Effects Of Glucocorticoid On The Migration And Invasion Of Ovarian Cancer Cells And Its Potential Mechanism

BackgroundMetastasis is one of the characteristics of malignant tumors,and it is also the main cause of death in patients with cancer.The process of tumor metastasis is extremely complex,including tumor cells shed from the primary tumor,tumor cells adhere to matrix and release of matrix hydrolase,invade through the basal membrane of blood or lymphatic vessels and enter the circulation via intravasation.Subsequently,cancer cells leave the circulation at the metastatic site via extravasation,establishing metastatic tumor nodules.Cell migration and invasion play key roles in tumor metastasis.Cell migration involves changes of cytoskeleton,morphology,ability if adhesion and motion,and a variety of signals,such as growth factors,cell adhesion molecules,small G proteins associated with cell motility(such as Rho subfamily members of RhoA,RhoB and RhoC),cytoskeleton regulatory protein and chemokine etc.In addition to the ability of proliferation and movement,the ability of degrading extracellular matrix,such as the secretion of matrix metalloproteinase(MMPs)and protease activator,such as urokinase type plasminogen activator(uPA)and so on,are very important for tumor cell invasion.Recent studies have demonstrated that the metastatic potential of tumor cells is also dependent on the microenvironment of the tumor.The process of tumor metastasis is extremely complex,and the mechanism is still not very clear.Epidemiological studies and animal model based studies have suggested that chronic stress affects tumor progression,therefore neuroendocrine changes due to stress affecting tumor progress receives more attention in recent years.Previous studies have indicated that the sympathetic and adrenergic systems play key roles on promoting tumor progression and metastasis.Glucocorticoid(GC)is another important stress hormone,which is significantly increased in plasma under physical and psychological stress.In addition,the synthetic GCs,such as dexamethasone(Dex)is widely used for the treatment of immune and inflammatory disease(such as lupus and rheumatoid arthritis),as well as the important auxiliary drug used to relieve adverse reactions caused by chemotherapy for cancer treatment(such as allergies,nausea and vomiting),these can also lead to increase GC levels in vivo.Glucocorticoid receptor(GR)is a hormone dependent transcription factor.Unliganded GR is found primarily in the cytoplasm of cells as part of a large multi-protein complex that includes various chaperone proteins such as hsp90.Binding of glucocorticoids triggers a conformational change in GR resulting in the dissociation of the heterocomplex,exposure of the nuclear localization signals,and translocates into the nucleus.Once in the nucleus,GR dimerizes and binds directly to GREs to stimulate the expression of target genes.GR can also regulate gene expression by physically associating with other transcription factors.For example,the interaction of GR with the proinflammatory transcription factors AP-1(activator protein-1)and NF-κ B inhibits their activity and accounts for the major anti-inflammatory and immunosuppressive effects of glucocorticoids.In recent years,foreign and our studies have indicated that GC can affect the expression and activity of many kinds of signaling molecules,and play an important role in the regulation of cellular signaling network.So far,the effect of GC on solid tumors is mainly confine to its strong suppression role in immune system,the effect and mechanism of GC on tumor cells migration,invasion and metastasis are very limited and ambiguous,so it is necessary to further study in this area.Ovarian cancer is the most lethal of gynecological tumors,and >70% of ovarian cancer cases are diagnosed at a late stage with pelvic cavity or distant metastasis.So far,the mechanism of ovarian cancer metastasis is not clear.Previous studies have indicated that the positive rate of GR in ovarian cancer tissue was 88%,higher than that of ER and PR,indicating that GCs/GR signaling pathway plays a very important role in the occurrence and development of ovarian cancer.Foreign and our laboratory studies have found that GC can slightly inhibit the proliferation of ovarian cancer cells,but promote ovarian cancer cell survival in the presence of multiple chemotherapy drugs and enhance cell adhesion to the extracellular matrix.The effect and mechanism of GCs on the metastasis of ovarian cancer are not clear.Therefore,in this study we investigated the effects of GCs on cell migration,invasion and metastasis of ovarian cancer in vitro and in vivo,and further explored the mechanism.MethodsOvarian cancer HO-8910,SKOV3 and A2780 cells are used in vitro experiments.100 nM Dex(synthetic GC)treated cells in medium containing 10% dextran-coated charcoal(DCC)-stripped fetal bovine serum(FBS)to avoid the influence of hormone in serum.Actin was fluorescence stained by rhodamine-phalloidin and photographed by confocal microscopy.Transwell chamber and cell scratch test were used to identify the ability of cell migration.Transwell chamber precoated with matrigel glue was used to detect the ability of cell invasion.Cell adhesion assay was used to detect adhesive ability between cells and extracellular matrix(ECM).The activity of Rho B and RhoC was detected by Rho-GTP pull down assay.Western-blot was used to detect the expression and phosphorylation levels of protein.Small interfering RNA(siRNA)was used to inhibit the expression of target genes.Flow cytometry was used to detect the distribution of integrin on cell membrane surface.ELISA assay was used for the detection of fibronectin(FN)secretion.In vivo experiment we used BABL/c nude mice to inject ovarian cancer cells to prepare the metastatic tumor model.Corticosterone(GC in mice)in drinking water increased plasma corticosterone levels in nude mice.Plasma corticosterone levels were detected by radioimmunoassay in nude mice.The pathologic characteristics of metastatic lesions were determined by H&E staining.Ovarian cancer cells with stable interference RhoB were screened by the method of the screening of purine.ResultsGCs promote ovarian cancer cell migration,invasion and metastasis in vitro and in vivo.In vitro dexamethasone(Dex)induced the change of morphology and cytoskeletal reorganization(stress fibers)of ovarian cancer cells,and enhanced the adhesion ability of cells to ECM and the ability of cell migration and invasion.In metastatic tumor model of BABL/c nude mice by intraperitoneal injection of ovarian cancer cells,administration of corticosterone(GC in mice)in drinking water(50mg/L)significantly increased the corticosterone levels in plasma and promote liver and peritoneal dissemination of ovarian cancer cells in vivo.GCs change the expression and / or activity of a variety of signaling pathway molecules to promote the migration,invasion and metastasis of ovarian cancer cells.Glucocorticoid receptor(GR)antagonist RU486 can completely block the promotion effect of GC on ovarian cancer cell migration.GC upregulated the express and secretion of fibronectin(FN),and enhanced the distribution of integrin beta 1 and alpha 5(FN receptor)on the surface of ovarian cancer cells.Inhibiting FN by siRNA can significantly inhibit cell adhesion,migration and invasion in ovarian cancer cells.GC can activate the PI3K/AKT signaling pathway and the activation effect is late,which mean it is not direct effect of Dex.Inhibiting FN by siRNA can inhibit the activation of the PI3K/AKT pathway,suggesting that cell adhesion enhanced by FN and its receptors(integrin alpha 5 beta 1)may be related to the activation of the PI3K/AKT signaling pathway.Inhibiting PI3K/AKT pathway by specific inhibitor wortmanin completely blocked Dex-induced cytoskeletal reorganization and the promotion effect of invasion and migration of ovarian cancer cells.We also found that Dex can activate RhoB and RhoC,and increase the phosphorylation of ROCK2,which is downstream of Rho.Inhibition ROCK2 by specific inhibitor Fasudil significantly reversed the promotion effect of GC on the migration and invasion in ovarian cancer cells.We next explored the role of RhoB in tumor metastasis and found inhibiting RhoB by siRNA decreased the secretion of FN and the activation of PI3K/AKT signaling pathway and inhibited the ability of cell adhesion,migration and invasion in vitro,as well the liver metastasis in vivo.GC can also upregulate the expression of matrix metalloproteinase(MMP)3 and MMP7 in ovarian cancer cells and inhibiting the activity of MMPs by BB-94 decreased invasion of ovarian cancer cells.ConclusionGC can promote the migration,invasion and metastasis of ovarian cancer cells in vitro and in vivo,which is mediated by GR.Activation of the PI3K/AKT signaling pathway plays an important role in GC induced cytoskeletal reorganization and the promotion effect of cell migration and invasion.GC can also promote the adhesion,migration,invasion and metastasis of ovarian cancer cells by activating the small G protein RhoB.In addition,Activation of the RhoC/ROCK2 signaling pathway and up regulation of MMP3 and MMP7 expression by GC are also associated with the promotion effect of migration and invasion in ovarian cancer cells.These results revealed that GC can promote the invasion and metastasis of ovarian cancer by affecting the expression and activity of a variety of important signaling molecules in the cells.