Abstract
2-(3,4-dihydroxyphenil)-ethanol (DPE), a polyphenol present in olive oil, has been found to attenuate the growth of colon cancer cells, an effect presumably related to its anti-inflammatory activity. To further explore the effects of DPE on angiogenesis and tumor growth we investigated the in vivo efficacy of DPE in a HT-29 xenograft model and in vitro activities in colon cancer cells exposed to interleukin-1beta (IL-1beta) and prostaglandin E-2 (PGE-2). DPE (10 mg/kg/day for 14 days) inhibited tumor growth, reducing vessel lumina and blood perfusion to tumor, and diminished expression of hypoxia inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), and microsomal prostaglandin-E synthase-1 (mPGEs-1). In vitro, DPE (100 mumol/L) neither affected cell proliferation nor induced apoptosis in HT-29 and WiDr cells. DPE prevented the IL-1beta-mediated increase of mPGEs-1 expression and PGE-2 generation, as it did the silencing of HIF-1alpha. Moreover, DPE blocked mPGEs-1-dependent expression of VEGF and inhibited endothelial sprouting induced by tumor cells in a coculture system. PGE-2 triggers a feed-forward loop involving HIF-1alpha, which impinges on mPGEs-1 and VEGF expression, events prevented by DPE via extracellular signal-related kinase 1/2. The reduction of PGE-2 and VEGF levels, caused by DPE, was invariably associated with a marked decrease in HIF-1alpha expression and activity, independent of proteasome activity, indicating that the DPE effects on tumor growth and angiogenesis are dependent on the inhibition of HIF-1alpha translation. We show that the in vivo DPE antitumor effect is associated with anti-inflammatory and antiangiogenic activities resulting from the downregulation of the HIF-1alpha/mPGEs-1/VEGF axis.
Highlights
MethodsReagents Reagents were as follows: prostaglandin E-2 (PGE-2), IL-1β, U0126, anti-βactin (Sigma); DPE, LY294002, anti-COX-2, anti-mPGEs1, anti-mPGEs-2, and anti-cPGEs (Cayman Chemicals); neutralizing anti-vascular endothelial growth factor (VEGF) (R&D Systems); anti-hypoxia inducible factor-1α (HIF-1α) (BD Transduction); anti-VEGF (RELIAtech), anti–cleaved caspase-3, anti phospho-p44/42 mitogen-activated protein kinase (MAPK), p44/42 MAPK, anti-phospho Akt, and anti-Akt (Cell Signalling); Hoechst 33342 (Invitrogen)
Hypoxia inducible factor-1α (HIF-1α) is a key player as it activates the transcription of Authors' Affiliations: 1Department of Molecular Biology, University of Siena, Siena, and Istituto Toscano Tumori (ITT), Italy; 2Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain; and 3SAIC Frederick, Inc., National Cancer Institute-Frederick, Frederick, Maryland
Multiple genes involved in tumor progression, which include vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2), hallmarks of angiogenesis and inflammation, respectively [1,2,3,4]
Summary
Reagents Reagents were as follows: PGE-2, IL-1β, U0126, anti-βactin (Sigma); DPE, LY294002, anti-COX-2, anti-mPGEs1, anti-mPGEs-2, and anti-cPGEs (Cayman Chemicals); neutralizing anti-VEGF (R&D Systems); anti-HIF-1α (BD Transduction); anti-VEGF (RELIAtech), anti–cleaved caspase-3, anti phospho-p44/42 mitogen-activated protein kinase (MAPK), p44/42 MAPK, anti-phospho Akt, and anti-Akt (Cell Signalling); Hoechst 33342 (Invitrogen). Cell lines HT-29 and WiDr, human colorectal adenocarcinoma cells, were obtained from the American Type Culture Collection and were cultured as recommended. CVEC, postcapillary venular endothelial cells, were cultured as described [9]. Caspase-3 activity Caspase-3 activity was measured using a commercially available kit (Molecular Probes, Invitrogen) following the manufacturer's instructions. Western blotting Cells (5 × 105) were plated in 60-mm dishes, serum deprived (0.1% FCS, 24 hours), exposed to IL-1β, PGE-2, or hypoxia in the presence or absence of DPE.
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