Elsevier

Biochemical Pharmacology

Volume 69, Issue 2, 15 January 2005, Pages 307-318
Biochemical Pharmacology

The novel targets for anti-angiogenesis of genistein on human cancer cells

https://doi.org/10.1016/j.bcp.2004.09.025 Get rights and content

Abstract

Genistein has been reported to be a natural chemopreventive in several types of human cancer. In our prior study, soy isoflavones were shown to induce cell cycle arrest and apoptosis of bladder cancer cells in the range of human urine excretion. This study was designed to identify the novel molecular basis underlying anti-angiogenic activities of soy isoflavones. An immortalized E6 and five human bladder cancer cell lines were studied by immunoassay, flow cytometry, functional activity, reverse transcription-polymerase chain reaction, immunoblotting, and transwell co-culture in vitro. The efficacy of soy isoflavones on angiogenesis inhibition in vivo was examined by nude mice xenograft and chick chorioallantoic membrane bioassay. Factors analyzed included angiogenic factors, matrix-degrading enzymes, and angiogenesis inhibitors. Genistein was the most potent inhibitor of angiogenesis in vitro and in vivo among the isoflavone compounds tested. It may also account for most of the reduced microvessel density of xenografts observed and the suppressed endothelial migration by soy isoflavones. Genistein exhibited a dose-dependent inhibition of expression/excretion of vascular endothelial growth factor165, platelet-derived growth factor, tissue factor, urokinase plasminogen activator, and matrix metalloprotease-2 and 9, respectively. On the other hand, there was an up-regulation of angiogenesis inhibitors—plasminogen activator inhibitor-1, endostatin, angiostatin, and thrombospondin-1. In addition, a differential inhibitory effect between immortalized uroepithelial cells and most cancer cell lines was also observed. Altogether, we discovered that tissue factor, endostatin, and angiostatin are novel molecular targets of genistein. The current investigation provides further evidence in support of soy-based foods as natural dietary inhibitors of tumor angiogenesis.

Introduction

Angiogenesis, the generation of new blood vessels, is required for the growth as well as expansion of solid tumors, especially those at 1–2 mm in diameter [1]. The initiation of this vascular phase marks a period of accelerated growth, local invasion, and, ultimately, metastasis of epithelial neoplasm. The prognostic importance of estimating the degree of angiogenesis in patients with carcinoma, including bladder cancer, supports the hypothesis that tumor angiogenesis is imperative in the development and progression of human cancer [2]. Hence, therapeutic agents are being devised either to interrupt the pathogenic steps of tumor angiogenesis or to directly destroy the tumor vasculature. Currently, chemicals that can cut off the tumor's blood supply, so-called angiogenesis inhibitors, are considered one of the most promising anticancer therapies.

A number of epidemiological studies show that the consumption of soy products may have a protective effect against human cancers of the breast, colon, or prostate in the Far East Asia [3], [4]. The beneficial effects of a traditional Japanese diet are thought to come from isoflavones, the plant pigments found in soybeans. Experiments both in vitro and in vivo for bladder cancer have supported the notion that isoflavonoids, flavonoids, or lignans are able to suppress tumor growth [5], [6], [7], [8]. We also found that isoflavone compounds tend to have a dose-dependent growth inhibition on human bladder cancer cells in vitro and a tumor suppressor effect in vivo [9]. It is interesting to note that the cooperative action of a mixture of isoflavone compounds generates greater anti-tumorigenic effects than any single compound does, and that the IC50 values of most cancer cell lines (3–5 μg/ml or 7.9–13.2 μM) are within the reach of the urine levels of daidzein (7,4′-dihydroxyisoflavone) (14.7 μM), and genistein (5,7,4′-trihydroxyisoflavone) (8.4 μM) following a soy challenge [10]. Nevertheless, genistein is one of the most potent soy isoflavones. These results have produced the hypothesis that soy isoflavones, especially the genistein, may be practical chemopreventives for human urinary tract cancer.

It is well-known that angiogenic activity can be detected in the urine of patients with bladder cancer [11], [12]. In cases of flat dysplasia or non-invasive transitional cell carcinoma, there are also increased numbers of sub-urothelial capillaries, suggesting the existence of an angiogenic stimulus in the early stage of bladder tumorigenesis [13]. For this reason, bladder cancer is a particularly good paradigm to use for investigating anti-angiogenic agents. Many molecules released by tumor and host cells are known to play a role in tumor angiogenesis, and the final outcome in vivo depends on the net balance between positive (angiogenic factors) and negative (angiogenesis inhibitors) regulatory elements. Factors known to be involved in human bladder carcinogenesis include (1) angiogenic growth factors, such as vascular endothelial growth factor (VEGF) [14], [15], [16] and platelet-derived growth factor (PDGF) [17], [18]; (2) other pro-angiogenic factors, such as cyclooxygenase-2 (COX-2) [19], [20], [21] and tissue factor (TF) [22]; (3) matrix-degrading enzymes, such as urokinase- and tissue-type plasminogen activator (uPA and tPA) [23], [24], [25], [26], the matrix metalloproteinase (MMP) family [27], [28], [29], [30], [31], [32], [33], [34], and their activator of transmembrane MMP (MT-MMP) [34]; and (4) angiogenesis inhibitors, such as tissue inhibitor of metalloproteinase-2 (TIMP-2) [28], [29], [30], [33], plasminogen activator inhibitor-1 (PAI-1) [35], and thrombospondin-1 (TSP-1) [15], [36], angiostatin [37], and endostatin. A number of biochemical targets and pharmacological actions have been proposed [38], [39], and anti-angiogenesis is one of the important mechanisms responsible for anti-cancer effects of soy isoflavones [5], [7], [40], [41]. Despite of the fact that in vitro anti-angiogenic mechanisms of genistein have been described in several cancer models, the information is far from complete. Using human bladder cancer as a paradigm, we performed this study to identify the molecular basis, especially the novel targets, for anti-angiogenesis of genistein in the range of urine excretion.

Section snippets

Cell culture

The E6 cell line was an immortalized human uroepithelium [42]. Human bladder cancer cell lines RT4 (grade 1 bladder cancer), J82 (grade 3 bladder cancer), 5637 (grade 3 bladder cancer), and T24 (grade 3 bladder cancer) were obtained from the American Type Culture Collection (Rockville, MD). TSGH8301 (grade 2 bladder cancer) was established locally and has been reported in detail previously [43]. The E6 cells were grown in F12 medium (GIBCO BRL), the RT4 cells in McCoy's 5A medium (GIBCO BRL),

Results

First, we used a previously described xenograft model to evaluate the direct anti-angiogenic effect of isoflavones on bladder cancer cells [9]. A significantly reduced MVD of TSGH8301 cells in SCID mice was observed at 10 μg/ml genistein (50.7 ± 4.0/mm2) or 10 μg/ml isoflavone mixture (28.4 ± 5.3/mm2) (P < 0.0001 by ANOVA, respectively) (Fig. 1A). The difference between treatment groups and the controls (70.2 ± 7.6/mm2) was also statistically significant (P < 0.001). The mean area of individual vessels

Discussion

Tumorigenesis of the human bladder is a multi-step and multifocal (field effect) process, possibly involving the spread of pre-malignant clones along the urothelial mucosa. As a result, human bladder cancer is uniquely suited to the development of chemoprevention, in which noncytotoxic drugs or nutrients are used to prevent, retard, or delay carcinogenesis. In this study, we demonstrated that both a cocktail mixture of isoflavones or genistein by itself drastically inhibit angiogenesis in vivo,

Acknowledgements

This study is supported by research grants NSC 89-2320-B-006-095 and NSC 90-2320-B-006-095 from the National Science Council, and MOE Program for Promoting Academic Excellent of Universities (grant number 91-B-FA09 -1-4), TAIWAN.

References (65)

  • Y. Mizutani et al.

    Synergistic cytotoxicity and apoptosis of jte-522, a selective cyclooxygenase-2 inhibitor, and 5-fluorouracil against bladder cancer

    J Urol

    (2002)
  • S.J. Su et al.

    Overexpression of HER-2/neu enhances sensitivity of human bladder cancer cells to urinary isoflavones

    Eur J Cancer

    (2001)
  • Y. Li et al.

    Down-regulation of invasion and angiogenesis-related genes identified by cDNA microarray analysis of PC3 prostate cancer cells treated with genistein

    Cancer Lett

    (2002)
  • Y. Li et al.

    Gene expression profiles of genistein-treated PC3 prostate cancer cells

    J Nutr

    (2002)
  • P. Nyberg et al.

    Endostatin inhibits human tongue carcinoma cell invasion and intravasation and blocks the activation of matrix metalloprotease-2, -9, and -13

    J Biol Chem

    (2003)
  • J. Folkman

    Tumor angiogenesis factor

    Cancer Res

    (1974)
  • B.H. Bochner et al.

    Angiogenesis in bladder cancer: relationship between microvessel density and tumor prognosis

    J Natl Cancer Inst

    (1995)
  • H. Adlercreutz

    Phytoestrogens: epidemiology and a possible role in cancer protection

    Environ Health Perspect

    (1995)
  • M. Messina et al.

    The role of soy products in reducing risk of cancer

    J Natl Cancer Inst

    (1991)
  • T. Fotsis et al.

    Genistein, a dietary-derived inhibitor of in vitro angiogenesis

    Proc Natl Acad Sci USA

    (1993)
  • J.R. Zhou et al.

    Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis

    Cancer Res

    (1998)
  • S.J. Su et al.

    The potential of soybean foods as a chemoprevention approach for human urinary tract cancer

    Clin Cancer Res

    (2000)
  • H. Adlercreutz et al.

    Urinary excretion of lignans and isoflavonoid phytoestrogens in Japanese men and women consuming a traditional Japanese diet

    Am J Clin Nutr

    (1980)
  • G.W. Chodak et al.

    Angiogenic activity as a marker of neoplastic and preneoplastic lesions of the human bladder

    Ann Surg

    (1980)
  • G.W. Chodak et al.

    Urine from patients with transitional-cell carcinoma stimulates migration of capillary endothelial cells

    N Engl J Med

    (1981)
  • S. Deen et al.

    Basement membrane and extracellular interstitial matrix components in bladder neoplasia—evidence of angiogenesis

    Histopathology

    (1994)
  • L.F. Brown et al.

    Increased expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in kidney and bladder carcinomas

    Am J Pathol

    (1993)
  • S.C. Campbell et al.

    Molecular mediators of angiogenesis in bladder cancer

    Cancer Res

    (1998)
  • N.H. Chow et al.

    Expression of vascular endothelial growth factor in primary superficial bladder cancer

    Anticancer Res

    (1999)
  • Y. Mizutani et al.

    Expression of platelet-derived endothelial cell growth factor in bladder carcinoma

    Cancer

    (1997)
  • T.S. O’Brien et al.

    Expression of the angiogenic factor thymidine phosphorylase/platelet-derived endothelial cell growth factor in primary bladder cancers

    Cancer Res

    (1996)
  • T. Shirahama

    Cyclooxygenase-2 expression is up-regulated in transitional cell carcinoma and its preneoplastic lesions in the human urinary bladder

    Clin Cancer Res

    (2000)
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