Inhibition of VEGF expression through blockade of Hif1α and STAT3 signalling mediates the anti-angiogenic effect of melatonin in HepG2 liver cancer cells

Abstract

Background:Hepatocellular carcinoma (HCC) growth relies on angiogenesis via vascular endothelial growth factor (VEGF) release. Hypoxia within tumour environment leads to intracellular stabilisation of hypoxia inducible factor 1 alpha (Hif1α) and signal transducer and activator of transcription (STAT3). Melatonin induces apoptosis in HCC, and shows anti-angiogenic features in several tumours. In this study, we used human HepG2 liver cancer cells as an in vitro model to investigate the anti-angiogenic effects of melatonin.Methods:HepG2 cells were treated with melatonin under normoxic or CoCl2-induced hypoxia. Gene expression was analysed by RT–qPCR and western blot. Melatonin-induced anti-angiogenic activity was confirmed by in vivo human umbilical vein endothelial cells (HUVECs) tube formation assay. Secreted VEGF was measured by ELISA. Immunofluorescence was performed to analyse Hif1α cellular localisation. Physical interaction between Hif1α and its co-activators was analysed by immunoprecipitation and chromatin immunoprecipitation (ChIP).Results:Melatonin at a pharmacological concentration (1 mℳ) decreases cellular and secreted VEGF levels, and prevents HUVECs tube formation under hypoxia, associated with a reduction in Hif1α protein expression, nuclear localisation, and transcriptional activity. While hypoxia increases phospho-STAT3, Hif1α, and CBP/p300 recruitment as a transcriptional complex within the VEGF promoter, melatonin 1 mℳ decreases their physical interaction. Melatonin and the selective STAT3 inhibitor Stattic show a synergic effect on Hif1α, STAT3, and VEGF expression.Conclusion:Melatonin exerts an anti-angiogenic activity in HepG2 cells by interfering with the transcriptional activation of VEGF, via Hif1α and STAT3. Our results provide evidence to consider this indole as a powerful anti-angiogenic agent for HCC treatment.

Melatonin-Induced Oncostasis, Mechanisms and Clinical Relevance

“…Melatonin decreases serum levels of VEGF in metastatic cancer patients [96]. Several studies have reported that melatonin-related anti-angiogenic activity in cancer cells is mediated, at least in part, by inhibition of HIF1a nuclear translocation, which is required for its transcriptional activation and subsequent VEGF expression [96,97]. Park et al [98] demonstrated that melatonin suppresses tumor angiogenesis by inhibiting HIF-1 and VEGF via sphingosine kinase 1 in colon cancer cells.…”

Effect of As2O3 and Ginsenoside Rg3 on Inhibiting Angiogenesis in Rabbit Liver VX2 Tumor

“…VEGF plays a key role in the formation and growth of normal blood vessels and in tumor angiogenesis [6]. In the present study, we investigated the levels of VEGF using immunohistochemical techniques.…”

Melatonin as an Anti-angiogenetic, Immunomodulatory and Antitumoral Agent

“…They suggested that melatonin exerts an anti-angiogenic activity in HepG2 cells by interfering the transcriptional activation of VEGF, thru HIF1 alpha and STAT3 [7]. Results offer evidence to recollect this indole as a strength antiangiogenic agent for hepatocellullar carcinoma treatment [7].…”

“…Melatonin induces apoptosis in liver cancer and suggests anti-angiogenic effect in numerous tumors. Carbajo-Pescador et al used human HepG2 liver cancer cells as an in vitro model to investigate the anti-angiogenic consequences of melatonin [7]. They suggested that melatonin exerts an anti-angiogenic activity in HepG2 cells by interfering the transcriptional activation of VEGF, thru HIF1 alpha and STAT3 [7].…”

“…Carbajo-Pescador et al used human HepG2 liver cancer cells as an in vitro model to investigate the anti-angiogenic consequences of melatonin [7]. They suggested that melatonin exerts an anti-angiogenic activity in HepG2 cells by interfering the transcriptional activation of VEGF, thru HIF1 alpha and STAT3 [7]. Results offer evidence to recollect this indole as a strength antiangiogenic agent for hepatocellullar carcinoma treatment [7].…”