RWPE-1 Cells Research Articles

Abstract 269: Reactivation of mutant p53 in human prostate cancer cells as a critical mechanism for inhibition of tumor growth by phenethyl isothiocyanate

Abstract Mutations in the p53 gene occur in a wide variety of human cancers at remarkably high frequencies. Prostate cancer (PCa), the most commonly diagnosed cancer and the second leading cause of cancer-related death in men in the United States, show a significantly high frequency of p53 mutations (~30%-70%). However, the role of mutant p53 as a target for the treatment or prevention of PCa remained to be investigated. In this study, we showed that cruciferous vegetable-derived phenethyl isothiocyanate (PEITC) can reactivate p53 mutants, irrespective of its site of mutation, in PCa cells in vitro. Importantly, this study showed that dietary supplementation of PEITC can significantly inhibit DU145 p53 mutant xenograft tumor in a nu/nu Balb/c mice. Interestingly, the animals in the PEITC-treated group showed a significant decrease in the mutant p53 expression levels as compared to the animals in the control group. DU145 cells harboring p53V274F/P223L mutant display maximum sensitivity to PEITC-induced growth inhibition as compared to the wild-type p53 LNCaP and p53 null PC-3 cells. PEITC treatment also caused a significant decrease in the p53 expression level in DU145 cells. No significant inhibition of cell proliferation was detected for the normal prostate epithelial RWPE1 cells treated with PEITC. Mechanistic studies revealed that PEITC induces a conformation change and restores the transactivation functions to the p53 mutant. Accordingly, in PEITC-treated cells the reactivated p53 mutant(s) induces apoptosis by activating canonical wild-type p53 targets, inducing a G1 and S phase delay and by phosphorylating ATM. PEITC treatment of isogenic PC-3 p53 null cells revealed that PC-3 cells expressing p53P223L mutant exhibit maximum inhibition of cell proliferation, suggesting that PEITC acts in a p53P223L mutant-dependent manner. Furthermore, PEITC treatment restores the transactivation functions to p53P223L mutant as shown by the activation of p53 regulated downstream target genes. Interestingly p73, a member of the p53 gene family that shares high functional homology and regulate cell-growth/ cell-death pathways in a manner similar to p53, does not play a role in p53-mediated pathway activation in PEITC treated cells. We further showed that PEITC inhibits cell proliferation and induces apoptosis in prostate cancer cells with hotspot mutants R248W (VCaP) and R175H (LAPC-4). Importantly, PEITC can induce a conformation change, reactivates p53 mutants and induces G1 and S-phase delays in these cells. These results demonstrate that PEITC can reactivate mutant p53, irrespective of the site of p53 mutation, in human prostate cancer cells. These studies provide the first example of targeting mutant p53 in prostate cancer cells by a dietary-related compound that may have impact on the prevention and treatment strategies of this disease. Citation Format: Monika Aggarwal, Rahul Saxena, Nasir Asif, Elizabeth Sinclair, Judy Tan, Idalia Cruz, Deborah Berry, Bhaskar Kallakury, Quynhchi Pham, Thomas Ty Wang, Fung-Lung Chung. Reactivation of mutant p53 in human prostate cancer cells as a critical mechanism for inhibition of tumor growth by phenethyl isothiocyanate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 269.

Abstract LB-358: Abnormal O-glycosylation promotes prostate cancer stemness and mediates metastatic niche

Abstract Oncofetal Galβ1-3GalNAc O-glycan, T antigen, is increased in about 60% carcinomas of breast, colon, stomach, and prostate. Notably, coexpression of T antigen with CD44 or CD133, markers of cancer stem cells, has been identified in lung, breast, and liver cancers. Using spontaneous metastasis model of 22Rv1 cells, we derived high metastatic 22Rv1-M4 cells and found altered O-glycosylation in the 22Rv1-M4 cells and also 22Rv1-M4-derived host microenvironment compared to parental cells. Our study indicated that high expressions of T antigen synthase C1GALT1 are associated with metastatic PCa and poor survival, probably contributed by the interaction of galectin-4 and glycan. Galectin-4 engaged with MYC-dependent C1GALT1 mediated O-glycan and resulted in RTK phosphorylation; activation of RTKs enhanced not only androgen-independent growth but also metastatic colonization which mediated by ERK-AP1-mediated SOX9 expression. Downregulation of SOX9 significantly inhibited the tumorsphere formation and in vivo metastasis. Moreover, coexpression of SOX9 and MYC in immortalized prostate epithelial cells RWPE1 generates the heterogeneous population presenting both epithelial type and cancer-like cells in mouse renal capsule. On the other hand, analyses of lung tissues from mice bearing tumor of 22Rv1-M4 cells relative to that bearing tumor of 22Rv1 parental cells exhibit the upregulated O-glycosylation enzymes, which associated with increased plant Jacalin lectin staining, CD45+ cells infiltration, and endothelial permeability indicated by dextran leakage. Tail-vein injection of 22Rv1-M4 cells exhibits aggressive systemic metastasis in a mouse conditioned by subcutaneous 22Rv1-M4 tumor compared to that in a mouse conditioned by subcutaneous 22Rv1 tumor These data suggest high metastatic prostate cancer cells adapt the lung microenvironment for seeding and colonization of cancer cells. Citation Format: Chin-Hsien Tsai, Sheue-Fen Tzeng. Abnormal O-glycosylation promotes prostate cancer stemness and mediates metastatic niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-358.

Basement membrane extract attenuates the more malignant gene expression profile accentuated by fibronectin in prostate cancer cells.

Prostate cancer (PCa) has high mortality rates, with most of the deaths resulting from the development of metastasis. Fibronectin (FN) plays key roles in cell adhesion and affects the migratory behavior of cells. In the tumor microenvironment and also in the blood plasma during metastasis, FN displays increased expression, however its role in prostate cancer remains poorly understood. This study aimed to unveil the specific roles of FN as a soluble component, alone or in combination with a complex basement membrane. To investigate the impact of FN in neoplastic prostate cells, we evaluated the gene expression of LNCaP cells by RT-qPCR after exposure to soluble FN (25µg/mL) either alone or in combination with a basement membrane. When FN was the predominant matrix element, such as in blood plasma, PCa tumor cells increased their expression of genes related to an invasive behavior and resistance to apoptosis, including CDH2, ITGA5, AKT1, and BCL2. However, the combined presence of FN and a complex basement membrane had the opposite effect on LNCaP cells, in which the expression levels of CDH2, ITGA5, AKT1, and BCL2 were reduced. Hierarchical clustering analysis with LNCaP and RWPE-1 cells showed that LNCaP cells exposed to an enriched extracellular matrix displayed an expression pattern more similar to that shown by RWPE-1 cells, a cell line that illustrates characteristics of the normal prostate epithelium. These findings provide the groundwork for future studies addressing the role of FN in tumor growth, particularly in the context of cancer evolution/progression from a solid primary tumor to a transitory circulating state.

Extracellular vesicles with altered tetraspanin CD9 and CD151 levels confer increased prostate cell motility and invasion

To facilitate intercellular communication, cells release nano-sized, extracellular vesicles (EVs) to transfer biological cargo to both local and distant sites. EVs are enriched in tetraspanins, two of which (CD9 and CD151) have altered expression patterns in many solid tumours, including prostate cancer, as they advance toward metastasis. We aimed to determine whether EVs from prostate cells with altered CD9 and CD151 expression could influence cellular behaviour and increase the metastatic capabilities of non-tumourigenic prostate cells. EVs were isolated by ultrafiltration and characterised for their tetraspanin expression and size distribution. iTRAQ was used to identify differences between RWPE1 and tetraspanin-modified RWPE1 EV proteomes, showing an enrichment in protein degradation pathways. Addition of EVs from RWPE1 cells with reduced CD9 or increased CD151 abundance resulted in increased invasion of RWPE1 cells, and increased migration in the case of high CD151 abundance. We have been able to show that alteration of CD9 and CD151 on prostate cells alters the proteome of their resultant EVs, and that these EVs can enhance the migratory and invasive capabilities of a non-tumourigenic prostate cellular population. This work suggests that cellular tetraspanin levels can alter EVs, potentially acting as a driver of metastasis in prostate cancer.

Multiple Tolerization Reduces Antibody Binding Against Tolerogen Cells: Implications for the Production of Monoclonal Antibodies.

We report an immunization technique that can update the production of monoclonal antibodies (mAbs): the multiple tolerization subtractive immunization (MTSI). A total of 10 BALB/C mice were used. Animals in group 1 received one inoculation of RWPE-1 cells (nontumoral), followed by cyclophosphamide, and then received serial inoculations of nonirradiated PC3 cells (tumoral). Animals in group 2 received our MTSI protocol, as follows: one inoculation of RWPE-1 cells, followed by cyclophosphamide (Cy). This whole tolerization step was repeated three other times, with 14-day intervals between the last Cy exposure and the next RWPE-1 cell inoculation. Finally, the animals received the same nonirradiated PC3 cell exposure as group 1. Blood was taken from each animal, and their polyclonal sera individually tested against the nontumoral RWPE-1 cells in flow cytometry. We found out that, after the MTSI was employed, the serum of the immunized animals, in group 2, contained considerably less antibodies that reacted against the tolerogenic cells, compared with the serum of the animals that underwent regular subtractive immunization. We showed that, by repeating the tolerization cycles, the polyclonal antibodies produced by mice have a reduced specificity toward common/immunodominant epitopes present at nontumoral cells, and thus this technique can be readily used by others in studies involving murine mAb protocols.

MiR-17 Regulates Prostate Cancer Cell Proliferation and Apoptosis Through Inhibiting JAK-STAT3 Signaling Pathway.

STAT3 is an important protein in Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway that facilitates B-cell lymphoma 2 (Bcl-2) expression. MiR-17 was found to be significantly reduced in prostate cancer tissues and cells, suggesting that it might be a tumor suppressor in prostate cancer tumorigenesis. Bioinformatics analysis showed the complementary binding site between miR-17 and STAT3. This study aimed to investigate the role of miR-17 in regulating JAK-STAT signaling pathway, as well as prostate cancer cell proliferation and apoptosis. Dual luciferase assay was used to verify the targeted relationship between miR-155 and STAT3. LNCaP cells were cultured in vitro and divided into four groups, including mimic NC, miR-17 mimic, si-NC, and si-STAT3 groups. STAT3, p-STAT3, and Bcl-2 expressions were tested by western blot. Cell apoptosis was detected by flow cytometry. Cell proliferation was assessed by EdU staining. MiR-17 mimic transfection significantly reduced the relative luciferase activity in HEK293T cells. MiR-17 targeted regulated STAT3 expression. MiR-17 expression and cell apoptosis were obviously declined, while STAT3 level and cell proliferation markedly were elevated in LNCaP cells compared with RWPE-1 cells. MiR-17 mimic and/or si-STAT3 transfection significantly downregulated the expression of STAT3, p-STAT3, and Bcl-2, attenuated cell proliferation, and enhanced cell apoptosis in LNCaP cells. Upregulation of miR-17 inhibited LNCaP cell proliferation and induced cell apoptosis by downregulating the expression of STAT3, p-STAT3, and Bcl-2.

Suppression of miR‐186 Attenuates Cellular Invasion, pAKT and β‐catenin in Metastatic Prostate Cancer Cell Models

BackgroundMicroRNA (miR) dysregulation alters the expression of cancer related genes and contributes to disease states in many cancers. For example, ectopic miR‐186 expression leads to enhanced cell proliferation and migration in pancreatic cancer. However, the role of miR‐186 in prostate cancer (PCa) remains unclear. Previously, we observed significant upregulation of miR‐186‐5p in PCa patient serum (stage III/IV) and PCa cell lines relative to appropriate controls. Moreover, inhibition of miR‐186 reduced cell proliferation and anchorage‐independent growth of PC‐3 PCa cells. However, the impact of miR‐186 inhibition on cell invasion and its corresponding mechanism is unknown.HypothesisWe speculated miR‐186 inhibition would repress cellular invasion in metastatic PCa cell lines, presumably by up‐regulating tumor suppressor gene(s) involved in at least one hallmark of cancer.MethodsAssessment of the impact of miR‐186 inhibition or overexpression on selected targets entailed microarray analysis, qRT‐PCR, and/or western blots. Candidate miR‐186 targets were selected using published reports on ‘miR‐186 and cancer’, availability of robust antibodies, and statistical filtering (false discovery ≥0.05 and ±1.2 fold change). Statistical evaluation used the modified t‐test, and ANOVA analysis.ResultsMir‐186 inhibition in PC‐3 cells significantly repressed proliferation and colony formation by 34–64% in PC3 and/or MDA‐PCA‐2b cells. Following miR‐186 inhibition in PC‐3 cells and overexpression in RWPE1 cells, 1,040 identified mRNA targets were differentially expressed compared to scramble controls (p < 0.05). The candidate miR‐186 target gene list focused on 11 up‐regulated candidates. One of the up‐regulated genes, AKAP12, plays a role in cell death, cell migration, cell proliferation, colony formation, and cell invasion. AKAP12 also possessed three miR‐186 binding sites. Notably, AKAP12 was upregulated in PC‐3 and MDA‐PCa‐2b cells transfected with a miR‐186 inhibitor. Conversely, ectopic miR‐186‐5p expression in HEK 293T cells decreased AKAP12 expression by 30%. Both pAKT and β‐catenin were down‐regulated in miR‐186 inhibited PCa cells. Conversely, overexpressing miR‐186‐5p in normal prostate epithelial RWPE1 cells increased pAKT expression by 30%.ConclusionsOur findings suggest miR‐186 plays an oncogenic role in PCa. The reduction in cell invasion in miR‐186 inhibited cell models corresponded with an up‐regulation of AKAP12, which in turn may reduce p‐AKT and β‐catenin. Repression of p‐AKT and β‐catenin favors a less invasive PCa phenotype. Future studies will further elucidate the mechanism by which miR‐186 inhibition suppresses cellular proliferation, migration, and invasion. Such efforts may lead to the identification of novel biomarkers to improve detection and clinical management strategies for aggressive PCa.Support or Funding InformationNational Cancer Institute Grant R25‐CA‐134283, Our Highest Potential Endowment in Cancer ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Androgen receptor modulation following combination exposure to brominated flame-retardants

Endocrine disrupting compounds can interfere with androgen receptor (AR) signaling and disrupt steroidogenesis leading to reproductive failure. The brominated flame-retardant (BFR) 1, 2-dibromo-4-(1, 2-dibromoethyl) cyclohexane (TBECH), is an agonist to human, chicken and zebrafish AR. Recently another group of alternative BFRs, allyl 2, 4, 6-tribromophenyl ether (ATE), and 2, 3-dibromopropyl 2, 4, 6-tribromophenyl ether (DPTE) along with its metabolite 2-bromoallyl 2, 4, 6-tribromophenyl ether (BATE) were identified as potent human AR antagonists. These alternative BFRs are present in the environment. The aim of the present study was to determine the effect of mixed exposures to the AR agonist and the AR antagonists at environmentally relevant concentrations. In vitro reporter luciferase assay showed that the AR antagonists, when present at concentration higher than TBECH, were able to inhibit TBECH-mediated AR activity. These AR antagonists also promoted AR nuclear translocation. In vitro gene expression analysis in the non-tumorigenic human prostate epithelial cell RWPE1 showed that TBECH induced AR target genes whereas DPTE repressed these genes. Further analysis of steroidogenic genes showed that TBECH up-regulated most of the genes while DPTE down-regulated the same genes. The results indicate that when TBECH and DPTE are present together they will antagonize each other, thereby reducing their individual effects.

TGFβ-induced epithelial-to-mesenchymal transition in prostate cancer cells is mediated via TRPM7 expression.

Growth factors, such as the transforming growth factor beta (TGFβ), play an important role in promoting metastasis of prostate cancer, thus understanding how TGFβ could induce prostate cancer cell migration may enable us to develop targeted strategies for treatment of advanced metastatic prostate cancer. To more clearly define the mechanism(s) involved in prostate cancer cell migration, we undertook a series of studies utilizing non‐malignant prostate epithelial cells RWPE1 and prostate cancer DU145 and PC3 cells. Our studies show that increased cell migration was observed in prostate cancer cells, which was mediated through epithelial‐to‐mesenchymal transition (EMT). Importantly, addition of Mg2+, but not Ca2+, increased cell migration. Furthermore, TRPM7 expression, which functions as an Mg2+ influx channel, was also increased in prostate cancer cells. Inhibition of TRPM7 currents by 2‐APB, significantly blocked cell migration in both DU145 and PC3 cells. Addition of growth factor TGFβ showed a further increase in cell migration, which was again blocked by the addition of 2‐APB. Importantly, TGFβ addition also significantly increased TRPM7 expression and function, and silencing of TRPM7 negated TGFβ‐induced cell migration along with a decrease in EMT markers showing loss of cell adhesion. Furthermore, resveratrol, which decreases prostate cancer cell migration, inhibited TRPM7 expression and function including TGFβ‐induced cell migration and activation of TRPM7 function. Together, these results suggest that Mg2+ influx via TRPM7 promotes cell migration by inducing EMT in prostate cancer cells and resveratrol negatively modulates TRPM7 function thereby inhibiting prostate cancer metastasis.

High glucose induces the proliferation of prostatic cells via downregulating MRE11.

The aim of the present study was to investigate the candidate genes and pathways associated with benign prostatic hyperplasia (BPH) and diabetes. In vitro experiments were performed using normal prostatic epithelial RWPE-1 and HPr-1 cells. The cell lines were treated with a high-glucose solution and MTS and bromodeoxyuridine assays were used to assess cell viability. Transcriptome sequencing was used to screen the candidate genes. The expression of candidate genes was further verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. A meiotic recombination 11 (MRE11) overexpression vector was designed and transfected into RWPE-1 cells to verify the function of MRE11. A streptozotocin-induced diabetic rat model was established and rat MRE11 levels were determined by RT-qPCR and immunohistochemical staining. High concentrations of glucose resulted in RWPE-1 and HPr-1 cells with high viability. Transcriptome sequencing revealed that MRE11 was downregulated when RWPE-1 cells were exposed to high-glucose conditions. When MRE11 was overexpressed, cell viability decreased and cell apoptosis was induced under high-glucose conditions. Prostatic tissues from rats were collected and assessed; MRE11 expression was observed to be decreased, which was consistent with the in vitro cell experiments. BPH may be associated with diabetes, as MRE11 expression in prostatic cells was decreased when exposed to high-glucose conditions. Therefore, MRE11 may have potential as a biomarker for the early diagnosis of BPH and diabetes.

Androgen receptor mediated epigenetic regulation of CRISP3 promoter in prostate cancer cells

Cysteine-rich secretory protein 3 (CRISP3) is one of the most upregulated genes in prostate cancer. Androgen receptor (AR) plays an important role not only in initial stages of prostate cancer development but also in the advanced stage of castration-resistant prostate cancer (CRPC). Role of AR in regulation of CRISP3 expression is not yet known. In order to understand the regulation of CRISP3 expression, various overlapping fragments of CRISP3 promoter were cloned in pGL3 luciferase reporter vector. All constructs were transiently and stably transfected in PC3 (CRISP3 negative) and LNCaP (CRISP3 positive) cell lines and promoter activity was measured by luciferase assay. Promoter activity of LNCaP stable clones was significantly higher than PC3 stable clones. Further in CRISP3 negative PC3 and RWPE-1 cells, CRISP3 promoter was shown to be silenced by histone deacetylation. Treatment of LNCaP cells with DHT resulted in increase in levels of CRISP3 transcript and protein. AR dependency of CRISP3 promoter was also evaluated in LNCaP stable clones by luciferase assay. To provide molecular evidence of epigenetic regulation of CRISP3 promoter and its response to DHT, ChIP PCR was performed in PC3 and LNCaP cells. Our results demonstrate that CRISP3 expression in prostate cancer cells is androgen dependent and in AR positive cells, CRISP3 promoter is epigenetically regulated by AR.

MicroRNA-1180 is associated with growth and apoptosis in prostate cancer via TNF receptor associated factor 1 expression regulation and nuclear factor-κB signaling pathway activation

In the present study, the aim was to investigate the role of microRNA-1180 (miR-1180) in the growth and apoptosis of prostate cancer, as well as to identify its direct targets. Initially, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to examine the expression of miR-1180 in the prostate cancer tissues and adjacent normal prostate tissues of 30 patients, as well as in DU145 and RWPE-1 cells. Next, DU145 cells were transfected with miR-1180 mimics, and the expression levels of associated proteins were determined by western blot assay. In addition, the role of miR-1180 in the proliferation, apoptosis, invasion and migration of DU145 cells was investigated by MTT, flow cytometry, cell invasion and wound healing assays, respectively. A dual-luciferase reporter assay was also performed to examine whether TNF receptor associated factor 1 (TRAF1) and B-cell lymphoma-2-associated athanogene 2 (BAG2) are direct targets of miR-1180. It was observed that miR-1180 expression was significantly decreased in the prostate cancer tissues compared with the normal prostate tissues, and was also inhibited in DU145 cells compared with RWPE-1 cells. Furthermore, transient overexpression of miR-1180 inhibited the proliferation, migration and invasion, and promoted the apoptosis of DU145 cells, as well as alleviated expression of associated proteins. The dual-luciferase reporter assay confirmed that TRAF1 and BAG2 are direct targets of miR-1180. These results suggested that miR-1180 contributed to prostate cancer by targeting TRAF1/BAG2 and by nuclear factor-κB signaling pathway activation.

Differential role of PTEN in transforming growth factor β (TGF-β) effects on proliferation and migration in prostate cancer cells.

Transforming growth factor-β (TGF-β) acts as a tumor suppressor in normal epithelial cells but as a tumor promoter in advanced prostate cancer cells. PI3-kinase pathway mediates TGF-β effects on prostate cancer cell migration and invasion. PTEN inhibits PI3-kinase pathway and is frequently mutated in prostate cancers. We investigated possible role(s) of PTEN in TGF-β effects on proliferation and migration in prostate cancer cells. Expression of PTEN mRNA and proteins were determined using RT-PCR and Western blotting in RWPE1 and DU145 cells. We also studied the role of PTEN in TGF-β effects on cell proliferation and migration in DU145 cells after transient silencing of endogenous PTEN. Conversely, we determined the role of PTEN in cell proliferation and migration after over-expression of PTEN in PC3 cells which lack endogenous PTEN. TGF-β1 and TGF-β3 had no effect on PTEN mRNA levels but both isoforms increased PTEN protein levels in DU145 and RWPE1 cells indicating that PTEN may mediate TGF-β effects on cell proliferation. Knockdown of PTEN in DU145 cells resulted in significant increase in cell proliferation which was not affected by TGF-β isoforms. PTEN overexpression in PC3 cells inhibited cell proliferation. Knockdown of endogenous PTEN enhanced cell migration in DU145 cells, whereas PTEN overexpression reduced migration in PC3 cells and reduced phosphorylation of AKT in response to TGF-β. We conclude that PTEN plays a role in inhibitory effects of TGF-β on cell proliferation whereas its absence may enhance TGF-β effects on activation of PI3-kinase pathway and cell migration.

The influence of endotoxin tolerance on the expression of IL-6 and IL-8 in normal human prostate epithelial cells

Objective Current studies have shown that tissue inflammation plays an important role in the course of development of benign prostate hyperplasia (BPH). Endotoxin tolerance (ET) was found to exist in human tissue cells, such as intestinal epithelial cells and human gingival epithelial cells, and could affect the tissue inflammation and immune responses. Lipopolysaccharide (LPS) generated by G-bacteria such as Escherichia coli, which was considered as the most important pathogen of tissue inflammation of prostate. We intended to analyze the inflammatory cytokines interleukin -6 (IL-6) and interleukin -8 (IL-8) expression of normal human prostate epithelial cells RWPE-1 which were stimulated by LPS repeatedly to find out whether ET exist in human prostate epithelial cells as well. Methods RWPE-1 cells were given stimulation of 1 μg/mL LPS which came from Escherichia coli for 24 hours.After collected the culture supernatant of REWP-1, 1 μg/mL LPS group stimulation was given again to form a model of ET. Expression level of cytokines IL-6 and IL-8 was detected by Millipore human cytokine detection kit instructions, in Luminex platform. Results Expression of IL-6 and IL-8 was significant rising at 6 hours and 12 hours after first LPS stimulation (P<0.05). Compared with first LPS stimulation, expression of IL-6 and IL-8 was significant lower after stimulated by LPS repeatedly (P<0.05). Conclusions Endotoxin tolerance phenomenon exists in normal prostate epithelial cells RWPE-1, and inhibits the expression of IL-6 and IL-8. It may influence the chronic inflammation and immune response of prostate tissue. Key words: Prostate; Endotoxins; Interleukin-6; Interleukin-8

IDH1R132H Promotes Malignant Transformation of Benign Prostatic Epithelium by Dysregulating MicroRNAs: Involvement of IGF1R-AKT/STAT3 Signaling Pathway

Risk stratification using molecular features could potentially help distinguish indolent from aggressive prostate cancer (PCa). Mutations in isocitrate dehydrogenase (IDH) acquire an abnormal enzymatic activity, resulting in the production of 2-hydroxyglutarate and alterations in cellular metabolism, histone modification, and DNA methylation. Mutant IDH1 has been identified in various human malignancies, and IDH1R132H constituted the vast majority of mutational events of IDH1. Most recent studies suggested that IDH1 mutations define a methylator subtype in PCa. However, the function of IDH1R132H in PCa development and progression is largely unknown. In this study, we showed that the prevalence of IDH1R132H in Chinese PCa patients is 0.6% (2/336). Of note, IDH1R132H-mutant PCa patients lacked other canonical genomic lesions (e.g., ERG rearrangement, PTEN deletion) that are common in most other PCa patients. The in vitro experiment suggested that IDH1R132H can promote proliferation of benign prostate epithelial cell RWPE-1 when under the situation of low cytokine. It could also promote migration capacity of RWPE-1 cells. Mechanistically, IDH1R132H was an important regulator of insulin-like growth factor 1receptor (IGF1R) by downregulating a set of microRNAs (miR-141-3p, miR-7-5p, miR-223-3p). These microRNAs were repressed by the alteration of epigenetic modification to decrease the enrichment of active marker H3K4me3 or to increase repressive marker H3K27me3 at their promoters. Collectively, we proposed a novel model for an IDH1R132H-microRNAs-IGF1R regulatory axis, which might provide insight into the function of IDH1R132H in PCa development.

Extracellular vesicles as drivers of epithelial-mesenchymal transition and carcinogenic characteristics in normal prostate cells.

There is increasing evidence that cancer dissemination and metastasis establishment may not only be due to the movement of tumor cells. Content of extracellular vesicles (EVs) secreted by tumor cells may also reflect the origin of these cells. Some molecules that constitute these EVs have already been used as targets for detection of specific tumors. However, to the best of our knowledge, EVs from biopsies and plasma have not yet been compared nor thoroughly investigated as triggers of malignant transformation and metastatic niche formation. To evaluate the role of EVs in the cellular microenvironment, we have treated the normal epithelial prostate cell lines, RWPE-1 and PNT-2, with a pool of EVs from biopsies of prostate primary tumors (bEVs), biopsies of benign prostate hyperplasia (hEVs), plasma of prostate cancer (PCa) patients (pEVs) or plasma of healthy individuals (pnEVs). Each of the four pools consisted of isolated EVs from several subjects, of which PCa patients were in different stages of cancer. Migration and proliferation profiles, cytokine release, and a panel of PCa-associated genes' expression of epithelial-mesenchymal transition in the cell lines were evaluated after 24 h incubation with EVs. When compared to the control groups, cells treated with the pool of EVs isolated from tumor biopsies and plasma of PCa patients showed greater migration and proliferation, significant alterations in gene expression, and high levels of IL-8, factors that are associated with cancer development. Specifically, isolated bEVs and pEVs may induce malignant features in non-tumor cells by activating several cellular events associated with cancer progression, suggesting that future PCa therapy may target multiple elements found in tumor-derived EVs.

Differential Modulation of Transcription Factors and Cytoskeletal Proteins in Prostate Carcinoma Cells by a Bacterial Lactone.

The present study tested the effect of a bacterial lactone N-(3-oxododecanoyl)-homoserine lactone (C12-HSL) on the cytoskeletal and transcriptional genes and proteins in prostate adenocarcinoma (PA) cells (DU145 and LNCaP) and prostate small cell neuroendocrine carcinoma (SCNC) PC3 cells including their cellular viability and apoptosis. Our data indicate that cell migration and colony formation were affected in the presence of C12-HSL. C12-HSL induced apoptosis and altered viability of both PA and SCNC cells in a concentration dependent manner as measured by fluorescence and chemiluminescence assays. Compared to PCa cells, noncancerous prostate epithelial cells (RWPE1) were resistant to modification by C12-HSL. Further, the viability of PC3 cells in 3D matrix was suppressed by C12-HSL treatment as detected using calcein AM fluorescence in situ. C12-HSL treatment induced cytoskeletal associated protein expression of vinculin and RhoC, which may have implications in cancer cell motility, adhesion, and metastasis. IQGAP protein expression was reduced in DU145 and RWPE1 cells in the presence of C12-HSL. C12-HSL decreased STAT3 phosphorylation in DU145 cells but increased STAT1 protein phosphorylation in PC3 and LNCaP cells. Overall, these studies indicate that C12-HSL can trigger changes in transcription factors and cytoskeletal proteins and thereby modulate growth and migration properties of PCa cells.

Agaricus blazei Mycelial Liquid Culture Extract Containing Eritadenine Improves Benign Prostatic Hyperplasia-related Biochemical Markers in RWPE-1 Cells through Anti-inflammatory and Anti-oxidative Actions

Agaricus blazei Mycelial Liquid Culture Extract Containing Eritadenine Improves Benign Prostatic Hyperplasia-related Biochemical Markers in RWPE-1 Cells through Anti-inflammatory and Anti-oxidative Actions

LncRNA HULC mediates radioresistance via autophagy in prostate cancer cells.

Prostate cancer (PCa) is the second leading cause of cancer death in men. Irradiation is one of the available options for treatment of PCa, however, approximately 10–45% of PCa are resistant to irradiation. We aimed to explore the role of long non-coding RNA highly upregulated in liver cancer (HULC) in the sensitivity of PCa cells to irradiation. Survival rate, cell apoptosis, cycle, expressions of related proteins, and caspase-3 activity were assessed to explore the effects of HULC on sensitivity of PCa cells to irradiation. Expression of HULC in DU-145, PC3, LNCaP, and RWPE-1 cells was determined and the influence of HULC on DU-145 cells was explored. Then, PC3 cells aberrantly expressing HULC were implanted into NOD-SCID mice for tumor xenograft study. Changes of autophagy after aberrant expression of HULC in vivo and in vitro were tested. Furthermore, the interacted protein of HULC and involved signaling pathway were investigated. In PC3 and LNCaP cells under irradiation, survival rate and cell cycle were decreased and apoptosis was increased by HULC knockdown. HULC knockdown arrested PC3 cells at G0/G1 phase. DU-145 was sensitive to irradiation, and resistance to irradiation of DU-145 cells was enhanced by HULC overexpression. Moreover, HULC knockdown enhanced the sensitivity of PC3 xenografts to irradiation. HULC knockdown promoted autophagy through interaction with Beclin-1 and inhibition of mTOR, resulting in increased apoptosis. HULC knockdown improved sensitivity of PCa cells to irradiation both in vivo and in vitro. HULC suppressed Beclin-1 phosphorylation, thereby reduced autophagy, involving the mTOR pathway.

Abstract A197: Synthesis and in vitro anti-prostate cancer activities of androgen receptor ligand-isothiocyanate hybrid drug

Abstract Prostate cancer (PCa) is the most common male malignancy in U.S.. In contrast to the localized early-stage disease, prognosis of castration-resistant prostate cancer (CRPC), i.e., the relapsed/metastatic advanced disease phenotype, is extremely poor. Because the sustained androgen receptor (AR) signaling remains one of the central driving forces of CRPC, new-generation nonsteroid AR antagonist has been developed in recent years, exemplified by the approval of enzalutamide (Enz) in 2012 for post-chemo and in 2014 for chemotherapy-naïve CRPC. However, drug resistance inevitably develops and survival benefits of Enz in CRPC patients are short-lived. Isothiocyanates (ITCs), e.g., sulforaphane (SFN) and phenethyl isothiocyanate (PEITC), are active metabolites of dietary glucosinolate from cruciferous vegetables with broad anti-PCa activities, such as suppressing AR expression, downregulating antiapoptotic proteins, inducing cell cycle arrest and apoptosis, and inhibiting PCa stem-like cells, etc. SFN-rich extract was recently tested in recurrent PCa patients and notably prolonged PSA doubling time without severe adverse events. By modifying Enz structure, we designed and synthesized AR ligand-ITC hybrid drug, aimed at antagonizing androgen-stimulated AR transcriptional activity and meanwhile downregulating AR expression and inducing other anticancer effects in PCa cells. To increase water solubility, ITCs were further reacted with N-acetyl cysteine (NAC) to obtain NAC conjugates. A representative hybrid drug NAC conjugate 2-63 was selected for in vitro study. In PBS buffer (pH 7.4), 2-63 gradually decomposes and releases free ITC with a half-life of about 3.4h. Measured using MTT assay, 2-63 was more tolerable in noncancerous RWPE-1 cell, significant differences were observed between RWPE-1 and PCa LNCaP cells, suggesting NAC conjugate could be a water-soluble carrier of AR ligand-ITC hybrid drug. The slower release of ITC constrains the concentration of electrophilic ITC within limit that is tolerated in healthy tissue. The newly designed hybrid drug chemical scaffold retains AR antagonist activity, evidenced by the inhibition of ARE-regulated luciferase activity in kb2 cell and the suppression of DHT-stimulated proliferation of LNCaP cell. 2-63 also dose- and time-dependently inhibited growth of CRPC VCaP cell and disrupted mitochondrial membrane potential, more effectively than SFN and Enz. Analyzed using Western blot, 2-63 downregulated the expression of full-length AR (fAR) and AR splice variants 7 (AR-V7). The AR downregulating properties were also observed in LNCaP and in 22Rv1 cells carrying different AR mutants. Since AR-V expression is one of the mechanisms causing clinical resistance to Enz treatment, targeting both fAR and AR-Vs using the newly discovered AR ligand-ITC hybrids may provide a new modality to improve the prognosis in CRPC patients. Citation Format: Siyu Ou, Liping Xu, Zhihui Qin. Synthesis and in vitro anti-prostate cancer activities of androgen receptor ligand-isothiocyanate hybrid drug [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A197.