Proliferation Of 22Rv1 Cells Research Articles

Anticancer Effect of Dihydroartemisinin via Dual Control of ROS-induced Apoptosis and Protective Autophagy in Prostate Cancer 22Rv1 Cells.

Dihydroartemisinin (DHA), a natural agent, exhibits potent anticancer activity. However, its biological activity on prostate cancer (PCa) 22Rv1 cells has not been previously investigated. In this study, we demonstrate that DHA induces anticancer effects through the induction of apoptosis and autophagy. Cell viability and proliferation rate were assessed using the CCK-8 assay and cell clone formation assay. The generation of reactive oxygen species (ROS) was detected by flow cytometry. The molecular mechanism of DHA-induced apoptosis and autophagy was examined using Western blot and RT-qPCR. The formation of autophagosomes and the changes in autophagy flux were observed using transmission electron microscopy (TEM) and confocal microscopy. The effect of DHA combined with Chloroquine (CQ) was assessed using the EdU assay and flow cytometry. The expressions of ROS/AMPK/mTOR-related proteins were detected using Western blot. The interaction between Beclin-1 and Bcl-2 was examined using Co-IP. DHA inhibited 22Rv1 cell proliferation and induced apoptosis. DHA exerted its antiprostate cancer effects by increasing ROS levels. DHA promoted autophagy progression in 22Rv1 cells. Inhibition of autophagy enhanced the pro-apoptotic effect of DHA. DHA-induced autophagy initiation depended on the ROS/AMPK/mTOR pathway. After DHA treatment, the impact of Beclin- 1 on Bcl-2 was weakened, and its binding with Vps34 was enhanced. DHA induces apoptosis and autophagy in 22Rv1 cells. The underlying mechanism may involve the regulation of ROS/AMPK/mTOR signaling pathways and the interaction between Beclin-1 and Bcl-2 proteins. Additionally, the combination of DHA and CQ may enhance the efficacy of DHA in inhibiting tumor cell activity.

Identification of key enzalutamide-resistance-related genes in castration-resistant prostate cancer and verification of RAD51 functions.

Patients with castration-resistant prostate cancer (CRPC) often develop drug resistance after treatment with enzalutamide. The goal of our study was to identify the key genes related to enzalutamide resistance in CRPC and to provide new gene targets for future research on improving the efficacy of enzalutamide. Differential expression genes (DEGs) associated with enzalutamide were obtained from the GSE151083 and GSE150807 datasets. We used R software, the DAVID database, protein-protein interaction networks, the Cytoscape program, and Gene Set Cancer Analysis for data analysis. The effect of RAD51 knockdown on prostate cancer (PCa) cell lines was demonstrated using Cell Counting Kit-8, clone formation, and transwell migration experiments. Six hub genes with prognostic values were screened (RAD51, BLM, DTL, RFC2, APOE, and EXO1), which were significantly associated with immune cell infiltration in PCa. High RAD51, BLM, EXO1, and RFC2 expression was associated with androgen receptor signaling pathway activation. Except for APOE, high expression of hub genes showed a significant negative correlation with the IC50 of Navitoclax and NPK76-II-72-1. RAD51 knockdown inhibited the proliferation and migration of PC3 and DU145 cell lines and promoted apoptosis. Additionally, 22Rv1 cell proliferation was more significantly inhibited with RAD51 knockdown than without RAD51 knockdown under enzalutamide treatment. Overall, six key genes associated with enzalutamide resistance were screened (RAD51, BLM, DTL, RFC2, APOE, and EXO1), which are potential therapeutic targets for enzalutamide-resistant PCa in the future.

The roles of mutated SPINK1 gene in prostate cancer cells.

SPINK1-positive prostate cancer (PCa) has been identified as an aggressive PCa subtype. However, there is a lack of definite studies to elucidate the underlying mechanism of the loss of SPINK1 expression in most PCa cells except 22Rv1 cells, which are derived from a human prostatic carcinoma xenograft, CWR22R. The aim of this study was to investigate the mechanisms of SPINK1 protein positive/negative expression and its biological roles in PCa cell lines. SPINK1 mRNA was highly expressed in 22Rv1 cells compared with LNCaP, C4-2B, DU145, and PC-3 cells, and the protein was only detected in 22Rv1 cells. Among these cell lines, the wild-type SPINK1 coding sequence was only found in 22Rv1 cells, and two mutation sites, the c.194G>A missense mutation and the c.210T>C synonymous mutation, were found in other cell lines. Our further research showed that the mutations were associated with a reduction in SPINK1 mRNA and protein levels. Functional experiments indicated that SPINK1 promoted PC-3 cell proliferation, migration, and invasion, while knockdown of SPINK1 attenuated 22Rv1 cell proliferation, migration, and invasion. The wild-type SPINK1 gene can promote the malignant behaviors of cells more than the mutated ones. Cell cycle analysis by flow cytometry showed that SPINK1 decreased the percentage of cells in the G0/G1 phase and increased the percentage of S phase cells. We demonstrated that the c.194G>A and c.210T>C mutations in the SPINK1 gene decreased the mRNA and protein levels. The wild-type SPINK1 gene is related to aggressive biological behaviors of PCa cells and may be a potential therapeutic target for PCa.

Isoflurane Promotes Cell Proliferation, Invasion, and Migration by Regulating BACH1 and miR-375 in Prostate Cancer Cells In Vitro.

The aim of this study was to investigate the mechanism of isoflurane in proliferation, invasion, and migration in prostate cancer (PC) cells in vitro by regulating BACH1 and miR-375. The effect of different concentrations of isoflurane (0%, 0.5%, 1%, and 2%) on PC cell proliferation (PC3 and 22RV1) was measured. After PC cells and normal human prostate stromal immortalized WPMY-1 cells were treated with isoflurane, BACH1 and miR-375 expression was measured. Subsequently, PC3 and 22RV1 cells underwent gain- and loss-of-function assays with or without 4-h 2% isoflurane pretreatment. The levels of miR-375, BACH1, and PTEN were assessed. The binding of BACH1 to miR-375 promoter was detected by ChIP assay. Dual-luciferase reporter assay detected the targeting relationship of miR-375 with BACH1 and PTEN. Isoflurane promoted PC3 and 22RV1 cell proliferation. In addition, isoflurane elevated the levels of BACH1 and miR-375 in a dosage-dependent manner in PC cells. Transfection with miR-375 inhibitor or sh-BACH1 repressed PC cell proliferation, invasion, and migration, while exposure to 2% isoflurane for 4 h before transfection counteracted the inhibitory effects of sh-BACH1 or miR-375 inhibitor on PC cells. PTEN expression was suppressed after 2% isoflurane treatment, but the transfection with miR-375 inhibitor partly abrogated this suppressive effect in PC cells. Moreover, BACH1 bound to miR-375 and miR-375 negatively targeted PTEN. miR-375 mimic could partially reverse the inhibitory effects of sh-BACH1 on the proliferation, invasion, and migration of isoflurane-treated PC cells. Isoflurane facilitated PC cell proliferation, migration, and invasion by activating BACH1 to upregulate miR-375.

Prognostic Value of Long Noncoding RNA SNHG11 in Patients with Prostate Cancer.

The present study was aimed to explore the prognostic value of long noncoding RNA SNHG11 in prostate cancer, study its expression, and assess its effect on tumor progression. One hundred and twenty prostate cancer patients and 45 cases of benign prostate hyperplasia (BPH) patients were collected. RT-qPCR was used to test the expression of SNHG11 in prostate cancer and BPH tissues, as well as in cell lines. Kaplan-Meier survival analysis and Cox regression assays were introduced to evaluate the prognostic meaning of SNHG11 in prostate cancer. The CCK-8 assays were performed to explore the effect of SNHG11 on prostate cancer cell proliferation, and a Transwell assay was conducted to access the influence of SNHG11 on prostate cancer cell migration and invasion. SNHG11 expression level was upregulated both in prostate cancer tissues and cell lines. Overexpression of SNHG11 was significantly associated with Gleason score, clinical T stage, surgical margin status, and lymph node metastasis. Patients with high SNHG11 expression levels led to a shorter overall survival time and biochemical recurrence-free survival when compared with those of low expression levels. Multivariate Cox regression results suggested that SNHG11 has the potential to act as a prognostic marker for prostate cancer patients. Knockdown of SNHG11 suppressed 22RV1 cell proliferation, migration, and invasion. In conclusion, SNHG11 is upregulated in prostate cancer patients and predicts an unfavorable prognosis for prostate cancer patients. Its knockdown can weaken prostate cancer cell metastasis and growth in vitro.

Enhanced antitumor efficacy by combining afatinib with MDV3100 in castration-resistant prostate cancer.

Background: Patients with prostate cancer often develop resistance to androgen deprivation therapy, a condition called castration-resistant prostate cancer (CRPC). Enzalutamide (MDV3100) can prolong the survival of patients with CRPC after chemotherapy, but ∼50% of patients eventually relapse and develop resistance to MDV3100. Thus, it is necessary to explore new treatment methods to improve the therapeutic effect of MDV3100. Tyrosine kinases play an essential role in the pathogenesis of CRPC. Methods: MTT assay was used to detect the inhibitory effects of MDV3100 and tyrosine kinase inhibitor on prostate cancer cells. CompuSyn version 1.0 was used to calculate the combination index (CI) values using the Chou-Talalay method. Clone formation and EdU assay were used to detect the effect of afatinib combined with MDV3100 on the proliferation of 22Rv1 cells. RT-qPCR and Western blot were used to explore the mechanism of drug combination. The aim of the present study was to determine the effects of several tyrosine kinase inhibitors (TKIs) when used in combination with MDV3100 in vitro. Results: The results demonstrated that TKIs combined with MDV3100 exerted a synergistic effect on a variety of PCa cells. Afatinib combined with MDV3100 could suppress the proliferation and migration of 22RV1 cells, as well as cause their cell cycle arrest and apoptosis. Mechanistically, afatinib effectively reduced the protein expression levels of HER2 and HER3 and inhibited EGFR phosphorylation, thereby enhancing the effect of MDV3100 and suppressing CRPC. Conclusions: These findings suggested that afatinib treatment improved the effect of MDV3100 on 22RV1 cells, highlighting this drug as a potential therapeutic strategy for patients with CRPC.

Utilizing RNA nanotechnology to construct negatively charged and ultrasound-responsive nanodroplets for targeted delivery of siRNA

Ultrasound nanodroplets (NDs) have been reported as a promising nanocarrier for siRNA delivery depending on its unique strengths of sonoporation. Presently, common means for NDs-mediated siRNA delivery is through electrostatic interaction, but challenges like cationic toxicity still exist. In this study, we demonstrated a novel strategy to construct negatively charged and ultrasound (US)-responsive O-carboxymethyl chitosan (O-CMS) NDs as a siRNA targeted delivery system through three-way junction of bacteriophage phi29 DNA packaging motor (3WJ-pRNA) nanotechnology. 39nt A10-3.2 aptamer targeting prostate specific membrane antigen (PSMA) and 21nt siRNA against cationic amino acid transporter 1 (siCAT-1) were annealed to 3WJ-pRNA scaffold via complementation with an extended sequence. The cholesterol molecule attached to one branch facilitates the 3WJ-pRNA nanoparticles anchoring onto NDs. The desired O-CMS NDs with siRNA-loading and RNA-aptamer modification (A10-3.2/siCAT-1/3WJ-NDs) were successfully prepared, which were with spherical shapes, core–shell structures and uniform in sizes (198 nm with PDI 0.3). As a main proportion of shell, O-CMC showed a certain anti-tumor effects. In vitro studies demonstrated that A10-3.2/siCAT-1/3WJ-NDs exhibited good contrast-enhanced US imaging, buffering capacity and high bio-safety, were able to deliver siCAT-1 to PSMA-overexpressed prostate cancer cells under US irradiation, thus silence the CAT-1 expression, and consequently suppressing 22RV1 cell proliferation and migration. Taken overall, our findings provide a promising strategy to develop negatively charged and US-responsive NDs for tumor-targeted siRNA delivery.

Identification of evodiamine as a suppressor of prostate cancer progression by reducing AR transcriptional activity via targeting Src.

Evodiamine (EVO) is a bioactive alkaloid that exerts antitumor activity in various cancers, including prostate cancer (PCa). In this paper, we further investigated the molecular mechanisms underlying the anti-PCa effect of evodiamine. In the present study, cell proliferation, colony formation, migration, and invasion were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, and transwell assays, respectively. Animal studies were used to evaluate the effect of evodiamine on the tumorigenicity of LNCaP cells in vivo. The expression levels of steroid receptor coactivator (Src), androgene receptor (AR), and prostate-specific antigen (PSA) were detected by western blot, quantitative real-time PCR (qRT-PCR) or ELISA assay. Association between Src and AR was examined by Co-Immunoprecipitation (CoIP). The impact of evodiamine on AR-mediated transcriptional activity was confirmed by dual-luciferase reporter assay. The results showed that evodiamine reduced LNCaP and 22Rv1 cell proliferation, colony formation, migration, and invasion induced by dihydrotestosterone (DHT) in vitro, as well as diminished tumor growth in vivo. Mechanistically, evodiamine directly targeted Src and reduced DHT-induced Src activation. Moreover, the restoration of Src activation abolished evodiamine-mediated suppression of proliferation, migration, and invasion of DHT-treated LNCaP and 22Rv1 cells. Furthermore, evodiamine inhibited DHT-induced AR transcriptional activity through targeting Src. As a conclusion, our findings demonstrate the antitumor property of evodiamine in PCa by blocking AR transcriptional activity through targeting Src and provide a rationale for developing evodiamine as a promising antitumor agent against PCa.

Design, Synthesis and Cytotoxicity Evaluation of Novel Indole Derivatives Containing Benzoic Acid Group as Potential AKR1C3 Inhibitors.

Castration-resistant prostate cancer (CRPC) is a fatal, metastatic form of prostate cancer, characterized by reactivation of the androgen axis. Aldo-keto reductase 1C3 (AKR1C3) converts androstenedione (AD) and 5α-androstanedione to testosterone (T) and 5α-dihydrotestosterone (DHT), respectively. In CRPC, AKR1C3 is upregulated and implicated in drug resistance and has been regarded as a potential therapeutic target. Here we examined a series of indole derivatives containing benzoic acid or phenylhydroxamic acid and found that 4-({3-[(3,4,5-trimethoxyphenyl)sulfanyl]-1H-indol-1-yl}methyl)benzoic acid (3e) and N-hydroxy-4-({3-[(3,4,5-trimethoxyphenyl)sulfanyl]-1H-indol-1-yl}methyl)benzamide (3q) inhibited 22Rv1 cell proliferation with IC50 values of 6.37 μM and 2.72 μM, respectively. In enzymatic assay, compounds 3e and 3q exhibited potent inhibitory effect against AKR1C3 (IC50 =0.26 and 2.39 μM, respectively). These results indicated that compounds 3e and 3q might be useful leads for further investigation of more potential AKR1C3 inhibitors used for CRPC.

Linalool inhibits 22Rv1 prostate cancer cell proliferation and induces apoptosis.

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.

Combined α-methylacyl-CoA racemase inhibition and docetaxel treatment reduce cell proliferation and decrease expression of heat shock protein 27 in androgen receptor-variant-7-positive prostate cancer cells.

BackgroundDisease progression in castrate-resistant prostate cancer (PCa) is most commonly driven by the reactivation of androgen receptor (AR) signaling and involves AR splice variants including ARV7.Materials and methodsWe used the ARV7-positive PCa cell line, 22Rv1, to study the relationship of the PCa marker α-methylacyl-CoA racemase (AMACR), AR, and ARV7 in PCa.ResultsDocetaxel addition but not AMACR inhibition decreased the proliferation of 22Rv1 cells. The combination of AMACR inhibition and docetaxel treatment resulted in a maximum reduction of cell proliferation. The Western blotting analysis revealed that both AR and ARV7 expression were significantly decreased with the use of charcoal-stripped serum following AMACR inhibition and docetaxel treatment. AMACR inhibition and docetaxel treatment in the charcoal-stripped serum condition reduced the proliferation of 22Rv1, possibly via the downregulation of the heat shock protein 27.ConclusionUsing cell proliferation and Western blot analysis, we demonstrated that AMACR inhibition and docetaxel treatment, under androgen deprivation conditions, significantly reduced the proliferation of ARV7 positive cancer cells and decreased the levels of AR and ARV7 expression, possibly via downregulation of heat shock protein 27.

MiR-31-5p Regulates 14-3-3 ɛ to Inhibit Prostate Cancer 22RV1 Cell Survival and Proliferation via PI3K/AKT/Bcl-2 Signaling Pathway.

IntroductionProstate cancer (PCa) is one of the most common malignancies, and almost all patients with advanced PCa will develop castration-resistant prostate cancer (CRPC) after receiving endocrine therapy. Effective treatment for patients with CRPC has not been established. Novel approaches are needed to identify therapeutic targets for CRPC.PurposeRecent research studies have found that members of the 14-3-3 family play an important role in the development and progression of PCa. Previous results have shown that 14-3-3 ɛ is significantly upregulated in several cancers. This study aimed to identify novel miRNAs that regulate 14-3-3 ɛ expression and therapeutic targets for CRPC.MethodsIn this study, we used computation and experimental approaches for the prediction and verification of the miRNAs targeting 14-3-3 ɛ, and investigated the potential roles of 14-3-3 ɛ in the survival and proliferation of 22RV1 cells.ResultsWe confirm that mir-31-5p is downregulated in 22RV1 cells and acts as a tumor suppressor by regulating 14-3-3 ɛ. Ectopic expression of miR-31-5p or 14-3-3 ɛ interference significantly inhibits cell proliferation, invasion, and migration in 22RV1 cells, as well as promotes cell apoptosis via the PI3K/AKT/Bcl-2 signaling pathway. Moreover, 14-3-3 ɛ is required for the miR-31-5p-mediated upregulation of the PI3K/AKT/Bcl-2 signaling pathway.ConclusionOur findings provide information on the underlying mechanisms of miR-31-5p/14-3-3 ɛ in 22RV1 cell proliferation and apoptosis through the PI3K/AKT/Bcl-2 signaling pathway. These results suggest that miR-31-5p and 14-3-3 ɛ may potentially be utilized as novel prognostic markers and therapeutic targets for PCa treatment.

PD10-01 PRECLINICAL EFFICACY & MECHANISM OF ACTION OF COMBINATION OF NITRIC OXIDE DONOR AND GROWTH HORMONE-RELEASING HORMONE ANTAGONISTS FOR CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) I (PD10)1 Apr 2020PD10-01 PRECLINICAL EFFICACY & MECHANISM OF ACTION OF COMBINATION OF NITRIC OXIDE DONOR AND GROWTH HORMONE-RELEASING HORMONE ANTAGONISTS FOR CASTRATION-RESISTANT PROSTATE CANCER Yash Soni, Manish Kuchakulla, Ranjith Ramasamy, and Himanshu Arora* Yash SoniYash Soni More articles by this author , Manish KuchakullaManish Kuchakulla More articles by this author , Ranjith RamasamyRanjith Ramasamy More articles by this author , and Himanshu Arora*Himanshu Arora* More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000844.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Castration resistant prostate cancer (CRPC) is unresponsive to androgen therapy and thus, other therapeutic agents and molecular events are being explored. We have shown in our studies that increased NO levels affect receptors for GnRH, LH and T through the hypothalamic-pituitary-gonadal axis, while simultaneously increasing overall oxidative/nitrosative stress. In the same context, several studies have used inhibition of GHRH-R (receptor) as an approach to target PCa. Therefore, considering that both increased NO levels and GnRH antagonists could be targeting the PCa via the same mechanistic channel, in the present study, we evaluated the impact of increased NO levels with or without GnRH antagonists on CRPC tumor progression to establish their preclinical efficacy for treatment of androgen independent prostate cancer. METHODS: Proliferation assay was performed using 22RV1 cells in the presence of GHRHR antagonist (MIA-602) at 1uM concentration, in the absence or presence of increasing concentration of GSNO (from 25, 50 or 100uM respectively). Expression of down terminal markers of CRPC (PSA, AR TMRPSS2, AR, ARV7 and pERK respectively) was checked at RNA and protein levels after treating 22RV1 cells with MIA-602 at 1uM concentration, in the absence or presence of increasing concentration of GSNO (from 10, 25, 50 or 100uM respectively). For in-vivo experiments, castrated SCID mice were grafted with 22RV1 cells to generate CRPC murine models. Once the tumors became palpable, the animals were divided into three groups. Group 1 received vehicle control, group 2 received GSNO treatment at the dosage of 10mg/kg/day intraperitonially (IP), Group 3 received GSNO (10mg/kg/day IP) and MIA-602 (5ug/kg/day IP) combination treatment. Treatment was continued for 30 days. Weight of the animals and tumor volume were measured 3 times per week during the 30 days treatment period. Post treatment, tumors and blood were extracted from the mice. Proteins were harvested from the tumors to study expression of markers of cancer progression. Animal experiments were carried out in compliance with the Institutional Animal Care and Use Committee of University of Miami. Molecular analyses were performed using standard procedures. GraphPad Prism (GraphPad Software) was used for statistical analysis. All data were presented as the means ± SEM. The statistical significance between two groups was estimated by unpaired two-tailed t test. RESULTS: MTT assay showed that MIA-602 compounds were able to significantly reduce the cell proliferation of 22RV1 cells (p<0.05). GSNO did not interfere with ability of MIA compounds towards reducing cell proliferation. Additionally, the increasing concentration of NO levels potentiated the effects of MIA-602 by decreasing PSA, AR and TMRPSS2 at RNA levels and AR, ARV7 and pERK at protein levels respectively. In-vivo results showed that both GSNO and the combinatorial treatment of GSNO and MIA-602 showed a significant decrease (p<0.05) in tumor volume and weight compared to the control. Proteins from tumors showed a reduction in androgen receptor (AR) and AR-V7 compared to individual treatments of control and GSNO respectively. CONCLUSIONS: This study shows that the combination of GSNO and MIA-602 work to decrease CRPC tumor burden and cellular proliferation. GSNO is known to act on the tumor microenvironment so when it is paired with a GHRHR antagonist the results show significant reduction in CRPC tumor burden. Further studies with active NO donors and GHRHR antagonists are in process to establish the preclinical efficacy and mechanism of action of the combination against CRPC. Source of Funding: American Cancer Society and CTSI © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e244-e244 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yash Soni More articles by this author Manish Kuchakulla More articles by this author Ranjith Ramasamy More articles by this author Himanshu Arora* More articles by this author Expand All Advertisement PDF downloadLoading ...

MP64-11 CHEMOPREVENTIVE AND CHEMOTHERAPEUTIC POTENTIALS OF LUTEOLIN VIA REGULATION OF OXIDATIVE STRESS IN CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2018MP64-11 CHEMOPREVENTIVE AND CHEMOTHERAPEUTIC POTENTIALS OF LUTEOLIN VIA REGULATION OF OXIDATIVE STRESS IN CASTRATION-RESISTANT PROSTATE CANCER Taku Naiki, Aya Naiki-Ito, Toshiki Etani, Keitaro Iida, Ryosuke Ando, Takashi Nagai, Noriyasu Kawai, Satoru Takahashi, and Takahiro Yasui Taku NaikiTaku Naiki More articles by this author , Aya Naiki-ItoAya Naiki-Ito More articles by this author , Toshiki EtaniToshiki Etani More articles by this author , Keitaro IidaKeitaro Iida More articles by this author , Ryosuke AndoRyosuke Ando More articles by this author , Takashi NagaiTakashi Nagai More articles by this author , Noriyasu KawaiNoriyasu Kawai More articles by this author , Satoru TakahashiSatoru Takahashi More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2056AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Identifying effective treatments for castration-resistant prostate cancer (CRPC) is important to prevent its persistent progression following androgen deprivation therapy. In a previous study, we showed that changes in the expression of genes concerned with oxidative stress regulation induced carcinogenesis and castration-resistant growth in prostate cancer. Herein, we investigated the chemotherapeutic potential of luteolin, an anti-oxidative flavonoid, on prostate cancer and CRPC. METHODS Male transgenic rats for adenocarcinoma of prostate (TRAP) were divided into three groups (n = 12 in each group) and fed the following for 8 weeks: basal diet, diet containing 20 ppm luteolin, and diet containing 100 ppm luteolin. Prostate tissues were collected from each animal for histopathological, gene, and protein expression analysis.PCai cells established from the TRAP prostate tumor and 22Rv1 cells were subcutaneously implanted in castrated and non-castrated nude mice, and in vivo tumor growth was analyzed. Subsequently, the effect of luteolin on cell proliferation and apoptosis in PCai1 and 22Rv1 cells was analyzed. RESULTS Luteolin decreased the incidence of adenocarcinoma and suppressed its progression from PIN to adenocarcinoma in TRAP. An increased apoptosis index with activation of caspase-3 and 7 was observed with high grade-PIN in the luteolin-fed TRAP. Production of reactive oxygen species and mRNA expression of the oxidative stress defense genes Gpx2 and Ho-1 were reduced by luteolin. Furthermore, luteolin inhibited tumor growth in the xenograft model via apoptosis induction and Gpx2 down-regulation. Luteolin also suppressed PCai1 and 22Rv1 cell proliferation in a dose-dependent manner in vitro by caspase-3 and 7-dependent apoptosis. CONCLUSIONS Luteolin decreased cell proliferation in a dose-dependent manner, and induced apoptosis with caspase activation in both, PCai1 and 22Rv1 cells. Dietary luteolin also suppressed tumor growth by increasing apoptosis and inhibiting angiogenesis in the PCai1 xenograft model. These results suggest that luteolin is a potential therapeutic agent for CRPC. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e854 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Taku Naiki More articles by this author Aya Naiki-Ito More articles by this author Toshiki Etani More articles by this author Keitaro Iida More articles by this author Ryosuke Ando More articles by this author Takashi Nagai More articles by this author Noriyasu Kawai More articles by this author Satoru Takahashi More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 1588: Effects of the selective CYP17-lyase and androgen receptor (AR) inhibitor, seviteronel, and the cyclin-dependent kinase (CDK) 4/6 inhibitor, G1T38, on tumor growth in an AR-V7+ castration-resistant prostate cancer (CRPC) xenograft model

Abstract Background: Castration-resistant prostate cancers that express the constitutively active androgen receptor (AR) variant AR-V7 exhibit significantly reduced progression-free and overall survival in response to the AR targeting agents, enzalutamide (ENZ) and abiraterone. Inhibition of Phosphoinositide-3-Kinase (PIK3CA) or CDK4/6 has been proposed as alternative therapies that directly target the cell cycle pathway in these tumors. However, the combined role of AR plus cell cycle inhibition in this treatment setting has not been established. G1T38 is a potent, selective clinical stage CDK 4/6 inhibitor. Seviteronel (SEVI) is a selective CYP17-lyase and AR inhibitor that blocks the growth of CRPC tumors with clinically relevant AR mutations, including T878A and AR-F876L. The activity of SEVI, ENZ and G1T38, alone or in combination, was evaluated using the 22Rv1 (AR-V7+) CRPC model in vitro and in vivo. Methods: Cellular proliferation (7 days) of 22Rv1 prostate cancer cells treated with SEVI, ENZ, and G1T38 alone or in combination with SEVI or ENZA was assessed by measuring DNA content. Orchiectomized male nu/nu mice bearing 22Rv1 were randomized to receive vehicle, SEVI (150mg/kg/day p.o.), ENZ (30 mg/kg/day p.o.), G1T38 (25-100 mg/kg/day p.o.), or SEVI or ENZ in combination with G1T38. A docetaxel (20 mg/kg weekly i.p.) group was included as a standard of care comparison. Time to progression (X-fold increase in tumor volume) and tumor volume changes were assessed over a 3-7-week dosing period. Blood and tissues were collected for analysis of exposure and pharmacodynamics markers of response. Results: SEVI and G1T38 alone or in combination, significantly reduced the proliferation of 22Rv1 cells in vitro (p&amp;lt;0.05). Compared to vehicle, SEVI and G1T38 monotherapy significantly inhibited tumor growth and time to progression by 1.5 and 2.5 fold, respectively (p&amp;lt;0.05); the combination of SEVI and G1T38 further decreased tumor growth and resulted in a 4-fold delay in progression (p&amp;lt;0.001). In contrast, ENZ alone was without effect on tumor growth and did not increase response to G1T38 as compared to G1T38 alone. Conclusions: SEVI and G1T38 mono- or combination therapy significantly decreased the growth of the AR-V7+ 22Rv1 CPRC model in vitro and in vivo, an activity that distinguishes SEVI from ENZ. Therefore, combining SEVI with a CDK4/6 inhibitor such as G1T38 may be an effective therapy for the treatment of CRPC that is resistant to current standards of care. Citation Format: Suzanne E. Wardell, Alexander P. Yllanes, John D. Norris, James P. Stice, Hannah White, Ronald A. Fleming, Jay C. Strum, William R. Moore, Donald P. McDonnell. Effects of the selective CYP17-lyase and androgen receptor (AR) inhibitor, seviteronel, and the cyclin-dependent kinase (CDK) 4/6 inhibitor, G1T38, on tumor growth in an AR-V7+ castration-resistant prostate cancer (CRPC) xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1588. doi:10.1158/1538-7445.AM2017-1588

Trichosanthes kirilowii Exerts Androgenic Activity via Regulation of PSA and KLK2 in 22Rv1 Prostate Cancer Cells.

Background:The androgen comprises a group of hormones that play roles in male reproductive activity as well as personal characteristics.Objective:We investigated the androgenic activity of various herbal medicines in human prostate cancer 22Rv1 cells.Materials and Methods:Herbal extracts of Trichosanthes kirilowii (TK), Asarum sieboldii (AS), Sanguisorba officinalis (SO), and Xanthium strumarium (XS) were selected to have androgenic effects based on a preliminary in vitro screening system.Results:TK, AS, SO, and XS enhanced the proliferation of 22Rv1 cells without having cytotoxic effects. All tested herbal extracts increased androgen receptor (AR)-induced transcriptional activity in the absence or presence of dihydrotestosterone (DHT). In an AR-binding assay, TK, but not AS, SO, or XS, produced a significant inhibition of AR binding activity, indicating it has androgenic activity. Additionally, TK treatment positively regulated mRNA expression of the AR-related molecular targets prostate-specific antigen (PSA) and kallikrein 2 (KLK2) compared with untreated control.Conclusion:Taken together, TK-enhanced AR-mediated transcriptional activity might be an attractive candidate drug for treating androgen-related diseases.SUMMARY Trichosantheskirilowii (TK), Asarumsieboldii (AS), Sanguisorbaofficinalis (SO), and Xanthium strumarium (XS) enhanced the proliferation of 22Rv1 cells without having cytotoxic effects.TK, AS, SO, and XS increased androgen receptor (AR)-induced transcriptional activity.TK, but not AS, SO, or XS, produced a significant inhibition against AR-binding activity.TK treatment positively regulated mRNA expression of the AR-related molecular targets prostate-specific antigen and kallikrein 2. Abbreviations used: BPH: benign prostatic hyperplasia; AR: androgen receptor; DHT: dihydrotestosterone; PSA: prostate-specific antigen; TK: Trichosanthes kirilowii; AS: Asarum sieboldii; SO: Sanguisorba officinalis; XS: Xanthium strumarium; ATCC: American Type Culture Collection; FBS: fetal bovine serum; PBS: phosphate-buffered saline; SD: standard deviation; ARE: androgenresponsive element; KLK: kallikrein

Lipocalin 2 over-expression facilitates progress of castration-resistant prostate cancer via improving androgen receptor transcriptional activity.

BackgroundCastration-resistant prostate cancer (CRPC) is the lethal phenotype of prostate cancer. Lipocalin 2 (LCN2) is aberrantly expressed in many cancers including primary prostate cancer (PCa), but its role in CRPC has not been reported.ResultsLCN2 expression was upregulated in human primary PCa and CRPC tissues. Overexpression of LCN2 promoted C4-2B and 22RV1 cell proliferation while knockdown of LCN2 markedly inhibited C4-2B and 22RV1 cell growth. LCN2 overexpression led to increased AR downstream gene SLC45A3 without upregulating AR expression. In the xenograft model, overexpression of LCN2 significantly promoted tumor growth.MethodsLCN2 expression was detected in primary PCa and CRPC tissues and cell lines C4-2B and 22RV1 using immunohistochemistry and western blotting, respectively. Serum LCN2 level was detected vi ELISA. Lentiviruses-mediated over-expression of LCN2 and LCN2 knockdown were performed in CRPC cell lines. Expressions of androgen receptor (AR) downstream genes was examined in cell lines, in CRPC tissues, and in animal models.ConclusionLCN2 could facilitate cell proliferation of CRPC via AR transcriptional activity. LCN2 could be a novel target in CRPC.

MP84-15 PREVENT METASTASIS BY DOUBLE CHIMERIC ANTIGEN RECEPTOR T CELLS TARGETING CIRCULATING CANCER CELLS FOR PROSTATE CANCER TREATMENT

INTRODUCTION AND OBJECTIVES: We and others have demonstrated that the androgen receptor (AR) is essential for the development and progression of prostate cancer. Although androgendeprivation therapy (ADT) is effective initially, unfortunately, metastatic prostate cancer often recurs as so-called castration resistant prostate cancer (CRPC). Overexpression of AR, AR hypersensitivity, c-terminal truncated AR variants, as well as altered steroid metabolism are considered the main mechanisms for CRPC. Current anti-AR treatments (Abiraterone and Enzalutamide) for CRPC only extend patient survival for approximately 5 months, indicating novel therapeutic agents are urgently needed. It has been shown that AR variants possess an intact and functional N-terminal domain that contains several key motifs and tyrosine residues involved in AR transactivation. METHODS: We recently designed a 14-amino acid peptide spanning the 355-368 residues (ARi14 peptide) on AR N-terminal, consisting of the partial transactivation activation unit-1 and three key tyrosine residues involved in CRPC progression. Multiple prostate cancer cell lines were used in the experiments. Trypan blue and Alamar Blue assays were used to assess cell proliferation. Western blot and anti-AR immunoprecipitation were conducted to assess AR protein levels and tyrosine phosphorylation. Quantitative RT-PCR was performed to assess PSA mRNA expression. RESULTS: Treatment of multiple AR-positive prostate cancer cells with this short peptide induced a significant reduction of AR protein, as well as AR transactivation in a luciferase reporter assay in a time-dependent manner. However, AR gene expression at the mRNA level was not altered. Further analysis revealed that ARi14 peptide abolished androgen-stimulated AR tyrosine phosphorylation in LNCaP cells, indicating a disruption of AR with its tyrosine kinase. In 22RV1 cells that harbor AR variants the ARi14 peptide dramatically reduced the protein levels of full length AR and AR variants, abolished AR-target PSA gene expression and also suppressed 22RV1 cell proliferation that Enzalutamide failed to do so. Sequence mutation analysis demonstrated that the C-terminal 8 residue portion (361-368aa) of the ARi14 is the key element for its anti-AR activities. CONCLUSIONS: Our data demonstrated that the novel short peptide ARi14 exerts a potent anti-AR activity by inducing AR protein degradation possibly through reduced tyrosine phosphorylation in Enzalutamide-resistant prostate cancer cells.

MP84-14 OVERCOMING ENZALUTAMIDE-RESISTANT WITH A NOVEL ANTI-AR PEPTIDE IN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2016MP84-14 OVERCOMING ENZALUTAMIDE-RESISTANT WITH A NOVEL ANTI-AR PEPTIDE IN PROSTATE CANCER CELLS Daniel Zainfeld, Ruibao Chen, J Brantley Thrasher, and Benyi Li Daniel ZainfeldDaniel Zainfeld More articles by this author , Ruibao ChenRuibao Chen More articles by this author , J Brantley ThrasherJ Brantley Thrasher More articles by this author , and Benyi LiBenyi Li More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2238AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We and others have demonstrated that the androgen receptor (AR) is essential for the development and progression of prostate cancer. Although androgen-deprivation therapy (ADT) is effective initially, unfortunately, metastatic prostate cancer often recurs as so-called castration resistant prostate cancer (CRPC). Overexpression of AR, AR hypersensitivity, c-terminal truncated AR variants, as well as altered steroid metabolism are considered the main mechanisms for CRPC. Current anti-AR treatments (Abiraterone and Enzalutamide) for CRPC only extend patient survival for approximately 5 months, indicating novel therapeutic agents are urgently needed. It has been shown that AR variants possess an intact and functional N-terminal domain that contains several key motifs and tyrosine residues involved in AR transactivation. METHODS We recently designed a 14-amino acid peptide spanning the 355-368 residues (ARi14 peptide) on AR N-terminal, consisting of the partial transactivation activation unit-1 and three key tyrosine residues involved in CRPC progression. Multiple prostate cancer cell lines were used in the experiments. Trypan blue and Alamar Blue assays were used to assess cell proliferation. Western blot and anti-AR immunoprecipitation were conducted to assess AR protein levels and tyrosine phosphorylation. Quantitative RT-PCR was performed to assess PSA mRNA expression. RESULTS Treatment of multiple AR-positive prostate cancer cells with this short peptide induced a significant reduction of AR protein, as well as AR transactivation in a luciferase reporter assay in a time-dependent manner. However, AR gene expression at the mRNA level was not altered. Further analysis revealed that ARi14 peptide abolished androgen-stimulated AR tyrosine phosphorylation in LNCaP cells, indicating a disruption of AR with its tyrosine kinase. In 22RV1 cells that harbor AR variants the ARi14 peptide dramatically reduced the protein levels of full length AR and AR variants, abolished AR-target PSA gene expression and also suppressed 22RV1 cell proliferation that Enzalutamide failed to do so. Sequence mutation analysis demonstrated that the C-terminal 8 residue portion (361-368aa) of the ARi14 is the key element for its anti-AR activities. CONCLUSIONS Our data demonstrated that the novel short peptide ARi14 exerts a potent anti-AR activity by inducing AR protein degradation possibly through reduced tyrosine phosphorylation in Enzalutamide-resistant prostate cancer cells. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1095 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Daniel Zainfeld More articles by this author Ruibao Chen More articles by this author J Brantley Thrasher More articles by this author Benyi Li More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

457 Preclinical pharmacology of AZD5312, a generation 2.5 antisense oligonucletotide targeting the androgen receptor with differentiated activity from enzalutamide

The androgen receptor is a clinically validated target in prostate cancer; signalling through the androgen receptor remains an important driver of disease progression even in castration resistant disease, as shown by the recent successful phase 3 trials of second generation androgen receptor antagonist enzalutamide and CYP17 inhibitor abiraterone. However, resistance to these agents inevitably emerges, due to a variety of mechanisms including mutations in the receptor and expression of spliced variants that lack the ligand binding domain but still drive AR signalling. Down-regulation of AR using an antisense oligonucleotide has the potential to overcome these resistance mechanisms. AZD5312 causes dosedependent down-regulation of full length AR mRNA and protein, inhibits AR-dependent gene expression, and induces apoptosis in prostate cancer cell lines including LNCaP and VCaP. In 22Rv1 cells, AZD5312 also downregulates the spliced variant AR-v7, and shows a differential effect on a 48gene AR transcriptome signature to enzalutamide, whereas this response is similar in LNCaP cells that only express full length AR. Moreover, AZD5312 inhibits proliferation of 22Rv1 cells, whereas enzalutamide does not. In vivo, AZD5312 down-regulates full length and spliced variant forms of AR, and inhibits PSA protein expression, in the LuCAP86.2 transplantable prostate cancer xenograft model. AZD5312 also reduces AR expression and inhibits the growth of AR-expressing MDA-MB-453 breast cancer xenografts in nude mice supplemented with dihydrotestosterone. In conclusion, AZD5312 is a potent antisense oligonuleotide targeting AR that is therapeutically active preclinically, with differentiated activity from enzalutamide.