Detection Of Adiponectin Level In Ovarian Cancer Patients And Healthy Volunteers And Regulation Role Of Adiponectin On Proliferation And Apoptosis Of Ovarian Cancer Cell

Objective:Ovarian cancer is one of the common gynecological malignant tumors.Because of its occult and progression,there are no obvious symptoms in the early stage.About 70%of the patients are in advanced stage,and the mortality rate is in the forefront.In view of the high incidence and mortality of ovarian cancer,and the close relationship between adiponectin and cancer in epidemiology,this study aims to explore whether adiponectin has an effect on the proliferation and apoptosis of ovarian cancer cells and explore its potential mechanism.Methods:1.Detection of adiponectin level in ovarian cancer patients and healthy volunteers1.A total of 46 serum samples and 25 ascites specimens from ovarian cancer patients admitted to the First Affiliated Hospital of China Medical University from January 2013 to December 2013 were collected.The specimens met the following conditions:（a）Patients were pathologically confirmed ovarian cancer patients,（b）patients with clear phase staging by FIGO international staging,and（c）no chronic history of hypertension,diabetes,etc.2.Seventeen normal control serum samples were collected from volunteers who were examined at the First Affiliated Hospital of China Medical University from January2013 to December 2013.Volunteers met the following conditions:no healthy women with a history of cancer and gynecological diseases,no hypertension,or diabetes were found in the physical examination.3.The adiponectin levels in serum and ascites samples were determined by ELISA.2.Effection of adiponectin on ovarian cancer cell proliferation1.Real-time PCR and Western Blotting were applied to detect the expression level of adiponectin receptors-1（AdipoR1）in ovarian cancer cell SKOV3 and Caov3.2.MTT assay was applied to evaluate the proliferation of the ovarian cancer cells SKOV3 and Caov3 after stimulated with exogenous adiponectin by different concentrations（0,5,10,50,100,200 ng/ml）for 48 hours.Similarly,evaluate the proliferation of the ovarian cancer cells that were stimulated with exogenous adiponectin（100 ng/ml）at different time points（24,48,72 hours）.3.Real-time PCR and Western Blotting were applied to detect the expression levels of cyclin B of the ovarian cancer cells SKOV3 and Caov3 after stimulated with exogenous adiponectin（100 ng/ml）for 48 hours.3.Effection of adiponectin on ovarian cancer cell apoptosisWestern Blotting was applied to detect the expression of casepase 3 and Bcl-2 of ovarian cancer cells SKOV3,Caov3 in different three subgroups（without H₂O₂ treatment group,H₂O₂（50μM）treatment group,H₂O₂（50μM）+adiponectin treatment group）.4.Effection of adiponectin on phosphorylation of Akt and ERKWestern Blotting was applied to detect phosphorylation of Akt,ERK in ovarian cancer cell Caov3 and SKOV3 after stimulated with adiponectin（100 ng/ml）at different time points（0,10,30,60 points）.Observe the effection of adiponectin on proliferation and apoptosis of ovarian cancer cell dealed with LY294002 and PD980591.human ovarian cancer cell lines Caov3 and SKOV3 cells were dealed with LY294002（inhibitors of PI3K which is the upstream protein of Akt）and PD98059（inhibitors of MEK which is the upstream protein of ERK）for 1 hour respectively,and then MTT assay was applied to detect the proliferation of cancer cells after stimulated with adiponectin（100 ng/ml）at different time points（24,48,72 hours）.2.Western Blotting was applied to detect the expression level of Cyclin B,caspase3and Bax of ovarian cancer cell Caov3 and SKOV3 in five groups（untreated group,H₂O₂（50μM）group,H₂O₂（50μM）+100 ng/ml adiponectin group,H₂O₂（50μM）+LY294002+100 ng/ml adiponectin group,H₂O₂（50μM）+PD98059+100 ng/ml adiponectin group）respectively.Results:1.Adiponectin concentrations in serum of all the diffenent stages of ovarian cancer patients were significantly higher than that of normal volunteers（p<0.05）.Adiponectin concentrations in serum of patients with FIGO stageⅣovarian cancer was higher than those patients with FIGO stageⅠovarian cancer.There were no significant difference among other FIGO stages of ovarian cancer patients.2.Adiponectin concentrations in serum of FIGO stageⅢand FIGO stageⅣovarian cancer patients were lower than ascites adiponectin concentration（p<0.05）.3.Expression of AdipoR1 mRNA and protein were deteced in both Caov3 and SKOV3 cells,and the expression level of SKOV3 cells were higher than Caov3 cells（p<0.05）.4.The cell vability of Caov3 and SKOV3 cells stimulated with adiponectin were increased obviously in dose dependent manner.The cell vability of cells stimulated with adiponectin（100 ng/ml）significantly increased at different time points（24,48,72hours）.5.The levels of mRNA and protein of Cyclin B in Caov3 and SKOV3 cells stimulated with adiponectin（100 ng/ml）increased significantly（p<0.01）.6.Expression of caspase 3,Bax in Caov3 and SKOV3 cell dealed with H₂O₂（50μM）raised obviously.But the expression levels of caspase 3,Bax were decreased after dealed with adiponectin.7.The expression of p-Akt in Caov3 and SKOV3 cells stimulated with adiponectin raised,and this expression was highest at 30 minutes in Caov3 cells,and at 10 minutes in SKOV3 cells.The expression of p-ERK also appeared to rise,and this expression h was highest at 60 minutes in Caov3 cells,and at 30 minutes in SKOV3 cells.8.Caov3 and SKOV3 cells increased obviously after stimulated with adiponectin（100 ng/ml）at different time points（24,48,72 hours）.Cell proliferation of LY294002group and PD98059 group were significantly lower than that of adiponectin stimulation group at 48 and 72 hours（P<0.05）,but there was no significant difference at 24 hours.We also observed that after H₂O₂（50μM）induced the apoptosis of Caov3 and SKOV3cell,adiponectin could upregulate the expression level of Cyclin B protein,and then this effect was inhibited by LY294002 and PD98059.9.H₂O₂ induced the apoptosis of Caov3 and SKOV3 cell and expression of caspase3,Bax raised,while adiponectin can inhibit this effect.And after treated with LY294002and PD98059,the inhibition effect of adiponectin were antagonized.Conclusion:1.The concentration of adiponectin in serum of ovarian cancer patients is lower than that in ascites and higher than that in the healthy people serum.2.Adiponectin can promote the proliferation of Caov3 and SKOV3 ovarian cancer cells.3.Adiponectin can activate the PI3K-Akt and Raf-MEK-ERK pathways of ovarian cancer cells,thereby inhibiting the apoptosis of tumor cells and promoting tumor cell proliferation.