Prostate Epithelial Cell Line RWPE-1 Research Articles

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.

Abstract 3238: Therapeutic targeting of the genome guardian sirtuins in metastatic prostate cancer

Abstract Introduction: The effective treatment paradigm for the lethal form of metastatic prostate cancer (mPCa) remains challenging. Almost all mPCas eventually develops therapy resistance against androgen receptor (AR) inhibitors (e.g., Enzalutamide, Abiraterone) or to poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors (Olaparib, Niraparib, Talazoparib) (HRRd) when used as monotherapy. Although the patients in the ARSi+PARPi combination produced significantly longer radiologic progression-free survival (rPFS), a substantial number had attained therapy resistance by the end of the study tenure. So, a newer therapy is urgently needed. In recent years, a new targetable therapeutics development domain has been unfolding with an expanding understanding of the role of SIRTuins in prostate cancer cell survival, invasion, and progression to an aggressive metastatic state. Here, we will investigate the therapeutic efficacy of SIRT1/7 inhibitor (SIRT1/7i) and determine synergy with DNA-damaging PARP inhibitor combinations through a battery of cell and molecular assays using various prostate cancer cellular models. Methods: We have treated the mPCa cell lines 22Rv1, LAPC4, PC3 and LnCAP as well as prostate epithelial cell line RWPE1 with SIRT1 inhibitor SIRT1-IN-2 (MedChemExpress, HY-146689), SIRT7 inhibitor 97491 (MedChemExpress, HY-135899) and AR inhibitor Enzalutamide individually as well as in combination of PARP inhibitor Olaparib. The treatment of the SIRT1 and SIRT7 monotherapy arm was 72 hours long, and combination therapy continued for 7 days. The cell viability was assessed with CellTiter-Glo® 2.0 Cell Viability Assay (Promega). The cell cycle progression and proliferation analysis were done using flow cytometry through Propidium iodide staining and AnnexinV-FITC apoptosis detection chemistry. Cell survival was orthogonally assessed using colony counting assays. We adopt RAD51 foci counting assays and COMET assays for evaluating treatment-induced genome instability. Results: The preliminary result suggested that the SIRT1i+Olaparib and SIRT7i+Olaparib combination significantly sensitized the mPCa cell lines 22Rv1, PC3, and LnCAP for cell death compared to the prostate epithelial cell RWPE1. Upon the combination treatment, the mPCa cell lines showed elevated apoptotic cell death, reconfirmed through colony counting assay. Conclusion: Therapeutic combination treatment of SIRT1 and SIRT7 inhibition with Olaparib could be an effective treatment option for aggressive metastatic prostate cancer. Citation Format: Subhajit Giri. Therapeutic targeting of the genome guardian sirtuins in metastatic 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 3238.

Comparison on blood-prostate barrier permeability of tanshinone extract and corresponding major monomers

In this study, the transmittance of tanshinone Ⅱ_A(Tan Ⅱ_A) and cryptotanshinone(CTS) through the blood-prostate barrier and their distributions in the prostate tissue were compared between tanshinone extract(Tan E) treatment group and the corresponding monomer composition group under the equivalent dose conversion in vitro and in vivo. First, the human prostate epithelial cell line RWPE-1 was cultured in vitro for 21 days for the establishment of a blood-prostate barrier model, and the transmission of Tan Ⅱ_A and CTS through the barrier model was investigated after administration of Tan E and corresponding single active components. Second, SD rats were administrated with 700 mg·kg~(-1) Tan E, 29 mg·kg~(-1) CTS, and 50 mg·kg~(-1) Tan Ⅱ_A by gavage, and plasma and prostate tissue samples were collected at the time points of 2, 4, 8, 12, and 24 h. The Tan Ⅱ_A and CTS concentrations in the samples were determined. The results showed that in the cell model, the cumulative transmission amounts of CTS and Tan Ⅱ_A in the extract at each time point were higher than those of the corresponding single active components(P<0.01). In rats, after the administration of Tan E, the concentrations of Tan Ⅱ_A and CTS in rat plasma and prostate were higher than those of the corresponding single active components. This study demonstrated that the coexisting components in Tan E promoted the penetration of its main pharmacological components Tan Ⅱ_A and CTS through the blood-prostate barrier. The findings provide a theoretical and experimental basis for the application of Tan E in the clinical treatment of prostate-related diseases.

Overexpression of YTHDC2 contributes to the progression of prostate cancer and predicts poor outcomes in patients with prostate cancer.

YTH domain-containing protein 2 (YTHDC2), a member of N6-methyladenosine (m6A) readers, has been reported to be closely associated with multiple cancer types. However, very little is known about the YTHDC2 gene and its involvement in prostate cancer. YTHDC2 protein expression level was analyzed and correlated to clinical outcomes in prostate cancer patients who underwent prostatectomy in Guizhou Provincial People's Hospital. The YTHDC2 expression level was also detected in prostate cancer cell lines and an immortalized prostate epithelial cell line BPH-1 and RWPE1 by quantitative real-time reverse transcription polymerase chain reaction. Furthermore, we established stable cell lines (DU145 and PC-3) transfected with either empty vector or the full-length YTHDC2 gene and conducted cell function assays in vitro. Fisher's exact test and Pearson χ2 test were employed, Kaplan-Meier method was used for the survival analysis. Of 32 patient samples who enrolled in this study, YTHDC2 was significantly upregulated in prostate cancer (PCa) patients with higher Gleason scores and serum prostate-specific antigen levels. YTHDC2 expression was significantly elevated in all PCa cell lines compared to BPH-1 and RWPE1 (all p < 0.05). Functionally, the enforced expression of YTHDC2 markedly promoted cell growth, migration, and invasion efficacies in prostate cancer cells. Our data indicate that YTHDC2 upregulation may be potentially associated with the prognosis of prostate cancer patients.

Identifying Mitochondrial Transcription Factor A As a Potential Biomarker for the Carcinogenesis and Prognosis of Prostate Cancer.

Aims: Mitochondrial functional transformation contributes to the carcinogenesis of the prostate by meeting the metabolic needs of cancer cells. Mitochondrial transcription factor A (TFAM) is a pivotal regulator that maintains homeostasis of mitochondrial function. However, its role in prostate carcinogenesis has not been well elucidated. Materials and Methods: In the present study, we analyzed the expression of TFAM in normal prostate tissue and prostate cancer using public databases; a prostate-tissue chip was used to verify the results. The expression of TFAM in normal cells and in prostate cancer cells was determined by western blotting analysis. We knocked down TFAM in the prostate cancer cell line PC3 using a specific shRNA to explore the potential effects of TFAM in prostatic carcinogenesis. Results: We observed higher expression levels of TFAM in prostate cancer tissue than in normal prostate tissue and tumor adjacent normal tissues. A receiver operating characteristic curve was drawn that demonstrated the diagnostic efficacy of using TFAM expression for prostate cancer prognoses. Elevated levels of TFAM may indicate poorer overall survival in prostate cancer patients. Western blotting assays also showed that relative to the normal prostatic epithelial cell line RWPE-1, prostate cancer cell lines PC3 and DU145 expressed more TFAM protein. Furthermore, knockdown of TFAM inhibited the colony-formation capability of PC3 cells. Conclusion: Collectively, these results suggest that TFAM promotes carcinogenesis of the prostate, and may constitute a marker to be used in the diagnosis and prognosis of prostate cancer.

Nonmalignant AR-positive prostate epithelial cells and cancer cells respond differently to androgen.

Prostate cancer research suffers from the lack of suitable models to study the role of normal cells in prostate carcinogenesis. To address this challenge, we developed a cell line model mimicking luminal prostate epithelial cells by modifying the immortalized prostate epithelial cell line RWPE-1 to constitutively express the androgen receptor (AR). RWPE-1-AR cells express known AR target genes, and exhibit coexpression of luminal and basal markers characteristic of transient amplifying cells, and an RNA signature resembling prostate luminal progenitor cells. Under unstimulated conditions, constitutive AR expression does not have a biologically significant effect on the proliferation of RWPE-1 cells, but when stimulated by androgens, growth is retarded. The transcriptional response of RWPE-1-AR cells to androgen stimulation involves suppression of the growth-related KRAS pathway and is thus markedly different from that of the prostate cancer cell line LNCaP and its derivative AR-overexpressing LNCaP-ARhi cells, in which growth- and cancer-related pathways are upregulated. Hence, the nonmalignant AR-positive RWPE-1-AR cell line model could be used to study the transformation of the prostate epithelium.

Upregulation of mir-1199-5p is associated with reduced type 2 5-α reductase expression in benign prostatic hyperplasia

Background5-α reductase inhibitors (5-ARIs) are first-line drugs for managing benign prostatic hyperplasia (BPH). Unfortunately, some patients do not respond to 5-ARI therapy and may even show worsening symptoms. The decreased expression of steroid 5-α reductase type 2(SRD5A2) in BPH tissues may explain the failure of 5-ARI therapy, however, the mechanisms underlying SRD5A2 decreased remained unelucidated.ObjectivesTo investigate microRNA-mediated regulation of the expression of SRD5A2 resulting in 5-ARI therapy failure.Materials and methodsThe expression of SRD5A2 and microRNAs in BPH tissues and prostate cells were detected by immunohistochemistry, western blotting, and quantitative real-time PCR. Dual-luciferase reporter assay was performed to confirm that microRNA directly combine to SRD5A2 mRNA. The apoptosis of prostatic cells was detected by flow cytometry.ResultsSRD5A2 expression was variable; it was negative, weak, and strong in 13.6%, 28.8%, and 57.6% of BPH tissues respectively. The normal human prostatic epithelial cell line RWPE-1 strongly expressed SRD5A2, whereas the immortalized human prostatic epithelial cell line BPH-1 weakly expressed SRD5A2. miR-1199-5p expression was remarkably higher in BPH-1 than in RWPE-1 cells(P<0.001), and miR-1199-5p expression was significantly upregulated in BPH tissues with negative SRD5A2 expression than those with positive SRD5A2 expression. Transfection of miR-1199-5p mimics in RWPE-1 cells led to a marked decrease in SRD5A2 expression, whereas miR-1199-5p inhibitor increased SRD5A2 expression in BPH-1 cells. Dual-luciferase reporter assay showed that miR-1199-5p could bind the 3′untranslated region of SRD5A2 mRNA. miR-1199-5p also decreased the RWPE-1 sensibility to finasteride, an inhibitor of SRD5A2.ConclusionOur results show that SRD5A2 expression varies in BPH tissues and miR-1199-5p might be one of the several factors contributing to differential SRD5A2 expression in BPH patients.

Silencing effects of FOXD1 inhibit metastatic potentials of the PCa via N-cadherin – Wnt/β-catenin crosstalk

The elucidation of the mechanisms controlling the metastatic processes is important for the development of new treatment methods to prevent the progression of localized disease to metastasis. Forkhead box D1 (FOXD1) is a member of the FOX transcription factor family and has been reported to play an important role in the development and progression of various cancers. However, its role in prostate cancer (PCa) remains only partially understood. Therefore, we aimed to explore the effects on the associated regulatory signal pathway of FOXD1 in prostate cancer. To clarify the roles of FOXD1 in prostate cancer, we used siRNA to suppress its expression in 22Rv1 cells with relatively higher expression of FOXD1. The effects of FOXD1 silencing on cell proliferation, migration and invasion were determined. WST-1 assays were used to determine cell proliferation. Cell migration and invasion were evaluated through wound healing and transwell assays. The possible underlying mechanism of FOXD1 silencing on 22Rv1 was evaluated by determining the expression of proteins related to EMT and Wnt/β-catenin signaling pathway. Our results showed that FOXD1 was highly expressed in prostate cancer cell lines -PC-3, DU145, LNCaP and 22Rv1- compared to normal prostate epithelial cell line RWPE-1. Additionally, silencing of FOXD1 significantly reduced proliferation, migration and invasion of 22Rv1 cells. Furthermore, silencing of FOXD1 decreased the expression of β-catenin and cyclin D1, which are involved in the Wnt/β-catenin signaling pathway. However, it did not appear to affect the expression of EMT-related proteins other than N-cadherin. Our results suggest that silencing of FOXD1 suppresses metastatic potentials of the PCa via N-cadherin - Wnt/β-catenin crosstalk. Therefore, the expression status of FOXD1 may be a new prognostic factor as well as a potential therapeutic target in prostate cancer treatment.

Fibroblast activation protein-alpha knockdown suppresses prostate cancer cell invasion and proliferation.

Prostate cancer is one of the most common malignant tumors of the male genitourinary system. Fibroblast activation protein alpha (FAP-α) overexpression has been shown to occur in a wide range of tumors. However, the specific mechanism of FAP-α in the development of prostate cancer has not been reported. In this study, real-time quantitative PCR (qRT-PCR) was used to detect the relative expression of FAP-α mRNA in prostate cancer cell lines (PC-3, LNCaP, and DU145) and human normal prostate epithelial cell line RWPE-1. Small interfering RNA (siRNA) targeting FAP-α and vectors expressing exogenous FAP-α were transfected to prostate cancer cells (LNCaP and DU145) to investigate the function of FAP-α. BALB/c nude mice were injected with DU145 cells which were transfected with NC-siRNA, FAP-α-siRNA-1, or FAP-α-siRNA-2. Compared to adjacent normal tissues, FAP-α protein and mRNA levels in prostate cancer tissues increased significantly (P<0.05). Compared to patients with high FAP-α mRNA levels, patients with low FAP-α mRNA levels had a significantly higher survival rate (χ²=5.050, log-rank P=0.025). Overexpression of FAP-α in LNCaP cells markedly inhibited cell apoptosis, and promoted cell invasion and proliferation. In contrast, knockdown of FAP-α expression in DU145 cells can significantly reduce invasion, proliferation, and promote apoptosis in prostate cancer. Immunofluorescence assay further indicated that down-regulation of FAP-α could suppress the nuclear translocation of β-catenin. An in vivo study found that compared with the NC-siRNA group, the tumor weight and tumor volume in the FAP-α-siRNA-1 and FAP-α-siRNA-2 groups were significantly decreased. In conclusion, down-regulation of FAP-α can inhibit the invasion and proliferation of prostate cancer. Our study provides a theoretical basis for the targeted treatment of prostate cancer.

MiR‑367‑3p downregulates Rab23 expression and inhibits Hedgehog signaling resulting in the inhibition of the proliferation, migration, and invasion of prostate cancer cells.

MicroRNAs play an important role in tumor cell proliferation, invasion, and Rab23 is a member of the Ras-related small GTPase family and plays a critical role in the progression of may types of tumors. The present study was designed to investigate the inhibitory effect of microRNA (miR)-367-3p on the proliferation, invasion, and metastasis of prostate cancer cells. qRT-PCR was used to detect the expression of miR-367-3p in prostate cancer and adjacent tissues. Cell proliferation, scratch, and Transwell assays were performed to verify the inhibitory effect of miR-367-3p overexpression or Ras-related protein Rab 23 (Rab23) knockdown on prostate cancer. Double luciferase reporter assay was utilized to verify whether miR-367-3p could target the Rab23 3′-untranslated region (UTR). The expression levels of Rab23, Gli1, and Gli2 in prostate cancer cells transfected with the miR-367-3p mimic were detected via qRT-PCR analysis. miR-367-3p expression in the prostate cancer tissues was downregulated compared with that in the para-cancer control tissues. miR-367-3p expression in DU145 and PC3 cells was also downregulated compared with that in the human prostate epithelial cell line RWPE-1. The overexpression of miR-367-3p or the knockdown of Rab23 inhibited the proliferation, invasion, and metastasis of prostate cancer cells. The results of the luciferase reporter assay confirmed that Rab23 was a target gene that was regulated by miR-367-3p. miR-367-3p specifically bound to the 3′-UTR of Rab23 mRNA. The overexpression of miR-367-3p inhibited Rab23 expression and the Hedgehog pathway. Cell function experiments confirmed that the overexpression of Rab23 reversed the anticancer effect of miR-367-3p. miR-367-3p was able to inhibit the Hedgehog pathway by targeting the expression of the Rab23 gene, thus inhibiting the proliferation, invasion, and metastasis of prostate cancer cells.

Long non-coding RNA-NEAT1, a sponge for miR-98-5p, promotes expression of oncogene HMGA2 in prostate cancer.

Increasing evidence demonstrated that noncoding RNAs (lncRNA, miRNA) play important roles in the cancer development. LncRNA NEAT1 functions as an oncogene in many cancers. However, the roles of NEAT1 in prostate cancer (PCa) remain largely unknown. In the present study, we aim to explore the molecular mechanism of NEAT1 in the development of PCa. We detected the expression levels of NEAT1 in a total of 16 benign prostatic hyperplasia tissues (BPH), 30 matched adjacent healthy control (HC) tissues and 30 PCa tissues, as well as PCa cell lines PC-3, DU-145, LNCaP and normal prostate epithelial cell line RWPE-1. The results showed that NEAT1 was significantly up-regulated in PCa tissues and PCa cell lines. Knockdown of NEAT1 can largely inhibit DU-145 and PC-3 cell growth and invasion. Bioinformatics analysis predicted NEAT1 has the binding site of miR-98-5p which can bind to the 3′UTR of HMGA2. And the expression level of NEAT1 has a positive correlation with HMAG2, while negative correlation with miR-98-5p in PCa cells. In addition, luciference assay and RNA immunoprecipitation (RIP) assay confirmed that NEAT1 can function as a competing endogenous RNA (ceRNA) by sponging miR-98-5p to active HMGA2. Moreover, silencing of HMGA2 can decrease the proliferation ability of PCa cells. Taken together, NEAT1/miR-98-5p/HMGA2 pathway is involved in the development and progression of PCa. NEAT1 could be recommended as a prognostic biomarker and inhibition of NEAT1 expression may be a promising strategy for PCa therapy.

CID-6033590 inhibits p38MAPK pathway and induces S-phase cell cycle arrest and apoptosis in DU145 and PC-3 cells

Metastatic prostate cancer, with no effective treatment, is among the leading causes of cancer-associated deaths in men. Overexpression of p38αMAPK has been observed in neuroendocrine prostate cancer patients and in both DU145 and PC-3 cell lines and represents a good drug target. Sulfonamide derivatives have shown biological activities against many human diseases, including cancer. CID-6033590, a sulfonylhydrazide compound, screened from PubChem database by molecular docking with p38αMAPK, was evaluated for anti-cancerous activities. CID-6033590 induced toxicity in both DU145 and PC-3 cells in a concentration and time-dependent manner with an IC50 value of 60 μM and 66 μM, respectively. Sub-cytotoxic concentrations of the compound significantly induced S-phase cell cycle arrest, inhibited cyclinA/CDK2 complex and blocked cell proliferation. Further, CID-6033590 downregulated phosphorylation of p38MAPK (P-p38) as well as its downstream targets, Activating transcription factor 2 (ATF-2) and Heat shock protein 27 (Hsp27). The compound increased ROS and decreased mitochondrial membrane potential (Δψm), downregulated Bcl-2 and survivin and cleaved poly ADP ribose polymerase (PARP) and caspase-3, indicating the induction of apoptosis. The evaluaion of the compound on noncancerous, human prostatic epithelial cell line RWPE-1, and healthy murine tissues yielded no significant toxicity. Taken together, we suggest CID-6033590 as a potential candidate for prostate cancer therapy.

Identification of reciprocal microRNA-mRNA pairs associated with metastatic potential disparities in human prostate cancer cells and signaling pathway analysis.

The major cause of mortality for prostate cancer (PCa) is metastasis; however, the metastatic mechanism remains unclear. MicroRNAs (miRNAs) alter the expression patterns of essential genes through posttranscriptional regulation during cancer development. The study was mainly aimed at identifying specific miRNA-messenger RNA (mRNA) interactions and signaling pathways associated with PCa distant metastasis. New analytical approaches were applied, combining miRNA and gene expression microarray, to screen differentially expressed miRNA-mRNA pairs in the normal prostate epithelial cell line RWPE-1, the highly-metastatic human PCa cell line PC-3M-1E8 (H-1E8 or 1E8) and the lowly metastatic cell line PC-3M-2B4 (L-2B4 or 2B4). Eight differentially expressed candidate miRNAs and their targets closely related to PCa metastasis were identified and validated in patients by using the Gene Expression Omnibus database. Among them, overexpression of hsa-miR-92b-3p and hsa-let-7a-5p and underexpression of their targets, such as glutathione-S-transferase M3 (GSTM3), baculoviral IAP repeat-containing 3, and cyclin-dependent kinase inhibitor 1 (CDKN1A), were also validated in H-1E8 cells compared with L-2B4 cells. Bioinformatics suggested that hsa-miR-92b-3p and hsa-let-7a-5p and their targets might promote PCa metastasis through platinum-based drug resistance and the JAK-STAT signaling pathway. H-1E8 and L-2B4 cells treated by cisplatin showed the greatly decreased levels of hsa-miR-92b-3p and hsa-let-7a-5p; however, in contrast to 2B4 cells, 1E8 cells did not negatively regulate the increase in the expression levels of the targets GSTM3 and CDKN1A. This finding suggests that the dysregulation between hsa-let-7a-5p/CDKN1A and hsa-miR-92b-3p/GSTM3 pairs is associated with platinum-based chemoresistance of metastatic cancer cells.

LncRNA UCA1 acts as a sponge of miR-204 to up-regulate CXCR4 expression and promote prostate cancer progression.

Prostate cancer (PCa) is a devastating malignant disease with a poor prognosis. The aim of current study is to investigate the role of lncRNA-urothelial carcinoma associated 1 (UCA1) in the progression of PCa. We evaluated the expression levels of UCA1 in a total of 16 benign prostatic hyperplasia tissues (BPH) and 40 PCa tissues, as well as PCa cells. The functional regulatory effects of UCA1 were investigated using a series of cell function approaches. Our data showed that UCA1 is frequently overexpressed in PCa tissues compared with BPH tissues (P<0.01). Moreover, the higher expression of UCA1 was observed in patients with Gleason score ≥8 (P<0.05). In consistent, we found the expression levels of UCA1 was higher in the PCa cell lines PC-3, LnCaP, and DU-145 than in the normal prostate epithelial cell line RWPE-1 (P<0.01). Functionally, we found knockdown of UCA1 in PC-3 significantly suppressed cell growth and invasion of PC-3, while overexpression of UCA1 in DU-145 cells promote cell growth and invasion. Mechanistically, UCA1 overexpression permitted activation of CXCR4 oncogenes through inhibition of miR-204 activity, as evidenced by the positive association of these two genes with UCA1 levels and inverse correlation with miR-204 expression in PCa tissues. Luciferase activity assay further confirmed the targetting relationship between UCA1 and miR-204, CXCR4, and miR-204. The up-regulation of UCA1 in PC-3 cells significantly impaired the inhibitory effect of miR-204 on CXCR4 expression. Taken together, our research revealed that UCA1 works as an oncogene by targetting miR-204. The UCA1-miR-204-CXCR4 regulatory network regulated the growth and metastasis of PCa, providing new insight in the management of patients with such malignancy.

Overexpression of lncRNA GAS5 suppresses prostatic epithelial cell proliferation by regulating COX-2 in chronic non-bacterial prostatitis

ABSTRACT Chronic non-bacterial prostatitis (CNP) is a common urologic disease that is linked to the development of prostate cancer. Long non-coding RNA (lncRNA) GAS5 has been identified to mediate cell proliferation in prostate cancer, although its role in CNP is still unclear. Human prostate epithelial cell line RWPE-1 was induced by lipopolysaccharide (LPS) to mimic CNP model in vitro. Real-time PCR was performed to determine the expression of GAS5 and COX-2, while western blotting was used to evaluate the protein expression of COX-2. The interaction between GAS5 and COX-2 was determined using RNA pull-down and RNA immunoprecipitation (RIP). Cell proliferation was determined using MTT assay. The expression of GAS5 was decreased, while COX-2 was increased in prostatitis tissues and in LPS-induced RWPE-1 cells. The overexpression of GAS5 suppressed the protein level of COX-2, and inhibited cell proliferation of LPS-induced RWPE-1 cells and HPECs, which was rescued by the co-transfection with pcDNA-GAS5 and pcDNA-COX-2. GAS5 was confirmed to promote the ubiquitination of COX-2, and the in vivo GAS5-overexpressed CNP rat model decreased the motor scores, the volume of prostate tissues, the average number of inflammatory cells, prostatic proliferation, and COX-2 expression. Our findings revealed that overexpression of GAS5 inhibited cell proliferation via negatively regulating the expression of COX-2, thus alleviating the progression of CNP.

Abi1 loss drives prostate tumorigenesis through activation of EMT and noncanonical WNT signaling.

280 Background: Prostate cancer is characterized by heterogeneity of mechanisms which are poorly understood but pointing to epithelial plasticity as the key mechanism in progression to metastatic disease. ABI1, a member of WAVE complex and actin cytoskeleton regulator and adaptor protein, is proposed to act as tumor suppressor in prostate cancer, but the mechanism of tumor progression due to Abi1 loss is not clear. Methods: To address Abi1’s role in prostate cancer we used CRISPR-based gene editing and retroviral expression to manipulate Abi1 levels in prostate cancer cell lines. Levels of Abi1 expression in prostate organoid tumor cell lines were evaluated by Western blotting and/or RNA sequencing. Association of Abi1 loss with tumor grade was evaluated by immunohistochemistry. Results: Abi1 expression is downregulated in tumor organoid cell lines from metastatic bone and lymph node biopsies. Moreover, low Abi1 expression is associated with high-grade prostate tumors (GG3 or higher, p &lt; 0.001). Disruption of Abi1 gene in a benign prostate epithelial cell line RWPE-1 resulted in a gain of invasive phenotype, which is characterized by loss of cell-cell adhesion markers and increased migratory ability of RWPE-1 Abi1 KO spheroids. Through RNA sequencing and protein expression analysis we discovered that Abi1 loss leads to activation of non-canonical WNT signaling and EMT pathways, which are rescued by re-expression of Abi1. Furthermore, increase in STAT3 phosphorylation upon Abi1 inactivation and evidence for high affinity interaction of FYN-SH2 domain with Abi1 pY421 support the model that Abi1 acts as a gatekeeper of the non-canonical WNT-EMT pathway activation downstream from FZD2 receptor. The gene expression profile of Abi1-EMT-WNT pathway overlaps with the reported gene signature of high-risk prostate tumors. Conclusions: Abi1 contributes to prostate cancer progression and epithelial plasticity through regulation of EMT-WNT pathways. Understanding of Abi1’s role may provide more mechanistic understanding of prostate cancer tumor progression.

PARP inhibitor veliparib and HDAC inhibitor SAHA synergistically co-target the UHRF1/BRCA1 DNA damage repair complex in prostate cancer cells

BackgroundThe poly ADP ribose polymerase (PARP) inhibitor olaparib has been approved for treating prostate cancer (PCa) with BRCA mutations, and veliparib, another PARP inhibitor, is being tested in clinical trials. However, veliparib only showed a moderate anticancer effect, and combination therapy is required for PCa patients. Histone deacetylase (HDAC) inhibitors have been tested to improve the anticancer efficacy of PARP inhibitors for PCa cells, but the exact mechanisms are still elusive.MethodsSeveral types of PCa cells and prostate epithelial cell line RWPE-1 were treated with veliparib or SAHA alone or in combination. Cell viability or clonogenicity was tested with violet crystal assay; cell apoptosis was detected with Annexin V-FITC/PI staining and flow cytometry, and the cleaved PARP was tested with western blot; DNA damage was evaluated by staining the cells with γH2AX antibody, and the DNA damage foci were observed with a fluorescent microscopy, and the level of γH2AX was tested with western blot; the protein levels of UHRF1 and BRCA1 were measured with western blot or cell immunofluorescent staining, and the interaction of UHRF1 and BRCA1 proteins was detected with co-immunoprecipitation when cells were treated with drugs. The antitumor effect of combinational therapy was validated in DU145 xenograft models.ResultsPCa cells showed different sensitivity to veliparib or SAHA. Co-administration of both drugs synergistically decreased cell viability and clonogenicity, and synergistically induced cell apoptosis and DNA damage, while had no detectable toxicity to normal prostate epithelial cells. Mechanistically, veliparib or SAHA alone reduced BRCA1 or UHRF1 protein levels, co-treatment with veliparib and SAHA synergistically reduced BRCA1 protein levels by targeting the UHRF1/BRCA1 protein complex, the depletion of UHRF1 resulted in the degradation of BRCA1 protein, while the elevation of UHRF1 impaired co-treatment-reduced BRCA1 protein levels. Co-administration of both drugs synergistically decreased the growth of xenografts.ConclusionsOur studies revealed that the synergistic lethality of HDAC and PARP inhibitors resulted from promoting DNA damage and inhibiting HR DNA damage repair pathways, in particular targeting the UHRF1/BRCA1 protein complex. The synergistic lethality of veliparib and SAHA shows great potential for future PCa clinical trials.

Vanillic acid attenuates testosterone-induced benign prostatic hyperplasia in rats and inhibits proliferation of prostatic epithelial cells.

Benign prostatic hyperplasia (BPH) is a common disease in the male population, especially in elderly men. Vanillic acid (VA), a dihydroxybenzoic derivative used as a flavoring agent, is reported to have an anti-inflammatory effect. However, there are no reports of its effects on BPH to date. BPH was induced with a pre-4-week treatment of daily subcutaneous injections of testosterone propionate (TP), and the normal control group received injections of ethanol with corn oil instead. Six weeks of further injections were done with (a) ethanol with corn oil, (b) TP only, (c) TP + finasteride, and (d) TP + VA. Finasteride was used as a positive control group. VA had protective effects on the TP-induced BPH. In the VA treatment group, the prostate weight was reduced, and the histological changes including the epithelial thickness and lumen area were restored like in the normal control group. Furthermore, in the VA treatment group, two proliferation related factors, high molecular weight cytokeratin 34βE12 and α smooth muscle actin, were significantly down-regulated compared to the TP-induced BPH group. The expressions of dihydrotestosterone and 5α-reductase, the most crucial factors in BPH development, were suppressed by VA treatment. Expressions of the androgen receptor, estrogen receptor α and steroid receptor coactivator 1 were also significantly inhibited by VA compared to the TP-induced BPH group. In addition, we established an in vitro model for BPH by treating a normal human prostatic epithelial cell line RWPE-1 with TP. VA successfully inhibited proliferation and BPH-related factors in a concentration-dependent manner in this newly established model. These results suggest a new and potential pharmaceutical therapy of VA in the treatment of BPH.

Oncolytic alphavirus SFV-VA7 efficiently eradicates subcutaneous and orthotopic human prostate tumours in mice

Background:Despite recent therapeutic and diagnostic advances, prostate cancer remains the second leading cause of cancer-related deaths among men in the Western world. Oncolytic viruses that replicate selectively in tumour cells represent a novel treatment candidate for these malignancies.Methods:We analysed infectivity of avirulent Semliki Firest virus SFV-VA7 in human prostate cancer cell lines VCaP, LNCaP and 22Rv1 and in nonmalignant prostate epithelial cell line RWPE-1. Therapeutic potency of SFV-VA7 was evaluated in subcutaneous and orthotopic mouse LNCaP xenograft models.Results:SFV-VA7 infected and killed the tested human prostate cancer cell lines irrespective of their hormone response status, while the nonmalignant prostate epithelial cell line RWPE-1 proved highly virus resistant. Notably, a single peritoneal dose of SFV-VA7 was sufficient to eradicate all subcutaneous and orthotopic LNCaP tumours.Conclusions:Our results indicate that SFV-VA7 is a novel, promising therapeutic virus against prostate cancer warranting further testing in early clinical trials.

Notch-4 silencing inhibits prostate cancer growth and EMT via the NF-κB pathway.

Epithelial-mesenchymal transition (EMT) is implicated in the metastasis of human prostate cancer (PCa). Notch signaling has been established as a regulator of EMT. Notch-4 has emerged as a mammary proto-oncogene and a target in several cancers. However, the role and the mechanism of action of Notch-4 in PCa are still unclear. In the present study, we first observed a marked increase in Notch-4 expression in the PCa cell lines DU145, PC3 and LnCAP compared with the non-malignant prostate epithelial cell line RWPE1. Knocking down the expression of Notch-4 suppressed the viability and proliferation in the PCa cell lines DU145 and PC3. Also, further study showed that a decline in Notch-4 significantly promoted apoptosis in PC3 cells. Notch-4 silencing also resulted in decreased cell migration and invasion and affected the expression of EMT markers. We hypothesized that Notch-4 ablation suppresses the activity of NF-κB, so we used PMA to stimulate NF-κB p50 and p65 activation in PC3 cells. The results indicate that PMA treatment impaired the action of Notch-4 ablation in the biology of PC3 cells including cell growth, apoptosis, migration, invasion and EMT. The results of the present study show that RNAi targeting against Notch-4 expression suppresses PCa progression.