Normal Prostate Epithelial Cells Research Articles

Distinctive expression and cellular localisation of zinc homeostasis-related proteins in breast and prostate cancer cells

BackgroundZinc transport proteins (ZIP and ZnT), metallothioneins (MT) and protein kinase CK2 are involved in dysregulation of zinc homeostasis in breast and prostate cancer cells. Following up our previous research, we targeted ZIP12, ZnT1, MT2A and CK2 in this study by investigating their expression levels and protein localisation. MethodsQuantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence confocal microscopy were employed to quantify the expression of ZIP12, ZnT1, MT2A and CK2 subunits in a panel of breast and prostate cell lines without or with extracellular zinc exposure. The cellular localisations of these target proteins were also examined by immunofluorescence confocal microscopy. ResultsIn response to the extracellular zinc exposure, the gene expression was elevated for SLC39A12 (ZIP12), SLC30A1 (ZnT1) and MT2A (MT2A) in normal prostate epithelial cells (RWPE-1) in contrast to their cancerous counterparts (PC3 and DU145), whilst the gene expression was higher for SLC39A12 (ZIP12) and SLC30A1 (ZnT1) in both normal (MCF10A) and basal breast cancer cells (MDA-MB-231) compared to luminal breast cancer cells (MCF-7). At the protein level, the expression for both ZIP12 and ZnT1 was trending lower in the time course for the breast cancer cells whilst their expression was remained constant in the normal breast epithelial cells. The expression of ZIP12 in prostate cancer cells was higher than the normal prostate cells. The protein expression for CK2 α/αꞌ and CK2β was markedly higher in prostate cancer cells than the normal prostate cells. Upon extracellular zinc exposure, ZIP12 was, for the first time, conspicuously localised in the plasma membrane of breast cancer cells but not in normal breast epithelial cells and prostate cells. ZnT1 is only localised in the plasma membrane of breast cancer cells. MT2A is distinctively seen close to the plasma membrane in breast cancer cells. CK2 is also for the first time shown to be localised in proximity to the plasma membrane of breast cancer cells. ConclusionThe findings, particularly the localisation of ZIP12 and CK2, are novel and significant for our understanding of zinc homeostasis in breast and prostate cancer cells.

Pentagalloyl Glucose (PGG) Exhibits Anti-Cancer Activity against Aggressive Prostate Cancer by Modulating the ROR1 Mediated AKT-GSK3β Pathway.

Androgen-receptor-negative, androgen-independent (ARneg-AI) prostate cancer aggressively proliferates and metastasizes, which makes treatment difficult. Hence, it is necessary to continue exploring cancer-associated markers, such as oncofetal Receptor Tyrosine Kinase like Orphan Receptor 1 (ROR1), which may serve as a form of targeted prostate cancer therapy. In this study, we identify that Penta-O-galloyl-β-D-glucose (PGG), a plant-derived gallotannin small molecule inhibitor, modulates ROR1-mediated oncogenic signaling and mitigates prostate cancer phenotypes. Results indicate that ROR1 protein levels were elevated in the highly aggressive ARneg-AI PC3 cancer cell line. PGG was selectively cytotoxic to PC3 cells and induced apoptosis of PC3 (IC50 of 31.64 µM) in comparison to normal prostate epithelial RWPE-1 cells (IC50 of 74.55 µM). PGG was found to suppress ROR1 and downstream oncogenic pathways in PC3 cells. These molecular phenomena were corroborated by reduced migration, invasion, and cell cycle progression of PC3 cells. PGG minimally and moderately affected RWPE-1 and ARneg-AI DU145, respectively, which may be due to these cells having lower levels of ROR1 expression in comparison to PC3 cells. Additionally, PGG acted synergistically with the standard chemotherapeutic agent docetaxel to lower the IC50 of both compounds about five-fold (combination index = 0.402) in PC3 cells. These results suggest that ROR1 is a key oncogenic driver and a promising target in aggressive prostate cancers that lack a targetable androgen receptor. Furthermore, PGG may be a selective and potent anti-cancer agent capable of treating ROR1-expressing prostate cancers.

Increased levels of thymidine kinase 1 in malignant cell-derived extracellular vesicles

Extracellular vesicles (EVs), whose main subtypes are exosomes, microparticles, and apoptotic bodies, are secreted by all cells and harbor biomolecules such as DNA, RNA, and proteins. They function as intercellular messengers and, depending on their cargo, may have multiple roles in cancer development. Thymidine kinase 1 (TK1) is a cell cycle-dependent enzyme used as a biomarker for cell proliferation. TK1 is usually elevated in cancer patients' serum, making the enzyme a valuable tumor proliferation biomarker that strongly correlates with cancer stage and metastatic capabilities. Here, we investigated the presence of TK1 in EVs derived from three prostate cancer cell lines with various p53 mutation statuses (LNCaP, PC3, and DU145), EVs from the normal prostate epithelial cell line RWPE-1 and EVs isolated from human seminal fluid (prostasomes). We measured the TK1 activity by a real-time assay for these EVs. We demonstrated that the TK1 enzyme activity is higher in EVs derived from the malignant cell lines, with the highest activity from cells deriving from the most aggressive cancer, compared to the prostasomes and RWPE-1 EVs. The measurement of TK1 activity in EVs may be essential in future prostate cancer studies.

Brucine Suppresses Malignant Progression of Prostate Cancer by Decreasing Sarcosine Accumulation via Downregulation of GNMT in the Glycine/sarcosine Metabolic Pathway.

Prostate cancer (PCa) remains a leading cause of cancer-related incidence and mortality in men. Disruptions in amino acid (AA) metabolism contribute to the disease progression, with brucine, a glycine antagonist, exhibiting antitumor effects. This study explores the antitumor impact of brucine on PCa and investigates its mechanisms in regulating AA metabolic pathways. The study employed the PCa cell line DU-145, characterized by high sarcosine (Sar) levels, for various assays including Cell Counting Kit-8 (CCK8), wound healing, Transwell, 5-Ethynyl-2'-deoxyuridine (EDU), TdT mediated dUTP Nick End Labeling (TUNEL), flow cytometry, Western blot, and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Network pharmacological analysis determined the anticancer mechanisms of brucine. Sar levels in DU-145 cells were significantly higher than in normal prostatic epithelial cells RWPE-1. Treatment with brucine resulted in a marked decrease in cell viability, proliferation, invasion, and migration, while promoting apoptosis in a dose-dependent manner. Sar levels decreased with increasing brucine concentration. Network pharmacology analysis linked brucine's anticancer effect to the AA metabolism and glycine N-methyltransferase (GNMT) pathways. GNMT expression in prostate cancer tissues and The Cancer Genome Atlas database was significantly elevated compared to controls. Treatment with brucine led to downregulation of GNMT expression in DU-145 cells without significant effect on sarcosine dehydrogenase (SARDH). Addition of recombinant GNMT partially reversed the inhibitory effects of brucine on DU-145 cells. Treatment with brucine downregulates GNMT expression in DU-145 cells, reducing Sar accumulation and inhibiting tumor progression. These findings provide new insights into the antitumor mechanisms of brucine in PCa.

FOXA2 rewires AP-1 for transcriptional reprogramming and lineage plasticity in prostate cancer

FOXA family proteins act as pioneer factors by remodeling compact chromatin structures. FOXA1 is crucial for the chromatin binding of the androgen receptor (AR) in both normal prostate epithelial cells and the luminal subtype of prostate cancer (PCa). Recent studies have highlighted the emergence of FOXA2 as an adaptive response to AR signaling inhibition treatments. However, the role of the FOXA1 to FOXA2 transition in regulating cancer lineage plasticity remains unclear. Our study demonstrates that FOXA2 binds to distinct classes of developmental enhancers in multiple AR-independent PCa subtypes, with its binding depending on LSD1. Moreover, we reveal that FOXA2 collaborates with JUN at chromatin and promotes transcriptional reprogramming of AP-1 in lineage-plastic cancer cells, thereby facilitating cell state transitions to multiple lineages. Overall, our findings underscore the pivotal role of FOXA2 as a pan-plasticity driver that rewires AP-1 to induce the differential transcriptional reprogramming necessary for cancer cell lineage plasticity.

Oncogenic Role of SATB2 In Vitro: Regulator of Pluripotency, Self-Renewal, and Epithelial-Mesenchymal Transition in Prostate Cancer.

Special AT-rich sequence binding protein-2 (SATB2) is a nuclear matrix protein that binds to nuclear attachment regions and is involved in chromatin remodeling and transcription regulation. In stem cells, it regulates the expression of genes required for maintaining pluripotency and self-renewal and epithelial-mesenchymal transition (EMT). In this study, we examined the oncogenic role of SATB2 in prostate cancer and assessed whether overexpression of SATB2 in human normal prostate epithelial cells (PrECs) induces properties of cancer stem cells (CSCs). The results demonstrate that SATB2 is highly expressed in prostate cancer cell lines and CSCs, but not in PrECs. Overexpression of SATB2 in PrECs induces cellular transformation which was evident by the formation of colonies in soft agar and spheroids in suspension. Overexpression of SATB2 in PrECs also resulted in induction of stem cell markers (CD44 and CD133), pluripotency-maintaining transcription factors (cMYC, OCT4, SOX2, KLF4, and NANOG), CADHERIN switch, and EMT-related transcription factors. Chromatin immunoprecipitation assay demonstrated that SATB2 can directly bind to promoters of BCL-2, BSP, NANOG, MYC, XIAP, KLF4, and HOXA2, suggesting SATB2 is capable of directly regulating pluripotency/self-renewal, cell survival, and proliferation. Since prostate CSCs play a crucial role in cancer initiation, progression, and metastasis, we also examined the effects of SATB2 knockdown on stemness. SATB2 knockdown in prostate CSCs inhibited spheroid formation, cell viability, colony formation, cell motility, migration, and invasion compared to their scrambled control groups. SATB2 knockdown in CSCs also upregulated the expression of E-CADHERIN and inhibited the expression of N-CADHERIN, SNAIL, SLUG, and ZEB1. The expression of SATB2 was significantly higher in prostate adenocarcinoma compared to normal tissues. Overall, our data suggest that SATB2 acts as an oncogenic factor where it is capable of inducing malignant changes in PrECs by inducing CSC characteristics.

Brevilin A Inhibits Prostate Cancer Progression by Decreasing PAX5-Activated SOX4.

Brevilin A possesses inhibitory effects on the development of prostate cancer (PCa); however, the underlying mechanism remains unclear. The present work aims to analyze how Brevilin A regulates PCa cell malignancy. RNA expression of paired box 5 (PAX5) and SRY-box transcription factor 4 (SOX4) was analyzed by quantitative real-time polymerase chain reaction. Protein expression of PAX5, SOX4, and nuclear proliferation marker (Ki67) was detected by western blotting or immunohistochemistry assay. The viability, proliferation, apoptosis, and migratory and invasive abilities of PCa cells were investigated by cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry, and transwell assays, respectively. The association between PAX5 and SOX4 was identified by dual-luciferase reporter assay and chromatin immunoprecipitation assay. Xenograft mouse model assay was used to reveal the effect of Brevilin A on tumor tumorigenesis in vivo. PAX5 and SOX4 expression were upregulated in PCa tissues and cells relative to normal prostate tissues and human prostate epithelial cells. Brevilin A treatment inhibited PAX5 protein expression in PCa cells. Additionally, Brevilin A inhibited proliferation, migration and invasion and induced apoptosis of PCa cells, whereas these effects were attenuated after PAX5 overexpression. SOX4 was transcriptionally activated by PAX5, and its introduction partially relieved the inhibitory effects of PAX5 knockdown on PCa cell malignancy. Moreover, Brevilin A delayed tumor formation in vivo. Brevilin A inhibited PCa progression by regulating SOX4 expression in a PAX5-dependent manner, providing a promising anti-tumor drug for PCa.

DSCAM-AS1 promotes the development of prostate cancer

PurposeThe purpose of this study was to investigate the role of lncRNA DSCAM-AS1 in prostate cancer to find new therapeutic targets and promote the research progress of prostate cancer.MethodsRT-qPCR was used to detect DSCAM-AS1 expression in prostate cancer tissues, normal tissues, human normal prostate epithelial cells (RWPE), and four prostate cancer cell lines. The clinical and prognostic role of DSCAM-AS1 was evaluated by the Kaplan–Meier curve and chi-square test. Secondly, a dual luciferase reporter gene assay was used to study the regulatory mechanism between miR-338-3p and DSCAM-AS1. Finally, the roles of DSCAM-AS1 and miR-338-3p in prostate cancer cell proliferation and metastasis were explored by CCK-8 and Transwell assays.ResultsIt was found that DSCAM-AS1 upregulation could serve as a warning of deterioration and poor prognosis in prostate cancer patients, and that knockdown of DSCAM-AS1 expression inhibited the progression of prostate cancer cells. In addition, miR-338-3p, a target of DSCAM-AS1, was found to be down-regulated in prostate cancer cells and miR-338-3p knockdown could reverse the inhibitory effect of DSCAM-AS1 silencing on prostate cancer.ConclusionDSCAM-AS1 is up-regulated in prostate cancer and regulates the progression of prostate cancer cells by targeting miR-338-3p.

Abstract LB324: STING protein expression is increased in intermediate luminal cells in proliferative inflammatory atrophy of the prostate (PIA) and is absent in the majority of primary prostatic adenocarcinomas

Abstract Unresolved chronic inflammation has been postulated to drive prostate cancer development. We have proposed that proliferative inflammatory atrophy (PIA) can lead to the development of preneoplastic high grade prostatic intraepithelial neoplasia (PIN) lesions and/or directly to adenocarcinoma at times. Additionally, some PIA cells harbor molecular alterations seen in PIN and invasive carcinoma lesions, with a low frequency of PIA cells showing MYC over-expression, partial promoter hypermethylation and silencing of GSTP1, and formation of ETS gene fusions. PIA appears to result from inflammatory injury and regeneration in which proliferative and atrophic epithelial cells have an intermediate cell phenotype with both luminal and basal cell features, including basal cell markers, keratin 5 and BCL2, as well as varying levels of prostate luminal enriched genes such as androgen receptor, PSA and NKX3.1. Also, a number of gene products that are apparently stress induced, which are not expressed in normal prostate are induced in at least a subset of intermediate luminal cells in PIA, including GSTA1, PTGS2, LTF and PIGR (PMIDs:17384581, 37550827). Stimulator of interferon genes (STING), encoded by TMEM173, is a multipass transmembrane ER associated protein, and is a critical hub for innate responses directed against numerous bacterial, viral and parasitic pathogens, and is important for the induction and maintenance of adaptive immunity in a number of situations. Here, we examined STING expression by IHC, using a genetically validated assay, and show that the majority of atrophic prostatic luminal epithelial cells in all PIA lesions (N=20) consistently express high levels of STING, which is present in normal prostatic basal epithelial cells yet absent in normal-appearing prostatic luminal cells. In primary prostatic adenocarcinomas (> 260 tumors), STING protein was either completely negative , or extremely low in ~ 95% of cases. That STING in PIA is activate is supported by single cell RNA sequencing (scRNAseq) data from human prostatectomy tissues that show cells with the phenotype of intermediate luminal cells, indicative of PIA, express a number of interferon response genes that are not expressed in normal luminal cells (PMID 37905029). To our knowledge this is the first systematic report of STING protein staining in human prostate cancer and benign tissues. We postulate that upregulation of STING in human intermediate cells in PIA represents an inflammatory response that can drive interferon stimulated gene expression and inflammation in PIA. This innate immune response is absent, potentially by gene silencing, in prostatic adenocarcinoma, which may help neoplastic cells evade the immune response and could help contribute to the long standing finding that most prostate cancers are immunologically cold. Citation Format: Thomas M. Steele, Alan K. Meeker, Mindy K. Graham, Rulin Wang, Carolina Gomes-Alexandre, William G. Nelson, Fernanda Caramella Pereiria, Sushant Kachhap, Srinivasan Yegnasubramanian, Angelo Michael De Marzo. STING protein expression is increased in intermediate luminal cells in proliferative inflammatory atrophy of the prostate (PIA) and is absent in the majority of primary prostatic adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB324.

Abstract 3035: Bridging the gap: Exploring the roles of ETS- coactivator interactions in promoting prostate cancer

Abstract The ETS family of transcription factors is comprised of 28 members that all share a common C- terminal DNA-binding domain. ETS play essential roles in developmental processes, such as stem cell maintenance, blood vessel formation, and cellular fate. While absent in the normal prostate, four members of the ETS family become expressed in prostate tumors: ETV1, ETV4, ETV5 and ERG. Our lab has previously classified these proteins as oncogenic ETS for their ability to promote cellular migration in normal prostate epithelial RWPE1 cells. Transcription factors may need to interact with additional proteins to serve oncogenic roles. My research explores the interactions between 1) ETV1 and EWS, as well as 2) ERG and p53. Our lab has established that ETV1, ETV4 and ETV5 form essential interactions with EWS, a transcriptional coactivator that promotes ETS activity. In prostate tumors, chromosomal rearrangements result in the N-terminal truncations of ETV1, ETV4 and ETV5. I hypothesize that the EWS interaction domain is retained and that these truncated ETVs will remain oncogenic. Pulldown assays using purified full-length and truncated ETV1 suggests EWS interacts with both N-terminal and C-terminal portions ETV1. I am attempting to express truncated ETV1, ETV4 and ETV5 in RWPE1 cells. Upon stable expression, I will evaluate their interactions with EWS and their ability to promote migration in vitro. To identify putative proteins that bridge EWS to ETV1, immunoprecipitation mass spectrometry will be used. Mutations in p53, a tumor suppressor protein, promote oncogenesis and occur in nearly half of all cancer cases. We hypothesize that mutant p53 interacts with ETS to promote its recruitment to new genomic sites. Previous studies demonstrate that mutant p53 binds ETS2 at a higher affinity than wildtype p53 and we have expanded this list of interacting ETS to include ERG. We seek to explore whether ERG and mutant p53 cooperate to promote oncogenic phenotypes. I successfully cloned wildtype p53, mutant p53 (R248W) and double-mutant p53 (R248W, T81A) into a retroviral backbone. I then transduced RWPE1-Vector and RWPE1-ERG expressing cells to express these constructs. Although additional replicates are needed, it appears that mutant p53 promotes colony formation in both cell types relative to wildtype p53. I plan to validate these observations and generate an appropriate negative control cell line before extending into a mouse model. Through the continuation of this work, I will provide critical insight into the molecular mechanisms by which a subset of ETS factors achieve oncogenic function. We address the contributions of DNA-binding specificity and coactivator interactions in promoting the phenotypes associated with prostate cancer. Citation Format: Kaitlyn M. Mills, Nicholas Downing, Stephanie Metcalf, Peter Hollenhorst. Bridging the gap: Exploring the roles of ETS- coactivator interactions in promoting prostate cancer [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 3035.

Abstract 773: Overexpression of phosphoserine phosphatase (PSPH) in prostate cancer and in tumor microenvironment promotes tumor cell proliferation

Abstract Reprogrammed cellular metabolism is one of the key features of cancer and tumor microenvironment. L-3-phosphoserine phosphatase (PSPH) is one of the five rate-limiting enzymes in the biosynthesis of serine from glucose that supports cell proliferation. Here, we aim to study the role of PSPH in prostate cancer (PCa) and its tumor microenvironment. We find that PSPH not only is overexpressed in prostate cancer cell lines compared to normal prostate epithelial cells (PrEC) and benign prostate epithelial cells (BPH-1), but also exhibits a higher level in patient-derived carcinoma-associated fibroblasts (CAFs) compared to the matched benign associated fibroblasts (BAFs), which are derived from fresh radical prostatectomy specimens of African American (AA) and European American (EA) men. Notably, MDAPCa2b cells derived from an AA prostate cancer patient and AA CAFs have even higher expression of PSPH protein. The mRNA levels of PSPH are also significantly elevated in prostate cancer tissues in AA men compared to EA men and predict poor survival in PCa patients. Knock-down of PSPH expression significantly inhibits the proliferation and colony formation of prostate cancer cells. Growth of xenograft tumors derived from PSPH knockdown MDAPCa2b cells has also been suppressed markedly. Gene expression profiling revealed that suppression of PSPH expression by shRNAs in LNCaP and MDAPCa2b cells is associated with the regulation of gene expression related to metabolism, immunity, extracellular matrix remodeling, and Ion channels. Similarly, analysis of publicly available PCa RNA seq databases also demonstrates that PSPH amplification or over-expression is associated with alterations of gene expression related to metabolism and immunity. Our results suggest that PSPH is upregulated in prostate cancer cells and their surrounding fibroblasts to support tumor growth via reprogramming metabolic pathways and transcriptomes. Citation Format: Liankun Song, Noriko Yokoyama, Jun Xie, Yunjie Hu, Zhenyu (Arthur) Jia, Beverly Wang, Dan Mercola, Edward Uchio, Thomas Ahlering, Michael Lilly, Xiaolin Zi. Overexpression of phosphoserine phosphatase (PSPH) in prostate cancer and in tumor microenvironment promotes tumor cell proliferation [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 773.

Abstract 3033: Oncogenic ETS transcription factors avoid repression by EZH2 and FOXO1 in prostate cancer and Ewing sarcoma

Abstract ETS transcription factors play roles throughout development, aiding in hematopoiesis, blood vessel formation and cell fate. The ETS family is composed of 28 members, all of which share an ETS DNA-binding domain. Chromosomal rearrangements that lead to the overexpression of specific ETS promote oncogenic transformation and tumor development. More than half of prostate tumors are driven by aberrant expression of an ETS protein. Our lab has demonstrated that oncogenic ETS form essential interactions with a ubiquitous RNA-binding protein, EWS, to promote prostate tumorigenesis. A similar mechanism exists in Ewing sarcoma (ES). ES tumors are driven by the expression EWS/ETS fusion proteins. This project focuses on the similarities and differences between oncogenic ETS in prostate cancer and EWS/ETS fusions in Ewing sarcoma. We compared biological functions of the most common oncogenic ETS in prostate cancer, ERG, and the most common fusion in ES, EWS/FLI1. Both ERG and EWS/FLI1 promoted migration and clonogenic survival in normal prostate epithelial cell lines, RWPE1 and PNT2. These data suggest that the EWS-ERG complex and EWS/FLI1 fusion protein can function similarly in prostate cells. Knockdown of endogenous EWS/FLI1 in the ES cell line, A673, reduced anchorage-independent colony formation in soft agar. Rescue with exogenous EWS/FLI1 or EWS/ERG restored colony formation relative to control. Wildtype ERG was unable to rescue colony formation in A673. This suggests that ERG and EWS/FLI1 function differently in A673 cells. Pulldown assays from A673 lysates reveal that ERG interacts with EZH2 and FOXO1. Our lab has established that ERG acts as a transcriptional repressor through the formation of an ERG-EZH2-PRC2 complex. Others demonstrate that FOXO1 represses ERG activity by reducing its recruitment to the target sites. Both the N-terminal truncation of ERG and phosphorylation of ERG at S96 disrupted these interactions and rescued anchorage independent growth in A673 ES cells. These data suggest that EWS/ETS fusions avoid transcriptional repression in Ewing tumors due to loss of the N-terminus of the ETS protein. Citation Format: Nicholas F. Downing, Kaitlyn M. Mills, Peter C. Hollenhorst. Oncogenic ETS transcription factors avoid repression by EZH2 and FOXO1 in prostate cancer and Ewing sarcoma [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 3033.

Multifaceted impact of adipose conditioned media: Obesity-driven promotion of prostate cancer and cancer stem cell dynamics.

Obesity is an established risk factor for the development and progression of prostate cancer (PC). This study used adipose conditioned media (ACM) from differentiated adipocytes to assess its effect on PC development and aggressiveness. Due to limited research on ACM's impact on isolated PC stem cells (PCSCs), we also examined CD44+ PCSCs. ACM notably boosted interleukin-1β (IL-1β), IL-6, and IL-8 production in normal prostate epithelial cells and LNCaP cells. It also increased IL-6 and IL-8 production in PC3 and CD44+ LNCaP cells, and IL-1β and IL-6 production in CD44+ PC3 cells. This indicates that ACM induces the production of inflammatory cytokines in both cancer and prostate epithelial cells. Furthermore, ACM promoted proliferation in androgen receptor (AR)-negative PC3 cells, CD44+ PC3 PCSCs, and nonmalignant RWPE cells, without affecting AR-positive LNCaP cells. In addition, ACM-enhanced invasion and migration potential in both PC3 and CD44+ PC3 cells. Western blot analysis indicated the involvement of NF-κB and AKT pathways in ACM-induced proliferation in PC3 cells and NF-κB in PCSCs. In ACM-treated PC3 cells, E-cadherin was downregulated, while N-cadherin, Snail, vimentin, fibronectin, and Twist were upregulated, suggesting ACM-induced invasion via classical epithelial-to-mesenchymal transition (EMT) pathways. In response to ACM, PCSCs exhibited increased expression of E-cadherin, Snail, and vimentin, which are partial EMT markers promoting stemness and resistance to apoptosis. In addition, increased expressions of Nanog, Oct3/4, survivin, and Bcl-2 were observed. Although the molecules we studied have diverse effects on cellular regulation, our data emphasize obesity's multifaceted role in promoting and aggressing PC, notably affecting PCSC populations.

New 2-oxoindole derivatives as multiple PDGFRα/ß and VEGFR-2 tyrosine kinase inhibitors

Two new series of N-aryl acetamides 6a-o and benzyloxy benzylidenes 9a-p based 2-oxoindole derivatives were designed as potent antiproliferative multiple kinase inhibitors. The results of one-dose NCI antiproliferative screening for compounds 6a-o and 9a-p elucidated that the most promising antiproliferative scaffolds were 6f and 9f, which underwent five-dose testing. Notably, the amido congener 6f was the most potent derivative towards pancreatic ductal adenocarcinoma MDA-PATC53 and PL45 cell lines (IC50 = 1.73 µM and 2.40 µM, respectively), and the benzyloxy derivative 9f was the next potent one with IC50 values of 2.85 µM and 2.96 µM, respectively. Both compounds 6f and 9f demonstrated a favorable safety profile when tested against normal prostate epithelial cells (RWPE-1). Additionally, compound 6f displayed exceptional selectivity as a multiple kinase inhibitor, particularly targeting PDGFRα, PDGFRβ, and VEGFR-2 kinases, with IC50 values of 7.41 nM, 6.18 nM, and 7.49 nM, respectively. In contrast, the reference compound Sunitinib exhibited IC50 values of 43.88 nM, 2.13 nM, and 78.46 nM against the same kinases. The derivative 9f followed closely, with IC50 values of 9.9 nM, 6.62 nM, and 22.21 nM for the respective kinases. Both 6f and 9f disrupt the G2/M cell cycle transition by upregulating p21 and reducing CDK1 and cyclin B1 mRNA levels. The interplay between targeted kinases and these cell cycle regulators underpins the G2/M cell cycle arrest induced by our compounds. Also, compounds 6f and 9f fundamentally resulted in entering MDA-PATC53 cells into the early stage of apoptosis with good percentages compared to the positive control Sunitinib. The in silico molecular-docking outcomes of scaffolds 6a-o and 9a-p in VEGFR-2, PDGFRα, and PDGFRβ active sites depicted their ability to adopt essential binding interactions like the reference Sunitinib. Our designed analogs, specifically 6f and 9f, possess promising antiproliferative and kinase inhibitory properties, making them potential candidates for further therapeutic development.

Enhanced Chemoprevention of Prostate Cancer by Combining Arctigenin with Green Tea and Quercetin in Prostate-Specific Phosphatase and Tensin Homolog Knockout Mice.

The low bioavailability of most phytochemicals limits their anticancer effects in humans. The present study was designed to test whether combining arctigenin (Arc), a lignan mainly from the seed of Arctium lappa, with green tea (GT) and quercetin (Q) enhances the chemopreventive effect on prostate cancer. We performed in vitro proliferation studies on different cell lines. We observed a strong synergistic anti-proliferative effect of GT+Q+Arc in exposing androgen-sensitive human prostate cancer LNCaP cells. The pre-malignant WPE1-NA22 cell line was more sensitive to this combination. No cytotoxicity was observed in normal prostate epithelial PrEC cells. For an in vivo study, 3-week-old, prostate-specific PTEN (phosphatase and tensin homolog) knockout mice were treated with GT+Q, Arc, GT+Q+Arc, or the control daily until 16 weeks of age. In vivo imaging using prostate-specific membrane antigen (PSMA) probes demonstrated that the prostate tumorigenesis was significantly inhibited by 40% (GT+Q), 60% (Arc at 30 mg/kg bw), and 90% (GT+Q+Arc) compared to the control. A pathological examination showed that all control mice developed invasive prostate adenocarcinoma. In contrast, the primary lesion in the GT+Q and Arc alone groups was high-grade prostatic intraepithelial neoplasia (PIN), with low-grade PIN in the GT+Q+Arc group. The combined effect of GT+Q+Arc was associated with an increased inhibition of the androgen receptor, the PI3K/Akt pathway, Ki67 expression, and angiogenesis. This study demonstrates that combining Arc with GT and Q was highly effective in prostate cancer chemoprevention. These results warrant clinical trials to confirm the efficacy of this combination in humans.

The Effect of Nano-Encapsulated Orlistat on Prostate Cancer: An In vitro Study

Background: The targeted inhibition of fatty acid synthase (FASN) by Orlistat, a potent FASN inhibitor, has been shown to block tumor proliferation and induce apoptosis in cultured tumor cells. Since Orlistat is insoluble, its solubility in blood circulation is limited. Cancer nanotherapeutics are rapidly progressing and are being implemented to solve several limitations of conventional drug delivery systems. Aim: In this in vitro study, we aimed to overcome the limitation of orlistat changes by designing a nanoparticle and investigating its selective cytotoxic effects on cancer cells. Method: To investigate the effects of encapsulated Orlistat on prostate cancer, we measured apoptosis and the distribution of cells via flow cytometry, confocal microscope, and Western blot. Results: There is an improvement in the solubility of Orlistat with the effect of nanoparticles; it was observed that Orlistat decreased the expression of FASN and caused dose-dependent cytotoxicity with Caspase-3, Bax, and PARP activation in PC-3 cells, while it did not have a significant effect on normal prostate epithelial cells. Conclusion: The present results indicate that the limitation of Orlistat can be overcome by assembling a nano-micellar formulation that can induce apoptotic death in the PC-3 cell line but normal prostate epithelial cells.

Sinularin stabilizes FOXO3 protein to trigger prostate cancer cell intrinsic apoptosis

Sinularin, a natural product that purified from soft coral, exhibits anti-tumor effects against various human cancers. However, the mechanisms are not well understood. In this study, we demonstrated that Sinularin inhibited the viability of human prostate cancer cells in a dose-dependent manner and displayed significant cytotoxicity only at high concentration against normal prostate epithelial cell RWPE-1. Flow cytometry assay demonstrated that Sinularin induced tumor cell apoptosis. Further investigations revealed that Sinularin exerted anti-tumor activity through intrinsic apoptotic pathway along with up-regulation of pro-apoptotic protein Bax and PUMA, inhibition of anti-apoptotic protein Bcl-2, mitochondrial membrane potential collapses, and release of mitochondrial proteins. Furthermore, we illustrated that Sinularin induced cell apoptosis via up-regulating PUMA through inhibition of FOXO3 degradation by the ubiquitin–proteasome pathway. To explore how Sinularin suppress FOXO3 ubiquitin–proteasome degradation, we tested two important protein kinases AKT and ERK that regulate FOXO3 stabilization. The results revealed that Sinularin stabilized and up-regulated FOXO3 via inhibition of AKT- and ERK1/2-mediated FOXO3 phosphorylation and subsequent ubiquitin–proteasome degradation. Our findings illustrated the potential mechanisms by which Sinularin induced cell apoptosis and Sinularin may be applied as a therapeutic agent for human prostate cancer.

Receptor tyrosine kinase-like orphan receptor 1 inhibitor strictinin exhibits anti-cancer properties against highly aggressive androgen-independent prostate cancer

Aim: It is important to identify anti-cancer compounds that can inhibit specific molecular targets to eradicate androgen-receptor negative (ARneg), androgen-independent (AI) prostate cancer, which is an aggressive form of prostate cancer with limited treatment options. The goal of this study was to selectively target prostate cancer cells that have high levels of oncogenic protein Receptor tyrosine kinase-like orphan receptor 1 (ROR1) by using strictinin, a small molecule ROR1 inhibitor. Methods: The methods performed in this study include western blots, methyl thiazolyl tetrazolium (MTT) proliferation assays, phosphatidylserine apoptosis assays, apoptosis flow cytometry (Annexin V, caspase 3/7), migration scratch assays, Boyden chamber invasion assays, and cell cycle flow cytometry. Results: Strictinin was most lethal against PC3 [half-maximal drug inhibitory concentration (IC50) of 277.2 µmol/L], an ARneg-AI cell type that expresses the highest levels of ROR1. Strictinin inhibited ROR1 expression, downstream phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-glycogen synthase kinase 3beta (GSK3β) pro-survival signaling, and epithelial-to-mesenchymal transition markers in PC3 cells. Additionally, strictinin decreased PC3 cell migration and invasion, while increasing S-phase cell cycle arrest. In ARneg-AI DU145 cells, strictinin inhibited ROR1 expression and modulated downstream AKT-GSK3β signaling. Furthermore, strictinin exhibited anti-migratory, anti-invasive, but minimal pro-apoptotic effects in DU145 cells likely due to DU145 having less ROR1 expression in comparison to PC3 cells. Throughout the study, strictinin minimally impacted the phenotype of normal prostatic epithelial cells RWPE-1 (IC50 of 658.5 µmol/L). Strictinin was further identified as synergistic with docetaxel [combination index (CI) = 0.311] and the combination therapy was found to reduce the IC50 of strictinin to 38.71 µmol/L in PC3 cells. Conclusions: ROR1 is an emerging molecular target that can be utilized for treating prostate cancer. The data from this study establishes strictinin as a potential therapeutic agent that targets ARneg-AI prostate cancer with elevated ROR1 expression to reduce the migration, invasion, cell cycle progression, and survival of prostate cancer.

A novel circular RNA, circMAML3, promotes tumor progression of prostate cancer by regulating miR-665/MAPK8IP2 axis

Many studies have now demonstrated that circRNAs are aberrantly expressed in cancer and are involved in the regulation of malignant tumor progression. However, the role of circMAML3 (hsa_circ_0125392) in prostate cancer has not been reported. circMAML3 was selected from public data through screening. The circMAML3 circular characterization was performed using Sanger sequencing, agarose gel electrophoresis assay, RNase R assay and actinomycin D assay. The expression of circMAML3 in prostate cancer tissues and cells was detected by qRT-PCR. In vivo and in vitro experiments were conducted to investigate the biological functions of circMAML3 in prostate cancer. Finally, the underlying mechanism of circMAML3 was revealed by qRT-PCR, luciferase reporter assay, miRNA Pulldown, RNA immunoprecipitation, western blotting, and rescue assay. Compared to normal prostate tissue and prostate epithelial cells, circMAML3 is highly expressed in prostate cancer tissues and cell lines. CircMAML3 overexpression promotes prostate cancer proliferation and metastasis, while knockdown of circMAML3 exerts the opposite effect. Mechanistically, circMAML3 promotes prostate cancer progression by upregulating MAPK8IP2 expression through sponge miR-665. Our research indicates that circMAML3 promotes prostate cancer progression through the circMAML3/miR-665/MAPK8IP2 axis. circMAML3 and MAPK8IP2 are upregulated in prostate cancer expression and play an oncogenic role, whereas miR-665 is downregulated in prostate cancer and plays an oncogenic role. Therefore, CircMAML3 may be a potential biomarker for prostate cancer diagnosis, treatment and prognosis.

Targeting Metabolic Vulnerabilities to Overcome Prostate Cancer Resistance: Dual Therapy with Apalutamide and Complex I Inhibition

Prostate cancer (PCa) often becomes drug-treatment-resistant, posing a significant challenge to effective management. Although initial treatment with androgen deprivation therapy can control advanced PCa, subsequent resistance mechanisms allow tumor cells to continue growing, necessitating alternative approaches. This study delves into the specific metabolic dependencies of different PCa subtypes and explores the potential synergistic effects of combining androgen receptor (AR) inhibition (ARN with mitochondrial complex I inhibition (IACS)). We examined the metabolic behaviors of normal prostate epithelial cells (PNT1A), androgen-sensitive cells (LNCaP and C4-2), and androgen-independent cells (PC-3) when treated with ARN, IACS, or a combination. The results uncovered distinct mitochondrial activities across PCa subtypes, with androgen-dependent cells exhibiting heightened oxidative phosphorylation (OXPHOS). The combination of ARN and IACS significantly curbed cell proliferation in multiple PCa cell lines. Cellular bioenergetics analysis revealed that IACS reduced OXPHOS, while ARN hindered glycolysis in certain PCa cells. Additionally, galactose supplementation disrupted compensatory glycolytic mechanisms induced by metabolic reprogramming. Notably, glucose-deprived conditions heightened the sensitivity of PCa cells to mitochondrial inhibition, especially in the resistant PC-3 cells. Overall, this study illuminates the intricate interplay between AR signaling, metabolic adaptations, and treatment resistance in PCa. The findings offer valuable insights into subtype-specific metabolic profiles and propose a promising strategy to target PCa cells by exploiting their metabolic vulnerabilities.