Prostate-specific Antigen Secretion Research Articles

Suppression of Human Solid Tumor Growth in Mice by Intratumor and Systemic Inoculation of Histidine-Rich and pH-Dependent Host Defense–like Lytic Peptides

Previously, we reported that intratumor or systemic inoculation of a cationic 15-mer, innate immunity-like lytic peptide composed of d- and l-amino acids ([D]-K(6)L(9)) caused growth arrest of 22RV1 prostate carcinoma xenografts in a mouse model. However, despite its therapeutic potential, this peptide has significant systemic toxicity at concentrations slightly higher than the therapeutic one. Here, we used the acidic environment created by solid tumors as a trigger to activate anticancer lytic peptides by making them cationic only at low pH levels. We achieved this selectivity by substituting lysines (pKa, approximately 10.5) for histidines (pKa, approximately 6.1) in the parental peptide [D]-K(6)L(9). Histidine is protonated below pH 7. For that purpose, we replaced either three or all six lysines in the parental peptide with histidines to obtain the peptides [D]-K(3)H(3)L(9) and [D]-H(6)L(9). Interestingly, in vitro experiments showed pH-dependent activity only with [D]-H(6)L(9) mainly toward cancer cell lines. However, both peptides showed reduced systemic toxicity compared with the parental peptide. Intratumor and systemic inoculation of these peptides resulted in a significant decrease in the 22RV1 prostate cancer tumor volume and systemic secretion of prostate-specific antigen in a xenograft mice model. Moreover, histologic modifications revealed a significant reduction in new blood vessels selectively in tumor tissues after treatment with the peptides compared with the untreated tumors. The lytic mode of action of these new peptides, which makes it difficult for the cancer cells to develop resistance, and their selective and pH-dependent activity make them potential candidates for treatment of solid cancer tumors.

Suppression of mutant androgen receptors by flutamide

To examine the effects of flutamide and hydroxyflutamide on the transactivation of mutant androgen receptors. Androgen-independent human prostate cancer cell line PC3 was transfected with plasmids expressing wild-type, W741C mutant, T877A mutant or W741C+T877A mutant androgen receptors. The effects of bicalutamide, hydroxyflutamide or flutamide on the basal and dihydrotestosterone-induced transcriptional activities of the wild-type and mutant androgen receptors were evaluated by luciferase assays using a reporter plasmid containing the prostate-specific antigen (PSA) promoter. The effects of the antiandrogens on the transcription and translation of the PSA gene in LNCaP cells expressing a mutant (T877A) androgen receptor were assessed by real-time reverse transcription-polymerase chain reaction and radioimmunoassays. Affinity of the antiandrogens to each androgen receptor construct was evaluated by ligand-binding assay. Flutamide, but not hydroxyflutamide, successfully suppressed the transcription of all of the mutant androgen receptors examined in this study and also showed suppressive effects on PSA secretion by LNCaP cells treated with dihydrotestosterone. These inhibitory effects were probably not the result of competitive inhibition by flutamide given its low affinity to the androgen receptor constructs. Flutamide, with its suppressive effects on mutant androgen receptors, may be an alternative to conventional antiandrogens for hormone refractory prostate cancer.

TRIM68 REGULATES LIGAND-DEPENDENT TRANSCRIPTION OF ANDROGEN RECEPTOR IN PROSTATE CANCER CELLS

The androgen receptor (AR) is a transcription factor belonging to the family of nuclear receptors that mediate the action of androgen. AR plays an important role in normal development of the prostate, as well as in the progression of prostate cancer. AR is regulated by several posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. In this study, we found that the putative E3 ubiquitin ligase TRIM68, which is preferentially expressed in prostate cancer cells, interacts with AR and enhances transcriptional activity of the AR in the presence of dihydrotestosterone. We also found that TRIM68 functionally interacts with TIP60 and p300, which act as coactivators of AR, and synergizes in the transactivation of AR. Overexpression of TRIM68 in prostate cancer cells caused an increase in secretion of prostate-specific antigen (PSA), one of the most reliable diagnostic markers for prostate cancer, whereas knockdown of TRIM68 attenuated the secretion of PSA and inhibited cell growth and colony-forming ability. Moreover, we showed that TRIM68 expression is significantly up-regulated in human prostate cancers compared with the expression in adjacent normal tissues. These results indicate that TRIM68 functions as a cofactor for AR-mediated transcription and is likely to be a novel diagnostic tool and a potentially therapeutic target for prostate cancer. [Cancer Res 2008;68(9):3486–94]

Overexpression of 5α‐reductase type 1 increases sensitivity of prostate cancer cells to low concentrations of testosterone

Conversion of testosterone to dihydrotestosterone (DHT) by the enzymes 5 alpha-reductase types 1 (5 alpha R1) and 2 (5 alpha R2) is important for normal and pathological growth of the prostate. The predominant isoenzyme in normal prostate is 5 alpha R2. However, prostate cancer (PCa) development is accompanied by a decrease in 5 alpha R2 and an increase in 5 alpha R1. The biological significance of increased 5 alpha R1 expression is not fully understood. Therefore, the aim of this study was to determine the effect of overexpression of 5 alpha R1 on growth and prostate-specific antigen (PSA) production in PCa cells. LNGK-9 PCa cells, transiently transfected with pTRE-5 alpha R1 or pTRE alone, were cultured in the presence or absence of testosterone at varying concentrations. Cell growth and PSA secretion were measured after 4-6 days. Cyclin E1, Ki67, and PSA mRNA levels were evaluated using RT-PCR after 24 hr of treatment. 10 pM testosterone increased growth of pTRE-5 alpha R1 transfectants by 54.1% over cells grown in the absence of testosterone, compared to 25.0% in pTRE transfectants (P < 0.01). Likewise, PSA secretion was increased by 56-fold in pTRE-5 alpha R1 transfectants treated with 10 pM testosterone, compared to 26-fold in pTRE transfectants (P < 0.01). At concentrations of testosterone above 10 pM, the stimulatory effect on growth and PSA secretion was not distinguishable between pTRE-5 alpha R1 and pTRE transfectants. These results demonstrate that upregulation of 5 alpha R1 enhances the cellular response to low, but not high, concentrations of testosterone. This explains one mechanism by which castration-recurrent PCa can proliferate in the presence of castrate levels of circulating testosterone.

Androgenic action of Tinospora cordifolia ethanolic extract in prostate cancer cell line LNCaP

Recently, Tinospora cordifolia (TC) was shown to affect prostate growth in rats. It is not known whether this is a direct effect of TC or whether it is induced by altered hormone release. To investigate the actions of TC on the prostate, human LNCaP cells were exposed to an ethanolic extract of TC. LNCaP cells were incubated with the test substances for 48 h. Proliferation was measured by MTT test and prostate-specific antigen (PSA) secretion was determined with ELISA. TC showed a dose-dependent stimulation of proliferation of LNCaP cells. Co-incubation with the anti-androgen flutamide (FLU) reversed the TC-induced stimulation of PSA secretion. The reference compound dihydrotestosterone (DHT) caused a significant increase of growth of LNCaP cells. Similarly, TC stimulated proliferation of these prostate cells. The anti-androgen FLU reversed the increase of PSA release caused by either DHT or TC. Thus, we suggest that TC may contain androgenic compounds, which appear to act via androgen receptor (AR).

Sesquilignans and dilignans from Campylotropis hirtella (Franch.) Schindl.

A new sesquilignan, erythro-Guaiacylglycerol-β-O-4′-(5′)-methoxylariciresinol, together with six known compounds including three sesquilignans and three dilignans, have been isolated from Campylotropis hirtella (Franch.) Schindl. that was used as traditional Chinese medicine for treatment of benign prostate hyperplasia. All these known compounds were found for the first time from the family leguminosae. The structure of the new compound and its relative configurations were elucidated on the basis of detailed spectroscopic analysis (NMR and MS). These compounds showed no obvious inhibition effect on prostate specific antigen secretion in LNCaP cells by ELISA method.

Enterolactone restricts the proliferation of the LNCaP human prostate cancer cell line in vitro

Ecological data suggest a long-term diet high in plant material rich in biologically active compounds, such as the lignans, can significantly influence the development of prostate cancer over the lifetime of an individual. The capacity of a pure mammalian lignan, enterolactone (ENL), to influence the proliferation of the LNCaP human prostate cancer cell line was investigated as a function of cell density, metabolic activity, expression and secretion of prostate specific antigen (PSA), cell cycle profile, and the expression of genes involved in development and progression of prostate cancer. Treatment with a subcytotoxic concentration of ENL (60 muM for 72 h) was found to reduce: cell density (57.5%, SD 7.23, p < 0.001), metabolic activity (55%, SD 0.03, p < 0.001), secretion of PSA (48.50% SD 4.74, p = 0.05) and induce apoptosis (8.33-fold SD 0.04, p = 0.001) compared to untreated cells. Cotreatment with 10 muM etoposide was found to increase apoptosis by 50.17% (SD 0.02, p < 0.001). Additionally, several key genes (e. g. MCMs, survivin and CDKs) were beneficially regulated by ENL treatment (p < 0.05). The data suggest that the antiproliferative activity of ENL is a consequence of altered expression of cell cycle associated genes and provides novel molecular evidence for the antiproliferative properties of a pure lignan in prostate cancer.

TRIM68 Regulates Ligand-Dependent Transcription of Androgen Receptor in Prostate Cancer Cells

The androgen receptor (AR) is a transcription factor belonging to the family of nuclear receptors that mediate the action of androgen. AR plays an important role in normal development of the prostate, as well as in the progression of prostate cancer. AR is regulated by several posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. In this study, we found that the putative E3 ubiquitin ligase TRIM68, which is preferentially expressed in prostate cancer cells, interacts with AR and enhances transcriptional activity of the AR in the presence of dihydrotestosterone. We also found that TRIM68 functionally interacts with TIP60 and p300, which act as coactivators of AR, and synergizes in the transactivation of AR. Overexpression of TRIM68 in prostate cancer cells caused an increase in secretion of prostate-specific antigen (PSA), one of the most reliable diagnostic markers for prostate cancer, whereas knockdown of TRIM68 attenuated the secretion of PSA and inhibited cell growth and colony-forming ability. Moreover, we showed that TRIM68 expression is significantly up-regulated in human prostate cancers compared with the expression in adjacent normal tissues. These results indicate that TRIM68 functions as a cofactor for AR-mediated transcription and is likely to be a novel diagnostic tool and a potentially therapeutic target for prostate cancer.

Leupaxin, a Novel Coactivator of the Androgen Receptor, Is Expressed in Prostate Cancer and Plays a Role in Adhesion and Invasion of Prostate Carcinoma Cells

In the present study, we demonstrate that leupaxin mRNA is overexpressed in prostate cancer (PCa) as compared with normal prostate tissue by using cDNA arrays and quantitative RT-PCR analyses. Moderate to strong expression of leupaxin protein was detected in approximately 22% of the PCa tissue sections analyzed, and leupaxin expression intensities were found to be significantly correlated with Gleason patterns/scores. In addition, different leupaxin expression levels were observed in PCa cell lines, and at the subcellular level, leupaxin was usually localized in focal adhesion sites. Furthermore, mutational analysis and transfection experiments of LNCaP cells using different green fluorescent protein-leupaxin constructs demonstrated that leupaxin contains functional nuclear export signals in its LD3 and LD4 motifs, thus shuttling between the cytoplasm and the nucleus. We could also demonstrate for the first time that leupaxin interacts with the androgen receptor in a ligand-dependent manner and serves as a transcriptional activator of this hormone receptor in PCa cells. Down-regulation of leupaxin expression using RNA interference in LNCaP cells resulted in a high rate of morphological changes, detachment, spontaneous apoptosis, and a reduction of prostate-specific antigen secretion. In contrast, knockdown of leupaxin expression in androgen-independent PC-3 and DU 145 cells induced a significant decrease of both the invasive capacity and motility. Our results therefore indicate that leupaxin could serve as a potential progression marker for a subset of PCa and may represent a novel coactivator of the androgen receptor. Leupaxin could function as a putative target for therapeutic interventions of a subset of advanced PCa.

Piper cubeba Targets Multiple Aspects of the Androgen-Signalling Pathway. A Potential Phytotherapy against Prostate Cancer Growth?

Despite the high prevalence of prostate cancer (PC) in the Western world, there is a dearth of effective medication. Since the androgen-signalling pathway is very much involved in PC growth and development, we investigated the potential of Piper cubeba L. extract, P9605, in targeting multiple events simultaneously within this pathway. This may be more effective compared to an antiandrogen monotherapy. Our results indicated that P9605 inhibited proliferation in androgen-dependent LNCaP human prostate cancer cells by reducing DNA synthesis and inducing apoptosis. This antigrowth effect was less pronounced in androgen-independent PC-3 prostate cancer cell lines. P9605 potently inhibited 5 alpha-reductase II activity, which is responsible for converting testosterone to its active form, dihydrotestosterone (DHT), in the prostate. It also acted as an antagonist at recombinant wild-type androgen receptors (AR). P9605 suppressed cell growth and prostate-specific antigen (PSA) secretion stimulated by physiological concentrations of DHT in LNCaP cells. Interestingly, it down-regulated AR levels. In conclusion, our findings suggest that P9605 may potentially retard the growth of androgen-dependent PC via several mechanisms.

Suppressor of cytokine signalling-3 is up-regulated by androgen in prostate cancer cell lines and inhibits androgen-mediated proliferation and secretion

Suppressors of cytokine signalling (SOCS) are induced by interleukins (ILs) and various peptide hormones and may prevent sustained activation of signalling pathways. We have previously shown that SOCS-3 antagonizes regulation of cellular events by cAMP and is expressed in human prostate cancer. To investigate possible effects of androgen on SOCS-3 protein expression, two prostate cancer cell lines (PC3-AR and LAPC4) were treated with different concentrations of R1881. Western blot analyses revealed induction of SOCS-3 protein expression in both cell lines by androgen, an effect which can be blocked by the anti-androgen bicalutamide. To further characterize the effects of R1881 on the SOCS-3 gene, promoter-reporter assay and real-time PCR were performed. We found no influence of androgen on promoter activity or SOCS-3 mRNA levels, thus suggesting a post-transcriptional effect of androgen. Concordant with our previous findings, we show a significant increase of SOCS-3 protein after androgen treatment in cells in which transcription was blocked, but not in those with impaired translation. In order to understand implications of SOCS-3 regulation by androgen, we used SOCS-3-negative LNCaP-IL-6 cells and stably transfected them with a tetracycline-responsive SOCS-3 Tet-On plasmid. We report that androgenic effects on cell proliferation and prostate-specific antigen secretion are significantly diminished following up-regulation of SOCS-3. In conclusion, androgen up-regulates SOCS-3 protein via post-transcriptional effects. SOCS-3 inhibits androgen-stimulated proliferation by influencing cell cycle regulation. Taken together with previous findings showing androgen receptor activation by IL-6, our results imply that androgen and cytokine signalling pathways interact at multiple levels in prostate cancer.

Wwox Suppresses Prostate Cancer Cell Growth through Modulation of ErbB2-Mediated Androgen Receptor Signaling

The expression of the WWOX tumor suppressor gene is lost or reduced in a large fraction of various cancers, including prostate cancer. We previously reported that Wwox overexpression induced apoptosis and suppressed prostate cancer growth in vitro and in vivo. In this study, pathways through which Wwox contributes to control of prostate cancer cell growth have been investigated. We found that Wwox interacts with Ap2gamma and prevents it from entering the nucleus to bind the ERBB2 promoter region to activate transcription of ERBB2, a mediator of androgen receptor activity and prostate cancer cell growth at limiting androgen concentration. Ectopic expression of Wwox reduced ErbB2 protein expression in vitro and expression of Wwox protein inversely correlated with expression of ErbB2 protein in prostate cancer tissues. Furthermore, Wwox suppressed Ap2gamma/ErbB2-induced prostate cancer cell growth and suppressed prostate-specific antigen secretion through interaction with Ap2gamma and down-modulation of ErbB2, an effect that required functional androgen receptor.

Ubiquitylation of ε-COP by PIRH2 and regulation of the secretion of PSA

Ubiquitylation appears to be involved in the membrane trafficking system including endocytosis, exocytosis, and ER-to-Golgi transport. We found that PIRH2, which was identified as an interacting protein for androgen receptor or p53, interacts with and ubiquitylates the epsilon-subunit of coatmer complex, epsilon-COP. PIRH2 promotes the ubiquitylation of epsilon-COP in vitro and in vivo and consequently promotes the degradation of epsilon-COP. The interaction between PIRH2 and epsilon-COP is affected by the presence of androgen, and PIRH2 in the presence of androgen promotes ubiquitylation of epsilon-COP in vivo. Furthermore, overexpression of the wild type of PIRH2 in prostate cancer cells causes downregulation of the secretion of prostate-specific antigen (PSA), a secretory protein in prostate epithelial cells and one of diagnostic markers for prostate cancer. Our results indicate that PIRH2 functions as a regulator for COP I complex.

Calcium/calmodulin-dependent kinase II plays an important role in prostate cancer cell survival

It has recently been shown that the androgen receptor (AR) is the main factor that required for prostate cancer cells survival. We show that knocking down AR expression by siRNA induces PI3K-independent activation of Akt, which was mediated by calcium/calmodulin-dependent kinase II (CaMKII). We further show, for the first time, that prostate cancer cells express β, γ, and δ CaMKII genes, and the expression of these genes is under the control of AR activity: active AR in the presence of androgens inhibits CaMKII gene expression whereas inhibition of AR activity results in elevated level of kinase activity and in enhanced expression of CaMKII-β and –γ genes. Overexpression of CaMKII genes results in resistance to apoptosis induced by KN-93, a CaMKII inhibitor, or wortmanninn, a PI3K/Akt inhibitor, in combination with doxorubicin, thapsigargin and TRAIL. Moreover, overexpression of CaMKII increases secretion of prostate specific antigen and promotes cell growth of LNCaP in steroid-free condition. Our data show that there is cross-talk between AR- and CaMKII-mediated pathways. The results of this study suggest that CaMKII is an important player in prostate cancer cells ability to escape apoptosis under androgen ablation and facilitate the progression of prostate cancer cells to an androgen-independent state.

Phytoestrogens from Belamcanda chinensis regulate the expression of steroid receptors and related cofactors in LNCaP prostate cancer cells

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. LNCaP cells were treated with 100, 400 or 1400 microg/mL BCE; proliferation was assessed with an Alamar Blue assay. We used real-time 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-beta (ER-beta) 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. 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-beta expression increased. Furthermore, AR protein and PSA secretion were markedly diminished after treatments with the BCE. 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-beta, a putative tumour-suppressor gene.

Petasiphenone, a Phenol Isolated from Cimicifuga Racemosa, in vitro Inhibits Proliferation of the Human Prostate Cancer Cell Line LNCaP

Extracts of Cimicifuga racemosa (L.) Nutt. (syn.: Actaea racemosa L.) (CR) inhibit the proliferation of the human prostate cancer cell line LNCaP. Recently, the phenylpropanoid ester 3,4-dihydroxyphenacyl caffeate (petasiphenone, 1) was isolated from CR. This substance is a structural homologue to petasiphenol ([3-(3,4-dihydroxyphenyl)-2-oxopropyl caffeate]), a compound produced by Petasites japonicus Sieb. & Zucc. which inhibits the growth of various human leukemia cell lines. Because of the structural similarity, we examined whether 1 affects the proliferation of LNCaP cells and the secretion of prostate-specific antigen (PSA). Under basal conditions as well as under co-incubation with 10 nM estradiol [E2 or 1 nM dihydrotestosterone (DHT)], 1 dose-dependently inhibited proliferation of LNCaP cells while PSA release per cell was not altered. We report for the first time that a defined compound isolated from CR inhibits the growth of the human prostate cancer cells LNCaP.

Differential effects of naturally occurring and synthetic organoselenium compounds on biomarkers in androgen responsive and androgen independent human prostate carcinoma cells

Epidemiological studies and clinical trials show that selenium supplementation results in reduction of prostate cancer incidence; however, the form of selenium and mechanisms underlying protection remain largely unknown. Toward this end, we compared the effects of naturally occurring selenomethionine (SM) and Se-methylselenocysteine (MSC) and synthetic 1,4-phenylenebis(methylene)selenocyanate (p-XSC) and p-xylylbis(methylselenide) p-XMS) organoselenium compounds in androgen responsive (AR) LNCaP and its androgen independent clone (AI) LNCaP C4-2 human prostate carcinoma cells on cell growth, secretion of prostate specific antigen (PSA), intracellular redox status and genomic profiles with emphasis on identifying redox sensitive genes. Both p-XSC and p-XMS reduced cell number and total protein concentration compared to control-treated AR and AI cells, while SM and MSC exhibited no effect on growth of AR and AI cells. SM, p-XSC and p-XMS but not MSC inhibited levels of secreted PSA in AR cells. SM, MSC and p-XMS increased glutathione (GSH) levels in AI LNCaP cells. By contrast, in both cell types, only p-XSC significantly decreased GSH concentrations to <50% of control suggesting either an increase in intracellular oxidative stress or a change in GSH/GSSG ratio. On the basis of RT-PCR analysis, SM and p-XSC increased p53 gene expression by 2-fold in AR cells but not in AI cells and only SM enhanced epidermal growth factor receptor in AR cells. Depending on the structure, organoselenium compounds exhibit differential effects on growth, PSA secretion, oxidative stress and selective gene responses in human prostate cancer cells and suggest the potential of developing novel organoselenium compounds as chemopreventive agents in models of human prostate cancer.

Down-regulation of prostate specific antigen in LNCaP cells by flavonoids from the pollen of Brassica napus L.

The pollen of Brassica napus L. has been used in China to treat benign prostatic hyperplasia (BPH) for over decades. In this study, the pollen of Brassica napus L. was extracted successively with chloroform, ethyl acetate and ethanol. The ethyl acetate extract showed strong activity in decreasing the secretion of prostate specific antigen (PSA) in LNCaP cells as compared to two other extracts, measured by ELISA with finasteride as positive control in the assay. Five flavonoids were subsequently isolated from the active extract using bioassay-guided fractionation. They were Naringenin (1); Luteolin (2); Kaempferol (3); Kaempferol 3-(3-E-p-coumaroyl-alpha-L-rhamnopyranoside) (4); and Kaempferol 3-(2,3-di-E-p-coumaroyl-alpha-L-rhamnopyranoside) (5). All these compounds inhibited PSA secretion significantly, with IC50 values in the range of 5-50 microM. Compounds 2, 4 and 5 showed moderate cytotoxicity to LNCaP cells within the active concentration range, while compounds 1 and 3 showed no cytotoxicity. Further studies on the mechanism action of these compounds were performed by evaluating their activation of estrogen receptor (ER) and antagonistic activities on androgen receptor (AR) in cell-based reporter gene assays. All compounds described here were first isolated from the pollen of Brassica napus L.

Prostate Specific Antigen Expression Does Not Necessarily Correlate With Prostate Cancer Cell Growth

The antiproliferative effects of pharmacological agents used for androgen ablative therapy in prostate cancer, including goserelin, bicalutamide and cyproterone acetate (Fluka Chemie, Buchs, Switzerland), were tested in vitro. It was determined whether they affected prostate specific antigen mRNA and protein expression independent of growth inhibition. Goserelin, bicalutamide (AstraZeneca, Zug, Switzerland) and cyproterone acetate were added to prostate specific antigen expressing, androgen dependent LNCaP and androgen independent C4-2 cell line (Urocor, Oklahoma City, Oklahoma) cultures. Proliferation was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assay (Roche, Mannheim, Germany). Prostate specific antigen mRNA expression was assessed by quantitative real-time polymerase chain reaction. Secreted prostate specific antigen protein levels were quantified by microparticle enzyme-immunoassay. Goserelin inhibited cell growth and prostate specific antigen protein secretion in LNCaP and C4-2 cells. Prostate specific antigen mRNA expression was not decreased. Bicalutamide did not affect cell growth or prostate specific antigen mRNA expression in LNCaP or C4-2 cells, although it significantly decreased prostate specific antigen protein secretion in LNCaP and to a lesser extent in C4-2 cells. Cyproterone acetate decreased the growth of C4-2 but not of LNCaP cells. It did not affect prostate specific antigen mRNA or protein expression in either cell line. Prostate specific antigen expression does not necessarily correlate with cell growth. Without a substantial effect on cell growth bicalutamide lowers prostate specific antigen synthesis, whereas cyproterone acetate decreases cell growth with no effect on prostate specific antigen secretion. Prostate specific antigen expression may be influenced by growth inhibition but also by altered mRNA and protein levels depending on the agent, its concentration and the cell line evaluated. For interpreting clinical trials prostate specific antigen is not necessarily a surrogate end point marker for a treatment effect on prostate cancer cell growth.

Androgen-Dependent Regulation of Her-2/neu in Prostate Cancer Cells

The mechanisms underlying the progression of prostate cancer to a state of resistance to hormone ablation remain poorly understood. Here, we have investigated the relationship between androgen receptor (AR) and Her-2/neu in prostate cancer cells. Overexpression of Her-2/neu (c-ErbB2) activates the AR pathway and confers a survival and growth advantage to prostate cancer cells in an androgen-deficient milieu. In vitro, the absence of androgens or AR blockade induced Her-2/neu protein expression and phosphorylation. In contrast, upon readministration of androgens, Her-2/neu mRNA, protein, and phosphorylation levels decreased linearly with increasing concentrations of dihydrotestosterone as LNCaP cells reentered the cell cycle. In vivo, induction of Her-2/neu by castration in orthotopically injected LNCaP cells resulted in a progressive increase in prostate-specific antigen secretion into the mouse serum, indicating that Her-2/neu-mediated, AR-dependent transcription occurs following castration and results in tumor cell growth. Finally, selection of LNCaP cells stably transfected with short hairpin RNA specific for AR resulted in Her-2/neu overexpression. Similarly, knockdown of Her-2/neu led to induction of AR. However, when Her-2/neu and AR were simultaneously targeted, we observed cell death, whereas surviving cells retained low level expression of Her-2/neu. Thus, induction and activation of Her-2/neu occurs in an androgen-depleted environment or as a result of AR inactivation, promoting ablation-resistant survival of prostate cancer cells. These data provide the biochemical rationale to target Her-2/neu in hormone-refractory prostate cancer.