| Epithelial ovarian cancer(EOC)is a common gynecological malignant tumor with a high mortality rate due to the initially diagnosis is advanced stage.The main cause of EOC's low survival rate is the high incidence of general dissemination metastasis.And tumor growth,invasion and metastasis depend on the tumor angiogenesis.The treatment of ovarian cancer is mainly based on surgery and postoperative chemotherapy,but the traditional chemotherapy drugs bring ovarian cancer recurrence,resistance,low survival rate and other issues.A large number of recent studies have shown that tumor angiogenesis plays an important role in the malignant biology of tumors.Finding new drugs of anti-angiogenesis can bring new directions to the treatment of ovarian cancer.Sphingosine-1-phosphate(S1P)is a kind of important metabolite of sphingomyelin from cell membrane,involved in cell proliferation,apoptosis,angiogenesis and regulation of tumor development.Sphingosine kinase(SphK),a key kinase in cells,catalyze sphingosine to generate S1P,involved in the regulation of cellular signaling pathways.S1P is a special ligand of G protein coupled receptor(GPCR)and mediates a variety of bioactive roles with the dual function as the first and second messenger.The biological roles of S1P are mediated by the action of five different S1PR(S1PR1-5).S1PR1,S1PR2 and S1PR3 are broadly expressed in various tissues and involved in the regulation of angiogenesis.There is no evidence that S1PR4 and S1PR5 paly a regulatory role in vascular function.In vitro and in vivo,it's confirmed that S1P play a crucial role in regulating angiogenesis in a variety of physiological and pathological models,such as tumor,embryonic vasculation and ischemic injury disease process.The role and mechanism of S1P/S1PR pathway in ovarian cancer angiogenesis has not been confirmed.Part 1.S1P and S1PR expression in serum and tissues of ovarian cancer patients and its clinical significanceObjective:We aimed to detect the level of S1P in serum of ovarian cancer patients and investigate the expression of S1PR in samples from ovarian cancer patients to explore the correlation between S1P/S1PR and tumor angiogenesis.Methods:The serum S1P concentration was detected by ELISA.The expression of S1PR1,S1PR2,S1PR3,CD34 and CD105 in tissues were determined by immunohistochemistry.Pearson correlation analysis was used to analyze the correlation between S1PR expression and microvascular density MVD in ovarian cancer.Results:1.The S1P level in serum of patients with ovarian cancer was higher than that in benign ovarian tumor(p<0.01),and the S1P level in ovarian cancer patients with stage?-? was significantly higher than that in stage ?-?(p<0.01);2.MVDCD34 and MVDCD105 in ovarian cancer tissue was significantly higher than that in benign ovarian tumor(p<0.01),MVDCD34 and MVDCD105 were significantly correlated with FIGO stage and tumor grade(p<0.01);3.High S1PR1 expression was detected in ovarian cancer tissues(p<0.05),significantly associated with FIGO stage,tumor grade,lymph node metastasis,distant metastasis and residual tumor size;4.S1PR3 was highly expressed in ovarian cancer(p<0.05),significantly correlated with FIGO stage and tumor grade;5.There was no difference in S1PR5 expression between ovarian cancer and benign ovarian tumor tissues;6.S1PR1 high expression was significantly correlated with MVDCD34 and MVDCD105(p<0.01),S1PR3 high expression was significantly correlated with MVDCD34 and MVDCD105(p<0.01,p<0.05).Conclusion:The S1P level in serum of patients with ovarian cancer was significantly higher than that in patients with benign ovarian tumor.MVDCD34 and MVDCD105 in ovarian cancer tissues were significantly higher.We detected high expression of S1PR1 and S1PR3 in ovarian cancer tissue.High S1PR1 expression was significantly associated with FIGO stage,tumor grade,lymph node metastasis and distant metastasis.High S1PR3 expression was significantly correlated with FIGO stage and tumor grade.The expression of S1PR1 and S1PR3 were positively correlated with MVDCD34 and MVDCD105 in ovarian cancer tissues.Part 2.Effects of S1P and S1PR on angiogenesis in ovarian cancer cellsObjective:The study was to investigate that ovarian cancer cells with S1P could promote tube formation and to explore the roles and mechanism of S1PR in ovarian cancer cells by detecting the expression of angiogenic factors.Methods:1.The tube formation assay of EA.hy926 cells was to explore the effect of cell culture supernatant of ovarian cancer cell lines(SKOV3,HO8910)with S1P;2.The qRT-PCR was used to identify the angiogenic factors expression changes in ovarian cancer cells incubation with S1P and cells were starved for 48h before S1P treatment in RPMI-1640 without FBS;3.SiRNA interference sequences silencing S1PR1,S1PR2 and S1PR3 gene were designed and synthesized.The angiogenic factors change levels in S1PR gene silencing ovarian cancer cell lines were detected by qRT-PCR.Results:1.The tube formation of EA.hy926 cells was significantly increased by the ovarian cancer cells culture supernatants with S1P(P<0.05);2.The mRNA expression of IL-8,IL-6,VEGF were significantly increased by S1P in SKOV3 and HO8910 cells(P<0.01);3.IL-8,IL-6 and VEGF mRNA expression levels were inhibited in S1PR1 and S1PR3 gene silencing SKOV3 cells.Conclusion:The study indicated that S1P could effect on ovarian cancer cells to promote tumor angiogenesis and IL-8,IL-6,VEGF may play a role in this process.When S1PR1 or S1PR3 was silenced,the expression level of IL-8,IL-6 and VEGF decreased.And S1PR2 was silenced,IL-8,IL-6,VEGF expression level was not affected by that.S1P may play a role in the regulation of tumor angiogenesis by acting S1PR1\S1PR3 in ovarian cancer cells.Part 3.Roles of SphKl and S1PR1/3 on angiogenesis of human ovarian cancer xenograft model in nude miceObjective:We aimed to explore the roles of SphKl and S1PR1/3 on angiogenesis of ovarian cancer cells in nude mice.Methods:Tumor xenografts were established by intraperitoneal injection of nude mice.After mice were sacrificed,the tumor metastasis were observed between control and SKI-?(SphKl inhibitor)group,VPC23019(S1PR1/3 antagonist)group.The MVDCD34 and MVDCD105 were evaluated in tumor samples by immunohistochemistry.Results:1.The peritoneal metastatic tumors were significantly inhibited by SKI-?(p<0.01),MVDCD34 and MVDCD105 were also reduced(p<0.01);2.The peritoneal metastatic tumors were significantly inhibited by VPC23019(p<0.01),MVDCD34 and MVDCD105 were also reduced(p<0.05,p<0.01).Conclusion:Ovarian cancer development dependeded on tumor angiogenesis.And the process could be inhibited by inhibition of SphKl or S1PR1/S1PR3.S1P/S1PR pathway was expected to become a new target for ovarian cancer treatment. |
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