Effect And Mechanism Of Melatonin's Action On The Proliferation And Apotosis In Human Umbilical Vein Cells

Effect and mechanism of melatonin's action on the proliferation and apotosis in human umbilical vein cellsObjecive Part one: At present there are no direct data available as to melatonin's 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, and the action on the cell proliferation and apoptosis in human umbilical vein cells (HUVECs). Part two: on the basis of previous study, this study was designed to explore the possible cell signalling pathways when melatonin inhibited the HUVECs proliferation and induced apoptosis.Methods Part one: Primary HUVECs were isolated from fresh human umbilical veins and cultured, after immunomagnetic separation by anti-CD31 antibody coated Dynabeads, the HUVECs were indentified by FactorⅧimmuocytochemical staining and eletroscope observation; MTT method was carried out to assay the proliferation of HUVECs when treated with melatonin in concentrations from 0.1nmol/L to 1mmol/L (final concetration in the medium); and the cell cycle and apoptosis were detect using propidium iodione (PI) staining; the expressions of P53, Bax and Bcl-2 mRNA were detected by RT-PCR, also the proteins by immunofluorescence and Westem Blot anaysis. Part Two: the melatonin's receptors type expression in HUVECs was detected at first, and the interactions was observed when treated with melatonin; the possible intracellular signaling pathways include MAPK/ERK, PI3K/AKT and PKC were detected whether contributed to the anti-proliferative and pro-apoptotic effect of the indolamine in HUVECs by using specific inhibitors and activators; EMSA was used to detected the NF-κB binding activity in HUVECs when treated with melatonin and the relationship between NF-κB and its possible up-stream trigger signaling pathways. Results Part one: (1) Primary HUVECs were isolated sucessfully from fresh human umbilical veins and cultured, afer purified by immunomagnetic separation. The endothelial origin was confirmed by the positive labeling of von Willebrand Factor (vWF), CD31 and electronmicropy observation. (2) At near physiological concentrations (1nmol/L), melatonin had no significant effect on cellular proliferation. However, melatonin in higher concentrations (greater than 10 nmol/L) significantly decreased cell proliferation, and the negative effect on HUVECs proliferation was enhanced with increasing melatonin concentrations. Melatonin of 1mmol/L had the greatest antiproliferative effect on HUVECs with the cell viability no difference between the control and the melatonin-treatment groups. (3) FCM showed HUVECs treated with melatonin in high concentrations presented a very distinct hypodiploid peak of apoptosis (0.64% vs 31.3%, P<0.01), and Cell cycle analyzed also showed a reduction of cells entering in the S phase (S phase arrest). (4) The Bax or P53 mRNA expression is increasing when treatment with 100nmol/L after 24hr, whereas no obvious changes to Bcl-2, but when incubated with 1mmol/L melatonin, a very early increasing expression of P53 and Bax mRNA at 3hr and a later decrease of Bcl-2 mRNA were shown. (4) The up-regulation of P53 and Bax and down-regulation of Bcl-2 proteins possibly contributed to the apoptotis in HUVECs with melatonin.Part two: (1) In normal cultured HUVECs, G- protein- coupled membrane receptors of melatonin (MT1 and MT2) also with the nuclear receptors of melatonin (RORa and RORb, especially RORb) are both expressed, whereas no obvious expression of RORc in HUVECs. (2) The membrane receptors was involved in melatonin antiproliferative properties in HUVECs, and the receptor antagonist Luzindole could partially prevent the indolamine's efficacy. (3) In cultured HUVECs, melatonin, even at physiological concentrations, could suppress the expression of melatonin receptors, and it may be the outcome of a direct or indirect melatonin-mediated activation of constitutive PKCα. (4) The survival and proliferation of HUVECs is dependent on the activation of several intracellular signaling pathways include mitogen-activated protein kinases (MAPK)/extracellualar signal-related kinases (ERK), phosphoinositol-3-kinase (PI3K)/AKT and protein kinases C (PKC), and the blockade of these pathways by specifical inhibitors could decrease the cell growth rate respectively. (5) High concentration melatonin reduced the proliferation and induced the apoptosis direct or indirect by inactivation the ERK, PI3K/AKT and PKC, with the fact that the antiproliferative effect was partially regulated by its special inhibitors and activators. (6) HUVECs showed basal activation of NF-κB transcription factor, and high concetration melatonin inhibited the NF-κB expression and its binding to DNA. (7) All the signaling pathways specical inhibior could partially inhbit the NF-κB binding to DNA, thus suggest melatonin inhibited NF-κB activity possibly through inactivating these pathways, which may be responsible for the antiproliferative and pro-apoptotic effect of melatonin in HUVECs.