Androgen-dependent Prostate Cancer Cells Research Articles

Innovative C2-symmetric testosterone and androstenedione dimers: Design, synthesis, biological evaluation on prostate cancer cell lines and binding study to recombinant CYP3A4.

The synthesis of two isomeric testosterone dimers and an androstenedione dimer is reported. The design takes advantage of an efficient transformation of testosterone leading to the synthesis of the key diene, 7α-(buta-1,3-dienyl)-4-androsten-17β-ol-3-one, through an elimination reaction. It was found that in some instances the same reaction led to partial epimerization of the 17β-hydroxyl group into the 17α-hydroxyl group. The specific orientation of the hydroxyl function was confirmed by NMR spectroscopy. Capitalizing on this unforeseen side reaction, several dimers were assembled using an olefin metathesis reaction with Hoveyda-Grubbs catalyst. This led to the formation of two isomeric testosterone dimers with 17α-OH or 17β-OH (14α and 14β) as well as an androstenedione dimer (14). The new dimers and their respective precursors were tested on androgen-dependent (LNCaP) and androgen independent (PC3 and DU145) prostate cancer cells. It was discovered that the most active dimer was made of the natural hormone testosterone (14β) with an average IC50 of 13.3μM. In LNCaP cells, 14β was ∼5 times more active than the antiandrogen drug cyproterone acetate (IC50 of 12.0μM vs. 59.6μM, respectively). At low concentrations (0.25-0.5μM), 14α and 14β were able to completely inhibit LNCaP cell growth induced by testosterone or dihydrotestosterone. Furthermore, cross-reactivity of androgen-based dimers with sterol-metabolizing cytochrome P450 3A4 was explored and the results are disclosed herein.

Alnus sibirica Compounds Exhibiting Anti-Proliferative, Apoptosis-Inducing, and GSTP1 Demethylating Effects on Prostate Cancer Cells.

Alnus sibirica (AS) is distributed in Korea, Japan, China, and Russia and has reported anti-oxidant, anti-inflammatory, and reducing activities on atopic dermatitis-like skin lesions, along with other beneficial health properties. In the present study, we tried to prove the cancer-preventive activity against prostate cancer. The extracted and isolated compounds, oregonin (1), hirsutenone (2), and hirsutanonol (3), which were isolated from AS, were tested for anti-proliferative activity. To do this, we used the MTT assay; NF-κB inhibitory activity, using Western blotting; apoptosis-inducing activity using flow cytometry; DNA methylation activity, using methylation-specific polymerase chain reaction in androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cell lines. The compounds (1–3) showed potent anti-proliferative activity against both prostate cancer cell lines. Hirsutenone (2) exhibited the strongest NF-κB inhibitory and apoptosis-inducing activities compared with oregonin (1) and hirsutanonol (3). DNA methylation activity, which was assessed for hirsutenone (2), revealed a concentration-dependent enhancement of the unmethylated DNA content and a reduction in the methylated DNA content in both PC-3 and LNCaP cells. Overall, these findings suggest that hirsutenone (2), when isolated from AS, may be a potential agent for preventing the development or progression of prostate cancer.

Receptor Tyrosine Kinase‐like Orphan Receptor 1 (ROR1) as a Selective Molecular Target in Androgen‐Dependent and Androgen‐Independent Prostate Cancer

Prostate cancer is a disease that has a high prevalence and mortality amongst men. The severity of the cancer is stratified by risk, which is based on the Gleason grade, prostate-specific antigen (PSA) level, and clinical staging. Unlike breast cancer which can be classified into subtypes based on molecular heterogeneity, prostate cancer has no such classification and is therefore treated with non-specific chemotherapy and radiation if local, and androgen deprivation therapy (ADT) if metastatic. It is of the utmost importance to investigate molecular targets that can specifically eradicate prostate cancer while retaining the functions of normal tissue. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a receptor that is highly expressed in embryogenesis and cancer, while being minimally expressed in normal tissue. In particular, prostate cancer exhibits moderate to high levels of ROR1 expression. The goal of this study is to elucidate the role of ROR1 in aggressive prostate cancer and selectively target it for treatment. Our laboratory had previously characterized Strictinin, a ROR1 inhibitor, as being selectively cytotoxic against TNBC via inhibition of the ROR1 receptor. We hypothesize that Strictinin can reduce the proliferation and invasion of prostate cancer and may potentially serve as a specific therapeutic agent. Preliminary results indicate that Strictinin is selectively lethal to androgen-dependent and androgen-independent prostate cancer cell lines. Strictinin treatment reduced expression of proteins downstream of ROR1 implicated in promoting migration, invasion, and anti-apoptosis. Furthermore, Strictinin treatment decreased migration and invasion while inducing apoptosis in PC3 and LnCAP cell lines, providing evidence that inhibition of the ROR1 molecular pathway is linked to reduction of cancerous phenotypes. These preliminary results indicate that ROR1 could be a novel molecular target that can be utilized for subtyping and treating prostate cancer. The data from this study will establish Strictinin as a potential therapeutic that targets ROR1 to reduce migration, invasion, and survival of prostate cancer.

The MYH9 Cytoskeletal Protein Is a Novel Corepressor of Androgen Receptors.

In the progression of castration-resistant prostate cancer (CRPC), the androgen receptor (AR) that serves as a transcription factor becomes the most remarkable molecule. The transcriptional activity of AR is regulated by various coregulators. As a result, altered expression levels, an aberrant location or activities of coregulators promote the development of prostate cancer. We describe herein results showing that compared with androgen-dependent prostate cancer (ADPC) cells, AR nuclear translocation capability is enhanced in androgen-independent prostate cancer (AIPC) cells. To gain insight into whether AR coregulators are responsible for AR translocation capability, we performed coimmunoprecipitation (CO-IP) coupled with LC-MS/MS to screen 27 previously reported AR cofactors and 46 candidate AR cofactors. Furthermore, one candidate, myosin heavy chain 9 (MYH9), was identified and verified as a novel AR cofactor. Interestingly, the distribution of MYH9 was in both the cytoplasmic and nuclear compartments yet was enriched in the nucleus when AR was knocked down by AR shRNA, suggesting that the nuclear translocation of MYH9 was negatively regulated by AR. In addition, we found that blebbistatin, an inhibitor of MYH9, not only promoted AR nuclear translocation but also enhanced the expression of the AR target gene PSA, which indicates that MYH9 represses nuclear AR signaling. Taken together, our findings reveal that MYH9 appears to be a novel corepressor of AR plays a pivotal role in the progression of CRPC.

Acetylation of interferon regulatory factor-5 suppresses androgen receptor and downregulates expression of Sox2.

Interferon regulatory factor-5 (IRF5) is a transcription factor and has essential cellular mechanisms as a tumour suppressor gene. IRF5 protein function is irregular in various human cancers, and its role in prostate cancer is also unknown. This study presents the first evidence that IRF5 expression is controlled with androgen receptor (AR) signalling interaction and stem cell factors (Nanog, Oct4, Sox2) in prostate cancer. Human prostate cancer cell lines (PC3, DU145 and LNCaP) were transfected plasmids and assessed for cellular localization of IRF5 and AR interaction with IF-staining. Co-immunoprecipitation and ChIP assay were used to detect the IRF5 and AR protein-protein interaction and IRF5 stem cell factors protein-gene interaction. The target relation between IRF5, AR, CREB, p300, ISRE, ARE and NF-кB was tested by luciferase assay. IRF5 was low expressed in androgen-dependent prostate cancer cells and tissues. The analysis of human prostate cancer clinical samples supports the interaction of IRF5 and AR in a pathological role, as IRF5 expression is down-regulated in the tumours' advanced stages. Tumour suppression mechanism of IRF5 and SOX2 levels in cells reduces and causes AR acetylation. Those affect the prostate cancer mechanism by modifying the cellular response in the signal pathway. IRF5 can be promising for treating androgen-dependent prostate cancers and is a therapeutic protein for new drug studies.

Cross-Resistance to Abiraterone and Enzalutamide in Castration Resistance Prostate Cancer Cellular Models Is Mediated by AR Transcriptional Reactivation.

Simple SummaryProstate cancer is the second most common cancer in males. In prostate cancer cells, androgens bind and activate the intracellular mediator called Androgen Receptor that control cell proliferation and survival. Hormone deprivation therapy is administrated to reduce androgen levels and consequently tumour growth. Unfortunately, most patients develop resistance to hormone treatment over the years and novel hormonal agents, such as Abiraterone or Enzalutamide, are administered. However, many patients do not initially respond or become resistant to these drugs quickly. Firstly, we demonstrated that in hormonal sensitive human prostate cancer cells the combination therapy of Abiraterone plus Enzalutamide reduced cell growth and survival. Moreover, starting from these prostate cancer cell lines, we generated cellular models of resistance to hormonal deprivation alone or in combination with the novel hormonal agents. In all the cases, resistant cell lines restore Androgen Receptor expression, Androgen Receptor functionality, cell proliferation and migration in the absence of androgens. Importantly, these novel cellular models acquire cross-resistance to each other. These results are consistent with clinical trials in castration resistant prostate cancer patients and suggest the biological rationale to test the combination therapy of Abiraterone plus Enzalutamide as first-line treatment in hormone-sensitive prostate cancer patients before becoming hormonal resistant.Androgen deprivation therapy (ADT) and novel hormonal agents (NHAs) (Abiraterone and Enzalutamide) are the goal standard for metastatic prostate cancer (PCa) treatment. Although ADT is initially effective, a subsequent castration resistance status (CRPC) is commonly developed. The expression of androgen receptor (AR) alternative splicing isoforms (AR-V7 and AR-V9) has been associated to CRPC. However, resistance mechanisms to novel NHAs are not yet well understood. Androgen-dependent PCa cell lines were used to generate resistant models to ADT only or in combination with Abiraterone and/or Enzalutamide (concomitant models). Functional and genetic analyses were performed for each resistance model by real-time cell monitoring assays, flow cytometry and RT-qPCR. In androgen-dependent PCa cells, the administration of Abiraterone and/or Enzalutamide as first-line treatment involved a critical inhibition of AR activity associated with a significant cell growth inhibition. Genetic analyses on ADT-resistant PCa cell lines showed that the CRPC phenotype was accompanied by overexpression of AR full-length and AR target genes, but not necessarily AR-V7 and/or AR-V9 isoforms. These ADT resistant cell lines showed higher proliferation rates, migration and invasion abilities. Importantly, ADT resistance induced cross-resistance to Abiraterone and/or Enzalutamide. Similarly, concomitant models possessed an elevated expression of AR full-length and proliferation rates and acquired cross-resistance to its alternative NHA as second-line treatment.

The lncRNA PTTG3P promotes the progression of CRPC via upregulating PTTG1

Overexpression of certain long non-coding RNAs (lncRNAs) promotes the progression of castration-resistant prostate cancer (CRPC). The significance and potential role of the lncRNA designated pituitary tumour-transforming 3, pseudogene (PTTG3P) in CRPC is unknown. We detected PTTG3P expression by qPCR. Upregulated PTTG3P expression was performed to explore the role of PTTG3P in PCa cells resistant to ADT (androgen deprivation therapy). The relationship among PTTG3P, mir-146a-3p and PTTG1 were validated by qPCR, western blot and luciferase assay. PTTG3P levels were significantly increased in the androgen-independent PC cell lines, as well as in CRPC tissues compared with those of the androgen-dependent prostate cancer cell line LNCaP and tumour tissues of patients with hormone-naive prostate cancers. Enforced expression of PTTG3P in androgen-deprived LNCaP cells significantly enhanced survival, clonogenicity, and tumorigenicity. Further, PTTG3P acted as a competing endogenous RNA (ceRNA, natural miRNA sponge) to upregulate PTTG1 expression by competing for mir-146a-3p in the progression to CRPC. Our findings suggest that PTTG3P promotes the resistance of prostate cancer cells to androgen-deprivation therapy via upregulating PTTG1. PTTG3P may therefore represent a potential target for therapy of CRPC.

Identification of novel androgen receptor degrading agents to treat advanced prostate cancer.

Prostate cancer (PCa) is one of the most common malignancies affecting men worldwide. Androgen receptor (AR) has been a target of PCa treatment for nearly six decades. AR antagonists/degraders can effectively treat PCa caused by increased AR overexpression. However, all approved AR antagonists have similar chemical structures and exhibit the same mode of action on the protein. Although initially effective, resistance to these AR antagonists usually develops. Therefore, this calls for the identification of novel chemical structures of AR antagonists to overcome the resistance. Herein, we employed the synergetic combination of virtual and experimental screening to identify a flavonoid compound which not only effectively inhibits AR transcriptional activity, but also induces the degradation of the protein. Based on this compound, we designed and synthesized a series of derivatives. We discovered that the most potent compound 10e could effectively inhibit AR transcriptional activity, and possessed a profound ability to cause degradation of both full length- and ARv7 truncated forms of human AR. Notably, 10e efficiently inhibited the growth of ARv7 dependent prostate cancer cell-lines, which are completely resistant to all current anti-androgens. Compound 10e also showed strong antitumor activity in the LNCaP (androgen dependent prostate cancer cell line) invivo xenograft model. These results provide a foundation for the development of a new class of AR antagonists.

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.

Abstract P07: Inhibition of androgen-regulated TMPRSS2 and lipogenic enzymes in prostate and lung carcinoma cell lines by a cisplatin prodrug

Abstract The main causative agent for the global pandemic of COVID-19 is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Developing therapeutic strategies to stop the virus is the hour of need. According to the recent clinical reports, it is seen that an androgen-regulated host cell serine protease TMPRSS2 acts on the spike protein of the SARS-CoV-2 virus which interacts with the host angiotensin-converting enzyme 2 (ACE2) and enters the host cell to cause the infection. Reports also suggest that TMPRSS2 is regulated by androgen present in prostate cells and it is highly expressed in PCa patients. Our lab has recently synthesized a new cisplatin prodrug which is a conjugate of lauric acid and cisplatin that potentially works very effectively in various androgen dependent and independent prostate cancer (PCa) cells. The cisplatin prodrug unlike other conventional platinum drugs is involved in inhibition of one of the major metabolic pathways of the PCa cells. Preliminary results show that, the prodrug in combination with the anti-androgen bicalutamide has an increased inhibition on the expression of TMPRSS2 in androgen dependent PCa and lung carcinoma cells along with down-regulation of some the lipogenic enzymes in-vitro. Here, we propose that the prodrug inhibits one of the mitochondrial metabolic pathways making the PCa cells sensitive towards cisplatin-based chemotherapy along with reducing the expression of TMPRSS2. Once completed, our work will provide an inside story of cisplatin prodrug mediated alteration of lipogenesis of cells in PCa tumor microenvironment resulting in a platform that has the potential to reduce the burden of cancer aggressiveness in both androgen dependent and independent PCa and also can be used as a potent chemotherapeutic agent against COVID-19. Citation Format: Subham Guin, Bapurao Surnar, Uttara Basu, Shanta Dhar. Inhibition of androgen-regulated TMPRSS2 and lipogenic enzymes in prostate and lung carcinoma cell lines by a cisplatin prodrug [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr P07.

Androgen-dependent prostate cancer cells reprogram their metabolic signature upon Glut-1 upregulation by Manganese Superoxide Dismutase (Mnsod/SOD2)

Androgen-dependent prostate cancer cells reprogram their metabolic signature upon Glut-1 upregulation by Manganese Superoxide Dismutase (Mnsod/SOD2)

Disulfiram-copper activates chloride currents and induces apoptosis with tyrosine kinase in prostate cancer cells.

Aim: To elucidates the mechanism that disulfiram/copper complex (DSF/Cu) treatment activates chloride channels and induces apoptosis in prostate cancer cells.Methods: Cellular membrane currents were measured by membrane clamp technique; western blot to detect protein expression; flow cytometry to detect apoptosis; immunofluorescence to detect target protein co‐localization, and further validated by a combination of protein‐protein interaction and mock protein molecular docking techniques.Results: DSF/Cu activated chloride channels and induced apoptosis in LNCaP (a type of androgen‐dependent prostate cancer cells) cells. The chloride currents activated by DSF/Cu were significantly reduced after knockdown of CLC3 with siRNA. In addition, DSF/Cu‐activated chloride currents were reduced to background current levels after perfusion with genistein, a highly specific tyrosine kinase inhibitor. Conversely, DSF/Cu failed to activate chloride currents in LNCaP cells after 30 minutes of pre‐incubation with genistein. When genistein was removed, and DSF/Cu was added, the activated currents were small and unstable, and gradually decreased. Immunofluorescence in LNCaP cells also showed co‐localization of the CLC3 protein with tyrosine kinase 2β (PTK2B).Conclusion: DSF/Cu can activate chloride channels and induce apoptosis in LNCaP cells with the involvement of tyrosine kinase. These results provide new insights into the target therapy of prostate cancer.

The potent AMPK inhibitor BAY-3827 shows strong efficacy in androgen-dependent prostate cancer models.

5' adenosine monophosphate-activated kinase (AMPK) is an essential regulator of cellular energy homeostasis and has been associated with different pathologies, including cancer. Precisely defining the biological role of AMPK necessitates the availability of a potent and selective inhibitor. High-throughput screening and chemical optimization were performed to identify a novel AMPK inhibitor. Cell proliferation and mechanistic assays, as well as gene expression analysis and chromatin immunoprecipitation were used to investigate the cellular impact as well as the crosstalk between lipid metabolism and androgen signaling in prostate cancer models. Also, fatty acid turnover was determined by examining lipid droplet formation. We identified BAY-3827 as a novel and potent AMPK inhibitor with additional activity against ribosomal 6 kinase (RSK) family members. It displays strong anti-proliferative effects in androgen-dependent prostate cancer cell lines. Analysis of genes involved in AMPK signaling revealed that the expression of those encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), fatty acid synthase (FASN) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), all of which are involved in lipid metabolism, was strongly upregulated by androgen in responsive models. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) analysis identified several androgen receptor (AR) binding peaks in the HMGCR and PFKFB2 genes. BAY-3827 strongly down-regulated the expression of lipase E (LIPE), cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) and serine-threonine kinase AKT3 in responsive prostate cancer cell lines. Also, the expression of members of the carnitine palmitoyl-transferase 1 (CPT1) family was inhibited by BAY-3827, and this was paralleled by impaired lipid flux. The availability of the potent inhibitor BAY-3827 will contribute to a better understanding of the role of AMPK signaling in cancer, especially in prostate cancer.

Exposure of androgen mimicking environmental chemicals enhances proliferation of prostate cancer (LNCaP) cells by inducing AR expression and epigenetic modifications

Exposure to environmental endocrine disrupting chemicals (EDCs) is highly suspected in prostate carcinogenesis. Though, estrogenicity is the most studied behavior of EDCs, the androgenic potential of most of the EDCs remains elusive. This study investigates the androgen mimicking potential of some common EDCs and their effect in androgen-dependent prostate cancer (LNCaP) cells. Based on the In silico interaction study, all the 8 EDCs tested were found to interact with androgen receptor with different binding energies. Further, the luciferase reporter activity confirmed the androgen mimicking potential of 4 EDCs namely benzo[a]pyrene, dichlorvos, genistein and β-endosulfan. Whereas, aldrin, malathion, tebuconazole and DDT were reported as antiandrogenic in luciferase reporter activity assay. Next, the nanomolar concentration of androgen mimicking EDCs (benzo[a]pyrene, dichlorvos, genistein and β-endosulfan) significantly enhanced the expression of AR protein and subsequent nuclear translocation in LNCaP cells. Our In silico studies further demonstrated that androgenic EDCs also bind with epigenetic regulatory enzymes namely DNMT1 and HDAC1. Moreover, exposure to these EDCs enhanced the protein expression of DNMT1 and HDAC1 in LNCaP cells. These observations suggest that EDCs may regulate proliferation in androgen sensitive LNCaP cells by acting as androgen mimicking ligands for AR signaling as well as by regulating epigenetic machinery. Both androgenic potential and epigenetic modulatory effects of EDCs may underlie the development and growth of prostate cancer.

Exosomes Promote the Transition of Androgen-Dependent Prostate Cancer Cells into Androgen-Independent Manner Through Up-Regulating the Heme Oxygenase-1.

BackgroundCastration-resistant prostate cancer (CRPC) is still considered incurable, even though the mechanisms of CRPC had been extensively researched. Studies have demonstrated that exosomes in the tumor microenvironment contribute to prostate cancer development and progression. However, the role of exosomes in the process of CRPC progression has not yet been determined.MethodsCo-culturing and exosome treatment assays combined with in vitro and in vivo assays were performed to determine the function of exosomes in the transformation of androgen-dependent prostate cancer (ADPC) cells into androgen-independent cells. Then, the mRNA expression profiles of ADPC cells and ADPC cells co-cultured with androgen-independent prostate cancer (AIPC) cell-derived exosomes were studied using microarrays. After silencing the expression of heme oxygenase-1 (HMOX1), Western blotting, quantitative real-time PCR, immunohistochemistry (IHC) studies, and MTS assay were used to confirm the mechanisms of exosome participation in CRPC progression.ResultsThe results showed that ADPC cells acquired tolerance for androgen deprivation due to the exosome-mediated communication between cells. AIPC cell-derived exosomes promoted the transformation of ADPC cells into androgen-independent cells in vivo and in vitro. Microarray analysis revealed that HMOX1 in ADPC cells was up-regulated after treatment with AIPC cell-derived exosomes. Further results showed that HMOX1 is overexpressed in human AIPC specimens and protects ADPC cells from androgen deprivation.ConclusionsOur findings revealed that exosomes contribute to CRPC progression via promoting the transition of prostate cancer cells into an androgen-independent growth stage by activating HMOX1.

MYBL2 disrupts the Hippo-YAP pathway and confers castration resistance and metastatic potential in prostate cancer.

Rationale: Resistance to androgen-deprivation therapy (ADT) associated with metastatic progression remains a challenging clinical task in prostate cancer (PCa) treatment. Current targeted therapies for castration-resistant prostate cancer (CRPC) are not durable. The exact molecular mechanisms mediating resistance to castration therapy that lead to CRPC progression remain obscure.Methods: The expression of MYB proto-oncogene like 2 (MYBL2) was evaluated in PCa samples. The effect of MYBL2 on the response to ADT was determined by in vitro and in vivo experiments. The survival of patients with PCa was analyzed using clinical specimens (n = 132) and data from The Cancer Genome Atlas (n = 450). The mechanistic model of MYBL2 in regulating gene expression was further detected by subcellular fractionation, western blotting, quantitative real-time PCR, chromatin immunoprecipitation, and luciferase reporter assays.Results: MYBL2 expression was significantly upregulated in CRPC tissues and cell lines. Overexpression of MYBL2 could facilitate castration-resistant growth and metastatic capacity in androgen-dependent PCa cells by promoting YAP1 transcriptional activity via modulating the activity of the Rho GTPases RhoA and LATS1 kinase. Importantly, targeting MYBL2, or treatment with either the YAP/TAZ inhibitor Verteporfin or the RhoA inhibitor Simvastatin, reversed the resistance to ADT and blocked bone metastasis in CRPC cells. Finally, high MYBL2 levels were positively associated with TNM stage, total PSA level, and Gleason score and predicted a higher risk of metastatic relapse and poor prognosis in patients with PCa.Conclusions: Our results reveal a novel molecular mechanism conferring resistance to ADT and provide a strong rationale for potential therapeutic strategies against CRPC.

Chemosensitizing effects of methyl jasmonate on paclitaxel-resistant androgen-dependent and independent prostate cancer cell lines

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 taxane-related 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.

Puerarin induces apoptosis in prostate cancer cells via inactivation of the Keap1/Nrf2/ARE signaling pathway

ABSTRACT In this study, we examined the antitumor effects of Puerarin (PEU) on androgen-independent (DU145 and PC-3) and androgen-dependent (LNCaP) prostate cancer cells, and explored its potential mechanisms. Supplement with PEU (2.5 μM, 5 μM, and 10 μM) exhibited a marked inhibitory effect against the growth of DU145 and PC-3 cells, especially beyond 24 h, whereas there is only slight growth inhibitory effect on LNCaP cells at the high concentration of 10 μM at 72 h. This loss of cell viability in DU145 and PC-3 cells by PEU was mediated by the induction of apoptosis via up-regulation of Bax and cleaved-caspase-3, but downregulation of Bcl-2. Moreover, more intracellular ROS and LDH production were observed in DU145 and PC-3 cells upon PEU treatment. Meanwhile, the amount of pro-inflammatory cytokines (IL-1β and IL-6) was increased, but the content of anti-inflammatory cytokines IL-10 was attenuated. Additionally, PEU pretreatment resulted in an increase of Keap1 protein expression, and a decline of Nrf2, HO-1 and NQO1 protein expression in DU145 and PC3 cells. The present findings indicated that PEU exerted its antitumor activities toward androgen-independent prostate cancer cells via inactivation of Keap1/NrF2/ARE signaling pathway.

Betel Nut Arecoline Induces Different Phases of Growth Arrest between Normal and Cancerous Prostate Cells through the Reactive Oxygen Species Pathway.

Prostate cancer (PCa) is a reproductive system cancer in elderly men. We investigated the effects of betel nut arecoline on the growth of normal and cancerous prostate cells. Normal RWPE-1 prostate epithelial cells, androgen-independent PC-3 PCa cells, and androgen-dependent LNCaP PCa cells were used. Arecoline inhibited their growth in dose- and time-dependent manners. Arecoline caused RWPE-1 and PC-3 cell cycle arrest in the G2/M phase and LNCaP cell arrest in the G0/G1 phase. In RWPE-1 cells, arecoline increased the expression of cyclin-dependent kinase (CDK)-1, p21, and cyclins B1 and D3, decreased the expression of CDK2, and had no effects on CDK4 and cyclin D1 expression. In PC-3 cells, arecoline decreased CDK1, CDK2, CDK4, p21, p27, and cyclin D1 and D3 protein expression and increased cyclin B1 protein expression. In LNCaP cells, arecoline decreased CDK2, CDK4, and cyclin D1 expression; increased p21, p27, and cyclin D3 expression; had no effects on CDK1 and cyclin B1 expression. The antioxidant N-acetylcysteine blocked the arecoline-induced increase in reactive oxygen species production, decreased cell viability, altered the cell cycle, and changed the cell cycle regulatory protein levels. Thus, arecoline oxidant exerts differential effects on the cell cycle through modulations of regulatory proteins.

MiR-199b-5p-DDR1-ERK signalling axis suppresses prostate cancer metastasis via inhibiting epithelial-mesenchymal transition.

BackgroundThe investigation of underlying mechanism and the exploitation of novel therapies for metastatic prostate cancer (PCa) are still urgently needed. miR-199b-5p has been suggested to function as tumour suppressor in various human cancers. However, the clinical significance and role of miR-199b-5p in PCa remain unclear.MethodsThe current study sought to investigate the expression status of miR-199b-5p in PCa and the involved molecular mechanisms in PCa metastasis by using bioinformatics analyses, loss-and gain-of-functions and rescue experiments in vitro and in vivo.ResultsWe demonstrated that miR-199b-5p was significantly downregulated in metastatic PCa tissues and cells when compared with the normal prostate tissue, the localised disease, the weakly metastatic and androgen-dependent PCa cell and the normal prostate epithelial cell. We also found that miR-199b-5p drastically suppressed PCa cell proliferation, migration and invasion in vitro and inhibited xenografts tumour growth and metastasis in vivo. Mechanistically, our results showed that miR-199b-5p could inhibit discoidin domain receptor tyrosine kinase 1 (DDR1) expression by directly targeting its 3’-UTR, thereby hindering epithelial-mesenchymal transition (EMT)-associated traits, which were induced by DDR1 activating ERK signalling pathway. Moreover, PCa patients with low miR-199b-5p expression level had a remarkably shorter overall survival than those with high miR-199b-5p level, indicating an association of miR-199b-5p loss with poor prognosis in patients with PCa. Furthermore, DDR1 was upregulated in PCa, and significantly correlated with high Gleason score, advanced pathological stage, tumour metastasis and shorter overall survival.ConclusionsOur study, for the first time, provide evidence of a tumour-suppressive function of miR-199b-5p in the invasion and metastasis of PCa, supporting the translational exploitation of miR-199b-5p-based therapeutic approaches for PCa metastases. Also, the miR-199b-5p-DDR1-ERK signalling axis identified in this study represents a novel mechanism of regulating EMT in PCa metastases.