Androgen-sensitive Prostate Cancer Cells Research Articles

Design, Synthesis, Characterization, and Cytotoxicity of New Pyrazolylmethylene-2-thioxoimidazolidin-4-one Derivatives towards Androgen-Sensitive LNCaP Prostate Cancer Cells

A new class of pyrazolylmethylene-2-thioxoimidazolidin-4-one derivatives 3a–p were rationally designed and synthesized with the aim of exploring their potential as treatments for prostate cancer. The synthesized compounds 3a–p were biologically analyzed for their anticancer effects against AR+LNCaP, AR-PC-3, and Wi38 cell lines. The observed IC50 values against AR+LNCaP ranged between 10.27 ± 0.14 and 109.72 ± 2.06 µM after 24 h of incubation. Compounds 3i–k, 3m, and 3o–p recorded IC50 values of 05.22 ± 0.12 to 11.75 ± 0.07 µM after 48 h incubation in the presence of 1 nM DHT, with higher selectivity towards AR+LNCaP. Moreover, compounds 3i and 3k significantly induced Caspase 3 accumulation, reduced DNA content at the various stages of the cell cycle, and ultimately caused AR+LNCaP cell growth arrest, as confirmed by cell apoptosis assays. These findings suggest that these analogues of androgen receptor blockers have promising potential for further investigation as effective treatments for prostate cancer.

Mitochondrial Elongation and ROS-Mediated Apoptosis in Prostate Cancer Cells under Therapy with Apalutamide and Complex I Inhibitor.

Metabolic reprogramming and mitochondrial dynamics are pivotal in prostate cancer (PCa) progression and treatment resistance, making them essential targets for therapeutic intervention. In this study, we investigated the effects of the androgen receptor antagonist apalutamide (ARN) and the mitochondrial electron transport chain complex I inhibitor IACS-010759 (IACS) on the mitochondrial network architecture and dynamics in PCa cells. Treatment with ARN and/or IACS induced significant changes in mitochondrial morphology, particularly elongation, in androgen-sensitive PCa cells. Additionally, ARN and IACS modulated the mitochondrial fission and fusion processes, indicating a convergence of metabolic and androgen-signaling pathways in shaping mitochondrial function. Notably, the combination treatment with ARN and IACS resulted in increased apoptotic cell death and mitochondrial oxidative stress selectively in the androgen-sensitive PCa cells. Our findings highlight the therapeutic potential of targeting mitochondrial metabolism in prostate cancer and emphasize the need for further mechanistic understanding to optimize treatment strategies and improve patient outcomes.

Purine-Rich Element Binding Protein Alpha, a Nuclear Matrix Protein, Has a Role in Prostate Cancer Progression.

Solid tumors as well as leukemias and lymphomas show striking changes in nuclear structure including nuclear size and shape, the number and size of nucleoli, and chromatin texture. These alterations have been used in cancer diagnosis and might be related to the altered functional properties of cancer cells. The nuclear matrix (NM) represents the structural composition of the nucleus and consists of nuclear lamins and pore complexes, an internal ribonucleic protein network, and residual nucleoli. In the nuclear microenvironment, the NM is associated with multi-protein complexes, such as basal transcription factors, signaling proteins, histone-modifying factors, and chromatin remodeling machinery directly or indirectly through scaffolding proteins. Therefore, alterations in the composition of NM could result in altered DNA topology and changes in the interaction of various genes, which could then participate in a cascade of the cancer process. Using an androgen-sensitive prostate cancer cell line, LNCaP, and its androgen-independent derivative, LN96, conventional 2D-proteomic analysis of the NM proteins revealed that purine-rich element binding protein alpha (PURα) was detected in the NM proteins and differentially expressed between the cell lines. In this article, we will review the potential role of the molecule in prostate cancer.

Abstract 2185: Mechanism of antitumor effects of saffron in human prostate cancer cells

Abstract Prostate cancer is the most common malignancy in men in the USA and the second leading cause of cancer deaths. Several studies have suggested the antitumor properties of saffron in many types of cancers including prostate. The objective of this study was to carry out in vitro studies of saffron-treated prostate cancer cells to ascertain the effects of saffron on key intermediates in prostate carcinogenesis. Our studies demonstrate significant inhibition of cell proliferation of androgen-sensitive prostate cancer cell lines via apoptotic pathways. We also demonstrate statistically significant down-regulation of DNA methyltransferase; COMT, MGMT, EHMT2, and SIRT1 in saffron-treated prostate cancer cells. In addition, saffron-treated prostate cancer cells displayed statistically significant dysregulation of DNA repair intermediates; WRN, p53, RECQ5, MST1R, and WDR70 in a time-dependent manner. Furthermore, Western blot analysis demonstrated that saffron treatment induced changes in the expression of other key genes; DNMT1, DNMT3b, MBD2, CD44, HDAC3, c-Myc, NF-kB, TNFα, AR, N-RAS and PTEN in prostate cancer cells. Collectively, our findings demonstrate important mechanistic targets of saffron mediating anti-tumor properties in prostate cancer. These findings suggest that the use of saffron supplements alongside standard treatment protocols may yield beneficial effects for individuals with prostate cancer. Citation Format: Mohammad Khan, Kaitlyn Hearn, Christian Parry, Mudasir Rashid, Hassan Brim, Hassan Ashktorab, Bernard Kwabi-Addo. Mechanism of antitumor effects of saffron in human prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2185.

Dual targeting of the androgen receptor and PI3K/AKT/mTOR pathways in prostate cancer models improves antitumor efficacy and promotes cell apoptosis.

Prostate cancer is a frequent malignancy in older men and has a very high 5-year survival rate if diagnosed early. The prognosis is much less promising if the tumor has already spread outside the prostate gland. Targeted treatments mainly aim at blocking androgen receptor (AR) signaling and initially show good efficacy. However, tumor progression due to AR-dependent and AR-independent mechanisms is often observed after some time, and novel treatment strategies are urgently needed. Dysregulation of the PI3K/AKT/mTOR pathway in advanced prostate cancer and its implication in treatment resistance has been reported. We compared the impact of PI3K/AKT/mTOR pathway inhibitors with different selectivity profiles on in vitro cell proliferation and on caspase 3/7 activation as a marker for apoptosis induction, and observed the strongest effects in the androgen-sensitive prostate cancer cell lines VCaP and LNCaP. Combination treatment with the AR inhibitor darolutamide led to enhanced apoptosis in these cell lines, the effects being most pronounced upon cotreatment with the pan-PI3K inhibitor copanlisib. A subsequent transcriptomic analysis performed in VCaP cells revealed that combining darolutamide with copanlisib impacted gene expression much more than individual treatment. A comprehensive reversal of the androgen response and the mTORC1 transcriptional programs as well as a marked induction of DNA damage was observed. Next, an in vivo efficacy study was performed using the androgen-sensitive patient-derived prostate cancer (PDX) model LuCaP 35 and a superior efficacy was observed after the combined treatment with copanlisib and darolutamide. Importantly, immunohistochemistry analysis of these treated tumors showed increased apoptosis, as revealed by elevated levels of cleaved caspase 3 and Bcl-2-binding component 3 (BBC3). In conclusion, these data demonstrate that concurrent blockade of the PI3K/AKT/mTOR and AR pathways has superior antitumor efficacy and induces apoptosis in androgen-sensitive prostate cancer cell lines and PDX models.

Mechanism of Antitumor Effects of Saffron in Human Prostate Cancer Cells.

Prostate cancer is the most common cancer and the second leading cause of cancer deaths among men in the USA. Several studies have demonstrated the antitumor properties of saffron in different types of cancers, including prostate cancer. The oral administration of saffron extract has been reported to have antitumor effects on aggressive prostate-cancer-cell-line-derived xenografts in nude male mice. The objective of this study was to carry out in vitro studies of saffron-treated prostate cancer cells to ascertain the effects of saffron on key intermediates in prostate carcinogenesis. Our studies demonstrated the significant inhibition of cell proliferation for androgen-sensitive prostate cancer cell lines via apoptotic pathways. We also demonstrate the statistically significant down-regulation of DNA methyltransferases (COMT, MGMT, EHMT2, and SIRT1 deacetylase) in saffron-treated prostate cancer cells. In addition, saffron-treated prostate cancer cells displayed a statistically significant dysregulation of DNA repair intermediates (WRN, p53, RECQ5, MST1R, and WDR70) in a time-dependent manner. Furthermore, Western blot analysis demonstrated that saffron treatment induced changes in the expression of other key genes (DNMT1, DNMT3b, MBD2, CD44, HDAC3, c-Myc, NF-kB, TNFα, AR, N-RAS, and PTEN) in prostate cancer cells. Collectively, our findings demonstrate the important mechanisms by which saffron mediates anti-tumor properties in prostate cancer. These findings suggest that the use of saffron supplements alongside standard treatment protocols may yield beneficial effects for individuals with prostate cancer.

Electrochemical Aptamer-Based Biosensor Developed to Monitor PCA3 and PSA Released by Prostate Cancer Cells

1. Introduction Detection of prostate cancer (PCa) in the early stage of the disease is fundamental for the prevention of metastases and for the initiation of treatment, which may bring greater chance of patient cured; so several research groups have sought the manufacture of a simple and non-invasive methodology [1]. The early diagnosis relies on prostate specific antigen (PSA) serum test, even if it showed clear limits [2]. Recently, prostate cancer gene 3 (PCA3), a non-coding mRNA which is overexpressed in tumor cells when compared with noncancerous prostate tissue [3]. Here, we report a new method for detection of prostate cancer-specific antigen 3 (PCA3) and prostate specific antigen (PSA) released by cancer cells using an aptasensor. Electrochemical impedance spectroscopy (EIS), SEM and EDS were used to evaluate biosensor surface. 2. Experimental Part The aptamers were modified through Biosearch Technologies at 5′-terminus in C6-disulfide linker to have it bond to the gold electrode; and at 3′-end in methylene blue (MB) (as redox probe) to perform the electrochemical analysis, which was followed by the HPLC purification process to remove truncated-synthesis products and to enrich the purity of the aptamer sequences. Aptamer immobilization was done by incubation of 1 μM thiolated aptamer on the top of the gold-coated silicon electrode overnight, in the dark, at 4°C. Subsequently, the electrodes were washed with PBS and a backfilling consisted of 3 mM of MCH aqueous solution for 30 minutes was done to displace the nonspecifically adsorbed aptamer molecules and to passivate the electrode surface [26]. Finally, the electrodes were once more rinsed with PBS to remove MCH excesses. The electrochemical characterization was done in a potentiostat μAutolabIII (Metrohn, NL), using a three-electrode system, which consisted of Ag/AgCl (3M KCl), as reference electrode; Pt wire, as a counter electrode; and gold-coated silicon wafer, as a working electrode. The prostate cell lines used were normal human prostate cells (RWPE-1; ATCC® CRL-11™), androgen-sensitive human prostate cancer cells (LNCaP; ATCC® CRL-1740™), and androgen-independent human prostate cancer cells (PC-3; ATCC® CRL-1435™). The three lineages were chosen to better understand PCA3 and PSA production patterns by different cell types. 3. Results and Discussion The gold-coated silicon wafe electrode was analyzed by SEM and EDS, which acts as working electrode and substrate to anchor the thiolated aptamer on the surface. By using square wave voltammetry, the present aptasensor was able of monitoring the PCA3 and PSA released from prostate cells (normal and cancerous). The PSA and PCA3 detection limits were 1 ng/mL and 9.2 ng/mL, respectively, at linear range up to 150 ng/mL, and it was also able to detect both biomarker even in presence of several interfering compounds (UA, AA, BSA and MUC1). In addition, the herein developed aptasensor was able to verify, at accurate and robust response, the patterns of the PCA3 and PSA released by different cell lines (RWPE-1, LNCaP and PC3). The detection of PSA and PCA3 by aptasensor agree with the cell lineage type, the LNCaP is an androgen-dependent lineage. In this way, an overexpressed both biomarkres was release was verified [49,50] and it can be compared to early-stage patients. On the other hand, the PC3 cells PSA and PCA3 releasing was nearly background signal, demonstrating almost absent production of studied targets, the PC3 lineage is androgen-independent which can be compared to advanced-disease patients. Funding This work has been supported by a grant from FAPESP-Brazil, CNPq and Capes.

Functional consequences of A-to-I editing of miR-379 in prostate cancer cells

Prostate cancer is the predominant cause of cancer in men, but there is still a lack of biomarkers and treatments for metastatic spread. The initial promise of microRNAs to provide avenues to solve these problems has been dampened by the realisation that microRNAs co-exist in multiple functionally distinct isoforms, for example due to A-to-I editing. We recently found that A-to-I-editing of microRNA-379 (miR-379) was associated with prostate cancer, and that only the unedited isoform was negatively correlated with aggressive disease. Here, we set out to decipher the biological effects of unedited and edited miR-379 in prostate cancer cells. After transfection of four different prostate cancer cell lines with isoform-specific miR-379 mimics, we performed assays for cell growth, colony formation, migration, cell–cell adhesion, and analysed epithelial–mesenchymal transition (EMT) and stemness markers. We found that unedited miR-379 affected cell growth, with a promoting function in androgen receptor (AR)-negative cells and an inhibiting effect in AR-positive cells. This is supported by our in silico analysis that found unedited miR-379 targets are predicted to be predominantly involved in cellular proliferation whereas the targets of edited miR-379 are not. We further found that both miR-379 isoforms could promote colony formation, migration, and cell–cell adhesion. Overall, our data suggests that editing of miR-379 attenuates the growth-suppressive function of unedited miR-379 in androgen-sensitive prostate cancer cells, thereby promoting tumor growth.

Design, Synthesis, and Biological Evaluation of Anti-prostate Cancer Agent

In search of more effective chemotherapeutics for the treatment of castration-resistant prostate cancer. The 1,3- thiazolidine-2,4-diones possess a wide diversity of important biochemical effects and interesting pharmacological properties. 1,9-diarylnona-1,3,6,8-tetraen-5-ones bearing two identical terminal heteroaromatic rings have been successfully synthesized through Wittig reaction followed by Horner-Wadsworth-Emmons reaction. The cell proliferation assay was employed to assess their anti-proliferative effects toward both androgen-sensitive and androgen-insensitive human prostate cancer cell lines. curcumin in inhibiting prostate cancer cell proliferation. It can be concluded from our data that 1,9-diarylnona-1,3,6,8-tetraen-5-one can serve as a new potential scaffold for the development of anti-prostate cancer agents.

Saw Palmetto may fight prostate cancer.

Saw Palmetto may fight prostate cancer.

Abstract A004: Investigating the function of ZFHX3 in hormone sensitive prostate cancer initiation and treatment resistance

Abstract Background: The zinc-finger homeobox ZFXH3 (also known as ATBF1 for AT motif-binding factor 1) is a transcription factor that is frequently downregulated or deleted in human prostate cancers. In a recent clinical trial (NCT02430480), patients with localized prostate cancer (PCa) received six months of neoadjuvant androgen deprivation therapy (ADT) with 10.8mg of the GnRH agonist goserelin every 12 weeks and 160mg of oral enzalutamide daily before a radical prostatectomy (RP). Throughout the six months, the patients had two multiparametric MRIs (mpMRI) at baseline and prior to surgery, along with targeted biopsies of the tumor extracted. DNA from laser capture microdissection (LCM) biopsy and RP tissues was extracted for whole exome sequencing with somatic mutation calling and copy number analysis. The most common alteration in all residual tumor samples shared a single-copy loss or deletion of chromosome 16q overlapping with ZFHX3. We hypothesized that ZFHX3 is necessary for the proper development of normal human prostates and drives tumor-suppressive activity. Methods: To mimic the reduced expression of ZFHX3 observed in patient samples, we first surveyed ZFHX3 expression by Western blotting and real-time qPCR and then developed shRNA knockdowns in androgen-sensitive (22Rv1 and LNCaP) and insensitive (C4-2) PCa cell lines. These cell lines were challenged by dose-response curve studies utilizing standard fetal bovine serum (FBS) growth media with the androgen receptor (AR) axis inhibitors abiraterone and enzalutamide. Continued treatment conditions included charcoal dextran-stripped fetal calf serum (CSS) to simulate androgen deprivation supplemented with either abiraterone or enzalutamide. Cell proliferation was determined at the end of the five-day dose-response curve treatment studies using the CellTiter Glo 2.0 cell viability assay. Results and Conclusions: In the three PCa cell lines with ZFHX3 knockdown, we observed increased sensitivity to AR-targeting drugs with a lower IC50 dose. In addition, ZFHX3 knockdown cell lines exhibited increased cell proliferation compared to scrambled control and parental cell lines using dose-response time course studies. These preliminary results form the scientific premise for this work and substantiate ongoing efforts to expand these assays to more cell lines and treatments. Our future efforts will provide mechanistic insights into the functions of ZFHX3 through chromatin immunoprecipitation sequencing with antibodies against AR and FOXA1 and the expansion of ZFHX3 knockdown into additional cell lines to investigate the transcriptional activity of multiple hormonal signaling pathways. We will further explore genomic interactions between the effects of ZFHX3 reduction and alterations to commonly-mutated genes in PCa, such as PTEN, TP53, and SPOP. Findings in this study will seek to determine how ZFHX3 exerts its tumor suppressor function and its role in the development and progression of prostate cancer. Citation Format: Isaiah M. King, John R. Bright, Nicholas T. Terrigino, Scott Wilkinson, Adam G. Sowalsky. Investigating the function of ZFHX3 in hormone sensitive prostate cancer initiation and treatment resistance [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A004.

Novel α-Trifluoromethyl Chalcone Exerts Antitumor Effects Against Prostate Cancer Cells.

Despite treating advanced prostate cancer (PCa) with androgen deprivation therapy, it eventually progresses to castration-resistant PCa. Subsequently, taxanes are administered, but when PCa becomes resistant to taxanes, another treatment is needed, which has not yet been established. We previously synthesized a novel α-trifluoromethyl chalcone, YS71, and reported its antitumor effects against PCa cells. In this study, we confirmed its efficacy against androgen-sensitive, androgen-independent, and taxane-resistant PCa cells. The PCa cell lines used were LNCaP, PC-3, DU145, PC-3-TxR (paclitaxel-resistant), PC-3-TxR/CxR (paclitaxel- and cabazitaxel-resistant), DU145-TxR, and DU145-TxR/CxR. The antiproliferative effects of YS71 were evaluated using proliferation assay. The reverse transcriptase transcription-polymerase chain reaction and western blot were performed to determine the expression level of androgen receptor (AR), whereas luciferase assay was performed to determine the AR activity. Furthermore, TUNEL assay and western blot were performed to investigate the mechanism of the antiproliferative effect. YS71 exerted a dose-dependent antitumor effect, inhibited AR activity, and induced apoptosis in all PCa cells in a dose-dependent manner. Western blot showed that YS71 increased the levels of apoptosis-related proteins, cleaved caspase-3, and cleaved PARP, and decreased the levels of the antiapoptotic proteins, Bcl-xL and Bcl-2. In addition, microarray analysis revealed that YS71 decreased several cancer-related genes. YS71 exhibits antitumor activity by inducing apoptosis in PCa cells, including taxane-resistant cells. It could be a potential future therapeutic option for hormone- and chemotherapy-resistant PCa.

Abstract 4816: Targeting GCN2 regulation of amino acid homeostasis sensitizes prostate cancer cells to senolytic therapies

Abstract The integrated stress response (ISR) features a family of eIF2 kinases that sense cellular stress, triggering translational and transcriptional modes of gene expression that enhance cell adaptation to the underlying stress. Previously we reported (Cordova et al., 2022 eLife) that prostate cancer (PCa) cells rely on one of these eIF2 kinases, GCN2, for maintenance of amino acid homeostasis and sustained proliferation. GCN2 functions to enhance expression of amino acid transporters and subsequent import of essential amino acids (EAAs) that are necessary to sustain PCa growth. Genetic loss or pharmacological inhibition of GCN2 results in decreased expression of amino acid transporters, severe depletion of intracellular EAAs, and decreased proliferation of PCa cells in culture and mouse xenograft models. Although loss of GCN2 in PCa cells reduces proliferation, depletion of this eIF2 kinase is suggested to lead to cell stasis and modest cell death. We hypothesize that the static phenotype by GCN2 inhibition in PCa cells renders these cells vulnerable to senolytic treatment and is a potential therapeutic target in combination therapies. To address this idea, we utilized models of androgen-sensitive and castration-resistant PCa and non-tumorigenic prostate epithelial cells. GCN2 inhibition induced a static phenotype in PCa cell lines, but not non-tumorigenic prostate epithelial cells, and this was accompanied by increased expression of the senescent markers p53, p21, p27, and MCL-1. Furthermore, induction of senescent markers following GCN2 inhibition in PCa cells was reversed by supplementation with EAAs, suggesting that limitation of EAAs is critical to the induction of cell stasis. Of importance, treatment of PCa cells with Navitoclax, a senolytic agent that targets Bcl-2 family of anti-apoptotic proteins, in combination with GCN2 inhibition resulted in enhanced cell death and apoptosis compared to each single treatment alone. Our results suggest that GCN2 inhibition in PCa cells leads to depletion of EAAs and induction of a static phenotype, rendering these cells vulnerable to targeting the Bcl-2 family of proteins to expedite cell death. As such, therapies integrating GCN2 inhibition and senolytic agents are proposed to be effective strategies for treatment of PCa. Citation Format: Noah R. Sommers, Ricardo A. Cordova, Angela J. Klunk, Roberto Pili, Ronald C. Wek, Kirk A. Staschke. Targeting GCN2 regulation of amino acid homeostasis sensitizes prostate cancer cells to senolytic therapies. [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 4816.

CACNA1D overexpression and voltage-gated calcium channels in prostate cancer during androgen deprivation

Prostate cancer is often treated by perturbing androgen receptor signalling. CACNA1D, encoding CaV1.3 ion channels is upregulated in prostate cancer. Here we show how hormone therapy affects CACNA1D expression and CaV1.3 function. Human prostate cells (LNCaP, VCaP, C4-2B, normal RWPE-1) and a tissue microarray were used. Cells were treated with anti-androgen drug, Enzalutamide (ENZ) or androgen-removal from media, mimicking androgen-deprivation therapy (ADT). Proliferation assays, qPCR, Western blot, immunofluorescence, Ca2+-imaging and patch-clamp electrophysiology were performed. Nifedipine, Bay K 8644 (CaV1.3 inhibitor, activator), mibefradil, Ni2+ (CaV3.2 inhibitors) and high K+ depolarising solution were employed. CACNA1D and CaV1.3 protein are overexpressed in prostate tumours and CACNA1D was overexpressed in androgen-sensitive prostate cancer cells. In LNCaP, ADT or ENZ increased CACNA1D time-dependently whereas total protein showed little change. Untreated LNCaP were unresponsive to depolarising high K+/Bay K (to activate CaV1.3); moreover, currents were rarely detected. ADT or ENZ-treated LNCaP exhibited nifedipine-sensitive Ca2+-transients; ADT-treated LNCaP exhibited mibefradil-sensitive or, occasionally, nifedipine-sensitive inward currents. CACNA1D knockdown reduced the subpopulation of treated-LNCaP with CaV1.3 activity. VCaP displayed nifedipine-sensitive high K+/Bay K transients (responding subpopulation was increased by ENZ), and Ni2+-sensitive currents. Hormone therapy enables depolarization/Bay K-evoked Ca2+-transients and detection of CaV1.3 and CaV3.2 currents. Physiological and genomic CACNA1D/CaV1.3 mechanisms are likely active during hormone therapy—their modulation may offer therapeutic advantage.

Euchromatic histone lysine methyltransferase 2 facilitates radioresistance in prostate cancer by repressing endoplasmic reticulum protein 29 transcription.

Prostate cancer is one of the most common cancers in men. This study was conducted to investigate the role of euchromatic histone lysine methyltransferase 2 (EHMT2) and endoplasmic reticulum protein 29 (ERP29) in the progression of radioresistance in prostate cancer. The expression of EHMT2 and ERP29 in prostate cancer cells and during the progression of radioresistance was detected using quantitative reverse transcription-polymerase chain reaction and western blotting, and the interaction between EHMT2 and ERP29 was investigated. The proliferation of transfected cells under x-ray irradiation was determined using the methyl thiazolyl tetrazolium and colony formation assays. Flow cytometry was used to analyze the apoptosis of the transfected cells under x-ray irradiation. Nude mice were subcutaneously injected with prostate cancer (DU145) cells stably transfected with sh-ERP29 or sh-NC. The effect of ERP29 expression on radioresistance in nude mice was assessed by x-ray irradiation. The expression of EHMT2 was upregulated and that of ERP29 was downregulated in prostate cancer cells during radioresistance progression. EHMT2 downregulation suppressed radioresistance in DU145 and androgen-sensitive prostate cancer (LNCaP) cells. In irradiated DU145 cells, EHMT2 inhibition decreased the number of colonies and accelerated apoptosis. The transcription of ERP29 was suppressed by EHMT2 by upregulating H3K9me2 and downregulating H3K4me3, thereby regulating radioresistance in prostate cancer cells. In addition, the downregulation of ERP29 promoted the progression of radioresistance in prostate cancer cells in nude mice. EHMT2 promotes radioresistance in prostate cancer cells by repressing ERP29 transcription.

Estrogens drive the endoplasmic reticulum-associated degradation and promote proto-oncogene c-Myc expression in prostate cancer cellsby androgen receptor/estrogen receptor signaling.

The tumorigenic properties of prostate cancer are regulated by advanced hormonal regulation-mediated complex molecular signals. Therefore, characterizing the regulation of these signal transduction systems is crucial for understanding prostate cancer biology. Recent studies have shown that endoplasmic reticulum (ER)-localized protein quality control mechanisms, including ER-associated degradation (ERAD) andunfolded protein response (UPR) signaling contribute to prostate carcinogenesis and to the development of drug resistance. It has also been determined that these systems are tightly regulated by androgens. However, the role of estrogenic signaling in prostate cancer and its effects on protein quality control mechanisms is not fully understood. Herein, we investigated the regulatory effects of estrogens on ERAD and UPR and their impacts on prostate carcinogenesis. We found that estrogens strongly regulated the ERAD components and IRE1⍺ branch of UPR by Er⍺/β/ARaxis. Besides,estrogenic signaling rigorously regulatedthe tumorigenicity of prostate cancer cells by promoting c-Myc expression and epithelial-mesenchymal transition (EMT). Moreover, estrogenic signal blockage significantly decreased the tumorigenic features of prostate cancer cells. Additionally, simultaneous inhibition of androgenic/estrogenic signals more efficiently inhibited tumorigenicity of prostate cancer cells, including proliferation, migration, invasion and colonial growth. Furthermore, computational-based molecular docking, molecular dynamics simulations and MMPBSA calculations supported the estrogenic stimulation of AR. Present findings suggested that ERAD components and IRE1⍺ signaling are tightlyregulated by estrogen-stimulated AR and Er⍺/β. Our data suggest that treatment approaches targeting the co-inhibition of androgenic/estrogenic signals may pave the way for new treatment approaches to be developed for prostate cancer. The present model of the impact of estrogens on ERAD and UPR signaling in androgen-sensitive prostate cancer cells.

Comparative Proteomic and Transcriptomic Analysis of the Impact of Androgen Stimulation and Darolutamide Inhibition.

Several inhibitors of androgen receptor (AR) function are approved for prostate cancer treatment, and their impact on gene transcription has been described. However, the ensuing effects at the protein level are far less well understood. We focused on the AR signaling inhibitor darolutamide and confirmed its strong AR binding and antagonistic activity using the high throughput cellular thermal shift assay (CETSA HT). Then, we generated comprehensive, quantitative proteomic data from the androgen-sensitive prostate cancer cell line VCaP and compared them to transcriptomic data. Following treatment with the synthetic androgen R1881 and darolutamide, global mass spectrometry-based proteomics and label-free quantification were performed. We found a generally good agreement between proteomic and transcriptomic data upon androgen stimulation and darolutamide inhibition. Similar effects were found both for the detected expressed genes and their protein products as well as for the corresponding biological programs. However, in a few instances there was a discrepancy in the magnitude of changes induced on gene expression levels compared to the corresponding protein levels, indicating post-transcriptional regulation of protein abundance. Chromatin immunoprecipitation DNA sequencing (ChIP-seq) and Hi-C chromatin immunoprecipitation (HiChIP) revealed the presence of androgen-activated AR-binding regions and long-distance AR-mediated loops at these genes.

Progesterone regulates the endoplasmic reticulum-associated degradation and Unfolded Protein Response axis by mimicking the androgenic stimulation in prostate cancer cells.

Today, androgen receptor(AR)-mediated signaling mechanisms in prostate cancer are intensively studied. However, the roles of other steroid hormones in prostate cancer and their effects on androgenic signaling still remain a mystery. Recent studies focused on the androgen-mediated regulation of protein quality control mechanisms such as endoplasmic reticulum-associated degradation (ERAD) and unfolded protein response (UPR) in prostate cancer cells. Present study, we investigated the action of progesterone signaling on ERAD and UPR mechanisms and analyzed the crosstalk of progesterone signaling with androgenic signal in prostate cancer cells. The mode of action of progesterone on ERAD, UPR and AR signaling in prostate cancer was investigated by cell culture studies using LNCaP and 22Rv1 cells. To this aim qRT-PCR, western-blotting assay, immunofluorescent microscopy, nuclear fractionation and bioinformatic analysis were used. Our results indicated that progesterone positively regulates mRNA and protein levels of ERAD components in LNCaP cells. Also, it induced the IRE⍺ and PERK branches of UPR signaling. Progesterone receptor antagonist effectively antagonized the progesterone-induced responses. We also had similar results in 22Rv1cells. Also,we tested the effect of the pharmacologically reducing of IRE⍺ and PERK signaling on progesterone-induced ERAD. Additionally, we determined the presence of putative progesterone response elements(PREs) in the promoter regions of ERAD members by bioinformatic tool. More strikingly, we found progesterone regulates AR signaling by modulating the nuclear transactivation of AR. Herein, we defined that progesterone hormone positively regulates ERAD and UPR mechanisms in prostate cancer cells and that progesterone contributes to the molecular biology of prostate cancer by regulating androgenic signaling. Mode of Action of Progesteron on Androgen sensitive prostate cancer cells.

Alteration of the HIF-1α/VEGF Signaling Pathway and Disruption of the Cell Cycle by Second Generation Carbosilan Dendrimers.

Current therapies against prostate cancer (PCa) disease, such as surgery, radiotherapy, or in last term chemical castration by androgen deprivation, have led to significant reduction of the incidence of PCa throughout the world. Worse prognosis is found in those patients which exhibit castration resistance, relapsing into the disease with even greater aggressiveness. Hypoxia cancer cell adaption has been observed to be closely connected to fatal prognostic tumor features. Therefore, hypoxia adaptive mechanisms of cancer cells have attracted large interest as a relevant biological target for treatment-resistant patients. Dendrimers have been established as a promising nanotechnological tool owing to their beneficial physicochemical features such as multivalency and monodispersity. Herein, we have completed a thorough study to better understand the effect within the cell of the already published ruthenium(II)-N-heterocyclic carbene metallodendrimer (G2Ru) that was able to drastically reduce HIF-1α stabilization and exhibited antiproliferative capability against androgen-sensitive (LNCaP) and androgen-resistant prostate cancer cells (LNFLU) in vitro. G2Ru, as well as its cationic imidazolium precursor (G2P), displayed scavenging properties against intracellular and externally stimulated ROS levels, which would presumably hinder the stabilization of HIF-1α by prolyl hydroxylase (PHD) inhibition. Furthermore, these dendrimers have shown considerably beneficial properties against tumor progression capability in terms of apoptosis, cell cycle, CSCs expression, and epithelial phenotype promotion. Taken all together, in this study we could demonstrate the extraordinary anticancer properties of NHC-based carbosilane dendrimers against androgen-resistant prostate cancer cells in vitro.

Apoptosis induction of essential oils from Artemisia arborescens L. in human prostate cancer cells

Ethnopharmacological relevanceProstate cancer originates from cells inside a gland, which begin to grow out of control. In the world, prostate cancer is the most common cancer in the male population. New therapeutic strategies are needed for this tumor which still has a high mortality. A. arborescens leaves and aerial parts have various ethnopharmacological uses such as anti-spasmodic, and their decoctions were used to resolve urticaria, neuralgia and several lung diseases. Often this species has been also used to treat different inflammatory-related diseases such as cancer. Aim of the studyIn a continuation of our research on essential oils from medicinal plants, we have selected, two essential oils from Artemisia arborescens L. (Compositae), an aromatic shrub widely used in traditional medicine. We evaluated their pro-apototic effect on androgen-sensitive (LNCaP) and androgen-insensitive (DU-145) human prostate cancer cells. In this study, we also evaluated the anti-Signal transducer and transcription factor 3 (STAT-3) activity of both essential oils in the human prostate cancer cell lines, and the treatment with Tumor necrosis factor (TNF)-Related Apoptosis (TRAIL). Materials and methodsThe cells were exposed to essential oils for 72 h and cell viability and cell membrane integrity were evaluated. Genomic DNA and the activity of caspase-3 was tested to confirm the cell death for apoptosis. Western blot analysis was employed to evaluate the expression of Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, Hsp70, STAT-3 and SOD proteins. Assays to evaluate reactive oxygen species (ROS) and GSH levels were also performed. ResultsThe results showed the capacity of two essential oils to activate an apoptotic process increasing the inhibition of Hsp70 and STAT-3 protein expression. In addition, our natural products sensitize LNCaP cells to Tumor necrosis factor (TNF)-Related Apoptosis (TRAIL)-induced apoptosis. ConclusionsIn summary, our study provides a further contribution to the hypothesis of the use of essential oils, from traditional medicinal plants, for the treatment of tumors, and suggests that the combination of our samples with other anti-prostate cancer therapies could be used to affect prostate cancer.