Prostate-specific Antigen mRNA Expression 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.

Epigallocatechin-3-gallate Enhances the Efficacy of MicroRNA-34a Mimic and MiR-93 Inhibitor Co-transfection in Prostate Cancer Cell Line

The potential role of microRNAs (miRNA) as therapeutic molecules has moved them from basic research to the field of cancer therapy. High expression of miR-93 and low expression of miR-34a have previously been indicated in prostate cancer (PC), which is the second leading cause of cancer-related death in men. Androgen receptor (AR) and prostate-specific antigen (PSA) play key roles in the initiation and progression of this cancer. Therefore, this study aimed to investigate the effects of the transfection and co-transfection of miR-34a mimic and miR-93 inhibitor with or without epigallocatechin-3-gallate (EGCG) on prostate cancer cell line and also to evaluate their effects on the expression of AR, PSA.
 Human lymph node carcinoma of the prostate (LNCaP) cells were treated with miR-34a mimic or/and miR-93 inhibitor with or without EGCG. Gene or protein expressions were assessed by real-time PCR or western blotting of lysates.
 The transfection with miR-34a mimics significantly reduced the mRNA expression of AR (p=0.0016), and PSA (p=0.038) compared to the control. Also, the miR-93 inhibitor led to a decrease in the mRNA expression of AR (p=0.0057) and PSA (p>0.05) compared to the control group. Furthermore, the co-transfection, along with EGCG, caused more decrease in both the AR (p<0.001) and the PSA (p=0.003) expression compared with the co-transfection without EGCG.
 Our study indicates that the reduced expression of AR and PSA in PC cells followed by treatment with miR-34a mimic and miR-93 inhibitor and their combination with EGCG as a natural substance may be a promising therapeutic way for controlling the growth of these malignant cells.

Estrogenic and antiandrogenic activities of methoxyflavans isolated from the fruit of Mauritia Flexuosa (Moriche Palm).

Moriche palm is consumed as both a fresh fruit and processed food in Peru and Brazil. Although its fruit contains phytoestrogens, the active compounds have not yet been identified. Therefore, we purified moriche palm extract (MPE) and identified compounds exhibiting estrogenic and antiandrogenic activities. Estrogenic activity was assessed by the estrogen-dependent growth of MCF-7 cells and increases in uterine weights in mice. Antiandrogenic activity was evaluated by 5α-reductase inhibitory activity and prostate-specific antigen (PSA) expression in LNCaP cells. In vivo antiestrogenic activity was also assessed based on testosterone-induced prostate growth in castrated mice. Four methoxyflavans were isolated from MPE and all, except for 7,4'-dihydroxy-5-methoxyflavan, promoted MCF-7 cell growth, indicating estrogenic activity. Uterine and ovary weights increased in mice orally administered MPE (400mg/kg) for 2weeks. Regarding antiandrogenic activity, among the four methoxyflavans isolated, 6,7,4'-trihydroxy-5-methoxyflavan (1 µg/ml) suppressed the mRNA and protein expression of PSA in LNCaP cells. Furthermore, prostate growth was suppressed in mice orally administered MPE (200mg/kg) for 2weeks. All methoxyflavans inhibited 5α-reductase activity with IC50 less than 10 µg/ml. Collectively, the present results demonstrated that orally administered MPE exhibited estrogenic and antiandrogenic activities. Methoxyflavans, particularly 6,7,4'-trihydroxy-5-methoxyflavan, appear to be the active compounds for these activities. PRACTICAL APPLICATIONS: The fruit of Mauritia flexuosa (moriche palm) has been used for beverages and processed foods. Although it is said to contain phytoestrogens, the active compounds have not yet been identified. In this study, we isolated and identified methoxyflavans exhibiting estrogenic and antiandrogenic activities. Among them, 6,7,4'-trihydroxy-5-methoxyflavan appeared to be the most effective compounds for these activities.

Prostatic aspirated cellular RNA analysis enables fast diagnosis and staging of prostate cancer

ObjectiveThe aim of this study is to investigate the potential application of prostatic aspirated cellular RNA analysis technique for fast diagnosing and staging prostate cancer. MethodsProstatic aspirated cells were obtained immediately after transrectal ultrasound (TRUS)-guided needle biopsy. Cellular RNA such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, prostate specific antigen (PSA) mRNA and prostate-specific RNA (PCA3) mRNA were analyzed by using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). PCA3 score was calculated as the ratio of PCA3 mRNA to PSA mRNA expression. Diagnostic performance of the fast-aspirated cellular RNA analysis technique for determining prostate cancer and metastatic status were evaluated by developing receiver operating characteristic curves (ROC), and the correlation between aspirated cellular RNA mRNA expressions and risk grouping was calculated, to investigate the underlying potential for PCa staging. ResultsPCA3 score was significantly higher in prostatic aspirated cells obtained from cancerous tissues than noncancerous tissues. The median aspirated cellular PCA3 score was higher in patients with PCa compared to BPH, and presenting an area under the ROC curve (AUC) of 0.87 (95%CI: 0.79–0.94) for PCa diagnosis. Multivariate regression analysis revealed that baseline median aspirated cellular PCA3 score (OR=9.316, 95%CI: 1.045–83.033, P<0.05) was an independent predictive factor for metastatic status in PCa patients. ConclusionThe ease of use and minimal complexity of fast prostatic aspirated cellular RNA analysis may be a valuable diagnostic technique, providing urgent diagnostic information for accurate PCa diagnosing and staging.

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.

PSA secretion and cell proliferation are affected by NCoR1 silencing in prostate cancer cells

The androgen receptor (AR) plays an important role in normal prostate gland development as well as in prostatic neoplasms. Transcriptional regulation by AR is modulated by its interaction with co-activators or co-repressors, such as NCoR1 (nuclear receptor co-repressor 1), which is involved in the reduction of AR activity over the target gene transcription. To identify the role of NCoR1 in the prostate cancer androgen independence in a cell line model, we aimed to evaluate the effects of silencing NCoR1 on prostate-specific antigen ( PSA) gene expression, the proliferative response and PSA secretion on the supernatant of C4-2B and LNCaP cells that were submitted to small interfering RNAs (siRNAs) transfection and to treatments with different androgen dosages. In LNCaP and C4-2B cells with no dihydrotestosterone (DHT) treatment, a decrease of PSA mRNA expression was observed 48 hours and 72 hours after gene silencing in the siNCoR group when compared to the control and siNC groups. LNCaP and C4-2B cells showed a biphasic pattern in response to dihydrotestosterone treatment in transfected groups (siNCoR and siNC) as well as in the control condition (no transfection). The secretion of PSA in cell supernatant of LNCaP and C4-2B cells was higher in the siNCoR group, and, regarding the hormonal treatment, higher in the 10 -8 M DHT group. A reduction in the levels of NCoR1 seems to have a double influence on the activity of AR in PCa cells. These results suggest that NCoR can act as AR a co-repressor depending upon hormonal stimulation.

Evaluation of E2F3 and survivin expression in peripheral blood as potential diagnostic markers of prostate cancer

Abstract Background Prostate cancer (PC) incidence has risen globally. As there are no current independent biomarkers with high diagnostic efficiency to detect PC, this study was performed to investigate the relative gene expression levels of E2F3 and survivin in the whole blood of PC, benign prostate hyperplasia (BPH), and normal control individuals and to explore their diagnostic value. Material and methods Participants of the study were divided into three groups; normal control group (n=25), BPH patients (n=25), and PC patients (n=75). The E2F3 and survivin gene expression levels were assessed using real-time qPCR in addition to the measurement of free and total levels of prostate-specific antigen (PSA) using electrochemiluminescence assays. Results Survivin relative gene expression was over-expressed in PC and BPH patients compared to the normal control group, whereas, E2F3 did not differ significantly among the studied groups. Compared to PSA, E2F3 and survivin mRNA expression levels had lower diagnostic efficacy to differentiate PC from normal and BPH individuals with an area under curve (AUC) of 0.471 and 0.727, respectively. Further, survivin expression level was associated with increased the risk of PC. Conclusion Survivin and E2F3 relative expression levels in peripheral blood had low diagnostic performance to detect PC and individuals with high survivin expression levels may have higher risk to develop PC.

Effect of Paecilomyces tenuipes Extract on Testosterone-Induced Benign Prostatic Hyperplasia in Sprague-Dawley Rats.

Benign prostatic hyperplasia (BPH) is one of the major public health concerns, which has a high prevalence rate and causes significant decline in men’s quality of life. BPH is highly related to sexual hormone metabolism and aging. In particular, dihydrotestosterone (DHT), to which testosterone is modified by 5α-reductase (5AR), has a significant effect on BPH development. DHT binds to an androgen receptor (AR) and steroid receptor coactivator 1 (SRC-1); then, it induces the proliferation of a prostate cell and expression of prostate specific antigen (PSA). Paecilomyces tenuipes (P. tenuipes) is a mushroom that has been popularized by the artificial cultivation of fruiting bodies based on silkworms by researchers from the Republic of Korea. In a previous study, we identified the effect of PE on PSA mRNA expression in LNCaP cells. This suggests that PE may have an inhibitory effect on androgen signaling. Therefore, we confirmed the expression of androgen signaling-related factors, such as AR, SRC-1, and PSA in LNCaP. Furthermore, we confirmed the androgen signaling inhibitory effect of PE using the testosterone propionate (TP)-induced BPH rat model. A BPH rat model was established with a four-week treatment of daily subcutaneous injections of testosterone propionate (TP, 3 mg/kg) dissolved in corn oil after castration. The rats in the treatment group were orally gavaged P. tenuipes extract (PE), finasteride (Fi), or saw palmetto extract (Saw) with TP injection. DHT induced an increase in the expression levels of AR, SRC-1, and PSA proteins in LNCaP cells. On the contrary, the PE treatment reduced the expression levels. In vivo, the BPH group showed an increase in prostate size compared with the control group. The PE gavaged group showed a decrease in prostate size compared with the BPH group. In addition, the protein expressions of AR, 5AR2, and PSA were significantly lower in the PE gavaged group than BPH group in prostate tissue. These results suggest the beneficial effects of PE on BPH via the modulation of AR signaling pathway.

Synergistic Anticancer Effect of Paclitaxel and Noscapine on Human Prostate Cancer Cell Lines.

Paclitaxel is one of the most common chemotherapeutic drugs used for the treatment of prostate cancer. However, its current clinical utility has been limited due to numerous serious side effects and drug resistance. Noscapine is an antitussive opium alkaloid that showed antitumor activity against a variety of cancer while it has not exhibited severe side effects. This study investigates the anticancer activity of noscapine in combination with paclitaxel against two LNCaP and PC-3 human prostate cancer cell lines. LNCaP and PC-3 cells were treated with noscapine or paclitaxel or combination. Cell viability was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test. Apoptosis was assessed by acridine orange/ ethidium bromide (AO/EB) staining. The mRNA expression of Bax, Bcl-2, AR, and PSA in the cellular response to treatments was investigated. MTT assay indicated the combination treatment of paclitaxel and noscapine significantly decreased viability compared to single-agent treatment and control groups. The results obtained with AO/EB double staining showed increased percentages of apoptotic cells in the presence of the combination of paclitaxel and noscapine. Furthermore, combinational treatment of paclitaxel and noscapine showed significant decrease in the mRNA expression of B-cell CLL/Lymphoma (Bcl-2) and increase in the mRNA expression of Bcl-2-associated X protein (Bax(, and Bax/Bcl-2 ratio in LNCaP and PC-3 cells (P<0.05.( The mRNA expression of androgen receptor (AR) and prostate specific antigen (PSA) decreased in paclitaxel and noscapine combination-treated of LNCaP cells (P<0.05). This study provides a novel concept of combination treatment of paclitaxel and noscapine to improve efficiency in human prostate cancer treatment.

Effects of inhibition of neural precursor cell-expressed developmentally down regulated 8 gene on biological behavior, and androgen receptor of human prostate cancer cells

Objective To explore the effect of lentivirus-mediated short hairpin RNA (shRNA) silencing neural precursor cell-expressed developmentally down regulated 8 (NEDD8) gene on androgen receptor (AR), prostate specific antigen (PSA), proliferation, apoptosis and invasion of human prostate cancer LNCap and VCaP cells. Methods LNCap and VCaP cells were transfected with lentivirus-mediated NEDD8-shRNA. The mRNA and protein expression of NEDD8, AR and PSA was detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting, respectively. The proliferation, apoptosis and invasion abilities were evaluated by cell counting kit-8 (CCK-8) assay, flow cytometry and Transwell TM assay, respectively. Results After transfection with NEDD8-shRNA, NEDD8 mRNA expression in LnCaP and VCaP was (0.11±0.03) fold and (0.16±0.04) fold, respectively, as compared to the blank control group; AR mRNA expression was (0.52±0.04) fold and (0.42±0.05) fold; PSA mRNA expression was (0.49±0.04) fold and (0.59±0.06) fold. NEDD8, AR and PSA protein expression in LnCaP and VCaP was (11.26±2.65)% and (6.26±2.04)%, (30.36±4.35)% and (15.36±3.37)%, and (13.37±2.70)% and (11.36±2.67)%, respectively. As compared with the control groups, the mRNA and protein expression levels of NEDD8, AR and PSA were significantly inhibited after transfection with NEDD8-shRNA (P=0.000). As compared with the control groups, the proliferation of LnCaP and VCaP was significantly inhibited after transfection with NEDD8-shRNA. The apoptosis rate of LNCap and VCaP cells was (15.08±1.23)% and (20.37±1.76)% respectively, which was significantly enhanced as compared with the control groups (P=0.001, P=0.000). After transfection, the number of LnCaP and VCaP cells invading the Matrigel was (55.5±12.5) and (60.8±11.3) respectively, which was significantly reduced as compared with the control groups (P=0.000, P=0.004). Conclusion NEDD8 may downregulate AR at the transcriptional level. The lentivirus-mediated shRNA silencing NEDD8 gene could inhibit the proliferation and invasion, and promote apoptosis of LNCap and VCaP cells. Key words: Prostate cancer; Neural precursor cell-expressed developmentally down regulated factor 8; Androgen receptor; RNA interference

Effects of 14 frequently used drugs on prostate-specific antigen expression in prostate cancer LNCaP cells.

Prostate cancer occurs more frequently among older males and such elderly individuals often have chronic underlying disorders for which various drugs are administered for treatment. The levels of prostate-specific antigen (PSA), a widely used prostate cancer marker, are influenced by a number of drugs, such as non-steroidal anti-inflammatory drugs and statins. In the present study, the drugs prescribed to patients on a repeat prescription collected at the pharmacy of the Gifu Pharmaceutical University (Gifu, Japan) were examined for their effects on the levels of PSA expression in prostate cancer LNCaP cells. Among the 14 drugs investigated, betamethasone, an agonist of the glucocorticoid receptor, was found to increase the levels of PSA mRNA expression in the LNCaP cells. This betamethasone-induced expression was mediated, at least in part, through androgen receptor (AR) transcriptional activation. Dexamethasone, a typical agonist of the glucocorticoid receptor, was also found to stimulate the AR transcriptional activity, however, to a lesser extent than betamethasone. Therefore, it would be interesting to examine in future studies whether the serum PSA levels in prostate cancer patients are influenced by betamethasone.

Inhibitors of vacuolar ATPase proton pumps inhibit human prostate cancer cell invasion and prostate-specific antigen expression and secretion.

Vacuolar ATPases (V-ATPases) comprise specialized and ubiquitously distributed pumps that acidify intracellular compartments and energize membranes. To gain new insights into the roles of V-ATPases in prostate cancer (PCa), we studied the effects of inhibiting V-ATPase pumps in androgen-dependent (LNCaP) and androgen-independent (C4-2B) cells of a human PCa progression model. Treatment with nanomolar concentrations of the V-ATPase inhibitors bafilomycin A or concanamycin A reduced the in vitro invasion in both cell types by 80%, regardless that V-ATPase was prominent at the plasma membrane of C4-2B cells and only traces were detected in the low-metastatic LNCaP parental cells. In both cell types, intracellular V-ATPase was excessive and co-localized with prostate-specific antigen (PSA) in the Golgi compartment. V-ATPase inhibitors reversibly excluded PSA from the Golgi and led to the accumulation of largely dispersed PSA-loaded vesicles of lysosomal composition. Inhibition of acridine orange staining and transferrin receptor recycling suggested defective endosomal and lysosomal acidification. The inhibitors, additionally, interfered with the AR-PSA axis under conditions that reduced invasion. Bafilomycin A significantly reduced steady-state and R1881-induced PSA mRNA expression and secretion in the LNCaP cells which are androgen-dependent, but not in the C4-2B cells which are androgen ablation-resistant. In the C4-2B cells, an increased susceptibility to V-ATPase inhibitors was detected after longer treatments, as proliferation was reduced and reversibility of bafilomycin-induced responses impaired. These findings make V-ATPases attractive targets against early and advanced PCa tumors.

Abstract A76: MDV3100, a novel androgen receptor signaling inhibitor, promotes cell apoptosis and tumor regression in both in vitro and in vivo models of castration-resistant prostate cancer.

Abstract Prostate tumors initially respond to androgen deprivation therapy but eventually progress despite low serum levels of testosterone. In this stage, known as castration-resistant prostate cancer (CRPC), abnormal androgen receptor (AR) signaling persists and classic anti-androgens such as bicalutamide are ineffective at controlling tumor growth. The novel AR signaling inhibitor MDV3100 has been demonstrated to inhibit AR signaling in preclinical models of CRPC. Here, we investigate further the cellular effects of MDV3100 treatment on AR signaling in in vitro models and in vivo xenograft models of CRPC. Our experiments reveal that MDV3100 (&amp;gt; 100 nM) inhibits dihydrotestosterone (DHT) induced AR-target gene expression in LNCaP cells and LNCaP/AR cells, a CRPC cell culture model in which AR is over-expressed. Inhibition of Prostate-Specific Antigen mRNA expression by MDV3100 was effective at concentrations 10-fold lower than with bicalutamide. As evidenced in other studies and in contrast to bicalutamide, MDV3100 did not display agonistic effects on AR signaling at 10 μM. In contrast to bicalutamide, MDV3100 (&amp;gt; 1 μM) inhibited androgen dependent nuclear translocation of AR, as measured with a quantitative optical based enzyme fragment complementation assay and AR-YFP localization in HEK-293 cells. Furthermore, in the presence of DHT, MDV3100 effectively reduced cell viability of LNCaP cells at concentrations as low as 0.1 μM, 10-fold lower than bicalutamide. MDV3100 also decreased cell viability of LNCaP/AR and W741C-LNcaP cells. We found that the apoptosis marker cleaved-caspase 3 was induced in LNCaP/AR cells treated with 1 M MDV3100 for 48 hours. Under these conditions, MDV3100 was more potent than bicalutamide in decreasing cell viability. Finally, we show that MDV3100 inhibited tumor growth and induced regression of tumor volume in a xenograft model of CRPC generated with LNCaP/AR cells. These results demonstrate that MDV3100, unlike anti-androgens, is effective in both in vitro and in vivo models of CRPC and that its mechanistic advantages over other anti-androgens could be explained by its ability to inhibit AR nuclear translocation and induce cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A76.

Insulin Increases De Novo Steroidogenesis in Prostate Cancer Cells

Androgen-dependent pathways regulate maintenance and growth of normal and malignant prostate tissues. Androgen deprivation therapy (ADT) exploits this dependence and is used to treat metastatic prostate cancer; however, regression initially seen with ADT gives way to development of incurable castration-resistant prostate cancer (CRPC). Although ADT generates a therapeutic response, it is also associated with a pattern of metabolic alterations consistent with metabolic syndrome including elevated circulating insulin. Because CRPC cells are capable of synthesizing androgens de novo, we hypothesized that insulin may also influence steroidogenesis in CRPC. In this study, we examined this hypothesis by evaluating the effect of insulin on steroid synthesis in prostate cancer cell lines. Treatment with 10 nmol/L insulin increased mRNA and protein expression of steroidogenesis enzymes and upregulated the insulin receptor substrate insulin receptor substrate 2 (IRS-2). Similarly, insulin treatment upregulated intracellular testosterone levels and secreted androgens, with the concentrations of steroids observed similar to the levels reported in prostate cancer patients. With similar potency to dihydrotestosterone, insulin treatment resulted in increased mRNA expression of prostate-specific antigen. CRPC progression also correlated with increased expression of IRS-2 and insulin receptor in vivo. Taken together, our findings support the hypothesis that the elevated insulin levels associated with therapeutic castration may exacerbate progression of prostate cancer to incurable CRPC in part by enhancing steroidogenesis.

Paxillin Regulates Androgen- and Epidermal Growth Factor-induced MAPK Signaling and Cell Proliferation in Prostate Cancer Cells

Although transcriptional effects of androgens have been extensively studied, mechanisms regulating transcription-independent (nongenomic) androgen actions are poorly understood. Previously, we have shown that paxillin, a multidomain adaptor protein, is a critical regulator of testosterone-induced MAPK-signaling during Xenopus oocyte maturation. Here we examine the nongenomic effects of dihydrotestosterone (DHT) in prostate cancer cells, focusing on how paxillin mediates Erk signaling and downstream physiologic actions. We show that in LnCAP cells DHT functions as a growth factor that indirectly activates the EGF-receptor (EGFR) via androgen receptor binding and matrix metalloproteinase-mediated release of EGFR ligands. Interestingly, siRNA-mediated knockdown of paxillin expression in androgen-dependent LnCAP cells as well as in androgen-independent PC3 cells abrogates DHT- and/or EGF-induced Erk signaling. Furthermore, EGFR-induced Erk activation requires Src-mediated phosphorylation of paxillin on tyrosines 31/118. In contrast, paxillin is not required for PKC-induced Erk signaling. However, Erk-mediated phosphorylation of paxillin on serines 83/126/130 is still needed for both EGFR and PKC-mediated cellular proliferation. Thus, paxillin serves as a specific upstream regulator of Erk in response to receptor-tyrosine kinase signaling but as a general regulator of downstream Erk actions regardless of agonist. Importantly, Erk-mediated serine phosphorylation of paxillin is also required for DHT-induced prostate-specific antigen mRNA expression in LnCAP cells as well as EGF-induced cyclin D1 mRNA expression in PC3 cells, suggesting that paxillin may regulate prostate cancer proliferation by serving as a liaison between extra-nuclear kinase signaling and intra-nuclear transcriptional signals. Thus, paxillin may prove to be a novel diagnostic or therapeutic target in prostate cancer.

Isolation and structure elucidation of a novel androgen antagonist, arabilin, produced by Streptomyces sp. MK756-CF1

In the course of screening for a new type of androgen receptor (AR) antagonist, we isolated a novel compound, arabilin, with two structural isomers, spectinabilin and SNF4435C, produced by Streptomyces sp. MK756-CF1. Structure elucidation on the basis of the spectroscopic properties showed that arabilin is a novel polypropionate-derived metabolite with a p-nitrophenyl group and a substituted γ-pyrone ring. Arabilin competitively blocked the binding of androgen to the ligand-binding domain of AR in vitro. In addition, arabilin inhibited androgen-induced prostate-specific antigen mRNA expression in prostate cancer LNCaP cells.

The helix‐loop‐helix Id‐1 inhibits PSA expression in prostate cancer cells

The inhibitor of basic helix-loop-helix transcription factors, Id-1, is an important gene whose expression increases during prostate cancer progression and that upregulates proliferation, migration and invasion. We used microarray analysis to identify the downstream genes whose transcriptional expression is modulated by Id-1 protein. We compared gene expression in control LNCaP cells and Id-1-transduced LNCaP cells, which become significantly more aggressive after Id-1 overexpression, thus mimicking the high levels of Id-1 detected in metastatic cell lines. We used the Affy HTA U133A Expression Arrays with 45,000 probe sets representing more than 39,000 transcripts. We found that one of the most significantly downregulated genes on Id-1 expression was kallikrein 3 [also called prostate specific antigen (PSA)], the most commonly used biomarker of prostate cancer. Here, we show that the reduction in PSA mRNA and protein expression associated with high-grade prostate cancers, which generally express high levels of Id-1, could be the consequence of Id-1 overexpression.

Detection of prostate specific transcripts in the peripheral blood during brachytherapy predicts postoperative PSA kinetics

We evaluated whether detection of prostate-specific antigen (PSA) and human kallikrein 2 (hK2) transcripts in the peripheral blood during brachytherapy could predict biochemical outcome. Eighty-one patients who underwent (125)Iodine-based brachytherapy for localized prostate cancer (Gleason score <8, PSA <20 ng/ml, stage <T3), participated in the study. Brachytherapy was given to 35 patients as monotherapy, to 36 in combination with androgen deprivation therapy (ADT), and to 10 in combination with external beam radiation and ADT. Blood samples from 80 patients were available for analysis. Nested RT-PCR means was used to detect mRNA expression of PSA and hK2 in the peripheral blood. Their expression was analyzed before, during and 1 month after brachytherapy. Patients' biochemical outcome (blood PSA levels) during 3 years of follow-up was correlated with the PCR results. The incidence of PSA and hK2 mRNA expression in the peripheral blood was significantly higher during than before or after the procedure. Patients with concurrent positive PSA and hK2 PCR results during brachytherapy had higher postoperative blood PSA values and a slower decline rate of PSA compared with patients with negative PSA and hK2 PCR results. No correlations were found between pre- and postbrachytherapy PCR results and biochemical outcome. ADT was the only significant factor that affected PSA and hK2 mRNA expression during brachytherapy. Our short-term results suggest that detection of PSA and hK2 transcripts in the peripheral blood of prostate cancer patients during brachytherapy could serve as a predictor of biochemical outcome.

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.

Human prostate stromal cells stimulate increased PSA production in DHEA-treated prostate cancer epithelial cells

Dehydroepiandrosterone (DHEA) is commonly used as a dietary supplement and may affect prostate pathophysiology when metabolized to androgens and/or estrogens. Human prostate LAPC-4 cancer cells with a wild type androgen receptor (AR) were treated with DHEA, androgens dihydrotestosterone (DHT), T, or R1881), and E 2 and assayed for prostate specific antigen (PSA) protein and gene expression. In LAPC-4 monocultures, DHEA and E 2 induced little or no increase in PSA protein or mRNA expression compared to androgen-treated cells. When prostate cancer-associated (6S) stromal cells were added in coculture, DHEA stimulated LAPC-4 cell PSA protein secretion to levels approaching induction by DHT. Also, DHEA induced 15-fold more PSA mRNA in LAPC-4 cocultures than in monocultures. LAPC-4 proliferation was increased 2–3-fold when cocultured with 6S stromal cells regardless of hormone treatment. DHEA-treated 6S stromal cells exhibited a dose- and time-dependent increase in T secretion, demonstrating stromal cell metabolism of DHEA to T. Coculture with non-cancerous stroma did not induce LAPC-4 PSA production, suggesting a differential modulation of DHEA effect in a cancer-associated prostate stromal environment. This coculture model provides a research approach to reveal detailed endocrine, intracrine, and paracrine signaling between stromal and epithelial cells that regulate tissue homeostasis within the prostate, and the role of the tumor microenvironment in cancer progression.