Contrary Regulation Of Bladder Cancer Cell Proliferation And Invasion By Dexamethasone-Mediated Glucocorticoid Receptor Signals

1objectives and contentTo investigate the effect and the underlying mechanism of glucocorticoid receptors in the proliferation and invasion of bladder cancer from the molecular, cellular and clinical level, explore new ideas for the treatment of bladder cancer.1) Detecting the expression of GR in clinical specimens and bladder cancer cell lines. Evaluating the correlation between intensity of GR expression and presence of muscle invasion, status of lymph node involvement and risk of progression after radical cystectomy in bladder cancer.2) Evaluating the effect of GC/GR signal activation on proliferation and invasion in bladder cancer cell lines, clarifying the underlying mechanism.3) Evaluating the effect of GC/GR signal activation on proliferation and invasion in xenograft model. 2Experimental methods2.1The expression level of GR in bladder cancer specimens clinicopathological correlationthe expression level of GR in bladder cancer, normal urothelia, metastatic lymph node specimens were examined by immunohistochemical analysis. The correlation between intensity of GR expression and presence of muscle invasion, status of lymph node involvement and risk of progression after radical cystectomy in bladder cancer was analysed.2.2Screening for GR positive bladder cancer cell lines and creation of GR knock-down cell lines.1) GR expression in bladder cancer cell lines:the mRNA and protein expression level of GR in4bladder cancer cell lines were examined by western-blotting and Real-time PCR, respectively.2) GR biological fuction detection:luciferase assay.3) Creation of GR knock-down cell lines:GR-shRNA was introducer into bladder cancer.cell.by.using.lentivirus.Cells.stablly.expressing.GR-shRNA.was.screend.under.selection.pressure.of.puromycin,confirmed.by. western-blotting.2.2Effect of GC/GR signal activation on proliferation and invasion of bladder cancer cell lines. 1) Proliferation:MTT.assay.for.viability,TUNEL.assay.forapoptosis,flowcytometry for.cell.cycle.Q-PCR.and.western-blotting.for.expression.level.of.cyclineD1/D2/D3,.CDK2/CDK4/CDK6,P21/P27and Caspase3.2) Invasion:Transwell.assay.for tumor cells invasion, Gel Zymography for activity of.MMP2/9,.Q-PCR.and.western-blotting.for.expression.level.of.MMP2/MM9, IL-6,VEGF.3) Epithelial-Mesenchymal.Transition：Morphology.of.cells.cultured.with.dexame-thasone.and/or.RU486.was.assessed,.using.the.NIH.Image.J.software.Paramete-rs included the area,perimeter,circularity,and roundness.Western-blotting for expression level of MMP2/MMP9.IL-6.VEGF.2.4GC/GR signals regulating NFkB signaling pathway.1) Key molecules expression:Expression of key molecules(Rel A,IkBα) in NFkB signaling pathway with dexamethasone and/or RU486were assessed, using western-blotting.2) Translocation of NFkB:Nuclear translocation of NFkB with dexamethasone and/or.RU486.was.assessed,.using.immunofluorescent.staining.andwestern-blo-tting.3) Coimmunoprecipitation：Interaction.between.NFkB.and.IkBa.with.Dexamethas-one and/or RU486was assessed, using coimmunoprecipitation.2.5Xenograft.model:Bladder.cancer.lines.(1×106.cells.in.100.μL.per.site) resuspended in Matrigel were inoculated subcutaneously into the right (GR-shRNA) and left (control-shRNA) flanks of7-week-old male severe combined immunodeficient (SCID) mouse. Slow-releasing pellets [dexamethasone (0.5mg/mouse) or placebo] were injected with a precision trochar when the sizes of all tumors in each group reached40mm3. Tumors were measured using calipers and tumor weight was calculated by the following formula:tumor weight (mg)=tumor length (mm) x[tumor width (mm)]2×0.5(9). After5weeks of treatment, the mice were killed and all the tumors including metastases were harvested for histologic and immunohistochemical assessment.3Results3.1All the4bladder cancer cell lines were found to express glucocorticoid receptor at both mRNA and protein levels. All the nonneoplastic and neoplastic bladders as well as metastases showed at least weak signals in urothelial cells. GR expression tended to be weaker in urothelial carcinoma than in benign urothelium.3.2Treatment of a synthetic glucocorticoid dexamethasone and/or a glucocorticoid receptor antagonist RU486Dexamethasone increased luciferase activity in UMUC3(23.6-fold), TCC-SUP (14.9-fold), J82(1.4-fold), and5637(3.1-fold), compared with respective mock treatments.3.3Silencing of glucocorticoid receptor expression in UMUC3-GR-shRNA and TCC-SUP-GRshRNA was then confirmed.3.4GC/GR signals activation stimulate proliferation and prevent apoptosis: Dexamethasone increased cell growth in a dose-dependent manner in UMUC3/TCC-SUP, and RU486at least partially antagonized the dexamethasone effect. Dexamethasone treatment led to significant increases in Gl-phase cell population in control UMUC3and TCC-SUP lines, and RU486abolished the dexamethasone effects. Dexamethasone-mediated glucocorticoid receptor signals likely prevent apoptosis of bladder cancer cells in the presence or absence of CDDP.3.5Dexamethasone-suppressed cell invasion:Dexamethasone treatment resulted in significant decreases in the invasive properties of both control lines, and RU486clearly abolished the dexamethasone effect. In glucocorticoid receptor knockdown lines, dexamethasone did not show significant suppressive effects on cell invasion.3.6Dexamethasone-induced mesenchymal-to-epithelial transition:Using the Image J software,the.area,perimeter,circularity,and roundness of the cells were compared among different.treatments.Dexamethasone. increased, these.parameters. in. glucocorticoid.recep-tor-positive.lines,compared.with.those.in.mock-treated.lines,.In.glucocorticoid. receptor-positive.lines,dexamethasone.up/down-regulated.the.expression.of.epithelial/mesench--ymal markers,respectively,compared with mock treatment.3.7Dexamethasone-induced.disruption.of NFkB:Western blot analysis showed increases in the level of IkBa, a natural cytoplasmic inhibitor of NFkB, but not in NFkB levels, in both of dexamethasone-treated glucocorticoid receptor-positive lines. Dexamethasone induced the interaction between NFkB and IkBa in control UMUC3cells, and RU486or GR-shRNA diminished the dexamethasone effect. Dexamethasone blocked nuclear translocation of NFkB induced by TNF-a in TCC-SUP.3.8Dexamethasone increased tumor size in mouse xenograft models:Control glucocorticoid receptor-positive tumors in dexamethasone-treated mice were larger/heavier than other tumors at5weeks of treatment.3.9Dexamethasone inhibited invasion/metastasis in mouse xenograft models:bloody ascites, suggestive of peritoneal dissemination of the tumors, and actual metastatic tumors in the peritoneum were identified in7(88%) and4(50%) of8placebo-treated mice, respectively, but in none of dexamethasone-treated mice. Histologic examination of the tumors revealed invasion into the skeletal muscle in all groups of mice except the control-shRNA/DEX group.3.10Key molecules in mouse xenograft models:Harvested tumor specimens were also assessed for cell proliferation (Ki-67), apoptosis (TUNEL), and angiogenesis or metastatic ability (MMP-9/VEGF/CD34). Dexamethasone treatment in control glucocortico(?)d receptor-positive tumors led to marginal changes in proliferation but decreased apoptosis and angiogenesis/metastasis-related factors, compared with DEX/GR-shRNA, placebo/control-shRNA, placebo/GR-shRNA, or placebo/metastasis.4Conclusions1) GR expression are100%positive in both bladder cancer and normal urothelium. High intensity of GR expression in bladder cancer may predict good prognosis.2) Dexamethasone-mediated.glucocorticoid.receptor.signals.prevent.apoptosis.and.induce.cell cycle arrest of bladder cancer cells, finally stimulate proliferation.3) Glucocorticoids inhibit bladder cancer cell invasion through the glucocorticoid receptor.pathway. Inhibition of NF-kB signaling pathway may be its underlying central mechanism.