Intra-prostatic androgen levels during various androgen-blockade regimens

Androgens are heavily involved in the development of prostate cancer. This article reviews the scenario of the androgen environment and androgen metabolism in the prostate during androgen-deprivation therapy (ADT) in patients with prostate cancer. Ways of altering the intra-prostatic androgen milieu during various androgen-blockade regimens include surgical castration, luteinizing hormone-releasing hormone analogues to block androgen secretion by the testes, anti-androgens, and 5alpha-reductase inhibitors. The levels of androgen precursors in the blood are different under different ADT regimens, and the androgen levels in the prostate also vary according to the ADT used. This may affect the therapeutic effect of ADT. We therefore discuss the subject of prostatic androgen levels during various androgen-blockade regimens, and we describe the prospects for the future of ADT.     

雄激素受体对前列腺癌PC3细胞株增殖能力的影响

目的研究雄激素受体（AR）对雄激素非依赖性前列腺癌细胞株PC3增殖能力的影响。方法将含有AR的质粒（Pbabe-AR）稳定转染到PC3细胞,建立起新的细胞系PC3-AR,并用实时逆转录聚合酶链反应（Q—PCR）、蛋白免疫印迹试验（Western blot）和荧光素酶法确认AR的表达水平和功能;用MTT法、软琼脂糖凝胶实验检测AR对体外培养PC3细胞增殖的影响。结果PC3-AR细胞能够稳定表达有功能的AR;MTT实验显示PC3-AR细胞体外生长慢于PC3-v细胞,加入DHT以后效果更加明显。相对于PC3-v,PC3-AR在软琼脂糖凝胶中形成的克隆明显减少。结论AR能够降低PC3-AR细胞在体外的增殖能力。     

Identification of an androgen receptor in the cytosol of the female Mastomys prostate

An in vivo and in vitro study was carried out on the prostate from the female Praomys (Mastomys) natalensis to identify and characterize the binding of androgens within the cytoplasm. The labelled cytosol was prepared and subjected to gel exclusion chromatography and density gradient centrifugation. A macromolecular protein associated with the radioactivity was isolated on Sephadex G-200. Subsequent analysis of the steroid receptor complex showed that the major part of the radioactive steroid (64 percent) was dihydrotestosterone. This binding was inhibited by unlabelled testosterone and could not be demonstrated in liver cytosol. Characterization of this dihydrotestosterone receptor complex revealed a sedimentation coefficient of 4.6 s in the presence of a high salt solution (0.4 M KCl). The complex aggregated in the absence of 0.4 M KCl and sedimented preferentially from 5.6-7.4 s together with polydisperse aggregates of higher sedimentation coefficients. The use of this animal as an experimental model for hormonal studies on the prostate is suggested.     

The role of androgen metabolism in the control of androgen action in the rat prostate

Recent results from a number of laboratories have led us to re-examine the role of 3 beta-androstanediol in the rat ventral prostate. Whereas previously 5 alpha-dihydrotestosterone and 3-beta androstanediol were thought to have distinctly separate effects on the prostate, we suggest that 3 beta-androstanediol serves only as an intermediate in the metabolism and removal of 5 alpha-dihydrotestosterone from the organ. In our view the action of androgens on the prostate are exerted exclusively through the binding of 5 alpha-dihydrotestosterone to the androgen receptor and its subsequent translocation to the nucleus. Differences in effects are related to the amount of 5 alpha-dihydrotestosterone available to the gland. 3 alpha-Hydroxysteroid dehydrogenase may play a critical role in modulating the level of 5 alpha-dihydrotestosterone available to the translocatable receptor.     

Morphological changes induced by androgen blockade in normal prostate and prostatic carcinoma

Maximal androgen blockade (MAB), combining a luteinizing hormone releasing hormone (LHRH) agonist and a pure or non-steroidal anti-androgen, induces significant morphologic changes in the prostate. The tumor volume, density, capsular penetration, and surgical margin involvement are strongly reduced following such treatment. On histology, normal prostate tissue and tumor undergo marked atrophy and shrinkage. Although residual cancer cells are readily identifiable in most cases, they may often be sparse and easily overlooked. The increased Gleason score apparent after MAB is most likely related to fragmentation of acinar structures, and grading is not recommended following MAB. Residual cancer cells show features of lower activity and increased apoptosis. Such therapy-induced changes may be reversible, although occasional clones of cancer cells are apparently not affected and have probably developed resistance. Finally, MAB leads to marked but reversible morphologic changes and reduction in prevalence and extent of prostatic intra-epithelial neoplasia (PIN). Monotherapy using a variety of agents causes comparable but often less extensive changes.     

Inhibition by estradiol-17 beta of porcine thecal androgen production in vitro.

Thecal preparations from medium-sized procine ovarian follicles (3.5-5 mm diameter) were incubated for 4 h in a chemically defined medium in the presence or absence of highly purified luteinizing hormone (LH) and/or estradiol-17 beta (estradiol). LH (1 microgram/ml) stimulated the thecal production of testosterone (T) and dihydrotestosterone (DHT) by 2- to 3-fold. Although estradiol (10 microgram/ml) alone had only a slight but non-significant inhibitory effect on basal testosterone production, it significantly inhibited the production of both T and DHT as well as decreasing the DHT/T ratio in a dose-related manner in the presence of LH. Production of cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone by the thecal cells was stimulated 50-to 200-fold and 2.5-fold, respectively, by LH. Estradiol had no significant effect on thecal cAMP and progesterone production in the presence or absence of the gonadotropin. These findings are consistent with the concept that estradiol produced by granulosa cells following hormonal stimulation may serve as a local negative feedback mechanism to control thecal androgen production.     

连翘三萜类化合物对前列腺癌PC-3细胞增殖抑制及放疗敏感性的实验研究

目的探讨连翘三萜类化合物达玛-24-烯-3β-乙酰氧基-20s-醇（DM）对人前列腺癌PC-3细胞的增殖抑制及放疗增敏作用。方法采用流式细胞术检测不同浓度DM液对PC-3细胞周期、凋亡的影响;PC-3细胞接受6-mVX线照射,用克隆形成法检测细胞存活分数、计算放疗增敏比;端粒重复序列扩增法检测DM对PC-3细胞端粒酶活性的抑制作用;实时定量PCR测定DM对PC-3细胞周期相关基因表达的影响。结果 DM可诱导PC-3细胞凋亡,其作用6 h后肿瘤细胞端粒酶活性降低,48 h后渐恢复至正常。DM可使细胞周期相关基因p21、TGF-β、Smad3表达增加,Cyclin D1、CDC25A表达降低（P均〈0.05）。DM有放疗增敏作用,其放疗增敏比为1.80。结论 DM可诱导PC-3细胞凋亡,抑制其细胞端粒酶活性,调节细胞周期相关基因表达,对前列腺癌PC-3细胞有放疗增敏作用。     

Regulation of androgen receptor expression in the human heterotransplantable prostate carcinoma PC-82.

In vivo effects of androgen withdrawal and substitution on human androgen receptor (hAR) expression were evaluated in the androgen-dependent human prostatic carcinoma tumor line PC-82. By application of several antibodies reactive with different epitopes of the hAR molecule, hAR protein expression was studied in tumor transplants by immunohistochemistry and immunoblotting. hAR messenger RNA (mRNA) levels were quantitated in PC-82 tumor tissue with a S1-nuclease protection assay. Most PC-82 tumor cells (> 97%) from testosterone-supplemented mice displayed nuclear hAR protein expression immunohistochemically. The almost complete reduction of nuclear hAR immunoreactivity within 5 days after androgen withdrawal (< 10%) was restored after androgen substitution within 1 day. The immunochemical data were confirmed by Western blot analysis. In contrast, no significant changes were observed in hAR mRNA content of PC-82 cells after 5 days of androgen withdrawal. Correlating hAR expression with proliferative activity of PC-82 tumor tissue during endocrine manipulation, a rapid, castration-induced decline of the percentage of bromodeoxyuridine-labeled cells accompanied the loss of hAR. Androgen substitution in castrated male mice restored the proliferative activity. However, this increase of proliferative activity lagged at least 24 h behind the normalization of the hAR protein level. In contrast to the steroid receptor down-regulation by homologous ligands observed in other experimental models, our data support the concept of hAR up-regulation by androgen. Since the hAR mRNA content of PC-82 tumor tissue was hardly affected by castration, expression of the hAR in PC-82 is thought to be modulated by translational and/or posttranslational mechanisms.     

Androgen biosynthetic pathways in the human prostate

It is well recognized that there are two androgens, namely testosterone (T) and dihydrotestosterone (DHT); T plays an important role in the testis and muscle, and DHT is crucial for the development, function and pathology of the prostate. It is generally thought that DHT is produced from the 5alpha-reduction of circulating T before being inactivated by 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) that converts DHT into 5alpha-androstane-3alpha,17beta-diol (3alpha-diol). However, the presence of various steroidogenic enzymes in the prostate as well as the availability at high levels of various steroid precursors such as dehydroepiandrosterone sulphate (DHEAS), dehydroepiandrosterone (DHEA) and 4-androstenedione (4-dione) strongly suggest the existence of additional pathways involved in the biosynthesis and metabolism of DHT. Because steroidogenesis could be different in different species, data from the literature obtained from various human, dog, rat and mouse prostate tissues, as well as primary cells and prostatic cancer cell lines, provide a somewhat confusing picture. In the present chapter, we review the data in order to provide a clearer picture of the pathways involved in DHT biosynthesis and metabolism in the human prostate.     

Carbon sources and pathways for citrate secreted by human prostate cancer cells determined by NMR tracing and metabolic modeling

Prostate epithelial cells have the unique capacity to secrete large amounts of citrate, but the carbon sources and metabolic pathways that maintain this production are not well known. We mapped potential pathways for citrate carbons in the human prostate cancer metastasis cell lines LNCaP and VCaP, for which we first established that they secrete citrate (For LNCaP 5.6 ± 0.9 nmol/h per 10⁶ cells). Using ¹³C-labeled substrates, we traced the incorporation of ¹³C into citrate by NMR of extracellular fluid. Our results provide direct evidence that glucose is a main carbon source for secreted citrate. We also demonstrate that carbons from supplied glutamine flow via oxidative Krebs cycle and reductive carboxylation routes to positions in secreted citrate but likely do not contribute to its net synthesis. The potential anaplerotic carbon sources aspartate and asparagine did not contribute to citrate carbons. We developed a quantitative metabolic model employing the ¹³C distribution in extracellular citrate after ¹³C glucose and pyruvate application to assess intracellular pathways of carbons for secreted citrate. From this model, it was estimated that in LNCaP about 21% of pyruvate entering the Krebs cycle is converted via pyruvate carboxylase as an anaplerotic route at a rate more than sufficient to compensate carbon loss of this cycle by citrate secretion. This model provides an estimation of the fraction of molecules, including citrate, leaving the Krebs cycle at every turn. The measured ratios of ¹³C atoms at different positions in extracellular citrate may serve as biomarkers for (malignant) epithelial cell metabolism.

Healthy prostate epithelial cells have the unique capability of secreting citrate into the ducts of the prostate (1). Citrate is formed by condensation of oxaloacetate and acetyl-CoA, catalyzed by citrate synthase as the first step of the Krebs cycle. A part of this citrate is diverted from the Krebs cycle into the cytosol, facilitated by a citrate transport protein in the inner mitochondrial membrane. It can then be secreted into the ducts of the prostate, involving an independent electrogenic transport system (2). Citrate in the lumen of the prostate can reach levels up to about 180 mM, which is thought to mainly serve as an energy source for sperm cells (3, 4). Citrate accumulation is promoted by inhibition of the citrate-converting enzyme m-aconitase through binding of zinc, which is taken up at relatively high levels in epithelial prostate cells (1, 5, 6) (Fig. 1A).Open in a separate windowFig. 1.Schematic overview of metabolic pathways relevant in the biosynthesis of citrate in the prostate and schematic representation of ¹³C atoms in compounds entering the Krebs cycle and their fate during multiple cycle turns to produce citrate. (A) In healthy prostate epithelial cells, zinc ions, imported via the ZIP1 transporter, inhibit aconitase, the enzyme catalyzing the stereospecific isomerization of citrate into isocitrate. The resulting excess of citrate is transported to the cytosol via the citrate transporter (CT) and excreted by the cells via the citrate transporter KCitT into the luminal space. Upon malignant transformation, Zn²⁺ uptake is decreased and consequently inhibition of aconitase is released and citrate becomes more oxidized to isocitrate instead of being secreted. Potential metabolites supplying the Krebs cycle with carbons are indicated in green. Anaplerosis is needed when citrate secretion drains carbons from this cycle. Aspartate can enter the Krebs cycle via oxaloacetate. Glutamine is converted into glutamate and its carbons enter the Krebs cycle as five-carbon metabolite α-ketoglutarate. Pyruvate can be converted by PC into four-carbon Krebs cycle intermediate oxaloacetate. Both reductive and oxidative metabolic pathways are indicated. (B) The distribution of ¹³C labels originating from [1,6-¹³C₂]glucose. (C) Idem from [2-¹³C]pyruvate. Pyruvate carbons converted to oxaloacetate by PC are indicated by blue circles and those converted into acetyl-CoA by PDC by red circles. (D) Distribution of ¹³C labels in possible anaplerosis involving [U-¹³C₄]aspartate. (E) Idem involving [5-¹³C]glutamine. Glutamine carbons converted by oxidative metabolism are indicated by red circles and those converted by reductive exchange by blue circles. In this figure the carbon numbers in each compound are indicated and for simplicity mitochondrial and cellular export of citrate is omitted. Citrate carbon numbering is chosen in such a way that C1 and C2 of acetyl-CoA end up at C1 and C2 of citrate.Prostate cancer is a major health burden worldwide (7). A remarkable metabolic change upon malignant transformation in the prostate is the local decrease of the tissue concentration of citrate. This can be a result of tumor cells occupying space in ducts, physically displacing luminal fluid, and/or of metabolic reprogramming toward the diversion of less citrate (8). A key metabolic event in the transformation of epithelial cells to cancer cells is considered to be down-regulation of the zinc transporter ZIP1, causing decreased zinc uptake and therefore increased m-aconitase activity and oxidation of citrate instead of secretion into ducts (Fig. 1A) (9). A decreased citrate signal in MR spectroscopic images of the prostate is employed as biomarker to identify cancer lesions (10–13). Understanding metabolic reprogramming in malignant transformation may help to better diagnose and treat prostate cancer (14–18).A major question is how a high level of citrate production is supported by the metabolic network of the prostate. Studies in epithelial cells and ventral tissue of the rat prostate point to glucose as a major carbon source for citrate (19, 20). Rat epithelial prostate cells only produced citrate with aspartate in the medium, suggesting that aspartate is an essential precursor (21). Its uptake is mediated by the amino acid transporter EAAC1, and subsequent transamination by mitochondrial aspartate aminotransferase (mAAT) provides oxaloacetate (22). The high EAAC1 expression in rat and human prostate further seems to support the importance of aspartate (23). None of these studies, however, provide direct evidence that carbon atoms from glucose or aspartate indeed end up in extracellular citrate and which metabolic pathways are followed by these carbons.Because citrate produced in the Krebs cycle flows to the luminal space and toward de novo lipogenesis (24), this cycle is depleted of carbon atoms needed for its maintenance and therefore requires anaplerotic supplementation. In the case that glucose is a major carbon source for citrate, aspartate may function as an anaplerotic supply. Anaplerosis can also occur via pyruvate and glutamine (25) (Fig. 1A). Pyruvate can be converted into oxaloacetate by pyruvate carboxylase (PC) and glutamine into glutamate by glutaminase and subsequently into the Krebs cycle intermediate α-ketoglutarate by glutamate dehydrogenase (GDH). Since prostate cells harbor a high number of glutamine transporters (26) and glutamine occurs at relatively high levels in blood, it may serve as carbon source for citrate.To determine which of the abovementioned substrates and metabolic pathways may contribute carbons in the production of citrate in the human prostate we searched for prostate epithelial cells of human origin that are able to secrete citrate in sufficient amounts for metabolic analysis. Previous studies indicated that the prostate cancer lymph node metastatic cell line LNCaP secretes citrate (27, 28). LNCaP cells also express the amino acid transporter EAAC1 and are commonly used as a model to study prostate cancer metabolism (23). VCaP is a prostate cancer cell line derived from a vertebral bone metastasis (29). Like LNCaP cells, the VCaP cells express the glutamine transporter ASCT2 (30, 31), are androgen-sensitive, and produce prostate-specific antigen (PSA), reflecting their prostatic origin and still-differentiated nature (32–34), and thus may also secrete citrate.An elegant method to follow metabolism in tissues and cells is by supplying them with ¹³C-labeled substrates and to monitor the fate of the ¹³C atoms by ¹³C NMR spectroscopy (35–37). The specific ¹³C labeling of metabolic products provides information on their synthetic route. In this study we focused on the specific ¹³C labeling pattern of secreted citrate as a readout of intracellular metabolic pathways contributing to its carbon skeleton. Eventually, the labeling pattern of extracellular citrate may be used as a fingerprint of intracellular metabolism for diagnostic purposes.After establishing that LNCaP and VCaP cells indeed secrete citrate, we investigated whether and how aspartate, asparagine, glucose, pyruvate, and glutamine can serve as carbon sources for this extracellular citrate. For this purpose, we employed one-dimensional (1D) and two-dimensional (2D) ¹³C and ¹H NMR of the growth media of the VCaP and LNCaP cells. We developed a metabolic model that uses specific ¹³C labeling of extracellular citrate, after ¹³C glucose and pyruvate supplementation, as input to provide quantitative information of intracellular metabolism supporting supply of citrate carbons. Ultimately, compounds and metabolic pathways involved in citrate production could act as biomarkers to characterize (malignant) epithelial cell metabolism and may be considered as targets for treatments (17, 38, 39).     

Heterogeneity in androgen receptor levels and growth response to dihydrotestosterone in sublines derived from human hepatocellular carcinoma line (KYN-1)

Abstract: Heterogeneity in the growth response to androgen and androgen receptor (AR) status in an AR-positive human hepatocellular carcinoma (HCC) line, KYN-1, was investigated in the present study. Seven sublines were obtained from KYN-1 by the limited dilution method. Four of them had no detectable amounts of AR and did not show any growth response to dihydrotestosterone (DHT) at the concentration up to 1000 nM, while the other three had varying amounts of both cytosolic and nucleosolic AR that were 5- to 7-fold higher than that of the parent cell. In the parent cell, the addition of DHT caused a modest but significant increase in the cell number (21%) in a 14-day culture. Such effect was increased by 2- to 7-fold in two AR-positive sublines. In addition, the sensitivity of the response in these two sublines was increased by up to 24-fold. The one remaining AR-positive subline did not show any growth response to DHT, although the behavior of its AR in sucrose gradient ultracentrifugation and Western blotting was the same as that of the other two sublines. These data clearly show that sublines derived from an established human HCC line have a broad heterogeneity in the growth response of HCC to androgen as well as in AR status and could be classified into three groups on the basis of the growth responsiveness to androgen and the AR expression.     

Intracrine androgen metabolism in prostate cancer progression: mechanisms of castration resistance and therapeutic implications

Residual tissue androgens are consistently detected within the prostate tumors of castrate individuals and are thought to play a critical role in facilitating the androgen receptor-mediated signaling pathways leading to disease progression. The source of residual tumor androgens is attributed in part to the uptake and conversion of circulating adrenal androgens. Whether the de novo biosynthesis of androgens from cholesterol or earlier precursors occurs within prostatic tumors is not known, but it has significant implications for treatment strategies targeting sources of androgens exogenous to the prostate versus 'intracrine' sources within the prostatic tumor. Moreover, increased expression of androgen-metabolizing genes within castration-resistant metastases suggests that up-regulated activity of endogenous steroidogenic pathways may contribute to the outgrowth of 'castration-adapted' tumors. These observations suggest that a multi-targeted treatment approach designed to simultaneously ablate testicular, adrenal and intracrine contributions to the tumor androgen signaling axis will be required to achieve optimal therapeutic efficacy.     

Vitamin D-mediated growth inhibition of an androgen-ablated LNCaP cell line model of human prostate cancer

1,25-(OH)(2) vitamin D(3) (1,25-(OH)(2) D), the active metabolite of vitamin D, exerts antiproliferative effects on a variety of tumor cells including prostate. This inhibition requires vitamin D receptors (VDRs) as well as downstream effects on the G1 to S phase checkpoint of the cell cycle. Recent data raise the possibility that androgen plays a role in the antiproliferative effects of 1,25-(OH)(2) D in prostate cancer cells; however, this hypothesis has been difficult to test rigorously as the majority of prostate cancer cell lines (unlike human prostate tumors) lack androgen receptors (ARs). We utilized two different models of androgen-independent prostate cancer that express functional ARs and VDRs to evaluate a possible role of androgen in 1,25-(OH)(2) D mediated growth inhibition. We stably introduced the AR cDNA into the human prostate cancer cell line ALVA 31, which expresses functional VDR but is relatively resistant to growth inhibition by 1,25-(OH)(2) D. Neither ALVA-AR nor the control cells, ALVA-NEO, exhibited substantial growth inhibition by 1,25-(OH)(2) D in the presence or absence of androgen. This observation suggests that the basis for the resistance of ALVA 31 to 1,25-(OH)(2) D-mediated growth inhibition is not the lack of AR. The second model was LNCaP-104R1, an AR-expressing androgen independent prostate cancer cell line derived from androgen dependent LNCaP. 1,25-(OH)(2) D inhibited the growth of LNCaP-104R1 cells in the absence of androgen and this effect was not blocked by the antiandrogen Casodex. As was observed in the parental LNCaP cells, this effect was correlated with G1 phase cell cycle accumulation and upregulation of the cyclin dependent kinase inhibitor (CKI) p27, as well as increased association of p27 with cyclin dependent kinase 2. These findings suggest that the antiproliferative effects of 1,25-(OH)(2) D do not require androgen-activated AR but do involve 1,25-(OH)(2) D induction of CKIs required for G1 cell cycle checkpoint control.     

原花青素对前列腺癌PC-3细胞增殖和凋亡的影响

目的探讨原花青素对人雄激素非依赖性前列腺癌细胞株PC-3细胞（PC-3细胞）增殖和凋亡的影响。方法体外培养的PC-3细胞与100、200、300μg／ml的原花青素共孵育,分别在24、48和72h时采用HE染色观察细胞形态学变化,噻唑蓝（MTT）比色法检测细胞增殖抑制率,流式细胞术（FCM）分析细胞凋亡情况。结果原花青素可引起PC-3细胞形态明显改变;抑制PC-3细胞增殖,不同浓度（100、200、300μg／ml）原花青素对PC-3细胞作用72h时的细胞增殖抑制率分别为25．2％、70．9％和85．2％;FCM检测表明原花青素可诱导PC-3细胞凋亡,300μg／ml原花青素处理7Zh引起36．3％的凋亡率。这3种作用均随原花青素浓度和作用时间的延长而增强。结论原花青素可在体外抑制前列腺癌PC-3细胞增殖,并促进其凋亡。     

The proliferative effect of "anti-androgens" on the androgen-sensitive human prostate tumor cell line LNCaP

The effect of steroidal and nonsteroidal "anti-androgens" on the proliferative capacity of androgen-sensitive LNCaP-FGC human prostate tumor cells in culture was studied using charcoal-dextran stripped human serum-supplemented media. Cyproterone and medroxyprogesterone acetates, flutamide, hydroxyflutamide, and anandron (R23908) were administered alone at concentrations between 3 X 10(-12) and 3 X 10(-6) M. Results indicated that although medroxyprogesterone induced maximal proliferation at 3 X 10(-9) M, the other "anti-androgens" (with the exception of flutamide that was ineffective) were effective at 3 X 10(-8) M and higher concentrations; the amplitude of the proliferative response by these compounds was comparable to that elicited by estradiol-17 beta (3 to 5-fold over control). None of the anti-androgens tested triggered the shutoff effect characteristic of androgen action. When 3 X 10(-10) M DHT and the above mentioned anti-androgens were administered simultaneously, a synergistic pattern was seen; on the contrary, 3 X 10(-8) M DHT cancelled the proliferative effect of each of the anti-androgens when administered simultaneously. The relative binding affinity of these anti-androgens to androgen receptors present in LNCaP-FGC cells did not correlate well with their proliferative efficiency. The data collected were interpreted within the premises of the negative control hypotheses for the regulation of cell proliferation in metazoans. Within those premises, results became compatible with the notion that first, "anti-androgens" elicited the proliferation of androgen-sensitive cells by neutralizing the effect of a serum-borne inhibitor (androcolyone-I); this event seems not to be mediated by androgens receptors. Second, anti-androgens did not trigger a proliferative shutoff response like androgens do, i.e. the proliferative pattern induced by anti-androgens was comparable to that elicited by estrogens and progestins. Third, when administered simultaneously with 3 X 10(-10) M DHT, anti-androgens behaved synergistically. Fourth, the DHT-induced shutoff effect consistently overrode the proliferative effect generated by anti-androgens and estrogens when added alone. Finally, taken together these results raise important questions regarding the therapeutic role of anti-androgens in prostate cancer.     

Growth hormone (GH) receptors in prostate cancer: gene expression in human tissues and cell lines and characterization, GH signaling and androgen receptor regulation in LNCaP cells

Various hormones and growth factors have been implicated in progression of prostate cancer, but their role and the underlying molecular mechanism(s) involved remain poorly understood. In this study, we investigated the role of human growth hormone (GH) and its receptor (GHR) in human prostate cancer. We first demonstrated mRNA expression of GHR and of its exon 9-truncated isoform (GHR_tr) in benign prostate hyperplasia (BPH) and prostate adenocarcinoma patient tissues, as well as in LNCaP, PC3 and DU145 human prostate cancer cell lines. GHR mRNA levels were 80% higher and GHR_tr only 25% higher, in the carcinoma tissues than in BPH. Both isoforms were also expressed in LNCaP and PC3 cell lines and somewhat less so in DU145 cells. The LNCaP cell GHR protein was further characterized, on the basis of its M_r of 120 kDa, its binding to two different GHR monoclonal antibodies, its high affinity and purely somatogenic binding to ¹²⁵I-hGH and its ability to secrete GH binding protein, all characteristic of a functional GHR. Furthermore, GH induced rapid, time- and dose-dependent signaling events in LNCaP cells, including phosphorylation of JAK2 tyrosine kinase, of GHR itself and of STAT5A (JAK2-STAT5A pathway), of p42/p44 MAPK and of Akt/PKB. No effect of GH (72 h) could be shown on basal or androgen-induced LNCaP cell proliferation nor on PSA secretion. Interestingly, however, GH caused a rapid (2–12 h) though transient striking increase in immunoreactive androgen receptor (AR) levels (≤5-fold), followed by a slower (24–48 h) reduction (≤80%), with only modest parallel changes in serine-phosphorylated AR. In conclusion, the GH-induced activation of signaling pathways, its effects on AR protein in LNCaP cells and the isoform-specific regulation of GHR in prostate cancer patient tissues, suggest that GH, most likely in concert with other hormones and growth factors, may play an important role in progression of human prostate cancer.     

Melatonin receptors in PC3 human prostate tumor cells

Melatonin, secreted nocturnally by the pineal gland, can bind to human benign prostate epithelial cells and attenuate their growth and viability. In the present study, melatonin binding and responses were explored in the human steroid-independent PC3 prostatic tumor cells. PC3 cells bound 125I-melatonin with low affinity (Kd ca. 0.9 nM) at high as well as low cell density. Melatonin enhanced cGMP and 3H-thymidine incorporation at low, but attenuated them at high cell density. In addition, melatonin inhibited cAMP at low, but augmented it at high cell density. These effects were associated with an increase in cell count at low- but not high-density cultures. Pertussis toxin treatment suppressed 125I-melatonin binding and ablated all the effects of melatonin on 3H-thymidine incorporation, cAMP, and cGMP at both cell densities. Cholera toxin treatment failed to block the effects of melatonin on 3H-thymidine incorporation, but prevented the modulation by melatonin of cAMP at low and cGMP at high cell density. The cGMP analog 8-Br-cGMP, inhibited melatonin's effects on 3H-thymidine incorporation at both cell densities. H89, a protein kinase A inhibitor, prevented melatonin's effects on 3H-thymidine incorporation at low but not high cell density. These results provide the first demonstration of direct interaction of melatonin with hormone-insensitive prostate tumor cells. The melatonin receptors in the PC3 cells are coupled to pertussis toxin-sensitive G proteins to induce cell density-dependent changes in cGMP, cAMP, and cell growth.