Androgen-independent Cell Lines Research Articles

Identification of ferroptosis related genes and pathways in prostate cancer cells under erastin exposure

BackgroundFew studies are focusing on the mechanism of erastin acts on prostate cancer (PCa) cells, and essential ferroptosis-related genes (FRGs) that can be PCa therapeutic targets are rarely known.MethodsIn this study, in vitro assays were performed and RNA-sequencing was used to measure the expression of differentially expressed genes (DEGs) in erastin-induced PCa cells. A series of bioinformatic analyses were applied to analyze the pathways and DEGs.ResultsErastin inhibited the expression of SLC7A11 and cell survivability in LNCaP and PC3 cells. After treatment with erastin, the concentrations of malondialdehyde (MDA) and Fe2+ significantly increased, whereas the glutathione (GSH) and the oxidized glutathione (GSSG) significantly decreased in both cells. A total of 295 overlapping DEGs were identified under erastin exposure and significantly enriched in several pathways, including DNA replication and cell cycle. The percentage of LNCaP and PC3 cells in G1 phase was markedly increased in response to erastin treatment. For four hub FRGs, TMEFF2 was higher in PCa tissue and the expression levels of NRXN3, CLU, and UNC5B were lower in PCa tissue. The expression levels of SLC7A11 and cell survivability were inhibited after the knockdown of TMEFF2 in androgen-dependent cell lines (LNCaP and VCaP) but not in androgen-independent cell lines (PC3 and C4-2). The concentration of Fe2+ only significantly increased in TMEFF2 downregulated LNCaP and VCaP cells.ConclusionTMEFF2 might be likely to develop into a potential ferroptosis target in PCa and this study extends our understanding of the molecular mechanism involved in erastin-affected PCa cells.

Oridonin attenuated human PC-3 cell activity by modulating the Wnt/β-catenin signaling.

Prostate cancer (PC) prevention is effectively achieved through its inhibition. Oridonin (ORD), an active diterpenoid isolated from Rabdosia rubescens, has been shown to have an inhibitory effect on PC cells, although its impact on PC is unknown. The present work investigated the actions and probable mechanisms of ORD on cellular proliferation, apoptosis, PC, and the wingless-type MMTV integration site family member 2 (Wnt)/β-catenin signaling pathway using the androgen-independent PC-3 cell line. In this study, cell viability was analyzed with MTT assay method, apoptotic morphology determined using DAPI dye method, while protein (CD1333, OCT-4, Nanog, SOX-2 & Aldh1A1) and mRNA expressions were analyzed with western blotting and real time polymerase chain reaction (PCR). We demonstrated a concentration-dependent ORD inhibition of PC-3 cell proliferation and inhibition of induction apoptosis. Furthermore, ORD decreased PC-3 Wnt-2, phosphorylated glycogen synthase kinase-3 (p-GSK3), and β-catenin protein levels and downregulated cyclin-D1 and c-myc messenger ribonucleic acid (mRNA). Oridonin inhibited proliferation and induced apoptosis in PC-3 cells, with the findings suggesting that it acted via the Wnt/β-catenin pathway to exert its effects. This study demonstrates that ORD may impact PC.

Autocrine Proteinase-Activated Receptor (PAR) signaling in PC3 prostate cancer cells.

Proteinase-activated receptors (PARs) are G protein-coupled receptors (GPCRs) activated by limited N-terminal proteolysis. PARs are highly expressed in many cancer cells including PCa and regulate various aspects of tumor growth and metastasis. Specific activators of PARs in different physiological and pathophysiological contexts remain poorly defined. In this study, we examined the androgen independent human prostatic cancer cell line PC3 and find functional expression of PAR1 and PAR2 but not PAR4. Using genetically encoded PAR cleavage biosensors, we show that PC3 cells secrete proteolytic enzymes that cleave PARs and trigger autocrine signaling. CRISPR/Cas9 targeting of PAR1 and PAR2 combined with microarray analysis revealed genes that are regulated through this autocrine signaling mechanism. We find several genes that are known PCa prognostic factors or biomarker to be differentially expressed in the PAR1-KO and PAR2-KO PC3 cells. We further examined PAR1 and PAR2 regulation of PCa cell proliferation and migration and find that absence of PAR1 promotes PC3 cell migration and suppresses cell proliferation while PAR2 deficiency showed opposite effects. Overall, these results demonstrate that autocrine signaling through PARs is an important regulator of PCa cell function.

Abstract 2858: Radiotherapy and NK cell-based immunotherapy: An evolutionary double-bind

Abstract Evolution-informed therapies exploit the ecological and evolutionary consequences of drug resistance to inhibit the expansion of treatment resistant populations and prolong time to progression. One strategy, termed an evolutionary double-bind, uses an initial therapy to elicit a specific adaptive mechanism that is then selectively targeted by a follow-on therapy. Here we examine combining radiation therapy followed by immunotherapy as a double bind strategy. Radiotherapy (RT) induces lethal double-strand DNA breaks in tumor cells and radiosensitivity is governed by the cells’ DNA repair capabilities. Radioresistant cancer cells upregulate DNA damage response pathways. However, successful adaptations to RT and other DNA damaging agents can increase natural killer (NK) cell ligand expression on tumor cells increasing their vulnerability to the immune system. We conducted a multidisciplinary investigation of this potential evolutionary double bind using an isogenic cell line model of radiation resistance derived from an androgen-independent human prostate carcinoma cell line (22Rv1). We demonstrate that RT-resistant cells maintain higher expression of NK cell ligands and have a 2-fold increase in sensitivity to NK cell mediated killing. Furthermore, we show that preferential targeting of the RT-resistant population by NK cells is preserved in competition assays with varying initial population frequencies. Scope of treatment responses are dependent on population frequencies in our in vitro analysis. Mathematical modeling quantified three key elements of a double-bind: cost of resistance, inter-specific competition between RT-sensitive and RT-resistant lines, and preferential targeting of RT-resistant lines by NK cells. We conclude that RT followed by immunotherapy produces an evolutionary double bind that can be exploited in heterogenous tumors to limit RT resistance. Furthermore, model simulations predict that extinction of both populations is achievable by sequential RT and NK cell therapy. Citation Format: Kimberly Luddy, Jeffery West, Mark Robertson-Tessi, Bina Desai, Taylor M. Bursell, Sarah Barrett, Joel Brown, Jacintha O'Sullivan, Robert Gatenby, Laure Marignol, Alexander R. Anderson, Cliona O'Farrelly. Radiotherapy and NK cell-based immunotherapy: An evolutionary double-bind [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2858.

THE INFLUENCE OF SPERMINE AND CHLORHEXIDINE ON THE SURVIVAL, ELECTROKINETIC AND CYTOMORPHOLOGICAL CHARACTERISTICS OF HUMAN PROSTATE CANCER CELLS

Summary. Aim: to study the effects of spermine and the spermine oxidase inhibitor chlorhexidine, applied alone or in combination, on the viability, electrokinetic and structural-functional characteristics of human prostate cancer cells in vitro. Object and methods: Studies were conducted on cell cultures: differentiated androgen-dependent LNCaP cell line and low-differentiated androgen-independent DU-145 cell line. Cell survival was determined by the method of exclusion of the vital dye trypan blue by living cells. The electrokinetic parameters of the cells (ζ-potential and total surface charge density) were determined according to the Smoluchovsky and Quincke-Helmholtz equations. Morphological changes were assessed by light microscopy of fixed preparations of cells stained with hematoxylin and eosin. Results: The fact established by previous studies that spermine at a concentration of 1.5 mM exhibits a pronounced inhibitory effect on the growth of differentiated human prostate cancer cells of the LNCaP line, which consists in reducing their survival to 48.5 ± 1.5% compared to intact control, and when this factor is used in a concentration exceeding 5.0 mM – in their death. It was shown that poorly differentiated cells of the DU-145 line were significantly more resistant to its influence: their survival decreased to 56.0 ± 1.9% only under the influence of spermine at a concentration of 5.0 mM; a concentration of 10.0 mM caused complete cell death of both lines. The trend persisted in the case of chlorhexidine: under its influence at a concentration of 15.0 μM, the survival of cells of the hormone-dependent LNCaP line was 33.0 ± 1.5%, and the hormone-independent DU-145 cell line was 79.0 ± 2.5%. The combined use of spermine and chlorhexidine (0.6 mM and 3.2 µM, respectively) caused a strong cytotoxic effect in cells of the LNCaP line (survival 19.0 ± 1.2%), while cells of the DU-145 line maintained a fairly high rate (76.0 ± 2.1%) even with a significantly higher content of spermine (3.0 mM) and chlorhexidine (5.0 μM) in the culture medium. It was established that under the influence of chlorhexidine, the cells of both studied lines show an inversion of the sign of the surface electric charge, changing it from negative to positive under physiological conditions, just as it was shown in experiments with spermine on cells of the LNCaP line. Certain quantitative differences caused by the investigated compounds in the electrokinetic indicators of cells of hormone-dependent and hormone-independent lines were revealed. Cells of the DU-145 line under the influence of spermine, or simultaneously spermine and chlorhexidine, showed a typical apoptotic morphology: they rounded and separated from each other and the substrate, chromatin condensation, blurred contours of the surface membrane, nuclear fragmentation and the presence of apoptotic bodies were noted. Conclusions: Spermine in concentrations of 1.5-10.0 mM and chlorhexidine in concentrations of 10.0-30.0 μM, applied separately, have a cytotoxic effect on human prostate cancer cells of LNCaP and DU-145 lines in culture. Cytotoxic properties of spermine, chlorhexidine or their combination are significantly more pronounced when acting on the hormone-dependent LNCaP line cells than on the hormone-independent DU-145 line. Their combined use demonstrates a significantly higher cytotoxic effect on cells of the LNCaP line, which is not observed in the DU-145 cell line. Chlorhexidine, like spermine, causes an inversion of the surface charge in the studied cell lines, changing its sign from negative to positive. The revealed differences like changes in survival and electrokinetic parameters of LNCaP and DU-145 cells can be explained by the presence or absence of androgen binding receptors on their surface membrane. Cells of the DU-145 line under the influence of spermine, or at the same time spermine and chlorhexidine, go to the state of apoptosis, as well as cells of the LNCaP line when they are exposed to spermine.

Acute effect of high-intensity interval aerobic exercise on serum myokine levels and resulting tumour-suppressive effect in trained patients with advanced prostate cancer.

Although skeletal muscle releases cytokines called myokines during exercise, the kinetics of the acute myokine response to exercise (exercise-induced circulatory myokine level alteration) is unknown in patients with advanced prostate cancer. We measured myokine levels in serum obtained from patients with metastatic castrate-resistant prostate cancer (mCRPC) before and after exercise and assessed the growth-suppressive effect of the serum by applying it to a PCa cell line. Nine patients with mCRPC (age = 67.8 ± 10.1 years, time since mCRPC diagnosis 36.2 ± 22.5 months) undertook 34 min of a high-intensity interval exercise session on a cycle ergometer. Blood was collected immediately pre, post and 30 min post. Serum levels of secreted protein acidic and rich in cysteine (SPARC), oncostatin M (OSM), interleukin-6 (IL-6), interleukin-15 (IL-15), decorin, irisin, and IGF-1 were determined. Growth of the androgen-independent PCa cell line DU-145 exposed to serum collected at three points was measured. There was a significant elevation of SPARC (19.9%, P = 0.048), OSM (11.5%, P = 0.001), IL-6 (10.2%, P = 0.02) and IL-15 (7.8%, P = 0.023) in serum collected immediately after exercise compared to baseline, returning to baseline after 30 min rest. A significant reduction in DU-145 Cell growth and the Cell Index area under the curve at 72 h incubation was observed with the presence of serum obtained immediately post-exercise (Cell Index at 72 h: 16.9%, P < 0.001; area under the curve: 15.2%, P < 0.001) and with the presence of serum obtained 30 min post-exercise compared to baseline (Cell Index at 72 h: 6.5%; area under the curve: 8.8%, P < 0.001). This study provides preliminary evidence for an acute exercise-induced myokine response and tumour growth suppression in serum after a bout of high-intensity interval exercise in patients with advanced PCa.

Lncap-AI prostate cancer cell line establishment by Flutamide and androgen-free environment to promote cell adherent

BackgroundTo establish castration-resistant prostate cancer (CRPC) - Lncap androgen-independent (AI) cell line from Lncap androgen-dependent (AD) cell line, and explore the different molecular biological between these two cell lines.MethodsThe Lncap-AD cell line was cultured and passaged 60 times over 16 months. The morphology of the Lncap-AI cell line was observed. AR levels identification were detected in qRT-PCR and Western Blot assay. CCK-8, EdU assay, wound healing assay and cell adhesion assays were used to observe the ability of proliferation, migration, and adhesion. SEM and TEM were used to observe microculture structure. At last, the PSA secrete ability was evaluated by Elisa assay.ResultsThe Lncap-AD cell line was cultured and passaged 60 times over 16 months. The Lncap-AI cell line showed a morphologic change at the end stage of culture, the cells turned slender and cell space turned separated compared to the Lncap-AD cell line. The relative levels of AR-related genes in the Lncap-AI cell line were up-regulation compared to the Lncap-AD cell line both in mRNA and protein levels. The expression of AR and HK2 proteins were influenced and down-regulation by Enzalutamide in the Lncap-AD cell line, but no obvious difference in Lncap-AI cell lines. Lncap-AI cell line showed strong viability of proliferation, migration, and adhesion by CCK-8, EdU assay, wound healing assay, and adhesion assay. The microstructure of Scanning Electron Microscopy (SEM) showed many synapses in the Lncap-AI cell line and PC3 cell line, but not in the Lncap-AD cell line. At last, the PSA secrete ability was evaluated by Elisa assay, and PCa cell lines showed no significant difference.ConclusionSimulation of CRPC progression, Lncap-AD cell line turned to Lncap-AI cell line with androgen deprivation therapy.

Bicalutamide Anticancer Activity Enhancement by Formulation of Soluble Inclusion Complexes with Cyclodextrins.

Bicalutamide (BCL) is a nonsteroidal antiandrogen drug that represents an alternative to castration in the treatment of prostate cancer, due to its relatively long half-life and tolerable side effects. However, it possesses a very low water solubility that can affect its oral bioavailability. In this work, we developed inclusion complexes of BCL with the highly soluble hydroxypropyl-β-cyclodextrin (HP-β-CyD) and sulfobutylether-β-cyclodextrin (SBE-β-CyD) to increase the water solubility and anticancer activity of BCL. The inclusion complexes were prepared using the freeze-drying method and were then characterized in a solid state via differential scanning calorimetry and X-ray analysis and in solution via phase-solubility studies and UV-vis and NMR spectroscopy. The BCL/HP-β-CyD and BCL/SBE-β-CyD inclusion complexes were amorphous and rapidly dissolved in water. Both the 1H-NMR spectra and molecular modeling studies confirmed the penetration of the 2-(trifluoromethyl)benzonitrile ring of BCL within the cavity of both cyclodextrins (CyDs). Due to the consistent improvement of the water solubility of BCL, the inclusion complexes showed higher antiproliferative activity toward the human prostate androgen-independent cell lines, DU-145 and PC-3, with respect to free BCL. These results demonstrate the ability of HP-β-CyD and SBE-β-CyD to complex BCL, permitting the realization of liquid formulations with potentially high oral bioavailability and/or possible parenteral administration.

Abstract 1543: Regulatory role of miR-410 in prostate cancer

Abstract Prostate cancer (PCa), a leading cause of male cancer related mortality, is dependent upon androgens acting via androgen receptor (AR) signaling. Androgen targeted therapies are often used as the first line of treatment for prostate cancer that are effective initially. However, PCa eventually progresses to castration-resistant stage that has limited treatment options. Castration-resistant prostate cancer (CRPC) is treated by second generation of AR pathway inhibitors such as Enzalutamide and Abiraterone. Resistance to these agents develop owing to various mechanisms such as evolution to androgen-independent neuroendocrine prostate cancer (NEPC). In this study, we examined the potential role of miR-410 in different stages of prostate cancer. Analyses of miR-410 in PCa clinical samples and patient-derived xenograft (PDX) models showed that miR-410 is frequently upregulated in PCa. Its expression was further increased in CRPC cases with neuroendocrine differentiation (NED). We examined the functional role of miR-410 by stably expressing miR-410 in androgen dependent and androgen independent cell lines. Our data suggests a context dependent role of miR-410 in prostate cancer cell lines. In androgen independent cell line, miR-410 plays an oncogenic role by impacting epithelial to mesenchymal transition (EMT) via directly targeting SNAIL. Also, it was found to increase the expression of neuronal genes such as Synaptophysin and neural transcription factor BRN2. However, in androgen dependent cell line, miR-410 was found to target PTEN. In conclusion, our data suggests that miR-410 play an important regulatory role in prostate cancer with effects on EMT and neuronal differentiation programs. Citation Format: Amritha Sreekumar, Nikhil Patel, Sharanjot Saini. Regulatory role of miR-410 in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1543.

Abstract 3311: Darolutamide potentiates the antitumor efficacy of a PSMA-targeted thorium-227 conjugate (PSMA-TTC) in a hormone-independent prostate cancer model

Abstract Androgen receptor (AR) inhibitors are standard of care for the treatment of advanced prostate cancer. Despite an initial response to treatment, patients eventually progress, and novel therapeutic approaches are required. Prostate-specific membrane antigen (PSMA; FOLH1) is an integral membrane glycoprotein that is highly expressed in prostate cancer but has limited expression in normal tissues. PSMA-TTC (227Th-pelgifatamab corixetan; BAY 2315497) consists of the alpha emitter thorium-227 complexed to a 3,2-HOPO chelator conjugated to a PSMA targeting antibody. PSMA-TTC delivers potent radiation to PSMA expressing cells. Here we evaluated the efficacy of PSMA-TTC in combination with darolutamide, a novel AR inhibitor in a hormone insensitive prostate cancer model and investigated the mode of action of the combination treatment. In vitro, darolutamide induced the expression of PSMA in the androgen-independent cell lines C4-2 and 22Rv1 more than 3-fold and 2-fold, respectively. Expression of the apoptosis marker CDKN1A was significantly induced in the PSMA-TTC alone and in the combination group compared to vehicle. Expression of the DNA repair genes BRCA1, XRCC2 and XRCC3 were significantly reduced in PSMA-TTC alone and in the combination group compared to vehicle. In vivo, a single i.v. injection of PSMA-TTC at 300 kBq/kg resulted in a tumor/control (T/C) ratio of 0.44 on day 19 in the 22Rv1 hormone-insensitive prostate cancer model while a twice daily oral treatment with 100 mg/kg darolutamide showed a T/C ratio of 0.82. The combination of darolutamide and PSMA-TTC resulted in a higher efficacy with a T/C ratio of 0.27. Tumor area on day 19 after treatment was significantly reduced in the PSMA-TTC alone group and more so in the combination group, compared to vehicle. As expected, darolutamide alone was not efficacious in this hormone insensitive model. Nine out of 10 mice showed disease control (CR, PR or SD) in the combination group whereas no animals showed disease control in either of the monotherapy groups. This difference was significant by Fisher’s exact test analysis. Treatments were well tolerated with no significant changes in body weight in any of the treatment groups. Using the tumor dissociation kit Miltenyi individual tumor cells were isolated from darolutamide treated and untreated 22Rv1 xenograft tumors. FACS analysis showed a 50fold increase in PSMA expression in the darolutamide treated 22Rv1 tumors compared to vehicle treated animals. These data indicate that darolutamide induces PSMA expression in the hormone independent 22Rv1 model, which may have contributed to the stronger efficacy observed when combined with PSMA-TTC. Altogether these results support the further evaluation of darolutamide combination with PSMA radionuclides. Citation Format: Christoph Schatz, Urs Hagemann, Sabine Zitzmann-Kolbe, Bernard Haendler, Hartwig Hennekes, Stefanie Hammer, Arne Scholz. Darolutamide potentiates the antitumor efficacy of a PSMA-targeted thorium-227 conjugate (PSMA-TTC) in a hormone-independent prostate cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3311.

PLK4 is upregulated in prostate cancer and its inhibition reduces centrosome amplification and causes senescence.

BackgroundIdentification of novel molecular target(s) is important for designing newer mechanistically driven approaches for the treatment of prostate cancer (PCa), which is one of the main causes of morbidity and mortality in men. In this study, we determined the role of polo‐like kinase 4 (PLK4), which regulates centriole duplication and centrosome amplification (CA), in PCa.Materials and MethodsEmploying human PCa tissue microarrays, we assessed the prevalence of CA, correlated with Gleason score, and estimated major causes of CA in PCa (cell doubling vs. centriole overduplication) by staining for mother/mature centrioles. We also assessed PLK4 expression and correlated it with CA in human PCa tissues and cell lines. Further, we determined the effects of PLK4 inhibition in human PCa cells.ResultsCompared to benign prostate, human PCa demonstrated significantly higher CA, which was also positively correlated with the Gleason score. Further, most cases of CA were found to arise by centriole overduplication rather than cell doubling events (e.g., cytokinesis failure) in PCa. In addition, PLK4 was overexpressed in human PCa cell lines and tumors. Moreover, PLK4 inhibitors CFI‐400945 and centrinone‐B inhibited cell growth, viability, and colony formation of both androgen‐responsive and androgen‐independent PCa cell lines. PLK4 inhibition also induced cell cycle arrest and senescence in human PCa cells.ConclusionsCA is prevalent in PCa and arises predominantly by centriole overduplication as opposed to cell doubling events. Loss of centrioles is cellular stress that can promote senescence and suggests that PLK4 inhibition may be a viable therapeutic strategy in PCa.

CYP1B1-catalyzed 4-OHE2 promotes the castration resistance of prostate cancer stem cells by estrogen receptor \u03b1-mediated IL6 activation

BackgroundResistance to androgen deprivation therapy remains a major challenge for the clinical treatment of patients with castration-resistant prostate cancer (CRPC). CYP1B1, a critical enzyme that catalyzes the conversion of estradiol to 4-Hydroxy-17β-estradiol (4-OHE2), has been reported to promote the development and progression of hormone-related cancer, but its role in CRPC is unclear.MethodsTo explore the underlying mechanism which CYP1B1 promotes the prostate cancer stem cells (PCSCs) characteristics, bioinformatics analyses of human clinical prostate cancer (PCa) datasets were performed. CYP1B1, IL6, and estrogen receptor-α (ERα) expression levels were evaluated in PCa and CRPC tissues via immunohistochemistry. The high-performance liquid chromatography-mass spectrometry assay was carried out to examine intracellular 4-OHE2 levels. Serum-free suspension culture and flow cytometry assays were performed to evaluate PCSCs. Chromatin immunoprecipitation was used to validate that 4-OHE2 recruited ERα to the IL6 promoter.ResultsCYP1B1 expression was significantly increased in CRPC tissues and androgen-independent PCa cell lines. CYP1B1+ PCa cells were significantly enriched in bicalutamide-treated LNCaP cells, and CYP1B1 knockdown reduced the cell viability under bicalutamide treatment. In addition, CYP1B1 knockdown decreased the intracellular 4-OHE2 concentration, accompanied by reduced PCSC characteristics. In PCa cells, 4-OHE2 stimulated ERα transcriptional activity and upregulated the expression of IL6 and downstream genes of the IL6-STAT3 signaling. 4-OHE2 increased cell viability under bicalutamide treatment and promoted PCSC characteristics, while IL6 neutralizing antibody reversed these effects. Mechanistically, siERα and the ER antagonist ICI182780 significantly attenuated 4-OHE2-induced IL6 expression, and 4-OHE2 promoted the binding of ERα to the estrogen response element of the IL6 promoter.ConclusionsOur findings indicate that CYP1B1-catalyzed 4-OHE2 enhanced PCSC characteristics and attenuated bicalutamide sensitivity by ERα-mediated the IL6-STAT3 pathway activation. Our study further emphasizes the role of CYP1B1 in castration resistance and illustrates a novel mechanism of CRPC development.Graphical 54dyAth8NatsYXqV4_5FMeVideo .

Molecular mechanism of inhibitory effects of melatonin on prostate cancer cell proliferation, migration and invasion.

The increasing incidence of prostate cancer (PCa) indicates an urgent need for the development of new effective drug therapy. There are limited options to treat the PCa, this study tried to determine a new therapy option for this acute cancer. Androgen-independent PCa cell lines PC3 and DU145 were treated with different melatonin concentrations (0.1~3.5 mM) for 1~3 days and assessed cell migration, cell invasion, cycle arrest in G0/G1 phase as well as apoptosis. We utilized RNA-seq technology to analyze the transcriptional misregulation pathways in DU145 prostate cancer cell line with melatonin (0.5 mM) treatment. Data revealed 20031 genes were up and down-regulated, there were 271 genes that differentially expressed: 97 up-regulated (P<0.05) and 174 down-regulated (P<0.05) genes. Furthermore, RNA-seq results manifested that the melatonin treatment led to a significant increase in the expression levels of HPGD, IL2Rβ, NGFR, however, IGFBP3 and IL6 (P <0.05) had decreased expression levels. The immunoblot assay revealed the expression of IL2Rβ and NGFR genes was up-regulated, qPCR confirmed the gene expression of HPGD and IL2RB were also up-regulated in Du145 cells. Consequently, we probed mechanisms that generate kinetic patterns of NF-κB-dependent gene expression in PCa cells responding to a NF-κB-activation, the significant results were indicated by the inhibition of the NF-kB pathway via IL2Rβ actions. Based on our investigation, it could be concluded that melatonin is a chemotherapeutic molecule against PCa and provides a new idea for clinical therapy of PCa.

Imatinib mesylate inhibits androgen-independent PC-3 cell viability, proliferation, migration, and tumor growth by targeting platelet-derived growth factor receptor-α

AimThe abnormal expression of oncogenic tyrosine kinase receptors such as platelet-derived growth factor receptors (PDGFRs) has been reported in cancer progression. However, the role of PDGFRs in the human androgen-independent prostate cancer PC-3 cell line is not well understood. Thus, this study examined the role of PDGFRs in androgen-independent PC-3 cells. Main methodsPDGFR mRNA and protein expression was determined by quantitative real-time PCR and western blotting, respectively. The effects of the tyrosine kinase inhibitor imatinib (imatinib mesylate) and small interfering RNAs (siRNAs) were determined by a Cell Counting Kit-8 assay, bromodeoxyuridine assay, and Transwell migration assay. The in vivo effect of imatinib was analyzed using a tumor formation assay in nude mice. Key findingsPDGFRα was upregulated in androgen-independent PC-3 cells compared with normal prostate epithelial cells. PDGF-BB induced the phosphorylation of PDGFRα and downstream signaling molecules, including Akt, in a dose-dependent manner. Imatinib reduced the phosphorylation of the PDGFRα/Akt axis. Imatinib also suppressed the viability, proliferation, migration, and tumor growth of PC-3 cells. PDGFRα knockdown by siRNA decreased the viability and migration of PC-3 cells. SignificanceThese results demonstrated the distinct contribution of PDGFRα signaling to the proliferation and migration of PC-3 cells and suggested the potential for PDGFRα as a therapeutic target for metastatic and androgen-independent prostate cancer.

Peri-Prostatic Adipocyte-Released TGFβ Enhances Prostate Cancer Cell Motility by Upregulation of Connective Tissue Growth Factor

Periprostatic adipose tissue (PPAT) has emerged as a key player in the prostate cancer (PCa) microenvironment. In this study, we evaluated the ability of PPAT to promote PCa cell migration, as well as the molecular mechanisms involved. Methods: We collected conditioned mediums from in vitro differentiated adipocytes isolated from PPAT taken from PCa patients during radical prostatectomy. Migration was studied by scratch assay. Results: Culture with CM of human PPAT (AdipoCM) promotes migration in two different human androgen-independent (AI) PCa cell lines (DU145 and PC3) and upregulated the expression of CTGF. SB431542, a well-known TGFβ receptor inhibitor, counteracts the increased migration observed in presence of AdipoCM and decreased CTGF expression, suggesting that a paracrine secretion of TGFβ by PPAT affects motility of PCa cells. Conclusions: Collectively, our study showed that factors secreted by PPAT enhanced migration through CTGF upregulation in AI PCa cell lines. These findings reveal the potential of novel therapeutic strategies targeting adipocyte-released factors and TGFβ/CTGF axis to fight advanced PCa dissemination.

KLF9 suppresses cell growth and induces apoptosis via the AR pathway in androgen-dependent prostate cancer cells

Kruppel-like factors (KLFs) play an important role in many biological processes including cell proliferation, differentiation and development. Our study showed that the level of KLF9 is lower in PCa cell lines compared to a benign prostate cell line; the androgen-independent cell line PC3 expresses significantly lower KLF9 than the androgen-dependent cell line, LNCaP. Forced overexpression of KLF9 suppressed cell growth, colony formation, and induced cell apoptosis in LNCaP cells. We also found that KLF9 expression was induced in response to apoptosis caused by flutamide, and further addition of dihydrotestosterone antagonized the action of flutamide and significantly decreased KLF9 expression. Furthermore, activation of the androgen receptor (AR) was inhibited by the overexpression of KLF9. Our research shows that KLF9 is lower in androgen-independent cell lines than in androgen-dependent cell lines; Overexpression of KLF9 dramatically suppresses the proliferation, anchorage-independent growth, and induces apoptosis in androgen-dependent cells; KLF9 inhibition on prostate cancer cell growth may be acting through the AR pathway. Our results therefore suggest that KLF9 may play a significant role in the transition from androgen-dependent to androgen-independent prostate cancer and is a potential target of prevention and therapy.

MP33-11 OBESITY-ASSOCIATED PROINFLAMMATORY CYTOKINES REGULATE PROSTATE CANCER CELL PROLIFERATION AND MIGRATION

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP33)1 Sep 2021MP33-11 OBESITY-ASSOCIATED PROINFLAMMATORY CYTOKINES REGULATE PROSTATE CANCER CELL PROLIFERATION AND MIGRATION Xiaobo Wu, Christina Sharkey, Zongwei Wang, and Aria Olumi Xiaobo WuXiaobo Wu More articles by this author , Christina SharkeyChristina Sharkey More articles by this author , Zongwei WangZongwei Wang More articles by this author , and Aria OlumiAria Olumi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002042.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Previous studies revealed that obesity is significantly associated with a higher incidence of prostate cancer (PCa). Meanwhile, obesity-associated inflammation plays an important role in tumorigenesis. Here we wished to identify the downstream signaling pathways of obesity-associated proinflammatory cytokines IL-6 and TNF-α’s effects on prostate cancer cell proliferation and migration. METHODS: Adipocytes and THP-1 macrophages were differentiated and treated with myristic acid (MA) to induce inflammatory cytokines, and conditioned media (CM) were collected to treat prostate epithelial cancer, androgen-dependent LNCaP and androgen-independent PC-3 cell lines. Cells were also treated with interleukin-6 (IL-6) 10ng/ml, tumor necrosis factor-alpha (TNF-α) 10ng/ml, oncometabolite R-2-hydroxyglutarate 50µM (R-2-HG) and Dutasteride 10µM (SRD5A1 inhibitor). Prostate cancer cell migration and proliferation were assessed by scratch assay (0h, 24h, 48h) and MTT assay (0h, 24h, 48h, 72h) and analyzed by 2-way ANOVA. Enzymes regulating steroid hormone metabolism SRD5A1, AKR1C1, AKR1C3, HSD3B2, CYP17A1 and UGT2B15 were determined by immunocytochemistry and qPCR. The protein level of SRD5A1 was measured by western blot. RESULTS: The proliferation of LNCaP and PC-3 cells was significantly stimulated by adding of CM for 24h, 48h, and 72h. The migration of PC-3 cells was also promoted by CM. Addition of testosterone did not change the effect of CM. Administration of IL-6, TNF-α and R-2-HG significantly increased the proliferation and migration of LNCaP and PC-3 cells. IL-6 also increased the expression of SRD5A1.. The expression of AKR1C1 and HSD3B2 was downregulated, and UGT2B15 was upregulated by TNF-α. SRD5A1 was upregulated either by R-2-HG alone or combining with IL-6 and TNF-α, but downregulated by Dutasteride alone. CONCLUSIONS: Our study suggests that obesity-associated proinflammatory cytokines, IL-6 and TNF-α, affect prostate cancer cells’ proliferation and migration in an androgen-independent manner. Synergistic targeting oncometabolite R-2-HG and proinflammatory cytokines may improve the management of androgen-independent prostate cancer. Source of Funding: NIH/NIDDK © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e610-e611 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Xiaobo Wu More articles by this author Christina Sharkey More articles by this author Zongwei Wang More articles by this author Aria Olumi More articles by this author Expand All Advertisement Loading ...

Abstract 2459: Altered bioenergetics profile in prostate cancer cells treated with andrographolide

Abstract Prostate cancer (PCa) is the second leading cause of cancer deaths in the United States alone. An emerging area of cancer therapeutics is the combination of the standard-of-care treatment with natural products. Andrographolide, a labdane diterpenoid and the main bioactive component of the medicinal plant Andrographis paniculata, has shown a wide range of therapeutic potential, including cancer. Previous studies from our laboratory have demonstrated that Andrographolide induces a DNA damage response, including ATM gene upregulation, involving cell growth suppression, cell cycle arrest, and apoptosis in PCa in vitro. The ATM gene is an important regulator of p53 and is consider a master switch for the p53-family member p63. The p63 is a natural marker of the basal lamina in the normal prostate, while its expression is lost in 99% of prostatic adenocarcinomas. Like p53, the p63 protein is implicated in tumor suppression and inhibition of metastasis, and also plays a role in energy metabolism. In this study, we aim to (1) determine the role of Andrographolide on the expression of p63, and (2) determine the bioenergetics profile of PCa cells. Androgen-dependent (22Rv1) and androgen-independent (PC3) PCa cell lines were treated with Andrographolide (25µM) for 24 h and 48 h. The gene expression of TP63 was assessed by real-time quantitative PCR (RT-qPCR). At the protein level, the expression of p63 was evaluated by immunofluorescence. The bioenergetics profile was performed using the Agilent Seahorse XF Real-Time ATP Rate Assay to determine the ATP production from glycolysis and mitochondria in living PC3 and 22Rv1 PCa cells. RT-PCR analysis showed a significant upregulation of the TP63 gene with a fold-change of 2.2 (p&amp;lt; 0.05) and immunofluorescence analysis showed an increase in expression of p63 protein with a fold-change of 1.8 (p&amp;lt; 0.05) in 22Rv1 cells treated with Andrographolide. The bioenergetics profile of PC3 cells, 48 h after Andrographolide treatment, revealed a shift in the cytoplasmic to mitochondrial ATP production rate from 54.4% to 72.6% and from 45.6% to 27.4%, respectively. Similar results were obtained in 22Rv1 cells. These results confirmed that Andrographolide induces the expression of the tumor protein p63 in androgen-dependent 22Rv1 PCa cells and alters the bioenergetics profile of both PC3 and 22Rv1 PCa cells. Our findings suggest a novel mechanism in which Andrographolide promotes the expression of p63 as in normal cells, and transforms the bioenergetics by increasing glycolytic-ATP. Citation Format: Jesus Sosa, María Sánchez Vázquez, Sylvette Ayala-Peña, Carlos Torres Ramos, Magaly Martínez Ferrer. Altered bioenergetics profile in prostate cancer cells treated with andrographolide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2459.

Periprostatic adipose tissue promotes prostate cancer resistance to docetaxel by paracrine IGF‐1 upregulation of TUBB2B beta‐tubulin isoform

Growing evidence supports the pivotal role played by periprostatic adipose tissue (PPAT) in prostate cancer (PCa) microenvironment. We investigated whether PPAT can affect response to Docetaxel (DCTX) and the mechanisms associated. Conditioned medium was collected from the in vitro differentiated adipocytes isolated from PPAT which was isolated from PCa patients, during radical prostatectomy. Drug efficacy was studied by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide citotoxicity assay. Culture with CM of human PPAT (AdipoCM) promotes DCTX resistance in two different human prostate cancer cell lines (DU145 and PC3) and upregulated the expression of BCL‐xL, BCL‐2, and TUBB2B. AG1024, a well‐known IGF‐1 receptor inhibitor, counteracts the decreased response to DCTX observed in presence of AdipoCM and decreased TUBB2B expression, suggesting that a paracrine secretion of IGF‐1 by PPAT affect DCTX response of PCa cell. Collectively, our study showed that factors secreted by PPAT elicits DCTX resistance through antiapoptotic proteins and TUBB2B upregulation in androgen independent PCa cell lines. These findings reveal the potential of novel therapeutic strategies targeting adipocyte‐released factors and IGF‐1 axis to overcome DCTX resistance in patients with PCa.

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.