The Role Of Sphingosine Kinase-1(SphK1)in Ovarian Cancer Angiogenesis

Ceramide(Cer),sphingosine(Sph)and sphingosine-1-phosphate(S1P)are three important sphingomyelin metabolites involved in cell proliferation and apoptosis,angiogenesis,thereby regulating tumor development.SphK is the key kinases of intracellular phospholipid metabolism,which catalyze the Sph generates S1 P,involved in regulating the delivery of many important intracellular signal transduction pathways,thus playing an important role in the maintenance of cellular homeostasis,regulation of cell proliferation,differentiation and apoptosis.SphK is an evolutionarily conserved lipid kinase family,comprising five conserved domains.SphK was expressed in human,mouse,yeast and plants.In 1998,Olivera and others extracted SphK from rat kidney cells first,with an relative molecular mass of 49,000.Currently,SphK family has been cloned and identified with seven isozymes.Of all the isozymes,SphK1 and SphK2 were human and mouse origining.SphK1 was expressed in most normal cells in human,mainly in the cytoplasm and perinuclear regions of the cell,with no transmembrane domain;and it was expressed abnormally in tumor cells.With a variety of external factors stimulating,SphK1 can lead to the increase of intracellular S1 P concentration,as well as the increase of SphK1 release in certain types of cells.S1 P is a special ligand of G protein coupled receptors(GPCR).S1 P is a metabolite of cell membrane sphingomyelin metabolic,functioned as a first messenger and a second messenger at the same time.As a result,S1 P may mediate different pathological and physiological processes,incluing the promotion of cell survival,tumor formation,lymphocyte responsion,immune responsion,and angiogenesis.The Tissue-specific expression and subtype of G protein receptors determines the diversity of biological effects of S1 P.At present,it has been found S1 P receptors(sphingosine-1-phoshate receptor,S1PR)have 5 subtypes,from S1PR1 to S1PR5.Among them,S1PR1,S1PR2,S1PR3 are the main receptors in vessels.Studies have shown that,S1 P regulates the downstream signaling pathways by S1PR1?S1PR2 and S1PR3,functioned as a bidirectional regulatory factor in angiogenesis.It can mediate cell adhesion and migration of endothelial and vascular smooth muscle cells,promote angiogenesis stabilizing,complete vascular endothelial cell barrier,and maintain the functional of neovascularization integrity and stability.S1 P has now been found overexpressed in ovarian cancer patients,while S1 P involved in a variety of biological behavior of ovarian cancer,but the mechanism of S1 P in ovarian cancer angiogenesis is still unclear. Angiogenesis is the formation of new blood vessels from existing capillaries or post-capillary venous.It is a complex process involving multiple molecules of a variety of cells,including the activated vascular basement membrane degradation;vascular endothelial cells activation? proliferation?migration;blood vessels and vascular network reconstruction multiple steps nets,affecting tumor growth and metastasis.SphK pathway plays an important role in angiogenesis.S1 P,which producted by SphK1,can be seen as a lipid angiogenic growth factor and is involved in many physiological and pathological processes of angiogenesis.The literature suggests,SphK/S1 P signaling pathway can promote the angiogenesis of liver fibrosis.SphK/S1 P can promote the development of breast cancer by stimulating angiogenesis and lymphangiogenesis of tumer.In addition,S1 P may induce a variety of tumor cells?immune cells to secrete vascular endothelial growth factor(VEGF),interleukin-8(IL-8),interleukin-6(IL-6)and other pro-angiogenic factors,which can promote endothelial cells migration,proliferation and angiogenesis through paracrine in further.Previous studies confirmed that,VEGF promoter activity is enhanced in MS-1 murine islet cells with S1 P simulating.Applications of SphK1 inhibitor DMS to block SphK1/S1 P signaling pathway can inhibit liver cancer cells VEGF expression and angiogenesis.Thus,regulating angiogenic growth factors and angiogenesis by SphK1/S1 P may become a new direction in tumor therapy.Epithelial ovarian cancer(EOC)is a common gynecological malignant tumor and serious harm to women's health,due to the clinical symptoms are often late to discover which makes the fatality rate ranks first because it is always later period when diagnosised.It is considered,one of the main reason for the low survival rate is that the epithelial ovarian cancer is easy to transfer and dissemination of a wide range.In other hand,the growth?invasion and metastasis of tumors are dependent on tumor angiogenesis.Currently,the role of SphK in angiogenesis and its mechanism is still unclear.Based on the above study,topic of the thesis was to study the expression of SphK1 in ovarian cancer and explore its relevance with angiogenesis.Studies found that MVD is positively correlated with SphK1 expression in ovarian cancer,SphK1 silencing can inhibit ovarian cancer cells in vitro angiogenesis and inhibit ovarian cancer cells S1 P secretion and IL-8 expression,thereby affecting on ovarian cancer angiogenesis.This research was divided into three parts:(1)The relationship between the expression of SphK1 and microvessel density in ovarian cancer tissue.(2)The role of SphK1 on ovarian cancer cells pro-angiogenesis effection research(3)The role of SphK1 on nude mice ovarian cancer angiogenesis research.Part 1.The relationship between the expression of SphK1 and microvessel density in ovarian cancer tissueObjective: We aimed to identify the expression of SphK1 in human ovarian tumor tissues,analyse the correlation between SphK1 and ovarian cancer microvessel density(MVD)to further explore the correlation between SphK1 and ovarian cancer angiogenesis.Methods: SphK1 protein and CD34 protein were examined by immunohistochemistry in human malignant ovarian tumor tissues and benign ovarian tumor tissues or ovarian tissues of uterine fibroids to determin the expression levels,and calculated MVD.Pearson correlation was used to analysis the correlation of SphK1 and MVD in ovarian cancer.Results: 1.The level of SphK1 expression was higher in human ovarian cancer tisues,the difference was statistically significant(P<0.05);2.The expression of SphK1 protein was interrelated with FIGO stage and distant metastasis(P<0.05),with the increase of clinical stage,the expression of SphK1 increased;and SphK1 protein expression has no relationship with the age of patient,tumor differentiation,lymph node metastasis and histological type;3.The MVD level was higher in human ovarian cancer tisues,and the difference was statistically significant(P<0.05);4.SphK1 protein and MVD was positively correlated(r=0.567,P<0.05).Conclusion: SphK1 was high expressed in human ovarian cancer tissues,related with FIGO stage and distant metastasis,and has no relationship with the age of patient,tumor differentiation,lymph node metastasis and histological type.MVD was highly raised in human ovarian cancer tissues,and the MVD level was positively correlated with SphK1 expression,Part 2.The role of SphK1 in ovarian cancer cells pro-angiogenesis effection researchObjective: We aimed to study sphingosine kinase 1(SphK1)altered expression on sphingosine-1-phosphate(S1P)secretion in ovarian cancer cell(SKOV3),and to explore its role and possible mechanisms in ovarian cancer cells pro-angiogenesis effection.Methods: 1.SphK1 gene was silenced by siRNA,the effect of SphK1 gene silencing on human umbilical vein endothelial cells fused cells(EA.hy926 cells)angiogenesis was tested by tube formation assay;2.S1 P secretion level was examined by enzyme-linked immune sorbent assay(ELISA)after SphK1 gene silencing in ovarian cancer cells;3.IL-8 mRNA expression was tested by Realtime-PCR,to determine the effect of S1 P treatment on human ovarian cancer cells pro-angiogenesis,and the ovarian cancer cells were cultivated with serum-free medium for 24 hours;4.S1PR1?S1PR2 and S1PR3 gene was silenced by siRNA.IL-8 mRNA expression was tested by Realtime-PCR,to determine the effect of S1 PR on human ovarian cancer cells pro-angiogenesis.Results: 1.The tube formation of EA.hy926 cells was significantly reduced by the ovarian cancer cells culture supernatants with SphK1 gene silenced(P<0.05);2.The level of S1 P secretion in ovarian cancer cells was significantly inhibited after SphK1 gene silencing(P<0.05);3.The IL-8 mRNA expression was promoted by S1 P in human ovarian cancer cells(P<0.05);4.The level of IL-8 mRNA expression in ovarian cancer cells was inhibited after S1PR1?S1PR3 gene silencing(P<0.05),but the change of IL-8 mRNA expression is not obvious wit S1PR2 gene silencing(P>0.05).Conclusion: SphK1 plays an important role in the process of angiogenesis in ovarian cancer.SphK1 gene silencing can inhibit ovarian cancer cells S1 P secretion and tumor angiogenesis,prompting that SphK1 may affect ovarian cancer angiogenesis by modulating S1 P.Part 3.The role of SphK1 on nude mice ovarian cancer angiogenesis researchObjective: We aimed to identify the expression of SphK1 in subcutaneous tumor tissues,and to explore the rple of SphK1 and its inhibitor on nude mice ovarian cancer microvessel density.Methods: We established nude mice subcutaneous tumor model of ovarian cancer,and parted them to SK-II group and control group(saline group).The weight of nude mice,the growth of subcutaneous tumor(including the tumor formation,tumor size,tumor volume,and tumor weight)were detected.SphK1 protein and CD34 protein were examined by immunohistochemistry in nude mice subcutaneous tumor tissues to determin the expression levels,and calculated MVD.Results: 1.Compared with control group,the ovarian cancer subcutaneous tumor formation rate?tumor volume,and tumor weight of SK-II drug treatment group were lower(P<0.05);2.Compared with control group,the level of SphK1 and MVD were lower in the subcutaneous tumor tissues of SK-II group nude mice(P<0.05).Conclusion: The development of ovarian cancer is dependent on tumor angiogenesis,while SphK1 can affect ovarian cancer angiogenesis.By decreasing the expression of SphK1 through SphK1 inhibitors can inhibit angiogenesis in ovarian cancer,and further affect the growth of ovarian cancer.