Study On Protection And Inhibitory Action Of Vitamin D On Ovarian Cancer In Vitro And Vivo

Objective: Ovarian cancer is the most frequent cause of death from gynecologicmalignancy. The models of ovarian cancer （in vitro and in vivo） are crucial forunderstanding the pathogenesis of the disease and for testing new treatment strategies.In this study, We isolated and characterized mouse ovarian surface epithelialcells（MOSEC）, and investigated the effect of1,25-dihydroxyvitamin D3on malignanttransformation of MOSEC. Meanwhile,7,12-dimethylbenz[a] anthracene （DMBA） wasapplied to the ovary to yield mice ovarian cancer, in order to explore the role of vitaminD on development of ovarian cancer and biological mechanism. Besidesly, wediscussed the influence of1,25（OH）₂D₃and γ ray combined application on SKOV-3ovarian cancer cells’ proliferation.Methods:（1） MOSE cells were separated, cultured and identified in vitro. All thepurified MOSECs were divided into four groups, including control group,1,25（OH）₂D₃group, DMBA-induced group and DMBA+1,25（OH）₂D₃group. We measured cellsinhibitory rate by CCK-8assay, identified the MOSEC by immunofluorescence,detected anchorage-independent growth capacity, and analyzed the expression ofVitamin D receptor, E-cadherin and β-catenin.（2） DMBA was injected to the ovarytomaximally preserve its structural integrity. DMBA-induced mice was divided intopure VD group, control group,0-20weeks VD group,0-4weeks VD group,8-20weeksVD group and model group. We observed the changes of mice appearance, carryed outpathological analysis of ovarian tissue, measured spleen index and determed leverl ofCA125,25-hydroxy vitamin D and Ca²⁺in serum, and quantified the expression ofVitamin D receptor, E-cadherin and β-catenin.（3） Cells were divided into1,25（OH）₂D₃group,⁶⁰Coγ ray group and their joint action group. We determined the survival rate ofcells in MTT method, analyzed the changes of cells cycle, cells active oxygen （ROS）and Ca²⁺with flow cytometry. Results:（1） The results showed that there was obvious transforming focus whenDMBA group were cultured in vitro to12passages in vitro, and the growth contactinhibition disappeared. However, the DMBA+1,25（OH）₂D₃group appeared the samephenotype in15passages, the cloning formation rate was also significantlyreduced（p<0.05）, which indicating that1,25（OH）₂D₃may delay malignanttransformation of MOSEC inducted by DMBA. The inhibition effect is relevant to theimprovement of VDR and E-cadherin expression and reduction of β-catenin expressionin nucleus.（2） VD can improve the appearance of the mice and reduce the incidence ofovarian cancer （p<0.05）. Experimental results showed that the incidence of ovariancancer had negative correlation with the level of25-hydroxy vitamin D inmice（r=-0.988, p<0.01）. Compared with DMBA induction group, vitamin D can reducethe content of CA125which is one of the valuable cancer marker in serum and ascites（p<0.05） and the expression of β-catenin （p<0.05）. Compared with the control group,the level of25-hydroxy vitamin D increased significantly in0-20weeks group while itreduced in DMBA group（p<0.05）. The Ca²⁺concentration in serum was unchanged ineach group.（3） The results show that each of the treatments alone displayedantiproliferative activity, and the growth inhibition of SKOV-3cells was furtherenhanced by the combination of1,25（OH）₂D₃with irradiation. The analysis of cell cycleindicates that1,25（OH）₂D₃enhanced the proportion of SKOV-3cells in G0/G1, γ-raysenhance the proportion in G2/M, while in the combined treatment group proportion ofSKOV-3cells significantly increased, which retards the cells in G2/M. The contents ofROS and Ca²⁺in the combined group cells were significantly higher than that of thesingle treatment groups （P<0.05）.Conclusion:（1）1,25（OH）₂D₃can reduce ability of malignant transformation ofMOSEC by delaying the time of MOSEC focal formation and growth contactinhibition, and decreaseinging cloning formation rate. This effect is relevant to theexpression of raising VDR and E-cadherin and reducing β-catenin expression.（2） Vitamin D can reduce the incidence of mice ovarian cancer and the content ofCA125in mice serum and ascites; the incidence of overian cancer has negatively relatedto the content of1,25（OH）₂D₃in serum. Its mechanism might be related to theincreasing expression of VDR and E-cadherin and the decreasing expression ofβ-catenin in ovarian tissues by VD. （3）1,25（OH）₂D₃can enhance the inhibition of human SKOV-3ovarian cancercells by γ-rays irradiation. This inhibition is related to the cells retardant of1,25（OH）₂D₃in G1/M as well as the content of ROS and Ca²⁺in cells.