Androgen-sensitive Prostate Cancer Cell Line Research Articles

Abstract 1254: Novel acetal and halogen oridonin analogues potently induce apoptosis in prostate cancer cell lines via induction of p53/p73 and PUMA expression

Abstract Isodon rubescens (a Chinese herb named “Donglingcao”) has been used as a folk, botanical medicine for treatment of inflammatory diseases and cancer for many years. Oridonin, a complex ent-kaurane diterpenoid that was isolated from this herb, has been demonstrated as a promising anti-cancer agent. However, the anti-cancer potency of oridonin and its in vivo stability remains unsatisfactory for its clinical development. Therefore, we have designed and synthesized a series of acetal and halogen oridonin analogues at the 20 position of the chemical skeleton. All these analogues have demonstrated improved potency over oridonin against the growth of bladder, breast, colon, lung and prostate cancer cell lines with low micromolar to submicromolar IC50 values. In particular, compound 10 potently induces apoptosis in both androgen-sensitive and castration-resistant prostate cancer cell lines, including LAPC4, LNCaP, 22Rv1, PC3 and DU145. Compound 10 increased the expression of YAP1 and its phosphorylation, as well as expression of p73 and p53, which further led to upregulation of p73/p53 target gene PUMA. The apoptotic effect of compound 10 is at least in part dependent on the existence of p53 and PUMA. These results demonstrated the potential of compound 10 as a novel apoptosis inducer for treatment of prostate cancer. The evaluation of the in vivo anti-tumor efficacy of compound 10 is in progress. Note: This abstract was not presented at the meeting. Citation Format: Yu Ke, Zhongbo Liu, Hong-Min Liu, Xiaolin Zi. Novel acetal and halogen oridonin analogues potently induce apoptosis in prostate cancer cell lines via induction of p53/p73 and PUMA expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1254. doi:10.1158/1538-7445.AM2014-1254

Identification of PRL1 as a novel diagnostic and therapeutic target for castration-resistant prostate cancer by the Escherichia coli ampicillin secretion trap (CAST) method

ObjectivesAlthough chemotherapy for castration-resistant prostate cancer (CRPC) has been applied clinically in recent years, the effects are not sufficient. It is urgently necessary to develop novel therapeutics for CRPC. We previously generated Escherichia coli ampicillin secretion trap libraries of 2 prostate cancer (PCa) cell lines and normal prostate. By comparing the E. coli ampicillin secretion trap libraries of CRPC cell lines with those of androgen-sensitive PCa cell lines and normal prostate, we focused on the protein-tyrosine-phosphatase of regenerating liver 1 (PRL1) gene and analyzed its expression and biological function. Materials and methodsThe expression of PRL1 was examined by quantitative reverse transcription polymerase chain reaction and immunohistochemistry in clinical PCa samples. The effects of PRL1 on PCa cells were evaluated by cell growth, migration, and invasion assays. To investigate the effect of PRL1 on epidermal growth factor receptor (EGFR) signaling, PRL1 knockdown PC3 cells were examined by Western blot and immunohistochemical analyses. ResultsQuantitative reverse transcription polymerase chain reaction revealed that PRL1 was expressed much more highly in PCa than in nonneoplastic prostate samples. High expression of PRL1 detected by immunohistochemistry correlated with poor prognosis after prostatectomy and combined androgen blockade therapy. Functional analysis indicated that PRL1 stimulated cell growth, migration, and invasion in PCa cell lines. Expression EGFR and matrix metalloproteinase 9 was reduced by knockdown of PRL1 in the PC3 cell line. ConclusionsPRL1 regulates expression of EGFR and modulates downstream targets. PRL1 has potential as a therapeutic target in PCa including CRPC.

P0224 A novel, small molecule inhibitor, MSU-1001, in androgen-sensitive prostate cancer cell lines

Introduction Prostate cancer is one of the leading causes of cancer-related death in men worldwide. Current treatment options are limited to traditional chemotherapy, surgery, radiation, or hormone therapy, which often have unwanted side effects. There is a need to develop safer molecules that attenuate tumour progression without adversely affecting the overall quality of life. We report the in vitro activity of a novel, synthetic, small molecule, with potential to be a lead candidate for the treatment of prostate cancer. Methods A library of novel small molecule inhibitors was developed through rational drug design. A pyrrolo-pyrimidine derivative (MSU-1001) was identified as a potential lead molecule. Prostate and colon cancer cell lines (LNCap, HT-29, and HCT-8) were obtained from the American Type Culture Collection (ATCC). Cells were incubated with various concentrations of MSU-1001 for 72 h. Growth inhibition was determined colorimetrically on a plate-reader using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Findings Colon cancer cell lines HCT-8 and HT-29 were used in conjunction with the androgen-sensitive human prostate adenocarcinoma cell line, LNCaP, to investigate the specificity of MSU-1001 on tumour type. Among the library of compounds tested, MSU-1001 was pursued based on its anti-proliferative activity in LNCaP cells. Addition of MSU-1001 to LNCaP cells resulted in a dose-dependent inhibition of proliferation (GI 50 = 161 nM) with a near-complete inhibition of cell growth noted at 10 μM. By contrast, GI 50 of gemcitabine was more than 6-fold higher (GI 50 = 1.21 μM) in LNCaP cells. Additionally, GI 50 of MSU-1001 in HCT-8 and HT-29 was 1.8 and 8.7 μM, respectively, indicating relative specificity of MSU-1001 against prostate cancer. Interpretation These data show the therapeutic potential of MSU-1001 in prostate cancer. Combination studies of MSU-1001 with testosterone, 17 β -estradiol, and dehydroepiandro-stenone in androgen-sensitive and insensitive prostate cancer cell lines are currently ongoing.

Abstract 1726: Androgenic regulation of the anti-apoptotic Bcl-2 family member Mcl-1 in prostate cancer cells.

Abstract Anti-apoptotic proteins of the Bcl-2 family are known to confer resistance against a variety of therapeutic agents. Among them is myeloid leukemia cell differentiation protein (Mcl-1), a highly regulated protein at both transcriptional and post-translational levels by a number of signaling pathways. Overexpression of Mcl-1 has been reported in biopsies of prostate cancer patients with high Gleason grades. Currently, novel anti-cancer approaches that aim to inhibit Mcl-1 are being developed. In this study we have analyzed the influence of androgens and androgen receptor (AR) signaling on Mcl-1 protein expression. Western Blot experiments with androgen-sensitive prostate cancer cell lines LNCaP and VCaP showed a consistent upregulation of Mcl-1 protein expression levels under androgen-depleted conditions, i.e. in steroid-free medium. Addition of 0.1-10 nM of the synthetic androgen R1881 decreased Mcl-1 expression in a concentration-dependent manner in LNCaP and VCaP cells. This effect could also be observed in the castration-resistant LNCaP-abl cell line, but not in AR-negative PC3 cells. Further analysis showed that regulation of Mcl-1 was dependent on the presence of functional AR since it could not be observed in cells co-treated with R1881 and antiandrogens bicalutamide or hydroxyflutamide or after AR downregulation by siRNA. Quantitative PCR on mRNA levels revealed that Mcl-1 regulation by androgens is at a transcriptional level, while downregulation of the three known Mcl-1 E3 ligases APC/C(CDC20), SCF(FBW7), and HUWE1 (MULE) by siRNA did not influence androgenic regulation of Mcl-1 at the post-translational level. To evaluate a possible role of Mcl-1 as a resistance mechanism in cancer stem cells, its expression in basal cells isolated from patient's biopsies was assessed. The highest expression of Mcl-1 mRNA was observed in tumors isolated from castration therapy resistant patients, followed by therapy naïve samples and was the lowest in prostatic cell lines. Interestingly, after isolation of stem cells (SC), transit amplifying (TA) and committed basal (CB) cells, the highest expression of Mcl-1 mRNA could be detected in SC. Our findings indicate that androgenic signaling leads to decreased Mcl-1 expression levels in prostate cancer cell lines, which could be mediated by E2F1 transcription factor known to inhibit Mcl-1 transcription. Vice versa, absent AR signaling leads to upregulated Mcl-1 expression levels. The finding of high Mcl-1 mRNA expression in prostate cancer stem cells that do not express AR supports the hypothesis that Mcl-1 could be involved in resistance mechanisms against first- and second-line therapies. Therefore, anti-Mcl-1 therapies might be considered to improve the efficacy of androgen ablation and could furthermore target prostate cancer stem cells. Citation Format: Frédéric R. Santer, Holger H. Erb, Birgit Luef, Florian Handle, Su Jung Oh, Susanne Lobenwein, Christian Ploner, Jayant Rane, Anne T. Collins, Norman J. Maitland, Zoran Culig. Androgenic regulation of the anti-apoptotic Bcl-2 family member Mcl-1 in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1726. doi:10.1158/1538-7445.AM2013-1726

Abstract 5068: Differential regulation of tissue inhibitor of metalloproteinase (TIMP-1) expression in primary stromal fibroblast cells by androgen sensitive and resistant prostate cancer cells.

Abstract Background: The tissue inhibitor of metalloproteinase 1 (TIMP-1) regulates matrix metalloproteinases (MMPs) activities and is involved in cancer invasion and metastasis. TIMP-1 level is significantly elevated in prostate cancer patient blood and the elevated plasma TIMP-1 level predicts decreased survival of metastatic castration-resistant prostate cancer (mCRPC) patients. Methods and findings: In this study, we examined serum TIMP-1 levels in 114 prostate cancer patient samples by ELISA and found mCRPC patients have significantly higher serum TIMP-1 (mean value of 373.5 ng/ml) than patients with recurrent hormone-sensitive disease (mean value of 304.8 ng/ml) (p<0.001). Then we examined TIMP-1 expression in androgen-sensitive (AS) and androgen resistant (AR) prostate cancer cell lines (LNCaP, VCaP, LAPC-4, 22RV1, DU145, PC-3 and PC-3M) and primary prostate stromal fibroblast cells (HPrF) by realtime PCR and ELISA. Our results showed that AS and AR cells regulate TIMP-1 expression differently. AS cells barely expressed any TIMP-1, whereas AR prostate cancer cells not only expressed moderate amount of TIMP-1, but also induced stromal fibroblast cells to secrete more TIMP-1. But overall, fibroblast cells secreted at least 10 times more TIMP-1 than AR prostate cancer cells, indicating that stromal fibroblasts are the major source of TIMP-1 in prostate cancer. To identify the soluble factors secreted by AR cells to induce TIMP-1 expression in HPrF, we analyzed cytokine and growth factor profiles of AR and AS cell condition medium (CM) by RayBio cytokine and growth factor arrays and found that multiple cytokines and growth factors were upregulated in AR cell line condition medium including IL-6, IL-10, GM-SCF, GRO alpha and etc. More investigation is underway to verify the roles of the cytokines and growth factors in TIMP-1 regulation in prostate cancer. In addition, using pharmaceutical inhibitors, we showed that AZD6244 (a MEK inhibitor) and BAY11-7082 (an NF-kB inhibitor) completely blocked prostate cancer cell line CM-induced TIMP-1 expression in stromal fibroblasts. Conclusion: Our studies have demonstrated that androgen resistance is associated with TIMP-1 overproduction in prostate cancer and suggested that we can potentially inhibit TIMP-1 overexpression in prostate cancer patients with MEK and NF-kB inhibitors. Citation Format: Yixuan Gong, Lan He, Matthew Galsky, William Oh. Differential regulation of tissue inhibitor of metalloproteinase (TIMP-1) expression in primary stromal fibroblast cells by androgen sensitive and resistant prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5068. doi:10.1158/1538-7445.AM2013-5068 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

A Competitive Inhibitor That Reduces Recruitment of Androgen Receptor to Androgen-responsive Genes

The androgen receptor (AR) has a critical role in the growth and progression of androgen-dependent and castration-resistant prostate cancers. To identify novel inhibitors of AR transactivation that block growth of prostate cancer cells, a luciferase-based high-throughput screen of ~160,000 small molecules was performed in cells stably expressing AR and a prostate-specific antigen (PSA)-luciferase reporter. CPIC (1-(3-(2-chlorophenoxy) propyl)-1H-indole-3-carbonitrile) was identified as a small molecule that blocks AR transactivation to a greater extent than other steroid receptors. CPIC inhibited AR-mediated proliferation of androgen-sensitive prostate cancer cell lines, with minimal toxicity in AR-negative cell lines. CPIC treatment also reduced the anchorage-independent growth of LAPC-4 prostate cancer cells. CPIC functioned as a pure antagonist by inhibiting the expression of AR-regulated genes in LAPC-4 cells that express wild-type AR and exhibited weak agonist activity in LNCaP cells that express the mutant AR-T877A. CPIC treatment did not reduce AR levels or alter its nuclear localization. We used chromatin immunoprecipitation to identify the site of action of CPIC. CPIC inhibited recruitment of androgen-bound AR to the PSA promoter and enhancer sites to a greater extent than bicalutamide. CPIC is a new therapeutic inhibitor that targets AR-mediated gene activation with potential to arrest the growth of prostate cancer.

Autophagy regulates lipolysis and cell survival through lipid droplet degradation in androgen‐sensitive prostate cancer cells

Androgen deprivation therapy, one of the standard treatments for prostate cancer (PCa) induces apoptosis, as well as autophagy in androgen-responsive PCa cells. As autophagy can promote either cell survival or death, it is important to understand its role in PCa treatment. The objective of this study was to elucidate the function of autophagy in lipid droplet (LD) homeostasis and survival in androgen-sensitive PCa cells. To produce androgen deprivation, charcoal filtered serum or the androgen inhibitor casodex were used in LNCaP and LAPC4 cells. Autophagy was monitored by immunofluorescence/confocal microscopy and immunoblot analysis. Levels of intracellular LDs and triacyglycerols after the inhibition of autophagy by 3-methyladenine, bafilomycin A(1) , or si-ATG5 were quantified by three independent methods, Oil Red O staining, triacyglycerols lipase assay, and nuclear magnetic resonance. Androgen deprivation induced autophagy and the depletion of LDs in both of the androgen-sensitive PCa cell lines examined, whereas the blockage of autophagy by pharmacological or genetic means inhibited LD degradation and therefore lipolysis and cell growth. In addition, under androgen deprivation, increased colocalization of LDs and autophagic vesicles was observed in LNCaP cells, which can be further enhanced by blocking the autophagic flux. Autophagy mediates LD degradation and lipolysis in androgen-sensitive PCa cells during androgen deprivation which aids the survival of PCa cells during hormone therapy.

Increased TGF‐β1‐mediated suppression of growth and motility in castrate‐resistant prostate cancer cells is consistent with Smad2/3 signaling

Elevated TGF-β levels are associated with prostate cancer progression. Although TGF-β is a tumor suppressor for normal epithelial and early-stage cancer cells, it may act paradoxically as a tumor promoter in more advanced cancers, although its effects are largely cell and context dependent. This study analyzed prostate cancer responses to TGF-β signaling in an isogenic model of androgen-sensitive and castration-resistant prostate cancer cells. Phosphorylation and nuclear translocation of Smad2 and Smad3 were analyzed using immunoblotting. Proliferation and cell cycle responses to TGF-β1 (5 ng/ml) were assessed using growth assays and flow cytometry for DNA content, as well as Western blot and immunoprecipitation of cell cycle proteins. Both androgen-sensitive (LNCaP) and castration-resistant (C4-2 and C4-2B) prostate cancer cell lines demonstrated TGF-β1-induced phosphorylation and nuclear translocation of Smad2/3 that was robust in metastatic lines. Smad phosphorylation was completely abrogated with inhibition of ALK-5 kinase activity using the kinase inhibitor, SB-431542. Increased sensitivity to TGF-β1-mediated growth inhibition was observed in C4-2 and C4-2B cells, as compared to LNCaP cells. This was paralleled with downregulation of Cyclin D and increased association of p15(Ink4b) or p27(Kip) with CDK's. Additionally, TGF-β1 inhibited motility and invasion of metastatic cell lines. TGF-β-mediated suppression of growth and motility is enhanced in metastatic, castration-resistant prostate cancer cells. Enhanced TGF-β1-induced Smad2 and -3 signaling in prostate cancer cells may correlate with tumor suppressive activity. Therefore, the direct effects of TGF-β1 on prostate cancer cells post-castration may be anti-tumorigenic and growth-suppressive.

Abstract B237: Sorafenib decreases proliferation and induces apoptosis of prostate cancer cells by inhibition of the androgen receptor and Akt signaling pathways.

Abstract Prostate cancer is one of the most common male cancers in the western world. When the tumor is organ-confined the disease can in most cases be cured by radical prostatectomy or radiotherapy. For non-organ-confined prostate cancer standard treatments are androgen ablation therapies by orchiectomy, luteinizing hormone releasing hormone agonists or anti-androgens (e.g. bicalutamide) and doxetaxel-based chemotherapy for castration-resistant prostate cancer. However, the response duration of these therapies is limited and the development of alternative approaches is in need. Sorafenib is an oral multikinase inhibitor that targets a number of serine/threonine and tyrosine kinases and has been approved for the treatment of unresectable hepatocellular and advanced renal cell carcinoma. In regard of a possible application of sorafenib in prostate cancer we set out to investigate its effects in a panel of androgen-sensitive (LNCaP and CWR22Rv1) and castration-resistant (PC3 and LNCaP-IL-6+) prostate cancer cell lines as well as in their derivatives mimicking anti-androgen (LNCaP-Bic) and chemotherapy (PC3-DR) resistance. Proliferation was measured by 3H-thymidine incorporation assay and apoptosis by Annexin V flow cytometry and caspase 3/7 activity assays. Cell cycle- and apoptosis-regulating proteins were determined by Western blotting. Secreted prostate-specific antigen was used to monitor androgen receptor activity. 3H-thymidine incorporation assays revealed that sorafenib causes a dose-dependent inhibition of proliferation of all cell lines associated with down-regulation of cyclin-dependent kinase 2 and cyclin D1 expression. Apoptosis was induced at 2 μM sorafenib in androgen-sensitive cells, whereas a higher dose of the drug was needed in castration-resistant cell lines. Apoptosis induction was associated with inhibition of Akt and GSK-3beta phosphorylation as well as down-regulation of the Bcl-2 family member Mcl-1. In contrast to treatments with bicalutamide or docetaxel, therapy-resistant cells were still sensitive to the anti-cancer effects of sorafenib. Yet, a higher dose was needed to induce apoptosis in the therapy-resistant sublines compared to parental cell lines. Most interestingly, sorafenib was able to reduce expression levels of androgen receptor and prostate-specific antigen. In cells in which androgen receptor expression was down-regulated by siRNA, treatment with sorafenib increased apoptosis in an additive manner. In summary, the results of the present study indicate that there is a potential to use sorafenib in prostate cancers as an adjuvant therapy option to current androgen ablation treatments, but also in progressed prostate cancers that become unresponsive to standard therapies. 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 B237.

Bisindole alkaloids and secoiridoids from Alstonia macrophylla Wall. ex G. Don

The ethanolic extract from stems of a Thai medicinal plant, Alstonia macrophylla Wall. ex G. Don (Apocynaceae) showed a significant inhibitory effect on acetylcholinesterase (AChE) determined by using Ellman assay. Four compounds i.e., a bisindole alkaloid, macralstonine (1), a new bisindole alkaloid, thungfaine (2), a secoiridoid glycoside, sweroside (3) and a new secoiridoid glycoside, naresuanoside (4) were isolated. Compound 4 showed moderate AChE and butyrylcholinesterase (BChE) inhibitory effects. Interestingly, compound 4 inhibited cell growth on human androgen-sensitive prostate cancer cell line (LNCaP) but no effect on viability of human foreskin fibroblast cells (HF).

IL-6 causes multiple effects in androgen-sensitive and -insensitive prostate cancer cell lines

The androgen receptor (AR) is expressed in most human prostate cancers. It can be activated in a hypersensitive manner by androgens, nonandrogenic steroids, nonsteroidal anti-androgens and in a ligand-independent manner. IL-6 is a proinflammatory cytokine that activates several signaling pathways. It can either stimulate or inhibit growth of prostate cancer cells. IL-6 is an important positive regulator of AR activity and can stimulate the expression of prostate-specific antigen in a ligand-independent manner. IL-6/AR interaction may either result in growth stimulation (MDA PCa 2b cells) or inhibition (LAPC-4 cells). IL-8 and IL-4 have also been observed to activate AR. Cells generated by prolonged treatment with IL-6 acquire a growth advantage. There are several therapeutic options to target IL-6 in prostate cancer and most laboratory studies have been performed with the monoclonal antibody siltuximab. Endogenous suppressors of cytokine signaling (SOCS)-3 and -1 are expressed in prostate cancer tissue. SOCS-3 inhibits apoptosis in AR-negative cells. However, in androgen-sensitive prostate cancer cell lines, SOCS-3 is induced by androgen and blocks its effects on proliferation and secretion. It is currently understood that there are numerous interactions between androgen and IL-6 signaling in human prostate cancer.

Novel Oligomeric Proanthocyanidin Derivatives Interact with Membrane Androgen Sites and Induce Regression of Hormone-Independent Prostate Cancer

Prostate cancer is the most common malignancy among men in Western societies, and current therapeutic approaches are evolving to manage growth, recurrence, and mortality neoplasia. Membrane androgen receptors (mARs) have been characterized in human prostate cancer, being preferentially expressed in tumor rather than benign gland areas. Furthermore, mAR agonists (protein-conjugated testosterone) decrease in vitro prostate cancer cell growth and induce apoptosis, whereas in vivo they regress growth of tumor xenografts alone or in combination with taxane drugs. In this respect, targeting mARs might be a novel therapeutic approach in prostate cancer. In our search for new small-molecule ligands of mAR, we report that flavanol dimers B1-B4 (oligomeric procyanidins) decrease in vitro growth of the androgen-sensitive (LnCaP) and androgen-resistant (DU145) human prostate cancer cell lines in the following order: B3 = B4 > B2 ≫ B1 (LnCaP) and B2 ≫ B3 = B4 ≫ B1 (DU145). Some of these analogs were previously shown to trigger signaling cascades similar to testosterone-bovine serum albumin (BSA) conjugate. Galloylation does not confer an additional advantage; however, oleylation increases the dimers' antiproliferative potency by a factor of 100. In addition, we report that B2, oleylated or not, displaces testosterone from mARs with an IC(50) value at the nanomolar range and induces DU145 tumor xenograft regression by 50% (testosterone-BSA 40%). In this respect, oleylated B2 is a potent small-molecule agonist of mAR and could be a novel therapeutic agent for advanced prostate cancer, especially when taking into account the absence of androgenic actions and (liver) toxicity.

Formation of AR-SMRT binding in prostate cancer cells treated with natural histone deacetylase inhibitor

Signaling through the androgen receptor (AR) plays a critical role in prostate cancer progression. The AR is a classical nuclear receptor (NR) providing a link between signaling molecule and transcription response. Histone deacetylase inhibitors (HDACI) have antiproliferative and proapoptotic effects on prostate cancer cells and their implication in silence AR signaling may have potential therapeutic use. We aimed to study the inhibitory effects of the corepressor SMRT (Silencing Mediator for Retinoid and Thyroid hormone receptors) which forms a complex together with nuclear receptor corepressor (N-CoR) and with histone deacetylase 3 (HDAC3) on AR activity. The androgen-sensitive prostate cancer cell line LNCaP and androgen-insensitive prostate cancer cell line C4-2 both AR-positive, and androgen-insensitive DU145 and PC3 prostate cancer cell lines were treated with two HDACIs, sodium butyrate (NaB) and/or trichostatin A (TSA). We amplified immunoprecipitated DNA by conventional PCR and in the following step we used the chromatin immunoprecipitation (ChIP) analysis coupled with quantitative PCR for monitoring NaB induced formation of AR-SMRT/N-CoR complex binding on the PSA promoter. The co-immunoprecipitation assay revealed increase in AR-SMRT formation in NaB treated cells. Simultaneously, the Western blot analysis showed a significant decrease in AR protein expression. Furthermore, we estimated the reduced presence of HDAC2 and HDAC3 proteins by NaB and TSA treatment in AR-negative DU145 cell line. In conclusion, the inhibitory effect of NaB on AR gene expression seems to be specific and unique for prostate cancer AR-positive cell lines and corresponds with its ability to stimulate AR-SMRT complex formation. We suggest that AR and SMRT/N-CoR corepressors may form a stable complex in vitro and NaB may facilitate the interaction between AR nuclear steroid receptor and SMRT corepressor protein.

Synthesis and initial tumor affinity testing of iodine-123 labelled EGFR-affine agents as potential imaging probes for hormone-refractory prostate cancer

The epidermal growth factor receptor (EGFR) is over-expressed in a variety of human cancers, including in hormone-refractory prostate carcinomas, in which the EGFR has been associated with advanced disease stage, resistance to standard treatment and poor prognosis. Therefore, the EGFR is considered to be a promising molecular target for molecular imaging and therapy for hormone-refractory prostate cancer. This work describes the synthesis and initial tumor affinity testing of the EGFR antagonist 123I-mAb425 and the EGF receptor tyrosine kinase (EGFR-TK) inhibitor 123I-PD153035 as potential imaging probes for studying EGFR-expressing prostate cancer using single photon emission tomography. Methods 123I-mAb425 and 123I-PD153035 were prepared, starting from the IgG2a antibody and EGFR antagonist mAb425, that binds to the external domain of the EGF receptors, and from the EGFR-TK inhibitor PD153035, targeting the intra-endothelial tyrosine kinase domain of the EGFR, respectively. The potential of 123I-mAb425 and 123I-PD153035 to target EGFR-positive prostate carcinoma was tested on androgen-insensitive PC-3 and DU-145 prostate carcinoma cell lines, and on the androgen-sensitive LNCaP prostate cancer cell line for comparison. In vivo, the capability of 123I-mAb425 and 123I-PD153035 to target hormone-refractory prostate cancer was assessed in RNU rats or nu/nu mice bearing human PC3 prostate cancer xenografts. Results 123I-mAb425 was obtained in >90% radiochemical yield using the IODO-GEN ® method. 123I-PD153035 was synthesized by a non-isotopic [ 123I]iodo-debromination of PD153035 in 50–60% radiochemical yield in a total synthesis time including HPLC separation of 70 min. In vitro 123I-mAb425 and 123I-PD153035 accumulated highly in human PC-3 and DU-145 prostate cancer cells. Radioactivity incorporation into PC-3 and DU-145 tumor cells following 15-min incubation at 37 °C varied from 25% to 48% of the total loaded activity per 10 6 tumor cells (560–1230 cpm/1000 cells). In comparison, the uptake of the EGFR-affine probes into LNCaP prostate carcinoma cells was significantly low (105 ± 25 cpm/1000 cells). Inhibition experiments revealed that 123I-mAb425 is taken up into tumor cells via the same pathway as the naturally occurring epidermal growth factor (EGF), while 123I-PD153035 accumulation in prostate cancer cells occurs presumably via the same pathway as the selective EGFR-Tyrosine kinase antagonist AG1418. In vivo, the human prostate cancer xenografts in mouse war accurately visualized after i.v. administration of 123I-mAb425 by a gamma camera. Conclusion These data suggest that 123I-mAb425 and 123I-PD153035 are promising candidates as imaging probes for EGFR-positive prostate cancer and warrant further in vivo validations to ascertain their potential as imaging agents for clinical used.

Abstract 1490: Overexpression of a calcium channel protein TRPM8 in prostate cancer cells enhances their anchorage-independent growth and resistance to paclitaxel-induced apoptosis

Abstract The non-voltaged-gated transient receptor potential protein melastatin member 8 (TRPM8) has been characterized to function as a cold- and menthol-activated calcium-permeable channel protein, which it was originally cloned in a prostate-specific substracted cDNA library and shown to be up-regulated in benign prostatic hyperplasia and prostate cancer as compared to normal prostatic epithelial cells. Recent studies in cultured prostate cancer cells and clinical prostate cancer tissues show that TRPM8 is an androgen receptor-regulated calcium channel which is required for the survival and androgen-dependent growth of prostate cancer cells. Based on this evidence, it is believed that TRPM8 may play an important role in the advanced growth of prostate cancer. However, the prostate-specific function of TRPM8 and the role of TRPM8-regulated calcium-influx in prostate function and its carcinogenesis remain unclear. Our preliminary studies showed that TRPM8 was significantly over-expressed in an androgen-independent and metastatic prostate cancer cell line C4-2B as compared to its parental line LNCaP and also in two animal models of metastatic prostate cancers (a transgenic mouse model of prostate cancer-TGMAP and xenograft model of C4-2B). In order to elucidate the functional significance of TRPM8 in prostate cancer cell growth, we over-expressed TRPM8 in an androgen-sensitive prostate cancer cell line LNCaP by lentiviral transduction and generated stable TRPM8 clones for phenotypic growth studies. Our findings showed that stable ectopic TRPM8 expression induced morphological changes in LNCaP cells with reduction of filopodial processes and also suppressed their in vitro cell proliferation as shown by MTT assay. However, stable LNCaP-TRPM8 clones demonstrated significantly enhanced colony formation efficiencies in soft agar assay. Moreover, stable TRPM8 expression also enhanced the in vivo tumor growth of LNCaP cells in immunodeficient SCID mice and resistance to paclitaxel-induced apoptosis, as compared to their parental line. The results of our present study suggest that altered calcium-homeostasis as disturbed by TRPM8 overexpression in prostate cancer cells could enhance their in vitro anchorage-independent and in vivo tumor growth, and also resistance to paclitaxel-induced apoptosis, while the involved calcium-dependent signaling pathways still remain to be elucidated. (Study supported by a RGC Competitive Earmarked Research Grant project code 461007) 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 1490.

Binding of AR to SMRT/N-CoR complex and its co-operation with PSA promoter in prostate cancer cells treated with natural histone deacetylase inhibitor NaB

Signaling through the androgen receptor (AR) plays a critical role in prostate cancer progression. The AR is a classical nuclear receptor (NR) providing a link between signaling molecule and transcription response. Histone deacetylase inhibitors- (HDACI) have antiproliferative and proapoptotic effects on prostate cancer cells and their implication in silence AR signaling may have potential therapeutic use. We aimed to study the inhibitory effects of the corepressor SMRT (Silencing Mediator for Retinoid and Thyroid -hormone receptors) which forms a complex together with nuclear receptor corepressor (N-CoR) and with histone deacetylase 3 (HDAC3) on AR activity.The androgen-sensitive prostate cancer cell line LNCaP and androgen-insensitive prostate cancer cell line C4-2 both AR-positive, and androgen-insensitive DU145 and PC3 prostate cancer cell lines were treated with two HDACIs, sodium butyrate (NaB) and/or trichostatin A (TSA). We amplified immunoprecipitated DNA by conventional PCR and in the -following step we used the chromatin immunoprecipitation (ChIP) analysis coupled with quantitative PCR for monitoring NaB induced formation of AR-SMRT/N-CoR complex binding on the PSA promoter. The co-immunoprecipitation assay revealed increase in AR-SMRT formation in NaB treated cells. Simultaneously, the Western blot analysis showed a significant decrease in AR protein expression. In conclusion, the inhibitory effect of NaB on AR gene expression seems to be specific and unique for prostate cancer AR-positive cell lines and corresponds with its ability to stimulate AR-SMRT complex formation. We suggest that AR and SMRT/N-CoR corepressors may form a stable complex in vitro and NaB may facilitate the interaction between AR nuclear steroid receptor and SMRT corepressor prote.

Lentivirus-mediated SMO RNA interference inhibits SMO expression and cell proliferation, and affects the cell cycle in LNCaP and PC3 cancer cell lines

Smoothened (SMO) is an important member of the Hedgehog signaling pathway. We constructed a specific recombinant lentiviral vector for RNA interference, targeting the SMO gene (NM_005631) to observe its effect on SMO expression, cell proliferation and the cell cycle in the human androgen-sensitive prostate cancer cell line, LNCaP, and in the androgen-independent prostate cancer cell line, PC3. Four siRNA sequences were designed and inserted into a lentiviral vector pGCSIL-GFP to construct four recombinant vectors. The vector with the highest interfering efficiency was co-transfected with packaging vectors (pHelper1.0 and pHelper2.0) in 293T cells to assemble lentivirus particles by liposome for infecting LNCaP and PC3 cell lines, respectively. The expression level of SMO mRNA, tumor cell proliferation and cell cycle were measured by quantitative realtime polymerase chain reaction (qRT-PCR), 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and flow cytometry, respectively. Sequence results showed that recombinant lentiviral vectors were constructed successfully. pGCSIL-GFP-723 had the highest interfering efficiency, named Lv-SIL-SMO723 after co-transfection, with which LNCaP and PC3 cell lines were infected. Compared with the control groups, results showed significantly decreased (P < 0.05) SMO mRNA expressions of LNCaP and PC3, lower mean percentage of S-phase cells and higher mean percentage of G(2)/M phase cells, as well as obviously slow proliferation (P < 0.01) of LNCaP in the infected group. Yet, the proliferation of PC3 was not altered (P > 0.05). In conclusion, the recombinant lentivirus particles were able to suppress SMO expression, regulate the cell cycle in the LNCaP and PC3 cell lines and markedly inhibit proliferation of LNCaP cells but not PC3 cells.

Expression, regulation and function of the ISGylation system in prostate cancer

The androgen receptor (AR) plays a crucial role in the modulation of prostate cell proliferation and is involved in the development and progression of prostate cancer (PCa). An understanding of the complex regulation of AR provides novel treatment options for PCa. Here, we show (i) that the ubiquitin-like modifier, interferon-stimulated gene 15 (ISG15), and most enzymes involved in ISG15 conjugation were upregulated in tumor samples versus in non-malignant tissues of PCa patients and (ii) that the expression of these components significantly differed between tumors in patients treated with and without androgen ablation. Using PCa cell lines as in vitro models, the specific androgen-mediated, AR-dependent regulation of the ISGylation components was confirmed. In addition, the ISGylation system controls AR mRNA and protein expressions, as overexpression of Ube1L as a limiting ISGylation factor in the AR(+) androgen-sensitive PCa cell line, LNCaP, results in significant AR upregulation, accompanied by an increased proliferation even under androgen deprivation. Accordingly, Ube1L knockdown decreased the AR expression. Thus, this study describes for the first time the modulation of AR expression by ISGylation components, which affects the proliferation of PCa cells, thereby providing evidence for a novel function of the ISGylation system in malignant transformation.

Androgens down-regulate myosin light chain kinase in human prostate cancer cells

Androgens play a major role in the growth and survival of primary prostate tumors. The molecular mechanisms involved in prostate cancer progression are not fully understood but genes that are regulated by androgens clearly influence this process. We searched for new androgen-regulated genes using the Affymetrix GeneChip Human Genome U95 Set in the androgen-sensitive LNCaP prostate cancer cell line. Analysis of gene expression profiles revealed that myosin light chain kinase (MLCK) mRNA levels were markedly down-regulated by the synthetic androgen R1881. The microarray data were confirmed by ribonuclease protection assays. RNA and protein analyses revealed that LNCaP cells express both long (non-muscle) and short (smooth muscle) isoforms, and that both isoforms are down-regulated by androgens. Taken together, these data identify MLCK as a novel downstream target of the androgen signalling pathway in prostate cells.

The Iroquois Homeobox Gene 5 Is Regulated by 1,25-Dihydroxyvitamin D3 in Human Prostate Cancer and Regulates Apoptosis and the Cell Cycle in LNCaP Prostate Cancer Cells

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the most active metabolite of vitamin D3, has significant antitumor activity in a broad range of preclinical models of cancer. In this study, we show that the Iroquois homeobox gene 5 (Irx5) is down-regulated by 1,25(OH)2D3 in human prostate cancer samples from patients randomly assigned to receive weekly high-dose 1,25(OH)2D3 or placebo before radical prostatectomy. Down-regulation of Irx5 by 1,25(OH)2D3 was also shown in the human androgen-sensitive prostate cancer cell line LNCaP and in estrogen-sensitive MCF-7 breast cancer cells. Knockdown of Irx5 by RNA interference showed a significant reduction in LNCaP cell viability, which was accompanied by an increase in p21 protein expression, G2-M arrest, and an increase in apoptosis. The induced apoptosis was partially mediated by p53, and p53 protein expression was increased as a result of Irx5 knockdown. Cell survival was similarly reduced by Irx5 knockdown in the colon cancer cell line HCT 116 and in MCF-7 breast cancer cells, each being derived from clinical tumor types that seem to be inhibited by 1,25(OH)2D3. Overexpression of Irx5 led to a reduction of p21 and p53 expression. This is the first report that Irx5 is regulated by 1,25(OH)2D3 in humans and the first report to show that Irx5 is involved in the regulation of both the cell cycle and apoptosis in human prostate cancer cells. Irx5 may be a promising new therapeutic target in cancer treatment.