Effect And Mechanism Of Dihydromyricetin On Ovarian Cancer Mediated By GRASP65 Through MAPK Pathway

Objectives1.To detect the expression of Golgi stacking protein GRASP65 in ovarian tissues with serous and mucous tumors and to analyze the difference of GRASP65 expression level and its relationship with clinical and pathological parameters.2.To analyze the effects of dihydromyricetin?DHM?on the invasive and migrative abilities,as well as cell apoptosis,cell autophagy and the ultrastructure in ovarian cancer SKOV3 and A2780 cells,and to further explore the possible effect and novel mechanism of DHM on ovarian cancer mediated by GRASP65 through JNK and ERK pathway.3.To explore the role and mechanism of DHM intervention in inhibiting the growth of transplanted tumor in nude mice with ovarian cancer.Methods1.118 cases of patients with ovarian tumors confirmed after the surgical removal and the specimens of paraffin wax were collected from Minda hospital of Hubei Minzu University,and the carcinoma and adjacent tissues from 6 cases of postoperative patients with ovarian serous carcinoma were selected as the research objects.GRASP65 expression was detected using immunohistochemical staining?IHC?and Western blot?WB?analysis.The correlation between the GRASP65 expression and clinical and pathological parameters was analyzed.2.After the intervention with various concentrations of DHM in ovarian cancer SKOV3 and A2780 cells,cell viability was detected by CCK8 assay.The abilities of cell migration and invasion were analyzed by Wound healing and Transwell assay.The apoptosis rate was analyzed using Flow cytometry?FCM?.The localized expression of GRASP65 and pro-apoptotic protein Caspase-3 were detected by Immunofluorescence staining?IF?.The expression of Caspase-3,Bax and Bcl-2,as well as the protein levels of MAPK pathway were tested using Western blot.siGRASP65 transfection was used to down-regulate GRASP65 expression and overexpression plasmid to up-regulate GRASP65 level,followed by DHM intervention,then,the cell vitality,migration and apoptosis were detected using CCK8 and wound healing assay,Western blot,and FCM.The expression levels of p-JNK/ERK and JNK/ERK proteins,GRASP65and Caspase-3 were detected using WB analysis after pretreatment with the inhibitors of JNK/ERK pathway and DHM intervention.The ultrastructural changes of Golgi apparatus and cell autophagy in DHM-treated cells were observed by transmission electron microscopy?TEM?.3.SKOV3 and A2780 cells were transplanted into subcutaneous tissue to construct two transplantation tumor models and then treated with DHM suspension by intraperitoneal injection,respectively.Tumor volume,weight and liver and kidney function in nude mice were detected and analyzed.The expression levels of Caspase-3,LC3 and GRASP65 proteins in tumor tissues were analyzed using WB and IHC staining.The Golgi apparatus and autophagosome in tumor tissues were observed by TEM.Results1.Among the 118 patients with ovarian tumors,82 were benign tumors,14 were borderline tumors,and 22 were malignant tumors.All cases were divided into 65 cases of serous tumor,48 cases of mucous tumor,and 5 cases of special type according to histological types.The IHC results in paraffin tissues showed that the expression of GRASP65 was brown yellow or brown and fine particles,which were dispersed in the cytoplasm.Statistical analysis showed that GRASP65 protein was highly expressed in ovarian borderline and malignant tumors compared with benign tumor tissues??²=12.57,P<0.01?.The expression of GRASP65 in mucous tumors was higher than that in serous tumors??²=5.23,P<0.05?.Among 6 cases with ovarian cancer after surgical resection,the expression of GRASP65 in cancer tissue and adjacent tissues from the same individual was detected using Western blot.The results showed that the expression of GRASP65 in the cancer tissues was significantly upregulated compared with the adjacent ovarian tissues.The above results indicated that GRASP65 may be involved in the development of ovarian tumors,and the abnormal expression of GRASP65 may be related to the character and histological type of ovarian tumor.2.Two ovarian cancer cells?A2780 and SKOV3?were treated with DHM at different concentrations?0,10,20,40,80,120,160,240,320?M?,and the results showed that DHM could reduce the cell viability,reduce the ability of cell migration and invasion,and induce apoptosis,implying that there was a certain dose relationship.According to the calculated IC₅₀ value of DHM on SKOV3 and A2780 cells,the DHM concentrations were 120and 80?M for 48 hours in the subsequent experiments,respectively.3.Western blot analysis showed that DHM treatment reduced the expression of GRASP65 in SKOV3 and A2780 cells?P<0.05,0.01?compared with the control group.The fluorescence intensity of GRASP65was reduced and dispersed after DHM treatment using IF staining,which was similarly to Golgi fragmentation morphologically.Compared with DHM treatment alone,the specific inhibitor of Caspase-3 pretreatment could reduce the activation of Caspase-3 induced by DHM?P<0.01?,and the level of GRASP65 expression increased?P<0.05,0.01?.FCM analysis also showed that compared with DHM treatment alone,pretreatment with Caspase-3 inhibitor could reduce the apoptosis rate?P<0.01?,indicating that DHM may activate Caspase-3 to cause GRASP65 protein cleavage and downregulation,and the activation of caspase-3 plays an important role in the downregulation of GRASP65induced by DHM.4.Downregulation of GRASP65 by siGRASP65 transfection in A2780cells decreased cell viability and the ability of cell migration and increased apoptotic rate.Compared with DHM treatment alone,the co-treatment with DHM and siGRASP65 transfection had an additive effect on the inhibition of cell viability and migration,as well as the induction of apoptosis.On the contrary,compared with DHM treatment alone,the inhibition of DHM on cell viability and migration,as well as the induction of apoptosis,could be attenuated after the co-treatment of DHM and overexpressed plasmid transfection.5.Western blot analysis showed that DHM?0,40,80,120?M?treatment induced an increase in the levels of p-JNK and p-ERK?P<0.05,0.01?in a dose-dependent manner,but no change in p-p38 level,suggesting that the ERK and JNK signaling pathway may be involved in the anti-cancer effect of DHM in A2780 cells.There was no significant difference in p-JNK and p-ERK levels between the siGRASP65 group and the control group,additionally,no difference between the DHM group and the DHM+siGRASP65 group.Therefore,GRASP65 depletion had no effects on the p-JNK and p-ERK levels in A2780 cells.Additionally,the inhibitors of JNK or ERK pathway were pretreated for 2 h in A2780 cells,respectively,and then interfered with DHM.The results showed that,compared with the DHM group,SP600125 and U0126significantly inhibited DHM-induced caspase-3 activation?P<0.05?and increased GRASP65 levels?P<0.01?after treatment with DHM,followed by the reduction of p-JNK and p-ERK levels?P<0.01?.These results demonstrated that JNK and ERK pathway might be involved in the activation of Caspase-3 and the downregulation of GRASP65 mediated by DHM in A2780 cells and suppression the ERK and JNK pathway could attenuate DHM-induced apoptosis.In addition,GRASP65 may be downstream of JNK and ERK pathway activation induced by DHM.6.The changes in the morphology and structure of the Golgi body in A2780 cells induced by DHM were observed with the use of TEM.The results showed there was Golgi body edema,fragmentation and more autophagosomes formed in the DHM group,the siGRASP65 group,and the co-treated group,indicating that DHM treatment may induce Golgi fragmentation and apoptosis via GRASP65 and improve the level of autophagy.7.SKOV3 and A2780 cells were implanted under the skin of nude mice,respectively,to construct transplant tumor models with ovarian cancer,and then treated with DHM suspension by intraperitoneal injection.Normal saline was set as the control group,respectively.Compared with the control group,DHM treatment inhibited tumor growth in both models?P<0.01?.Moreover,the results showed that GRASP65 expression down-regulated?P<0.05?,expression of Cleaved caspase-3,pro-apoptotic protein,and LC3II level upregulated after DHM treatment by IHC staining and western blot analysis?P<0.05,0.01?.Compared with the control group,Golgi body swelled and the number of autophagosomes increased significantly in the tumor tissues in DHM-treated group using TEM,accompanied by obvious endoplasmic reticulum edema and expansion.Conclusions1.GRASP65 expression was upregulated in ovarian borderline and malignant tumors compared with benign tumors,also increased in mucous tumors compared with serous tumors,and its positive high expression was correlated with tumor character and pathological type to some extent.2.DHM treatment could downregulate the expression of GRASP65protein and induce Golgi edema and fragmentation and cell apoptosis in ovarian cancer cells through JNK and ERK signaling pathways,to play an anti-cancer role.