© 2007 THE AUTHORS JOURNAL COMPILATION © 2 0 0 7 B J U I N T E R N A T I O N A L | 1 0 0 , 1 9 9 – 2 0 3 | doi:10.1111/j.1464-410X.2007.06924.x 199 Investigative Urology PHYTOESTROGENS FROM BELAMCANDA CHINENSIS AND PROSTATE CANCER CELLS THELEN et al. Phytoestrogens from Belamcanda chinensis regulate the expression of steroid receptors and related cofactors in LNCaP prostate cancer cells Paul Thelen, Thomas Peter, Anika Hünermund, Silke Kaulfuß*, Dana Seidlová-Wuttke†, Wolfgang Wuttke†, Rolf-Hermann Ringert and Florian Seseke Department of Urology, *Institute of Human Genetics and †Department Clinical and Experimental Endocrinology, Georg-August-University, Göttingen, Germany Accepted for publication 25 January 2007 PT and TP contributed equally to this study reverse transcription-polymerase chain reaction to quantify mRNA expression of the androgen receptor (AR), the AR coactivator prostate derived Ets transcription factor (PDEF), NKX3.1, prostate specific antigen (PSA) and oestrogen receptor- β (ER- β ) compared with the expression of the housekeeping gene porphobilinogen deaminase (PBGD). PSA secretion from LNCaP cells was measured and protein expression of the AR investigated by Western blot analysis. RESULTS Concomitant with a marked decrease of tumour cell proliferation BCE down-regulated the expression of the AR, PDEF, NKX3.1 and PSA. In the same experiments, the expression of PBGD was unaltered, whereas ER- β expression increased. Furthermore, AR protein and PSA secretion were markedly diminished after treatments with the BCE. CONCLUSION BCE, comprising 13 different phytoestrogens, decreases the expression of the AR and its co- activator PDEF concomitant with diminished cell proliferation and PSA secretion. NKX3.1 expression was also reduced by BCE. We hypothesise that the positive effects of BCE are initiated by up-regulation of the ER- β , a putative tumour-suppressor gene. KEYWORDS prostate cancer, LNCaP, phytoestrogens, androgen receptor, oestrogen, proliferation OBJECTIVE To investigate the changes in expression underlying the marked reduction of tumour growth in vivo , by analysing the effect of Belamcanda chinensis extract (BCE) on LNCaP cells in vitro , as phytoestrogens are chemopreventive in prostate cancer, and in previous studies we examined the effects of the isoflavone tectorigenin isolated from B. chinensis on LNCaP prostate cancer cells, and a BCE consisting of 13 phytoestrogenic compounds on tumour-bearing nude mice. MATERIALS AND METHODS LNCaP cells were treated with 100, 400 or 1400 μ g/mL BCE; proliferation was assessed with an Alamar Blue assay. We used real-time INTRODUCTION In Western societies prostate cancer is the most common malignancy in men; in the USA each year > 230 000, and in Germany > 40 000, new cases are currently diagnosed. Prostate cancer is characterized by strong dietary influences and a long disease latency period. This long latency affords opportunities for intervention with therapies that are designed to delay disease initiation or progression [1]. Diets are regarded as important in the transformation from latent into more aggressive prostate cancer, considering that the frequency of latent prostate cancer is evenly distributed among populations [2]. Progression from latent stages into clinically significant prostate cancer is a process that generally requires several years. During this period, dietary phytochemicals might have chemopreventive effects that could slow or obviate the development of hormone- dependent cancer. This is supported by epidemiological evidence that populations consuming diets rich in soya have lower incidence of prostate cancer [3]. Several studies have evaluated the chemopreventive potential of phytochemicals or phytoestrogens. Phytoestrogens are polyphenolic nonsteroidal plant compounds with oestrogen-like biological activity that are currently under intensive investigation for their role in human health, and the reasons for the geographical differences in prostate cancer incidence rates [4]. Phytoestrogens can act as selective oestrogen receptor (ER) modulators and they have been evaluated for potential androgen-blocking activity [5]. One indicator of androgen- blocking is the inhibition of androgen- regulated proteins such as PSA, which is used as a surrogate marker of disease progression in clinical studies. PSA secretion and other actions of androgens are mediated by the androgen receptor (AR). Chen et al. [6] showed that the AR is the crucial factor in the process by which prostate cancer cells become hormone-refractory and therapy- resistant. They showed that cancer progression in hormone-refractory tumours is controlled by traces of androgen that remain even under antiandrogen therapy, and that can be detected by an overexpressed AR. Moreover, their experiments showed that AR antagonists under these conditions acted as AR agonists. This antiandrogen withdrawal syndrome is shown by a PSA decrease when