Inhibition of VEGF Expression Through Blockade of Hif1α and STAT3 Signalling Mediates the Anti-angiogenic Effect of Melatonin in HepG2 Liver Cancer Cells

Overview

Journal Br J Cancer

Specialty Oncology

Date 2013 Jun 13

PMID 23756865

Citations 103

Authors

S Carbajo-Pescador

R Ordonez

M Benet

R Jover

A Garcia-Palomo

J L Mauriz

J Gonzalez-Gallego

Affiliations

Soon will be listed here.

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.

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References

Farriol M, Venereo Y, Orta X, Castellanos J, Segovia-Silvestre T . In vitro effects of melatonin on cell proliferation in a colon adenocarcinoma line. J Appl Toxicol. 2000; 20(1):21-4. DOI: 10.1002/(sici)1099-1263(200001/02)20:1<21::aid-jat623>3.0.co;2-m. View

Lissoni P, ROVELLI F, Malugani F, Bucovec R, Conti A, Maestroni G . Anti-angiogenic activity of melatonin in advanced cancer patients. Neuro Endocrinol Lett. 2001; 22(1):45-7. View

Kitamura H, Koike S, Nakazawa K, Matsumura H, Yokoi K, Nakagawa K . A reversal in the vascularity of metastatic liver tumors from colorectal cancer after the cessation of chemotherapy plus bevacizumab: contrast-enhanced ultrasonography and histological examination. J Surg Oncol. 2012; 107(2):155-9. DOI: 10.1002/jso.23244. View

Carbajo-Pescador S, Martin-Renedo J, Garcia-Palomo A, Tunon M, Mauriz J, Gonzalez-Gallego J . Changes in the expression of melatonin receptors induced by melatonin treatment in hepatocarcinoma HepG2 cells. J Pineal Res. 2009; 47(4):330-8. DOI: 10.1111/j.1600-079X.2009.00719.x. View

Sun H, Tang Z . Angiogenesis in hepatocellular carcinoma: the retrospectives and perspectives. J Cancer Res Clin Oncol. 2004; 130(6):307-19. DOI: 10.1007/s00432-003-0530-y. View

Cho S, Lee H, Jeong S, Lee H, Kim H, Chen C . Sphingosine kinase 1 pathway is involved in melatonin-induced HIF-1α inactivation in hypoxic PC-3 prostate cancer cells. J Pineal Res. 2011; 51(1):87-93. DOI: 10.1111/j.1600-079X.2011.00865.x. View

Wang X, Crowe P, Goldstein D, Yang J . STAT3 inhibition, a novel approach to enhancing targeted therapy in human cancers (review). Int J Oncol. 2012; 41(4):1181-91. DOI: 10.3892/ijo.2012.1568. View

Tischer E, Mitchell R, Hartman T, Silva M, GOSPODAROWICZ D, Fiddes J . The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem. 1991; 266(18):11947-54. View

Gray M, Zhang J, Ellis L, Semenza G, Evans D, Watowich S . HIF-1alpha, STAT3, CBP/p300 and Ref-1/APE are components of a transcriptional complex that regulates Src-dependent hypoxia-induced expression of VEGF in pancreatic and prostate carcinomas. Oncogene. 2005; 24(19):3110-20. DOI: 10.1038/sj.onc.1208513. View

10.

Lissoni P . Is there a role for melatonin in supportive care?. Support Care Cancer. 2002; 10(2):110-6. DOI: 10.1007/s005200100281. View

11.

Dai M, Cui P, Yu M, Han J, Li H, Xiu R . Melatonin modulates the expression of VEGF and HIF-1 alpha induced by CoCl2 in cultured cancer cells. J Pineal Res. 2008; 44(2):121-6. DOI: 10.1111/j.1600-079X.2007.00498.x. View

12.

Zha L, Fan L, Sun G, Wang H, Ma T, Zhong F . Melatonin sensitizes human hepatoma cells to endoplasmic reticulum stress-induced apoptosis. J Pineal Res. 2012; 52(3):322-31. DOI: 10.1111/j.1600-079X.2011.00946.x. View

13.

Shin J, Lee H, Jung D, Jung J, Lee H, Lee E . Suppression of STAT3 and HIF-1 alpha mediates anti-angiogenic activity of betulinic acid in hypoxic PC-3 prostate cancer cells. PLoS One. 2011; 6(6):e21492. PMC: 3123343. DOI: 10.1371/journal.pone.0021492. View

14.

Park S, Jang W, Yi E, Jang J, Jung Y, Jeong J . Melatonin suppresses tumor angiogenesis by inhibiting HIF-1alpha stabilization under hypoxia. J Pineal Res. 2010; 48(2):178-84. DOI: 10.1111/j.1600-079x.2009.00742.x. View

15.

Tabernero J . The role of VEGF and EGFR inhibition: implications for combining anti-VEGF and anti-EGFR agents. Mol Cancer Res. 2007; 5(3):203-20. DOI: 10.1158/1541-7786.MCR-06-0404. View

16.

Carbajo-Pescador S, Garcia-Palomo A, Martin-Renedo J, Piva M, Gonzalez-Gallego J, Mauriz J . Melatonin modulation of intracellular signaling pathways in hepatocarcinoma HepG2 cell line: role of the MT1 receptor. J Pineal Res. 2011; 51(4):463-71. DOI: 10.1111/j.1600-079X.2011.00910.x. View

17.

Rathinavelu A, Narasimhan M, Muthumani P . A novel regulation of VEGF expression by HIF-1α and STAT3 in HDM2 transfected prostate cancer cells. J Cell Mol Med. 2011; 16(8):1750-7. PMC: 3822688. DOI: 10.1111/j.1582-4934.2011.01472.x. View

18.

Jung J, Lee H, Cho I, Chung D, Yoon S, Yang Y . STAT3 is a potential modulator of HIF-1-mediated VEGF expression in human renal carcinoma cells. FASEB J. 2005; 19(10):1296-8. DOI: 10.1096/fj.04-3099fje. View

19.

Hanahan D, Weinberg R . Hallmarks of cancer: the next generation. Cell. 2011; 144(5):646-74. DOI: 10.1016/j.cell.2011.02.013. View

20.

Riddell J, Maier P, Sass S, Moser M, Foster B, Gollnick S . Peroxiredoxin 1 stimulates endothelial cell expression of VEGF via TLR4 dependent activation of HIF-1α. PLoS One. 2012; 7(11):e50394. PMC: 3503895. DOI: 10.1371/journal.pone.0050394. View