Objectives: 

Hepatocellular carcinoma (HCC) as solid tumor which response to hypoxia with aberrant visualization. Hypoxia-inducible factor 1 (Hif1) controls oxygen homeostasis inducing the expression of angiogenic factors like vascular endothelial growth factor (VEGF). Signal transducer and activator of transcription 3 (STAT3) has been reported to control angiogenesis by promoting Hif1 stability and transcriptional activity. Melatonin induces apoptosis in HCC and has shown anti-angiogenic features in some tumors. In this study, we examined the role of STAT3/Hif1 alpha/VEGF in melatonin anti-angiogenic effects using an in vitro model of HCC.

Materials: 

HepG2 human liver cancer cells where treated with physiologic and pharmacologic concentration of melatonin under normoxic or hypoxic conditions induced by CoCl2. Gene expression of Hif1 alpha and VEGF analyzed by RT-qPCR, their protein levels as well as phosphorylation status of STAT3 by western blot. Immunofluorescence was performed to analyze Hif1 alpha subcellular localization. Additionally, we used Stattic to analyze whether melatonin effects on Hif1 alpha and VEGF where mediated trough phospho STAT3 inhibition.Statistical analysis were assayed with One-way ANOVA followed by Student-Newmann-Keuls post hoc test.

Results: 

Pharmacologic concentration of melatonin decreased Hif1 alpha mRNA, protein expression and nuclear localization, accompanied by reduction of VEGF also at mRNA and protein levels under hypoxia. Furthermore, melatonin suppressed hypoxia mediated STAT3 phosphorylation, and treatment with either melatonin or the selective STAT3 inhibitor, Stattic reduced the Hif1 alpha, phospho-STAT3 and VEGF protein expression compared with untreated control.

Conclusions: 

Together, our results provide evidences that point to melatonin as a powerful anti-angiogenic agent in HCC, through downregulation of STAT3/Hif1 alpha/VEGF pathway.