Elsevier

Oral Oncology

Volume 37, Issue 1, January 2001, Pages 77-83
Oral Oncology

Hypoxia-induced angiogenesis of cultured human salivary gland carcinoma cells enhances vascular endothelial growth factor production and basic fibroblast growth factor release

https://doi.org/10.1016/S1368-8375(00)00062-2Get rights and content

Abstract

The angiogenic activity of two human salivary gland tumor cell lines, ACCS from adenoid cystic carcinoma and IT-2 from mucoepidermoid carcinoma, was examined by stimulating tube formation by bovine capillary endothelial cells (BCE). ACCS and IT-2 were cultured in 20 or 3% oxygen, representing normoxic and hypoxic conditions, respectively, and conditioned medium (CM) was obtained from each culture. The BCE tubes stimulated by hypoxic CM were 1.59 (ACCS) and 1.42 (IT-2) times longer than those stimulated by normoxic CM. The tube-forming activity of CM was inhibited by preincubation with either anti-vascular endothelial growth factor (VEGF) IgG or anti-basic fibroblast growth factor (bFGF) IgG, suggesting that both VEGF and bFGF with angiogenic activity were present in the CM. This was confirmed by ELISA, which also demonstrated increased concentrations of both proteins in the hypoxic CM. Northern blot analysis showed an increased VEGF mRNA level in both carcinoma cells with hypoxia, while hypoxia did not affect the bFGF mRNA level in either cell line. The results suggest that both VEGF and bFGF are major angiogenesis factors in salivary gland tumors, and hypoxia-induced angiogenesis results from upregulation of VEGF and increased release of bFGF.

Introduction

Some types of salivary gland tumors, such as adenoid cystic carcinoma and high grade mucoepidermoid carcinoma, are highly invasive and frequently result in distant metastasis, which is the main cause of treatment failure. The reasons for the invasiveness and aggressive metastatic dissemination of these tumors are uncertain. A possible mechanism may involve angiogenesis. Angiogenesis is essential for tumor growth, which is postulated to be fundamentally dependent on the induction of stromal neovascularization [1], [2], [3]. The initial growth of solid tumors is thought to be limited to a few millimeters (about 106 cells) without the infiltration of newly formed blood vessels from stromal tissue, but growth can continue after neovascularization by increasing the opportunity for tumor cells to enter the circulation [4]. Specific angiogenic molecules (so-called angiogenic factors) produced by tumor cells stimulate surrounding capillary endothelial cells in a paracrine manner, resulting in angiogenesis into tumor tissues. These angiogenic factors include transforming growth factors-β and [5], angiogenin [6], epidermal growth factor [7], tumor necrosis factor-α [8], and platelet-derived endothelial cell growth factor [9]. Of these, vascular endothelial growth factor (VEGF) is the most potent factor in the neovascularization of several neoplasms [10], [11]. VEGF is a particularly selective mitogen for vascular endothelial cells, which promotes angiogenesis and induces vascular permeability under some circumstances [12], [13]. VEGF expression is also increased by a hypoxic microenvironment [14], [15], so expression is elevated in a variety of hypoxic solid tumors and functions as a general tumor angiogenic factor. VEGF stimulates the growth of endothelial cells by binding to specific receptors. Two tyrosine kinases, Flt-1 [16] and KDR [17], have been identified as high affinity receptors for VEGF in human endothelial cells. Recently, tumor growth and metastasis were reported to be inhibited by suppressing VEGF with antisense oligonucleotides [18], neutralizing antibody [19], and soluble receptors for VEGF [20].

Basic fibroblast growth factor (bFGF) is also a well-known angiogenic factor [21], [22] that stimulates cell proliferation, migration, and protease synthesis [23].

There is little information on angiogenesis in salivary gland tumors. This study examined angiogenic factors produced by human salivary tumor cell lines; ACCS from adenoid cystic carcinoma [24] and IT-2 from mucoepidermoid carcinoma [25], using an in vitro angiogenic assay model.

Section snippets

Reagents

Human recombinant VEGF and rabbit anti-human VEGF IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). bFGF purified from bovine brain was purchased from R&D Systems (Minneapolis, MN, USA). Rabbit anti-human recombinant bFGF IgG was purchased from Biomedical Technologies (Stoughton, MA, USA). The lactate dehydrogenase assay kit was obtained from Sigma (St. Louis, MO, USA) and the bovine type I collagen was from Koken (Tokyo, Japan).

Cell culture

Bovine capillary endothelial cells (BCE) were

Cell damage under hypoxic conditions

ACCS and IT-2 cells cultured under normoxic or hypoxic conditions for 12 or 24 h were observed under a phase contrast microscope. No morphological alteration suggesting cell damage was found in either line cultured under hypoxic conditions for 12 or 24 h. The LDH levels and number of viable cells in 24 h cultures under hypoxic conditions were comparable to those of normoxic controls.

BCE tube formation

When seeded on the surface of collagen gels, BCE cells attached within 0.5–1 h and formed a monolayer within 24

Discussion

This study examined the production of angiogenic factors by salivary gland carcinoma cell lines ACCS and IT-2, using an in vitro angiogenic assay model [22]. The BCE tube-formation assay indicated that both ACCS and IT-2 cells secreted angiogenic factors into the medium in a time-dependent manner. Inhibition of the activity by neutralizing antibodies suggested the presence of VEGF and bFGF in both ACCS and IT-2 CM. The angiogenic activity, as estimated by the length of the BCE tube, was higher

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