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Tumor cells induce COX-2 and mPGES-1 expression in microvascular endothelial cells mainly by means of IL-1 receptor activation

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Abstract

Prostaglandin (PG) E2 plays a key role in immune response, tumor progression and metastasis. We previously showed that macrovessel-derived endothelial cells do not produce PGE2 enzymatically because they do not express the inducible microsomal PGE-synthase-1 (mPGES-1). Nevertheless, differences between macro- and micro-vessel-derived endothelial cells regarding arachidonic acid (AAc) metabolism profile have been reported. The present work was conducted to evaluate the expression of PGE2-pathway-related enzymes in human microvascular endothelial cells (HMVEC) in culture and to test the hypothesis that the tumor cell-HMVEC cross talk could increase mPGES-1 expression in HMVEC. We treated HMVEC in culture with human recombinant IL-1β. IL-1β induced PGE2 release and COX-2 and mPGES-1 expression in terms of mRNA and protein, determined by real-time PCR and immunoblotting, respectively. HMVEC constitutively expressed mPGES-2 and cytosolic PGES (cPGES) and the IL-1β treatment did not modify their expression. PGE2 synthesized by HMVEC from exogenous AAc was linked to mPGES-1 expression. Immunohistochemistry analysis confirmed mPGES-1 expression in microvessels in vivo. COX-2 and mPGES-1 were also induced in HMVEC by the conditioned medium from two squamous head and neck carcinoma cell lines. Conditioned medium from tumor cell cultures contained several cytokines including the IL-1β and IL-1α. Tumor cell-induced COX-2 and mPGES-1 in HMVEC was strongly inhibited by the IL-1-receptor antagonist, indicating the important implication of IL-1 in this effect. HMVEC could therefore contribute directly to PGE2 formed in the tumor. Our findings support the concept that mPGES-1 could be a target for therapeutic intervention in patients with cancer.

Graphical abstract

Research Highlights

►Unlike macrovessel endothelial cells (EC), microvessel EC express mPGES-1. ►IL-1β induces PGE2 and COX-2 and mPGES-1 expression in microvessel EC. ►Tumor cells induce mPGES-1 in microvessel EC mainly by IL-1 receptor activation. ►mPGES-1 could be a target for therapeutic intervention in patients with cancer

Introduction

Prostaglandin (PG) E2 is an important prostanoid derived from arachidonic acid (AAc). There are a wide range of autocrine and paracrine biological activities related to immune response and inflammation (Griffiths, 1999, Phipps et al., 1991). Several lines of evidence indicate that PGE2 is also involved in malignant growth through the activation of several growth factor receptor signaling cascades (Pai et al., 2002), inhibiting tumor cell apoptosis (Liu et al., 2001) and modulating production of immune-regulatory cytokines that allow cancer cells to escape the immune system (Harris et al., 2002). There is strong evidence for a link between PGE2 and tumor angiogenesis associated with production of pro-angiogenic factors (Gallo et al., 2001, Ziche et al., 1982, Seno et al., 2002). Additionally, PGE2 induces the expression of matrix metalloproteinases (Corcoran et al., 1992), enzymes regarded crucial in tumor progression and metastasis (Dohadwala et al., 2002).

The substrate for PGE2 biosynthesis, PGH2, is generated by the enzyme cyclooxygenase (COX) from AAc. Two isoforms of COX have been characterized. COX-1 generally is regarded as a constitutively expressed isoform and is present in almost all cell types, whereas COX-2 is an inducible enzyme normally expressed in few tissues, such as endothelium (Vila, 2004, McAdam et al., 1999). COX-2 is induced in response to hormones, growth factors, pro-inflammatory cytokines, bacterial endotoxin and tumor promoters and is typically over-expressed at inflammatory sites and tumors (Jabbour et al., 2001, Camacho et al., 2008, Greenhough et al., 2009). Conversion of PGH2 to PGE2 is catalyzed by PGE-synthases (PGES). Three PGES isoenzymes have been characterized so far, two microsomal isoforms (mPGES-1 and mPGES-2) and one cytosolic isoform (cPGES) (Murakami et al., 2002). The inducible PGES (mPGES-1) seems to be the essential PGES isoenzyme involved in PGE2 biosynthesis by human vascular cells (Soler et al., 2000, Camacho et al., 2007, Salvado et al., 2009).

COX-2/mPGES-1 has been widely regarded as the main contributing enzymatic tandem for PGE2 biosynthesis under pathological conditions. Genetic inactivation or pharmacological inhibition of COX-2 reduces tumor-induced neovascularization (Iñiguez et al., 2003). Besides, mPGES-1 also appears to be critical for tumor angiogenesis (Murakami and Kudo, 2006).

Endothelial cells play a pivotal role in a wide range of biological processes including haemostasis, wound healing, angiogenesis, diabetic retinopathy, inflammatory disorders, and tumor progression and metastasis. They produce AAc metabolites, which play key roles in the regulation of coagulation, vascular tone, immune response and angiogenesis. In a previous study, we found that macrovessel-derived endothelial cells from fetal and adult samples did not produce PGE2 enzymatically (Camacho et al., 1998). In a later study, we showed that these cells did not express mPGES-1 (Soler et al., 2000). In support of these findings, Redondo and cols (Salvado et al., 2009) recently reported that umbilical vein-derived endothelial cells (HUVEC) only express cPGES and mPGES-2 and that these cells are unable to produce PGE2. Nevertheless, early reports showed that microvessel derived endothelial cells produce PGE2 as the main prostanoid under basal and stimulated conditions, and possessed a glutathione-dependent PGES activity (Charo et al., 1984, Gerritsen and Cheli, 1983).

A growing body of evidence shows that interactions between tumor cells and stromal cells, which include fibroblasts, myofibroblasts and endothelial cells, may dramatically influence tumor progression (Bhowmick and Moses, 2005, Tlsty and Coussens, 2006, Hu and Polyak, 2008, West and van de Rijn, 2007). Furthermore, we have recently shown that tumor cells release pro-inflammatory factors that are able to induce cytokine and adhesion molecules in human microvascular endothelial cells (HMVEC) in culture (Brú et al., 2009). However, it remains to be determined which PGES isoforms are involved in PGE2 synthesis in HMVEC. The present work was conducted to evaluate the expression of COX-2 and mPGES-1 under basal and stimulated conditions in HMVEC. We also aimed to test the hypothesis that the tumor cell-HMVEC cross talk could result in COX-2 and mPGES-1 over-expression in HMVEC.

Section snippets

Isolation and culture of human microvascular endothelial cells

HMVEC were isolated from human adult foreskins using a previously described technique (Brú et al., 2009). In brief, foreskins obtained from adult circumcisions were placed in phosphate buffered saline (PBS) supplemented with 200 units/ml penicillin, 200 μg/ml streptomycin and 0.5 μg/ml amphotericin B (all from Biological Industries, Kibbutz Beit Haemek, Israel). Foreskins were cut into 3 mm squares and placed in PBS containing 0.3% trypsin and 1% EDTA at 37 °C for 30 min. Segments were then washed

Characterization of HMVEC

Primary cultures, established by plating dermal cells released from human foreskin, consisted of a mixture of dermal microvascular endothelial cells (majority), dermal fibroblasts and several epidermal keratinocytes. Endothelial cells were purified from subconfluent primary cultures using magnetic beads coupled to an anti-CD31 antibody. After an immunomagnetic purification step, first-passage cultures usually consisted of > 99% HMVEC. HMVEC cultures displayed characteristic cobblestone

Discussion

In this study we found that HMVEC express mPGES-1. The IL-1β treatment of HMVEC in culture induced PGE2 release and COX-2 and mPGES-1 expression in terms of mRNA and protein. In vivo, mPGES-1 expression in microvessels was confirmed by immunohistochemistry. COX-2 and mPGES-1 were also induced in HMVEC by the conditioned medium from two squamous head and neck carcinoma cell lines.

Unlike endothelial cells from macrovessels, which produce PGI2 as the main prostanoid (Camacho et al., 1998), PGE2 is

Acknowledgments

This work was supported by grants from the Ministerio de Ciencia e Innovación (SAF2008-01777, FIS08-0537 and SAF2009-09598) and by grants from the Red Temática de Investigación Cardiovascular RECAVA (RD06/0014/1005). The authors wish to thank Dr. Rosa Antón and Sonia Alcolea for their assistance with tumor cells protein analysis.

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