Prostate Cell Lines Research Articles

Periprostatic adipose tissue (PPAT) supernatant from obese mice releases anticontractile substances and increases human prostate epithelial cell proliferation: the role of nitric oxide and adenosine.

Background: The prostate gland is surrounded by periprostatic adipose tissue (PPAT) that can release mediators that interfere in prostate function. In this study, we examined the effect of periprostatic adipose tissue supernatant obtained from obese mice on prostate reactivity in vitro and on the viability of human prostatic epithelial cell lines. Methods: Male C57BL/6 mice were fed a standard or high-fat diet after which PPAT was isolated, incubated in Krebs-Henseleit solution for 30min (without prostate) or 60min (with prostate), and the supernatant was then collected and screened for biological activity. Total nitrate and nitrite (NOx-) and adenosine were quantified, and the supernatant was then collected and screened for biological activity. NOx- and adenosine were quantified. Concentration-response curves to phenylephrine (PE) were obtained in prostatic tissue from lean and obese mice incubated with or without periprostatic adipose tissue. In some experiments, periprostatic adipose tissue was co-incubated with inhibitors of the nitric oxide (NO)-cyclic guanosine monophosphate pathway (L-NAME, 1400W, ODQ), adenylate cyclase (SQ22536) or with adenosine A2A (ZM241385), and A2B (MRS1754) receptor antagonists. PNT1-A (normal) and BPH-1 (hyperplasic) human epithelial cells were cultured and incubated with supernatant from periprostatic adipose tissue for 24, 48, or 72h in the absence or presence of these inhibitors/antagonists, after which cell viability and proliferation were assessed. Results: The levels of NOx- and adenosine were significantly higher in the periprostatic adipose tissue supernatant (30min, without prostate) when compared to the vehicle. A trend toward an increase in the levels of NOX was observed after 60min. PPAT supernatant from obese mice significantly reduced the PE-induced contractions only in prostate from obese mice. The co-incubation of periprostatic adipose tissue with L-NAME, 1400W, ODQ, or ZM241385 attenuated the anticontractile activity of the periprostatic adipose tissue supernatant. Incubation with the supernatant of periprostatic adipose tissue from obese mice significantly increased the viability of PNT1-A cells and attenuated expression of the apoptosis marker protein caspase-3 when compared to cells incubated with periprostatic adipose tissue from lean mice. Hyperplastic cells (BPH-1) incubated with periprostatic adipose tissue from obese mice showed greater proliferation after 24h, 48h, and 72h compared to cells incubated with culture medium alone. BPH-1 cell proliferation in the presence of PPAT supernatant was attenuated by NO-signaling pathway inhibitors and by adenosine receptor antagonists after 72h. Conclusion: NO and adenosine are involved in the anticontractile and pro-proliferative activities of periprostatic adipose tissue supernatant from obese mice. More studies are needed to determine whether the blockade of NO and/or adenosine derived from periprostatic adipose tissue can improve prostate function.

Antitumoral photoinduced effects of crude extract, fractions, and naphthoquinones from Sinningia magnifica (Otto & A. Dietr.) Wiehler (Gesneriaceae) in a bioguided study.

Photodynamic therapy (PDT) has been used for various purposes, including as an antitumor resource in a noninvasive therapy with minimal side effects. Sinningia magnifica (Otto & A. Dietr.) Wiehler is a rupicolous plant found in rock crevices in Brazilian tropical forests. Initial studies indicate the presence of phenolic glycosides and anthraquinones in species of the genus Sinningia (Generiaceae family). It is known that anthraquinones are natural photosensitizers with potential PDT applications. This led us to investigate the potential compounds of S. magnifica for use as a natural photosensitizer against the melanoma (SK-MEL-103) and the prostate cancer (PC-3) cell lines in a bioguided study. Our results showed that singlet oxygen production by the 1,3-DPBF photodegradation assay greatly increased in the presence of crude extract and fractions. The biological activity evaluation showed photodynamic action against melanoma cell line SK-MEL-103 and prostate cell line PC-3. These results suggest the presence of potential photosensitizing substances, as demonstrated in this in vitro antitumor PDT study by the naphthoquinones Dunniol and 7-hydroxy-6-methoxy-α-dunnione for the first time. Naphthoquinones, anthraquinones and phenolic compounds were identified in the crude extract by UHPLC-MS/MS analysis, motivating us to continue with the bioguided phytochemical study aiming to discover more photochemically bioactive substances in Gesneriaceae plants.

Investigation of the Antifungal and Anticancer Effects of the Novel Synthesized Thiazolidinedione by Ion-Conductance Microscopy

In connection with the emergence of new pathogenic strains of Candida, the search for more effective antifungal drugs becomes a challenge. Part of the preclinical trials of such drugs can be carried out using the innovative ion-conductance microscopy (ICM) method, whose unique characteristics make it possible to study the biophysical characteristics of biological objects with high accuracy and low invasiveness. We conducted a study of a novel synthesized thiazolidinedione’s antimicrobial (for Candida spp.) and anticancer properties (on samples of the human prostate cell line PC3), and its drug toxicity (on a sample of the human kidney cell line HEK293). We used a scanning ion-conductance microscope (SICM) to obtain the topography and mechanical properties of cells and an amperometric method using Pt-nanoelectrodes to register reactive oxygen species (ROS) expression. All data and results are obtained and presented for the first time.

Abstract A031: A fat lot of good: A novel monounsaturated fatty acid promotes prostate cancer growth and survival

Abstract Advanced prostate cancer (PCa) is currently incurable, and development of novel treatments requires a greater understanding of key pathways that are altered during PCa progression. We have focussed on fatty acid metabolism, as the major source of energy for PCa cells, and have implicated a monounsaturated fatty acid (MUFA), cis-vaccenic acid (cVA), as a critical regulator of PCa cell homeostasis. Production of cVA requires stearoyl-CoA desaturase 1 (SCD1), the enzyme attributed to general MUFA production, and whose activity is important in a range of cancer types. Nonetheless, the specific role of cVA has not previously been studied, in PCa nor cancer more generally. To elucidate the role of cVA in PCa more precisely, we used SCD1 inhibition to study PCa responses to broad MUFA depletion and specific MUFA supplementation, comparing the responses of cVA with its more common structural isomer oleate. To accomplish these aims, we utilised cell line models of PCa (LNCaP, MR49F and V16D) and a benign prostate cell line (PNT1A) to perform cell viability, lipidomic and flow cytometric assays. In addition, responses in clinically-relevant patient-derived tumor explants were measured by proliferative (Ki67) and cell death (CC3) marker staining. Pharmacological SCD1 inhibition (SCDi; A939572) decreased cell viability and increased cell death in PCa cell lines and patient-derived tumor explants, while only minimally affecting the benign prostate cell line PNT1A. Phenotypically, analysis of mitochondrial function and lipidomic changes under SCDi suggested a role for MUFAs in regulating mitochondrial membrane composition via cardiolipins, and oxidative stress levels. This in turn has the potential to regulate cell death, control tumor burden, and influence overall PCa patient survival. Supplementation of cVA or oleate under SCDi demonstrated that only cVA was successful at rescuing cell viability, and cVA, but not oleate, could rescue changes observed in mitochondrial-related lipids, suggesting that cVA regulation of mitochondrial function was a major determinant of its effect on cell viability. Additionally, cVA alone dose-dependently increased PCa cell viability, implicating cVA as an important oncogenic factor. Together, these data identify cVA as a novel substrate required for PCa cell viability, likely via regulation of mitochondrial homeostasis and associated cell death pathways. In summary, this research has revealed more precise insights into the oncogenic effects of an overlooked MUFA, cVA, in PCa, and may uncover new druggable targets that are more selective, to improve treatment options for advanced PCa. Citation Format: Julia S. Scott, Reuben Young, Swati Irani, Jonas Dehairs, Stephen Blanksby, Johannes V. Swinnen, Zeyad D. Nassar, Lisa M. Butler. A fat lot of good: A novel monounsaturated fatty acid promotes prostate cancer growth and survival [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 A031.

Abstract A020: Unraveling the role of ANO7 in prostate cancer metabolism

Abstract Prostate cancer (PCa), the most common male cancer worldwide, causes about 10% of cancer related deaths in Europe. It has a wide spectrum of clinical behavior that ranges from decades of indolence to rapid metastatic progression and lethality. However, the molecular mechanisms involved in aggressive PCa progression are still poorly understood. We have previously linked single-nucleotide polymorphisms in the anoctamin 7 (ANO7) gene to the risk of aggressive prostate cancer and shown that carriers of a rs77559646 variant have improved treatment response to docetaxel. The expression of ANO7 is lost when prostate cancer progresses and is completely lacking in prostate cancer metastases. ANO7 is a prostate specific gene and is highly expressed in the luminal cells of the human prostate. ANO7 belongs to a family of calcium-activated chloride channels and select members of the anoctamin family have phospholipid scramblase activity. Whether ANO7 has any of these capabilities is unclear. To uncover the cellular functions of ANO7 has proven challenging because the expression of ANO7 at protein level is either minimal or nonexistent in all commercially available cell lines. To overcome this and to study the cellular functions of ANO7, we have generated stable prostate cell lines overexpressing ANO7 using lentiviral transduction. The characterization of the RWPE-1 cells overexpressing ANO7 showed that the viability and proliferation was increased both in 2D and 3D. To gain information into what pathways ANO7 is affecting, we performed RNA-sequencing and pathway analysis. Interestingly, we found enrichment of mitochondrial genes participating in oxidative phosphorylation in cells overexpressing ANO7. The genes expressed from the mitochondrial genome were upregulated and the nuclear genes encoding for proteins involved in mitochondrial functions were mostly downregulated. This suggests that the mitochondrial metabolism has changed. We assessed the mitochondrial function by measuring the oxidative phosphorylation capability of the cells and showed that the maximal respiratory capacity of ANO7 overexpressing cells is indeed reduced compared to control cells. We also analyzed which of the three major mitochondrial fuels (glucose, glutamine and fatty acids) are not used as efficiently for oxidative phosphorylation in ANO7 overexpressing cells. The result proves that ANO7 cells are not utilizing glucose as effectively as control cells as there were no differences in maximal respiratory capacity when we treated the cells with the UK5099 inhibitor, which inhibits pyruvate import to mitochondria. Furthermore, preliminary results from the glycolysis stress assay indicates that glycolysis is increased in ANO7 overexpressing cells. In addition, electron microscopy study suggests that ANO7 overexpressing cells have less cristae compared to control. This study shows for the first time that ANO7 rewires prostate mitochondrial functions and thus cellular metabolism. Citation Format: Nasrin Sultana, Christoffer Lof, Neha Goel, Johanna Schleutker. Unraveling the role of ANO7 in prostate cancer metabolism [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 A020.

Abstract A016: Mechanism of hypoxia-induced centrosome loss in early prostate cancer

Abstract PURPOSE: We aim to understand cellular and microenvironmental determinants of chromosomal instability in prostate cancer and use these findings as prognostic biomarkers portending aggressive disease. Disease transitions during prostate cancer progression are accompanied by bursts of chromosomal instability (CIN), including chromosomal translocations, oncogene amplifications, and chromothripsis. While high CIN correlates with most metrics of aggressive disease, we lack a mechanistic understanding of how CIN originates in prostate cancer. Recently, we discovered that low Gleason score, primary prostate tumors lack centrosomes, cytoplasmic organelles that ensure the fidelity of chromosome segregation by organizing the shape of the mitotic spindle. Experimental elimination of centrosomes in immortalized, non-tumorigenic prostate cell lines was sufficient to generate extensive CIN resulting in oncogenic transformation of these lines when sub-cutaneously injected into NSG mice. Because we identified centrosome loss as a potential driver of CIN, we sought to understand mechanisms that can trigger centrosome loss in the prostate. Although centrosome loss naturally occurs during the development of some tissues, for example centrosome elimination during oogenesis, the mechanisms that trigger loss are not known. Therefore, we first investigated microenvironmental changes in early prostate cancer as a potential cause. Hypoxia, pathologically low oxygen concentration, is common in the aging prostate due to loss of vasculature and is associated with poor prostate cancer prognosis and high CIN. Therefore, we hypothesized that hypoxic exposure induces centrosome loss in prostate cells. Indeed, we found that exposure to 1% oxygen concentrations leads to progressive centrosome loss in non-tumorigenic, immortalized prostate epithelial cell lines. Using functional assays in cultured cells, we found that hypoxia-induced centrosome loss is an active disassembly process mediated by confluence-dependent Hippo signaling but independent of Hypoxia-Inducible Factor (HIF) transcription. We conclude that hypoxia-induced centrosome disassembly is a plausible driver of CIN in early prostate cancer. At present, we are developing immunohistochemistry assays to identify centrosome loss and hypoxia in biopsy samples to confirm these discoveries. Recently, we have described a combinatorial staining pattern (GLUT3/GLUT1/CA-9) that reliably identifies hypoxic tissue in patient-derived xenografts from prostate tumors. We are coupling this with a new assay to identify the structural component of the centrosome, called the centriole, which is identifiable during both mitosis and interphase. Together, our in situ assays will allow us to determine the penetrance of hypoxia-induced centrosome loss and its association with aggressive prostate cancer. Citation Format: John M. Ryniawec, Gregory C. Rogers, Anne E. Cress. Mechanism of hypoxia-induced centrosome loss in early prostate cancer [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 A016.

Abstract B069: SPOP loss places the prostate luminal epithelial cells at a selective disadvantage

Abstract Background: Prostate cancer (PC) is the single most common and second-most lethal cancer in men, with over 268,000 estimated cases and over 34,500 estimated deaths in the US in 2022. The Speckle-Type POZ protein (SPOP) mutant subclass of PC accounts for 10% to 15% of all primary PC cases. SPOP is an adaptor for Cullin3/Ring (CUL3-RING)-type E3 ubiquitin ligase complexes and provides substrate specificity. The Cancer Genome Atlas (TCGA) studies show that SPOP is the most frequently mutated gene in primary prostate cancer (PC). Interestingly, PC-associated SPOP mutations are always missense and occur in a heterozygous fashion. The current gap in knowledge is the lack of understanding of the role of wildtype SPOP in PC. Methods: By utilizing prostate specific SPOP knockout (KO) mice, we recently reported increased levels of AR and MYC protein and increased cellular turnover (both proliferation and apoptosis) in the prostate luminal epithelium compared to wildtype prostates. We now characterized these mice for the expression of Cre protein and SPOP mRNA at different age using immunohistochemistry and RNA in situ hybridization. Furthermore, we performed RNA-sequencing analysis in the SPOP knockout mice and matched control littermates. Moreover, we performed RNA-seq in LNCaP, LNCaP-Abl, and RWPE1 cells following SPOP inhibition via siRNA targeting SPOP. Finally, we compared our SPOP inhibition signature from in vitro cell lines and prostate specific SPOP knockout murine model to gain insights about the role of wildtype SPOP protein in the prostate epithelium. Result: Using our Spopfl/fl;PBCre+ model, we observed SPOP floxed cells are rapidly lost and the murine prostate epithelium was repopulated with SPOP wildtype carrying cells. Similarly, knockdown (KD) of SPOP through siRNA treatment in a panel of PC cell lines resulted in a significant reduction in cell viability. These observations suggest that SPOP is important for the normal prostate cell viability. Further transcriptomic profiling of SPOP KO (from transgenic murine model) as well as siSPOP treated in vitro prostate cell lines revealed a significant reduction in the transcriptional activity of the AR. Conclusion: Our data illustrate for the first time a critical role for SPOP in the growth and survival of the prostate epithelium and prostate cancer cell. Our findings further validates SPOP as a important therapeutic target for the treatment of prostate cancer. Citation Format: Kinza Rizwan, Darlene Skapura, Cammy Mason, Cristian Coarfa, Nicholas Mitsiades, Damian Young, Salma Kaochar. SPOP loss places the prostate luminal epithelial cells at a selective disadvantage [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 B069.

Improved Analysis of Prostate Cancer: VIM3, ATG7 and P53 Form a Complex and Activate miRNA 371a-3p.

It is not possible to differentiate prostate carcinomas sufficiently to ensure that every patient receives the right therapy. New molecular markers are needed. Our objective was to identify a complex consisting of vimentin variant 3 (VIM3), autophagy-related protein 7 (ATG7) and tumor protein p53 (TP53) in prostate cancer cells and its effect on microRNA (miR)-371a-3p. Prostate cancer cell lines (PC3, DU145, LNCaP) and the benign prostatic hyperplasia cell line BPH-1 were cultured in growth medium for 24 h, then stimulated with endothelin 1 (EDN1) (50 nM) and withaferin A (2 nM) for 24 h. Cell extracts were then analyzed by western blot. The localization of VIM3, ATG7 and TP53 in the nucleus was demonstrated with immunofluorescence staining and complex formation was demonstrated by immunoprecipitation. Cancer cell migration was analyzed with a scratch assay and agarose drop analysis. The binding of the complex to the promoter of pri-miR-371a-3p was analyzed with a non-radioactive electrophoretic mobility shift assay. VIM3 knockdown using small interfering RNA and quantitative real-time polymerase chain reaction for miR-371a-3p were performed. The complex was present in the nucleus of prostate cancer cells and in the BPH-1 cell line. EDN1 increased the levels of the complex partners and cell migration, whereas withaferin A reduced the levels of the complex partners and migration. The complex bound to the promoter of pri-miR-371a-3p and might be involved in its transcription. Transfection with miR-371a-3p increased migration of prostate cancer cells. VIM3 knockdown reduced miR-371a-3p expression. The VIM3-ATG7-P53 complex, with its stimulatory effect on miR-371a-3p, may have the potential to be a marker for improved differentiation between prostate carcinomas, allowing tailored therapy.

A novel thieno[2,3-d]pyrimidine derivative inhibiting vascular endothelial growth factor receptor-2: A story of computer-aided drug discovery.

Following the pharmacophoric features of vascular endothelial growth factor receptor 2 (VEGFR-2) inhibitors, a novel thieno[2,3-d]pyrimidine derivative has been designed and its activity against VEGFR-2 has been demonstrated by molecular docking studies that showed an accurate binding mode and an excellent binding energy. Furthermore, the recorded binding was confirmed by a series of molecular dynamics simulation studies, which also revealed precise energetic, conformational, and dynamic changes. Additionally, molecular mechanics with generalized Born and surface area solvation and polymer-induced liquid precursors studies were conducted and verified the results of the MD simulations. Next, in silico absorption, distribution, metabolism, excretion, and toxicity studies have also been conducted to examine the general drug-like nature of the designed candidate. According to the previous results, the thieno[2,3-d]pyrimidine derivative was synthesized. Fascinatingly, it inhibited VEGFR-2 (IC50 = 68.13 nM) and demonstrated strong inhibitory activity toward human liver (HepG2), and prostate (PC3) cell lines with IC50 values of 6.60 and 11.25 µM, respectively. As well, it was safe and showed a high selectivity index against normal cell lines (WI-38). Finally, the thieno[2,3-d]pyrimidine derivative arrested the growth of the HepG2 cells at the G2/M phase inducing both early and late apoptosis. These results were further confirmed through the ability of the thieno[2,3-d]pyrimidine derivative to induce significant changes in the apoptotic genes levels of caspase-3, caspase-9, Bcl-2 associated X-protein, and B-cell lymphoma 2.

Synthesis, Biological Activity Evaluation and Molecular Docking of Imidazole Derivatives Possessing Hydrazone Moiety.

In an attempt to identify potential active anticancer agents with low cytotoxic properties and CA inhibitors, a new series of hybrid compounds incorporating imidazole ring and hydrazone moiety as part of their structure were synthesized by aza-Michael addition reaction followed by intramolecular cyclization. The structure of synthesized compounds was elucidated using various spectral techniques. Synthesized compounds were evaluated for their in vitro anticancer (prostate cell lines; PC3) and CA inhibitory (hCA I and hCA II) activity. Among them, some compound displayed remarkable anticancer activity and CA inhibitory activity with Ki values in range of 17.53±7.19-150.50±68.87 nM against cytosolic hCA I isoform associated with epilepsy, and 28.82±14.26-153.27±55.80 nM against dominant cytosolic hCA II isoforms associated with glaucoma. Furthermore, the theoretical parameters of the bioactive molecules were calculated to establish their drug-likeness qualities. The proteins used for the calculations are prostate cancer protein (PDB ID: 3RUK and 6XXP). ADME/T analysis was carried out to examine the drug properties of the studied molecules.

Targeting apoptotic anticancer response with natural glucosinolates from cell suspension culture of Lepidium sativum

BackgroundFinding natural products with anticancer activity is an effective strategy to fight this disease. In this respect, Lepidium sativum or garden cress (family Brassicaceae) has been widely used worldwide for its wide therapeutic application, including anticancer and chemoprotective agents. Plant tissue culture techniques hold great promise for natural product enhancement without any climatic boundaries. In this study, glucosinolates and petroleum ether fractions were isolated from in vitro cell cultures and used against different carcinoma cell lines to investigate their anticancer potential. MethodsIn this study, callus cultures from leaf and root explants were initiated, cell suspension cultures were established, and cell growth and viability profiles were characterized. Different amino acids were added as precursors to the cell suspension cultures to enhance glucosinolates accumulation. Gas chromatography–mass spectrometric analysis (GC–MS) of glucosinolates and petroleum ether fractions was performed, and all fractions were tested against different carcinoma cell lines. ResultsThe findings clarified that the maximum callus initiation percentage was obtained in the medium containing 1.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) + 1.0 mg/l kinetin (Kin) (C1). The viable cell number of cell suspension cultures from leaves and roots increased until it reached the maximum values on day 15. Adding tyrosine and methionine to the cell suspension cultures was the most influential and recorded high glucosinolate percentages. 1H-Cyclopenta (b) pyridine-3-carbonitrile-4,5,6,7-tetrahydro-2-methylthio-4-spirocyclohexane was the main glucosinolate compound found in tyrosine-treated leaf suspension (GLT). Fifteen compounds were detected in the petroleum ether fraction in both cell suspensions initiated from the leaf and root (OL and OR). The major compounds were benzene-1,3,5-trimethyl (12.99%) in root cell suspension (OR), and benzene-2-ethyl-1,4-dimethyl (10.66%) in leaf cell suspension (OL). All glucosinolate extracts demonstrated significant anticancer activity against the prostate (PC3), lung (A-549), colorectal (caco2), and liver (HepG2) cell lines. Glucosinolates extracted from leaf cell suspension (GL) were the most active on the hepatocellular carcinoma cell line (HepG2) among all remaining glucosinolate extracts. Treated hepatocellular carcinoma with an IC50 of GL extract (47.5 ug/ml) upregulates pro-apoptotic BAX and downregulates anti-apoptotic BCL2, which disrupts the BAX/BCL2 ratio, leading to activation of caspase 3 inside treated HepG2 cells. ConclusionsThe anticancer action of the GL extract was validated by the cell cycle study of its glucosinolates, which successfully promoted apoptosis and reduced hepatocellular growth by causing S-phase arrest.

Overexpression of DDX49 in prostate cancer is associated with poor prognosis

BackgroundThere is increasing evidence that DEAD-box helicases (DDX) can act either as promoters or suppressors in various cancer types. Nevertheless, the function of DDX49 in prostate cancer (PCa) is unknown. This study reveals the prognostic and predictive value of DDX49 in PCa.MethodsFirst, we evaluated the expression of DDX49 between PCa and normal tissues based on TCGA and GEO databases. Univariate and multivariate regression analyses were conducted to reveal the risk factors for PCa recurrence. A K–M curve was employed to assess the relationship between DDX49 and recurrence-free survival. In vitro, DDX49 expression was evaluated in PCa and normal prostate cell lines. Furthermore, we constructed a shDDX49 lentivirus to knock down the expression of DDX49. Celigo® Image Cytometer and MTT assay were performed to analyse cell proliferation in PC-3 cells. Cell cycle distribution was detected with flow cytometry analysis. Apoptosis affected by the lack of DDX49 was metred with the PathScan® Stress and Apoptosis Signalling Antibody Array Kit.ResultsThis study shows a high increase in DDX49 in PCa tissues in comparison with normal tissues and that increased DDX49 indicates a poor prognosis among PCa patients. Meanwhile, DDX49 knockdown suppressed the proliferation and migration of PC-3 cells, causing cell cycle arrest in the G1 phase. Stress and apoptosis pathway analysis revealed that the phosphorylation of HSP27, p53, and SAPK/JNK was reduced in the DDX49 knockdown group compared with the control group.ConclusionsIn summary, these results suggest that high expression of DDX49 predicts a poor prognosis among PCa patients. Downregulation of DDX49 can suppress cell proliferation, block the cell cycle, and facilitate cell apoptosis. Therefore, knockdown of DDX49 is a promising novel therapy for treating patients with PCa.

Versatile Platinum(IV) Prodrugs of Naproxen and Acemetacin as Chemo-Anti-Inflammatory Agents.

Developing new and versatile platinum(IV) complexes that incorporate bioactive moieties is a rapidly evolving research strategy for cancer drug discovery. In this study, six platinum(IV) complexes (1-6) that are mono-substituted in the axial position with a non-steroidal anti-inflammatory molecule, naproxen or acemetacin, were synthesised. A combination of spectroscopic and spectrometric techniques confirmed the composition and homogeneity of 1-6. The antitumour potential of the resultant complexes was assessed on multiple cell lines and proved to be significantly improved compared with cisplatin, oxaliplatin and carboplatin. The platinum(IV) derivatives conjugated with acemetacin (5 and 6) were determined to be the most biologically potent, demonstrating GI50 values ranging between 0.22 and 250 nM. Remarkably, in the Du145 prostate cell line, 6 elicited a GI50 value of 0.22 nM, which is 5450-fold more potent than cisplatin. A progressive decrease in reactive oxygen species and mitochondrial activity was observed for 1-6 in the HT29 colon cell line, up to 72 h. The inhibition of the cyclooxygenase-2 enzyme was also demonstrated by the complexes, confirming that these platinum(IV) complexes may reduce COX-2-dependent inflammation and cancer cell resistance to chemotherapy.

SERPINA1 long transcripts produce non-secretory alpha1-antitrypsin isoform: In vitro translation in living cells

SERPINA1 is a well-studied serpin gene due to its dramatic impact on human health. Translation initiation at the main SERPINA1 start codon produces the only known alpha1-antitrypsin (AAT) isoform intended for secretion. AAT performs essential functions by inhibiting proteases and modulating immunity. However, SERPINA1 expression at the level of translation is not sufficiently studied. Here we hypothesize that the main SERPINA1 ORF can be alternatively translated, producing a non-secretory AAT isoform by either masking or excluding a signal peptide. We defined SERPINA1 long mRNA isoforms specific for prostate (DU145) and liver (HepG2) cell lines and studied their individual expression by in vitro assay. We found that all long transcripts produce both glycosylated secretory AAT-eGFP fusion protein and non-glycosylated intracellular AAT-eGFP (initiated from an alternative AUG-2 start codon), with the proportion regulated by the SERPINA1 5’-UTR. Both fusion proteins localize to distinct cellular compartments: in contrast to a fusion with the secretory AAT accumulating in the ER, the intracellular one exhibits nuclear-cytoplasmic shuttling. We detected putative endogenous AAT isoform enriching the nuclear speckles. ConclusionAlternative translation initiation might be a mechanism through which SERPINA1 expands the biological diversity of its protein products. Our findings open up new prospects for the study of SERPINA1 gene expression.

Antibacterial and Cytotoxic Silica-Polycaprolactone-Chlorogenic Acid Hybrids by Sol-Gel Route.

Organic-inorganic hybrid materials were synthesized by a sol-gel route, using silicon alkoxide together with low molecular weight polycaprolactone and caffetannic acid. The synthesized hybrids were characterized by scanning Fourier-transform infrared (FTIR) spectroscopy, and their surface morphology was acquired by scanning electron microscopy (SEM) analysis. The hybrids were investigated for their antiradical capacity using the DPPH and ABTS tests, while the Kirby-Bauer test was used to evaluate their effects on the growth of Escherichia coli and Enterococcus faecalis. Furthermore, a biologically active hydroxyapatite layer has been observed to form on the surface of intelligently synthesized materials. The MTT direct test showed that the hybrid materials are biocompatible with NIH-3T3 fibroblast cells, while they were cytotoxic towards colon, prostate, and brain tumor cell lines. These results shed new light on the suitability of the synthesized hybrids in the medical field, thus affording knowledge on the features of the bioactive silica-polycaprolactone-chlorogenic acid hybrids.

Abstract 3509: 1,25-dihydroxyvitamin D3-mediated suppression of genes associated with cell cycle regulation and actin organization in a non-malignant African American prostate cell line

Abstract Vitamin D is a steroid hormone that confers anti-proliferative and anti-inflammatory properties in prostate cells. Vitamin D deficiency is associated with advanced prostate cancer (PCa) stage, grade, and increased mortality. Since AA men are more likely to be vitamin D deficient compared to EA men, elucidating the pleiotropic effects of vitamin D may provide additional drug targets to mitigate disparate outcomes for AA men with PCa. Total RNA sequencing (RNAseq) was performed on an AA prostate cell line, RC-77N/E, derived from non-malignant epithelial tissue obtained from a PCa patient. We compared untreated RC-77N/E cells with RC-77N/E cells treated with 10 nM of vitamin D3 metabolite, 1α,25(OH)2D3 at 24h. Bioinformatic analysis, public cohort data mining, and RT-qPCR was performed to filter and validate selected statistically significant differentially expressed genes from RNAseq analysis. Our comparison of 1α,25(OH)2D3 treated versus untreated control RC-77N/E cell replicates revealed, 1601 significantly differentially expressed genes (DEGs) (FDR p < 0.05). Ingenuity Pathway Analysis software was used to conduct pathway enrichment analysis, which predicted repression of signaling pathways involved in actin cytoskeleton organization, epithelial-mesenchymal transitioning, adherens junction pathway, and RhoA pathway signaling with high enrichment z-score and statistically significant p-value. We additionally report predicted repression of PCa cell viability, invasion, cell proliferation and organismal death, for disease and function. We prioritized differentially expressed genes by identifying genes with 1) vitamin D response elements (VDREs) and 2) predicted poor prognosis, by performing overall survival analyses using The Cancer Genome Atlas (TCGA) Prostate Adenocarcinoma (PRAD) Cohort. We identified ANLN (Anillin) and ECT2 (Epithelial Cell Transforming 2), which were further validated using an AA tumor/benign contralateral matched clinical cohort, and a public cohort study using a non-malignant European prostate cell line, RWPE1. Our data suggest 1α,25(OH)2D3 significantly downregulates ANLN and ECT2 (FDR p < 0.05) in the RC-77N/E cell line, where survival analyses in the TCGA PRAD cohort show low expression of both ANLN and ECT2 are associated with significantly better overall survival outcome (p < 0.05). In our clinical cohort validations, both ANLN and ECT2 also show significantly decreased expression in non-malignant compared to tumor (p < 0.05). In conclusion, our study provides more insight of vitamin D3 regulation of PCa biomarkers and their potential roles in AA prostate cancer prevention, adjuvant treatment, as well as improving cancer survivor outcome Citation Format: Jabril R. Johnson, Rachel N. Martini, Yate Yuan-Ching, Leanne Woods-Burnham, Mya Walker, Greisha L. Ortiz-Hernandez, Dorothy Galloway, Melissa B. Davis, Sean K. Kimbro, Adam B. Murphy, Rick A. Kittles. 1,25-dihydroxyvitamin D3-mediated suppression of genes associated with cell cycle regulation and actin organization in a non-malignant African American prostate cell line [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 3509.

Abstract 1620: Identification of a molecular degrader targeting the AR-V7 splice variant

Abstract Prostate cancer is the most common cancer in males and the fifth leading cause of cancer mortality worldwide. A variety of therapeutic approaches exist including steroid synthesis inhibitors and next-generation ligand binding domain (LBD) antagonists [e.g., enzalutamide (Enza)]. However, these approaches are only effective against tumors bearing the full-length androgen receptor (AR-FL). In the course of their disease, many patients acquire androgen receptor splice variant 7 (AR-V7), a mutant form of the receptor which lacks the LBD and is constitutively activated. To date, no effective therapy has been demonstrated for patients with metastatic prostate cancer bearing the AR-V7 signature. We have recently developed a high throughput platform that allows for the identification of ligands that can bind to wide variety of protein targets. Using this approach, we screened a diverse chemical library of 100,000 small molecules to identify compounds that bound to AR-V7. One such molecule, CL-AR-100, directly bound to AR-V7 and moreover, induced proteasomal-mediated targeted protein degradation of both AR-V7 and AR-FL. This compound did not affect the expression of other structurally related ligand-activated nuclear receptors. In prostate cancer cell lines expressing AR-FL, CL-AR-100 induced a gene expression profile similar to cells treated with Enza including a marked reduction in KLK3 (PSA) expression. Moreover, CL-AR-200 inhibited the growth of 22RV1 cells (IC50=0.3uM), a cell line that predominantly expresses AR-V7. In contrast, the compound did not affect the growth of a wide variety of non-tumorigenic cells, non-prostate tumor cell lines or PC3 cells, a prostate cell line that exhibits AR-independent cell growth. These results thereby identify a small molecule molecular degrader that can specifically induce the targeted protein degradation of the AR-V7 splice variant. Given the strong association of this isoform with treatment resistance, this approach would appear to provide an attractive strategy for patients with metastatic castration resistant prostate cancer. Citation Format: Yuan Liu, Bo Lin, Mads B. Larsen, Irene Alfaras, Jason R. Kennerdell, Daniel P. Camarco, Ferhan Tuncer, Toren Finkel, Bill B. Chen. Identification of a molecular degrader targeting the AR-V7 splice variant [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 1620.

Abstract 1705: SPOP: An essential gene for normal and prostate tumor cells

Abstract Background: Prostate cancer (PC) is the single most common and second-most lethal cancer in men, with over 268,000 estimated cases and over 34,500 estimated deaths in the US in 2022. The Speckle-Type POZ protein (SPOP) mutant subclass of PC accounts for 10% to 15% of all primary PC cases. SPOP is an adaptor for Cullin3/Ring (CUL3-RING)-type E3 ubiquitin ligase complexes and provides substrate specificity. The Cancer Genome Atlas (TCGA) studies show that SPOP is the most frequently mutated gene in primary prostate cancer (PC). Interestingly, PC-associated SPOP mutations are always missense and occur in a heterozygous fashion. The current gap in knowledge is the lack of understanding of the role of wildtype SPOP in PC. Methods: By utilizing prostate specific SPOP knockout (KO) mice, we recently reported increased levels of AR and MYC protein and increased cellular turnover (both proliferation and apoptosis) in the prostate luminal epithelium compared to wildtype prostates. We now characterized these mice for the expression of Cre protein and SPOP mRNA at different age using immunohistochemistry and RNA in situ hybridization. Furthermore, we performed RNA-sequencing analysis in the SPOP knockout mice and matched control littermates. Moreover, we performed RNA-seq in LNCaP, LNCaP-Abl, and RWPE1 cells following SPOP inhibition via siRNA targeting SPOP. Finally, we compared our SPOP inhibition signature from in vitro cell lines and prostate specific SPOP knockout murine model to gain insights about the role of wildtype SPOP protein in the prostate epithelium. Result: Using our Spopfl/fl;PBCre+ model, we observed SPOP floxed cells are rapidly lost and the murine prostate epithelium was repopulated with SPOP wildtype carrying cells. Similarly, knockdown (KD) of SPOP through siRNA treatment in a panel of PC cell lines resulted in a significant reduction in cell viability. These observations suggest that SPOP is important for the normal prostate cell viability. Further transcriptomic profiling of SPOP KO (from transgenic murine model) as well as siSPOP treated in vitro prostate cell lines revealed a significant reduction in the transcriptional activity of the AR. Conclusion: Our data illustrate for the first time a critical role for SPOP in the growth and survival of the prostate epithelium and prostate cancer cell. Our findings further validates SPOP as a important therapeutic target for the treatment of prostate cancer. Citation Format: Kinza Rizwan, Darlene Skapura, Cammy Mason, Cristian Coarfa, Nicholas Mitsiades, Damian Young, Salma Kaochar. SPOP: An essential gene for normal and prostate tumor cells [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 1705.

Abstract 316: Engineering large chromosomal deletions to advance precision oncology for prostate cancer

Abstract Human prostate cancer is notable for its high number of somatic copy number alterations (CNAs), which include both focal and broad arm-level amplifications and deletions. Up to 90% of prostate tumors harbor aberrant CNAs, and genomic deletions affect about 40% of these tumors. Many chromosomal deletions are highly recurrent and occur in more than 10% of prostate tumors, including 8p (40~50%), 6q14-16, 10q23, 13q14, and 16q22-24 (20~30% each). Most of these deletions are large, often including hundreds of genes. Notably, several large deletions, including 6q14-16 and 16q22-24, are significantly associated with poor patient outcomes, suggesting that these large chromosomal deletions play a functional role in prostate tumorigenesis, and highlighting the critical need for elucidation of the mechanisms underlying large-deletion driven tumorigenesis. However, due to the limited cloning capacity of targeting vectors and the rarity of on-target homologous recombination events in traditional gene-targeting technology, modelling large chromosomal deletions has proven highly challenging, and the role of such lesions in tumorigenesis has therefore been significantly understudied. Here, we developed an innovative approach to engineering the chromosome over large genetic distances through CRISPR/Cas9 technology in both mouse embryonic stem (ES) cells and human prostate cancer cells. Upon completion of the desired genome edits, the resulting mouse ES clones make possible the creation of conditional knockout mice that accurately mimic prostate cancer-associated large deletions, while the engineered human cancer cell clones serve as an indispensable in vitro model to validate the biological relevance of large chromosomal deletions in prostate tumorigenesis. Further characterization of the human prostate cell line bearing 10q23 deletion revealed that 10q23 loss conferred RWPE1 cells growth advantage. The development and characterization of these in vivo and in vitro models will contribute greatly to the implementation of precision oncology research, open new opportunities for modelling human diseases associated with large chromosomal deletions, and lead to novel insights into the biology of prostate cancer-associated large deletions, which will facilitate the identification of deletion-specific vulnerabilities that can be exploited for prostate cancer treatment. Citation Format: Jinjin Wu, Chen Wu, Joel A. Lawitts, Cheryl B. Bock, Jiaoti Huang, Ming Chen. Engineering large chromosomal deletions to advance precision oncology for prostate cancer [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 316.

PD16-01 SYNERGISTIC EFFECTS OF CURCUMIN AND LACTOFERRIN TO INHIBIT THE PROSTATIC CANCER CELLS GROWTH AND MIGRATION ABILITY

You have accessJournal of UrologyCME1 Apr 2023PD16-01 SYNERGISTIC EFFECTS OF CURCUMIN AND LACTOFERRIN TO INHIBIT THE PROSTATIC CANCER CELLS GROWTH AND MIGRATION ABILITY Erica Costantini, Michele Marchioni, Lisa Aielli, Simone Ferretti, Antonio De Palma, Davide Ciavarella, Giulio Litterio, Marcella Reale, Marta Di Nicola, and Luigi Schips Erica CostantiniErica Costantini More articles by this author , Michele MarchioniMichele Marchioni More articles by this author , Lisa AielliLisa Aielli More articles by this author , Simone FerrettiSimone Ferretti More articles by this author , Antonio De PalmaAntonio De Palma More articles by this author , Davide CiavarellaDavide Ciavarella More articles by this author , Giulio LitterioGiulio Litterio More articles by this author , Marcella RealeMarcella Reale More articles by this author , Marta Di NicolaMarta Di Nicola More articles by this author , and Luigi SchipsLuigi Schips More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003271.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Castration resistant prostate cancer (CRPC) represents a challenge in urologic oncology. Several treatments are available for this tumor, but their efficacy tends to reduce in time. In recent years, several natural compounds have been tested as possible adjuvant treatment in CRPC patients. Among the other curcumin (CU) showed interesting bioactive properties, but low penetration within cells. We hypothesized that the combination with lactoferrin (LF) might exert a synergistic effect on prostatic cancer cells as anti-cancer. Their effects have been evaluated in PC3 and DU145 prostate cancer cells, that are in vitro models of CRPC. METHODS: Human CRPC cell lines (DU-145, PC3) were cultured in medium added with or without LF (175 µM) ± CU (2.5 µg/ml and 5 µg/ml). Cell viability was determined using a standard colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) reduction assay. To test ability of cell migration observation at 6 and 24 h after scratch wounds were made. Quantitative Real-time PCR was performed to evaluate gene expression of death receptors (DR) and integrins (α3/β1). The human annexin V ELISA assay was used for the quantitative detection of annexin V, involved in apoptosis regulation. RESULTS: In PC3 cells, the proliferation rate is reduced by treatments alone or in association, with a significant reduction in presence of CU, both at 2.5 (p=0.007) and 5 ug/ml (p=0.001), in a dose-dependent manner. In DU145 cells, the inhibitory effect of CU is more significant in presence of LF. In PC3 migration, LF significantly weakened invasion capacity with an increase of the cell-free area of 24.9%, 31.8%, and 24% respectively in presence of LF alone, CU2.5+LF and CU5+LF, after 6 h, with similar results in DU145 cells. In accord the integrins α3/β1, showed a significant reduction in both prostate cell lines, in presence of CU2.5, CU5, and LF175 and their combination (p<0.005). Moreover, in PC3 and DU145 an upregulation of pro-apoptotic DR4 and DR5 gene expression have been observed. Finally, in both PC3 and DU145 cells line an increased concentration of human annexin V, a marker of apoptosis, have been recorded (p<0.005). CONCLUSIONS: Our results showed that the association of CU and LF might led to beneficial effect on CRPC prostate cancer reducing the proliferative activity of cancerous cells and their ability to migrate and produce metastases. In addition, we can suggest a potential pro-apoptotic effect of CU and LF leading to a potential application as an adjuvant treatment for the treatment of CRPC. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e489 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Erica Costantini More articles by this author Michele Marchioni More articles by this author Lisa Aielli More articles by this author Simone Ferretti More articles by this author Antonio De Palma More articles by this author Davide Ciavarella More articles by this author Giulio Litterio More articles by this author Marcella Reale More articles by this author Marta Di Nicola More articles by this author Luigi Schips More articles by this author Expand All Advertisement PDF downloadLoading ...