Genistein increases estrogen receptor beta expression in prostate cancer via reducing its promoter methylation
Introduction
Prostate cancer (PCa) is the most frequently diagnosed malignancy and the second most common cause of cancer death in men in U.S. [1]. Over 186,000 patients are diagnosed each year and greater than 27,000 will succumb to death by PCa [2]. Despite the high incidence of PCa, little is known about its etiology. Accepted risk factors are age, race, ethnicity, and geographical location. Currently, there is no effective cure for this disease once it has spread beyond the prostate, and more efforts should be devoted for developing preventive strategies to reduce PCa occurrence and impact.
The incidence of PCa is much lower in Asian than in Western populations [1] and Asian migrants to the U.S. have an increased incidence [3]. Therefore, environmental factors including diet have been presumed to play a key role in prostate carcinogenesis. Unlike Western populations, many Asians consume large amounts of soy foods rich in isoflavones. Average concentrations of soy isoflavones in serum and prostatic fluid of Asian men are much higher than those in Western men [4], [5]. Over the past decade, researchers have generated data demonstrating that soy isoflavones and their metabolites have actions that may be useful for the prevention or treatment of PCa [6], [7], [8]. Genistein is the predominant and most biologically active isoflavone in soy. It has a structural similarity to 17β-estradiol and it binds to estrogen receptor (ER), with higher affinity to ER-β than to ER-α [9], [10]. Accordingly, it has been suggested that genistein exerts some of its anti-cancer effects through binding to ER-β [11]. ER-β is expressed in prostate epithelial cells, and has a role in the cellular homeostasis that is anti-proliferative [12], pro-differentiative [13], and pro-apoptotic [14]. ER-β expression declines in localized PCa with increasing grade from prostatic intraepithelial neoplasia (PIN) through low to high Gleason scores [15], [16]. This expression pattern supports the tumor suppressor role of ER-β. Therefore, ER-β agonists, such as genistein, have potential as chemopreventive and therapeutic agents for PCa.
One of the proposed mechanisms by which ER-β is modulated in PCa is promoter methylation [17], [18], [19]. DNA methylation is an essential regulator of gene transcription and aberrant DNA methylation can result in an unscheduled silencing of genes, such as tumor suppressor genes, which have been associated with a large number of human malignancies. In PCa, the extent of ER-β promoter methylation has been reported to correlate with the degree of aggressiveness of the disease; the more aggressive the tumor is the greater the methylation of the ER-β promoter and consequently the lower the ER-β gene expression [20]. In addition, PCa cells that have been treated with demethylating agents showed increased expression of ER-β relative to untreated cells [21].
Although a number of studies support the notion that genistein can reverse promoter hypermethylation of tumor suppressor genes in PCa cells [22], [23], [24], [25], there is no data for the effect of genistein on ER-β gene promoter methylation. Furthermore, data about the effect of genistein on ER-β expression in PCa are inconsistent. In some in vitro studies, ER-β levels increased in response to genistein [26], whereas in others ER-β did not exhibit any apparent changes [11]. Thus, the main objective of this study was to determine the effect of genistein on ER-β methylation and subsequently ER-β expression levels and transcriptional activity. We hypothesize that: (1) genistein is capable of reversing ER-β promoter hypermethylation resulting in an increase in ER-β expression and transcriptional activity in PCa; (2) ER-β is mediating the protective effects of genistein in PCa. The second part of our hypothesis is built on observations from recent studies showing that dietary soy reduced the incidence of PCa in ER-β wild-type TRAMP transgenic mice, but not in ER-β knockout TRAMP mice [27].
In the current study, we tested our hypothesis using three PCa cell lines: LNCaP, LAPC-4 and PC-3. Cells were treated with a physiological range of genistein (0.5–10 μmol/L) and 5-aza-2′-deoxycytidine (5-aza-dC), a demethylating agent, was used as a positive control. Relative fractions of methylated and unmethylated ER-β promoter were determined in each PCa cell line in basal states and after treatment. ER-β mRNA and protein expression and transcriptional activity were subsequently determined. PCa cell proliferation was analyzed in response to genistein in the presence or absence of the ER-β specific antagonist, 4-(2-phenyl-5,7-bis (trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenol (PHTPP), in order to verify the mediating role of ER-β to the action of genistein in PCa cells.
Section snippets
Chemicals and antibodies
Genistein, 5-aza-2′deoxycytidine, MPP dihydrochloride, 17β-estradiol (E2) and ERB-041 were purchased from Sigma–Aldrich (St. Louis, MO). PHTPP (4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ER-β (N-19) goat polyclonal, p-ER-β (serine 87) rabbit polyclonal, DNMT1 (H-300) rabbit polyclonal, DNMT3a (H-295) rabbit polyclonal and DNMT3b (N-19) goat polyclonal antibodies and a pool of five target-specific siRNAs
Identification of three CpG islands in the promoter and exon 0N region of ER-β
DNA methylation is a crucial regulator of gene transcription; genes with high levels of 5-methylcytosine in their promoter region are transcriptionally silent. Aberrant DNA methylation represses transcription of tumor suppressor genes in malignancy. Methylation groups are added to the DNA at the CpG sites and the most important CpG islands are those in the promoter region since they are located at the site of initiation of transcription of the gene. Methylation of these CpG islands causes gene
Discussion
This study revealed two important features regarding the effects of genistein on PCa. First, genistein is capable of inducing epigenetic modifications in PCa through reversing ER-β promoter hypermethylation and subsequently increasing ER-β gene expression. Second, ER-β is a major mediator for genistein’s anti-cancer activity in PCa.
Genistein is known to possess several anti-cancer properties in PCa, which have recently been shown to involve epigenetic mechanisms such as DNA methylation.
Acknowledgements
We gratefully acknowledge Dr. R. Reiter (UCLA) for providing us with LAPC-4 cells. This work was supported by NIH Grant No. CA 116195 and by Grant No. GM 0842 from the Egyptian Ministry of Higher Education.
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