Inhibition of trichostatin A-induced antiangiogenesis by small-interfering RNA for thrombospondin-1

Expression of thrombospondin-1 (TSP-1), which is a known inhibitor of tumor growth and angiogenesis, is reciprocally regulated by positive regulators, such as VEGF. Additionally, trichostatin A (TSA) suppresses tumor progression by altering VEGF levels and VEGF-mediated signaling. Thus, understanding TSA-regulated TSP-1 expression and the effects of altered TSP-1 levels might provide insights into the mechanism of action of TSA in anti-tumorigenesis, and provide an approach to cancer therapy. Here, we examined the effect of TSA on TSP-1 expression, and the effects of TSA-induced TSP-1 on cell motility and angiogenesis, in HeLa and bovine aortic endothelial cells. TSA remarkably increased TSP-1 expression at the mRNA and protein levels, by controlling the TSP-1 promoter activity. Both TSA and exogenous TSP-1 reduced cell migration and capillary-like tube formation and these activities were confirmed by blocking TSP-1 with its neutralizing antibody and small-interfering RNA. Our results suggest that TSP-1 is a potent mediator of TSA-induced anti- angiogenesis.

The Effect of Epicatechin on the High Glucose-induced TSP-1 Expression and MMP-2 Activity in Rat Vascular Smooth Muscle Cells / 대한내분비학회지

BACKGROUND: The incidence of atherosclerosis is well correlated with the progression of type 2 diabetes mellitus. High plasma glucose in uncontrolled diabetic patients evokes many vascular complications such as atherosclerosis. Specifically, high glucose was reported to induce thrombospondin-1 (TSP-1), which activates matrix metalloproteinase-2 (MMP-2) and leads to the invasion of vascular smooth muscle cells (VSMCs) into the intima. Catechins with antioxidant effects are known to inhibit MMP-2 activity. Therefore, this study was aimed at revealing the effect of epicatechin, one of catechins, on high glucose-induced TSP-1 and the invasiveness of VSMCs. METHODS: VSMCs were primarily isolated from Sprague-Dawley rat aorta. The VSMCs were incubated with different doses (30, 100 and 300 micrometer) of epicatechin under high glucose concentration (30 mM). The TSP-1 protein and mRNA expressions were analyzed by performing Western blotting and Northern blot analyses, respectively. RT-PCR was performed to observe the MMP-2 mRNA expression. Gelatin zymography was performed for the measurement of MMP-2 activity. Invasion assays were performed to evaluate the invasiveness of VSMCs. RESULTS: Epicatechin inhibited the high glucose-induced TSP-1 expression and the MMP-2 activity in a dose-dependent manner. Also, epicatechin inhibited the high glucose-induced invasiveness of VSMCs across the matrix barrier in a dose-dependent fashion. CONCLUSION: Collectively, epicatechin may prevent the high glucose-induced proliferation and invasion of VSMCs by inhibiting the TSP-1 expression and the MMP-2 activity. Therefore, epicatechin appears to play a protective role in the development of atherosclerosis.

The Effect of Epicatechin on the High Glucose-induced TSP-1 Expression and MMP-2 Activity in Rat Vascular Smooth Muscle Cells / 대한내분비학회지

BACKGROUND: The incidence of atherosclerosis is well correlated with the progression of type 2 diabetes mellitus. High plasma glucose in uncontrolled diabetic patients evokes many vascular complications such as atherosclerosis. Specifically, high glucose was reported to induce thrombospondin-1 (TSP-1), which activates matrix metalloproteinase-2 (MMP-2) and leads to the invasion of vascular smooth muscle cells (VSMCs) into the intima. Catechins with antioxidant effects are known to inhibit MMP-2 activity. Therefore, this study was aimed at revealing the effect of epicatechin, one of catechins, on high glucose-induced TSP-1 and the invasiveness of VSMCs. METHODS: VSMCs were primarily isolated from Sprague-Dawley rat aorta. The VSMCs were incubated with different doses (30, 100 and 300 micrometer) of epicatechin under high glucose concentration (30 mM). The TSP-1 protein and mRNA expressions were analyzed by performing Western blotting and Northern blot analyses, respectively. RT-PCR was performed to observe the MMP-2 mRNA expression. Gelatin zymography was performed for the measurement of MMP-2 activity. Invasion assays were performed to evaluate the invasiveness of VSMCs. RESULTS: Epicatechin inhibited the high glucose-induced TSP-1 expression and the MMP-2 activity in a dose-dependent manner. Also, epicatechin inhibited the high glucose-induced invasiveness of VSMCs across the matrix barrier in a dose-dependent fashion. CONCLUSION: Collectively, epicatechin may prevent the high glucose-induced proliferation and invasion of VSMCs by inhibiting the TSP-1 expression and the MMP-2 activity. Therefore, epicatechin appears to play a protective role in the development of atherosclerosis.

Weakening of the repressive YY-1 site on the thrombospondin-1 promoter via c-Jun/YY-1 interaction

Thrombospondin-1 (TSP-1) level is tightly regulated at the transcriptional level. To determine the detailed molecular mechanisms of TSP-1 expression, nine serial 5'-deletion constructs of the human genomic tsp-1 promoter (nucleotides -2,220 to +756) were prepared, inserted into luciferase reporter plasmids, and transiently transfected into the Hep3B human hepatocarcinoma cell. Among the nine 5'-deletion constructs, pTSP-Luc-4 (-767~+756) had consistently decreased luciferase activity with or without PMA stimulation, whereas a further truncated construct [pTSP-Luc-4' (-407~+756)] had increased levels of expression. By searching the nucleotides from -767 to -407, a consensus binding sequence (5'-CCATTTT-3') for the repressor Yin Yang-1 (YY-1) at nucleotide -440 was identified. The suppression induced by this site was weakened in the presence of the region upstream of nucleotide -767 (pTSP-Luc-1 and -2). Nuclear protein directly bound to an oligonucleotide containing the repressive YY-1 sequence but the binding capacity of the sequence was decreased by the increased c-Jun levels. Moreover, proteins immunoprecipitated with anti-YY-1 revealed an interaction between c-Jun and YY-1 factor. These data suggest that the repressive YY-1 site of the tsp-1 promoter could not be functional via activating positive cis-elements on the upstream from this site and weakened via c-Jun/YY-1 interactions.

Cell-type specific regulation of thrombospondin-1 expression and its promoter activity by regulatory agents

Thrombospondin-1 (TSP-1), a multifunctional protein that is able to function as a negative regulator of solid tumor progression and angiogenesis, is normally present at a very low level but rapidly elevated in pathological tissues. To understand the cellular regulation of TSP-1 expression, the mode of it's expression in Hep3B, SK-HEP-1, and porcine aortic endothelial (PAE) cells was examined in the presence of all-trans retinoic acid (ATRA), interleukin-6 (IL-6), interferon-gamma (IFN-gamma), or phorbol 12-myristate 13-acetate (PMA). ATRA or IL-6 induced a dose-dependent increase of TSP-1 protein and mRNA levels in PAE cells, while they negatively regulated TSP-1 expression in the Hep3B and SK-HEP-1 cells. In contrast, PMA showed just the opposite effects on the TSP-1 expression in the same cells. IFN-gamma had little effect on TSP-1 level in Hep3B and PAE cells. The TSP-1 expression in SK-HEP-1 cells by these agents showed a close resemblance to that of liver cells rather than that of the endothelial cell line. Possible TSP-1 promoter-mediated responses by ATRA, IL-6, IFN-gamma, or PMA in Hep3B and PAE cells examined with luciferase activity of TSP-LUC reporter plasmid showed that levels of TSP-1 promoter activity were lower than that of the expressed TSP-1 protein and mRNA levels. Transfection of c-Jun and/or RARalpha expression vectors into Hep3B and PAE cells resulted in the enhanced TSP-1 promoter activity as well as the increments of of its protein and mRNA level. These results suggest that regulatory agents-induced TSP-1 expression may be attributed to mRNA stability and/or translational activation in concert with transcriptional activation and TSP-1 expression may be independently controlled via each signal pathway stimulated by PMA or ATRA.

Responsive site on the thrombospondin-1 promotor to down-regulation by phorbol 12-myristate 13-acetate in porcine aortic endothelial cells

Thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein, inhibits neovascularization and is implicated in the regression of tumor growth and metastasis. We found that the synthesis of TSP-1 in porcine aortic endothelial (PAE) cells was decreased in a dose-dependent manner by phorbol 12-myristate 13-acetate (PMA) treatment in porcine aortic endothelial (PAE) cells. In this study, a responsive site on the TSP-1 promotor affected by PMA treatment in PAE was characterized. The level of TSP-1 mRNA was also decreased by PMA after 1 h and persisted that way for at least 24 h. PMA treatment and c-Jun overexpression suppressed the transcription of TSP-1 promotor-luciferase reporter gene. A deletion between -767 and -657 on the TSP-1 promotor neutralized the PMA-induced down-regulation. In addition, oligo a (-767 approximately -723) was responsive to PMA-induced repression, while oligo b (-734 approximately -689) and c (-700 approximately -656) was not. Electrophoretic mobility shift assays showed that this PMA responsive element specifically bound a nuclear protein and that the binding activity was diminished by PMA treatment in PAE cells but not in Hep 3B cells. In supershift assay, potential regulatory elements in this region, SP1 and GATA-1, were not responsive to the inhibition of TSP-1 expression by PMA. Our results suggest that the repression of TSP-1 synthesis by PMA is mediated by blocking a particular unknown nuclear protein binding to the responsive site (-767 approximately -735), which is regulated by c-Jun.

Growth Regulation of Ovarian Cancer Cells through the Inactivation of AP-1 by Retinoid Derivatives / 대한암학회지

PURPOSE: The growth regulatory effect of retinoid derivatives could be mediated by the transcriptional inactivation of AP-1 oncogenic transcription factor. By using ovarian cancer cell lines we were to investigate the cross-regulation mechanism between retinoids and AP-1. MATERIALS AND METHODS: Cell proliferation assays were performed in 4 ovarian cancer cells (A2774, PA-1, OVCAR-3, SKOV-3) by increasing the concentrations of all-trans retinoic acid (ATRA), 9-cis retinoic acid (9RA), 13-cis RA (13RA), 4-hydroxyphenyl retinamide (4-HPR). Transient transfection and CAT ELISA were done to determine the selective activity of each retinoid on the RAR (alpha, beta, gamma), RXR (alpha, beta, gamma). and the negative activity on AP-1 (c-Jun). RESULTS: Antiproliferative effect of 4-HPR (IC50; 0.7~2.7 micrometer) was more potent than those of other retinoid derivatives (IC50; 2.7~9.0 micrometer). To assess the anticancer mechanism, we examined the effect of 4-HPR on the transriptional activity of retinoic acid receptors (RAR/RXR) and of c-jun. Contrary to other retinoid derivatives that are active for RAR and RXR with some different levels, 4-HPR showed weak activity only for RARgamma. However, 4-HPR exerted the strongest suppression on AP-1 (c-Jun) activity. CONCLUSION: Based on our results showing much 4-HPR's potent antiproliferative activity coupled with the most effectively inhibiting activity on AP-1 and minimum activity on RA receptor (selective for RARgamma) than other retinoid derivatives, we suggest that 4-HPR may be a novel, and very effective anticancer drugs for ovarian cancer.