Development of β-amino-carbonyl compounds as androgen receptor antagonists

Aim： Androgen receptor （AR） antagonists have proven to be useful in the early control of prostate cancer. The aim of this study was to identify and characterize a novel β-amino-carbonyl-based androgen receptor antagonist. Methods Different isomers of the β-amino-carbonyl compounds were obtained by chiral separation. The bioactivities of the isomers were evaluated by AR nuclear translocation, mammalian two-hybrid, competitive receptor binding and cell proliferation assays. The expression of genes downstream of AR was analyzed with real-time PCR. The therapeutic effects on tumor growth in vivo were observed in male SClD mice bearing LNCaP xenografts. Results： Compound 21 was previously identified as an AR modulator by the high-throughput screening of a diverse compound library. In the present study, the two isomers of compound 21, termed compounds 21-1 and 2.1.-2, were characterized as partial AR agonists in terms of androgen-induced AR nuclear translocation, prostate-specific antigen expression and cell proliferation. Further structural modifications led to the discovery of a androgen receptor antagonist （compound 6012）, which blocked androgen receptor nuclear translocation, androgen-responsive gene expression and androgen-dependent LNCaP cell proliferation. Four stereoisomers of compound 6012 were isolated, and their bioactivities were assessed. The pharmacological effects of 6012, including AR binding, androgen-induced AR translocation, NH2- and COOH-terminal interaction, growth inhibition of LNCaP cells in vitro and LNCaP xenograft growth in nude mice, were mainly restricted to isomer 6012-4 （1R, 3S）. Conclusion： Compound 6012-4 was determined to be a novel androgen receptor antagonist with prostate cancer inhibitory activities comparable to bicalutamide both in vitro and in vivo.     

Non-THC cannabinoids inhibit prostate carcinoma growth in vitro and in vivo: pro-apoptotic effects and underlying mechanisms

BACKGROUND AND PURPOSE

Cannabinoid receptor activation induces prostate carcinoma cell (PCC) apoptosis, but cannabinoids other than Δ⁹-tetrahydrocannabinol (THC), which lack potency at cannabinoid receptors, have not been investigated. Some of these compounds antagonize transient receptor potential melastatin type-8 (TRPM8) channels, the expression of which is necessary for androgen receptor (AR)-dependent PCC survival.

EXPERIMENTAL APPROACH

We tested pure cannabinoids and extracts from Cannabis strains enriched in particular cannabinoids (BDS), on AR-positive (LNCaP and 22RV1) and -negative (DU-145 and PC-3) cells, by evaluating cell viability (MTT test), cell cycle arrest and apoptosis induction, by FACS scans, caspase 3/7 assays, DNA fragmentation and TUNEL, and size of xenograft tumours induced by LNCaP and DU-145 cells.

KEY RESULTS

Cannabidiol (CBD) significantly inhibited cell viability. Other compounds became effective in cells deprived of serum for 24 h. Several BDS were more potent than the pure compounds in the presence of serum. CBD-BDS (i.p.) potentiated the effects of bicalutamide and docetaxel against LNCaP and DU-145 xenograft tumours and, given alone, reduced LNCaP xenograft size. CBD (1–10 µM) induced apoptosis and induced markers of intrinsic apoptotic pathways (PUMA and CHOP expression and intracellular Ca²⁺). In LNCaP cells, the pro-apoptotic effect of CBD was only partly due to TRPM8 antagonism and was accompanied by down-regulation of AR, p53 activation and elevation of reactive oxygen species. LNCaP cells differentiated to androgen-insensitive neuroendocrine-like cells were more sensitive to CBD-induced apoptosis.

CONCLUSIONS AND IMPLICATIONS

These data support the clinical testing of CBD against prostate carcinoma.

LINKED ARTICLE

This article is commented on by Pacher et al., pp. 76–78 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02121.x     

Indirect activation of the SV23 and SV24 splice variants of human constitutive androstane receptor: analysis with 3-hydroxyflavone and its analogues

BACKGROUND AND PURPOSE

Naturally occurring splice variants of human CAR (hCAR), including hCAR-SV23 (insertion of amino acids SPTV) and hCAR-SV24 (APYLT), have been shown to be expressed in liver. However, little is known regarding how hCAR-SV23 and hCAR-SV24 are activated. Therefore, we investigated the mode of activation of these hCAR splice variants.

EXPERIMENTAL APPROACH

Cell-based reporter gene assays, including ligand-binding domain transactivation assays and coactivator recruitment assays, were conducted on cultured HepG2 cells transfected with various constructs and treated with 3-hydroxyflavone or a hydroxylated (galangin, datiscetin, kaempferol, morin, quercetin or myricetin) or methylated (isorhamnetin, tamarixetin, or syringetin) analogue.

KEY RESULTS

Among the flavonols investigated, only 3-hydroxyflavone increased hCAR-SV23 and hCAR-SV24 activities. 3-Hydroxyflavone did not transactivate the ligand-binding domain of these isoforms or recruit steroid receptor coactivators (SRC-1, SRC-2, or SRC-3). By comparison, 3-hydroxyflavone, galangin, datiscetin, kaempferol, quercetin, isorhamnetin and tamarixetin activated hCAR-WT, whereas none of the flavonols activated hCAR-SV25 (both SPTV and APYLT insertions). The flavonols 3-Hydroxyflavone, galangin, quercetin and tamarixetin transactivated the ligand-binding domain of hCAR-WT, but only 3-hydroxyflavone recruited SRC-1, SRC-2 and SRC-3 to the receptor.

CONCLUSION AND IMPLICATIONS

hCAR-SV23 and hCAR-SV24 can be activated by a mechanism that does not involve the ligand-binding domain of the receptor or recruitment of SRC-1, SRC-2, or SRC-3. 3-Hydroxyflavone and its structural analogues activated hCAR in an isoform-selective and chemical-specific manner. Overall, our study provides insight into a novel mode of ligand activation of hCAR-SV23 and hCAR-SV24.     

cGMP-independent anti-tumour actions of the inhibitor of soluble guanylyl cyclase, ODQ, in prostate cancer cell lines

Background and purpose:

Soluble guanylyl cyclase (sGC) is a receptor for nitric oxide that generates cGMP. This second messenger molecule has established roles in cellular physiology; however, less is known about its effects in tumour cells.

Experimental approach:

The effects of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS2028), both selective sGC inhibitors on proliferation, death and migration were determined in prostate cancer cell lines.

Key results:

Western blot analysis confirmed the presence of α1 and β1 subunits of sGC in LNCaP and PC-3 cells. Sodium nitroprusside (SNP) increased cGMP accumulation in LNCaP and PC-3, but not DU-145 cells. SNP-stimulated cGMP production in LNCaP cells was dose-dependently reduced by ODQ, with more than 90% inhibition being observed at 0.1 μM. ODQ activated caspase-3 in all three cell lines, but not in normal prostate epithelial cells, at concentrations over 10 μM. High concentrations of ODQ also promoted DNA fragmentation and nucleosome accumulation in the cytosol of LNCaP cells. Interestingly, the chemically related inhibitor, NS2028 was without effect on caspase-3. In addition, ODQ inhibited LNCaP, Du145 and PC-3 cell growth. Finally, although fibroblast growth factor-2 did not enhance cGMP levels in LNCaP cells, its ability to stimulate LNCaP motility was abolished by ODQ.

Conclusions and implications:

These observations taken together suggest that the action of ODQ in LNCaP cells did not reflect sGC inhibition. We conclude that ODQ promotes cell death and inhibits growth and migration of prostate cancer cells and that these actions are independent of its effects on GMP levels.     

Tanshinones from Chinese Medicinal Herb Danshen (Salvia miltiorrhiza Bunge) Suppress Prostate Cancer Growth and Androgen Receptor Signaling

Purpose

To test whether tanshinones inhibit prostate cancer (PCa) growth at least in part through inhibiting androgen receptor (AR) signaling.

Methods

We evaluated cell growth, survival and AR signaling parameters of PCa cells after exposure to tanshinones in in vitro models. We also tested the in vivo inhibitory efficacy of tanshinone IIA (TIIA) against LNCaP xenograft model in athymic nude mice.

Results

For androgen-dependent LNCaP cells, a colony growth assay showed strong inhibitory potency following the order of TIIA??cryptotanshinone>tanshinone I, being 10?C30 folds higher than Casodex (racemic). TIIA inhibited growth of LNCaP cells more than several androgen-independent PCa cell lines. All 3 tested tanshinones were devoid of AR agonist activity under castrate condition. Mechanistically, tanshinones inhibited AR nuclear translocation within 2?h, decreased protein and mRNA abundance of AR and its target prostate-specific antigen within 12?h, and stimulated proteosomal degradation of AR. Oral administration of TIIA (25?mg/kg, once daily) retarded LNCaP xenograft growth and down-regulated tumor AR abundance in athymic nude mice.

Conclusion

AR targeting action of tanshinones was distinct from Casodex and contributed to prostate cancer growth suppression in vitro and in vivo.     

Retigeric acid B-induced mitophagy by oxidative stress attenuates cell death against prostate cancer cells in vitro

Aim:

Retigeric acid B (RAB), a pentacyclic triterpenic acid from Lobaria kurokawae Yoshim, has been found to induce apoptosis in prostate cancer cells. The aim of this study was to investigate the roles of mitochondrial damage-caused mitophagy in RAB-induced prostate cancer cell death in vitro.

Methods:

Human prostate cancer PC3 and LNCaP cells were tested. Cell viability was analyzed with MTT assay. Cell apoptosis, ROS level and mitochondrial transmembrane potential (mtΔψ) were measured with flow cytometry. Autophagy- and apoptosis-related proteins were studied using Western blotting. GFP-LC3B puncta, mitochondrial swelling and mitophagy were examined morphologically. Quantitative RT-PCR was used to measure LC3B mRNA level, and siRNA was used to knock down LC3BII.

Results:

In both PC3 and LNCaP cells, RAB (15 μmol/L) increased ROS accumulation and decreased mtΔψ in a time-dependent manner. Furthermore, RAB induced mitochondrial swelling and mitophagy, significantly increased LC3B expression and conversion of LC3BI to LC3BII, and the elimination of mitochondria by LC3BII-containing autophagolysosomes. In addition, RAB suppressed the PI3K/Akt/mTOR pathway activation. Pretreatment of PC3 cells with autophagy inhibitor 3-MA (5 mmol/L) or the lysosomal protease inhibitor CQ (10 μmol/L) significantly increased RAB-induced apoptosis. Similar results were obtained in RAB-treated PC3 cells with LC3B knocked down.

Conclusion:

RAB induces mitochondrial damage and mitophagy that attenuates RAB-induced prostate cancer cell death. Thus, suppression of mitophagy might be a potential strategy for improving the chemotherapeutic effects of RAB.     

Reactive oxygen species up-regulate p53 and Puma; a possible mechanism for apoptosis during combined treatment with TRAIL and wogonin

Background and purpose:

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptotic death in a variety of cancer cells without marked toxicity to most normal cells. We previously reported that wogonin, a potent anticancer agent from a Chinese herb, up-regulates p53 in prostate cancer cells. In this study, the effects of combinations of TRAIL and wogonin on a human prostate cancer cell line LNCaP, resistant to TRAIL, was evaluated for evidence of synergy in triggering apoptosis.

Experimental approach:

Western blot assay and the ‘comet’ assay were used to study the underlying mechanisms of cell death and search for any mechanisms of enhancement of TRAIL-induced apoptosis in the presence of wogonin.

Key results:

During combined treatment with wogonin and TRAIL, cytotoxicity, poly(ADP-ribose) polymerase cleavage and caspase activation were associated with up-regulation of p53 through DNA damage and reactive oxygen species (ROS) generation. N-acetylcysteine (NAC), an antioxidant, inhibited ROS generation and synergistic interaction between TRAIL and wogonin. Experimental results in human colon cancer HCT116 cells demonstrated that p53-dependent Puma up-regulation played an important role; deficiency in either p53 or Puma prevented wogonin-enhanced TRAIL-induced apoptosis.

Conclusions and implications:

The present studies suggest that wogonin enhances TRAIL-induced cytotoxicity through up-regulation of p53 and Puma, mediated by ROS.     

Highly frequent anti-idiotype antibody in cynomolgus monkeys developed against mouse-derived regions of anti-Fas antibody humanized by complementarity determining region grafting

Background and purpose:

We investigated the immunogenicity of a humanized anti-human Fas monoclonal antibody, R-125224, in cynomolgus monkeys to estimate its efficacy, as well as its toxicity in clinical situations.

Experimental approach:

R-125224 was intravenously administered to cynomolgus monkeys at single doses of 0.4, 1.2, 6 and 30 mg·kg⁻¹, and the plasma concentrations of R-125224 and anti-R-125224 antibody (ARA) were measured. We conducted a competitive enzyme-linked immunosorbent assay to determine which part of R-125224 was recognized by ARA. We also examined the retention of radioactivity in mononuclear cells and granulocytes after the injection of [¹²⁵I]-R-125224 to a collagen-induced arthritis monkey model.

Key results:

After i.v. administration of R-125224, the elimination of the plasma R-125224 concentrations was accelerated at around 10 days post-dose, and 10 of 12 monkeys were ARA positive. From an epitope analysis of ARA, the ARA produced in monkeys recognized the mouse-derived regions located in complementarity determining regions, but could not recognize the human IgG. After the injection of [¹²⁵I]-R-125224 to a collagen-induced arthritis monkey model, a significantly longer retention of the radioactivity in mononuclear cells compared to granulocytes was observed.

Conclusions and implications:

In monkeys, the development of antibodies against R-125224 is rapid and highly frequent. Our hypothesis is that this highly frequent development of ARA might be due to the binding of R-125224 to immune cells, and its circulation in monkey blood might contribute to an increase in its chances of being recognized as an immunogen.     

A comparison of androgen deprivation therapy versus surgical castration for patients with advanced prostatic carcinoma

Aim:

To examine the outcomes of patients with advanced prostate carcinoma who underwent medical or surgical castration.

Methods:

A hundred twenty one consecutive cases of patients with advanced prostate carcinoma who underwent medical or surgical castration between 2001 and 2006 were retrospectively reviewed. Associations between clinical outcomes and prognostic scoring factors were determined based on the Reijke study. In the surgical and medical castration groups, the impact on the prostate-specific antigen (PSA) normalization rate, the rebound rate and the disease-free survival rate were evaluated. The mean follow-up was 36.1 months.

Results:

In the initial 12 months, there were no statistical differences in the PSA normalization rate and the PSA rebound rate between the two groups. However, the PSA rebound rate after the 12th month (20.90% vs 40.74%, P=0.0175) and the 18th month PSA normalization rate (59.70% vs 37.04%, P=0.0217) differed significantly between the two groups, and these differences were maintained to the end of the study. When comparing patients grouped according to Reijke prognosis scores, there was no difference between medical and surgical castration for the good prognosis group. However, among the patients given a poor prognosis, surgical castration was superior in terms of the PSA normalization rate, the PSA rebound rate, the tumor progression-free survival rate (P<0.001) and the overall survival rate (P<0.001).

Conclusion:

Advanced prostate carcinoma patients with poor pretreatment prognosis scores should undergo surgical castration rather than medical castration for better PSA rebound rates and overall survival.     

Differential inhibition of tumour cell-induced platelet aggregation by the nicotinate aspirin prodrug (ST0702) and aspirin

BACKGROUND AND PURPOSE

Tumour cell-induced platelet aggregation (TCIPA) facilitates cancer cell invasion, angiogenesis and the formation of metastatic foci. TCIPA can be modulated by pharmacological inhibitors of MMP-2 and ADP; however, the COX inhibitor aspirin did not prevent TCIPA. In this study, we have tested the pharmacological effects of a new group of isosorbide-based aspirin prodrugs on TCIPA.

EXPERIMENTAL APPROACH

TCIPA was induced in human platelets by mixing with human adenocarcinoma or fibrosarcoma cells under no flow and flow conditions. The release of gelatinases and P-selectin expression during TCIPA were studied by zymography and flow cytometry respectively.

KEY RESULTS

Tumour cells caused platelet aggregation. This aggregation resulted in the release of MMP-2 and a significant up-regulation of P-selectin on platelets, indicative of platelet activation. Pharmacological modulation of TCIPA revealed that ST0702, one of the aspirin prodrugs, down-regulated TCIPA while aspirin was ineffective. The deacetylated metabolite of ST0702, 5-nicotinate salicylate (ST0702 salicylate), down-regulated both ADP-stimulated platelet aggregation and TCIPA.

CONCLUSIONS AND IMPLICATIONS

Our results show that ST0702 was an effective inhibitor of TCIPA in vitro. Its deacetylated metabolite may contribute to the effects of ST0702 by inhibiting ADP-mediated TCIPA.     

Synthetic (+)-antroquinonol exhibits dual actions against insulin resistance by triggering AMP kinase and inhibiting dipeptidyl peptidase IV activities

BACKGROUND AND PURPOSE

The fungal product (+)-antroquinonol activates AMP kinase (AMPK) activity in cancer cell lines. The present study was conducted to examine whether chemically synthesized (+)-antroquinonol exhibited beneficial metabolic effects in insulin-resistant states by activating AMPK and inhibiting dipeptidyl peptidase IV (DPP IV) activity.

EXPERIMENTAL APPROACH

Effects of (+)-antroquinonol on DPP IV activity were measured with a DPPIV Assay Kit and effects on GLP-1-induced PKA were measured in AR42J cells. Translocation of the glucose transporter 4, GLUT4, induced either by insulin-dependent PI3K/AKT signalling or by insulin-independent AMPK activation, was assayed in differentiated myotubes. Glucose uptake and GLUT4 translocation were assayed in L6 myocytes. Mice with diet-induced obesity were used to assess effects of acute and chronic treatment with (+)-antroquinonol on glycaemic control in vivo.

KEY RESULTS

The results showed that of (+)-antroquinonol (100 μM ) inhibited the DPP IV activity as effectively as the clinically used inhibitor, sitagliptin. The phosphorylation of AMPK Thr¹⁷² in differentiated myotubes was significantly increased by (+)-antroquinonol. In cells simultaneously treated with S961 (insulin receptor antagonist), insulin and (+)-antroquinonol, the combination of (+)-antroquinonol plus insulin still increased both GLUT4 translocation and glucose uptake. Further, (+)-antroquinonol and sitagliptin reduced blood glucose, when given acutely or chronically to DIO mice.

CONCLUSIONS AND IMPLICATIONS

Chemically synthesized (+)-antroquinonol exhibits dual effects to ameliorate insulin resistance, by increasing AMPK activity and GLUT4 translocation, along with inhibiting DPP IV activity.     

Involvement of microRNA-21 in mediating chemoresistance to docetaxel in androgen-independent prostate cancer PC3 cells

Aim:

To investigate whether microRNA-21 was involved in mediating the chemoresistance of prostate cancer cells to docetaxel.

Methods:

A microarray technique was used to determine the miRNA profile in docetaxel-resistant PC3 cells. Real-time PCR was used to confirm the array results. miR-21 mimics and inhibitors were synthesized and introduced to cells using Lipofectamine 2000. Cell proliferation was examined with the CCK-8 assay. Luciferase reporter containing PDCD 3′UTR was constructed and the activity was detected by a dual luciferase assay. PDCD4 protein expression was evaluated using Western blot.

Results:

A docetaxel-resistant prostate cancer PC3 cell line (PC3R) was established . Using microarrays, miR-21 was found to be up-regulated in PC3R cells. Ectopic expression of miR-21 increased the resistance to docetaxel in PC3 wild type cells. In contrast, silencing of miR-21 in PC3R cells sensitized the cells to docetaxel. The IC₅₀ values for miR-21-silencing cells and control cells were 28.31 and 35.89 nmol/L, respectively. PDCD4, a direct target gene of miR-21, could mediate chemoresistance to docetaxel in PC3 cells.

Conclusion:

Our findings suggest that miR-21 contributed to the resistance of PC3 cells to docetaxel, and that targeting miR-21 may offer a promising therapeutic approach in sensitizing prostate cancer to docetaxel treatment.     

Cyclic bisbibenzyls induce growth arrest and apoptosis of human prostate cancer PC3 cells

Aim:

To investigate the cytotoxic effects of four cyclic bisbibenzyls, Riccardin C (Ric), Pakyonol (Pak), Marchantin M (Mar), and Plagiochin E (Pla) against chemoresistant prostate cancer PC3 cells.

Methods:

Cell growth was assayed by MTT method, and apoptotic related protein Bcl-2 and Bax, poly(ADP-ribose) polymerase (PARP) were examined by Western blotting. Cell cycle and apoptosis of PC3 cells were evaluated with flow cytometry and morphologic examinations.

Results:

The four compounds inhibited proliferation and elicited cell death in a dose- and time-dependent manner with IC₅₀ values of 3.22 μmol/L for Ric, 7.98 μmol/L for Pak, 5.45 μmol/L for Mar, and 5.99 μmol/L for Pla, respectively. Furthermore, exposed to these chemicals caused a decrease in the antiapoptotic protein Bcl-2 and an increase in proapoptotic Bax expression. PARP cleavage and caspase-3 activity were also observed.

Conclusion:

The results suggest that cyclic bisbibenzyls could be used for the development of novel therapeutic chemicals against prostate cancer.