Prostate-specific Antigen mRNA Levels Research Articles

Association of serum prostate-specific antigen (PSA) level and circulating tumor cell-based PSA mRNA in prostate cancer.

BackgroundProstate-specific antigen (PSA) is used for diagnosing prostate cancer, but does not reflect the characteristics of prostate cancer cells to allow assessment of cancer progression. PSA mRNA and circulating tumor cells (CTCs) could be potential biomarkers. However, the relationship between serum PSA levels and PSA mRNA in CTCs is unclear, and this study aimed to investigate this relationship.MethodsHealthy donors (HD, n = 9), and patients with local non-metastatic stage prostate cancer (n = 30), metastatic hormone–sensitive prostate cancer (mHSPC, n = 10), and metastatic castration–resistant prostate cancer (mCRPC, n = 75), were included. The expression of PSA mRNA in CTCs was measured by droplet digital PCR. Serum PSA (ng/mL) levels and PSA mRNA (copies/μL) in CTCs were then compared using Spearman correlation coefficients.ResultsPSA mRNA expression in CTCs was observed in 30% (9/30) of patients with localized cancer, 60.0% (6/10) among patients with mHSPC, 65.3% (49/75) among patients with mCRPC, and 0% among patients with HD, indicating that the detection rate of PSA mRNA increased with cancer stage. PSA mRNA expression in CTCs also increased from localized to metastatic stages. PSA mRNA levels rapidly increased in the mHSPC and mCRPC stages. Interestingly, PSA mRNA expression in CTCs was not correlated with serum PSA levels at the localized stage (R = 0.064, P = 0.512). However, there were significant correlations between serum PSA levels and PSA mRNA expression in mHSPC (R = 0.532, P = 0.041) and mCRPC (R = 0.566, P = 0.025). The number of CTCs isolated from mHSPC and mCRPC was not proportional to serum PSA and PSA mRNA levels.ConclusionCTC PSA mRNA has the potential to be used as a biomarker to complement serum PSA protein analysis or replace serum PSA in metastatic stages of prostate cancer.

Anti-androgenic effect of astaxanthin in LNCaP cells is mediated through the aryl hydrocarbon-androgen receptors cross talk.

The aim of this study was to investigate the anti-androgenic effects of astaxanthin (AST) on human prostatic cancer cell growth, and its impact on androgen receptor (AR) signaling using prostate cancer (PCa) cell line LNCaP. LNCaP cells were treated with AST alone and in combination with CH223191 and flutamide (Flu) in the presence and absence of testosterone. MTT assay, cellular prostate-specific antigen (PSA) and dihydrotestosterone (DHT) production, mRNA levels of CYP1A1, PSA, Kallikrein-Related Peptidase 2 (KLK2), Transmembrane Serine Protease 2 (TMPRSS2), and AR genes were measured as endpoints. The expression of CYP1A1, PSA, KLK2, TMPRSS2, and AR mRNA levels was decreased which results in reducing the production of PSA and DHT in the presence of testosterone. Our data clearly demonstrate that AST has a potential ability to suppress the human prostate LNCaP cells growth at high concentrations. AST was able to repress the testosterone-induced transcription of AR-target genes. PRACTICAL APPLICATIONS: Astaxanthin is a natural compound with the most potent antioxidant activity among other antioxidants. In the current study, ASX suppressed the LNCaP cells at high concentrations. Furthermore, AST inhibited testosterone-induced transcriptional activation of androgen-related genes. AST induced the expression of CYP1A1, which is able to metabolize the steroid hormones. It seems that AST can act as AhR exogenous ligand by induction of CYP1A1, which results in testosterone metabolism and consequent suppression of AR genes. So that, AST could prevent the growth of testosterone-dependent PCa cells, downregulate downstream genes in testosterone pathways, and enhance the metabolism of testosterone via AhR pathway. Collectively, AST could be considered as a potential candidate for the treatment of PCa.

Abstract 5378: A comparative analysis on the expression of PSA protein in serum and PSA mRNA in circulating tumor cells in prostate cancer

Abstract Introduction Unlike other cancers, prostate cancer has a distinguished biomarker for diagnosis and prognosis which is known as prostate specific antigen (PSA) protein in serum. In the early stage of prostate cancer, PSA protein in serum is highly expressed from primary tumor sites and thereby, the expression of PSA protein can be used in prognosis of prostate cancer during whole progression. However, due to low specificity of serum PSA protein, it raised concerns in over-diagnosis. Due to the advantages of noninvasive, real-time and pain-free for patients, circulating tumor cells (CTCs) is being researched as complementary alternative biomarkers for clinical and molecular analysis of cancer. Some studies reported that the levels of PSA protein and PSA mRNA in prostate cancer tissue are proportional to each other. Other studies showed that the expression level of PSA mRNA in peripheral blood and biochemical recurrence of PSA protein in serum are significant relationship. However, the relevance between PSA protein in serum and PSA mRNA in CTCs has not been clearly researched. In this study, we first report the relevance of the expression levels of PSA protein in serum and PSA mRNA in CTCs. Experimental method and analysis Prostate cancer patients were recruited from healthy donor (HD, n=5), local with non-metastatic stage (n=25), metastatic hormone sensitive prostate cancer (mHSPC, n=9) and metastatic castration-resistance prostate cancer (mCRPC, n=28). CTCs were isolated by high efficiency and purity using microfluidic technology based on lateral magnetophoresis, developed in our laboratory. Total mRNA was extracted from isolated CTC and the expression level of PSA mRNA in CTCs was measured by droplet digital PCR (ddPCR). Then, the expression levels of PSA protein (ng/ml) in serum and PSA mRNA (copies/µl) in CTCs were compared by statistical analysis. Results and discussion The expression of PSA mRNA in CTCs was observed in 40% of localized cancer, 55.56% of mHSPC, 89.29% of mCRPC patients and 0% of HD group. It indicates that the detection rate of PSA mRNA increases with stage of prostate cancer. The expression level of PSA mRNA in CTCs also increased from localized to metastatic stages. Interestingly, it increased very rapidly in mHSPC and mCRPC stages. In 7 of the blood samples of localized stage, the expression levels of PSA mRNA in CTCs were detected and they were not related to serum PSA protein. In mHSPC and mCRPC stage, the expression levels of PSA mRNA in CTCs were respectively detected in 5 and 25 samples, implicating that their expression levels are significantly related to the level of PSA protein in serum. Conclusions This result shows that the expression level of PSA mRNA in CTCs could be used as a new marker along with serum PSA protein in metastatic stages of prostate cancer, and that high-quality CTCs can be used as complementary alternative biomarkers for precision medicine in prostate cancer. Citation Format: Hyungseok Cho, Ki-Ho Han, Jae-Seung Chung. A comparative analysis on the expression of PSA protein in serum and PSA mRNA in circulating tumor cells in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5378.

Cell-based evidence regarding the role of FSH in prostate cancer

IntroductionConversion of androgen-responsive prostate cancer (CaP) to castration-resistant CaP is associated with an acceleration of the disease that often requires treatment modalities other than androgen deprivation therapy only. Recently, follicle-stimulating hormone (FSH) has been shown to play a role in CaP growth, and clinical data showed that high serum concentration of FSH in chemically castrated CaP patients was associated with a shorter time of progression to castration-resistant CaP. In this study, we sought to investigate if FSH could have direct effects on CaP cells, possibly through the androgen receptor and androgen receptor regulated genes, such as prostate-specific antigen (PSA). Materials and methodsThe human CaP cell lines PC-3, LNCaP and C4-2, and nonmalignant PNT1A cells, were utilized to investigate the effects of FSH. qPCR, Western blotting analysis, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymetoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium assays were performed in order to analyze the FSH effects. ResultsThe FSH receptor was present in all cell lines except PNT1A. FSH significantly increased PSA mRNA (P < 0.01) and protein (P < 0.03) levels in C4-2 cells in a dose-dependent manner. In LNCaP cells, FSH also increased PSA protein level, although to a lesser extent than in C4-2 cells, and the expression was reduced by the antiandrogen enzalutamide. In PC-3 cells, FSH was shown to increase their proliferation (P < 0.03) and β-catenin expression. ConclusionThese findings demonstrate that FSH may have a direct effect in CaP in an androgen-depleted environment. However, further research is needed to understand the significance of direct FSH action in the maintenance of CaP growth at the different phases of transition from androgen dependence to androgen independence.

Abstract 2990: Sulforaphane enhances the anti-cancer efficacy of anti-androgens in prostate cancer cells (LNCaP and C4-2B) by increasing androgen receptor (AR) degradation

Abstract Introduction: Prostate Cancer (PC) cells utilize androgen for their growth. Therefore, androgen deprivation therapy (ADT) that blocks both systemic androgen production and androgen receptor (AR) signaling remains the mainstay of PC treatment. However, despite the efficacy of AR antagonists such as bicalutamide (BIC) and enzalutamide (ENZ), their sub-therapeutic efficacy in tumor microenvironments enable the selection and outgrowth of castration resistant PC (CRPC). Interestingly, CRPC cells continue to express AR and exploit the intratumoral androgen. Hence, alternate strategies to suppress AR expression in PC cells will be needed to enhance the efficacy of BIC and ENZ. The phytochemical, sulforaphane (SFN) is known to decrease AR protein levels. We hypothesize that co-exposure to SFN will increase the anti-cancer efficacy of BIC/ENZ. Methods: The AR expressing PC lines, both androgen-dependent (AD) LNCaP cells and androgen-independent CRPC cells (C4-2B), were used to investigate the effects of BIC or ENZ, alone and in combination with SFN. The following cellular parameters were evaluated: (a) cell proliferation by MTT-assay; (b) clonogenic ability by colony forming unit (CFU); (c) AR expression by immunoblot analysis; (d) subcellular AR localization by immunofluorescence microscropy (IFM); (e) AR and prostate specific antigen (PSA) gene expression by qRT-PCR; and (e) PSA protein secretion by ELISA. Studies were carried out under hormone deprived (charcoal-stripped FBS) conditions and/or in presence of AR agonist, R1881. Results: Co-exposure to SFN (10μM) significantly (p&amp;lt;0.05) enhanced the anti-proliferative effects of BIC (50μM) or ENZ (20μM) at 48 h post treatment, and suppressed R1881 stimulated PC growth, as well. At much lower concentrations, SFN (0.2μM) co-exposure enhanced the suppressive effects of BIC (0.5μM) or ENZ (0.2μM) on both the number and size of CFUs at 14 days. Western blot analysis revealed that SFN decreases AR protein levels in a time- and dose-dependent manner. IFM analysis indicated that SFN suppresses R1881-stimulated AR nuclear localization. Cycloheximide treatment suggested that SFN's effect on AR is primarily regulated at the translational level. Experiments with MG-132, a proteasomal inhibitor indicated that the SFN-induced AR degradation is partially regulated by the proteasomal pathway. Studies with qRT-PCR showed that SFN enhances the efficacy of BIC in suppressing PSA mRNA levels and decreased PSA protein secretion was also corroborated by ELISA. Conclusion: Sulforaphane decreases AR levels in both LNCaP and C4-2B cells to increase the anti-cancer efficacy of AR antagonists in a synergistic manner. This implies that adjuvant therapy with this safe phytochemical may be very promising. Citation Format: Namrata Khurana, Sudha Talwar, Partha Chandra, Asim B. Abdel-Mageed, Debasis Mondal, Suresh Sikka. Sulforaphane enhances the anti-cancer efficacy of anti-androgens in prostate cancer cells (LNCaP and C4-2B) by increasing androgen receptor (AR) degradation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2990.

Systems Pharmacology Modeling of Prostate-Specific Antigen in Patients With Prostate Cancer Treated With an Androgen Receptor Antagonist and Down-Regulator.

First‐in‐human (FIH) studies with AZD3514, a selective androgen receptor (AR) down‐regulator, showed decreases of >30% in the prostate‐specific antigen (PSA) in some patients. A modeling approach was adopted to understand these observations and define the optimum clinical use hypothesis for AZD3514 for clinical testing. Initial empirical modeling showed that only baseline PSA correlated significantly with this biological response, whereas drug concentration did not. To identify the mechanistic cause of this observation, a mechanism‐based model was first developed, which described the effects of AZD3514 on AR protein and PSA mRNA levels in LNCaP cells with and without dihydrotestosterone (DHT). Second, the mechanism‐based model was linked to a population pharmacokinetic (PK) model; PSA effects of clinical doses were subsequently simulated under different clinical conditions. This model was used to adjust the design of the ongoing clinical FIH study and direct the backup program.

P,p′-Dichlorodiphenyltrichloroethane (p,p′-DDT) and p,p′-dichlorodiphenyldichloroethylene (p,p′-DDE) repress prostate specific antigen levels in human prostate cancer cell lines

Despite stringent restrictions on their use by many countries since the 1970s, the endocrine disrupting chemicals, DDT and DDE are still ubiquitous in the environment. However, little attention has been directed to p,p′-DDT and the anti-androgen, p,p′-DDE on androgen receptor (AR) target gene transcription in human cells. Inhibitors of androgenic activity may have a deleterious clinical outcome in prostate cancer screens and progression, therefore we determined whether environmentally relevant concentrations of p,p′-DDT and p,p′-DDE negatively impact AR-regulated expression of prostate-specific antigen (PSA), and other AR target genes in human LNCaP and VCaP prostate cancer cells. Quantitative real-time PCR and immuno-blotting techniques were used to measure intracellular PSA, PSMA and AR mRNA and protein levels. We have shown for the first time that p,p′-DDT and p,p′-DDE repressed R1881-inducible PSA mRNA and protein levels in a dose-dependent manner. Additionally, we used the fully automated COBAS PSA detection system to determine that extracellular PSA levels were also significantly repressed. These chemicals achieve this by blocking the recruitment of AR to the PSA promoter region at 10μM, as demonstrated by the chromatin immunoprecipitation (ChIP) in LNCaP cells. Both p,p′-DDT and p,p′-DDE repressed R1881-inducible AR protein accumulation at 10μM. Thus, we conclude that men who have been exposed to either DDT or DDE may produce a false-negative PSA test when screening for prostate cancer, resulting in an inaccurate clinical diagnosis. More importantly, prolonged exposure to these anti-androgens may mimic androgen ablation therapy in individuals with prostate cancer, thus exacerbating the condition by inadvertently forcing adaptation to this stress early in the disease.

Prostate cancer stem cells: molecular characterization for targeted therapy

Prostate cancer stem cells: molecular characterization for targeted therapy

Abstract 941: Suppression of the androgen receptor function by c-Jun in prostate cancer via a dual mechanism

Abstract Androgen receptor (AR) signaling plays a pivotal role in growth and survival of prostate cancer cells. Upon ligand binding, AR translocates into the nucleus and regulates expression of multiple genes through binding their promoter regions. Thus, altered expression and activity of AR contribute to disease progression. Consequently, significant effort has been undertaken to identify and characterize numerous coactivators and corepressors of AR. Here we report that the transcription factor c-Jun, a basic region leucine zipper (bZIP) transcription factor and is an important member of the AP-1 family, acts as a transcriptional repressor to inhibit the AR transcriptional activity. Furthermore, we have found that c-Jun also represses the transcription of AR. Overexpression of c-Jun in LNCaP prostate cancer cells inhibited luciferase reporters driven by the androgen-induced prostate-specific antigen (PSA) promoter, probasin promoter, mouse mammary tumor virus (MMTV) promoter and tandem repeats of androgen response elements in a dose-dependent manner. Conversely, knockdown of c-Jun enhanced androgen-stimulated AR activity in LNCaP cells. To further confirm the results, we generated a doxycycline-inducible c-Jun overexpression LNCaP stable cell line using lentivrial system. Consistent with the results of promoter activity assay, induction of c-Jun by doxycycline inhibited both steady state and androgen-induced PSA mRNA and protein levels. Interestingly, the protein and mRNA of AR were also downregulated when c-Jun was induced, suggesting that c-Jun suppresses AR expression at the transcriptional level. Moreover, c-Jun induction resulted in a significant reduction in cell number, suggesting that c-Jun suppresses LNCaP cell proliferation by antagonizing AR signaling. In summary, our results suggest that c-Jun negatively regulates the AR signaling by a dual mechanism. Future identification of negative regulators of c-Jun expression will provide a new opportunity to develop novel therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 941. doi:1538-7445.AM2012-941

Antiandrogenic and growth inhibitory effects of ring‐substituted analogs of 3,3′‐diindolylmethane (Ring‐DIMs) in hormone‐responsive LNCaP human prostate cancer cells

Cruciferous vegetables protect against prostate cancer. Indole-3-carbinol (I3C) and its major metabolite 3,3'-diindolylmethane (DIM), exhibit antitumor activities in vitro and in vivo. Several synthetic ring-substituted dihaloDIMs (ring-DIMs) appear to have increased anticancer activity. Inhibition of LNCaP prostate cancer cell growth was measured by a WST-1 cell viability assay. Cytoplasmic and nuclear proteins were analyzed by immunoblotting and immunofluorescence. Androgen receptor (AR) activation was assessed by measuring prostate-specific antigen (PSA) expression and using LNCaP cells containing human AR and an AR-dependent probasin promoter-green fluorescent protein (GFP) construct. Like DIM, several ring-substituted dihaloDIM analogs, namely 4,4'-dibromo-, 4,4'-dichloro-, 7,7'-dibromo-, and 7,7'-dichloroDIM, significantly inhibited DHT-stimulated growth of LNCaP cells at concentrations ≥1 µM. We observed structure-dependent differences for the effects of the ring-DIMs on AR expression, nuclear AR accumulation and PSA levels in LNCaP cells after 24 hr. Both 4,4'- and 7,7'-dibromoDIM decreased AR protein and mRNA levels, whereas 4,4'- and 7,7'-dichloroDIM had minimal effect. All four dihaloDIMs (10 and 30 µM) significantly decreased PSA protein and mRNA levels. Immuofluorescence studies showed that only the dibromoDIMs increased nuclear localization of AR. All ring-DIMs caused a concentration-dependent decrease in fluorescence induced by the synthetic androgen R1881 in LNCaP cells transfected with wild-type human AR and an androgen-responsive probasin promoter-GFP gene construct, with potencies up to 10-fold greater than that of DIM. The antiandrogenic effects of ring-DIMs suggest they may form the basis for the development of novel agents against hormone-sensitive prostate cancer, alone or in combination with other drugs.

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.

PCA3 Urine mRNA Testing for Prostate Carcinoma: Patterns of Use by Community Urologists and Assay Performance in Reference Laboratory Setting

Multiple trials have shown the high specificity of urine prostate cancer gene 3 (PCA3) compared with serum prostate-specific antigen (PSA) for biopsy detection of prostate carcinoma. We characterized the patterns of use of PCA3 by community urologists and determined the performance of PCA3 testing as a laboratory-developed test in a reference laboratory setting. The urine PCA3 and PSA mRNA levels after digital rectal examination were determined using transcription-mediated amplification. The cutoff for a positive PCA3 score (PCA3/PSA mRNA x 10(3)) were pre-established at > or = 35. The PCA3 results were correlated with the serum PSA level, previous biopsy history, and the prostate biopsy findings. A total of 278 PCA3 tests were performed from December 2006 to June 2007. Of the PCA3 tested patients, 55.5% had previously undergone > or = 1 prostate biopsy; 92.7% had a PSA level > or = 2.5 ng/mL. The PCA3 test informative rate was 97.5%. For 50 samples that were also analyzed at a separate laboratory, concordance was achieved in 94%. The mean and median PCA3 score was 44.3 and 21.1, respectively. No correlation was found with the serum PSA level. The PCA3 test was negative in 16 of 19 patients with negative concurrent biopsy findings and positive in 8 of 11 with positive concurrent biopsy findings (sensitivity 72.7% and specificity 84.2%). Of 32 patients (70% with previous biopsy) who had undergone biopsy an average of 56 days after positive PCA3 test results, prostate carcinoma was detected in 41%. Urine PCA3 testing on the transcription-mediated amplification platform performed well as a laboratory-developed test. The high specificity of PCA3 was confirmed. In patients with elevated PSA levels and negative biopsy findings, PCA3 testing might be useful in choosing between repeat biopsy and more conservative follow-up.

Quantification of PSA mRNA levels in peripheral blood of patients with localized prostate adenocarcinoma before, during, and after radical prostatectomy by quantitative real-time PCR (qRT-PCR)

To evaluate the clinical value of detected prostate-specific antigen (PSA) mRNA expression levels in circulating cells in patients with prostate cancer by quantitative real-time polymerase chain reaction (qRT-PCR) and to determine the effect of surgical manipulation on hematogenous dissemination. Blood samples before, during, and after (24 h) surgery were obtained from 34 patients with prostate cancer who underwent radical prostatectomy for detecting PSA mRNA expression levels. PSA mRNA expression levels, PSA levels, and Gleason scores were then compared. PSA mRNA expression levels were detected in 14 of 34 (41%) patients, and biochemical recurrence of blood PSA was observed in 6 of 34 (17%) patients. Differences of PSA mRNA expression levels in pre-, per-, and postoperative periods were statistically significant (P < 0.001) and differences of PSA mRNA expression levels in pre- and peroperative samples (P < 0.008), pre- and postoperative samples (P < 0.031), and per- and postoperative samples (P < 0.001) were also statistically meaningful. There was significant relationship between the detectable PSA mRNA expression levels in preoperative samples and biochemical recurrence of blood PSA levels. We demonstrated that surgical manipulation had no effect on biochemical recurrence of blood PSA. We also showed that there was significant correlation between high Gleason score and the detectable PSA mRNA expression levels (P < 0.027). Our results suggest that highly detectable PSA mRNA expression levels in preoperative samples seem to be a significant predictable factor for prostate cancer recurrence.

Methyl 2-Cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate Is a Peroxisome Proliferator-Activated Receptor-γ Agonist That Induces Receptor-Independent Apoptosis in LNCaP Prostate Cancer Cells

Methyl 2-cyano-3,11-dioxo-18beta-olean-1,12-diene-30-oate (beta-CDODA-Me) is a synthetic analog of the naturally occurring triterpenoid glycyrrhetinic acid, which contains a 2-cyano substituent in the A-ring. beta-CDODA-Me was a potent inhibitor of LNCaP prostate cancer cell growth (IC(50) approximately 1 muM) and activated peroxisome proliferator-activated receptor gamma (PPARgamma), whereas analogs without the cyano group were weakly cytotoxic and did not activate PPARgamma. beta-CDODA-Me induced p21 and p27, down-regulated cyclin D1 protein expression, and induced two other proapoptotic proteins, namely nonsteroidal anti-inflammatory drug-activated gene-1 and activating transcription factor-3. However, induction of these responses by beta-CDODA-Me was PPARgamma-independent and due to activation of phosphatidylinositol-3-kinase, mitogen-activated protein kinase, and jun N-terminal kinase pathways by this compound. In contrast, beta-CDODA-Me also decreased androgen receptor (AR) and prostate-specific antigen (PSA) mRNA and protein levels through kinase-independent pathways. beta-CDODA-Me repressed AR mRNA transcription, whereas decreased PSA mRNA levels were dependent on protein synthesis and were reversed by cycloheximide. Thus, potent inhibition of LNCaP cell survival by beta-CDODA-Me is due to PPARgamma-independent activation of multiple pathways that selectively activate growth-inhibitory and proapoptotic responses.

Augmented suppression of androgen receptor signaling by a combination of α‐tocopheryl succinate and methylseleninic acid

Previous reports showed that alpha-tocopheryl succinate (alphaTS) and methylseleninic acid (MSA) independently reduce the abundance of androgen receptor (AR) in prostate cancer cells. The response to MSA happens quickly, whereas the response to alphaTS takes much longer. The present study was designed to investigate whether a combination of alphaTS and MSA would produce an additive or a greater than additive effect in suppressing AR level, AR transactivation, and prostate-specific antigen (PSA). LNCaP cells were treated with alphaTS alone for 31 hours, MSA alone for 3 hours, or alphaTS first for 28 hours and alphaTS/MSA together for the last 3 hours. AR and PSA mRNA levels were quantitated by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). AR transactivation was determined by the ARE-luciferase reporter assay. Both cellular and secretory PSA was also measured by the enzyme-linked immunosorbent assay (ELISA) method. Different doses of alphaTS were evaluated in combination with MSA. Some striking results are highlighted below for alphaTS alone, MSA alone, or alphaTS/MSA (presented in that order). AR mRNA level was depressed by 0%, 20%, or 60%, respectively; AR transactivation was inhibited by 35%, 10%, or 60%, respectively; whereas the PSA mRNA level was decreased by 40%, 60%, or 90%, respectively. Interestingly, secretory PSA was consistently reduced to a greater extent than cellular PSA. A combination of alphaTS/MSA produced a greater than additive effect in suppressing AR signaling compared with the single agent. Decreased AR abundance is a major factor, but not necessarily the sole factor, in diminishing the transcriptional activity of AR by alphaTS or MSA.

Vav3, a Rho GTPase Guanine Nucleotide Exchange Factor, Increases during Progression to Androgen Independence in Prostate Cancer Cells and Potentiates Androgen Receptor Transcriptional Activity

The progression of prostate cancer from androgen dependence to androgen independence is often accompanied by enhanced androgen receptor (AR) transcriptional activity. We observed a marked increase in the expression of Vav3, a Rho GTPase guanine nucleotide exchange factor (GEF), during the progression of human prostate cancer LNCaP cells to the androgen-independent derivative, LNCaP-R1. GEFs activate Rho family GTPases by promoting the exchange of GDP for GTP. Reporter gene assays showed that Vav3 potentiated AR transcriptional activity, and knock down of Vav3 resulted in decreased AR transactivation. Vav3 also increased androgen-induced levels of prostate-specific antigen mRNA. Furthermore, Vav3 enhanced AR activity at subnanomolar concentrations of androgen. This finding is particularly relevant because low androgen levels may be present in prostate tissue of patients undergoing androgen deprivation therapy. Enhancement of AR activity by Vav3 required amino terminal activation function 1 (AF1) of AR; however, Vav3 did not interact with AR or increase AR levels. Neither GEF function nor the C-terminal domains of Vav3 were required for Vav3-mediated enhancement of AR activity; however, the pleckstrin homology domain was obligatory. These data show that Vav3 levels rise during progression to androgen independence and support continued AR signaling (even under conditions of low androgen) by a novel GEF-independent cross-talk mechanism.

The Magnitude of Early Castration-Induced Primary Tumour Regression in Prostate Cancer Does Not Predict Clinical Outcome

IntroductionThis study was designed to test whether early castration-induced short-term cellular changes in primary prostate tumours could predict clinical outcome in advanced disease. Patients and methodsBiopsies from 83 patients obtained before and within two weeks after surgical castration were investigated. Tumour epithelial cell apoptosis, proliferation, and prostate specific antigen (PSA) levels were quantified using immunohistochemistry, laser capture micro-dissection, and real time RT-PCR. Cellular effects were related to changes in serum PSA levels and clinical outcome. ResultsDecreased proliferation and PSA mRNA levels, and increased apoptosis were observed in most tumours. These early cellular responses were not correlated to each other and did not predict serum PSA response or cancer-specific survival. A nadir PSA level below 1ng/ml predicted a longer cancer-specific survival after castration therapy. ConclusionCastration therapy causes primary tumour regression in most patients with advanced prostate cancer, but these primary tumour effects are not predictive for systemic disease control. Studies of early changes in metastases during hormonal therapy will probably give more predictive information for clinical outcome than further studies in primary tumours.

Genistein affects androgen‐responsive genes through both androgen‐ and estrogen‐induced signaling pathways

This study examined the mechanisms by which the prostate cancer chemopreventive agent genistein modulates gene expression in LNCaP human prostate cancer cells. Expression of androgen- and estrogen-regulated genes was measured in LNCaP cells cultured in the presence or absence of hormonal stimulation and the presence or absence of genistein. Genistein strongly suppressed basal expression of androgen-responsive gene (ARG) mRNAs, including prostate-specific antigen (PSA) and Ste20-related proline-alanine-rich kinase (SPAK). However, genistein had little or no effect on basal expression of two other ARGs, beta2-microglobulin (B2M) or selenoprotein P (SEPP1). Culturing LNCaP cells in the presence of the synthetic androgen R1881-induced increases in PSA, SPAK, B2M, and SEPP1 mRNA levels. The R1881-induced expression of these genes was uniformly blocked by genistein. For PSA and SPAK, genistein also blocked or downregulated 17beta-estradiol-induced increases in mRNA expression. These results indicate that genistein selectively alters expression of ARG mRNAs in LNCaP cells through modulation of both androgen- and estrogen-induced signaling pathways.

Stress Kinase Signaling Regulates Androgen Receptor Phosphorylation, Transcription, and Localization

Activation of signal transduction kinase cascades is known to alter androgen receptor (AR) activity, but the molecular mechanisms are still poorly defined. Here we show that stress kinase signaling regulates Ser 650 phosphorylation and AR nuclear export. In LNCaP prostate cancer cells, activation of either MAPK kinase (MKK) 4:c-Jun N-terminal kinase (JNK) or MKK6:p38 signaling pathways increased Ser 650 phosphorylation, whereas pharmacologic inhibition of JNK or p38 signaling led to a reduction of AR Ser 650 phosphorylation. Both p38alpha and JNK1 phosphorylated Ser 650 in vitro. Small interfering RNA-mediated knockdown of either MKK4 or MKK6 increased endogenous prostate-specific antigen (PSA) transcript levels, and this increase was blocked by either bicalutamide or AR small interfering RNA. Stress kinase inhibition of PSA transcription is, therefore, dependent on the AR. Similar experiments involving either activation or inhibition of MAPK/ERK kinase:ERK signaling had little effect on Ser 650 phosphorylation or PSA mRNA levels. Ser 650 is proximal to the DNA binding domain that contains a nuclear export signal. Mutation of Ser 650 to alanine reduced nuclear export of the AR, whereas mutation of Ser 650 to the phosphomimetic amino acid aspartate restored AR nuclear export. Pharmacologic inhibition of stress kinase signaling reduced wild-type AR nuclear export equivalent to the S650A mutant without affecting nuclear export of the S650D mutant. Our data suggest that stress kinase signaling and nuclear export regulate AR transcriptional activity.

The Use of Real-Time Reverse Transcription-PCR for Prostate-Specific Antigen mRNA to Discriminate between Blood Samples from Healthy Volunteers and from Patients with Metastatic Prostate Cancer

A clinical role for nonquantitative reverse transcription-PCR (RT-PCR) using prostate-specific antigen in blood samples from patients with prostate cancer remains undefined. Assay variation and detection of prostate-specific antigen mRNA illegitimate transcription may explain inconsistent results between studies. Defining levels of prostate-specific antigen mRNA expression in blood samples from healthy volunteers and patients with prostate cancer would allow cutoffs to be established to distinguish the two groups. Quantitative real-time RT-PCR for prostate-specific antigen mRNA was established and levels of prostate-specific antigen mRNA measured in bloods samples from healthy volunteers (n=21) and patients with localized (n=27) and metastatic (n=40) prostate cancer. Levels of prostate-specific antigen mRNA were significantly higher in blood samples from patients with metastatic prostate cancer than in blood samples from patients with localized prostate cancer (P <0.001) or in blood samples from healthy volunteers (P <0.01); levels between patients with localized prostate cancer and healthy volunteers were no different. Assay sensitivity to detect patients with metastatic prostate cancer was 68% with specificity of 95%. In patients with newly diagnosed metastatic prostate cancer, monitoring response to hormonal therapy was possible with this assay. No correlation between levels of prostate-specific antigen mRNA and serum prostate-specific antigen protein levels was found, suggesting that prostate-specific antigen mRNA and serum prostate-specific antigen protein levels reflect different features of prostate cancer, i.e., circulating tumor cells and total tumor bulk, respectively. Quantitative RT-PCR discriminates patients with metastatic prostate cancer from healthy volunteers and patients with localized prostate cancer but cannot discriminate patients with localized prostate cancer from healthy volunteers. A role for quantitative RT-PCR has been identified in the assessment and monitoring of patients with metastatic prostate cancer.