Human Prostate Epithelial Cell Line Research Articles

Radiotherapy Metastatic Prostate Cancer Cell Lines Treated with Gold Nanorods Modulate miRNA Signatures.

MicroRNA (miRNA) modulation has been identified as a promising strategy for improving the response of human prostate cancer (PCa) to radiotherapy (RT). Studies have shown that mimics or inhibitors of miRNAs could modulate the sensitivity of PCa cells to RT. In addition, pegylated gold nanoparticles have been studied as a therapeutic approach to treat PCa cells and/or vehicles for carrying miRNAs to the inside of cells. Therefore, we evaluated the capacity of hypofractionated RT and pegylated gold nanorods (AuNPr-PEG) to modulate the miRNA signature on PCa cells. Thus, RT-qPCR was used to analyze miRNA-95, miRNA-106-5p, miRNA-145-5p, and miRNA-541-3p on three human metastatic prostate cell lines (PC3, DU145, and LNCaP) and one human prostate epithelial cell line (HprEpiC, a non-tumor cell line) with and without treatment. Our results showed that miRNA expression levels depend on cell type and the treatment combination applied using RT and AuNPr-PEG. In addition, cells pre-treated with AuNPr-PEG and submitted to 2.5 Gy per day for 3 days decreased the expression levels of miRNA-95, miRNA-106, miRNA-145, and miRNA-541-3p. In conclusion, PCa patients submitted to hypofractionated RT could receive personalized treatment based on their metastatic cellular miRNA signature, and AuNPr-PEG could be used to increase metastatic cell radiosensitivity.

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.

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

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

Gallium and indium complexes with isoniazid-derived ligands: Interaction with biomolecules and biological activity against cancer cells and Mycobacterium tuberculosis

Gallium and indium octahedral complexes with isoniazid derivative ligands were successfully prepared. The ligands, isonicotinoyl benzoylacetone (H2L1) and4-chlorobenzoylacetone isonicotinoyl hydrazone (H2L2), and their respective coordination compounds with gallium and indium [GaL1(HL1)] (GaL1), [GaL2(HL2)] (GaL2), [InL1(HL1)] (InL1) and [InL2(HL2)] (InL2) were investigated by NMR, ESI-MS, UV–Vis, IR, single-crystal X-ray diffraction and elemental analysis. In vitro interaction studies with human serum albumin (HSA) evidenced a moderate affinity of all complexes with HSA through spontaneous hydrophobic interactions. The greatest suppression of HSA fluorescence was caused by GaL2 and InL2, which was associated to the higher lipophilicity of H2L2. In vitro interaction studies with CT-DNA indicated weak interactions of the biomolecule with all complexes. Cytotoxicity assays withMCF-7 (breast carcinoma), PC-3 (prostate carcinoma) and RWPE-1 (healthy human prostate epithelial) cell lines showed that complexes with H2L2 are more active and selective against MCF-7, with the greatest cytotoxicity observed for InL2 (IC50 = 10.34 ± 1.69 μM). H2L1 and H2L2 were labelled with gallium-67, and it was verified that 67GaL2 has a greater lipophilicity than 67GaL1, as well as higher stability in human serum or in the presence of apo-transferrin. Cellular uptake assays with 67GaL1 and 67GaL2 evidenced that the H2L2-containing radiocomplex has a higher accumulation in MCF-7 and PC-3 cells than the non-halogenated congener 67GaL1. The anti-Mycobacterium tuberculosis assays revealed that both ligands and metal complexes are potent growth inhibitors, with MIC90 (μg mL−1) values observed from 0.419 ± 0.05 to 1.378 ± 0.21.

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.

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.

Tumorigenic transformation of human prostatic epithelial cell line RWPE-1 by growth hormone-releasing hormone (GHRH).

BackgroundGrowth hormone‐releasing hormone (GHRH) and its receptors have been implicated in the progression of various tumors. In this study, we analyzed the carcinogenetic potential of exposure to GHRH of a nontumor human prostate epithelial cell line (RWPE‐1) as well as its transforming effect in a xenograft model.MethodsWe performed cell viability, cell proliferation, adhesion and migration assays. In addition, metalloprotease (MMP)−2 activity by means gelatin zymography, GHRH‐R subcellular location using confocal immunofluorescence microscopy and vascular endothelial growth factor (VEGF) levels by enzyme‐linked immunoassay were assessed. Besides, we developed an in vivo model in order vivo model to determine the role of GHRH on tumorigenic transformation of RWPE‐1 cells.ResultsIn cell cultures, we observed development of a migratory phenotype consistent with the gelatinolytic activity of MMP‐2, expression of VEGF, as well as E‐cadherin‐mediated cell‐cell adhesion and increased cell motility. Treatment with 0.1 µM GHRH for 24 h significantly increased cell viability and cell proliferation. Similar effects of GHRH were seen in RWPE‐1 tumors developed by subcutaneous injection of GHRH‐treated cells in athymic nude mice, 49 days after inoculation.ConclusionsThus, GHRH appears to act as a cytokine in the transformation of RWPE‐1 cells by mechanisms that likely involve epithelial‐mesenchymal transition, thus reinforcing the role of GHRH in tumorigenesis of prostate.

Antioxidant and Anti-Inflammatory Activity of Combined Phycocyanin and Palmitoylethanolamide in Human Lung and Prostate Epithelial Cells

Inflammation involving the innate and adaptive immune systems is a normal response to infection; however, when allowed to continue unchecked, inflammation may result in several pathologies. Natural molecules with antioxidant properties can target the key players of inflammation and exert beneficial health effects. In this study, human normal bronchial (Beas-2B) and prostate (HPrEpiC) epithelial cell lines were exposed to infectious stimulation and treated with phycocyanin (PC) and palmitoylethanolamide (PEA), with the aim of demonstrating the enhanced antioxidant and anti-inflammatory properties of the combination. The cotreatment protected from cytotoxicity and greatly abated both the production of radical oxygen species (ROS) and the transcription of several inflammatory cytokines. Oxidative stress and inflammation were curtailed by affecting three main pathways: (1) inhibition of cyclooxygenase-2 enzyme and consequent decrease of signaling generating ROS; (2) increased synthesis of glutathione and therefore strengthening of the natural antioxidant defenses of the cells; (3) decreased infection-driven mitochondrial respiratory burst which generates oxidative stress. Based on the mounting interest in using nutraceuticals as adjuvants in the clinical practice, the present study unveils new mechanisms of action and enhanced efficacy of PC and PEA, supporting the possible exploitation of this combination in human disorders.

Inhibition of the proliferation, invasion, migration, and epithelial-mesenchymal transition of prostate cancer cells through the action of ATP1A2 on the TGF-β/Smad pathway.

BackgroundProstate cancer (PC) is one of the major male malignancies worldwide. Because Na+-K+-ATPase is widely involved in various pathological processes, but the action of its α2 subtype (ATP1A2) in PC is unclear, we investigated the role of ATP1A2 in the invasion and migration of PC cells.MethodsWe measured the expression levels of ATP1A2 in human normal prostate epithelial cell line (RWPE-1) and PC cell lines (PC-3 and DU145) by quantitative real-time PCR (qRT-PCR) and western blot. Cell proliferation, apoptosis, migration, and invasion of PC-3 and DU145 cells were investigated through clone formation assay, EdU assay, flow cytometry and transwell assay, respectively. The effect of ATP1A2 on a tumor-inhibitory pathway [transforming growth factor-β (TGF-β)/Smad] was assessed using western blot. In addition, tumor formation was detected using in vivo xenograft model in male BALB/c nude mice.ResultsThe Cancer Genome Atlas (TCGA) analysis showed that ATP1A2 expression was reduced in PC patients (P<0.05), and patients with low ATP1A2 expression had a lower survival rate (P<0.05). ATP1A2 levels were significantly reduced in PC-3 and DU145 cells, compared with RWPE-1 cells (P<0.01). We also demonstrated that overexpression of ATP1A2 significantly inhibited the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of PC-3 and DU145 cells (P<0.01) and promoted apoptosis (P<0.01). However, silencing ATP1A2 had the opposite effect (P<0.01). In addition, overexpression of ATP1A2 significantly inhibited the TGF-β/Smad pathway (P<0.01), whereas silencing ATP1A2 activated the TGF-β/Smad pathway (P<0.01). Meanwhile, the effect of ATP1A2 silencing on the proliferation, apoptosis, migration and invasion was reversed by TGF-β/Smad pathway inhibitor (LY364947). Furthermore, ATP1A2 inhibited tumor growth in vivo.ConclusionsATP1A2 inhibited proliferation, apoptosis, migration, invasion, and EMT in PC by inhibiting the TGF-β/Smad pathway.

Exosomal lncAY927529 enhances prostate cancer cell proliferation and invasion through regulating bone microenvironment

ABSTRACT Exosomes mediate the interaction between cancer cells and their microenvironment, and play a key role in tumor development. Although exosomes can package lncRNAs to mediate extracellular communication, the role of exosomal lncRNA AY927529 in prostate cancer (PCa) remains unclear. Exosomes were extracted from normal human prostatic epithelial cell lines (BPH-1 and RWPE-1) and PCa cell lines (VCaP and LNCaP, DU145, PC3) by ultrahigh speed centrifugation. Results of Western blot indicated that Alix, HSC70 and TSGl01 protein levels were upregulated in exosomes derived from PCa cells. LncAY927529 level was upregulated in PCa cells and exosomes derived from PCa patient serum and human PCa cells. CCK-8, Transwell and Flow cytometry assays demonstrated that bone marrow stromal cell line (ST2) conditioned medium (ST2-CM), treated with exosomes derived from PCa cells with high lncAY927529 level, promoted proliferation and invasion of PC3 and DU145 cells, and inhibited cell apoptosis. RT-qPCR assay indicated that lncAY927529 level was downregulated in PC3 and DU145 cells, exosomes derived from PCa cells (PCa-Exo) and ST2-CM treated with PCa-Exo with low expression of lncAY927529, and overexpression of lncAY927529 had the opposite results. In addition, Western blot assay showed that the autophagy related protein LC3II level was increased in ST2 cells treated with exosomes derived from DU145 cells with high expression of lncAY927529, and LC3I protein level was decreased. CXCL14 acted as a RNA-binding protein of lncAY927529, and exosome-mediated lncAY927529 positively regulated CXCL14 levels in ST2 cells. In general, exosome-mediated lncAY927529 could promote PCa cell proliferation and invasion by regulating bone microenvironment, suggesting that exosomal lncAY927529 may be a potential molecular diagnostic marker of PCa.

Detection of rapidly accumulating stress-induced SUMO in prostate cancer cells by a fluorescent SUMO biosensor.

SUMO conjugates and SUMO chains form when SUMO, a small ubiquitin-like modifier protein, is covalently linked to other cellular proteins or itself. During unperturbed growth, cells maintain balanced levels of SUMO conjugates. In contrast, eukaryotic cells that are exposed to proteotoxic and genotoxic insults mount a cytoprotective SUMO stress response (SSR). One hallmark of the SSR is a rapid and massive increase of SUMO conjugates in response to oxidative, thermal, and osmotic stress. Here, we use a recombinant fluorescent SUMO biosensor, KmUTAG-fl, to investigate differences in the SSR in a normal human prostate epithelial cell line immortalized with SV40 (PNT2) and two human prostate cancer cell lines that differ in aggressiveness and response to androgen (LNCaP and PC3). In cells that grow unperturbed, SUMO is enriched in the nuclei of all three cell lines. However, upon 30 min of exposure to ultraviolet radiationor oxidative stress, we detected significant cytosolic enrichment of SUMO as measured by KmUTAG-fl staining. This rapid enrichment in cytosolic SUMO levels was on average fivefold higher in the LNCaP and PC3 prostate cancer cell lines compared to normal immortalized PNT2 cells. Additionally, this enhanced enrichment of cytosolic SUMO was reversible as cells recovered from stress exposure. Our study validates the use of the fluorescent KmUTAG-fl SUMO biosensor to detect differences of SUMO levels and localization between normal and cancer cells and provides new evidence that cancer cells may exhibit an enhanced SSR.

Synthesis and morphological studies of Tc-99m-labeled lupulone-conjugated Fe3O4@TiO2 nanocomposite, and in vitro cytotoxicity activity on prostate cancer cell lines.

The purpose of this study was to develop a multifunctional theranostic probe for imaging (magnetic resonance imaging [MRI] and single-photon emission computed tomography [SPECT]) and therapy (photodynamic therapy). For this purpose, Tc-99m-labeled lupulone-conjugated Fe3O4@TiO2 nanocomposites (99mTc-DTPA-Fe3O4@TiO2-HLP and 99mTc-DTPA-Fe3O4@TiO2-ALP nanocomposites) were synthesized. The average diameter of the nanocomposites was 171 ± 20 nm as seen on transmission electron microscopy images. Fe3O4@TiO2 nanocomposites exhibited fluorescence spectra at an emission wavelength of 314 nm. Lupulone-conjugated Fe3O4@TiO2 nanocomposites were spherical-shaped with a suitable dispersion and without visible aggregation, and their radiolabeling yields were over 85%. Healthy (RWPE-1 normal human prostate epithelial cell line) and cancer prostate cell lines (PC-3 human prostate cancer cell line) were used to determine the in vitro biological behavior of the nanocomposites. The PC-3 cells treated with lupulone-conjugated Fe3O4@TiO2 nanocomposites showed a lower cell viability compared with RWPE-1 cells treated with lupulone-conjugated Fe3O4@TiO2 nanocomposites. Lupulone-modified Fe3O4@TiO2 nanocomposites may serve in the future as a multifunctional probe for positron emission tomography (PET)/MRI, photodynamic therapy, and hyperthermia therapy of cancer.

Prognostic Value of miR-1826 in Prostate Cancer and Its Regulatory Effect on Tumor Progression.

PurposemiRNAs can act as oncogenes or tumor suppressors and participate in the development and progression of tumors, thus affecting the prognosis and survival of cancer patients. In this paper, we mainly studied the role of miR-1826 in prostate cancer.Patients and MethodsThe expression of miR-1826 was studied by quantitative real-time PCR (qRT-PCR). Kaplan–Meier curves were used to analyze the relationship between the expression of miR-1826 and the survival rate of PC patients. Cox regression analysis was used to study the risk factors affecting the prognosis of PC patients. PC cells were transfected with miR-1826 mimic, mimic negative control (mimic NC), miR-1826 inhibitor, or inhibitor NC. The effect of miR-1826 on the proliferation of PC cells was studied by the CCK-8 method and colony formation assay. Transwell assays were used to detect the effect of miR-1826 on the migratory and invasive abilities of tumor cells.ResultsThe expression of miR-1826 in PC tissues was lower than that in adjacent normal tissues, and that the expression levels of miR-1826 in four PC cell lines were all lower than normal human prostate epithelial cell lines. Patients with low expression of miR-1826 had shorter overall survival compared with those with high expression. The downregulation of miR-1826 promoted PC cell proliferation, migration, and invasion.ConclusionIn summary, the low expression of miR-1826 may promote the progression of PC, and the low expression of miR-1826 is also associated with a poor prognosis in PC patients.

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.

The Roles of ZnT1 and ZnT4 in Glucose-Stimulated Zinc Secretion in Prostate Epithelial Cells.

We have previously demonstrated by MRI that high glucose stimulates efflux of zinc ions from the prostate. To our knowledge, this phenomena had not been reported previously and the mechanism remains unknown. Here, we report some initial observations that provide new insights into zinc processing during glucose-stimulated zinc secretion (GSZS) in the immortalized human prostate epithelial cell line, PNT1A. Additionally, we identified the subtypes of zinc-containing cells in human benign prostatic hyperplasia (BPH) tissue to further identify which cell types are likely responsible for zinc release in vivo. An intracellular fluorescence marker, FluoZin-1-AM, was used to assess the different roles of ZnT1 and ZnT4 in zinc homeostasis in wild type (WT) and mRNA knockdown PNT1A cell lines. Additionally, Bafilomycin A1 (Baf) was used to disrupt lysosomes and assess the role of lysosomal storage during GSZS. ZIMIR, an extracellular zinc-responsive fluorescent marker, was used to assess dynamic zinc efflux of WT and ZnT1 mRNA knockdown cells exposed to high glucose. Electron microscopy was used to assess intracellular zinc storage in response to high glucose and evaluate how Bafilomycin A1 affects zinc trafficking. BPH cells were harvested from transurtheral prostatectomy tissue and stained with fluorescent zinc granule indicator (ZIGIR), an intracellular zinc-responsive fluorescent marker, before being sorted for cell types using flow cytometry. Fluorescent studies demonstrate that ZnT1 is the major zinc efflux transporter in prostate epithelial cells and that loss of ZnT1 via mRNA knockdown combined with lysosomal storage disruption results in a nearly 4-fold increase in cytosolic zinc. Knockdown of ZnT1 dramatically reduces zinc efflux during GSZS. Electron microscopy (EM) reveals that glucose stimulation significantly increases lysosomal storage of zinc; disruption of lysosomes via Baf or ZnT4 mRNA knockdown increases multi-vesicular body (MVB) formation and cytosolic zinc levels. In human BPH tissue, only the luminal epithelial cells contained significant amounts of zinc storage granules. Exposure of prostate epithelial cells to high glucose alters zinc homeostasis by inducing efflux of zinc ions via ZnT1 channels and increasing lysosomal storage via ZnT4. Given that prostate cancer cells undergo profound metabolic changes that result in reduced levels of total zinc, understanding the complex interplay between glucose exposure and zinc homeostasis in the prostate may provide new insights into the development of prostate carcinogenesis.

Evaluation of intercellular communication between normal human prostate epithelial cells and prostate cancer cell lines in co-culture model

Evaluation of intercellular communication between normal human prostate epithelial cells and prostate cancer cell lines in co-culture model

Tight junction protein claudin-1 is downregulated by TGF-β1 via MEK signaling in benign prostatic epithelial cells.

Benign prostatic hyperplasia (BPH) is arguably the most common disease in aging men. Although the etiology is not well understood, chronic prostatic inflammation is thought to play an important role in BPH initiation and progression. Our recent studies suggest that the prostatic epithelial barrier is compromised in glandular BPH tissues. The proinflammatory cytokine transforming growth factor beta 1 (TGF-β1) impacts tight junction formation, enhances epithelial barrier permeability, and suppresses claudin-1 messenger RNA expression in prostatic epithelial cells. However, the role of claudin-1 in the prostatic epithelial barrier and its regulation by TGF-β1 in prostatic epithelial cells are not clear. The expression of claudin-1 was analyzed in 22 clinical BPH specimens by immunohistochemistry. Human benign prostate epithelial cell lines BPH-1 and BHPrE1 were treated with TGF-β1 and transfected with small interfering RNAs specific to claudin-1. Epithelial monolayer permeability changes in the treated cells were measured using trans-epithelial electrical resistance (TEER). The expression of claudin-1, E-cadherin, N-cadherin, snail, slug, and activation of mitogen-activated proteins kinases (MAPKs) and AKT was assessed following TGF-β1 treatment using Western blot analysis. Claudin-1 expression was decreased in glandular BPH tissue compared with adjacent normal prostatic tissue in patient specimens. TGF-β1 treatment or claudin-1 knockdown in prostatic epithelial cell lines increased monolayer permeability. TGF-β1 decreased levels of claudin-1 and increased levels of snail and slug as well as increased phosphorylation of the MAPK extracellular signal-regulated kinase-1/2 (ERK-1/2) in both BPH-1 and BHPrE1 cells. Overexpression of snail or slug had no effect on claudin-1 expression. In contrast, PD98059 and U0126, inhibitors of the upstream activator of ERK-1/2 (ie, MEK-1/2) restored claudin-1 expression level as well as the epithelial barrier. Our findings suggest that downregulation of claudin-1 by TGF-β1 acting through the noncanonical MEK-1/2/ERK-1/2 pathway triggers increased prostatic epithelial monolayer permeability in vitro. These findings also suggest that elevated TGF-β1 may contribute to claudin-1 downregulation and compromised epithelial barrier in clinical BPH specimens.

Differential impact of paired patient-derived BPH and normal adjacent stromal cells on benign prostatic epithelial cell growth in 3D culture.

Benign prostatic hyperplasia (BPH) is an age-related disease characterized by nonmalignant abnormal growth of the prostate, which is also frequently associated with lower urinary tract symptoms. The prostate with BPH exhibits enhanced growth not only in the epithelium but also in the stroma, and stromal-epithelial interactions are thought to play an important role in BPH pathogenesis. However, our understanding of the mechanisms of stromal-epithelial interactions in the development and progression of BPH is very limited. Matched pairs of glandular BPH and normal adjacent prostate specimens were obtained from BPH patients undergoing simple prostatectomy for symptomatic BPH. Tissues were divided further into fresh specimens for culture of primary prostatic stromal cells, and specimens were embedded in paraffin for immunohistochemical analyses. Proliferation assays, immunohistochemistry, and immunoblotting were used to characterize the primary prostate stromal cells and tissue sections. Coculture of the primary stromal cells with benign human prostate epithelial cell lines BHPrE1 or BPH-1 was performed in three-dimensional (3D) Matrigel to determine the impact of primary stromal cells derived from BPH on epithelial proliferation. The effect of stromal-conditioned medium (CM) on BHPrE1 and BPH-1 cell growth was tested in 3D Matrigel as well. BPH stromal cells expressed less smooth muscle actin and calponin and increased vimentin, exhibiting a more fibroblast and myofibroblast phenotype compared with normal adjacent stromal cells both in culture and in corresponding paraffin sections. Epithelial spheroids formed in 3D cocultures with primary BPH stromal cells were larger than those formed in coculture with primary normal stromal cells. Furthermore, CM from BPH stromal cells stimulated epithelial cell growth while CM from normal primary stromal cells did not in 3D culture. These findings suggest that the stromal cells in BPH tissues are different from normal adjacent stromal cells and could promote epithelial cell proliferation, potentially contributing to the development and progression of BPH.

Castration-induced stromal remodeling disrupts the reconstituted prostate epithelial structure

AbstractThe normal prostate epithelial structure is maintained by homeostatic interactions with smooth muscle cells. However, structural alterations of the stroma are commonly observed in prostatic proliferative diseases, leading to the abnormalities of prostate epithelial structure. A decrease in the androgen level experimentally induces stromal remodeling, i.e., replacement of smooth muscle cells with fibroblasts or myofibroblasts. In this study, we investigated the effects of castration-induced stromal remodeling and subsequent aberrant activation of epithelial–stromal interactions on the reconstituted human prostate-like epithelial structure. We performed in vivo experiments using the human prostate epithelial cell line BPH-1 and fetal rat urogenital sinus mesenchyme to generate heterotypic tissue recombinants that form human prostate-like epithelial structure (i.e., solid- and canalized-epithelial cords). Host mice were castrated at 12 weeks post transplantation (castration) and implanted with a dihydrotestosterone pellet at 14 days post castration (androgen replacement treatment; ART). In the castration group, the percentages of fibrotic area and disrupted prostate epithelial structure without the basement membrane (BM) increased proportionally in a time-dependent manner, but were suppressed by ART. In the castration group, tenascin-C (TNC)-positive fibroblasts were abundant in the stroma surrounding disrupted prostate epithelial structure without the BM. TGF-β1 secretion from BPH-1 cells was increased by co-culturing with human primary cultured prostate fibroblasts. TNC mRNA expression was increased in fibroblasts co-culturing with BPH-1 cells and was suppressed by treatment with a TGF-β RI kinase inhibitor. Moreover, in the castration group, the percentage of p-Smad2-positive cells was significantly higher in the stroma surrounding disrupted prostate epithelial structure without the BM. Our results demonstrate that castration-induced stromal remodeling disrupted the reconstituted human prostate-like epithelial structure and induced the appearance of TNC-positive fibroblasts accompanied by activation of TGF-β signaling. The alteration of prostate stromal structure may be responsible for loss of the BM and epithelial cell polarity.

Co-carcinogenic effects of vitamin E in prostate

A large number of basic researches and observational studies suggested the cancer preventive activity of vitamin E, but large-scale human intervention trials have yielded disappointing results and actually showed a higher incidence of prostate cancer although the mechanisms underlying the increased risk remain largely unknown. Here we show through in vitro and in vivo studies that vitamin E produces a marked inductive effect on carcinogen-bioactivating enzymes and a pro-oxidant status promoting both DNA damage and cell transformation frequency. First, we found that vitamin E in the human prostate epithelial RWPE-1 cell line has the remarkable ability to upregulate the expression of various phase-I activating cytochrome P450 (CYP) enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), giving rise to supraphysiological levels of reactive oxygen species. Furthermore, our rat model confirmed that vitamin E in the prostate has a powerful booster effect on CYP enzymes associated with the generation of oxidative stress, thereby favoring lipid-derived electrophile spread that covalently modifies proteins. We show that vitamin E not only causes DNA damage but also promotes cell transformation frequency induced by the PAH-prototype benzo[a]pyrene. Our findings might explain why dietary supplementation with vitamin E increases the prostate cancer risk among healthy men.