells:Skov-3 showed lower level of autophagy than A2780 cell,with up-regulated expressions of autophagy-related protein(P62),mesenchymal markers(Vimentin,N-cadherin),transcript factor(Zeb 1)and down-regulated expressions of autophagy-related proteins(Beclin-1,LC3Ⅱ/Ⅰ),epithelial marker(Keratin)(P<0.01).2.Effect of autophagy on the proliferation of A2780 and Skov-3 cells.CCK-8 was used to detect the cell viability of ovarian cancer cells after autophagy induction(incubation with EBSS and 20nM Rapamycin):Proliferation was inhibited in a time-dependent manner(P<0.05).Proliferation was promoted at 3h,and proliferation inhibition began at 6h.The survival rates at 24h were as followed,EBSS:A2780:86.8%±2.6%,Skov-3:88.8%±4.3%;Rapamycin:A2780:89.0%±2.9%,Skov-3:91.3%±3.6%.The survival rates of both cells were above 70%after 48h.3.Effect of autophagy on the EMT process of A2780 and Skov-3 cells.Western blotting was used to examine the expressions of autophagy related proteins and EMT associated proteins after autophagy induction:The expressions of LC3Ⅱ/Ⅰ and Beclin-1 were up-regulated and P62 were inhibited in a time-dependent manner(P<0.01).With the induction of autophagy,the expressions of mesenchymal markers(Vimentin,N-cadherin)and transcript factor(Zeb1)were down-regulated and epithelial marker(Keratin)was up-regulated(P<0.05).Meanwhile,After incubating with EBSS for 24 h,immunostaining of Keratin increased with an decreased expression of Vimentin in both cells.4.Effect of autophagy inhibition on EMT process,the migration and invasion abilities of A2780 and Skov-3 cells.Transwell chamber was used to test the migration and invasion abilities:After incubation with EBSS,EBSS+Chloroquine,Rapamycin,Rapamycin+Chloroquine for 24h,the migration and invasion abilities were inhibited by incubating with EBSS or 20nM Rap for 24 h(P<0.01);however,they were significantly reversed by incubating with 20μtM CQ together(P<0.01).Western blotting was used to examine the expressions of EMT associated proteins:The expressions of mesenchymal markers(Vimentin,N-cadherin)and transcript factor(Zeb1)were down-regulated and Keratin,the epithelial marker was up-regulated after incubating with EBSS or 20 nM Rap for 24 h(P<0.01).However,they were also significantly reversed by incubating with 20μM CQ together(P<0.01).Conclusions:1.Autophagy level was related with migration and invasion abilities of ovarian cancer cells.2.Autophagy inhibited migration and invasion abilities of ovarian cancer cells through reversing epithelial-mesenchymal transition.Part Ⅱ The study of effect and mechanism of ROS/HO-1 pathway in EMT induced by autophagy defect in ovarian cancer cellsObjective:1.To investigate the effect of autophagy defect on migration and invasion abilities of A2780 and Skov-3 ovarian cancer cells.2.To investigate the effect and mechanism of autophagy defect on EMT of A2780 and Skov-3 ovarian cancer cells.Methods:Western blotting was used to examine the expressions of autophagy related proteins(Atg7,P62,LC3)and EMT associated proteins(Keratin,Vimentin,N-cadherin,Zebl)and HO-1 protein of ovarian cancer cells.Transwell chamber was used to test the migration and invasion abilities of ovarian cancer cells.Small interfering RNA(siRNA)was used to generate autophagy defect cells(A2780 Atg7siRNA and Skov-3 Atg7siRNA cells).Reactive oxygen species(ROS)were examined by flow cytometry.N-acetylcysteine(NAC)was used to inhibit ROS accumulation.Zinc-protoporphyrin(Znpp)was used to inhibit the expression of HO-1.Results:1.Knocking down Atg7 and generation of ovarian cancer autophagy defect cells.The autophagy defect cells(A2780 Atg7siRNA and Skov-3 Atg7siRNA cells)were generated by knockdown of Atg7 and the effect on autophagy was evaluated by western blotting for P62 and LC3Ⅱ/Ⅰ.After knockdown of Atg7,expression of P62 was up-regulated,while,ratio of LC3II/I was down-regulated(P<0.05).2.Effect of autophagy defect on migration,invasion abilities and EMT process of A2780 and Skov-3 cells.Transwell chamber was used to test the migration and invasion abilities of Atg7siRNA(autophagy defect cells),negative control and control cells:The migration and invasion abilities of autophagy defect cells(A2780 Atg7siRNA and Skov-3 Atg7siRNA cells)were increased than A2780 and Skov-3 cells(P<0.01).Western blotting was used to examine the expressions of EMT associated proteins in Atg7siRNA(autophagy defect cells),negative control and control cells:The expressions of mesenchymal markers(Vimentin,N-cadherin)and transcript factor(Zebl)were increased,while,the expression of Keratin,an epithelial marker,was suppressed in A2780 Atg7siRNA and Skov-3 Atg7siRNA cells than A2780 and Skov-3 cells(P<0.01).3.Effect of autophagy defect on intracellular ROS and expression of HO-1 in A2780 and Skov-3 cells.Flow cytometry was used to examine the expressions of ROS in Atg7siRNA(autophagy defect cells),negative control and control cells:The intercellular ROS were higher in A2780 Atg7siRNA and Skov-3 Atg7siRNA cells than A2780 and Skov-3 cells.Western blotting was used to examine the expression of HO-1 in Atg7siRNA(autophagy defect cells),negative control and control cells:The expressions of HO-1 were up-regulated in A2780 Atg7siRNA and Skov-3 Atg7siRNA cells(P<0.01).4.Effect of ROS on EMT process and expression of HO-1 in A2780 Atg7siRNA and Skov-3 Atg7siRNA cells.After incubation A2780 Atg7siRNA and Skov-3 Atg7siRNA cells with 10mM NAC for 24h,transwell chamber was used to examine the migration and invasion abilities and western blotting was used to examine the expressions of EMT associated proteins:The migration and invasion abilities were significantly inhibited(P<0.001).Meanwhile,the mesenchymal markers(Vimentin,N-cadherin)and transcript factor(Zebl)were down-regulated and the epithelial marker(Keratin)was up-regulated(P<0.05).Furthermore,the expression of HO-1 was significantly suppressed after incubating with NAC(P<0.001).5.Effect of HO-1 on EMT process in A2780 Atg7siRNA and Skov-3 Atg7siRNA cells.After incubation A2780 Atg7siRNA and Skov-3 Atg7siRNA cells with 10μM Znpp for 24h,transwell chamber was used to examine the migration and invasion abilities and western blotting was used to examine the expressions of EMT associated proteins:The migration and invasion abilities were significantly inhibited(P<0.01).Meanwhile,the mesenchymal markers(Vimentin,N-cadherin)and transcript factor(Zebl)were down-regulated and the epithelial marker(Keratin)was up-regulated(P<0.01).Conclusions:1.Autophagy defect enhanced the migration and invasion abilities of ovarian cancer cells.2.Autophagy defect promoted the EMT of ovarian cancer cells.3.HO-1 promoted the EMT process of ovarian cancer cells induced by autophagy defect through ROS/HO-1 pathway.Part Ⅲ The study of the clinical significance and biological behaviors of HO-1 in ovarian cancerObjective:1.To detect the expressions of HO-1 in ovarian cancer tissues.2.To analyze the relationship between HO-1 expressions and clinicopathological characteristics and prognosis of ovarian cancer patients.3.To investigate the effects of HO-1 on biological behaviors of ovarian cancer cells.Methods:Immunochemistry was used to detect the expressions of HO-1 in 201 ovarian tissue species(117 serous ovarian cancer,16 mucinous ovarian cancer,25 endometrioid ovarian cancer,21 clear cell ovarian carcinoma and 22 other pathologic type ovarian cancer).The association between HO-1 expressions and clinicopathological characteristics was analyzed by X2 test/Fisher ’s exact test.Survival curves were generated by Kaplan-Meier method and compared by Log-rank test.Western blotting was used to examine the expressions of EMT associated proteins(Keratin,Vimentin,Zeb1)and apoptosis associated proteins(Bcl-2,Bax)of ovarian cancer cells.Transwell chamber was used to test the migration and invasion abilities of ovarian cancer cells.CCK-8 was used to investigate cell viability of A2780 and Skov-3 cells.Zinc-protoporphyrin(Znpp)was used to inhibit expression of HO-1.Hemin was used to induce expression of HO-1.Results:1.Expressions of HO-1 in ovarian cancer tissues.Immunochemistry results of 201 ovarian cancer tissues(117 serous ovarian cancer,16 mucinous ovarian cancer,25 endometrioid ovarian cancer,21 clear cell ovarian carcinoma and 22 other pathologic type ovarian cancer)showed HO-1 immunoreactivity was predominantly found in the cytoplasm.Besides,HO-1 expression was significantly different between various pathologic type ovarian cancer tissues,highly expressed in serous ovarian cancer(P=0.0359)and lowly expressed in mucinous ovarian cancer(P=0.0124).2.Relationship between HO-1 expressions and clinicopathological characteristics of ovarian cancer patients.High expression of HO-1 was significantly associated with serous ovarian cancer(OR=1.859,95%CI=1.038-3.327),advanced FIGO stage(OR=3.656,95%CI=1.602-8.344),lymph node metastasis(OR=7.779,95%CI=2.280-26.540)and non-optimal debulking(OR=4.464,95%CI=2.137-9.323).In the meantime,low expression of HO-1 was associated with mucinous ovarian cancer(OR=0.235,95%CI=0.078-0.705).No relationship was found between age,differentiation and HO-1 expression.3.Relationship between HO-1 expressions and prognosis of ovarian cancer patients.During the follow-up,22 deaths occurred in the HO-1 low expression group(n=49)and 63 deaths occurred in the HO-1 high expression group(n=101).Moreover,the median survival for patients of HO-1 low expression was 59 month and 36 months for HO-1 high expression.Kaplan-Meier analysis and the log-rank test were used to analyze censored survival data.Patients with high expression of HO-1 had poorer overall survival(OS)compared to patients with low expression of HO-1(P=0.0002),with HR=1.639 and 95%CI= 1.023-2.254(high expression versus low expression).4.Effects of HO-1 on biological behaviors of ovarian cancer.CCK-8 was used to detect the effect of HO-1 expression on proliferation in A2780 and Skov-3 cells.The results showed cell proliferation was significantly improved after incubating with Hemin(P<0.05),while inhibited with Znpp in a dose-and time-dependent manner(P<0.05).Transwell chamber was used to examine the migration and invasion abilities:The migration and invasion abilities were increased after incubating with Hemin(P<0.01),while decreased with Znpp(P<0.05).The increased cell aggressivity by Heme was reversed by incubating together with Znpp(P<0.01).Western blotting was used to examine the expressions of proteins influenced by HO-1 expression:The expressions of mesenchymal marker(Vimentin),EMT-transcription factor(Zeb1)and anti-apoptotic protein(Bcl-2)were up-regulated after incubating with Hemin,while down-regulated with Znpp for 24 h(P<0.05).When incubating together with Heme and Znpp for 24 h,the up-regulated expressions of Vimentin,Zebl and Bcl-2 were reversed(P<0.05).On the contrary,the expressions of epithelial marker(Keratin)and pro-apoptotic protein(Bax)were down-regulated after incubating with Heme,while up-regulated with Znpp for 24 h(P<0.05).When incubating together with Heme and Znpp for 24 h,the down-regulated expressions of Keratin and Bax were reversed(P<0.05).Conclusions:1.High expression of HO-1 was associated with advanced FIGO stage and lymph node metastasis,indicating that HO-1 participated in ovarian cancer progression.2.High expression of HO-1 was associated with poor prognosis of ovarian cancer patients,indicating that HO-1 might be a predict marker of ovarian cancer prognosis.3.HO-1 enhanced ovarian cancer cells proliferation,migration and invasion through inhibiting cell apoptosis and promoting EMT process.