Androgen-dependent Human Prostate Cancer Research Articles

New mode of action of curcumin on prostate cancer cells: Modulation of endoplasmic reticulum-associated degradation mechanism and estrogenic signaling.

Prostate cancer is leading to cancer-related mortality in numerous men each year worldwide. While there are several treatment options, acquired drug resistance mostly limits the success of treatments. Therefore, there is a need for the development of innovative treatments. Curcumin is one of the bioactive polyphenolic ingredients identified in turmeric and has numerous biological activities, such as anti-inflammatory and anticancer. In the present study, we investigated the effect of curcumin on the ER-associated degradation (ERAD) and estrogenic signaling in prostate cancer cells. The antiproliferative effect of curcumin on human androgen-dependent prostate cancer cell lines LNCaP and VCaP was estimated by WST-1 assay. Morphological alterations were investigated with an inverted microscope. We investigated the effect of curcumin on ERAD and estrogen signaling proteins by immunoblotting assay. To evaluate the impact of curcumin on endoplasmic reticulum (ER) protein quality-related, the expression level of 32 genes was analyzed by quantitative reverse transcription polymerase chain reaction. The nuclear translocation of estrogen receptor was examined by nuclear fractionation and immunofluorescence microscopy. We found that curcumin effectively reduced the proliferation rates of LNCaP and VCaP cells. ERAD proteins; Hrd1, gp78, p97/VCP, Ufd1 and Npl4 were strongly induced by curcumin. Also, the steady-state level of polyubiquitin was increased in a dose-dependent manner in both cell lines. Curcumin administration remarkably decreased the protein levels of estrogen receptor-alfa (Erα), whereas estrogen receptor-beta unaffected. Additionally, curcumin strongly restricted the nuclear translocation of Erα. Present data suggest that curcumin may be effectively used in therapeutic approaches associated with the targeting ER protein quality control mechanism and modulation of estrogen signaling in prostate cancer.

Investigation of the Effect of Myricetin On Human Androgen Dependent Prostate Cancer Cells

Prostat kanseri dünyada erkekler arasında en sık görülen ikinci kanser türüdür. Prostat kanserinin morbidite ve mortalitesi son zamanlarda artmıştır. Tedavisi için birçok alternatif yaklaşımlar geliştirilmeye çalışılsa da, prostat kanseri hala kötü prognoz sergilemekte ve yüksek ölüm oranları ile karşılaşılmaktadır. Myricetin, antikanser özelliği ile ilgi çeken doğal bir flavonoid bileşiktir. Yapılan in vitro ve in vivo çalışmalar myricetinin çeşitli mekanizmalar yoluyla prostat kanserini etkili bir şekilde inhibe ettiğini göstermektedir. Bu çalışmanın amacı artan dozlarda myricetin uygulamasının androjen reseptör bağımlı insan prostat kanser hücre hattı olan LNCaP hücrelerinin canlılığı üzerindeki etkilerini belirlemek ve apoptozla ilişkili BAX ve BCL2 genlerinin ekspresyon seviyelerini tespit etmektir. LNCaP hücreleri myricetinin 10 μM, 25 μM, 50 μM, 100 μM, 150 μM’lık konsantrasyonları ile 24 ve 48 saat süresince inkübe edilmiş ve hücre canlılığındaki değişimler 2,3-bis-(2-metoksi-4-nitro-5-sulfofenil)-2Htetrazolyum-5-karboksanilid (XTT) yöntemiyle belirlenerek IC50 değerleri hesaplanmıştır. BAX ve BCL2 gen ifadelerindeki değişimler ise Real-Time PCR metoduyla belirlenmiş ve elde edilen verilerin analizinde ΔΔCT metodu kullanılmıştır. Myricetinin uygulanan bütün dozlarda kontrole göre LNCaP hücre canlılığını azalttığı gösterilmiş olup IC50 değeri 24. saat için 123.76 μM, 48. saat için ise 84.79 μM olarak tespit edilmiştir. Ayrıca, myricetin uygulamasının apoptoz ilişkili BAX gen ifadesini istatistiksel olarak anlamlı bir düzeyde artırırken BCL2 gen ifadesini ise azalttığı görülmüştür. Myricetinin LNCaP hücrelerindeki antiproliferatif ve apoptotik etkileri daha detaylı olarak araştırılmalıdır.

Follicle-stimulating hormone promotes growth of human prostate cancer cell line-derived tumor xenografts.

Chemical castration in prostate cancer can be achieved with gonadotropin-releasing hormone (GnRH) agonists or antagonists. Their effects differ by the initial flare of gonadotropin and testosterone secretion with agonists and the immediate pituitary-testicular suppression by antagonists. While both suppress luteinizing hormone (LH) and follicle-stimulating hormone (FSH) initially, a rebound in FSH levels occurs during agonist treatment. This rebound is potentially harmful, taken the expression of FSH receptors (R) in prostate cancer tissue. We herein assessed the role of FSH in promoting the growth of androgen-independent (PC-3, DU145) and androgen-dependent (VCaP) human prostate cancer cell line xenografts in nude mice. Gonadotropins were suppressed with the GnRH antagonist degarelix, and effects of add-back human recombinant FSH were assessed on tumor growth. All tumors expressed GnRHR and FSHR, and degarelix treatment suppressed their growth. FSH supplementation reversed the degarelix-evoked suppression of PC-3 tumors, both in preventive (degarelix and FSH treatment started upon cell inoculation) and therapeutic (treatments initiated 3weeks after cell inoculation) setting. A less marked, though significant FSH effect occurred in DU145, but not in VCaP xenografts. FSHR expression in the xenografts supports direct FSH stimulation of tumor growth. Testosterone supplementation, to maintain the VCaP xenografts, apparently masked the FSH effect on their growth. Treatment with the LH analogue hCG did not affect PC-3 tumor growth despite their expression of luteinizing hormone/choriongonadotropin receptor. In conclusion, FSH, but not LH, may directly stimulate the growth of androgen-independent prostate cancer, suggesting that persistent FSH suppression upon GnRH antagonist treatment offers a therapeutic advantage over agonist.

MicroRNA-337-3p suppresses cell viability, apoptosis, and autophagy by modulating PPARγ expression in androgen-dependent human prostate cancer

We proposed that autophagy activated through PPARγ in prostate cancer could protect cancer against androgen deprivation. Our MTT and western blotting assay results showed that mimics of miR-337-3p repressed autophagy flux under androgen deprivation, whereas inhibitors had the opposite effect. Neither mimics nor inhibitors influenced cell motility and invasion. miR-337-3p downregulated PPARγ expression in dual luciferase reporter assays and promoted AMPK phosphorylation. The overexpression of PPARγ or AMPK agonists partly diminished the adverse influence of mimics on proliferation and cell autophagy. In a mouse xenograft model, tumors transplanted with LNCap cells having a miR-337-3p deficiency had a significantly larger volume and lower rate of apoptosis than in controls after orchidectomy. Our findings showed that the negative effects of miR-337-3p on cell survival after androgen deprivation through PPARγ degeneration and modulated autophagy could serve as a novel therapeutic target for androgen-dependent prostate cancer and provide a strategy against resistance to anti-androgen therapy.

Glutamate Decarboxylase 65 Signals through the Androgen Receptor to Promote Castration Resistance in Prostate Cancer.

The transition from an androgen-dependent to a castration-resistant state is a critical event in the progression of prostate cancer. In this study, we compared metabolic pathways between isogenic human androgen-dependent and castration-resistant prostate cancer (CRPC) patient-derived xenograft models, and found consistent activation of the γ-aminobutyric acid (GABA) shunt in CRPC. This difference was the result of phosphorylation and activation of glutamate decarboxylase 65 (GAD65), which synthesizes GABA from glutamate by decarboxylation. Mechanistic investigation showed that GABA binds to and retains the androgen receptor (AR) in the nucleus by facilitating AR association with the nuclear zinc finger protein ZNHIT3. GAD65 knockdown decreased the growth of multiple established CRPC xenografts and markedly delayed the time to emergence of castration resistance. These data encourage exploring GAD65 as a therapeutic target for CRPC. SIGNIFICANCE: This study reports metabolic alterations that could be responsible for the development of CRPC and identifies the GABA-producing enzyme GAD65 as a potential new therapeutic target.See related commentary by Taylor and Watt, p. 4580.

Effect of propofol on androgen receptor activity in prostate cancer cells

Androgen receptor is a nuclear receptor and transcription factor activated by androgenic hormones. Androgen receptor activity plays a pivotal role in the development and progression of prostate cancer. Although accumulating evidence suggests that general anesthetics, including opioids, affect cancer cell growth and impact patient prognosis, the effect of those drugs on androgen receptor in prostate cancer is not clear. The purpose of this study was to investigate the effect of the general anesthetic propofol on androgen receptor activity in prostate cancer cells. An androgen-dependent human prostate cancer cell line (LNCaP) was stimulated with dihydrotestosterone (DHT) and exposed to propofol. The induction of androgen receptor target genes was investigated using real-time reverse transcription polymerase chain reaction, and androgen receptor protein levels and localization patterns were analyzed using immunoblotting and immunofluorescence assays. The effect of propofol on the proliferation of LNCaP cells was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Propofol significantly inhibited DHT-induced expression of androgen receptor target genes in a dose- and time-dependent manner, and immunoblotting and immunofluorescence assays indicated that propofol suppressed nuclear levels of androgen receptor proteins. Exposure to propofol for 24h suppressed the proliferation of LNCaP cells, whereas 4h of exposure did not exert significant effects. Together, our results indicate that propofol suppresses nuclear androgen receptor protein levels, and inhibits androgen receptor transcriptional activity and proliferation in LNCaP cells.

Pioglitazone, a Peroxisome Proliferator-Activated Receptor γ Agonist, Suppresses Rat Prostate Carcinogenesis.

Pioglitazone (PGZ), a peroxisome proliferator-activated receptor γ agonist, which is known as a type 2 diabetes drug, inhibits cell proliferation in various cancer cell lines, including prostate carcinomas. This study focused on the effect of PGZ on prostate carcinogenesis using a transgenic rat for an adenocarcinoma of prostate (TRAP) model. Adenocarcinoma lesions as a percentage of overall lesions in the ventral prostate were significantly reduced by PGZ treatment in a dose-dependent manner. The number of adenocarcinomas per given area in the ventral prostate was also significantly reduced by PGZ treatment. The Ki67 labeling index in the ventral prostate was also significantly reduced by PGZ. Decreased cyclin D1 expression in addition to the inactivation of both p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)κB were detected in PGZ-treated TRAP rat groups. In LNCaP, a human androgen-dependent prostate cancer cell line, PGZ also inhibited cyclin D1 expression and the activation of both p38 MAPK and NFκB. The suppression of cultured cell growth was mainly regulated by the NFκB pathway as detected using specific inhibitors in both LNCaP and PC3, a human androgen-independent prostate cancer cell line. These data suggest that PGZ possesses a chemopreventive potential for prostate cancer.

Abstract 5173: Role of Gli2 in mediating the progression of prostate cancer to CRPC

Abstract Background: Prostate cancer is a leading cause of cancer-related death in the US men, largely due to the development of castration resistant prostate cancer (CRPC) in the patients treated with androgen deprivation therapy. There is increasing evidence indicating that Hedgehog pathway is involved in the malignancy of prostate cancer, which includes the finding of two prostate tumors with loss-of-function mutations in the SuFu gene, correlation of tumor grade with high expression of Sonic hedgehog, Gli2 and/or hedgehog target genes including Ptch1 and HIP, and increased Gli2 expression in therapy-resistant tumor cells from patients. In addition, treatment of mice with hedgehog inhibitors significantly attenuated the growth of prostate tumor xenografts. Some recent studies also implicated a crosstalk between Hedgehog/Gli signaling and androgen signaling pathways in prostate cancer. However, the role of Gli2 in CRPC progression has not been investigated. Methods: A human Gli2 specific small hairpin RNA (shRNA) expression vector was constructed using Tet-PLKO-puro lentivector, which was delivered into the androgen-dependent human prostate cancer LNCaP cells. RT-PCR, Western Blot and Luciferase Reporter Assay were performed to confirm doxycycline-inducible Gli2 knockdown. The effect of Gli2 knockdown on anchorage-dependent or -independent growth was assessed with MTT and Soft Agar Assays respectively. Cells were cultured in androgen depleted medium or treated with the anti-androgen, Casodex, to mimic androgen deprivation condition. Mouse xenograft models were used to assess the role of Gli2 in the development of CRPC in vivo. Results: Androgen deprivation promoted the expression of Hedgehog signaling components including Gli2 in the LNCaP cells. Gli2 knockdown inhibited anchorage-dependent and -independent growth of LNCaP cells. Casodex complemented the inhibition of Gli2 knockdown on the growth of LNCaP cells. Gli2 knockdown prevented the outgrowth of androgen-independent cells from LNCaP cells cultured in androgen depleted medium. Induced-knockdown of Gli2 by doxycycline inhibited the growth of castration-resistant tumors in vivo in mice. Conclusions: Our study demonstrated the important role of Gli2 in the development of CRPC. Therefore, it may have the potential to serve as a novel therapeutic target for the prevention of CRPC. Citation Format: Lu Xia, Tai Qin, Luzhe Sun. Role of Gli2 in mediating the progression of prostate cancer to CRPC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5173.

The role of mesenchymal stem cells in promoting the transformation of androgen-dependent human prostate cancer cells into androgen-independent manner.

Mesenchymal stem cells (MSCs) play an important role in the development of human prostate cancer (PCa). However, the role of MSCs in the transformation of androgen-dependent human PCa cells into androgen-independent manner has been poorly understood. In this study, we investigated the underlying mechanism of MSCs in promoting PCa cells from androgen-dependent into androgen-independent manner. Firstly, we demonstrated that MSCs could affect the transformation of androgen-dependent human PCa cells into androgen-independent manner in vivo and in vitro. Then we found a substantial expression of TGF-β in MSCs. TGF-β blockade could significantly inhibit the promotive function of MSCs in PCa cells. Besides that, we also demonstrated androgen might inhibit the expression of TGF-β in MSCs. Furthermore, we found that either overexpression of SSEA-4 or the number of SSEA-4 positive MSCs in PCa tissues was associated with a shorter cancer-free survival interval (CFSI) and a worse overall survival (OS). Our results suggest that androgen blockade treatment in clinical PCa therapy may elicit the expression of TGF-β in MSCs, which will result in the transformation of androgen-dependent human PCa cells into androgen-independent manner.

Silibinin derivatives as anti-prostate cancer agents: Synthesis and cell-based evaluations

This study aims to systematically explore the alkylation effect of 7-OH in silibinin and 2,3-dehydrosilibinin on the antiproliferative potency toward three prostate cancer cell lines. Eight 7-O-alkylsilibinins, eight 7-O-alkyl-2,3-dehydrosilibinins, and eight 3,7-O-dialkyl-2,3-dehydrosilibinins have been synthesized from commercially available silibinin for the in vitro cell-based evaluation. The WST-1 cell proliferation assay indicates that nineteen out of twenty-four silibinin derivatives have significantly improved antiproliferative potency when compared with silibinin. 7-O-Methylsilibinin (2) and 7-O-ethylsilibinin (3) have been identified as the most potent compounds with 98- and 123-fold enhanced potency against LNCaP human androgen-dependent prostate cancer cell line. Among 2,3-dehydrosilibinin derivatives, 7-O-methyl-2,3-dehydrosilibinin (10) and 7-O-ethyl-2,3-dehydrosilibinin (11) have been identified as the optimal compounds with the highest potency towards both androgen-dependent LNCaP and androgen-independent PC-3 prostate cancer cell lines. 7-O-Ethyl-2,3-dehydrosilibinin (11) was demonstrated to arrest PC-3 cell cycle at the G0/G1 phase and to induce PC-3 cell apoptosis. The findings in this study suggest that antiproliferative potency of silibinin and 2,3-dehydrosilibinin can be appreciably enhanced through suitable chemical modifications on the phenolic hydroxyl group at C-7 and that introduction of a chemical moiety with the potential to improve bioavailability through a linker to 7-OH in silibinin and 2,3-dehydrosilibinin would be a feasible strategy for the development of silibinin derivatives as anti-prostate cancer agents.

DAG-Lactone Radiosensitization of Human Prostate Cancer Cells Is Mediated by ATM Down-regulation But Not Due to Abnormal DNA Repair

DAG-Lactone Radiosensitization of Human Prostate Cancer Cells Is Mediated by ATM Down-regulation But Not Due to Abnormal DNA Repair

Effects of treatment with androgen receptor ligands on microRNA expression of prostate cancer cells.

Post-transcriptional regulation by microRNA (miRNA) is an important aspect of androgen receptor (AR) signalling in prostate cancer cells. However, the global profiling of miRNA expression in prostate cancer cells following treatment with AR ligands has not been reported so far. In this study we examined the effect of treatment with two AR agonists (mibolerone (MIB) and dihydrotestosterone (DHT)) and an AR antagonist (bicalutamide (BIC)) on miRNA expression in the human androgen-dependent LNCaP prostate cancer cell line using microarray technology and verification of selected miRNA using quantitative real-time PCR (qRT-PCR). No miRNA was identified as differentially expressed following treatment with the AR antagonist BIC. In contrast, a number of common and compound-specific alterations in miRNA expression were observed following treatment with AR agonists. Unexpectedly it was found that treatment with the AR agonists resulted in the repression of miR-221, a miRNA previously established to be involved with prostate cancer development. This observation indicates that this miRNA may have a more complex role in prostate cancer development than considered previously. Treatment with MIB led to an induction of miR-210 expression, a hypoxia-related miRNA. This miRNA is reported to be involved in cell adaptation to hypoxia and thus induction in conditions of normoxia may be important in driving metabolic changes observed in prostate cancer. Thus examining the effect of AR agonists and antagonists on miRNA expression can provide novel insights into the response of cells to AR ligands and subsequent downstream events.

Phase 1/2a, open-label, dose-escalation and safety study of APC-100 in men with advanced prostate cancer.

234 Background: Normal human prostate epithelium and prostate carcinoma exhibit high levels of oxidative stress, which is thought to play a role in the pathogenesis of prostate cancer. APC-100 (2,2,5,7,8-Pentamethyl-6-chromanol) is an oxidative stress modulator that reduces reactive oxygen species (ROS) in prostate cancer cells and inhibits growth of cultured androgen-dependent and independent human prostate cancer cells. We aimed to determine the maximum tolerated dose (MTD), dose-limiting toxicities (DLT), adverse events (AEs), clinical activity and pharmacokinetic (PK) parameters of APC-100 in men with castrate-resistant prostate cancer (CRPC). Methods: This open label phase 1/2a study utilizes a time-to-event reassessment method (TITE-CRM) design. Patients (pts) in cohorts of 3 were treated with escalating doses of APC-100 (900mg-2400mg) orally once daily continuously in 150 mg capsules. Cycles were 28 days. All patients were evaluable with PSAs and imaging studies. Results: Twenty pts (median age 72 [range 54-87]) with metastatic and non-metastatic CRPC were enrolled in the dose escalation cohort. Pts remained on study for a median of 12 weeks (range 4-36) and received therapy for a median of 3 cycles (range 1-9). One possible DLT (elevated ALT) was seen at dose level 1 and the patient was taken off study. No other DLTs at least possibly related to therapy were seen and no dose reductions were required. Most frequent AEs at least possibly related to therapy included nausea (grade 1 in 6 pts) and elevated transaminases (grade 1-3 in 5 patients). After enrolment of 20 pts the MTD was not reached, however the maximal feasible dose was exceeded due to the number of capsules required. Five of the 20 patients had stable disease per PSA and/or RECIST 1.1 as their best response. The median progression free survival (PFS) for the cohort was 2.8 months (range 1-8). Conclusions: APC-100 appears to be well tolerated with the MTD not reached. Limited PK assessment supports the need for alternative formulation to allow dose escalation to doses necessary to achieve exposures required for response. Once MTD is established, future studies will explore the activity of APC-100 as a therapeutic and chemopreventive intervention. Clinical trial information: NCT01436214.

Cytotoxicity of obacunone and obacunone glucoside in human prostate cancer cells involves Akt-mediated programmed cell death.

Obacunone and obacunone glucoside (OG) are naturally occurring triterpenoids commonly found in citrus and other plants of the Rutaceae family. The current study reports the mechanism of cytotoxicity of citrus-derived obacunone and OG on human androgen-dependent prostate cancer LNCaP cells. Both limonoids exhibited time- and dose-dependent inhibition of cell proliferation, with more than 60% inhibition of cell viability at 100 μM, after 24 and 48 h. Analysis of fragmentation of DNA, activity of caspase-3, and cytosolic cytochrome-c in the cells treated with limonoids provided evidence for activation of programmed cell death by limonoids. Treatment of LNCaP cells with obacunone and OG resulted in dose-dependent changes in expression of proteins responsible for the induction of programmed cell death through the intrinsic pathway and down-regulation of Akt, a key molecule in cell signaling pathways. In addition, obacunone and OG also negatively regulated an inflammation-associated transcription factor, androgen receptor, and prostate-specific antigen, and activated proteins related to the cell cycle, confirming the ability of limonoids to induce cytotoxicity through multiple pathways. The results of this study provided, for the first time, an evidence of the cytotoxicity of obacunone and OG in androgen-dependent human prostate cancer cells.

Abstract 3312: The antibiotic salinomycin cotargets two pivotal pathways in prostate cancer

Abstract Background: The androgen receptor (AR) and PI3K/AKT/mTOR signaling axis are two key pathways that are activated in prostate cancer (PC). Although interference with the AR axis is the common first-line treatment for controlling metastatic recurrent PC, fast-growing PC progresses rapidly on hormone therapy and chemotherapy to a terminal condition within 18 to 24 months. Inhibitors of the PI3K/AKT/mTOR axis, such as rapamycin and the dual inhibitor BEZ-235 (Novartis), are ineffective in treating metastatic PC in part due to feedback regulations that activate the AR axis. Reciprocally, inhibition of AR activity reduces the expression of an AKT phosphatase, which is an AR target gene, with consequent increase of the AKT kinase activity. The antibiotic salinomycin, a product of the bacterium Streptomyces albus, has known anti-neoplastic activity, and in a screen of 16,000 small molecules, salinomycin was found to be the most potent in causing apoptosis of breast cancer stem cells. We previously reported that salinomycin treatment elevated intracellular oxidative stress and induced growth arrest of PC cells due to apoptosis. Design and Results: Here we report further elaboration of the underlying mechanism for growth arrest of androgen-dependent (LNCaP) and castration-resistant (C4-2B) human prostate cancer cells by salinomycin at sub-micromolar concentrations. We report that salinomycin can significantly reduce mTOR activity in both LNCaP and C4-2B cells without altering the mTOR activity of non-malignant RWPE-1 prostate epithelial cells. The specificity of the salinomycin effect is evidenced by the result that other anti-cancer agents such as docetaxel, the plant lignan deoxypodophyllotoxin and vitamin D did not alter mTOR activity. Preliminary results suggest that salinomycin may also induce autophagy in PC cells. Importantly, AR expression was markedly reduced in salinomycin-treated prostate cancer cells, suggesting dual targeting of the AR pathway and PI3K/AKT/mTOR axis by this antibiotic. Conclusion: We conclude that salinomycin targets two pivotal pathways that promote prostate cancer progression. Unlike other PI3K/AKT/mTOR inhibitors, the AR-activating feedback cross regulation did not compromise the therapeutic potential of salinomycin. Salinomycin-targeted interventions of the survival signals for castration-resistant PC will further avoid chemotherapy-induced toxicity. Preclinical validation of our results will pave the way for testing the efficacy of salinomycin against prostate cancer in a clinical setting. Supported by a Merit-Review grant from the Department of Veterans Affairs. Citation Format: Nooshin Mirkheshti, Chung Song, Bandana Chatterjee. The antibiotic salinomycin cotargets two pivotal pathways in prostate cancer. [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 3312. doi:10.1158/1538-7445.AM2014-3312

Antischistosomal versus Antiandrogenic Properties of Aryl Hydantoin Ro 13-3978

In the early 1980s, the antischistosomal aryl hydantoin Ro 13-3978 (AH01), a close structural analogue of the androgen receptor antagonist nilutamide, was discovered. Administration of 100 mg/kg oral doses of AH01 to mice infected with adult and juvenile Schistosoma mansoni produced 95% and 64% total worm burden reductions, confirming its high activity against adult worms, and showing that AH01 is also effective against juvenile infections. AH01 had no measureable interaction with the androgen receptor in a ligand competition assay, but it did block dihydrotestosterone-induced cell proliferation in an androgen-dependent human prostate cancer cell line. For AH01, nilutamide, and three closely related aryl hydantoin derivatives, there was no correlation between antischistosomal activity and androgen receptor interaction.

Preclinical efficacy of growth hormone-releasing hormone antagonist MIA-602 for androgen-dependent and castration-resistant human prostate cancer.

221 Background: Advanced hormone-sensitive prostate cancer (PCa) responds to androgen deprivation therapy (ADT). However, therapeutic options for castration-resistant disease are limited. As growth hormone-releasing hormone receptor (GHRH-R) and ligand GHRH are regulated in an autocrine fashion in PCa, GHRH-R inhibition represents a novel approach to PCa treatment. We investigated the effects of a new, highly potent GHRH antagonist, MIA-602, on growth of androgen-dependent and castration-resistant PCa cells in vitro/in vivo. Methods: All three cell lines used in this study expressed androgen receptors (ARs). 22Rv1 cells are castration-resistant and also express clinically relevant AR splice variants. LNCaP and VCaP lines are androgen dependent models that progress to castration resistance following ADT. Protein and mRNA levels of GHRH-R and its biologically active splice variant, SV1, were evaluated in cell lines and tumors by immunoblot and real-time RT-PCR. The influence of MIA-602 on cell proliferation and tumor formation was examined. Results: GHRH-R and SV1 were present in 22Rv1, LNCaP, and VCaP. LNCaP and VCaP cells expressed higher levels of GHRH-R protein compared to 22Rv1. However, 22Rv1 expressed higher levels of SV1. Inhibition of GHRH-R using MIA-602 decreased cell proliferation in vitro of 22Rv1, LNCaP, and VCaP PCa cell lines respectively by 70.4%, 60.7% and 20.3% (P<0.05 for all). MIA-602 decreased 22Rv1 xenograft volumes in mice by 63% after 3 weeks of treatment. VCaP showed a substantial inhibition of xenograft growth following therapy with MIA-602 in vivo. MIA-602 effectively inhibited VCaP xenografts as a single agent or in combination with ADT by surgical castration. Conclusions: The effectiveness of the novel Miami class GHRH antagonist, MIA-602, in inhibiting growth of androgen-dependent and castration-resistant PCa models in vitro and in vivo was demonstrated. The inhibitory effect of GHRH antagonists appear to be due to effects exerted through GHRH receptors on cancer cells and/or possibly by indirect mechanisms. Further investigations of GHRH antagonists for PCa treatment are warranted.

Activation of RhoA,B,C by Yersinia Cytotoxic Necrotizing Factor (CNFy) Induces Apoptosis in LNCaP Prostate Cancer Cells

Prostate cancer is the most common malignancy, accounting for about 25% of all incident cases among men in industrialized countries. The human androgen-dependent prostate cancer cell line LNCaP, which is derived from a metastatic lesion of human prostatic adenocarcinoma, is frequently used to study prostate cancer associated signaling pathways in vitro. Recently it was described that Rho GTPase activation in these cells leads to apoptotic responses. We used the bacterial toxins CNFy and CNF1, which specifically and directly activate Rho GTPases by deamidation of a single glutamine. We asked whether these Rho activators could induce apoptosis in LNCaP cells. Our results indicate that RhoA activation, induced by CNFy, does lead to intrinsic apoptosis of the cells. Analysis of the underlying signaling pathway reveals that apoptosis induction requires the activity of Rho kinase (ROCK) and myosin activation, an apoptotic pathway previously identified in cancer stem cells.

Antiproliferative, antiandrogenic and cytotoxic effects of novel caffeic acid derivatives in LNCaP human androgen-dependent prostate cancer cells

Caffeic acid and its naturally occurring derivative caffeic acid phenethyl ester (CAPE) have antiproliferative and cytotoxic properties in a variety of cancer cell lines without displaying significant toxicity toward healthy cells, and are considered to be potential anticancer agents. However, little is known about their effects on prostate cancer cells. We synthesized and evaluated the effects of caffeic acid, CAPE (2) and 18 synthetic derivatives on cell viability and androgen-dependent cell proliferation, subcellular localisation and expression of androgen receptor (AR) and secretion of prostate-specific antigen (PSA) in LNCaP human hormone-dependent prostate cancer cells. Several synthetic derivatives of CAPE were strong, concentration-dependent cytotoxic agents in LNCaP cells with IC50 values in the 6.8–26.6μM range, potencies that were up to five-fold greater than that of CAPE (33.7±4.0μM). A number of caffeic acid derivatives were inhibitors of androgen-stimulated LNCaP cell proliferation with concomitant inhibition of DHT-stimulated PSA secretion. Compound 24 was the most cytotoxic and antiproliferative caffeic acid derivative (IC50 values of 6.8±0.3 and 2.4±0.8μM, respectively) inhibiting DHT-stimulated cell proliferation and PSA secretion statistically significantly at concentrations as low as 0.3μM. Exposure to DHT increased cytoplasmic and nuclear AR levels and co-treatment with increasing concentrations of compound 24 or CAPE (2), notably, further increased these levels. In conclusion, a number of synthetic derivatives of caffeic acid are potent inhibitors of androgen-dependent prostate cancer cell proliferation and viability, acting, at least in part, via an antiandrogenic mechanism that involves increased nuclear accumulation of (presumably inactive) AR.

Of mice and men‐warning: Intact versus castrated adult male mice as xenograft hosts are equivalent to hypogonadal versus abiraterone treated aging human males, respectively

Immune deficient male mice bearing human prostate cancer xenografts are used to evaluate therapeutic response to novel androgen ablation approaches and the results compared to surgical castration based upon assumption that testosterone microenvironment in intact and castrated adult male mice mimics eugonadal and castrated aging adult human males. To test these assumptions, serum total testosterone (TT) and free testosterone (FT) were determined longitudinally in groups (n > 20) of intact versus castrated adult male nude, NOG, and immune competent C57BL/6 mice. In adult male mice, TT and FT varies by 30- to 100-fold within the same animal providing a microenvironment that is only equivalent to hypogonadal, not eugonadal, adult human males (TT is 1.7 ± 1.2 ng/ml [5.8 ± 4.1 nM] in nude and 2.5 ± 1.3 ng/ml [8.7 ± 4.4 nM] in NOG mice versus >4.2 ng/ml [14.7 nM] in eugonadal humans). This was confirmed based upon enhanced growth of androgen dependent human prostate cancer xenografts inoculated into mice supplemented with exogenous testosterone to elevate and chronically maintain serum TT at a level (5 ng/ml [18 nM]) equivalent to a 50-year-old eugonadal human male. In castrated mice, TT and FT range from 2 to 20 pg/ml (7-70 pM) and <0.8 pg/ml (<2.6 pM), respectively, which is equivalent to castrate resistant prostate cancer (CRPC) patients treated with abiraterone. This was confirmed based upon the inability of another CYP17A1 inhibitor, ketoconazole, to inhibit the growth of CRPC xenografts in castrated mice. Adult male mice supplemented with testosterone mimic eugonadal human males, while unsupplemented animals mimic standard androgen ablation and castrated animals mimic abiraterone treated patients. These studies confirm what is claimed in Robert Burns' poem "To a Mouse" that "The best laid schemes of mice and men/often go awry.".