Research article
Suppression of VEGF-mediated autocrine and paracrine interactions between prostate cancer cells and vascular endothelial cells by soy isoflavones,☆☆

https://doi.org/10.1016/j.jnutbio.2006.08.006 Get rights and content

Abstract

Angiogenesis is an essential process involved in the development and progression of prostate cancer. Vascular endothelial growth factor (VEGF) is hypothesized to be a critical regulator of angiogenesis during prostate carcinogenesis. We have reported that dietary soy products inhibit prostate tumor progression in animal models, in association with a reduction in tumor microvessel density. The goal of the present study is to investigate potential antiangiogenic mechanisms of genistein, the major soy isoflavone, using in vitro systems. Genistein (5–50 μM) significantly inhibited the growth of human umbilical vein endothelial cells (HUVECs) in control media when stimulated by supplemental VEGF or when cultured in hypoxia-exposed PC-3 prostate adenocarcinoma cell conditioned media. These in vitro studies suggest detectable inhibitory effects by 5–10 μM genistein (P<.05) with an IC50 of approximately 20 μM or less. Genistein (10–50 μM) caused significant inhibition of basal VEGF expression and hypoxia-stimulated VEGF expression in both human prostate cancer PC-3 cells and HUVECs based on semiquantitative reverse transcription–polymerase chain reaction (P<.05). In parallel, VEGF secretion by PC-3 cells quantitated by enzyme-linked immunosorbent assay was significantly (P<.05) reduced by genistein (10–50 μM). Furthermore, genistein (10–50 μM) significantly (P<.05) reduced PC-3 nuclear accumulation of hypoxia-inducible factor-1α, the principle transcription factor that regulates VEGF expression in response to hypoxia. Expression of the VEGF receptor fms-like tyrosine kinase-1, but not kinase insert domain-containing kinase, in HUVECs was also reduced (P<.05) by genistein (10–50 μM). These observations support the hypothesis that genistein may inhibit prostate tumor angiogenesis through the suppression of VEGF-mediated autocrine and paracrine signaling pathways between tumor cells and vascular endothelial cells.

Introduction

Tumor angiogenesis is a complex biological process involving a dynamic interaction between cancer cells and those of the host microenvironment and is considered essential for tumor development and progression [1], [2]. An array of hormones, growth factors and cytokines orchestrates tumor-associated angiogenesis by shifting the homeostatic balance in the microenvironment towards vessel formation [3], [4]. Vascular endothelial growth factor (VEGF), originally described as a vascular permeability factor, has been implicated as one of the most important proangiogenic growth factors during carcinogenesis [5]. VEGF stimulates endothelial cell proliferation and differentiation, leading to primitive vessel formation [6], [7]. VEGF is known to elicit its biological roles through interaction with two classes of VEGF receptors, fms-like tyrosine kinase-1 (FLT-1) and kinase insert domain-containing region (KDR), which are found in vascular endothelial cells and propagate signals through receptor-associated protein tyrosine kinases [8], [9]. The transcription factor hypoxia-inducible factor-1α (HIF-1α) is one of the most important regulators of VEGF gene expression in response to hypoxia in tumor cells [7], [10]. Reduced oxygen tension is characteristic of the tumor microenvironment due to imbalance between accumulating tumor cells and lagging angiogenic responses. This imbalance contributes to sluggish blood flow caused by an irregular and poorly formed vasculature [11]. HIF-1α protein is constitutively synthesized and continuously degraded under normoxic conditions. However, the cellular content of HIF-1α is dramatically increased by hypoxia through a combination of increased synthesis and decreased degradation, thereby contributing to increased VEGF expression [9], [12], [13]. In addition to the paracrine stimulation of endothelial cells by VEGF secreted from tumor cells, vascular cells may also produce VEGF and thus stimulate angiogenesis through an autocrine fashion [14].

Accumulated evidence suggests that VEGF-stimulated angiogenesis is a component of human and experimental prostate carcinogenesis. Increased vascular density has been documented in prostatic intraepithelial neoplasia (PIN) relative to benign epithelium and is greater in locally advanced cancers or metastasis compared with organ-confined tumors [15]. Similarly, vascular density is increased during rodent prostate carcinogenesis and is associated with areas within tumors that show increased proliferation [16]. Several studies suggest that VEGF expression is enhanced in tumor cells, vascular endothelial cells and stromal cells within a developing prostate cancer, whereas normal prostate tissues show lower expression [17]. HIF-1α immunohistochemical (IHC) staining is significantly increased in high-grade PIN lesions compared with normal epithelium, stromal cells and benign prostatic hyperplasia tissues, which is further enhanced in prostate cancer lesions [18]. In addition, increased plasma VEGF concentrations are found in prostate cancer patients compared with healthy controls [19]. Plasma VEGF levels are increased with the progression of prostate cancer to locally advanced and metastatic disease and with the development of androgen independence [19], [20]. Consequently, anti-VEGF therapy is now being evaluated in prostate cancer clinical trials [21].

Our laboratory is evaluating angiogenesis as a target for dietary or chemopreventive interventions that may inhibit prostate carcinogenesis. For example, we have observed that dietary restriction reduces prostate tumor growth in association with reduced vascular density and lower VEGF expression in the tumor microenvironment [22]. We have also shown that diets containing soy protein or a phytochemical-rich extract also inhibited prostate tumor growth in murine transplantable models, in parallel with reduced intratumor vascularity [23].

In the present in vitro study, we examine the ability of genistein to act upon specific aspects of VEGF-mediated autocrine and paracrine interactions between prostate tumor cells and vascular endothelial cells. We observed that genistein inhibited endothelial cell proliferation and tube formation in an angiogenic assay, reduced hypoxia-induced VEGF expression in both prostate cancer and endothelial cells, reduced VEGF receptor expression in endothelial cells and decreased HIF-1α expression in prostate cancer cells in response to hypoxia. These studies establish mechanisms whereby soy isoflavones may impact interactions between tumor cells and the host microenvironment, which can be evaluated in future rodent or human translational studies.

Section snippets

Cell culture

Androgen-insensitive PC-3 human prostate cancer cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained as monolayer cultures in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 20,000 IU/L penicillin and 20 mg/L streptomycin. Human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics (San Diego, CA) and maintained as monolayer cultures in an endothelial cell growth

Genistein inhibits the growth of HUVECs with or without VEGF stimulation

We first measured the ability of genistein to directly modulate the in vitro growth of endothelial cells incubated in the presence or absence of VEGF. MTS assay data showed that genistein treatment at 10, 20 and 50 μM inhibited the growth of HUVECs by 25% (P<.01), 55% (P<.0001) and 69% (P<.0001), respectively (Fig. 1A). HUVEC growth, with the addition of 10 ng/ml supplemental VEGF, significantly increased to 138±8% (P<.01) compared to vehicle controls (Fig. 1A). The treatment of

Discussion

Angiogenesis is hypothesized to be indispensable for the development and progression of prostate cancer. Tumor angiogenesis is dependent upon complex autocrine and paracrine interactions between malignant tumor cells and components of the tumor microenvironment that include matrix and vascular cells, as well as modulators derived from the circulation [1], [7]. VEGF has emerged as one of the critical proangiogenic growth factors associated with prostate carcinogenesis [15], [16], [17], [18], [19]

References (45)

  • G.L. Wang et al.

    Effect of protein kinase and phosphatase inhibitors on expression of hypoxia-inducible factor 1

    Biochem Biophys Res Commun

    (1995)
  • K.B. Sandau et al.

    Induction of hypoxia-inducible-factor 1 by nitric oxide is mediated via the PI 3K pathway

    Biochem Biophys Res Commun

    (2000)
  • S. Wang et al.

    Tomato and soy polyphenols reduce insulin-like growth factor-I-stimulated rat prostate cancer cell proliferation and apoptotic resistance in vitro via inhibition of intracellular signaling pathways involving tyrosine kinase

    J Nutr

    (2003)
  • D. Guo et al.

    Vascular endothelial cell growth factor promotes tyrosine phosphorylation of mediators of signal transduction that contain SH2 domains. Association with endothelial cell proliferation

    J Biol Chem

    (1995)
  • J. Folkman

    Fundamental concepts of the angiogenic process

    Curr Mol Med

    (2003)
  • T. Tonini et al.

    Molecular basis of angiogenesis and cancer

    Oncogene

    (2003)
  • N. Ferrara

    The role of VEGF in the regulation of physiological and pathological angiogenesis

    Exs

    (2005)
  • A. Bikfalvi et al.

    Interaction of vasculotropin/vascular endothelial cell growth factor with human umbilical vein endothelial cells: binding, internalization, degradation, and biological effects

    J Cell Physiol

    (1991)
  • G. Bergers et al.

    Tumorigenesis and the angiogenic switch

    Nat Rev Cancer

    (2003)
  • A. Kaipainen et al.

    Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development

    Proc Natl Acad Sci U S A

    (1995)
  • I. Zachary

    Vascular endothelial growth factor: how it transmits its signal

    Exp Nephrol

    (1998)
  • L.E. Huang et al.

    Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin–proteasome pathway

    Proc Natl Acad Sci U S A

    (1998)
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      Therefore, VEGF expression specifically in the scalp tissue should be examined in conjunction with adrenergic receptor agonism. It is already known that tissue specific mediation of VEGF expression occurs with soy phytoestrogens, where downregulation occurs in prostate cancer cells (Guo et al., 2007), but upregulation occurs in the hair follicles (Lachgar et al., 2003). Phytoestrogen mediated VEGF expression in the hair follicle promotes perifollicular vascularisation and angiogenesis, subsequently enhancing hair growth, whereas suppression of VEGF expression in organ tissue has no undesirable effects due to a lack of a vascular system (Yano et al., 2001).

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    This work was supported by American Institute for Cancer Research 00B106-REV (to S.K.C.); Department of Defense Congressionally Directed Medical Research Program, Prostate Cancer Program DAMD17-02-1-0116 (to S.W.); National Institutes of Health, National Cancer Institute RO1 CA72482 NCI (to S.K.C.); The Bremmer Foundation (to S.K.C.); The Prostate Cancer Prevention Fund of the Arthur G. James Cancer Hospital and Richard S. Solove Research Institute(to S.K.C.); and National Institutes of Health, National Cancer Institute P30CA16058 (to The Ohio State University Comprehensive Cancer Center).

    ☆☆

    Yanping Guo and Shihua Wang contributed equally to this manuscript.

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