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Androgen deprivation therapy is commonly used for the treatment of prostate cancer. Enzalutamide is a next-generation androgen receptor inhibitor, initially approved to treat castration-resistance prostate cancer. Lupeol, a triterpene present in various fruits, vegetables, has anti-oxidant and anti-proliferative activity. The present study aimed to evaluate the Enzalutamide-induced toxicity and its possible amelioration by Lupeol. We performed multiple in vitro and in vivo experiments to conclude our hypothesis. The results revealed that both Enzalutamide and Lupeol interact with DNA through electrostatic interactions. Enzalutamide (5–20 μM) caused cytotoxicity in both normal (PNT2) and cancer cells (LNCaP and 22Rv1). However, Lupeol (10–50 μM) specifically killed the cancer cells while sparing normal cells. The study further revealed that Lupeol could attenuate Enzalutamide-induced cytotoxicity and genotoxicity (chromosomal aberrations and micronucleus formation) to normal cells and potentially induce cytotoxicity to transformed cells. We further observed that Lupeol (40 mg/kg) mediated attenuation of the Enzalutamide (10 mg/kg) induced oxidative and DNA damages. Our study also revealed that Lupeol reverses the Enzalutamide-induced hepatic and renal damages. In conclusion, our study indicates that Lupeol can be used as an adjuvant for reducing the toxic effects and enhancing the effectiveness of Enzalutamide. [ABSTRACT FROM AUTHOR]

Simple Summary: Prostate cancer (PCa) is one of the major cancers affecting men. Localized and loco-regional PCa is usually treated by radical prostatectomy, radiation therapy, cryosurgery or with HIFU (High-Intensity Focused Ultrasound). Advanced or metastatic cancers are most often treated with androgen deprivation therapy, but too often such patients progress to lethal castration-resistant PCa. IL-6 plays a key role in prostate cancer but no data on ciliary neurotrophic factor (CNTF), a member of interleukin-6 cytokine family, are known. We detected CNTF and its receptor CNTFRα in human androgen-responsive and in castration-resistant prostate cancer (CRPC) tissues. In addition, we showed that CNTF downregulated MMP-2 and GLUT-1 expression by MAPK/ERK, AKT/PI3K and Jak/STAT pathways in a CRPC in vitro model. This suggests a pivotal role of CNTF as negative modulator of invasion processes in this PCa model. Background: Prostate cancer (PCa) remains the most common diagnosed tumor and is the second-leading cause of cancer-related death in men. If the cancer is organ-confined it can be treated by various ablative therapies such as RP (radical prostatectomy), RT (radiation therapy), brachytherapy, cryosurgery or HIFU (High-Intensity Focused Ultrasound). However, advanced or metastatic PCa treatment requires systemic therapy involving androgen deprivation, but such patients typically progress to refractory disease designated as castration-resistant prostate cancer (CRPC). Interleukin-6 (IL-6) has been established as a driver of prostate carcinogenesis and tumor progression while less is known about the role of ciliary neurotrophic factor (CNTF), a member of the IL-6 cytokine family in prostate cancer. Moreover, MAPK/ERK, AKT/PI3K and Jak/STAT pathways that regulate proliferative, invasive and glucose-uptake processes in cancer progression are triggered by CNTF. Methods: We investigate CNTF and its receptor CNTFRα expressions in human androgen-responsive and castration-resistant prostate cancer (CRPC) by immunohistochemistry. Moreover, we investigated the role of CNTF in proliferative, invasive processes as well as glucose uptake using two cell models mimicking the PCa (LNCaP cell line) and CRPC (22Rv1 cell line). Conclusions: Our results showed that CNTF and CNTFRa were expressed in PCa and CRPC tissues and that CNTF has a pivotal role in prostate cancer environment remodeling and as a negative modulator of invasion processes of CRPC cell models. [ABSTRACT FROM AUTHOR]

The taxane-based therapy provides survival benefit in patients with metastatic prostate cancer; however, the average survival is less than 20 months due to the partial taxanerelated chemoresistance. Innovative strategies are needed to overcome the chemoresistance for improved patient survival. In this project, paclitaxel-resistance was developed on androgen-independent PC3 and androgen-dependent 22Rv1 and LNCaP human prostate cancer (PCa) cell lines to investigate the efficacy of methyl jasmonate (MeJa), an anti-cancer drug, in overcoming the chemoresistance. The PCa cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C under 5% CO2. The cell lines were exposed to the gradually increasing doses of paclitaxel. Since the resistance on LNCaP could not be achieved, the study was continued with 22Rv1 cell line. It was demonstrated that paclitaxel-resistant cell lines overexpress ABCB1. Resistance levels of cells and MeJa activity in all resistant and parental lines were measured using CellTiter-Glo® luminescent assay. Test results were compared with Student's t-test or analysis of variance (ANOVA). P=0.05 (two-tailed) was considered to be significant. In conclusion, MeJa showed more cytotoxicity on paclitaxel resistant PC3 (PC3-PtxR) cells than resistant 22Rv1 (22Rv1-PtxR) cells. Detection of cytotoxic effects of MeJa in overcoming paclitaxel resistance may contribute to the development of alternative new compounds for the prevention or chemosensitization of resistance to chemotherapeutics such as paclitaxel. [ABSTRACT FROM AUTHOR]

Objective: The present work aimed at developing novel hybrid molecules for targeting the prostate cancer. It is observed that two human shock proteins Hsp70 and Hsp90 are over-expressed in prostate cancer making them one of the important drug targets. We have designed and developed twelve new hybrid molecules 6a-j for targeting these proteins. Methods: The designed molecules were prepared following a four step reaction protocol and characterized on the basis of proton NMR and Mass spectrometry. These were subjected to in vitro studies by means of Oncotest and CCK-8 assays with two cell lines DU145 and 22Rv1. The selected molecules 6b and 6i were subjected to molecular docking and then for SPR based affinity assay. Results: Compounds 6b and 6i were found to be highly active anticancer compounds comparable to standard drug enzalutamide. They have significant IC50 and high dock score for the Hsp70 and Hsp90. These compounds are selective and have good binding affinity for the Hsp70 due to high Kd. Conclusion: Compound 6b and 6i can serve as lead molecules for the development of antiprostate cancer drugs with Hsp70 as target. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is the most frequently diagnosed cancer and the third highest cause of cancer-related deaths in men in the U.S. The development of chemotherapeutic agents that can bind PCa tumor cells with high specificity is critical in order to increase treatment effectiveness. Integrin receptors and their corresponding ligands have different expression patterns in PCa cells. They have been identified as promising targets to inhibit pathways involved in PCa progression. Currently, several compounds have proven to target specific integrins and their subunits in PCa cells. In this article, we review the role of integrins inhibitors in PCa and their potential as therapeutic targets for PCa treatments. We have discussed the following: natural compounds, monoclonal antibodies, statins, campothecins analog, aptamers, D-aminoacid, and snake venom. Recent studies have shown that their mechanisms of action result in decrease cell migration, cell invasion, cell proliferation, and metastasis of PCa cells. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is one of the most common malignancies among men and is the second leading cause of cancer-associated mortality in the developed world. Androgen deprivation therapy (ADT) is the most common treatment for PCa. However, the majority of androgen-sensitive PCa patients will eventually develop resistance to ADT and the disease will become androgen-independent. There is, therefore, an immediate requirement to develop effective therapeutic techniques towards the treatment of recurrent PCa. Oxibendazole (OBZ) is an anthelmintic drug that has also shown promise in the treatment of malignancies. In the present study, the capability of OBZ to repress the growth of PCa cells was assessed in human androgen-independent PCa 22Rv1 and PC-3 cell lines. The growth of the 22Rv1 and PC-3 cell lines, as assessed with a trypan blue exclusion assay, was markedly inhibited by OBZ treatment in vitro, with half-maximal inhibitory concentration values of 0.25 and 0.64 µM, respectively. The mean size of 22Rv1 tumors in nude mice treated with OBZ (25 mg/kg/day) was 47.96% smaller than that of the control mice. Treatment with OBZ increased the expression of microRNA-204 (miR-204), as determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the level of p53 as determined with western blotting, two well-characterized tumor suppressor genes. When miR-204 expression was knocked down by introduction of an miR-204 inhibitor, the inhibitory effect of OBZ was markedly reduced; however, when it was overexpressed, the inhibitory efficiency of OBZ was markedly higher, indicating that upregulation of miR-204 is key for the efficacy of OBZ. Additionally, OBZ was demonstrated with RT-qPCR to repress the expression of the androgen receptor, and by western blotting to reduce prostate-specific androgen in 22Rv1 cells. The results suggest that OBZ has potential for clinical use in the treatment of recurrent PCa. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is a prevalent cause of morbidity and mortality. DHRS2-modified human umbilical cord mesenchymal stem cells-derived exosomes (hUC-MSCs-derived exos) function in PCa. We explored the mechanism of DHRS2-modified hUC-MSCs-derived exos in PCa cell malignant behaviors. DHRS2 expression levels in WPMY-1 cells and 4 PCa cell lines were detected by RT-qPCR and Western blot. 22Rv1/DU145 cells with high/low DHRS2 expression were selected to establish the low/high DHRS2 expression models by transfection. Cell proliferation and apoptosis were detected by CCK-8, colony formation assays, and flow cytometry. hUC-MSCs were identified by oil red O, alizarin staining, and flow cytometry. Exos were extracted from hUC-MSCs by ultracentrifugation and identified by transmission electron microscopy, Nano series-Nano-ZS, and Western blot. DU145 cells were selected for in vitro study to further study the effects of DHRS2-modified exos on cell proliferation and apoptosis. The effect of DHRS2-modified exos on cell cycle distribution was detected by flow cytometry. DHRS2 was repressed in PCa cells. DHRS2 overexpression suppressed PCa cell proliferation and promoted apoptosis. Exos were successfully isolated from hUC-MSC. DHRS2-modified hUC-MSCs-derived exos carried DHRS2 into PCa cells and blocked malignant behaviors. Briefly, DHRS2 was repressed in PCa cells. DHRS2-modified hUC-MSCs-derived exos blocked PCa cell proliferation and enhanced apoptosis. • DHRS2 is lowered in PCa cells. • DHRS2 overexpression represses PCa cell proliferation and promotes apoptosis. • DHRS2-modified hUC-MSCs-exos internalized by PCa cells increase DHRS2 level. • DHRS2-modified hUC-MSCs-derived exos inhibit Pca cell malignant behaviors. • DHRS2-modified hUC-MSCs-exos induce DU145 cell cycle G0/G1 arrest. [ABSTRACT FROM AUTHOR]

Abstract: Prostate cancer (PCa) progression is enhanced by androgen and treatment with antiandrogens represents an alternative to castration. While patients initially respond favorably to androgen ablation therapy, most experience a relapse of the disease within 1–2 years by expressing androgen receptor (AR) mutants. Such mutations, indeed, promote unfavorable agonistic behavior from classical antagonists. Here, we have synthesized and screened 37 novel compounds derived from dihydrotestosterone (DHT), cyanolutamide and hydroxyflutamide. These derivatives were tested for their potential antagonistic activity using a luciferase reporter gene assay and binding properties were determined for wild type (WT) and mutant ARs (T877A, W741C, W741L, H874Y). In the absence and presence of antiandrogens, androgen dependent cellular proliferation and prostate specific antigen (PSA) expression were assayed in the prostate cancer cell line LNCaP by crystal violet, real time PCR and by Western blots. Also, cellular proliferation and PSA expression were assayed in 22Rv1. A novel compound RB346, derived from DHT, was found to be an antagonist for all tested AR forms, preventing DHT induced proliferation and PSA expression in LNCaP and 22Rv1 cells. RB346 displayed no agonistic activity, in contrast to the non-steroidal antiandrogen bicalutamide (Casodex®) with unfavorable agonistic activity for W741L-AR. Additionally, RB346 has a slightly higher binding affinity for WT-AR, T877A-AR and H874Y-AR than bicalutamide. Thus, RB346 is the first potent steroidal antiandrogen with efficacy for WT and various AR mutants. [Copyright &y& Elsevier]

Integrins are transmembrane proteins that facilitate the interaction of cells with the extracellular environment. They have also been implicated in cancer progression. The effects of nutrients thought to be involved in the prevention of prostate cancer on integrin expression have not been determined. Prostate cancer cell lines representing a range of malignancy from normal (RWPE-1) to highly invasive phenotypes (22Rv1 < LNCaP < PC-3) were cultured with or without lycopene (10 nm), vitamin E (5 μm) or fish oil (100 μm) for 48 h. Growth and integrin (α2β1, αvβ3 and αvβ5) expression were assessed using Trypan Blue exclusion and monoclonal antibodies combined with flow cytometry. Vitamin E enhanced (P < 0·001) whereas fish oil reduced the growth of all the cell lines tested (P < 0·001). Lycopene had no effect on growth. All the malignant cell lines exhibited lower expression of α2β1 with the addition of lycopene to culture media. Supplemental fish oil reduced α2β1 in most invasive cell lines (LNCaP and PC-3). Each nutrient at physiological levels reduced integrins αvβ3 and αvβ5 in most invasive cell lines (PC-3). The results suggest that integrins may represent an additional target of bioactive nutrients and that the effects of nutrients may be dependent on the type of cell line used. [ABSTRACT FROM PUBLISHER]

Abstract: In order to select a better prostate cancer cell model for androgen receptor (AR)-mediated reporter gene assays, we assessed the androgen response characteristics of three cell lines, LNCaP, PC3/AR+ and 22Rv1, in this study. Both the mRNA and the proteins of AR and glucocorticoid receptor (GR) were expressed in all three cell lines. Among the three cell lines, only in LNCaP cells, DHT concentration-dependently stimulated proliferation. DHT induced the luciferase activity in three cell lines which were transiently transfected with pMMTV-Luc, in a concentration-dependent manner. The maximum induction was 24.0-fold and 13.4-fold in 22Rv1 and in the LNCaP respectively. PC3/AR+ were more sensitive to respond to DHT at a minimal concentration of 10−12 M by 14.0-fold induction. The transcriptional activity induced with 10−8 M DHT was inhibited about 50–75% in the PC3/AR+ and 22Rv1, and 98% in the LNCaP, by vinclozolin. Dexamethasone concentration-dependently induced the luciferase activity in PC3 and 22Rv1, but not in the LNCaP. However, the response to dexamethasone in 22Rv1 was very weak compared to DHT. The (anti)androgencity of seven pyrethroids was assessed via an AR-mediated luciferase reporter assay. None of them showed the androgenic action in all three cell lines. Permethrin inhibited the DHT induced luciferase activity about 22%, 35.8% and 75.5% in 22Rv1, PC3/AR+ and LNCaP, respectively. Based on results from in this study and cell line character, 22Rv1 cells seemed to be an appropriate model for the screening of androgenic endocrine disruptors, although it needs further studies with other steroid receptor and thyroid receptor. [Copyright &y& Elsevier]

Abstract: The androgen receptor (AR) is a member of the nuclear receptor superfamily and mediates the physiological effects of androgens. Androgens are essential for male development and disruption of androgen signaling may cause androgen-dependent developmental defects and/or tumors. Here we present a comparative analysis of various model systems for the investigation of endocrine active compounds in human cell lines. We generated reporter plasmids containing androgen response elements derived from the human secretory component or the rat probasin genes as well as the glucocorticoid consensus response element and compared their activities to that of the mouse mammary tumor virus promotor. Additionally, we generated an expression plasmid containing the AR cDNA derived from LNCaP cells. In 22Rv1 cells transiently transfected with human AR, all reporters displayed a dose-dependent, high activity when treated with androgens. Interestingly, the potency of testosterone and its metabolite dihydrotestosterone was very low in HepG2 but not in 22Rv1 cells, independent of the reporter used. The efficacies of the androgens tested were comparable in both cell lines but highly dependent on the reporter used. Based on these results, 22Rv1 cells provide a highly sensitive in vitro test system to analyze endocrine activities of xenobiotics. Furthermore, this study highlights the need to investigate the (anti-) androgenic activity of compounds in dependence of the cellular and promoter context. [Copyright &y& Elsevier]

2,4-Dichlorophenoxyacetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (DCP) are used extensively in agriculture as herbicides, and are suspected of potential endocrine disruptor activity. In a previous study, we showed that these compounds exhibited synergistic androgenic effects by cotreatment with testosterone in the Hershberger assay. To elucidate the mechanisms of the synergistic effects of these compounds on the androgenicity of testosterone, the androgenic action of 2,4-D and DCP was characterized using a mammalian detection system in prostate cancer cell lines. In in vitro assay systems, while 2,4-D or DCP alone did not show androgenic activity, 2,4-D or DCP with 5α-dihydroxytestosterone (DHT) exhibited synergistic androgenic activities. Cotreatment of 10 nM 2,4-D or DCP with 10 nM DHT was shown to stimulate the cell proliferation by 1.6-fold, compared to 10 nM DHT alone. In addition, in transient transfection assays, androgen-induced transactivation was also increased to a maximum of 32-fold or 1.28-fold by cotreatment of 2,4-D or DCP with DHT, respectively. However, 2,4-D and DCP exerted no effects on either mRNA or protein levels of AR. In a competitive AR binding assay, 2,4-D and DCP inhibited androgen binding to AR, up to 50% at concentrations of approximately 0.5 μM for both compounds. The nuclear translocation of green fluorescent protein-AR fusion protein in the presence of DHT was promoted as the result of the addition of 2,4-D and DCP. Collectively, these results that 2,4-D and DCP enhanced DHT-induced AR transcriptional activity might be attributable, at least in part, to the promotion of AR nuclear translocation. [ABSTRACT FROM AUTHOR]

[Display omitted] Multiple Splice variants of AR have been reported in the past few years. These splice variants are upregulated in most cases of CRPC resulting in poor prognosis. Most of these variants lack the ligand binding domain (LBD) but still bind to DNA resulting in constitutive activation of downstream targets. The AR-V7 splice variant has been characterized extensively and current clinical trials in CRPC are exploring the use of AR-V7 as a biomarker. New therapeutic molecules that selectively target AR-V7 are also being explored. However, there is a dearth of information available on the selectivity, phenotypic responses in AR-V7 dependent cell lines and pharmacokinetic properties of such molecules. Using our proprietary computational algorithms and rational SAR optimization, we have developed a potent and selective AR-V7 degrader from a known AR DNA binding domain (DBD) binder. This molecule effectively degraded AR-V7 in a CRPC cell line and demonstrated good oral bioavailability in mouse PK studies. This tool compound can be used to evaluate the pharmacological effects of AR-V7 degraders. Further exploration of SAR can be pursued to develop more optimized lead compounds. [ABSTRACT FROM AUTHOR]

Prostate-specific membrane antigen (PSMA)-directed radioligand therapy (RLT) prolongs overall survival in men with metastatic castration-resistant prostate cancer (mCRPC). However, men with low PSMA expression are excluded from RLT. We explored the effect of androgen receptor blockade with enzalutamide on PSMA expression. Assessment of PSMA and androgen receptor (AR) expression on the human PC cell lines 22Rv1, C4-2, and LNCaP by immunohistochemistry and flow cytometry revealed low (22Rv1) and high (C4-2 and LNCaP) PSMA expression, and high, comparable AR positivity. Treatment with enzalutamide increased PSMA levels in 22Rv1, C4-2, and LNCaP (2.2/2.3/2.6-fold, p = 0.0005/0.03/0.046) after one week compared to DMSO-treated controls as assessed by flow cytometry. NOD/Scid mice bearing 22Rv1 tumors were treated with enzalutamide for two weeks. Positron emission tomography/computed tomography (PET/CT) demonstrated higher tumor uptake of 68Ga-PSMA after enzalutamide treatment (p = 0.004). Similarly, a clinical case with low baseline PSMA avidity demonstrated increased uptake of 68Ga-PSMA after enzalutamide on PET/CT and post-therapeutic 177Lu-PSMA scintigraphy in a patient with mCRPC. Enzalutamide induced PSMA expression in the 22Rv1 xenograft model and in an mCRPC patient, both with low baseline tumoral PSMA levels. Therefore, enzalutamide pre-treatment might render patients with low PSMA expression eligible for 177Lu-PSMA RLT. [ABSTRACT FROM AUTHOR]

Linalool is an unsaturated terpene that can be found in several plants and exhibits various biological activities. The aim of the present study was to investigate the anticancer activity of linalool using the human prostate cancer 22Rv1 cell line. Flow cytometry was employed to study the effects of linalool on the induction of apoptosis, cell cycle progression, loss of mitochondrial membrane potential and cytochrome c release, whereas the effects of linalool on apoptosis-associated proteins were investigated by western blot analysis. An efficacy study was conducted using 22Rv1 tumor-bearing mice. The expression of the cell proliferation markers Ki-67 and proliferating cell nuclear antigen (PCNA) in xenograft tumors was evaluated by immunohistochemistry. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to study the induction of apoptosis in an in vivo model. Linalool exerted an inhibitory effect on 22Rv1 cell proliferation and induced apoptosis in both in vitro and in vivo models. Western blot analysis indicated that both the mitochondria-mediated intrinsic and death-receptor-mediated extrinsic pathways were involved in the induction of apoptosis. Furthermore, linalool significantly reduced the expression of Ki-67 and PCNA in the 22Rv1 ×enograft model. The findings of the present study provide evidence supporting the anti-proliferative effects of linalool on 22Rv1 human prostate cancer cells, and suggest that linalool may be an effective agent for prostate cancer treatment. [ABSTRACT FROM AUTHOR]

Elongation factor for RNA polymerase II 2 (ELL2) and ELL-associated factor 2 (EAF2) are two functionally related androgen responsive gene-encoded proteins with prostate tumor suppressor characteristics. EAF2 and ELL2 have both been shown to be down-regulated in advanced prostate cancer, and mice with either Eaf2 or Ell2 deficiency developed murine prostatic intraepithelial neoplasia (mPIN), increased cellular proliferation and increased vascularity. Functional studies have revealed that EAF2 and ELL2 can bind to each other and have similar roles in regulating cell proliferation, angiogenesis and prostate homeostasis. Here, cell line experiments showed that knockdown of EAF2 or ELL2 induced an increase in proliferation and migration in C4-2 and 22Rv1 prostate cancer cells. Concurrent knockdown of EAF2 and ELL2 increased proliferation and migration similarly to the loss of EAF2 or ELL2 alone. Mice with homozygous deletion of Ell2 or heterozygous deletion of Eaf2 developed mPIN lesions characterized by increased epithelial proliferation, intraductal microvessel density, and infiltrating intraductal CD3-positive T-cells compared to wild-type controls. Mice with combined heterozygous deletion of Eaf2 and Ell2 developed mPIN lesions that were similar to those observed in mice with deficiency in Eaf2 or Ell2 alone. These results suggest that EAF2 and ELL2 have similar functions and are likely to require each other in their regulation of prostate epithelial cell proliferation and migration in prostate cancer cells as well as their tumor suppressive properties in the murine prostate., Competing Interests: None.

Mycophenolate Mofetil (MYC) is a transplant drug used to prevent rejection in heart and kidneys transplant patients. Inosine monophosphate dehydrogenase (IMPDH), an enzyme involved in de novo synthesis of guanosine nucleotides, was considered as a primary target for MYC. Recently, we described that MYC was activates aryl hydrocarbon receptor and it antagonizes glucocorticoid receptor. Here we describe an androgen receptor (AR) as another off-target for MYC. We found that MYC increased basal and dihydrotestosterone (DHT)-inducible AR-dependent luciferase activity in AIZ-AR cells. In the same manner it induced or augmented mRNA level of KLK3 (prostate specific antigen; PSA) in 22Rv1 cells. Herein it displayed a hormetic effect on proliferation activity, since it significantly stimulated proliferation in lower concentrations but inhibited in higher (>1 µg/ml) concentrations in the presence of DHT. In contrast, MYC suppressed DHT-inducible KLK3 mRNA expression and cell proliferation in androgen-dependent LNCaP cells. MYC augmented DHT-inducible nuclear translocation of AR and increased the expression of MAPK8/9 (JNK46/54) resulting in the drop of their phosphorylation status. Moreover, MYC sensitized DHT-treated 22Rv1 cells to JNK-IN-8 mediated growth inhibition with the drop of IC 50 from 1425 nM to 84 nM within 24 hrs. In conclusion, we suggest that, castrate-resistant prostate cancers progression might be retarded with the combination of MYC and chemical JNK inhibitors, involving AR-dependent mechanism., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.