Induction of metallothionein by CdCl2 administration in rat prostate

Metallothionein (MT) induction in the rat prostate gland was investigated by means of cadmium chloride administration. Ten-week-old male Wistar rats housed with cadmium free food were divided into three groups of six rats each and castrated. After seven days, 1 mg of testosterone propionate per rat was injected subcutaneously once a day until the end of the experiment. After three weeks, rats were injected daily for six days with a physiological saline, 0.3 mg/kg CdCl₂, and 0.9 mg/kg CdCl₂. MT concentration of the ventral and dorsal lobes was significantly increased in the three groups in proportion to the dose of CdCl₂. MT content of the lateral lobe in three groups was also increased, but was not significantly different. Immunohistochemically, MT was induced mainly in the ventral lobe in the basal cells, and in the lateral and dorsal lobes in the epithelial cells. The weights of the prostatic lobes were similar in the three groups, and no histological change was identified. These results suggested that MT in the prostate was induced by cadmium administration, and that it may prevent cellular damage from harmful metals. © 1993 Wiley-Liss, Inc.     

Sex hormone-induced alterations in the activities of antioxidant enzymes and lipid peroxidation status in the prostate of Noble rats

BACKGROUND: Oxidative stress has been implicated in prostate carcinogenesis. In the Noble rat model, treatment of rats with testosterone (T) plus 17beta-estradiol (E(2)) induced dysplasia and adenocarcinoma. Previous reports from us and other have indicated a linkage between steroid hormones and oxidative status in prostate cells in vivo and in vitro. Here, we provide further evidence that androgens and estrogens could induce a lobe-specific shift of prooxidant-antioxidant balance and alterations in antioxidant enzyme activities, leading to oxidative stress in the rat prostate gland in vivo. METHODS: Male Noble rats were subjected to single (T, E(2), or diethylstilbestrol [DES] alone) or combined (T + E(2) or T + DES) hormone treatments. Lipid peroxidation status and antioxidant enzyme activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD) were assayed spectrophotometrically. RESULTS: Treatment of rats with T did not alter lipid peroxidation in either the ventral prostate (VP) or the dorsolateral prostate (DLP). In contrast, exposure of rats to DES or E(2) modestly elevated lipid peroxidation in rat VP or DLP, respectively. Of importance, T + DES and T + E(2) treatments of rats induced marked increases in lipid peroxidation in the VP and the DLP, respectively. Antioxidant enzyme activities in the VP and DLP exhibited differential responses to sex hormone challenges. The activities of catalase, GPx, GR, and G6PD were mostly suppressed in either single or dual hormone-treated DLP, whereas there is a general increase of GR and G6PD activities in the VP after hormonal exposures. The changes in T + DES-treated VP were most dramatic with a marked activation of GPx (by one-fold), GR (by one-fold), and G6PD (by five-fold). CONCLUSION: The lobe-specific differential responses of hormone-induced oxidative stress and modulations of antioxidant enzymatic defenses in the rat prostate suggest that reactive oxygen species may play a role in hormone-induced prostate carcinogenesis.     

Prolactin and testosterone regulation of mitochondrial zinc in prostate epithelial cells

The prostate gland of many animals accumulates extremely high levels of zinc and citrate. Evidence currently exists in support of a concept that zinc might be an important regulator of m-aconitase and citrate oxidation of prostate epithelial cells. No information concerning the mitochondrial levels of zinc has been available. The roles of testosterone and prolactin in the regulation of zinc have not been established. In this report, we determined the levels of tissue and mitochondrial zinc of rat lateral prostate (LP), ventral prostate (VP), dorsal prostate (DP), liver, and kidney. The results demonstrate that the mitochondrial zinc levels of the prostate cells were higher than levels of nonprostatic cells. The LP contained severalfold higher zinc levels than DP and VP. The effects of testosterone and prolactin in vivo and in vitro on the zinc levels were also determined. Both hormones significantly increased cellular and mitochondrial zinc levels of LP cells; decreased the zinc levels of VP cells; and had no effect on the zinc levels of DP, liver, or kidney cells. These effects are direct and physiological effects of the hormones on the targeted prostate epithelial cells. The hormonal effects on mitochondrial zinc of LP and VP epithelial cells correlate perfectly with their effects on citrate oxidation. The results support the concept that mitochondrial zinc is an inhibitor of m-aconitase and citrate oxidation; and that prolactin and testosterone regulation of mitochondrial zinc provides a mechanism for their regulation of citrate oxidation in citrate-producing prostate epithelial cells. Prostate 30:26–32, 1997 © 1997 Wiley-Liss, Inc.     

Androgen regulation of spermidine synthase expression in the rat prostate

BACKGROUND: Spermidine synthase, an essential enzyme in the polyamine synthesis pathway, was identified as one of the androgen-response genes in the rat ventral prostate. Characterization of androgen regulation of spermidine synthase is important to the understanding of androgenic regulation of polyamine synthesis. METHODS: Full-length cDNA encoding rat spermidine synthase was isolated from a lambdaZAP cDNA phage library. Young male adult Sprague-Dawley rats were used for castration and androgen replacement. Northern blot and in situ hybridization were used to characterize gene expression. RESULTS: The amino acid sequence of rat spermidine synthase shares 99% and 94% identity with that of mouse and human spermidine synthase, respectively. Spermidine synthase gene is abundantly expressed and regulated by androgens in the ventral, dorsal, and lateral lobes of the rat prostate, and its expression is localized to the epithelial cells. Spermidine synthase also is regulated by androgens in the seminal vesicles but not in the muscle, brain, kidney, thymus, heart, or liver, suggesting that this enzyme is responsive to androgen in the male sex accessory organs only. The expression of spermidine synthase and two other enzymes involved in polyamine synthesis, S-adenosylmethionine decarboxylase and ornithine decarboxylase, are regulated by androgens coordinately. CONCLUSIONS: Spermidine synthase is most abundantly expressed and regulated by androgens in the prostatic epithelial cells, suggesting that regulation of spermidine synthase is likely a key step in coordinated androgen regulation of polyamine synthesis in the prostate.     

Androgens upregulate aquaporin 9 expression in the prostate

Objectives: Aquaporins (AQP) function as selective pores allowing water, glycerol and other small solutes to pass through the cell membrane. We previously reported that AQP are expressed in the prostates of both humans and rats. The present study investigated the androgen‐dependent expression of AQP9 in the prostate in vivo and in vitro. Methods: Rat ventral prostate tissue specimens and a normal human prostatic epithelial cell line (PNT2) were used. AQP9 messenger ribonucleic acid (mRNA) and protein expression were examined using real‐time polymerase chain reaction, Western blotting and immunohistochemical methods. Androgen modulation was achieved by surgical castration, treatment with testosterone propionate (5 µg/g bodyweight) or bicalutamide (20 µg/g bodyweight), and the ribonucleic acid interference (RNAi) method of the androgen receptor (AR). The synthetic small interfering RNA was transfected into PNT2 at a concentration of 10 nM, and the RNAi effect was evaluated using a Western blotting analysis. Results: AQP9 mRNA and protein were expressed in the rat prostate. Surgical castration or bicalutamide treatment significantly decreased their expression. In addition, the treatment with testosterone propionate after castration restored the expression to the level of the controls. An RNAi experiment in PNT2 also decreased the expression. Conclusions: AQP9 expression in the prostate is controlled by androgens.     

Androgen receptor-dependent regulation of Bcl-xL expression: Implication in prostate cancer progression

BACKGROUND: Recently we reported that silencing the androgen receptor (AR) gene reduced Bcl-xL expression that was associated with a profound apoptotic cell death in prostate cancer cells. In this study we further investigated AR-regulated Bcl-xL expression. METHODS: Prostate cancer cell line LNCaP and its sublines, LNCaP/PURO and LNCaP/Bclxl, were used for cell proliferation assay and xenograft experiments in nude mice. Luciferase gene reporters driven by mouse or human bcl-x gene promoter were used to determine androgen regulation of Bcl-xL expression. RT-PCR and Western blot assays were conducted to assess Bcl-xL gene expression. Chromatin immunoprecipitation assay was performed to determine AR interaction with Bcl-xL promoter. Bcl-xL-induced alteration of gene expression was examined using cDNA microarray assay. RESULTS: In cultured prostate cancer LNCaP cells, androgen treatment significantly increased Bcl-xL expression at mRNA and protein levels via an AR-dependent mechanism. Promoter analyses demonstrated that the AR mediated androgen-stimulated bcl-x promoter activation and that the AR interacted with bcl-x promoter. Enforced expression of Bcl-xL gene dramatically increased cell proliferation in vitro and promoted xenograft tumor growth in vivo. Genome-wide gene profiling analysis revealed that Bcl-xL expression was significantly higher in metastatic and castration-resistant diseases compared to normal prostate tissues or primary cancers. Bcl-xL overexpression significantly increased the expression of cyclin D2, which might be responsible for Bcl-xL-induced cell proliferation and tumor growth. CONCLUSIONS: Taken together, our data strongly suggest that androgen stimulates Bcl-xL expression via the AR and that increased Bcl-xL expression plays a versatile role in castration-resistant progression of prostate cancer.     

Differential localization of MT1-MMP in human prostate cancer tissue: role of IGF-1R in MT1-MMP expression

BACKGROUND: MT1-MMP is a metalloproteinase involved in prostate cancer metastasis. The IGF-1R is a tyrosine kinase receptor involved with tumor progression and metastasis. The purpose of this investigation was to examine MT1-MMP and IGF-1R expression and localization in prostate cancer tissues and explore the role of IGF-1R in regulating MT1-MMP in prostate cancer cell lines. METHODS: Immunohistochemistry was utilized to study MT1-MMP and IGF-1R expression in human prostate tissues. IGF-1R regulation of MT1-MMP expression was determined by gene promoter analysis, quantitative RT-PCR and Western blot analysis following pharmacological inhibition of the receptor in PC-3N cells and treatment of LNCaP cells with androgen and IGF-1. RESULTS: MT1-MMP expression was high in the apical regions of the luminal cells in PIN and prostate cancer and less intense in the basalateral regions of benign tissues. IGF-1R was expressed primarily in the basal cells of normal glands and highly expressed in prostate cancer. Inhibition of IGF-1R in PC-3N cells decreased MT1-MMP expression and treatment of LNCaP cells with a synthetic androgen and IGF-1 increased MT1-MMP expression. CONCLUSIONS: These data demonstrate that MT1-MMP is highly expressed in the apical cytoplasmic regions of the luminal cells in PIN and prostate cancer when compared to basalateral cytoplasmic membrane staining in benign glands. Additionally, we demonstrate that IGF-1R is highly expressed in human prostate carcinoma. These findings suggest that MT1-MMP localization and IGF-1R expression in prostate carcinoma could be predictive biomarkers for aggressive disease and support IGF-1R as a promising therapeutic target to decrease processes of prostate cancer metastasis.     

Ventral prostate predominant l, a novel mouse gene expressed exclusively in the prostate

BACKGROUND: Despite the region-specific nature of human prostate disease, there is a paucity of information regarding the molecular basis of prostate regionalization and patterning. To elucidate genetic mechanisms that underlie prostate growth and development, we investigated differential gene expression in mouse prostate lobes. METHODS: mRNA differential display analysis was used to identify differentially expressed genes during development of ventral, anterior, and dorsolateral prostate lobes. Differential gene expression was confirmed by Northern blot analysis and RT-PCR. RESULTS: A novel gene, Ventral prostate predominant1 (Vpp1) was identified. Vpp1 mRNA was evident in all lobes but accumulated predominantly in the ventral prostate, and was detected on postnatal day 7 through adulthood exclusively in the prostate gland. The steady-state level of Vpp1 mRNA decreased markedly in response to castration, suggesting androgen regulation of Vpp1 expression. Analysis of TRAMP tumors demonstrated a dramatic decrease in the level of Vpp1 mRNA. CONCLUSIONS: The spatial distribution and early postnatal onset of Vpp1 expression is consistent with a role for this gene in prostate regionalization. The absolute prostate specificity of Vpp1 expression may allow this gene to serve as a paradigm to study the molecular basis of gene expression that is restricted exclusively to the prostate gland.     

Finasteride treatment alters MMP-2 and -9 gene expression and activity in the rat ventral prostate

The safety of using finasteride as a prevention of prostate cancer is still under debate. In this study, we investigated the effects of finasteride on the location, gene expression and activities of matrix metalloproteinases -2 and -9, which are involved in the degradation of extracellular matrix components during tissue remodelling and prostate cancer progression, invasion and metastasis. Ventral prostates (VP) from Wistar rats treated with finasteride (25 mg/kg/day) for 7 and 30 days and age-matched controls were evaluated using histology, immunohistochemistry, semi-quantitative RT-PCR and gelatin zymography. Finasteride treatment reduced the epithelial immunostaining of MMP-2 but increased MMP-9 immunostaining in the epithelial cells and in the stroma. The mRNA expression of both MMP-2 and MMP-9 were significantly increased on day 7 of finasteride treatment, mainly for MMP-9 and returned to the control levels by day 30. However, gelatin zymography showed that MMP-9 activity was significantly increased on day 7 of finasteride treatment and remained elevated on day 30 ( p  < 0.05), while MMP-2 activity was reduced after 30 days of treatment. Finasteride increases MMP-9 and reduces MMP-2 activities in the prostate, which may affect negatively and positively both normal and tumoural prostatic cell behaviour during the treatment. Studies on expression of MMPs in the prostate during different androgen manipulation or cancer chemoprevention strategies can contribute to understand the tissue's overall response and clinical data.     

Fast Facts: Prostate Cancer

OBJECTIVE

To characterize the changes in androgen levels in the prostate after castration, as androgens are critical in the progression of prostate cancer after castration, but the time at which the androgen remaining in the prostatic cancer tissue after castration exerts its effects is poorly understood.

MATERIALS AND METHODS

The ventral prostate (VP) in adult male spontaneously hypertensive rats was excised at 2, 4 and 8 h, 1, 2, 4 and 7 days, and 2, 4 and 8 weeks after castration. The dihydrotestosterone (DHT), testosterone, dehydroepiandrosterone (DHEA) and androstenedione (4‐dione) levels in the VP were measured simultaneously using gas chromatography/tandem mass spectrometry.

RESULTS

Within 2 days of castration, the DHT and testosterone levels in the VP decreased sharply, while there were no significant changes in the DHEA or 4‐dione levels. From 2 days to 2 weeks after castration (2–7 days for 4‐dione), there was a sharp peak in tissue androgen levels in the VP (P < 0.05 for all androgens); during the subsequent 6 weeks after castration, all of the tissue DHT, testosterone, DHEA and 4‐dione levels gradually increased with time.

CONCLUSIONS

These data show the changes which occur in androgen levels in rat VP after castration and support the concept that the adrenal glands compensate for the loss of testicular androgen.     

Regulation of rat prostate stromal cell myodifferentiation by androgen and TGF-beta1

BACKGROUND: Myodifferentiation of stromal cells is a key step in prostate development and is a hallmark of reactive stroma in prostate cancer. Little is known about regulatory mechanisms, however, prostate stromal cells are androgen-regulated and TGF-beta1 is a known stimulator of stromal myodifferentiation. The PS-1 rat prostate stromal cell line expresses androgen receptor, and exhibits androgen-regulated gene expression and proliferation. TGF-beta1 inhibits androgen action in PS-1 cells through translocation of androgen receptor from the nucleus to the cytoplasm. The present study was conducted to determine whether myodifferentiation of PS-1 cells is regulated by androgen and TGF-beta1, and how myodifferentiation affects androgen receptor localization and cell proliferation. METHODS: PS-1 cell cultures were exposed to physiological concentrations of dihydrotestosterone, TGF-beta1, and combinations of both in chemically defined medium. Immunocytochemistry and Western blotting for smooth muscle alpha-actin filament formation, smooth muscle alpha-actin protein levels, calponin expression, PCNA index, and androgen receptor localization were performed. RESULTS: Dihydrotestosterone (DHT) and TGF-beta1 each separately promoted PS-1 myodifferentiation. A combination did not affect the rate of differentiation, however, the level of alpha-actin protein was elevated and PCNA was decreased in co-stimulated conditions. TGF-beta1 induction resulted in a transient translocation of androgen receptor from the nucleus to the cytoplasm during differentiation followed by a resumed nuclear localization in myodifferentiated cells. CONCLUSIONS: These data indicate that a complex cross-talk mechanism exists between androgen and TGF-beta1 signaling in prostate stromal cells that affects cell proliferation and myodifferentiation. These findings also suggest that androgen and TGF-beta1 interactions may cooperatively regulate myodifferentiation of stromal cells in the stromal response in prostate cancer.     

High fat diet increases the weight of rat ventral prostate

BACKGROUND: Understanding the mechanisms by which diet influences the prostate may eventually lead to novel approaches for preventing prostate cancer. The objective of this research is to examine the impact of dietary fat, vitamin D, and genistein on prostate weight, serum and intraprostatic androgen levels, and the expression of several androgen-response genes. METHODS: Sprague-Dawley rats were fed, beginning at 21 days of age, for 1 or 3 months of experimental diets with high saturated fat (32.2% calories from fat), low saturated fat (3.6% calories from fat), genistein plus (20 mg/kg), genistein deficient, vitamin D surplus (4,000 U/kg), or vitamin D deficient. The body weight, food intake, the weights of the ventral prostate and dorsolateral prostate, and the levels of testosterone and dihydrotestosterone (DHT) in the serum and in the prostate were determined. The expression of androgen-response genes was characterized by Northern blot analysis. RESULTS: The pilot experiments showed that high dietary fat appeared to consistently increase the weight of the ventral prostate, while vitamin D or genistein did not have a consistent effect on prostate weight. Further analysis confirmed that the ventral prostate is 15% (P < 0.001) heavier in the rat on a high fat diet as compared to a low fat diet. Dietary fat had no significant influence on the levels of serum and intraprostatic androgens and the expression of androgen-response genes. CONCLUSIONS: Our results suggested that the ventral prostate weight of the rat is increased without affecting the androgen axis by feeding the animals with high fat diet beginning at 21 days of age. This observation is potentially important since epidemiological data suggest that saturated fat consumption is a major risk factor associated with prostate cancer incidence rate.     

Androgen receptor binding characteristics in the cytosol of the rat dorsolateral prostate gland and the dunning R-3327 prostatic adenocarcinoma

The incidence of prostatic cancer is highly correlated with advanced age, and it has been suggested that changes in androgen binding may be important in age-associated alterations in growth regulatory mechanisms of prostatic epithelial cells. In this study the effects of age on androgen binding characteristics in the dorsolateral prostate glands of young and aged Copenhagen rats were determined and the binding properties in the Dunning R3327/130 subline of rat prostatic adenocarcinoma were characterized. Tritium-labeled and nonlabeled methyltrienolone analogs (R1881) were used to study the binding properties of 5α-dihydrotestosterone receptor in the cytosol of tumors and prostate glands. Binding of R1881 was low but specific for the androgen receptor as shown by competition studies in which nonlabeled R1881, 5α-dihydrostestosterone, and testosterone competed successfully with ³H-R1881 for binding sites, but 17β-estradiol and low levels of progesterone did not. In Copenhagen dorsolateral prostate, Scatchard analysis suggested a single class of binding sites. In young animals (three to five months) the average binding capacity was 10.36 fmol/mg cytosol protein with a dissociation constant (K_d) of 2.28 nmol/L. The dorsolateral prostate of aged rats (11–16 months) showed no significant difference in specific binding characteristics as compared to the younger age group. Specific binding of ³H-R1881 in R3327/130 tumor was saturable with a single class of high-affinity binding sites having an average binding capacity of 64.77 fmol/mg cytosol protein and a K_d of 2.76 nmol/L. These data show that the tumor had approximately 6.5 times the number of binding sites as did the normal Copenhagen rat dorsolateral prostate gland. However, no age-related changes were detected through 11–16 months of age in the androgen binding characteristics of normal rat dorsolateral prostate gland that could be correlated with the higher concentration of androgen binding sites in the R3327/130 tumor subline.