Silibinin Down-Regulates Prostate Epithelium-Derived Ets Transcription Factor in LNCaP Prostate Cancer Cells

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Cell culture
• Expression analysis
• Statistical analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

The androgen-sensitive human prostate cancer cell line LNCaP expresses the estrogen receptor β and androgen receptor and can be stimulated by androgens to secrete prostate-specific antigen (PSA). In this study we demonstrate the cancer protective potential of silibinin, a flavolignan derived from the fruits of Silybum marianum, which down-regulates the co-activator of the androgen receptor, the prostate epithelium-derived Ets transcription factor (PDEF) and consequently the secretion of PSA. LNCaP cells were treated with various concentrations of silibinin in the presence or in the absence of 5α-dihydrotestosterone (DHT). We used real-time RT-PCR to quantify mRNA expression of PDEF and PSA with gene-specific dual-labelled fluorescence probes. PSA secretion from LNCaP cells in conditioned media was measured with the Elecsys® System 2010. Silibinin down-regulated PSA mRNA expression and PSA secretion in conditioned medium under basal and DHT (10 ^{- 8} M) stimulated conditions, which was paralleled by PDEF down-regulation. DHT alone stimulated PDEF and PSA gene expression and PSA secretion. The down-regulation of basal as well as DHT stimulated PDEF and PSA by silibinin demonstrates the antiproliferative potential of this agent. These effects underline the possible therapeutic use of silibinin in the management of prostate cancer.

Abbreviations

Ets:E twenty-six transcription factor

PDEF:prostate epithelium-derived Ets factor

DHT:5α-dihydrotestosterone

PSA:prostate-specific antigen

IGFBP-3:insulin-like growth factor-binding protein 3

ER:estrogen receptor

cRNA:complementary RNA

Introduction

Prostate cancer is the most prevalent male neoplastic disease in Western countries. Androgen deprivation is an effective therapy for metastatic prostate cancer provided that it has not progressed to an androgen-insensitive phenotype. LNCaP cells express high levels of androgen receptor and can be stimulated by androgens to secrete prostate-specific antigen (PSA). PSA is a serine protease that cleaves insulin-like growth factor-binding protein-3 (IGFBP-3), thereby decreasing its function to regulate the bioavailability of IGF-1. IGF-1 is then liberated and available to bind to the IGF-1 receptor which is stimulatory to cellular proliferation [1]. Many prostate cancers as well as the prostate cancer cell line LNCaP express the estrogen receptor β (ER β), whereas the expression of the estrogen receptor α (ER α) is silenced by DNA hypermethylation [2]. To investigate new drugs in the prevention or therapy of prostate cancer also necessitates studies of alternative therapeutics. Soy-derived isoflavones, particularly genistein, have been shown to weakly inhibit proliferation of LNCaP cells. This isoflavone has a preferential action on ER β but still binds to and transactivates via the ER α. ER β-mediated mechanisms were proposed to prevent androgen-mediated effects in the prostate [3]. We showed recently that the flavolignan mixture silymarin of which silibinin (Fig. [1]) is the main component is a pure ER β agonist with no binding or transactivating activity via the ER α [4]. Silymarin has been used clinically for the treatment of liver diseases and it shows anti-carcinogenic activity in human prostate, breast, and cervical carcinoma cells [5]. Earlier approaches to impair proliferation of prostate cancer cells included phytoestrogens like those contained in traditional Asian food. In androgen-independent PC-3 prostate carcinoma cells, silibinin acted as an antiproliferative agent by up-regulating IGFBP-3 [6], thereby reducing the bioavailability of the proliferation promoting IGF-1. Silibinin was reported to down-regulate PSA gene expression and production and to cause a complete inhibition of cell growth via a G1 arrest in cell cycle progression in LNCaP [7]. Silibinin and silymarin were found to reduce the nuclear localization of the androgen receptor in LNCaP cells [8]. The androgen receptor is a nuclear transcription factor and there are several co-regulatory proteins which can either enhance or inhibit its transcriptional activity. Among these co-regulators of the androgen receptor is the prostate epithelium-specific Ets transcription factor (PDEF) which was found to be involved in PSA gene regulation [9].

In this study we show that a striking effect of silibinin in inhibiting LNCaP cell proliferation is to down-regulate PDEF expression and consequently PSA mRNA expression and PSA secretion.

Materials and Methods

Cell culture

All chemicals were purchased from Sigma (Taufkirchen, Germany) unless stated otherwise. LNCaP cells were maintained in phenol red-free DMEM (Invitrogen, Karlsruhe, Germany) supplemented with 10 % fetal calf serum (PAA, Coelbe, Germany), 2 % glutamine, 1 % sodium pyruvate and 1 % penicillin-streptomycin. Cells were plated in culture flasks or in 6-well plates at 100,000 cells per well for silibinin {[2,3-dihydro-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-1,4-benzodioxin-6-yl]-2,3-dihydro-3,5,7-trihydroxy-4H-1-benzopyran-4-one, purity by HPLC 98 %, Tocris, Köln, Germany}, and DHT treatments in concentrations and durations as indicated. Silibinin was dissolved in ethanol which yielded a concentration of 0.2 % (v/v) in all experiments. Cells were harvested by trypsination and washed with phosphate-buffered saline (BioWhittaker, Verviers, Belgium) prior to RNA extraction. Cell viability under hormone treatments was measured with an MTT test.

Expression analysis

Total cellular RNA from pelleted LNCaP cells was extracted with RNeasy Mini Kit (QIAGEN, Hilden, Germany). RNA integrity and quantity was assessed on an Agilent Bioanalyzer 2100 with an RNA 6000 Nano LabChip^-Kit (Agilent Technologies, Waldbronn, Germany). Reverse transcription of 500 ng total cellular RNA with random hexamer primers was performed with Omniscript RT Kit (QIAGEN). Expression of PDEF, PSA, androgen receptor, and of porphobilinogen deaminase (PBGD) was quantitated on an iCycler iQ real time detection system (BIORAD, Munich, Germany) with HotStar Taq DNA Polymerase Kit (QIAGEN). The 20 μL reaction from the kit was supplemented with 2 μL cDNA, 0.6 μM gene-specific primers and 0.2 μM dual-labeled fluorescent probes. Primers and probes (Operon, QIAGEN) were designed using primer3 on-line primer design program (www.genome. wi.mit.edu/cgi-bin/primer/primer3_www.cgi) except PDEF and PBGD [10], [11]. Impairment of optimal PCR conditions due to secondary DNA structure could be ruled out using Dr. Zuker’s site (http://www.bioinfo.rpi.edu/applications/mfold/old/dna/) and an optimal choice of non-dimer forming primers was made using the Operon oligo tool kit (http://www.operon. com).

• PSA forward primer: 5′-TGAACCAGAGGAGTTCTTGAC-3′

• PSA reverse primer: 5′-CCCCAGAATCACCCGAGCAG-3′

• PSA probe: 5′-FAM-GCGCAAGTTCACCCTCA-TAMRA-3′

• androgen receptor forward primer: 5′-AGGAACTCGATCCTATCATTGC-3′

• androgen receptor reverse primer: 5′-CTGCCATCATTTCCGGAA-3′

• androgen receptor probe: 5′-FAM-CGCTTCTACCAGCTCACCAAGCTCCT-TAMRA-3′

Standard curves for quantitative PCR were generated with the same reaction set-up using gene-specific standard cRNA (0.001 to 1000 attomoles) instead of total cellular RNA. In vitro transcription of gene-specific standard cRNA was performed as previously described [12]. PBGD served as an internal control. Acquisition of fluorescence signals was monitored on the iCycler and terminated when all reactions reached an amplification plateau while template-free controls remained at basal levels. Data analysis was done with iCycler iQ real-time detection system software (BIORAD). To verify that only specific PCR products evoked fluorescence signals PCR products were run on 2 % agarose gels and were analyzed with E.A.S.Y. Win 32 software (HEROLAB, Wiesloch, Germany).

PSA secretion from LNCaP cells was measured in conditioned media with the Elecsys® System 2010 (Roche Diagnostics, Mannheim, Germany).

Statistical analysis

Data are expressed as means and SEM. The statistical significance of differences was determined by Mann-Whitney U tests. P values < 0.01 were considered statistically significant.

Results

To study the effects of silibinin on the genes involved in prostate cancer proliferation the expression of PSA and factors affecting PSA activation were analyzed. During silibinin treatments of 24 to 48 hours no cytotoxic effects of the drug were detected in MTT tests. Since PSA secretion is strongly androgen-dependent in LNCaP cells we also investigated the effects of silibinin in the presence of 10 ^{- 8} M DHT. mRNA expression of prostate epithelium-specific Ets transcription factor PDEF, a co-regulator of the androgen receptor in PSA gene regulation was significantly reduced, when the cells were treated with silibinin in concentrations between 10 and 100 μM (Fig. [2] A). This diminished expression of PDEF was paralleled by PSA mRNA down-regulation (Fig. [3] A) and the PSA secretion was reduced (Fig. [4] A). When cells were pre-treated with 10 ^{- 8} M DHT for 24 hours and then treated for another 24 hours with silibinin in the presence of DHT the flavonoid also could down-regulate PDEF and PSA expression. PDEF and PSA mRNA expressions were increased by DHT alone but significantly reduced with increasing concentrations of silibinin (Figs. [2] B and [3] B). PSA secretion also was stimulated by DHT alone and again silibinin with increasing concentrations could counteract the DHT effect and reduce PSA secretion (Fig. [4] B). Fig. [5] shows the increase of PSA secretion within 48 hours with or without DHT and how increasing silibinin concentrations could diminish PSA secretion over this period of time. These results show a concentration-dependent parallel decrease of PDEF and PSA by silibinin with or without DHT treatment. This indicates a silibinin-induced decrease of PSA expression initiated by a decreased activation of the androgen receptor from PDEF. However, the quantification of the androgen receptor mRNA revealed no significant changes upon silibinin treatments which was also true for the mRNA expression of the housekeeping gene PBGD (data not shown).

Discussion

This study demonstrates that the flavolignan from Silybum marianum silibinin inhibits PSA secretion in vitro. PSA secretion is down-regulated concomitantly with the gene expression of the co-activator of the androgen receptor PDEF which is known to activate PSA gene expression. These results indicate that silibinin has a strong potential to interact with the expression of these genes and their protein products which play significant roles in carcinogenesis and cancer cell proliferation. Tyagi et al. found antiproliferative and apoptotic effects of silibinin in rat prostate cancer cells [13]. Zi et al. reported an up-regulation of IGFBP-3 and a strong inhibition of cell proliferation after treatment of prostate cancer cells with silibinin [6]. Zi and Agarwal also reported a strong inhibition of cell growth via a G1 arrest in cell cycle progression and a significant decrease in both intracellular and secreted forms of PSA in LNCaP after silibinin treatment [5]. Our results on PSA expression complement these findings by demonstrating that this inhibition of PSA expression by silibinin is also present under DHT stimulated conditions. PSA is a protease which cleaves IGFBP-3 and thereby prevents binding of IGF1. The reduced PSA production will result in less inactivation of IGFBP-3. Hence more IGF-1 can be bound by IGFBP-3 and thereby be made biologically inactive. Thus, the activity of IGF to cause cell proliferation will be reduced which is a highly desired effect for cancerous tissue. Furthermore, we showed that the silibinin-induced decrease in PSA secretion may be due to decreased PDEF gene and protein expression. PDEF is the prostate epithelium-specific Ets transcription factor which interacts with the androgen receptor by enhancing the androgen-mediated activation of the PSA promoter. Hence a reduction of PDEF will result in less androgen receptor activation. NKX-3.1, a homeobox gene defined as a tumor modulator [14] counteracts PDEF activity. NKX-3.1 is frequently deleted in prostate cancer. In animal models over-expression of NKX-3.1 leads to reduced cell growth and tumorigenicity. Homozygous and heterozygous NKX-3.1 knock-out mice are predisposed to prostate cancer [15]. Thus, the silibinin-induced down-regulation of PDEF can compensate for the loss of function of the PDEF suppressor NKX-3.1 in case it is deleted in prostate cancer.

We conclude that the initial effect of silibinin after binding to ERβ is to down-regulate PDEF which prevents its activating role on the androgen receptor and thereby reduces PSA expression. Thus, silibinin which is a well accepted drug to treat liver diseases offers new perspectives in prostate cancer treatment. The anti-proliferative effects of silibinin and the down-regulation of genes which strongly affect tumor cell proliferation reported here underline the potential of this compound as a putativel useful tool in prostate cancer therapy.

Acknowledgements

The authors thank Jasmin Held, Marion Striepe and Dorothee Ziegler for excellent technical assistance.

References

• 1 Sutkowski D M, Goode R L, Baniel J, Teater C, Cohen P, McNulty A M. et al . Growth regulation of prostatic stromal cells by prostate-specific antigen.  J Natl Cancer Inst.. 1999;  91 1663-9
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• 4 Seidlová-Wuttke D, Becker T, Christoffel V, Jarry H Wuttke W. Silymarin is a selective estrogen receptor β (ERβ) agonist and has estrogenic effects in the metaphysis of the femur but no or antiestrogenic effects in the uterus of ovariectomized (ovx) rats.  J Steroid Biochem Mol Biol. 2003;  86 179-88
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  CrossrefPubMedSearch in Google Scholar
• 9 Oettgen P, Finger E, Sun Z, Akbarali Y, Thamrongsak U, Boltax J. et al . PDEF, a novel prostate epithelium-specific ets transcription factor, interacts with the androgen receptor and activates prostate-specific antigen gene expression.  J Biol Chem. 2000;  275 1216-25
  CrossrefPubMedSearch in Google Scholar
• 10 Ghadersohi A, Sood A K. Prostate epithelium-derived Ets transcription factor mRNA is overexpressed in human breast tumors and is a candidate breast tumor marker and a breast tumor antigen. Clin.  Cancer Res. 2001;  7 2731-8
  PubMedSearch in Google Scholar
• 11 Finke J, Fritzen R, Ternes P, Lange W, Dolken G. An improved strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, paraffin-embedded tissues by PCR.  Biotechniques. 1993;  14 448-53
  PubMedSearch in Google Scholar
• 12 Grzmil M, Thelen P, Hemmerlein B, Schweyer S, Voigt S, Mury D. et al . Bax inhibitor-1 is overexpressed in prostate cancer and its specific down-regulation by RNA interference leads to cell death in human prostate carcinoma cells.  Am J Pathol. 2003;  163 543-52
  PubMedSearch in Google Scholar
• 13 Tyagi A, Bhatia N, Condon M S, Bosland M C, Agarwal C, Agarwal R. Antiproliferative and apoptotic effects of silibinin in rat prostate cancer cells.  Prostate. 2002;  53 211-17
  CrossrefPubMedSearch in Google Scholar
• 14 Chen H, Nandi A K, Li X, Bieberich C J. NKX-3.1 interacts with prostate-derived Ets factor and regulates the activity of the PSA promoter.  Cancer Res. 2002;  62 338-40
  PubMedSearch in Google Scholar
• 15 Abate-Shen C. Deregulated homeobox gene expression in cancer: cause or consequence? Nat. Rev.  Cancer. 2002;  2 777-85
  PubMedSearch in Google Scholar

Dr. Paul Thelen

Abt. Urologie

Georg-August-Universität

Robert Koch-Str. 40

37075 Göttingen

Germany

Phone: +49-551-398-651

Fax: +49-551-396-165

Email: pthelen@gwdg.de

References

• 1 Sutkowski D M, Goode R L, Baniel J, Teater C, Cohen P, McNulty A M. et al . Growth regulation of prostatic stromal cells by prostate-specific antigen.  J Natl Cancer Inst.. 1999;  91 1663-9
  CrossrefPubMedSearch in Google Scholar
• 2 Lau K M, LaSpina M, Long J, Ho S M. Expression of estrogen receptor (ER)-alpha and ER-beta in normal and malignant prostatic epithelial cells: regulation by methylation and involvement in growth regulation.  Cancer Res.. 2000;  60 3175-82
  PubMedSearch in Google Scholar
• 3 Adams J Y, Leav I, Lau K M, Ho S M, Pflueger S M. Expression of estrogen receptor beta in the fetal, neonatal, and prepubertal human prostate.  Prostate.. 2002;  52 69-81
  CrossrefPubMedSearch in Google Scholar
• 4 Seidlová-Wuttke D, Becker T, Christoffel V, Jarry H Wuttke W. Silymarin is a selective estrogen receptor β (ERβ) agonist and has estrogenic effects in the metaphysis of the femur but no or antiestrogenic effects in the uterus of ovariectomized (ovx) rats.  J Steroid Biochem Mol Biol. 2003;  86 179-88
  CrossrefPubMedSearch in Google Scholar
• 5 Bhatia N, Agarwal R. Detrimental effect of cancer preventive phytochemicals silymarin, genistein and epigallocatechin 3-gallate on epigenetic events in human prostate carcinoma DU145 cells.  Prostate. 2001;  46 98-107
  CrossrefPubMedSearch in Google Scholar
• 6 Zi X, Zhang J, Agarwal R, Pollak M. Silibinin up-regulates insulin-like growth factor-binding protein 3 expression and inhibits proliferation of androgen-independent prostate cancer cells.  Cancer Res. 2000;  60 5617-20
  PubMedSearch in Google Scholar
• 7 Zi X, Agarwal R. Silibinin decreases prostate-specific antigen with cell growth inhibition via G1 arrest, leading to differentiation of prostate carcinoma cells: implications for prostate cancer intervention.  Proc Natl Acad Sci USA. 1999;  96 7490-5
  CrossrefPubMedSearch in Google Scholar
• 8 Zhu W, Zhang J S, Young C Y. Silymarin inhibits function of the androgen receptor by reducing nuclear localization of the receptor in the human prostate cancer cell line LNCaP.  Carcinogenesis. 2001;  22 1399-403
  CrossrefPubMedSearch in Google Scholar
• 9 Oettgen P, Finger E, Sun Z, Akbarali Y, Thamrongsak U, Boltax J. et al . PDEF, a novel prostate epithelium-specific ets transcription factor, interacts with the androgen receptor and activates prostate-specific antigen gene expression.  J Biol Chem. 2000;  275 1216-25
  CrossrefPubMedSearch in Google Scholar
• 10 Ghadersohi A, Sood A K. Prostate epithelium-derived Ets transcription factor mRNA is overexpressed in human breast tumors and is a candidate breast tumor marker and a breast tumor antigen. Clin.  Cancer Res. 2001;  7 2731-8
  PubMedSearch in Google Scholar
• 11 Finke J, Fritzen R, Ternes P, Lange W, Dolken G. An improved strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, paraffin-embedded tissues by PCR.  Biotechniques. 1993;  14 448-53
  PubMedSearch in Google Scholar
• 12 Grzmil M, Thelen P, Hemmerlein B, Schweyer S, Voigt S, Mury D. et al . Bax inhibitor-1 is overexpressed in prostate cancer and its specific down-regulation by RNA interference leads to cell death in human prostate carcinoma cells.  Am J Pathol. 2003;  163 543-52
  PubMedSearch in Google Scholar
• 13 Tyagi A, Bhatia N, Condon M S, Bosland M C, Agarwal C, Agarwal R. Antiproliferative and apoptotic effects of silibinin in rat prostate cancer cells.  Prostate. 2002;  53 211-17
  CrossrefPubMedSearch in Google Scholar
• 14 Chen H, Nandi A K, Li X, Bieberich C J. NKX-3.1 interacts with prostate-derived Ets factor and regulates the activity of the PSA promoter.  Cancer Res. 2002;  62 338-40
  PubMedSearch in Google Scholar
• 15 Abate-Shen C. Deregulated homeobox gene expression in cancer: cause or consequence? Nat. Rev.  Cancer. 2002;  2 777-85
  PubMedSearch in Google Scholar

Dr. Paul Thelen

Abt. Urologie

Georg-August-Universität

Robert Koch-Str. 40

37075 Göttingen

Germany

Phone: +49-551-398-651

Fax: +49-551-396-165

Email: pthelen@gwdg.de