Normal Human Prostate Epithelial Cells Research Articles

Dihydroartemisinin attenuates benign prostatic hyperplasia in rats by inhibiting prostatic epithelial cell proliferation.

BackgroundBenign prostatic hyperplasia (BPH) is a common urological condition in aging men. While dihydroartemisinin (DHA) exhibits a wide range of pharmacological activities, to date, there have been no studies examining the effects of DHA on BPH.MethodsAn in vivo BPH model was constructed in rats via daily subcutaneous injection of testosterone propionate (TP) for 28 consecutive days. Rats were randomly distributed into four groups and treated as follows: (I) control; (II) TP treatment; (III) TP and finasteride treatment (positive control); and (IV) TP and DHA treatment. At the end of the experiment, rats were sacrificed and the prostate weight, prostate index, thickness of the epithelium, collagen deposition, serum dihydrotestosterone (DHT) levels, 5α-reductase 2 (5AR-2) expression, and proliferating cell nuclear antigen (PCNA) levels in the prostate were examined. Normal human prostatic epithelial RWPE-1 cells were used in in vitro experiments to further investigate the anti-proliferative effects of DHA.ResultsTP increased the prostate weight and prostate index in rats, and this effect was reduced with DHA treatment. In addition, DHA attenuated the morphological changes and collagen deposition in the prostate tissue induced by TP. Furthermore, DHA reduced the expression of PCNA, serum DHT, and prostatic 5AR-2 in rats with TP-induced BPH. In vitro analysis revealed that DHA significantly inhibited the proliferation of TP-treated RWPE-1 cells.ConclusionsDHA significantly inhibited the development of BPH by suppressing serum DHT levels, prostatic 5AR-2 expression, and the proliferation of benign prostatic epithelial cells. Thus, DHA is a novel medicinal agent with potential therapeutic efficacy in the treatment of patients with BPH.

Abstract 2442: Chondroitin sulfatases and transcription factors Gli, Tcf/Lef, and c-Myc in prostate stem cells

Abstract The enzymes arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) and galactose-6-sulfate sulfatase (GALNS; N-acetylgalactosamine-6-sulfatase) remove either the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate (C4S) and dermatan sulfate, or the 6-sulfate group of chondroitin 6-sulfate, chondroitin 4,6-disulfate (chondroitin sulfate E), or keratan sulfate. In normal and malignant prostate tissues obtained by laser-capture microdissection, ARSB expression is greater in stroma than epithelium and GALNS expression is greater in epithelium than stroma. In malignant epithelium, GALNS expression is greater and ARSB expression is less than in normal epithelium. In cultured, normal human prostate stem and epithelial cells, ARSB silencing and GALNS overexpression exert similar signaling effects, attributable to enhanced binding of C4S with SHP2 (PTPN11), leading to SHP2 inhibition and sustained phosphorylation of phospho-ERK1/2 and other critical signaling molecules. Experiments in developing rat prostate showed that exposure to estrogen significantly reduced ARSB activity, whereas GALNS activity increased at post-natal day 30. To further assess how changes in sulfatases may contribute to prostate development, transcription factor activation profiling plate arrays (Signosis) were performed in cultured human prostate stem cells (CRL-2887, ATCC). Cells were silenced for ARSB (EC 3.1.6.12) or GALNS (EC 3.1.6.4) by specific siRNA (Qiagen) or ARSB was overexpressed [for ARSB (NCBI NM_000046, transcript variant 1; for GALNS (NCBI NM_000512; TrueClone, Origene] by transfection of specific untagged plasmids. Biotin-labeled transcription factor probes based on the known DNA-binding consensus sequences were detected in nuclear extracts of the treated and control stem cell preparations. Nuclear DNA bound transcription factor Gli declined 35% when ARSB was silenced and 63% when GALNS was overexpressed. Inversely, nuclear bound Gli increased 62% when ARSB was overexpressed and 42% when GALNS was silenced. These findings are consistent with the observed inverse signaling effects of ARSB and GALNS in the prostate cells. Also, Gli mRNA expression was 3.50 ± 0.22 times greater in non-malignant epithelium than in malignant epithelium, consistent with decline when GALNS is greater and ARSB is reduced. Nuclear binding of TCF/Lef and c-Myc showed inverse effects in relation to GALNS silencing and overexpression, declining when GALNS was silenced and increasing when GALNS was overexpressed. These effects are consistent with the observed effects on Wnt signaling. These observations indicate that changes in chondroitin sulfates mediated by chondroitin sulfatases may have profound consequences on developmental programming in prostate tissue and suggest that hormonal-mediated effects may be transcribed through changes in sulfatases and chondroitin sulfates. Citation Format: Joanne Kramer Tobacman, Sumit Bhattacharyya, Leo Feferman. Chondroitin sulfatases and transcription factors Gli, Tcf/Lef, and c-Myc in prostate stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2442.

Abstract 3054: Synergistic activity of DNA damage response kinase inhibitors in combination with the targeted alpha therapy radium-223 dichloride for metastatic castration-resistant prostate cancer

Abstract Radium-223 dichloride (Ra-223, Xofigo®) is the first approved α-particle-emitting radionuclide for the treatment of symptomatic bone metastases in patients with castration-resistant prostate cancer (CRPC) with no known evidence of visceral metastases. Ra-223 is a calcium-mimetic that preferentially binds with the bone mineral hydroxyapatite at areas of high bone turnover, such as bone metastases. This highly localized radiotherapy causes a high frequency of unrepairable double-stranded DNA breaks (DSBs), resulting in a potent antitumor effect on bone metastases. Recent evidence has suggested that patients with mutations in the DNA damage response pathway (DDR), may have differential outcomes to Ra-223 treatment (1).DDR comprises a dynamic network of signaling pathways for the maintenance of genomic integrity. Ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) are critical proteins which orchestrate the DDR and their activation is dependent on the type of DNA lesion. ATM is the primary responder to DSBs whilst ATR is activated by a range of lesions including single strand DNA structures at resected ends of DBSs and after replication fork stalling. In this study, we evaluated the impact of combining DDR kinase inhibitors with Ra-223 to investigate whether a greater radiosensitization response occurs in comparison to standard X-rays in PC3 and DU145 human prostate cell lines and normal prostate epithelial RWPE-1 cells. Cell assays including clonogenic survival, DNA damage assays and flow cytometry were used to assess the effect of DDR kinase inhibitors in combination with ionizing radiation. Cells were pre-treated with DDR inhibitors one-hour before exposure to 2Gy X-rays or an equivalent dose of 0.25Gy Ra-223. Our data show that, in all prostate models, DDR kinase inhibitors in combination with Ra-223 significantly enhanced radiosensitivity (p<0.005) response in comparison to combined treatment with X-rays. Furthermore, a greater quantity of residual DSBs at 24 hours post combination treatment was observed after Ra-223 exposure in comparison to X-ray exposure (p<0.001). Promisingly, this combined treatment had minimal effect on RWPE-1 normal cells. Our findings strongly support the combination of DNA damage induction by Ra-223 with DDR kinase inhibitors as a novel potential treatment option for mCRPC patients in order to improve clinical outcome. {{References}}:1.Velho PI, Qazi F, Hassan S, et al. Efficacy of Radium-223 in Bone-metastatic Castration-resistant Prostate Cancer with and Without Homologous Repair Gene Defects. European Urology. 2019; 76: 170-176. Citation Format: Victoria L. Dunne, Timothy Wright, Francisco Liberal, Joe M. O'Sullivan, Kevin M. Prise. Synergistic activity of DNA damage response kinase inhibitors in combination with the targeted alpha therapy radium-223 dichloride for metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3054.

Human Prostate Epithelial Cells Activate the AIM2 Inflammasome upon Cellular Senescence: Role of POP3 Protein in Aging-Related Prostatic Inflammation.

Increased levels of type I (T1) interferon (IFN)-inducible POP3 protein in myeloid cells inhibit activation of the AIM2 inflammasome and production of IL-1β and IL-18 proinflammatory cytokines. The AIM2 mRNA levels were significantly higher in benign prostate hyperplasia (BPH) than the normal prostate. Further, human normal prostate epithelial cells (PrECs), upon becoming senescent, activated an inflammasome. Because in aging related BPH senescent PrECs accumulate, we investigated the role of POP3 and AIM2 proteins in pre-senescent and senescent PrECs. Here we report that the basal levels of the POP3 mRNA and protein were lower in senescent (versus young or old) PrECs that exhibited activation of the T1 IFN response. Further, treatment of PrECs and a BPH cell line (BPH-1) that expresses the androgen receptor (AR) with the male sex hormone dihydrotestosterone (DHT) increased the basal levels of POP3 mRNA and protein, but not AIM2, and inhibited activation of the AIM2 inflammasome. Of interest, a stable knockdown of POP3 protein expression in the BPH-1 cell line increased cytosolic DNA-induced activation of AIM2 inflammasome. These observations suggest a potential role of POP3 protein in aging-related prostatic inflammation.

Olfactomedin 4 mediation of prostate stem/progenitor-like cell proliferation and differentiation via MYC

Olfactomedin 4 (OLFM4) is expressed in normal prostate epithelial cells and immortalized normal human prostate epithelial cells (RWPE1), but the identity of OLFM4-expressing cells within these populations and OLFM4’s physiological functions in these cells have not been elucidated. Using single-cell RNA sequencing analysis, we found here that OLFM4 was expressed in multiple stem/progenitor-like cell populations in both the normal prostate epithelium and RWPE1 cells and was frequently co-expressed with KRT13 and LY6D in RWPE1 cells. Functionally, OLFM4-knockout RWPE1 cells exhibited enhanced proliferation of the stem/progenitor-like cell population, shifts stem/progenitor-like cell division to favor symmetric division and differentiated into higher levels PSA expression cells in organoid assays compared with OLFM4-wild RWPE1 cells. Bulk-cell RNA sequencing analysis pinpointed that cMYC expression were enhanced in the OLFM4-knockout RWPE1 cells compared with OLFM4-wild cells. Molecular and signaling pathway studies revealed an increase in the WNT/APC/MYC signaling pathway gene signature, as well as that of MYC target genes that regulate multiple biological processes, in OLFM4-knockout RWPE1 cells. These findings indicated that OLFM4 is co-expressed with multiple stem/progenitor cell marker genes in prostate epithelial cells and acts as a novel mediator in prostate stem/progenitor cell proliferation and differentiation.

Chronic exposure to microcystin-LR increases the risk of prostate cancer and induces malignant transformation of human prostate epithelial cells

Microcystins-LR (MC-LR) acts as a possible carcinogen for humans and causes a serious risk to public environmental health. The current study aimed to evaluate the interaction between MC-LR exposure and prostate cancer development and elucidate the underlying mechanism. In this study, mice were exposed to MC-LR at various doses for 180 days. MC-LR was able to induce the progression of prostatic intraepithelial neoplasia (PIN) and microinvasion. Furthermore, MC-LR notably increased angiogenesis and susceptibility to prostate cancer in vivo. In vitro, over 25 weeks of MC-LR exposure, normal human prostate epithelial (RWPE-1) cells increased secretion of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and colony formation, features typical for cancer cells. These MC-LR-transformed prostate epithelial cells displayed increased expression of forkhead box M1 (FOXM1) and cyclooxygenase-2 (COX-2); abrogation of FOXM1 or COX-2 activity by specific inhibitors could abolish the invasion and migration of MC-LR-treated cells. In conclusion, we have provided compelling evidence demonstrating the induction of a malignant phenotype in human prostate epithelial cells and the in vivo development of prostate cancer by exposure to MC-LR, which might be a potential tumor promoter in the progression of prostate cancer.

Synergistic anticancer activity of resveratrol in combination with docetaxel in prostate carcinoma cells.

BACKGROUND/OBJECTIVESThe study was conducted to investigate the efficacy of the combination treatment of phytochemical resveratrol and the anticancer drug docetaxel (DTX) on prostate carcinoma LNCaP cells, including factors related to detailed cell death mechanisms.MATERIALS/METHODSUsing 2-dimensional monolayer and 3-dimensional spheroid culture systems, we examined the effects of resveratrol and DTX on cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential, apoptosis, and necroptosis by MTT, flow cytometry, and Western blotting.RESULTSAt concentrations not toxic to normal human prostate epithelial cells, resveratrol effectively decreased the viability of LNCaP cells depending on concentration and time. The combination treatment of resveratrol and DTX exhibited synergistic inhibitory effects on cell growth, demonstrated by an increase in the sub-G0/G1 peak, Annexin V-phycoerythrin positive cell fraction, ROS, mitochondrial dysfunction, and DNA damage response as well as concurrent activation of apoptosis and necroptosis. Apoptosis and necroptosis were rescued by pretreatment with ROS scavenger N-acetylcysteine.CONCLUSIONSWe report resveratrol as an adjuvant drug candidate for improving the outcome of treatment in DTX therapy. Although the underlying mechanisms of necroptosis should be investigated comprehensively, targeting apoptosis and necroptosis simultaneously in the treatment of cancer can be a useful strategy for the development of promising drug candidates.

Silencing of microRNA-3175 represses cell proliferation and invasion in prostate cancer by targeting the potential tumor-suppressor SCN4B.

MicroRNA-3175 (miR-3175) expression is upregulated in prostate cancer, but its roles and the underlying mechanisms in prostate cancer cell growth and invasion need to be elucidated. This study aimed to uncover the roles of miR-3175 in regulating cell growth and migration, as well as the expression of its predicted target gene cardiac sodium channel β4-subunit gene (SCN4B). Real-time quantitative PCR (RT-qPCR) and/or western blotting techniques were used to measure miR-3175 and SCN4B expression levels in prostate cancer cells. Inhibitor or mimics transfections were used to overexpress or silence miR-3175 in prostate cancer cells. MTT and Edu assays were applied to assess cell viability. Scratch assay and transwell chambers were used to examine cell migration and invasion abilities. The interaction between miR-3175 and SCN4B was determined by means of luciferase gene reporter, RT-qPCR, and western blotting assays. The results showed that miR-3175 expression was increased and SCN4B expression was decreased in prostate cancer cell lines as compared with normal human prostatic epithelial cells. Compared with the control group, knockdown of miR-3175 resulted in strong inhibitions of cell growth, migration, invasion, and N-cadherin expression, together with an increase in E-cadherin expression. In addition, knockdown of miR-3175 dramatically increased the luciferase activity of the luciferase vector of SCN4B, and increased SCN4B expression. Together, this study illustrated that downregulation of miR-3175 repressed the proliferation and invasion of prostate cancer cells, which might be induced by SCN4B downregulation.

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

Increased CHST15 follows decline in arylsulfatase B (ARSB) and disinhibition of non-canonical WNT signaling: potential impact on epithelial and mesenchymal identity.

Expression of CHST15 (carbohydrate sulfotransferase 15; chondroitin 4-sulfate-6-sulfotransferase; BRAG), the sulfotransferase enzyme that adds 6-sulfate to chondroitin 4-sulfate (C4S) to make chondroitin 4,6-disulfate (chondroitin sulfate E, CSE), was increased in malignant prostate epithelium obtained by laser capture microdissection and following arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) silencing in human prostate epithelial cells. Experiments in normal and malignant human prostate epithelial and stromal cells and tissues, in HepG2 cells, and in the ARSB-null mouse were performed to determine the pathway by which CHST15 expression is up-regulated when ARSB expression is reduced. Effects of Wnt-containing prostate stromal cell spent media and selective inhibitors of WNT, JNK, p38, SHP2, β-catenin, Rho, and Rac-1 signaling pathways were determined. Activation of WNT signaling followed declines in ARSB and Dickkopf WNT Signaling Pathway Inhibitor (DKK)3 and was required for increased CHST15 expression. The increase in expression of CHST15 followed activation of non-canonical WNT signaling and involved Wnt3A, Rac-1 GTPase, phospho-p38 MAPK, and nuclear DNA-bound GATA-3. Inhibition of JNK, Sp1, β-catenin nuclear translocation, or Rho kinase had no effect. Consistent with higher expression of CHST15 in prostate epithelium, disaccharide analysis showed higher levels of CSE and chondroitin 6-sulfate (C6S) disaccharides in prostate epithelial cells. In contrast, chondroitin 4-sulfate (C4S) disaccharides were greater in prostate stromal cells. CSE may contribute to increased C4S in malignant epithelium when GALNS (N-aceytylgalactosamine-6-sulfate sulfatase) is increased and ARSB is reduced. These effects increase chondroitin 4-sulfates and reduce chondroitin 6-sulfates, consistent with enhanced stromal characteristics and epithelial-mesenchymal transition.

Isorhamnetin inhibited the proliferation and metastasis of androgen-independent prostate cancer cells by targeting the mitochondrion-dependent intrinsic apoptotic and PI3K/Akt/mTOR pathway.

The present study investigated the effects of Isorhamnetin on two types of prostate cancer cells (androgen-independent and androgen-dependent) and explored its possible mechanisms underlying such effects. Treatment with Isorhamnetin significantly inhibited cell growth and induced lactate dehydrogenase (LDH) release of androgen-independent DU145 and PC3 prostate cancer cells, but exhibited almost no toxicity effect on androgen-dependent LNCaP prostate cancer cell line or normal human prostate epithelial PrEC cells, which was achieved by the induction of apoptosis in a mitochondrion-dependent intrinsic apoptotic pathway. Furthermore, Isorhamnetin inhibited cell migration and invasion in concentration-dependent manners by enhancing mesenchymal−epithelial transition (MET) and inhibiting matrix metalloproteinase (MMP) 2 (MMP-2) and MMP-9 overexpression. In addition, Isorhamnetin also down-regulated the expression of phosphorylated PI3K (p-P13K), Akt (p-Akt), and mTOR (p-mTOR) proteins in both cancer cells, revealing Isorhamnetin to be a selective PI3K–Akt–mTOR pathway inhibitor. In summary, these findings propose that Isorhamnetin might be a novel therapeutic candidate for the treatment of androgen-independent prostate cancer.

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution. HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

HOXC6 in the prognosis of prostate cancer

Aims Our study aimed to investigate the expression and prognostic role of homeobox C6 (HOXC6) in prostate cancer (PCa). Methods Relative expression of HOXC6 at mRNA and protein levels in tissues and cell lines of PCa were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis, respectively. Association between HOXC6 expression and clinical factors was analyzed by Chi-square test. HOXC6 effects on the proliferation, invasion and metastasis of PCa cells were severally examined through CCK-8 and transwell assays. Results Relative expressions of HOXC6 at mRNA and protein levels were obviously higher in both PCa tissues and cells than in adjacent non-cancerous tissues and normal human prostate epithelial cells (p < .05). Chi-square test demonstrated that high expression of HOXC6 was significantly associated with PSA concentration, Gleason score and TNM stage (p < .05). The down-regulation of HOCX6 remarkably inhibited the proliferation, migration and invasion of PCa cells. Kaplan-Meier analysis showed that patients with high HOXC6 expression had shorter overall survival than those with low HOXC6 expression (log rank test, p < .001). Conclusion Up-regulated HOXC6, in PCa patients, could not only participate in the progression of PCa but also function as an independent prognostic marker for the cancer.

Triptonide acts as a novel antiprostate cancer agent mainly through inhibition of mTOR signaling pathway.

The increasing incidence of prostate cancer (PCa) indicates an urgent need for the development of new effective drugs in PCa therapy. Triptonide has been reported to have a strong inhibition activity in cancers through screening of Chinese herbal medicine. This study aims to investigate the effects of triptonide on anti-PCa activity and its mechanisms. Three human advanced PCa cell lines PC3, DU145, and LNCap, and a human normal prostate epithelial cell line RWPE were treated with a range (0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, and 320 nM) of triptonide concentrations for 72 hours respectively. Then, cell viability was assessed by cell counting kit-8. PCa cells were treated with different doses (0-20 nM) of triptonide for 72 hours. Cell cycle and apoptosis were assessed by flow cytometry assays. Nude mice bearing human PCa xenografts were intraperitoneally injected daily with either triptonide (10 mg/kg/d) or phosphate-buffered saline as a control for 35 days. RNA-sequencing (RNA-seq) was performed by an Illumina Hiseq Sequencing platform and confirmed by a real-time polymerase chain reaction. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway analysis, and ingenuity pathway analysis were used to analyze RNA-seq results. Triptonide effectively inhibits the proliferation of human PCa cells PC3, DU145, and LNCap in vitro with their IC50 values as 11.961, 10.259, and 12.012 nM, respectively. Triptonide (10 mg/kg) potently inhibits the growth of PCa cell xenografts in vivo at an inhibition rate of over 97.95%. Treatment with triptonide (5 nM) significantly promotes cell apoptosis and retaining cell-cycle arrest in the G2/M phase. RNA-seq data revealed that total of 936 genes were upregulated or downregulated in triptonide treated. Moreover, the phosphorylation of mechanistic target of rapamycin (mTOR) and the downstream protein p70S6K were both inhibited, most obviously in PCa cells. Our findings suggest that triptonide can efficaciously suppress PCa growth in vitro and in vivo via inhibiting the phosphorylation of mTOR and the activities of related downstream signaling pathways.

Inflammatory mediators of prostate epithelial cells stimulated with Trichomonas vaginalis promote proliferative and invasive properties of prostate cancer cells.

Trichomonas vaginalis (Tv) is the most common sexually transmitted parasite. It is detected in prostatic tissue of benign prostatic hyperplasia, prostatitis, and prostate cancer (PCa) and has been suggested to cause chronic prostatitis. Moreover, up to 20% of all cancers worldwide are associated with chronic inflammation. Here, we investigated whether inflammatory mediators produced by normal human prostate epithelial cells (RWPE-1) stimulated with Tv could promote growth and invasiveness of PCa cells. Conditioned medium of RWPE-1 cells was prepared by stimulating them with Tv (trichomonad-conditioned medium [TCM]) and without Tv (conditioned medium [CM]). Promotion of PCa cells (PC3, DU145, and LNCaP) was assessed by wound healing, proliferation, and invasion assays. We observed that the production of interleukin (IL)-1β, IL-6, CCL2, CXCL8, prostaglandin-E2 (PGE2 ), and COX2 by RWPE-1 cells was increased by stimulating them with Tv. When PCa cells were incubated with TCM, their proliferation, invasion, and migration increased. Moreover, they showed increased epithelial-mesenchymal transition (EMT)-related markers by a reduction in epithelial markers and an increase in mesenchymal markers. In vivo, xenograft tumor tissues injected with TCM also showed increased expression of cyclin D1 and proliferating cell nuclear antigen, as well as induction of EMT. Receptors and signal molecules of PCa cells increased in response to exposure to TCM, and blocking receptors (CXCR1, CXCR2, C-C chemokine receptor 2, glycoprotein 130, EP2, and EP4) reduced the proliferation of PCa cells with decreased production of cytokines (CCL2, IL-6, and CXCL8) and PGE2 , and expression of NF-κB and Snail1. Our results suggest that Tv infection may be one of the factors creating the supportive microenvironment to promote proliferation and invasiveness of PCa cells.

Further sesquiterpenoids and phenolics from two species of Taraxacum F.H. Wigg. and cytotoxic activity of taraxinic acid and its derivatives

From roots of Taraxacum flavostylum and Taraxacum lucidum, the species included in the section Taraxacum F. H. Wigg. of the genus Taraxacum F. H. Wigg., eight sesquiterpene lactones, including taraxinic acid (1) and 11β,13-dihydrotaraxinic acid (2), taraxinic acid β-glucopyranosyl ester (3) and its 11β,13-dihydroderivative (4), ainslioside (5), deacetylmatricarin (6), 3-epi-11β,13-dihydrodeacylcynaropicrin (7) and ixerin D (8) together with six phenolic metabolites (9‒14) were isolated. 3-Epi-11β,13-dihydrodeacylcynaropicrin (7), 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (9) and 5-methoxy-eugenyl-4-O-β-glucopyranoside (11) were found for the first time in Taraxacum spp. Compounds 1‒14 were isolated from T. flavostylum roots, whereas only three germacranolides (3‒5), a guaianolide (8) and dihydrosyringin (13) were present in the extract from T. lucidum. Structures of the compounds were elucidated based on spectroscopic data.In vitro cytotoxic activity of taraxinic acid (1) and its derivatives (2‒4) against three human melanoma cell lines (A375, HTB140 and WM793), human normal keratinocytes (HaCaT), human normal prostate epithelial cells (PNT-2), and human prostate carcinoma cell line (DU145) was also examined in this study.

MiR-515-5p acts as a tumor suppressor via targeting TRIP13 in prostate cancer

MiR-515-5p has been suggested to function as tumor suppressor in various human cancers. However, the role of miR-515-5p in prostate cancer was still unclear. In this study, we observed miR-515-5p expression was reduced in prostate cancer tissues and prostate cancer cell lines compared with paired adjacent normal prostatic tissues and normal human prostate epithelial cell lines, respectively. Furthermore, we found miR-515-5p was negatively correlated with TRIP13 mRNA and protein expression in prostate cancer tissue samples, and miR-515-5p directly targeted TRIP13 3'-UTR and negatively regulated TRIP13 mRNA and protein expression. Moreover, miR-515-5p acted as a tumor suppressor to regulate cell proliferation, migration and invasion via targeting TRIP13 in prostate cancer. Besides, we observed that miR-515-5p expression in prostate cancer patients with advanced T stage subgroup or high Gleason score was significantly lower than its expression in prostate cancer patients with early T stage or low Gleason score. Survival analysis suggested that prostate cancer cases with high miR-515-5p expression had better prognosis than prostate cancer cases with low miR-515-5p expression. In conclusion, our study highlights the clinical and biological role of miR-515-5p as tumor suppressive microRNA in prostate cancer.

Changes in receptor location affect the ability of oxytocin to stimulate proliferative growth in prostate epithelial cells.

In normal prostate cells, cell membrane receptors are located within signalling microdomains called caveolae. During cancer progression, caveolae are lost and sequestered receptors move out onto lipid rafts. The aim of this study was to investigate whether a change in the localisation of receptors out of caveolae and onto the cell membrane increased cell proliferation invitro, and to determine whether this is related to changes in the cell signalling pathways. Normal human prostate epithelial cells (PrEC) and androgen-independent (PC3) cancer cells were cultured with 10nM dihydrotestosterone (DHT). The effects of oxytocin (OT) and gonadal steroids on proliferation were assessed using the MTS assay. Androgen receptor (AR) and oxytocin receptor (OTR) expression was identified by immunofluorescence and quantified by western blot. OTR and lipid raft staining was determined using Pearson's correlation coefficient. Protein-protein interactions were detected and the cell signalling pathways identified. Treatment with OT did not affect the proliferation of PrEC. In PC3 cells, OT or androgen alone increased cell proliferation, but together had no effect. In normal cells, OTR localised to the membrane and AR localised to the nucleus, whereas in malignant cells both OTR and AR were identified in the cell membrane. Colocalisation of OTR and AR increased following treatment with androgens. Significantly fewer OTR/AR protein-protein interactions were seen in PrEC. With OT treatment, several cell signalling pathways were activated. Movement of OTR out of caveolae onto lipid rafts is accompanied by activation of alternative signal transduction pathways involved in stimulating increased cell proliferation.

Upregulation of Circular RNA Itchy E3 Ubiquitin Protein Ligase Inhibits Cell Proliferation and Promotes Cell Apoptosis Through Targeting MiR-197 in Prostate Cancer.

Objective:This study aimed to investigate the effect of circular RNA itchy E3 ubiquitin protein ligase on cell proliferation and apoptosis and to explore its target micro-RNAs in prostate cancer cells.Methods:Circular RNA itchy E3 ubiquitin protein ligase expression in human prostate cancer cells and normal prostate epithelial cells was determined by real time-quantitative polymerase chain reaction assay. Circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids (circular RNA itchy E3 ubiquitin protein ligase(+) group and control overexpression plasmids group were transfected with PC-3 cells. Rescue experiment was performed by transfection of circular RNA itchy E3 ubiquitin protein ligase overexpression and micro-197 overexpression plasmids (circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids/micro RNA (+) group) into PC-3 cells. Cell Counting Kit-8 and annexin V/propidium iodide assays were conducted to evaluate cell proliferation and apoptosis, respectively. Western blot was performed to determine the expressions of apoptotic-related markers.Results:Circular RNA itchy E3 ubiquitin protein ligase expression was decreased in DU 145, 22RV1, VCaP, and PC-3 cells compared to RWPE cells. In PC-3 cells, cell proliferation rate was reduced in circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids group compared to control overexpression plasmids group at 48 hours and 72 hours. Cell apoptosis rate was elevated in circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids group compared to control overexpression plasmids group at 48 hours, and Western blot showed the similar results. Micro RNA-197 but not micro RNA-31 or micro RNA-432 was the target micro-RNA of circular RNA itchy E3 ubiquitin protein ligase. In rescue experiments, cell proliferation rate was elevated, but apoptosis rate was reduced in circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids/micro RNA (+) group compared to circular RNA itchy E3 ubiquitin protein ligase overexpression plasmids group, indicating that circular RNA itchy E3 ubiquitin protein ligase upregulation inhibited cell proliferation but promoted apoptosis through downregulating micro RNA-197.Conclusion:Circular RNA itchy E3 ubiquitin protein ligase upregulation suppresses cell proliferation but promotes apoptosis through targeting micro RNA-197 in prostate cancer. Our study may provide a new insight for the treatment of prostate cancer.

AMD3100 inhibits epithelial-mesenchymal transition, cell invasion, and metastasis in the liver and the lung through blocking the SDF-1α/CXCR4 signaling pathway in prostate cancer.

Stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) have been found to be tightly correlated with the progression of prostate cancer (PC). In this study, we investigated the effects of an SDF-1α/CXCR4 inhibitor, AMD3100, on cell progression and metastasis potential of human PC cells. Human PC cell lines (LNCaP, PC3, and DU145) were cultured to detect SDF-1α/CXCR4, which showed higher SDF-1α and CXCR4 expression than the normal human prostate epithelial cell line, RWPE-1. AMD3100 was confirmed to be an inhibitor of SDF-1α, and to detect the effect of SDF-1α/CXCR4 inhibition on PC, PC cells were treated with AMD3100 or/and CXCR4 siRNA. The results suggested that inhibition of the SDF-1α/CXCR4 pathway could promote the E-cadherin level but inhibit the levels of invasion and migration of vimentin, N-cadherin and α5β1 integrin. Finally, tumor formation in nude mice was conducted, and the cell experiment results were verfied. These data show that AMD3100 suppresses epithelial-mesenchymal transition and migration of PC cells by inhibiting the SDF-1α/CXCR4 signaling pathway, which provides a clinical target in the treatment of PC.