Epigallocatechin-3-gallate inhibits the PDGF-induced VEGF expression in human vascular smooth muscle cells via blocking PDGF receptor and Erk-1/2.

Platelet-derived growth factor (PDGF) has been known to induce vascular endothelial growth factor (VEGF) expression in human vascular smooth muscle cells (hVSMCs). We previously reported that Erk-1/2 and AP-1 pathways are crucial in the PDGF-induced VEGF expression in hVSMCs . In this study, we investigated the effect of epigallocatechin-3-gallate (EGCG), the major green tea catechin, on the PDGF-induced VEGF expression in hVSMCs and the underlying mechanisms. EGCG were found to inhibit dose-dependently the VEGF expression and activation of PDGF receptor, Erk-1/2 and AP-1 induced by PDGF. In addition, cell free studies demonstrated that EGCG could directly inhibit the Erk-1/2 activity. Conditioned media from the hVSMCs treated with PDGF could remarkably stimulate the in vitro growth of human umbilical vein endothelial cells (HUVECs) but the media from the EGCG-pretreated hVSMCs lost its stimulatory activity for HUVEC proliferation. These results suggest that EGCG may exert the anti-angiogenic effect by inhibiting the PDGF-induced VEGF expression at multiple signaling levels.

Extracellular signal-regulated kinases and AP-1 mediate the up-regulation of vascular endothelial growth factor by PDGF in human vascular smooth muscle cells.

Endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) have been shown to communicate with each other via cytokine signaling during neovascularization. In this study, we investigated the effect of platelet-derived growth factor (PDGF), a cytokine released from tumors and ECs, on vascular endothelial growth factor (VEGF) expression in human VSMCs and underlying signal transduction pathways. PDGF induced VEGF expression in a time- and concentration-dependent manner. PDGF induced the activation of extra-cellular signal-regulated kinase-1/2 (ERK-1/2), but not the activation of c-jun amino terminal kinase (JNK) and P38 mitogen-activated protein kinase (MAPK). Specific inhibitor of mitogen-activated protein kinase kinase (MEK)-1 was found to suppress VEGF expression and promoter activity. The expression of vectors encoding a mutated-type MEK-1 decreased the VEGF promoter activity. Electrophoretic mobility shift assay revealed that PDGF dose-dependently increased the DNA binding activity of AP-1. Transient transfection studies using an AP-1 decoy oligonucleotide confirmed that the activation of AP-1 is involved in PDGF-induced VEGF upregulation. Conditioned media from the human VSMCs pretreated with PDGF could remarkably stimulate the in vitro growth of human umbilical vein endothelial cells and this effect was partially abrogated by VEGF neutralizing antibodies. The above results suggest that ERK-1/2 and AP-1 signaling pathways are involved in the PDGF-induced VEGF expression in human VSMCs and that these paracrine signaling pathways induce endothelial cell proliferation.

Pretreatment of acetylsalicylic acid promotes tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by down-regulating BCL-2 gene expression.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to be selective in the induction of apoptosis in cancer cells with minimal toxicity to normal tissues. However, not all cancers are sensitive to TRAIL-mediated apoptosis. Thus, TRAIL-resistant cancer cells must be sensitized first to become responsive to TRAIL. In this study, we observed that pretreatment by acetylsalicylic acid (ASA) augmented TRAIL-induced apoptotic death in human prostate adenocarcinoma LNCaP and human colorectal carcinoma CX-1 cells. Western blot analysis showed that pretreatment of ASA followed by TRAIL treatment activated caspases (8, 9, and 3) and cleaved poly(ADP-ribose) polymerase, the hallmark feature of apoptosis. Most interestingly, at least 12 h of pretreatment with ASA was prerequisite for promoting TRAIL-induced apoptosis and was related to down-regulation of BCL-2. Biochemical analysis revealed that ASA inhibited NF-kappaB activity, which is known to regulate BCL-2 gene expression, by dephosphorylating IkappaB-alpha and inhibiting IKKbeta activity but not by affecting the HER-2/neu phosphatidylinositol 3-kinase-Akt signal pathway. Overexpression of BCL-2 suppressed the promotive effect of ASA on TRAIL-induced apoptosis and changes in mitochondrial membrane potential. Taken together, our studies suggested that ASA-promoted TRAIL cytotoxicity is mediated through down-regulating BCL-2 and by decreasing mitochondrial membrane potential.

Role of HER-2/neu signaling in sensitivity to tumor necrosis factor-related apoptosis-inducing ligand: enhancement of TRAIL-mediated apoptosis by amiloride.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in numerous transformed cell lines but not in most normal cells. Although this selectivity offers a potential therapeutic application in cancer, not all cancers are sensitive to TRAIL-mediated apoptosis. In this study, we observed that amiloride, a current clinically used diuretic drug, which had little or no cytotoxicity, sensitized TRAIL-resistant human prostate adenocarcinoma LNCaP and human ovarian adenocarcinoma SK-OV-3 cells. The TRAIL-mediated activation of caspase, and PARP cleavage, were promoted in the presence of amiloride. Western blot analysis showed that combined treatment with TRAIL and amiloride did not change the levels of TRAIL receptors (DR4, DR5, and DcR2) and anti-apoptotic proteins (FLIP, IAP, and Bcl-2). However, amiloride dephosphorylated HER-2/neu tyrosine kinase as well as Akt, an anti-apoptotic protein. Interestingly, amiloride also dephosphorylated PI3K and PDK-1 kinases along with PP1alpha phosphatase. In vitro kinase assay revealed that amiloride inhibited phosphorylation of kinase as well as phosphatase by competing with ATP. Taken together, the present studies suggest that amiloride enhances TRAIL-induced cytotoxicity by inhibiting phosphorylation of the HER-2/neu-PI3K-Akt pathway-associated kinases and phosphatase.

Amiloride augments TRAIL-induced apoptotic death by inhibiting phosphorylation of kinases and phosphatases associated with the P13K-Akt pathway.

We have previously shown that low extracellular pH (pHe) promotes cell killing by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we examined whether amiloride, an inhibitor of the Na(+)/H(+) antiporter capable of lowering the intracellular pH (pHi), can potentiate TRAIL-induced apoptotic death. Human prostate adenocarcinoma DU-145 cells were treated with various concentrations of TRAIL (10-200 ng/ml) and/or amiloride (0.1-1 mM) for 4 h. Amiloride, which caused little or no cytotoxicity by itself, enhanced TRAIL-induced apoptosis. The TRAIL-mediated activation of caspase, and PARP (poly (ADP-ribose) polymerase) cleavage were both promoted by amiloride. Western blot analysis showed that combined treatment with TRAIL and amiloride did not change the levels of TRAIL receptors (death receptor (DR)4, DR5, and DcR2 (decoy recepter 2) or antiapoptotic proteins (FLICE-inhibitory protein (FLIP), inhibitor of apoptosis (IAP), and Bcl-2). However, unlike pHe, amiloride promoted the dephosphorylation of Akt. Interestingly, amiloride also induced the dephosphorylation of P13K (phosphatidylinositol 3-kinase) and PDK-1 (phosphoinositide-dependent kinase-1) kinases along with PTEN (phosphatase and tensin homolog deleted on chromosome 10) and PP1alpha phosphatases. In vitro kinase assays revealed that amiloride inhibited phosphorylation of kinases and phosphatases by competing with ATP. Taken together, the present studies suggest that amiloride enhances TRAIL-induced cytotoxicity by inhibiting phosphorylation of the PI3K-Akt pathway-associated kinases and phosphatases.