Prostate Epithelial Cell Apoptosis Research Articles

Aberrant activated Notch1 promotes prostate enlargement driven by androgen signaling via disrupting mitochondrial function in mouse

The prostate is a vital accessory gonad in the mammalian male reproductive system. With the ever-increasing proportion of the population over 60 years of age worldwide, the incidence of prostate diseases, such as benign prostatic hyperplasia (BPH) and prostate cancer (PCa), is on the rise and is gradually becoming a significant medical problem globally. The notch signaling pathway is essential in regulating prostate early development. However, the potential regulatory mechanism of Notch signaling in prostatic enlargement and hyperplasia remains unclear. In this study, we proved that overactivation of Notch1 signaling in mouse prostatic epithelial cells (OEx) led to prostatic enlargement via enhancing proliferation and inhibiting apoptosis of prostatic epithelial cells. Further study showed that N1ICD/RBPJ directly up-regulated the androgen receptor (AR) and enhanced prostatic sensitivity to androgens. Hyper-proliferation was not found in orchidectomized OEx mice without androgen supply but was observed after Dihydrotestosterone (DHT) supplementation. Our data showed that the number of mitochondrion in prostatic epithelial cells of OEx mice was increased, but the mitochondrial function was impaired, and the essential activity of the mitochondrial respiratory electron transport chain was significantly weakened. Disordered mitochondrial number and metabolic function further resulted in excessive accumulation of reactive oxygen species (ROS). Importantly, anti-oxidant N-Acetyl-L-Cysteine (NAC) therapy could alleviate prostatic hyperplasia caused by the over-activation of Notch1 signaling. Furthermore, we observed the incremental Notch signaling activity in progenitor-like club cells in the scRNA-seq data set of human BPH patients. Moreover, the increased number of TROP2+ progenitors and Club cells was also confirmed in our OEx mice. In conclusion, our study revealed that over-activated Notch1 signaling induces prostatic enlargement by increasing androgen receptor sensitivity, disrupting cellular mitochondrial metabolism, increasing ROS, and a higher number of progenitor cells, all of which can be effectively rescued by NAC treatment.

Benign prostatic hyperplasia treatment using plasmonic nanoparticles irradiated by laser in a rat model

ObjectiveIn the current study we have stimulated the efficacy of plasmonic nanoparticles (NPs) by laser hyperthermia to achieve a less invasive method for tumor photothermal therapy of benign prostatic hyperplasia (BPH). MethodsThe levels of apoptosis on induced BPH in rats were assessed after treatment and revealed and recorded by various assayed. Moreover, the expression of caspases was considered to demonstrate the apoptotic pathways due to laser induced plasmonic NPs. ResultsIn the Laser + NPs group prostate size of induced BPH decreased. Laser + NPs also decreased prostate specific antigen in comparison with the BPH groups. Furthermore, Laser + NPs attenuated BPH histopathologic indices in the rats. Laser + NPs induced apoptosis in prostatic epithelial cells via caspase-1 pathway. ConclusionsAltogether, the approach and findings from this study can be applied to introduce the laser irritated NPs method as a novel and less invasive therapy for patients suffering from BPH.

Inhibition of p53 alleviates prostate cell apoptosis in Escherichiacoli‑induced bacterial prostatitis.

Previous studies demonstrated that uropathogenic Escherichiacoli infection contributes to human bacterial prostatitis. Apoptosis of prostate epithelial cells is closely associated with the progression of bacterial prostatitis. The aim of the present study was to investigate the effect of cellular tumor antigen p53(p53) on the apoptosis of bacterial prostatitis cells. The prostate epithelial RWPE‑1 cell line was infected with Escherichiacoli, and treated cells and the culture supernatant were obtained at specific time points. The cell apoptosis rates, protein and mRNA of p53 were detected in the different treatment groups. Flow cytometry and terminal deoxynucleotidyl‑transferase‑mediated dUTP nick end labeling assays were used for the detection of cell apoptosis, and cell proliferation was determined by a Cell Counting Kit‑8 assay. The expression of p53 was inhibited by small interfering (si)RNA, and its mRNA and protein were detected. An ELISA was used for detecting cytokines in the culture supernatant. The result demonstrated that Escherichiacoli infection led to an increase in prostate epithelial cell apoptosis (P<0.05), and resulted in increases of interleukin (IL)‑4, IL‑6 and IL‑8, and decrease in IL‑10. p53, apoptosis regulator BAX (Bax), caspase‑9 and Caspase‑3 expression were upregulated upon Escherichiacoli exposure (P<0.05). Following transfection with p53 siRNA, the promotion of cell apoptosis induced by Escherichiacoli infection was decreased, and the p53 and Bax protein expression were additionally decreased. Therefore, it was suggested that Escherichiacoli increases cell apoptosis in bacterial prostatitis by activating the death receptor pathway involving p53. Inhibition of p53 alleviated prostate cell apoptosis induced by Escherichiacoli.

Castration-induced prostate epithelial cell apoptosis results from targeted oxidative stress attack of M1142 -macrophages.

Prostate development and function are regulated by androgens. Epithelial cell apoptosis in response to androgen deprivation is caspase-9-dependent and peaks at Day 3 after castration. However, isolated epithelial cells survive in the absence of androgens. Znf142 showed an on-off expression pattern in intraepithelial CD68-positive macrophages, with the on-phase at Day 3 after castration. Rats treated with gadolinium chloride to deplete macrophages showed a significant drop in apoptosis, suggesting a causal relationship between macrophages and epithelial cell apoptosis. Intraepithelial M1-polarization was also limited to Day 3, and the inducible nitric oxide synthase (iNOS) knockout mice showed significantly less apoptosis than wild-type controls. The epithelial cells showed focal DNA double-strand breaks (DSB), 8-oxoguanine, and protein tyrosine-nitrosylation, fingerprints of exposure to peroxinitrite. Cultured epithelial cells induced M1-polarization and showed focal DSB and underwent apoptosis. The same phenomena were reproduced in LNCaP cells cocultured with Raw 264.7 macrophages. In conclusion, the M1 142 -macrophage (named after Znf142) attack causes activation of the intrinsic apoptosis pathway in epithelial cells after castration.

SENP1 regulates PTEN stability to dictate prostate cancer development.

SUMO protease SENP1 is elevated in multiple carcinomas including prostate cancer (PCa). SENP1 exhibits carcinogenic properties; it promotes androgen receptor-dependent and -independent cell proliferation, stabilizes HIF1a, increases VEGF, and supports angiogenesis. However, mice expressing an androgen-responsive promoter driven SENP1-transgene (SENP1-Tg) develop high-grade prostatic intraepithelial neoplasia, but not carcinoma. We now show that tumor suppressive PTEN signaling is induced in SENP1-Tg to enhance prostate epithelial cell apoptosis. SENP1 blocks SUMO1-dependent ubiquitylation and degradation of PTEN. In the absence of SENP1, SUMO1-modified PTEN is sequestered in the cytosol, where binding to ubiquitin-E3 ligase WWP2 occurs. Concurrently, WWP2 is also SUMOylated, which potentiates its interaction with PTEN. Thus, SENP1 directs ubiquitin-E3-substrate association to control PTEN stability. PTEN serves as a barrier for SENP1-mediated prostate carcinogenesis as SENP1-Tg mice develop invasive carcinomas only after PTEN reduction. Hence, SENP1 modulates multiple facets of carcinogenesis and may serve as a target specifically for aggressive PTEN-deficient PCa.

Notch promotes tumor metastasis in a prostate-specific Pten-null mouse model.

Although Notch signaling is deregulated in prostate cancer, the role of this pathway in disease development and progression is not fully understood. Here, we analyzed 2 human prostate cancer data sets and found that higher Notch signaling correlates with increased metastatic potential and worse disease survival rates. We used the Pten-null mouse prostate cancer model to investigate the function of Notch signaling in the initiation and progression of prostate cancer. Disruption of the transcription factor RBPJ in Pten-null mice revealed that endogenous canonical Notch signaling is not required for disease initiation and progression. However, augmentation of Notch activity in this model promoted both proliferation and apoptosis of prostate epithelial cells, which collectively reduced the primary tumor burden. The increase in cellular apoptosis was linked to DNA damage-induced p53 activation. Despite a reduced primary tumor burden, Notch activation in Pten-null mice promoted epithelial-mesenchymal transition and FOXC2-dependent tumor metastases but did not confer resistance to androgen deprivation. Notch activation also resulted in transformation of seminal vesicle epithelial cells in Pten-null mice. Our study highlights a multifaceted role for Notch signaling in distinct aspects of prostate cancer biology and supports Notch as a potential therapeutic target for metastatic prostate cancer.

27-Hydroxycholesterol stimulates cell proliferation and resistance to docetaxel-induced apoptosis in prostate epithelial cells.

Although the causes of prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are not known, the role of oxidative stress, aging, and diet are suspected to increase the incidence of prostate complications. The cholesterol oxidation derivative (oxysterol) 27-hydroxycholesterol (27-OHC) is the most prevalent cholesterol metabolite in the blood. As aging, oxidative stress, and hypercholesterolemia are associated with increased risk of PCa and BPH, and because 27-OHC levels are also increased with aging, hypercholesterolemia, and oxidative stress, determining the role of 27-OHC in the progression of PCas and BPH is warranted. In this study, we determined the effect of 27-OHC in human prostate epithelial cells RWPE-1. We found that 27-OHC stimulates proliferation and increases androgen receptor (AR) transcriptional activity. 27-OHC also increased prostate-specific antigen expression and enhanced AR binding to the androgen response element compared to controls. Silencing AR expression with siRNA markedly reduced the 27-OHC-induced proliferation. Furthermore, 27-OHC blocked docetaxel-induced apoptosis. Altogether, our results suggest that 27-OHC may play an important role in PCa and BPH progression by promoting proliferation and suppressing apoptosis.

FOXP3-microRNA-146-NF-κB as oncotarget.

The X-linked FOXP3 gene is a member of the forkhead box/winged helix family. As a transcription factor, FOXP3 plays an important role in the immune system by acting as a master regulator of transcription for the development and function of regulatory T cells. Additionally, its tumor suppressive activity has been observed during tumor initiation [1]. MicroRNA (miR) plays a role in RNA silencing and in post transcriptional regulation of gene expression. Accumulating data suggest that the miR-146 family, including miR-146a/b, inhibits cancer cell proliferation, invasion and metastasis in human breast and prostate cancers [2-5]. Since miR-146a is overexpressed in Foxp3+ regulatory T cells and is critical for its function in the immune system, there is thought to be a link between miR-146a and the FOXP3 gene [6]. Additionally, nuclear factor-kappaB (NF-κB), a protein complex that controls transcriptional activity of DNA and helps regulate cell survival, was instrumental to the discovery of a novel pathway that may serve as a target of drug therapy for cancer patients. In these two papers, a new axis was identified and is known as the FOXP3-miR-146-NF-κB axis (Fig. ​(Fig.1)1) [7, 8]. In breast cancer cells, chromatin immunoprecipitation sequencing was used to identify a series of potential FOXP3-targeted miRs [7]. In particular, FOXP3 dramatically induced the expression of miR-146a/b that prevented tumor cell proliferation while also enhancing apoptosis. Functional analyses showed that FOXP3-induced miR-146a/b downregulated NF-κB activation by inhibiting the expression of two miR-146 target genes IRAK1 (involved in Toll/IL-1 signaling) and TRAF6 (a member of a family of proteins involved in the regulation of inflammation and apoptosis) [7]. Furthermore, chromatin immunoprecipitation assays revealed that FOXP3 directly bound to the promoter region of miR-146a but not to that of miR-146b. In addition, a direct interaction of FOXP3 with NF-κB p65 was identified to regulate a miR-146a-NF-κB negative feedback regulation loop in normal breast epithelial cells as well as in breast cancer cells (Figure ​(Figure1).1). In vivo analyses also validated that FOXP3-mediated induction of miR-146a resulted in the downregulation of IRAK1 and TRAF6 and subsequently inhibited NF-κB activation, thus leading to tumor suppression in breast cancer cells. Conversely, FOXP3-mediated inhibition of cell proliferation and tumor growth and its induction of apoptosis were partially blocked by miR-146a/b inhibitors, indicating a contribution of miR-146a/b to FOXP3-triggered tumor suppression. Figure 1 FOXP3 directly targets miR-146a and regulates the miR-146a and NF-κB feedback loop in cancer cells It has been determined that the FOXP3-miR-146-NF-κB axis serves as a target pathway for diminishing the pre-prostate cancerous state in the animal model [8]. In prostate cancer cells, it was also validated that FOXP3 transcriptionally inhibits IRAK1 and TRAF6 through an induced expression of miR-146a/b [8]. Hybridization analysis indicated that a low expression of miR-146a/b was observed in prostate cancer cell lines, while a high expression of miR-146a/b was seen in normal prostate tissues [4]. Transfection of miR-146a into prostate cancer cells resulted in a marked reduction of cell invasion, proliferation and metastasis to bone marrow [4, 5], suggesting that miR-146a functions as a tumor suppressor in prostate cancer cells. The functional FOXP3-miR-146-NF-κB axis during tumor initiation in prostate cancer cells was identified in prostate Foxp3 conditional knockout (Foxp3cKO) mice. It was further noted that miR-146a KO mice and Foxp3cKO mice developed prostatic intraepithelial neoplasia (PIN, a pre-prostate cancerous state), but not prostate cancer, suggesting that miR-146a as well as FOXP3 have a tumor-suppressive role during tumor initiation [1, 8]. Since FOXP3 can functionally interact with NF-κB to induce transcriptional activity of miR-146a, the FOXP3-miR-146-NF-κB axis could be a potential therapeutic target for cancers with FOXP3 defects. In fact, the NF-κB inhibitor bortezomib induced apoptosis in prostate epithelial cells of Foxp3cKO mice [8], suggesting that NF-κB-related apoptosis is involved in FOXP3-mediated tumor suppression. Although bortezomib did not completely eliminate PIN formation, its administration inhibited mouse prostate tumor growth and reduced PIN incidences. This was most likely due to an increase in apoptotic cells and shows the potential target of the FOXP3-miR-146-NF-κB axis in patients experiencing a precancerous state. Ultimately, FOXP3-induced miR-146a/b inhibited NF-κB activation by repressing IRAK1 and TRAF6, which led to tumor suppression and apoptosis during tumor initiation in breast and prostate epithelial cells. Additionally, the identification of the FOXP3-miR-146-NF-κB axis provides a mechanism for NF-κB activation as well as miR-146a/b disruption in breast and prostate cancer cells, allowing for a new therapeutic target in FOXP3 defect-related cancers.

FOXP3-miR-146-NF-κB Axis and Therapy for Precancerous Lesions in Prostate.

The tumor-suppressive activity of FOXP3 has been observed in tumor initiation, but the underlying mechanism still remains largely unknown. Here, we identified a FOXP3-microRNA-146 (miR-146)-NF-κB axis in vitro and in vivo in prostate cancer cells. We observed that FOXP3 dramatically induced the expression of miR-146a/b, which contributed to transcriptional inhibition of IRAK1 and TRAF6, in prostate cancer cell lines. Tissue-specific deletion of Foxp3 in mouse prostate caused a significant reduction of miR-146a and upregulation of NF-κB activation. In addition, prostatic intraepithelial neoplasia lesions were observed in miR-146a-mutant mice as well as in Foxp3-mutant mice. Notably, the NF-κB inhibitor bortezomib inhibited cell proliferation and induced apoptosis in prostate epithelial cells, attenuating prostatic intraepithelial neoplasia formation in Foxp3-mutant mice. Our data suggest that the FOXP3-miR-146-NF-κB axis has a functional role during tumor initiation in prostate cancer. Targeting the miR-146-NF-κB axis may provide a new therapeutic approach for prostate cancers with FOXP3 defects.

Abstract 1804: New insights in the induction of cell death by the imidazolium based compound YM155

Abstract The inhibitor of apoptosis protein (IAP) Survivin is robustly over-expressed in a variety of cancers including prostate carcinomas, and has been shown to play pivotal roles in cancer progression and resistance to chemotherapy. The imidazolium based small molecule YM155, which was first identified as a robust suppressor of the Survivin promoter of PC-3 prostate carcinoma and under clinical investigation for the treatment of multiple cancers, is a potent apoptosis inducer of castration-resistant prostate cancer (CRPC) cells. However, the mechanisms by which it inhibits the expression of Survivin and kills cancer cells remain unclear. Here we studied early signaling responses to YM155 in a panel of prostate and kidney cancer cell lines in standard cell culture conditions and showed that YM155 rapidly decreases the expression of cyclin Ds 1, 2 and 3, and activates the retinoblastoma protein (Rb), leading to the downregulation of the Survivin promoter. However, despite the robust lost of cyclin Ds, YM155 promotes an S-phase arrest rather than arrests cells at either G1 or G2/M. This suggests cell killing by YM155 involves S-phase arrest, consistent with a DNA damage response. In addition, we show that YM155 also downregulates the expression of the anti-apoptotic protein Mcl-1 and induces the expression of proapoptotic proteins Bad and Bim followed by the activation of caspases -3 and -8. Moreover, early time course experiments show that YM155 induces rapid (10 - 60 min) but transient activation of Akt and Erk1/2 followed by a marked suppression by 4 to 8 h likely via the induced expression of phosphatases 1 and 5 (DUSP1 and 5). Taken together we suggest YM155 induces apoptosis of prostate epithelial cells through a complex mechanism involving Akt, Erk1/2, DUSPs, cyclin Ds, Rb, S-phase arrest, Bim, Bad, Mcl-1,Survivin, and activation of caspases -3 and -8. Citation Format: David Danielpour, Eswar Shankar, Sarah L. Corum. New insights in the induction of cell death by the imidazolium based compound YM155. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1804. doi:10.1158/1538-7445.AM2014-1804

Inhibition of androgen induces autophagy in benign prostate epithelial cells

5-α Reductase inhibitor can reduce the volume of benign prostatic hyperplasia by lowering benign prostatic hyperplasia level and consequently inducing epithelial cells apoptosis. The present study investigated whether autophagy and apoptosis of benign prostatic hyperplasia epithelial cells are influenced by low benign prostatic hyperplasia levels. PWR-1E prostate epithelial cells transfected with GFP-LC3 plasmid were subjected to androgen deprivation conditions. Then the autophagic puncta were evaluated by fluorescence microscopy, and the cellular apoptosis rate was detected by 4, 6-diamidino-2-phenylindole staining after blocking of autophagic process by 3-methyladenine. Furthermore, autophagy status was also determined in hyperplasia prostate tissues from 5-α reductase inhibitor-treated patients by immunohistochemistry. In the androgen deprivation medium, autophagic punta increased markedly in PWR-1E cells, and blockage of autophagy by 3-methyladenine significantly promoted PWR-1E cells' apoptosis rate. In vivo, the expression of LC3 protein (an important autophagic marker) in hyperplasia prostate tissue significantly increased after 5-α reductase inhibitor treatment. Meanwhile, the prostate-specific antigen, as an inner control, decreased. 5-α Reductase inhibitor treatment increases autophagy and possibly decreases the apoptosis of prostate epithelial cells.

Abstract 2695: Olfactomedin 4 gene is associated with progression of prostate cancer: Evidence from an OLFM4 knockout mouse model

Abstract The olfactomedin 4 gene (OLFM4) encodes an olfactomedin-related glycoprotein whose physiological and pathological functions are largely unknown. We have previously reported that expression of OLFM4 is down regulated during the progression of human prostate cancer, and that reduced expression is due to gene deletions of OLFM4 (36.7%) and/or hyper-methlytion of CpG sites in the promoter region. We also demonstrated that ectopic expression of OLFM4 in several human prostate cancer cell lines inhibits proliferation, invasion and metastatic potential. In this study, we further investigated the potential role of OLFM4 in the carcinogenesis of prostate cancer utilizing an OLFM4 knock out mouse model. Histopathological analysis revealed that homozygous OLFM4 mutant mice develop prostatic epithelial lesions in an age-dependent manner. The prostatic epithelial hyperplasia was seen at 6 months of age, prostatic intraepithelial neoplasia (PIN) was found at 10-12 months of age, and prostate adenoma was observed from 18-24 months of ages in the both anterior prostate (AP) and dorsal-lateral prostate (DLP) of OLFM4-null mice. The frequency of PIN lesions at the AP and DLP was 0 % (0/20) at 3-6 months, 38% (5/13) at 10-12 months and 44% (11/25) at 18-24 months. The frequency of prostatic adenoma was 28% (7/25), and 8% (2/25) of OLFM4-null mice developed lung metastases at 18-24 months of age. To determine whether OLFM4 affects proliferation or apoptosis of prostatic epithelial cells in the OLFM4-null mice, we carried out Ki-67 immunohistochemical staining and TUNEL assays. The ratio of Ki-67 positive cells was significantly higher at 3-6 months (p=0.04180), 10-12 months (P=0.002) and 18-20 months (p=0.0038) compared to age-matched controls, OLFM4 wild type mice, or heterozygous mice, thus indicating an increase in proliferation in OLFM4-null prostate tissues. There was no difference in the number of apoptotic cells in the control versus null mice. Since we have previously found that OLFM4 blocks CXCL12 induced AKT activity in the human prostate cancer cell PC-3, and inhibits cell invasion, we studied the phosphorylation of AKT and downstream signaling pathway components by Western blot analysis of prostate samples from 3-4 month old mice. Compared with OLFM4 wild type mice, pAKT, pGSK3β, β-catenin, cyclin D1 and c-Myc were increased in the OLFM4-null mice tissue even in the absence of morphological abnormalities. Elevated β-catenin protein in OLFM4-null mice prostate epithelial cells was also observed in PIN and tumor cells at 18 months of age, and positive staining with Cyclin D1 in OLFM4-null tumor was also detected. Taken together, the data show OLFM4-null prostatic epithelial cells exhibit higher levels of AKT and β-catenin, suggesting this is the mechanism leading to their enhanced cellular proliferation and carcinogenetic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2695. doi:1538-7445.AM2012-2695

Abstract C10: Tumor necrosis factor is necessary but not sufficient for castration-induced prostate regression

Abstract TNF, a pro-inflammatory and immune-regulatory cytokine, is a potent apoptotic stimulus in vitro. There are few examples of a physiologic role for TNF induced apoptosis in vivo, but here we describe a novel role for TNF in prostate epithelial cell apoptosis following androgen withdrawal. We demonstrate, using high resolution serial MRI (magnetic resonance imaging) to measure mouse prostate volume changes over time, that castration induced prostate regression is significantly reduced in mice null for either the Tnf or Tnfr1 genes, but not mice deficient for TRAIL or Fas signaling. Wild type mice treated with soluble TNFR2 (to bind TNF and block signaling) prior to castration showed a similar reduction. Additionally, consistent with a paracrine role for TNF in prostate regression, membrane-bound TNF protein and stromal cell specific TNF mRNA levels increase in rat prostate following castration. Injection of soluble TNF restores normal levels of prostate regression of Tnf −/− mice. Together, these data indicate that uniquely among these extrinsic death signals, TNF is required for castration induced prostate regression. However, wild type mice receiving soluble TNF in the absence of castration do not exhibit prostate regression, indicating that TNF alone is not sufficient, but acts in the context of additional castration induced signals. A preliminary investigation using ALK5 inhibitors prior to castration suggests that TGFβ signaling may also be important. Understanding the physiologic role for TNF, and the interaction with other signaling pathways active in prostate regression following androgen withdrawal may lead to novel therapies for prostate cancer. Citation Format: Jennifer S. Davis, Kent L. Nastiuk. Tumor necrosis factor is necessary but not sufficient for castration-induced prostate regression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C10.

Opposing roles of ERα and ERβ in the genesis and progression of adenocarcinoma in the rat ventral prostate

Prostate cancer is a common malignancy in men and although hormone ablation therapy is effective, men develop hormone resistance. There is need for therapies applicable earlier, such as treatment of prostatic intraepithelial neoplasia (PIN). Estrogens besides androgens play a role in prostate cancer pathogenesis via two receptors ERα and ERβ and both receptors are thought to play different, opposing, roles with ERα having proliferative properties and ERβ having anti-proliferative properties. To differentiate between the roles both receptors play in prostate cancer an ERα and an ERβ agonist, ERA-45 and ERB-26, have been tested in a rodent model for prostate carcinogenesis. The influence of ERα on prostate cancer progression was studied in intact male rats treated with testosterone in combination with the ERα agonist, ERA-45 for either a long-term (20-week) period or a shorter term (6-week) period. The ERβ agonist was tested in the shorter term model in intact male rats treated with testosterone in combination with the ERα agonist, ERA-45, followed by administration of the ERβ agonist, ERB-26, during the last 2 weeks. Treatment of rats with testosterone in combination with ERA-45 induced mild PIN lesions at 6 weeks and severe precancerous PIN lesions at 20 weeks. The ERβ agonist prevented the onset of PIN lesions at 6 weeks. Moreover, prostate epithelial cell apoptosis was increased and proliferation was decreased. These findings confirm the opposing roles ERα and ERβ play in prostate carcinogenesis and suggest a therapeutic opportunity of ERβ for treating precancerous PIN lesions.

TNF Is Necessary for Castration-Induced Prostate Regression, Whereas TRAIL and FasL Are Dispensable

TNF, a proinflammatory and immune-regulatory cytokine, is a potent apoptotic stimulus in vitro. However, there have been few examples of a physiologic role for TNF-induced apoptosis in vivo. Here, we describe a novel role for TNF in prostate epithelial cell apoptosis after androgen withdrawal. Employing high-resolution serial magnetic resonance imaging to measure mouse prostate volume changes over time, we demonstrate that the extent of castration-induced prostate regression is significantly reduced in mice null for either the Tnf or Tnfr1 genes but not mice deficient for TNF-related apoptosis-inducing ligand or Fas signaling. Wild-type mice receiving soluble TNF (sTNF) receptor 2 (to bind TNF and block signaling) before castration exhibit an identical reduction of prostate regression. Together, these data indicate that uniquely among known extrinsic death signals, TNF is required for castration-induced prostate regression. Additionally, membrane-bound TNF protein and stromal cell specific TNF mRNA levels increase in rat prostate after castration. This is consistent with a paracrine role for TNF in prostate regression. When injected into the peritoneum of Tnf(-/-) mice at the time of castration, sTNF restores normal levels of prostate regression. However, wild-type mice receiving sTNF in the absence of castration do not exhibit prostate regression, indicating that TNF alone is not sufficient but acts in the context of additional castration-induced signals. These findings support a physiologic role for TNF in prostate regression after androgen withdrawal. Understanding this role may lead to novel therapies for prostate cancer.

Prostate epithelial stem cells are resistant to apoptosis after α1-antagonist treatment. The impact for BPH patients

IntroductionInduction of apoptosis in prostatic epithelial cells by doxazosin, terazosin and prazosin has been well documented. However, the biochemical pathways of doxazosin action is still unclear. Aforementioned drugs should lead to decrease of prostate volume, although this effect was never observed in patients suffering from BPH after treatment with α1-antagonists. Probably, it is connected with cancer stem cells’ resistance on chemotherapeutic agents. The aim of this study was to compare incidence of apoptosis induced by doxazosin in progenitor and differentiated cells isolated from human prostate epithelium.Material and methodsFor this purpose tissue specimens were obtained from 10 patients suffering from BPH, the primary cultures of prostate epithelium were established and CD133 MicroBeads sorting was prepared. Both, CD133(+)/CD133(-) co-cultures and CD133(+) cells were incubated with different concentration of doxazosin for 12 h. Cell viability and apoptosis was estimated with Annexin V-FITC.Results12 h incubation of CD133(+)/CD133(-) co-cultures with doxazosin resulted in increase of apoptotic cells, while in CD133(+) cultures no changes were observed. Correlation between apoptotic cell number and doxazosin concentration in CD133(+)/ CD133(-) co-cultures group was high (R = 0.99).ConclusionDoxazosin induced apoptosis in co-cultures of progenitor and differentiated epithelial cells. However, progenitor cells were not susceptible to apoptosis, what can be a reason of treatment failure in BPH patients.

DHT Selectively Reverses Smad3-Mediated/TGF-β-Induced Responses through Transcriptional Down-Regulation of Smad3 in Prostate Epithelial Cells

Androgens suppress TGF-β responses in the prostate through mechanisms that are not fully explored. We have recently reported that 5α-dihydrotestosterone (DHT) suppresses the ability of TGF-β to inhibit proliferation and induce apoptosis of prostatic epithelial cells and provided evidence that such suppression was fueled by transcriptional down-regulation of TGF-β receptor II (ΤβRII). We now show that androgen receptor (AR) activated by DHT suppresses the TGF-β-induced phosphorylation of Sma- and Mad-related protein (Smad)3 in LNCaP cells overexpressing TβRII under the control of a cytomegalovirus promoter, which is not regulated by DHT, suggesting that transcriptional repression of TβRII alone does not fully account for the impact of DHT on TGF-β responses. Instead, we demonstrate that such suppression occurs through loss of total Smad3, resulting from transcriptional suppression of Smad3. We provide evidence that DHT down-regulates the promoter activity of Smad3 in various prostate cancer cell lines, including NRP-154+AR, DU145+AR, LNCaP, and VCaP, at least partly through androgen-dependent inactivation of Sp1. Moreover, we show that overexpression of Smad3 reverses the ability of DHT to protect against TGF-β-induced apoptosis in NRP-154+AR, supporting our model that loss of Smad3 by DHT is involved in the protection against TGF-β-induced apoptosis. Together, these findings suggest that deregulated/enhanced expression and activation of AR in prostate carcinomas may intercept the tumor suppressor function of TGF-β through transcriptional suppression of Smad3, thereby providing new mechanistic insight into the development of castration-resistant prostate cancer.

Abstract 3967: Dihydrotestosterone downregulates expression of smad3 in prostate epithelial cells through a transcriptional mechanism, promoting androgenic protection against TGF-beta-induced apoptosis

Abstract Androgens suppress TGF-beta responses in the prostate through mechanisms that are not fully explored. We have recently reported that 5a-dihydrotestosterone (DHT) suppresses the ability of TGF-beta to inhibit proliferation and to induce apoptosis of prostatic epithelial cells, and provided evidence that such suppression was fueled by the transcriptional downregulation of TGF-beta receptor II (TbRII). We now show that AR activated by DHT suppresses the TGF-beta-induced phosphorylation of Smad3 in LNCaP cells overexpressing TbRII under the control of a CMV promoter, suggesting that transcriptional repression of TbRII alone does not fully account for the impact of DHT on TGF-beta responses. Instead, we demonstrate that such suppression occurs through loss of total Smad3, resulting from transcriptional suppression of Smad3. Consistent with this mechanism, we report that loss of Smad3 mRNA inversely correlates with elevated AR mRNA in a cohort of human prostate tissues (p &amp;lt; 0.0001). We provide evidence that DHT downregulates the promoter activity of Smad3 in various prostate cancer cell lines, including NRP-154+AR, DU145+AR, LNCaP, and VCaP, at least partly through androgen-dependent inactivation of Sp1. Moreover, we show that overexpression of Smad3 reverses the ability of DHT to protect against TGF-beta-induced apoptosis in NRP-154, supporting our model that loss of Smad3 by DHT is involved in the protection against TGF-beta-induced apoptosis. Together, these findings suggest that deregulated/enhanced expression and activation of AR in prostate carcinomas may intercept the tumor suppressor function of TGF-beta through transcriptional suppression of Smad3, thereby providing new mechanistic insight on the development of castration-resistant prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3967.

Effectiveness and significance of prostatic ischemia in rabbit

To evaluate the effect of prostatic ischemia in rabbit and approach the significance of ischemia in the development of benign prostatic hyperplasia. Male rabbits underwent surgical prostatic ischemia for durations of 1, 2, 4, 8 and 12 weeks and sham-operated rabbits served as controls. The weight of prostate was examined and a histological examination conducted. Ki67 immunohistochemical stain for the generation of prostatic cells and TUNEL test for the apoptosis of prostatic cells were used. In sham-operated rabbits, the mean (SD) weight of prostate were 0.831(0.127) g, the mean (SD) positive Ki67 staining prostatic epithelial cells 2.1(0.8) in 100 prostatic epithelial cells and stromal cells 2.6(1.1) in 100, mean (SD) positive TUNEL test prostatic epithelial cells 2.0(1.0) in 100 and stromal cells 2.5(0.7) in 100. In operated rabbits, the generation of prostatic stromal cells increased and the apoptosis of prostatic stromal cells decreased over 1 - 12 weeks. The generation of prostatic epithelial cells increased and the apoptosis of prostatic epithelial cells decreased over 2 - 12 weeks, but in the first week, there were no significantly difference between operated and sham-operated rabbits in the generation and apoptosis of prostatic epithelial cells. The weight of prostate in operated animals increased at Weeks 4, 8 and 12, were significantly heavier than sham-operated rabbits, 1 week post-op and 2 weeks post-op rabbits, there were no significantly difference between the latter 3 groups. Ischemia can induce the gain of prostatic weight by improving the generation and inhibiting the apoptosis of prostatic cells. And the effect of ischemia to prostate is correlated with ischemic time. Ischemia may play an important role in the development of benign prostatic hyperplasia. The first response to ischemia is the change of prostatic stromal cells.

Critical Role of Smad2 in Tumor Suppression and Transforming Growth Factor-β–Induced Apoptosis of Prostate Epithelial Cells

Transforming growth factor-beta (TGF-beta) functions as a tumor suppressor of the prostate through mechanisms that remain unresolved. Although TGF-beta receptors directly activate both Smads 2 and 3, to date, Smad3 has been shown to be the essential mediator of most Smad-dependent TGF-beta responses, including control of gene expression, cell growth, apoptosis, and tumor suppression. Using a robust lentiviral short hairpin RNA system to silence Smads 2 and/or 3 in the NRP-152 nontumorigenic rat prostate basal epithelial cell line, we provide the first evidence for Smad2 as a critical mediator of TGF-beta-induced apoptosis and gene expression. Parallel analyses revealed that Smad3 is the major mediator of TGF-beta-induced transcriptional and apoptotic responses in the NRP-154 rat prostate carcinoma cell line. Remarkably, silencing Smad2 alone caused malignant transformation of NRP-152 cells, as assayed by s.c. tumor growth in athymic mice, whereas silencing Smad3 alone did not induce tumors. Nevertheless, tumors induced by silencing both Smads 2 and 3 were larger than those from silencing Smad2 alone. Given previous reports that NRP-152 cells have a stem cell phenotype, we speculate a critical role for Smad2 as a tumor suppressor in the basal epithelial or stem cell compartment of the prostate.