Cancer Letters

Cancer Letters

Volume 186, Issue 2, 5 December 2002, Pages 157-164
Cancer Letters

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

https://doi.org/10.1016/S0304-3835(02)00349-X Get rights and content

Abstract

Prostate cancer is the second leading cause of cancer related deaths in men in the United States and for many years the treatment results for metastatic prostate cancer have been disappointing. Our previous studies have shown that genistein elicits pleiotropic effects on prostate cancer cells; however, its role in invasion and metastasis has not been fully elucidated. In order to better understand the precise molecular mechanism(s) by which genistein exerts its effects on PC3 cells, we have utilized cDNA microarray to interrogate 12 558 known genes to determine the gene expression profile altered by genistein treatment. We found a total of 832 genes which showed >2-fold change after genistein treatment. Among these genes, we found down-regulation of 11 genes (MMP-9, protease M, uPAR, VEGF, neuropilin, TSP, BPGF, LPA, TGF-β2, TSP-1, PAR-2) and up-regulation of two genes (connective tissue growth factor, connective tissue activation peptide), which are related to angiogenesis, tumor cell invasion and metastasis. Reverse transcription–polymerase chain reaction, Western blot, and zymographic analysis were conducted to confirm the data of microarray at the level of mRNA, protein, and biological function. The results were in direct agreement with the microarray data. From these results, we conclude that genistein down-regulates the transcription and translation of genes critically involved in the control of angiogenesis, tumor cell invasion and metastasis, suggesting the possible therapeutic role of genistein for metastatic prostate cancer. Thus, genistein-induced alternations of gene expressions may be exploited for devising chemopreventive or therapeutic strategies, particularly for chemosensitization of metastatic prostate cancer to existing chemotherapeutic agents.

Introduction

Prostate cancer ranks as the second leading cause of adult male deaths from cancer in the United States [1]. In contrast, Asian men who consume a traditional diet high in soy products, have a relatively low incidence and mortality of prostate cancer [2]. It has also been reported that high consumption of soymilk are associated with reduced risk of prostate cancer [3]. Genistein is a prominent isoflavone found in soybean, and has been proposed to be responsible for lowering the incidence of prostate cancer in Asian men [2], [4]. Our previous studies have shown that genistein inhibits the growth of PC3 prostate cancer cells and induces apoptosis by inhibiting NF-κB and Akt signaling pathways [5], [6]. Moreover, we found that genistein inhibits the expression of c-erbB2 and MMP-9 which play important roles in the processes of tumor cell invasion and metastasis [7], suggesting that genistein elicits pleiotropic effects on cancer cells.

Once prostate cancer is diagnostically confirmed, the most significant threat to patient's survival and quality of life is primarily contributed by the presence of occult metastasis, which eventually kills the patient [8]. This is partly because of the disappointing results of chemotherapy for the treatment of prostate cancer [9]. Thus, there is a tremendous need for the development of mechanism-based and targeted therapeutic strategies for prostate cancer, particularly for metastatic prostate cancer. cDNA microarray analysis permits the analysis for the expression of tens of thousands of genes to be monitored simultaneously and rapidly [10], which provides a window of opportunity for determining the effects of anti-cancer agent on cancer cells. This will contribute in devising therapeutic strategies more accurately and to determine the molecular mechanism(s) of action of therapeutic agents. In order to better understand the precise molecular mechanism(s) by which genistein exerts its effects on PC3 prostate cancer cells, we have utilized cDNA microarray to determine the gene expression profile altered by genistein treatment. In this report, we focused on the genes which are known to play important roles in the control of angiogenesis, tumor cell invasion and metastasis.

Section snippets

Cell culture and growth inhibition

PC3 human prostate cancer cells (ATCC, Manassas, VA) were cultured in RPMI-1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in a 5% CO2 atmosphere at 37 °C. Genistein (Toronto Research Chemicals, North York, Ontario, Canada) was dissolved in 0.1 M Na2CO3 to make a 10 mM stock solution for experiments. For growth inhibition, PC3 cells were treated with 5, 15, 30, and 50 μM genistein or 0.5 mM Na2CO3 (vehicle control) for 1–3 days.

Cell growth inhibition by genistein treatment

MTT assay showed that the treatment of PC3 prostate cancer cells with genistein resulted in a dose and time-dependent inhibition of cell proliferation (Fig. 1), demonstrating the inhibitory effect of genistein on PC3 cell growth. These results are consistent with our previous results [11]. Inhibition of cell proliferation observed by MTT could be due to the regulation of gene expression by genistein treatment. We, therefore, investigated the gene expression profile of PC3 prostate cancer cells

Discussion

Genistein is known as a protein tyrosine kinase inhibitor [12]. In addition, our previous studies along with the studies reported by other investigators have shown that genistein may exert multiple inhibitory effects on cancer cells through regulation of several cell signal transduction pathways including NF-κB, Akt, MEKK, etc. [5], [6], [13]. In the present study, the gene expression profile of PC3 prostate cancer cells exposed to genistein revealed that genistein regulated the transcription

Acknowledgements

This work was partly funded by a grant from the National Cancer Institute, NIH (CA83695-O1A2 awarded to F.H.S.) and was also supported by an unrestricted grant from RGK foundation.

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