Up-regulation Of Transforming Growth Factor-β1 Type Ⅱ Receptor By Glucocorticoid And Its Biological Significance In Cancer Cells

Glucocorticoid (GC) inhibits the proliferation of many tumor cells of epithelial origin, including androgen-independent prostate cancer (AIPC) and ovarian cancer cells, but the mechanism remained unclear. Several previous studies have indicated that Dex induced the expression of TGF-β1 in AIPC cell line PC-3 cells. We also found that Dex enhanced the inhibitory effect of TGF-β1 on the proliferation of PC-3 cells. So we speculated that Dex may inhibit PC-3 cell proliferation not only through increasing the expression of TGF-β1, but also through positively regulating TGF-β1 signaling transduction pathway, therefore enhancing the sensitivity of cells to TGF-β1.Previous work in our laboratory showed that GC could significantly inhibit the proliferation of HO-8910 cells. The work also showed that HO-8910 cells secreted TGF-β1 protein, and the cell also expressed two types receptor of TGF-β1, suggesting that TGF-β1 may regulate the proliferation, differentiation or apoptosis of HO-8910 cells. In the present study, we examined the effects of Dex on the expression of TGF-β1 receptors in PC-3 and HO-8910 cells. Based on the results that GC could up-regulation of the expression of TpRII, we further examined the effect of GC on TGF-β1 signaling transduction pathway and its relationship with the antiproliferative action by GC.Part I Up-regulation of TpRII by GC and Its Biological Significancein Prostate Cancer Cell Line PC-31. Dex Enhance the Inhibitory Effect on the Proliferation of PC-3 Cells by TGF-β1. The inhibitory effects of Dex or TGF-β1 on the growth of PC-3 prostate cancer cells were examined by the method of cell counting. Incubation of PC-3 cells with 100nM Dex or 10ng/ml TGF-β1 resulted in decreased proliferation in a time-dependent manner. Dex and TGF-β1 reduced cell number by 35±1.6% and 50±2.1% at 6 days, respectively. Combined treatment of PC-3 cells with TGF-β1 and Dex caused more obvious decrease in cell number (up to about 70+1.7%) as compared with TGF-β1 alone.2. Dex Treatment Increases Expression of TPRII in the PC-3 Cells. To determine whether Dex potentiates the inhibitory effect of TGF-β1 by regulating the expression of TGF-β1 receptors, we examined the effect of Dex on mRNA and protein level of TGF-β1 receptors in PC-3 cells by RT-PCR and Western Blot assay. Dex increased the expression of TβRII mRNA, but not TβRI mRNA in a time-dependent manner. Dex increased the expression of TβRII in a time-dependent manner. 2.5 ± 0.16 (p<0. 05) fold increasing of TβRII mRNA were obtained following treatment PC-3 cells with 100nM of Dex for 16 hr. RU486, a glucocorticoid receptor antagonist, blocked the inducing effect of Dex the expression of TβRII mRNA, indicating that the up-regulation effect of Dex on the expression of TβRII was mediated by glucocorticoid receptor.3. Dex Increases TβRII Promoter Activity in PC-3 Cells. We found that Dex increased TβRII promoter activity in PC-3 cells using dual luciferase reporter gene assay. A fusion construct containing the full-length TβRII promoter( - 1883/ + 50) attached to a luciferase reporter was transiently transfected into PC-3 cells to observe the effect of Dex on the expression of reporter gene. Dex increased the expression of TβRII-Luc in a dose-dependent manner and the induction level of luciferase was similar to the increased level of TβRII mRNA, indicating that Dex inducement of TβRII expression occurred at transcription level by increasing TβRII promoter activity in PC-3 cells.4. Dex and TGF-β1 induce the activation of reporter gene p3TP-Luc. Promoter region of reporter gene plasmid p3TP-Luc contained TGF-β1 responsive elements and was the generally used reporter gene for examing the biological activity of TGFβ-1. PC-3 cells were transient transfected with p3TP-Luc, then treated with Dex and/or TGF-β1. Cells were harvested 24 hours later and assayed for luciferase activity using the dual luciferase assay system. Result showed that Dex or TGF-β1 could induce the activation of p3TP-Luc, combined treatment with Dex and TGF-β1 had a more effectively activation.5. Dex and TGF-β1 Up-regulates Expression of Cell Cycle Negative Regulator p15~INK4B an(j p27Kn>1 in pc-3 Cells. To examine whether increased expression of TPRII induced by Dex treatment enhance TGF-pi signaling and lead to change of its down-stream events, effect of Dex on expression of plS?*48 and p27KIP1 were examined. Result showed that both TGF-pi and Dex induced expression of plS?*48 mRNA and p27K1P1 protein in a time-dependent fashion. Combined treatment with TGF-pi and Dex potentiate the induction of the expression of plS1?48 mRNA and p27ICIP1 protein.6. TpRII-neutralizing Antibody Partially Blocks the Growth inhibitory Action of Dex on PC-3 Cells.We further examined the ability of a neutralizing antibody specific for the extracellular domain of TpRII to block the growth-inhibitory action of Dex on PC-3 cells by MTT reduction method. The result showed that anti-TpRII polyclonal antibody almost completely blocked the inhibitory by TGF-pi, but partially blocked the growth-inhibitory action of Dex in these cells, suggesting that TGF-pi signaling was involved in the proliferative inhibition of Dex in PC-3 cells.In summary, the present study presents a new information that Dex inhibit PC-3 cells proliferation not only through increasing the expression of TGF-pi, but also through up-regulating the expression of TPRII, then enhanced the sensitivity to TGF-pi, and increased the expression of CKI such as p 1S11^48 and p27KIP'.Part II Up-regulation of TpRII by GC and Its Biological Significance inOvarian Cancer Cell Line HO-89101. Dex Treatment Increases Expression of TpRII in HO-8910 Cells. We found that Dex treatment increased expression of TpRII mRNA and protein in HO-8910 cells, but had no effect on the expression of TGF-pi and TpRI. The effect was mediated by GR.2. TGF-pl Can not Inhibit the Proliferation of HO-8910 Cells. We repeated the inhibitory effect on the proliferation of HO-8910 by TGF-pi. Contrast to what have reported by other laboratory, our result showed that TGF-pi can not inhibit the proliferation of HO-8910 cells. We got the samilar result in the other ovarian cancer cell line 3AO.3. Dex and TGF-pl Induce Smad Nuclear Translocation in HO-8910 Cells. As wefound that TGF-pi can not inhibit the proliferation of HO-8910 cells, we considered if the TGF-pi signaling transduction pathway is defective, then the cells lost the sensitivity to TGF-pi. We examined the nuclear translocation of Smad2/3 using confocal microscope and found that TGF-pi induced the phosphorylation of the Smad2/3 and the subsequent nuclear translocation. Dex itself not only induced the nuclear translocation of Smad2/3 but also enhanced the inducement of TGF-pi on phosphorylation and translocation of Smad2/3.4. Dex and TGF-pi Induce the Activation of Reporter Gene p3TP-Luc TGF-pl...