Ruijuan Xiu

Melatonin modulates the expression of VEGF and HIF‐1α induced by CoCl2 in cultured cancer cells

Abstract:  Melatonin is an important natural oncostatic agent. At present there are no data available as to its possible influence on tumor angiogenesis, which is a major biological mechanism responsible for tumor growth and dissemination. It is well known that vascular endothelial growth factor (VEGF) is crucial to a solid tumor’s higher vascularization and development. To investigate the possible influence of melatonin on angiogenesis, we studied the effect of melatonin on endogenous VEGF expression in three human cancer cell lines (PANC‐1, HeLa and A549 cells). In this study, we report that physiologic concentrations of melatonin have no obvious impact on the VEGF expression, whereas pharmacologic concentrations of melatonin suppress the VEGF mRNA and protein levels induced by hypoxia mimetic cobalt chloride (CoCl2). Melatonin also decreases hypoxia‐inducible factor (HIF)‐1α protein levels, suggesting a role for transcription factor HIF‐1 in the suppression of VEGF expression. The effect of pharmacologic concentrations of melatonin on VEGF and HIF‐1α under normoxia is uncertain, which indicates that the regulatory mechanisms of VEGF in the absence or presence of CoCl2 are different and other or additional transcription factors may be involved. Taken together, our data show that melatonin in high concentrations markedly reduces the expression of endogenous VEGF and HIF‐1α induced by CoCl2 in cultured cancer cells.

Intracellular signaling pathways involved in cell growth inhibition of human umbilical vein endothelial cells by melatonin.

Abstract:  Melatonin, an indolamine mainly produced in the pineal gland, has received a great deal of attention in the last decade because of its oncostatic effects, which are due to its immunomodulatory, antiproliferative, antioxidant and its possible antiangiogenesis properties. Herein, we document its antiproliferative action on human umbilical vein endothelial cells (HUVECs). Moreover, the possible cell signaling pathways when melatonin inhibited HUVEC proliferation were explored in this study. Primary HUVECs were isolated, cultured, purified and identified before the studies were performed. HUVECs were found to possess G‐protein‐coupled membrane receptors for melatonin (MT1 and MT2) and also nuclear melatonin receptors (RORα and RORβ, especially RORβ). No obvious expression of RORγ was found. We investigated the membrane receptors and several intracellular signaling pathways including mitogen‐activated protein kinases (MAPK)/extracellular signal‐related kinases (ERK), phosphoinositol‐3‐kinase (PI3K)/Akt and protein kinases C (PKC) involved in antiproliferative action of melatonin on HUVECs. The blockade of these pathways using special inhibitors decreased cell growth. Furthermore, the constitutive activation of nuclear factor kappa B (NF‐κB) contributed to the proliferation of HUVECs. High concentrations of melatonin inhibited both NF‐κB expression and its binding ability to DNA, possibly through inactivation of ERK/Akt /PKC pathways. Taken together, high concentrations of melatonin markedly reduced HUVEC proliferation; the antiproliferative action of melatonin was closely correlated with following pathway: melatonin receptors/ERK/PI3K/Akt/PKC/ NF‐κB.

Effect and mechanism of melatonin's action on the proliferation of human umbilical vein endothelial cells.

Abstract:  Melatonin is the major secretory product of the pineal gland and is considered an important natural oncostatic agent. The anticancer activity of melatonin is due to its immunomodulatory, anti‐proliferative and antioxidative effects. At present there are no direct data available as to melatonins possible influence on angiogenesis, which is a major biological mechanism responsible for tumor growth and dissemination. The current study investigated the influence of melatonin on angiogenesis. Human umbilical vein endothelial cells (HUVECs) were cultured, identified, and purified. Cell growth and viability, DNA fragmentation and cell cycle analyses were determined. To elucidate the mechanism of action of melatonin, Western blot analyses for P53, Bax and Bcl‐2 expression were carried out. The results demonstrate the anti‐proliferative and apoptosis‐inducing effects of melatonin; these changes were associated with cell cycle arrest, upregulation of P53 and Bax and downregulation of Bcl‐2. Taken together, our data showed that melatonin in high concentrations markedly reduces HUVECs proliferation, induces cellular apoptosis, and modulates cell cycle length. P53 and Bax/Bcl‐2 expression changes may be involved in these actions of melatonin.

Cisplatin up-regulates ICAM-1 expression in endothelial cell via a NF-κB dependent pathway

The anticancer drug cis‐diammindichloroplatin (cisplatin) can cause severe side‐effects, but to date, the mechanisms of action of these dangerous side‐effects have not been completely elucidated. Since cellular adhesion molecules (CAMs), by mediating the recruitment of circulating leukocytes to the blood vessel wall and their subsequent migration into the subendothelial spaces, play a crucial role in several pathophysiologic processes, we sought potential proof for CAMs in the pathophysiology of cisplatin‐induced vascular damage. In vitro, human umbilical vein endothelial cells (HUVECs) were subjected to various concentrations of cisplatin, considerable up‐regulation of intercellular adhesion molecule‐1 (ICAM‐1) but not P‐selectin, E‐selectin, and vascular cell adhesion molecule 1 at both messenger mRNA and protein expression levels were observed. Electrophoretic mobility shift assays and Western blotting analysis revealed that cisplatin up‐regulates ICAM‐1 expression in HUVECs via an NF‐kappaB‐dependent pathway. Further intravital microscopy study demonstrated that significantly higher (P < 0.01) numbers of rolling and sticking leukocytes on the wall of postcapillary venules in male Golden Syrian hamsters cheek pouch bearing a human cervical carcinoma were observed, while inhibition of ICAM‐1 by using specific anti‐ICAM‐1 antibody can attenuate cisplatin‐stimulated leukocyte/endothelium interactions. These data suggest that ICAM‐1 involves in the pathophsiologic process of cisplatin‐induced vascular toxicity and may be exploited for therapeutic advantage. (Cancer Sci 2008; 99: 391–397)

Melatonin inhibits IL-1β-induced monolayer permeability of human umbilical vein endothelial cells via Rac activation

Abstract:  Melatonin is a key factor in the coordination of circadian rhythms and seasonal reproduction. Melatonin and its metabolites directly scavenge free radicals, increase the expression of antioxidant enzymes, and play a role in the anti‐inflammatory phase of defense responses. At present, there are no direct data available as to melatonin’s possible influence on endothelial cell monolayer permeability, which is a major biological process responsible for vascular diseases. The aim of this study was to investigate the effect of melatonin on IL‐1β‐induced human umbilical vein endothelial cells (HUVECs) monolayer permeability and then to test the involvement of small GTPase Rac in the melatonin‐induced endothelial barrier‐protective effects as well as cell contact reorganization. It was found that IL‐1β treatment increased the permeability of HUVECs monolayer, disrupted adherens junctions, and down‐regulated the expression of VE‐cadherin which is the main functional protein of adherens junctions. Melatonin, however, decreased dextran permeability and increased intercellular adherens junction areas reflecting an endothelial cell barrier‐protective response. Furthermore, melatonin dramatically improved IL‐1β‐induced Rac inactivation. Our results show that the barrier‐protective effects of melatonin on endothelial cells are mediated by Rac activation and leads to enhancement of adherens junctions.

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-γ LIGANDS 15-DEOXY-δ12,14-PROSTAGLANDIN J2 AND PIOGLITAZONE INHIBIT HYDROXYL PEROXIDE-INDUCED TNF-α AND LIPOPOLYSACCHARIDE-INDUCED CXC CHEMOKINE EXPRESSION IN NEONATAL RAT CARDIAC MYOCYTES

Ligands for peroxisome proliferator-activated receptor &ggr; (PPAR-&ggr;) such as prostaglandin metabolite 15-deoxy-&dgr;12,14-prostaglandin J2 (15d-PGJ2) or thiazolidinedione pioglitazone have been identified as a new class of anti-inflammatory compounds with possible clinical applications. Reactive oxygen species play an important role in the generation of cellular damage by induction of proinflammatory cytokines and chemokines during myocardial I/R. These events were preceded by activation of the transcription factors nuclear factor (NF)-&kgr;B pathway. It has been suggested that myocardium overproduces TNF-&agr; after I/R, and locally produced TNF-&agr; is sufficient to cause severe impairment of cardiac function. LPS-induced CXC chemokine (LIX) is a rodent chemokine with potent neutrophil-chemotactic activity. Based on this concept, we examined the effects of 15d-PGJ2 and pioglitazone on oxidative stress-induced TNF-&agr; and LIX expression in neonatal rat cardiac myocytes. Pretreatment of myocytes with 15d-PGJ2 or pioglitazone decreased hydrogen peroxide-induced TNF-&agr; and LIX production (mRNA and protein) in a concentration-dependent manner. The beneficial effects of both ligands were associated with reduction of hydrogen peroxide-induced NF-&kgr;B activation. Treatment with 15d-PGJ2, but not pioglitazone, caused dose-dependent activation of heat shock factor 1, which could render cells unresponsive to stimulation of NF-&kgr;B. The cytoprotection afforded by pioglitazone was attenuated by the PPAR-&ggr; antagonist GW9662, which failed to affect the beneficial effects afforded by 15d-PGJ2. Taken together, these results demonstrate that treatment with these chemically distinct ligands of PPAR-&ggr; results in diverse anti-inflammatory mechanisms.ABBREVIATIONS-15d-PGJ2-15-deoxy-&dgr;12,14-prostaglandin J2; HSE-heat shock element; HSF 1-heat shock factor 1; C-cytokine-induced neutrophil chemoattractant; LIX-LPS-induced CXC chemokine; NF-&kgr;B-nuclear factor-&kgr;B; PPAR-&ggr;-peroxisome proliferator-activated receptor &ggr;

Enhanced matrix metalloproteinases-2 activates aortic endothelial hypermeability, apoptosis and vascular rarefaction in spontaneously hypertensive rat.

Microvascular rarefaction with endothelial cells apoptosis is a common characteristic of various microvascular complications in the spontaneously hypertensive rat (SHR). Elevated levels of proteolytic (e.g. matrix metalloproteinase, MMPs) activity and apoptosis in aortic endothelial cells of SHR were found when compared to its normotensive control. However, the exact mechanisms of microvascular rarefaction and the role of MMPs in this process remain poorly understood. Besides cleavage of VEGFR2 via unbalanced MMPs, we hypothesize that selected cleavage of Beta-Catenin and VE-cadherin by MMPs could induce apoptosis of rat aortic endothelial cells (RAECs) and rarefaction. Primary RAECs were isolated, identified and used in a in-vitro model. Transwell system was used to analyze the permeability of Wistar RAECs, SHR RAECs and SHR RAECs with pretreatment by doxycycline. Qualitative and semi-quantitative analysis of major endothelial adhesion molecules were detected by immunofluorescence technique and Western blot, respectively. MMP-2 activity of SHR RAECs was increased significantly and doxycycline (50 μM) effectively reduced the level of MMP-2 and hyper-permeability in SHR RAECs. SHR RAECs showed enhanced cleavage of VEGFR2, VE-cadherin and B-catenin, which could be prevented by doxycycline (50 μM). Doxycycline (50 μM) attenuated hyper-permeability via decreased MMP-2 by protecting VEGFR2, VE-cadherin, Beta-catenin from cleavage and inhibited the reduction of mitochondrial transmembrane potential (MTP), thus prevented mitochondria-mediated apoptotic signaling and capillary rarefaction in the SHR. It might be a novel insight into the mechanisms of SHR microvascular rarefaction that is independent of pressure but relevant to MMP-2.

Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2.

Matrix metalloproteinase-2 (MMP-2), also known as gelatinase A, is involved in vascular calcification. Another member of gelatinases is MMP-9 (gelatinase B). However, the role of gelatinases in the pathogenesis of vascular calcification is not well understood. The current study aims to clarify the relationship between gelatinases and vascular calcification and to elucidate the underlying mechanism. Beta-glycerophosphate (β-GP) was used to induce calcification of vascular smooth muscle cells (VSMCs) with or without 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane (SB-3CT), a specific gelatinases inhibitor. Levels of calcification were determined by assessing calcium content and calcification area of VSMCs. Phenotype transition of VSMCs was observed by assessing expressions of alkaline phosphatase (ALP), smooth muscle α-actin (SM-α-actin) and desmin. Gelatin zymography was applied to determine the activities of gelatinases, and western blot was applied to determine expressions of gelatinases, bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX2) and msh homeobox homolog 2 (Msx-2). Gelatinases inhibition by SB-3CT alleviated calcification and phenotype transition of VSMCs induced by β-GP. Increased gelatinases expression and active MMP-2 were observed in calcifying VSMCs. Gelatinases inhibition reduced expression of RUNX2, Msx-2 and BMP-2. BMP-2 treatment increased expressions of RUNX2 and Msx-2, while noggin, an antagonist of BMP-2, decreased expressions of RUNX2 and Msx-2. Gelatinases promote vascular calcification by upregulating BMP-2 which induces expression of RUNX2 and Msx-2, two proteins associated with phenotype transition of VSMCs in vascular calcification. Interventions targeting gelatinases inhibition might be a proper candidate for ameliorating vascular calcification.

Decreased expression of MDR1 in PEG-conjugated hemoglobin solution combined cisplatin treatment in a tumor xenograft model

Abstract This study aims to examine the contribution of PEG-conjugated hemoglobin combined with cisplatin to the expression of HIF-1α and MDR1 in a tumor xenograft model. Cervical carcinoma models were assigned to 4 groups and treated respectively: group 1(control); group 2, cisplatin; group 3, PEG-Hb; group 4 cisplatin plus PEG-Hb. 4 weeks later, tumor volume and MVD was significantly decreased in group 4 compared with other groups. Lower expression of HIF-1α and MDR1 were detected in group4. Taken together, our data indicated that PEG-Hb plus cisplatin can promote tumor tissue oxygenation and enhance the chemotherapy sensitivity. HIF-1α regulated MDR1 pathway correlated with this process.

The distinct abilities of tube-formation and migration between brain and spinal cord microvascular pericytes in rats

Pericytes are contractile cells that wrap around the endothelial cells of capillaries throughout the body. They play an important role in regulating the blood brain barrier (BBB) and blood spinal cord barrier (BSCB). The differences between brain and spinal cord microvascular endothelial cells have been investigated. However, no report has elucidated the similarities and differences between brain microvascular pericytes (BMPs) and spinal cord microvascular pericytes (SCMPs) in vitro. The similarities were found between the two types of pericytes not only in the proliferation ability but also in the expression of toll like receptor 4. On the other hand, BMPs showed more than 2 fold in tubular length formation compared with SCMPs. The number of migratory SCMPs was larger than that of migratory BMPs. The expressions of connexin 43 and vascular endothelial growth factor (VEGF) in BMPs were increased compared with those in SCMPs, while SCMPs expressed more desmin and N-cadherin than BMPs. The abilities of tube-formation and migration between BMPs and SCMPs were markedly different, which might be mediated by VEGF, connexin 43, N-cadherin and desmin. These distinguishing features may reflect the more widespread differences between the BBB and BSCB which directly impact pathophysiological processes in various major diseases.