Androgen-dependent Prostate Carcinoma Research Articles

Decursin inhibits EGFR-ERK1/2 signaling axis in advanced human prostate carcinoma cells.

We have shown that decursin, a coumarin compound, induces cell cycle arrest and apoptosis in human prostate cancer cells (PCa); however, its molecular mechanisms are largely unexplored. We studied the mechanisms associated with its anticancer activity in advanced human prostate carcinoma cells. We found that decursin inhibited epidermal growth factor receptor (EGFR) signaling by inhibiting its activating phosphorylation at tyrosine 1068 residue in DU145 and 22Rv1 cells. This inhibition of EGFR was associated with the downregulation of ERK1/2 phosphorylation. Both EGFR and ERK1/2 are known to be deregulated/activated in many human malignancies. Consistent with our earlier study, decursin (25-100 µM) treatment for 24-72 h inhibited DU145 cell proliferation by 49%-87% (p < 0.001) which was associated with strong G1 phase arrest and cell death. It also decreased (p < 0.001) the number of surviving colonies. Decursin moderately increased the expression of Rb-related proteins p107 and p130 but decreased the levels of E2F family transcription factors including E2F-3, E2F-4 and E2F-5. Further, decursin strongly inhibited the growth of androgen-dependent prostate carcinoma 22Rv1 cells from 61% to 79% (p < 0.001) and arrested these cells at G1 phase via induction of cyclin-dependent kinase inhibitor p27/Kip1 and downregulation of CDK2 and CDK4 protein expression. Additionally, EGFR inhibitor erlotinib- and EGF ligand-modulated EGFR activation validated EGFR signaling as a target of decursin-mediated cell growth inhibition and cytotoxicity. Decursin decreased EGF ligand-induced phosphorylation of EGFR (Y-1068) as well as activation of its downstream mediator, ERK1/2. Furthermore, inhibitory targeting of EGFR-ERK1/2 axis by combinatorial treatment of decursin and erlotinib further sensitized DU145 cells for the decursin-induced growth inhibition and cell death. Overall, these findings strongly suggest that anticancer efficacy of decursin against human PCa involves inhibitory targeting of EGFR-ERK1/2 signaling axis, a pathway constitutively active in advanced PCa.

Serotonin receptor 4 (5-hydroxytryptamine receptor Type 4) regulates expression of estrogen receptor beta and cell migration in hormone-naive prostate cancer.

Estrogens are considered to potentially play some roles in the development and progression of prostate cancer through estrogen receptor beta (ERβ). However, additional factors which could influence the clinical outcome of the patients through modulating these steroid signalings have also been proposed. Among these, increased expression of serotonin receptor especially that of 5-hydroxytryptamine receptor Type 4 (5-HTR4) has been recently proposed to be involved in autocrine/paracrine mechanisms of castration-resistant prostate cancer, but the presence and clinical significance of 5-HTR4 in hormone-naive prostate cancer (HNPC) and its interaction with hormonal signaling pathways have remained virtually unknown. We evaluated the status of 5-HTR4 in 112 human HNPC cases (acinar adenocarcinoma) using immunohistochemistry and correlated the findings with clinicopathological features of individual patients and the status of androgen receptor (AR) and ERβ. To further elucidate its underlying mechanisms, androgen-dependent human prostate carcinoma cell line, LNCaP, expressing 5-HTR4, was treated by 5-HTR4 agonist. 5-HTR4 immunoreactivity was detected in 34% of prostate cancer cases examined (38/112) and was significantly correlated with the status of ERβ but not with that of AR and other clinicopathological factors of the patients. Results of in vitro studies demonstrated that 24 h incubation with 5-HTR4 agonist (10 nM) increased the expression level of ERβ messenger RNA compared to controls. 5-HTR4 agonist (100 nM) significantly inhibited LNCaP carcinoma cell migration (P < 0.05). Results of our present study indicated that 5-HTR4 signaling upregulated ERβ expression in HNPCs and could impact on biological processes in HNPC.

Intracellular Elemental Patterns of Apoptosis Resistance in Transdifferentiated Androgen-Dependent Prostatic Carcinoma Cells.

The acquisition of neuroendocrine (NE) characteristics by prostate cancer (PC) cells relates to tumor progression and hormone resistance. PC cells may survive and function in androgen-deprived environments, where they could establish paracrine signaling networks, providing stimuli for the propagation of local carcinoma cells. We previously demonstrated, using electron probe X-ray microanalysis (EPXMA), in LNCaP, PC-3, and Du 145 cell lines that apoptosis is associated with intracellular elemental changes, and that the NE secretory products, bombesin and calcitonin, inhibit etoposide-induced apoptosis, as well as some of these elemental changes. In this study, LNCaP cells were induced in vitro to transdifferentiate under androgen deprivation, to mimic the role of NE cells in the apoptotic activity of transdifferentiated androgen-dependent PC cells. Changes in intracellular ion content associated with apoptosis, assessed by EPXMA, demonstrate that the transdifferentiated LNCaP cells are resistant to etoposide-induced apoptosis and also to the etoposide-induced elemental changes. The aggressive malignant potential of PC with neuroendocrine differentiation, associated with hormonal independence, is partly because of the ability that most NE tumor cells have to escape apoptosis, which can enhance the malignant properties of tumor cells and may have therapeutic implications as tumor cells are usually resistant to cytotoxic drugs as etoposide.

Expression and potential role of the peptide orexin-A in prostate cancer

The peptides orexin-A and orexin-B and their G protein-coupled OX1 and OX2 receptors are involved in multiple physiological processes in the central nervous system and peripheral organs. Altered expression or signaling dysregulation of orexins and their receptors have been associated with a wide range of human diseases including narcolepsy, obesity, drug addiction, and cancer. Although orexin-A, its precursor molecule prepro-orexin and OX1 receptor have been detected in the human normal and hyperplastic prostate tissues, their expression and function in the prostate cancer (PCa) remains to be addressed. Here, we demonstrate for the first time the immunohistochemical localization of orexin-A in human PCa specimens, and the expression of prepro-orexin and OX1 receptor at both protein and mRNA levels in these tissues. Orexin-A administration to the human androgen-dependent prostate carcinoma cells LNCaP up-regulates OX1 receptor expression resulting in a decrease of cell survival. Noteworthy, nanomolar concentrations of the peptide counteract the testosterone-induced nuclear translocation of the androgen receptor in the cells: the orexin-A action is prevented by the addition of the OX1 receptor antagonist SB-408124 to the test system. These findings indicate that orexin-A/OX1 receptor interaction interferes with the activity of the androgen receptor which regulates PCa onset and progression, thus suggesting that orexin-A and its receptor might represent novel therapeutic targets to challenge this aggressive cancer.

A single-chain fragment against prostate specific membrane antigen as a tool to build theranostic reagents for prostate cancer

Prostate carcinoma is the most common non-cutaneous cancer in developed countries and represents the second leading cause of death. Early stage androgen dependent prostate carcinoma responds well to conventional therapies, but relatively few treatment options exist for patients with hormone-refractory prostate cancer. One of the most suitable targets for antibody-mediated approaches is prostate specific membrane antigen (PSMA) which is a well known tumour associated antigen. PSMA is a type II integral cell-surface membrane protein that is not secreted, and its expression density and enzymatic activity are increased progressively in prostate cancer compared to normal prostate epithelium, thereby making PSMA an ideal target for monoclonal antibody imaging and therapy.To obtain a small protein that can better penetrate tissue, we have engineered a single-chain variable fragment (scFv) starting from the variable heavy and light domains of the murine anti-PSMA monoclonal antibody D2B. scFvD2B was analysed in vitro for activity, stability, internalisation ability and in vivo for targeting specificity. Maintenance of function and immunoreactivity as well as extremely high radiolabelling efficiency and radiochemical purity were demonstrated by in vitro assays and under different experimental conditions. Despite its monovalent binding, scFvD2B retained a good strength of binding and was able to internalise around 40% of bound antigen. In vivo we showed its ability to specifically target only PSMA expressing prostate cancer xenografts. Due to these advantageous properties, scFvD2B has the potential to become a good theranostic reagent for early detection and therapy of prostate cancers.

7-epi-nemorosone from Clusia rosea induces apoptosis, androgen receptor down-regulation and dysregulation of PSA levels in LNCaP prostate carcinoma cells

The aim of this work was to characterize the antitumoral activity of the plant compound 7-epi-nemorosone in prostate carcinoma cell lines. Prostate cancer is the most frequently diagnosed malignancy and the second-leading cause of cancer death in men. In spite of the current therapeutic options for this cancer entity, many patients die due to metastases in distant organs and acquired chemotherapy resistance. Thus, approaches to provide improvements in outcome and quality of life for such patients are urgently needed. Recently, the polyisoprenylated benzophenone 7-epi-nemorosone, originally collected by honeybees from Clusia rosea and Clusia grandiflora (Clusiaceae), has been described to be a potent antitumoral agent. Here, its activity in prostate carcinoma is reported. 7-epi-nemorosone was isolated from Caribbean propolis employing RP-HPLC techniques. Its cytotoxicity was assessed using the MTT proliferation assay in human androgen-dependent prostate carcinoma LNCaP cells including an MDR1+ sub-line. No cross-resistance was detected. FACS-based cell cycle analysis revealed a significant increase in the sub-G0/G1, G1, and depletion in the S phase populations. A concomitant down-regulation of cyclins D1/D3 and CDK 4/6 in LNCaP cells was detected by Western blot. Annexin-V-FITC labeling and caspase-3 cleavage assays showed that 7-epi-nemorosone induced apoptotic events. Major signal transduction elements such as p38 MAPK and Akt/PKB as well as androgen receptor AR and PSA production were found to be down-regulated after exposure to the drug. ERK1/2 protein levels and phosphorylation status were down-regulated accompanied by up-regulation but inhibition of the activity of their immediate upstream kinases MEK1/2. Additionally, Akt/PKB enzymatic activity was effectively inhibited at a similar concentration as for MEK1/2. Here, we demonstrate for the first time that 7-epi-nemorosone exerts cytotoxicity in an androgen-dependent prostate carcinoma entity by targeting the MEK1/2 signal transducer.

Vinorelbine plus prednisone versus docetaxel plus prednisone as first-line treatment in metastatic hormone-refractory prostate cancer.

e15129 Background: Metastatic hormone refractory prostate cancer (mHRPC) is not curable and all attempts at therapeutic intervention have been based on palliating the disease. Androgen dependent prostate carcinoma responds poorly to chemotherapy. Recent protocols using vinorelbine, mitoxantrone and docetaxel have significantly improved response rate, survival and pain control for those patients. The study aims to compare the therapeutic benefit of the use of vinorelbine versus docetaxel both plus prednisone in patients with mHRPC. Methods: Sixty pts (50% gpA, 50% gpB) were enrolled between Mar 2005 and Sep 2007. All patients had histologically confirmed prostatic adencarcinoma with evidence of metastatic disease and progression on hormonal therapy. WHO PS O-2, adequate marrow, liver and renal functions. Patients were randomized to gpA; Vinorellrine (V) IV 30mg/m2 d1 and 8, or gpB; Docetaxe (D) IV 75mg/m2 d1. All patients received 5mg of prednisone orally twice daily for 5 days starting on day 1 of the tudy. Cycles repeated every 3 weeks. Patients with PD went off the study while those with CR, PR or SD continued treatment for 8 cycles maximum. Results: All pts were evaluable for response, toxicity and survival. The median age (gpA, gpB): 59 and 58 years, median WHO PS 1 (range 0-2) in both groups , Median basal PSA were 110 (90-780) and 120 (80-850) in group A&amp;B respectively. The overall response rates were 53.3% (gpA) and 56.7% (gpB) P=0.24, the rates of PSA decline were higher in gpA than gpB (80% vs 70%) (p=0.27). Reduction of pain was better in patients receiving (V) than those treated with (D) (60% vs 50%) (p= 0.23). The improvement in the quality of life was higher in gp A than in gpB (50% vs 40%) but the difference was statistically insignificant. No WHO G3 or 4 toxicities in gp A. G3 alopecia (60%), G3 neutropenia (20%) were noted in gpB. Conclusions: Our results suggest that vinorelbine and docetaxel demonstrate similar efficacy as first line treatment for mHRPC. Vinorelbine is however better tolerated besides being a less costly therapeutic option in Egypt. Comparative phase III trial is needed to confirm these results.

Predicting microRNA modulation in human prostate cancer using a simple String IDentifier (SID1.0)

To make faster and efficient the identification of mRNA targets common to more than one miRNA, and to identify new miRNAs modulated in specific pathways, a computer program identified as SID1.0 (simple String IDentifier) was developed and successfully applied in the identification of deregulated miRNAs in prostate cancer cells. This computationally inexpensive Fortran program is based on the strategy of exhaustive search and specifically designed to screen shared data (target genes, miRNAs and pathways) available from PicTar and DIANA-MicroT 3.0 databases. As far as we know this is the first software designed to filter data retrieved from available miRNA databases. SID1.0 takes advantage of the standard Fortran intrinsic functions for manipulating text strings and requires ASCII input files. In order to demonstrate SID1.0 applicability, some miRNAs expected from the literature to associate with cancerogenesis (miR-125b, miR-148a and miR-141), were randomly identified as main entries for SID1.0 to explore matching sequences of mRNA targets and also to explore KEGG pathways for the presence of ID codes of targeted genes. Besides genes and pathways already described in the literature, SID1.0 has proven to useful for predicting other genes involved in prostate carcinoma. These latter were used to identify new deregulated miRNAs: miR-141, miR-148a, miR-19a and miR-19b. Prediction data were preliminary confirmed by expression analysis of the identified miRNAs in androgen-dependent (LNCaP) and independent (PC3) prostate carcinoma cell lines and in normal prostatic epithelial cells (PrEC).

Abstract 5648: Influence of gallate esterification on the activity of procyanidin B2 in androgen-dependent human prostate carcinoma LNCaP cells

Abstract Prostate cancer (PCA) is the most common cancer in elderly men with one out of six men afflicted with this malignancy in the United States. In spite of improvements in diagnostic and therapeutic strategies for PCA, prognosis of this malignancy remains poor at advanced stages suggesting that newer preventive/interventive strategies are required to control this malignancy. Plant based therapies are often used as alternative and complementary treatments for various diseases including cancer. Grape seed extract (GSE) is a rich source of procyanidins and is used as a health supplement in United States. In our previously published studies, we have demonstrated chemopreventive efficacy of this extract per se and of biologically active constituents present in the extract such as gallic acid against PCA. We also demonstrated anti-cancer efficacy of B2-3,3′-di-O-gallate (B2-G2), a di-gallate ester of procyanidin B2 against androgen-independent human PCA DU145 cells. Present study assessed the influence of gallate esterification on the anti-cancer activity of procyanidin B2 (B2) in human PCA LNCaP cells employing B2-3,3′-di-O-gallate (B2-G2), two mono-gallate esters B2-3-O-gallate (B2-3G) and B2-3′-O-gallate (B2-3′G) and the parent compound B2. Since, androgen receptor (AR) is recognized as an important molecular target in early as well as advanced stages of PCA, in the present study, we used human prostate carcinoma LNCaP cells that harbor functional AR and are dependent on androgen for their growth, to investigate the anticancer activity of these compounds. All these compounds were isolated from grape seed extract (GSE) following several chromatographic steps and structures determined by a combination of enzymatic hydrolysis, mass spectrometry and comparisons with standards. We observed that treatment of LNCaP cells with gallate esters B2-G2, B2-3G and B2-3′G significantly decreased LNCaP cell viability; however, B2 and gallic acid were ineffective. Furthermore, only B2-G2 also significantly decreased cell growth. Decreases in cell viability were largely due to apoptosis induction with B2-G2 and B2-3′G exhibiting comparable effects, whereas B2-3G was less effective. In mechanistic studies, B2-G2 and B2-3′G treatments caused caspases-9 and −3 and PARP cleavage, and down-regulated Bcl-2, Bcl-Xl and AR levels. Taken together, our findings demonstrate anti-PCA efficacy of B2-G2 and suggest that a gallate ester moiety at 3′ position of procyanidin B2 contributes more extensively toward the biological activity of the di-gallate ester than esterification of position 3. This study was supported by the National Cancer Institute, NIH RO1 grant CA091883. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5648.

Influence of Gallate Esterification on the Activity of Procyanidin B2 in Androgen-Dependent Human Prostate Carcinoma LNCaP Cells

Present study assessed the influence of gallate esterification on the anti-cancer activity of procyanidin B2 (B2) in androgen-dependent human prostate carcinoma LNCaP cells employing B2-3,3'-di-O-gallate (B2-G(2)), two mono-gallate esters B2-3-O-gallate (B2-3G) and B2-3'-O-gallate (B2-3'G) and the parent compound B2, all isolated from grape seed extract (GSE). Study compounds were isolated from GSE by several chromatographic steps and structures determined by a combination of enzymatic hydrolysis, mass spectrometry and comparisons with standards. Cells, treated with these compounds, were assessed for viability and apoptosis and examined by western blotting. Gallate esters B2-G(2), B2-3G and B2-3'G significantly decreased LNCaP cell viability; however, B2 and gallic acid were ineffective. Furthermore, only B2-G(2) also significantly decreased cell growth. Decreases in cell viability were largely due to apoptosis induction with B2-G(2) and B2-3'G exhibiting comparable effects, whereas B2-3G was less effective. In mechanistic studies, B2-G(2) and B2-3'G treatments caused caspases-9 and -3 and PARP cleavage, and down-regulated Bcl-2, Bcl-Xl and androgen receptor levels. Together, our findings demonstrate anti-PCA efficacy of B2-G(2) and suggest that a gallate ester moiety at 3' position of procyanidin B2 contributes more extensively toward the biological activity of the di-gallate ester than esterification of position 3.

Triiodothyronine modulates cell proliferation of human prostatic carcinoma cells by downregulation of the B‐Cell translocation gene 2

Studies suggest that triiodothyronine (T3) and cognate nuclear receptors (hTR) are involved in regulation of prostatic cell growth and differentiation. To probe mechanisms for T3 effects, we studied prostate carcinoma cells, investigating the effect of T3 on expression of the B-cell translocation gene 2 (BTG2), which regulates the G1/S transition of the cell cycle. Effects of T3 on cell proliferation were determined by (3)H-thymidine incorporation. T3 modulation of BTG2 expression was investigated using immunoblots, Northern blots, and transient gene expression assays. The putative T3 response element was determined by electrophoretic mobility shift assay. T3 (0.1-1,000 nM) enhanced threefold the proliferation of prostate carcinoma cells and human androgen-dependent prostate carcinoma cells (LNCaP), but not PC-3 cells. T3 also inhibited BTG2 gene expression in LNCaP cells. Reporter assays showed that T3 downregulates by 50% promoter activity of the BTG2 gene in LNCaP cells but not PC-3 cells or thyroid-hormone receptor (TRbeta1)-overexpression PC-3 cells. Deleting the putative thyroid hormone response element (TRE; AGCGATGACCTCAGCG) blocked the inhibitory effect of T3 on BTG2 promoter activity. Electrophoretic mobility shift assays with purified TRbeta1 from in vitro translation, or with nuclear extracts from LNCaP cells and PC-3 cells, demonstrated the presence of T3 receptor binding sites in the TRE region. These results suggested that the T3 upregulates proliferation of LNCaP cells by downregulating BTG2 gene expression through the consensus TRE pathway.

Phosphodiesterase (PDE) inhibitors reduce in vitro proliferation of prostate primary cells but do not interfere with growth of prostate carcinoma cell lines

14604 Background: PDE 5 is highly expressed in cavernosal and prostatic tissue. The mechanism of PDE-5 inhibitors on cavernosal tissue is well established but the effects of PDE-5 inhibitors on prostatic cells are unknown. The aim of this study was to analyze in vitro effects of PDE-5 inhibitors on prostate primary cells, fibroblasts (PrSC), basal epithelial cells (PrEC) and prostate cancer cell lines. Methods: Cultivated PrEC and PrSC, immortalized BPH cells (BPH 1), androgen dependent (LNCaP) and androgen independent (PC3) prostate carcinoma cell lines were exposed to increasing concentrations of commercially available PDE 5 inhibitors Sildenafil (Sil), Tadalafil (Tad), Vardenafil (Var). After incubation for 3 days cell viability was determined by a WST-1 assay (Roche-Biochemicals). Cells were evaluated morphologically by invert-light microscopy. PDE-5 protein concentrations were determined by western blot analyses and tissue distribution of PDE-5 by immunohistochemistry (IHC) with a polyclonal antiserum. Results: None of the PDE-5 inhibitors induced cell proliferation. Significant decreases in proliferation and viability were observed at high concentrations (1 mg/ml) of all substances in PrSC and PrEC. In PrSC, proliferation rate decreased to 37.7% in Sil, to 16.9% in Tad and to 63.7% for Var as compared to controls. In PrEC, proliferation decreased to 72.7%, 21.6% and 84.4% for Sil, Tad and Var, respectively. At 0.1 mg/ml only Tad reduced proliferation significantly to 57.4%. Moreover, Tad induced neuroendocrine-like morphology in some PrEC. High protein concentrations of PDE 5 were observed in PrEC, low concentrations in PrSC but none in cancer cells. Conclusions: Sil, Tad and Var inhibit proliferation of prostate primary cells in vitro. Tad showed highest inhibition. Tumor cells were insensitive to PDE-5 inhibitors, due to the lack of PDE-5 protein. It seems unlikely that any of these substances increases proliferation of prostate carcinoma. Tad induced neuroendocrine-like morphology in some basal PrEC indicating effects on cellular differentiation. No significant financial relationships to disclose.

Expression of Prostate‐Specific Membrane Antigen in Normal and Malignant Human Tissues

Prostate-specific membrane antigen (PSMA) is upregulated in androgen-dependent prostate carcinoma and it has been targeted for immunotherapy and diagnosis of this cancer. However, this protein is also expressed in other tissues. The objective of this study is to investigate its expression in normal and malignant human tissues. Using monoclonal antibodies 24.4E6 (specific for residues 638-657) and 7E11.C5 (specific for the transmembrane domain of PSMA), immunohistochemical detection of PSMA was performed in surgical specimens. Prostate-specific membrane antigen was detected in the epithelium of prostate, urinary bladder, proximal tubules of kidney, liver, esophagus, stomach, small intestine, colon, breast, fallopian tubes and testicular seminiferous tubules, hippocampal neurons and astrocytes, ependyma, cortex and medulla of the adrenal gland, and ovary stroma. It was also detected in neoplasms of the prostate, kidney, urinary bladder, stomach, small intestine, colon, lung, adrenal gland, and testis. It was not detected in normal seminal vesicles or the lung. These findings demonstrate that PSMA is widely distributed in normal tissues, and, depending on the tumors, its expression is up- or down-regulated, or unchanged. The broad distribution of PSMA may make it suitable for the diagnosis and therapy of a wide variety of tumors.

D‐TMPP: A novel androgen‐regulated gene preferentially expressed in prostate and prostate cancer that is the first characterized member of an eukaryotic gene family

The understanding of the molecular biology of the prostate and the process of prostate carcinogenesis is brought forward by the identification and characterization of new genes specifically expressed in prostate tissue. The encoded proteins may, in addition, provide novel diagnostic and therapeutic tools in prostate carcinoma (PCa). Here, we identify the novel gene Dresden-transmembrane protein of the prostate (D-TMPP) that is overexpressed in human prostate and prostate cancer. Proceeding from a prostate-specific expressed sequence tag identified with an Affymetrix chip-based expression database, the full-length cDNA of the novel gene was isolated from prostate tissue. The potential protein-coding function of the open reading frame (ORF) was tested by in vitro transcription-coupled translation and recombinant expression in transfected prostate cancer cells. The expression pattern of D-TMPP in malignant and nonmalignant tissues and tumor cell lines was analyzed by hybridization of a radioactively labeled cDNA probe with a multiple tissue expression array and by a quantitative real-time PCR assay. The D-TMPP-mRNA encodes a putative seven-span transmembrane protein of 883 amino acids and is selectively overexpressed in prostate tissue. D-TMPP represents the first cloned and characterized transcript of a family of eukaryotic genes. D-TMPP transcripts were detected in all analyzed pairs (n = 25) of malignant and nonmalignant prostate tissues. In the androgen-dependent PCa cell line LNCaP, D-TMPP was upregulated by methyltrienolone. We describe the novel prostate-restricted molecule D-TMPP widely expressed in prostate cancer tissues.

Effects of 5 alpha reductase inhibitors on androgen-dependent human prostatic carcinoma cells

To investigate the effects of MK906, a selective 5 alpha reductase (5alphaR) type 2 (5alphaR2) inhibitor, and of MK386, a specific 5alphaR1 inhibitor, on the cellular proliferation of androgen-dependent human prostatic cancer (PCa) cells in cultures of cells derived from bioptic and surgical tissues. In this study we tested the effects of MK906 and MK386 in 30 cultures derived from PCa, 6 from PIN and 10 from benign prostatic hyperplasia specimens. Prostate primary cultures under short-term conditions (with <4 subcultures) represent a mixture of epithelial and stromal cells. Epithelial cells require testosterone (T) for optimal growth, but were not able to grow in the presence of T under long-term conditions even if DHT was able to induce cellular proliferation to a similar extent in both conditions, suggesting that 5alphaR can be lost in long-term cultures. Therefore, our studies were performed under short-term conditions. Both 5alphaR inhibitors decreased cell proliferation significantly and dose-dependently in all the samples tested. MK906 was more efficient than MK386 in 7 out of 10 cultures derived from BPH tissues, in 4 out of 6 cultures derived from PIN and in 18 out of 30 cultures derived from PCa. In 3 out of 10 BPH, in 2 out of 6 PIN and in 5 out of 30 PCa-derived cultures, both inhibitors presented similar efficacy, whereas in 1 out of 10 BPH and 7 out of 30 PCa-derived cultures MK386 was more efficient than MK906. In addition, MK386 was more efficient than MK906 in 4 out of 15 non-metastatic PCa and 2 out of 7 metastatic PCa-derived cultures. Considering that 5alphaR1 (responsible primarily for androgenic catabolism) is mostly expressed in epithelial cells and that 5alphaR2 (responsible for local DHT synthesis and release) is expressed in the stromal cells (which provides several paracrine growth factors and DHT itself to the epithelial cells), our experiments suggest that the inhibition of both 5alphaR1 and 5alphaR2 by MK386 and MK906, respectively, may have therapeutic potential in order to reduce the growth and progression of human prostatic cancers, through the inhibition of autocrine or paracrine mechanisms involving the stromal cell compartment. In addition, some effects of 5alphaR inhibitors could be mediated by estrogens, which are synthesized by the aromatase enzyme present in the epithelial cells. These aspects could be considered in order to improve the therapeutical management of PCa and for future clinical trials.

Inositol Hexaphosphate Inhibits Growth and Induces G1 Arrest and Apoptotic Death of Androgen-Dependent Human Prostate Carcinoma LNCaP Cells

Prostate cancer (PCA) is the most common invasive malignancy and the second leading cause of cancerrelated deaths in the US male population. One approach to control this malignancy is its preventive intervention by dietary agents. Inositol hexaphosphate (IP6), a dietary constituent, has shown promising efficacy against various cancers; however, limited studies have been performed with IP6 against PCA. Here, we investigated the growth-inhibitory effect and associated mechanisms of IP6 in androgen-dependent human prostate carcinoma LNCaP cells. IP6 treatment of cells resulted in a strong growth inhibition and an increase in G1 cell population. In mechanistic studies, IP6 resulted in an increase in cyclin-dependent kinase inhibitors (CDKIs) Cipi/p21 and Kip1/p27 levels, together with a decrease in cyclin-dependent kinase (CDK) 4 and cyclin D1 protein levels. An increase in CDKI levels by IP6 also led to a concomitant increase in their interactions with CDK2 and CDK4, together with a strong decrease in the kinase activity of both CDKs. Downstream in CDKI-CDK-cyclin cascade, consistent with its inhibitory effect on CDK kinase activity, IP6 treatment of cells increased hypophosphorylated levels of retinoblastoma (Rb) with a decrease in Rb phosphorylation at serine 780, 807, and 811 sites, and caused a moderate to strong decrease in the levels of transcription factors E2F1, E2F4, and E2F5. In other studies, IP6 caused a dose- and a time-dependent apoptotic death of LNCaP cells, and a decrease in Bcl2 levels, causing a strong increase in Bax versus Bcl2 ratio, as well as an inhibition of constitutively active AKT phosphorylation. Taken together, these molecular alterations provide an insight into IP6-caused growth inhibition, G1 arrest, and apoptotic death of human prostate carcinoma LNCaP cells. Because early clinical PCA growth is an androgen-dependent response, the results of the present study employing androgendependent LNCaP cells suggest that IP6 has promise and potential to be effective against PCA.

Suppression of human prostate tumor growth in mice by a cytolytic D-, L-amino Acid Peptide: membrane lysis, increased necrosis, and inhibition of prostate-specific antigen secretion.

Gene-encoded host defense peptides are used as part of the innate immunity, and many of them act by directly lysing the cell membrane of the pathogen. A few of these peptides showed anticancer activity in vitro but could not be used in vivo because of their inactivation by serum. We designed a 15-amino acid peptide, composed of D- and L-amino acids (diastereomer), which targets both androgen-independent and androgen-dependent human prostate carcinoma cell lines (CL1, 22RV1, and LNCaP). Most importantly, we observed a complete arrest of growth in CL1 and 22RV1 xenografts treated intratumorally with the diastereomer. This was also accompanied by a lowering of prostate-specific antigen serum levels secreted by the 22RV1 xenograft. Furthermore, the diastereomer synergized with conventional chemotherapeutics. In contrast, the parental all l-amino acids peptide was highly active only in vitro and could not discriminate between tumor and nontumor cells. Fluorescent confocal microscopy, histopathologic examination, and cell permeability studies (depolarization of transmembrane potential and release of an encapsulated dye) suggest a necrotic mechanism of killing, after a threshold concentration of peptide has been reached. Its destructive killing effect and the simple sequence of the diastereomer make it an attractive chemotherapeutic candidate possessing a new mode of action, with potential to be developed additionally for the treatment of prostate carcinoma.

Phenotypic Switch from Paracrine to Autocrine Role of Hepatocyte Growth Factor in an Androgen-Independent Human Prostatic Carcinoma Cell Line, CWR22R

Support mechanisms involved in growth of androgen-independent prostate cancer are primarily unknown. Hepatocyte growth factor (HGF)/Met has been suggested to be one of them based primarily on immunohistochemical studies. We conducted a series of experiments to assess the role of the HGF/Met system in an androgen-dependent human prostate carcinoma, CWR22 and its androgen-independent derivative, CWR22R. We found that action of HGF changed from paracrine to autocrine in progression to androgen-independent state. CWR22 tumors did not express HGF but expressed Met, whereas prostate stromal cells expressed HGF at a high level. Growth of CWR22 was stimulated either by addition of HGF to the culture or by the presence of prostate stromal cells. On the other hand, CWR22R cells expressed both HGF and Met. Knockdown of Met expression by RNA interference method suppressed the growth of CWR22R cells. Our data suggest that HGF is intimately involved in growth of human prostate cancer and that progression from the androgen-dependent to the androgen-independent state is associated with an adaptive switch in support mechanism from paracrine to autocrine. Our data offer one mechanism to account for androgen-independent human cancer growth.

Effects of the herbal extract PC-SPES on microtubule dynamics and paclitaxel-mediated prostate tumor growth inhibition.

PC-SPES is a botanical preparation shown to have efficacy in patients with androgen-dependent and androgen-independent prostate carcinoma. Several herbal constituents in PC-SPES inhibit tumor growth through cell cycle arrest and apoptosis, although the mechanisms of these activities are poorly defined. We sought to identify PC-SPES-induced changes in gene expression, specifically in those genes encoding cytoskeletal proteins that could be associated with PC-SPES-induced cytoxicity. LNCaP prostate carcinoma cells were treated with PC-SPES, and changes in gene expression were determined by complementary DNA (cDNA) microarray hybridization and northern blot analyses. PC-SPES and paclitaxel, a microtubule-stabilizing drug, effects on microtubules were assessed by immunofluorescence of treated cells and by in vitro tubulin polymerization assays. In vivo effects of PC-SPES and paclitaxel were assessed using CWR22R androgen-independent prostate cancer xenografts. All statistical tests were two-sided. PC-SPES treatment of LNCaP cells for 24 hours altered the expression of 17 cytoskeletal genes. mRNA levels of alpha-tubulin decreased sevenfold. Although paclitaxel stabilized and PC-SPES treatment disrupted microtubule architecture in LNCaP cells, the combination of both agents had an intermediate effect. PC-SPES inhibited tubulin polymerization in vitro, even in the presence of paclitaxel. Compared with tumors in control mice (mean tumor volume = 2983 mm(3), 95% confidence interval [CI] = 2380 to 3586 mm(3)), tumors were statistically significantly smaller in mice that received PC-SPES (mean tumor volume = 2018 mm(3), 95% CI = 1450 to 2568 mm(3); P =.028), paclitaxel (mean tumor volume = 1340 mm(3), 95% CI = 697 to 1983 mm(3); P<.001), or the combination of PC-SPES and paclitaxel (mean tumor volume = 1955 mm(3), 95% CI = 1260 to 2650 mm(3); P =.034). PC-SPES may interfere with microtubule polymerization. This activity has implications for the clinical management of patients with advanced prostate cancer who may be taking PC-SPES concurrently with microtubule-modulating chemotherapeutic agents, such as paclitaxel.

HER‐2 profiling and targeting in prostate carcinoma

The clinical effects of targeting HER-2 in prostate carcinoma are not known. This study explored the feasibility of molecular profiling to determine the correlation between HER-2 expression, hormonal sensitivity, and the antitumor effects of trastuzumab and paclitaxel in patients with prostate carcinoma. Patients with progressive androgen dependent (AD) and androgen independent (AI) prostate carcinoma were eligible to participate in the study. HER-2 expression was assessed on pretreatment tissue specimens, and patients were then assigned to one of four treatment groups: AD HER-2 positive, AD HER-2 negative, AI HER-2 positive, and AI HER-2 negative. They were treated with weekly trastuzumab at a dose of 2 mg/kg (after a 4 mg/kg loading dose) until they experienced disease progression, when weekly paclitaxel at 100 mg/m(2) was added. The authors screened 130 patients for HER-2 expression. In total, 23 patients were treated. Six eligible patients had HER-2 positive disease; therefore, only the AI HER-2 negative arm accrued to completion. All patients (100%) experienced disease progression on trastuzumab alone at or before the first 12 weeks of treatment. Fifteen patients received combined therapy: Seven patients (47%) experienced disease progression, 5 patients (33%) had stable disease, and 3 patients (20%) had a decline > or = 50% in prostate specific antigen PSA level or in soft tissue disease. HER-2 overexpression was found in significant proportions only in AI metastatic tissue samples (42% HER-2 positive; 95% confidence interval, 14-60%). In three of nine matched pairs, the AD prostate biopsy was HER-2 negative, and the AI metastatic sample was HER-2 positive. Trastuzumab is not effective as a single agent for the treatment of patients with AI HER-2 negative tumors. HER-2 expression varies by clinical state in patients with prostate carcinoma: Accurate HER-2 profiling requires sampling metastatic tissue in patients with metastatic disease. Further development of trastuzumab for the treatment of patients with metastatic prostate carcinoma is not feasible until more reliable and practical methods of sampling metastatic disease are developed to identify patients with HER-2 positive tumors.