AR Protein Expression Research Articles

Effects of chronic exposure to cadmium on prostate lipids and morphology

Cadmium is an environmental toxic metal implicated in human prostate carcinogenesis. The mechanism of its toxicity is not fully understood. Previously, we showed that cadmium exposure induces oxidative stress, especially lipid peroxidation. This study evaluates the effect of chronic exposure to 0.886 mM of cadmium (Cd) per liter in the drinking water on prostate lipid content and metabolism in Wistar rats. We determined the lipid profile and measured the expression of lipogenic enzymes: FAS, GPAT, LPL, DGAT-1, DGAT-2, ACO, CPT-1 and CT, and of certain factors involved in lipid regulation and fatty acid transporters: FAT/CD36, E-FABP, SREBP-2, PPAR-gamma and PPAR-alpha by RT-PCR. Ultrastructure was analyzed by electron microscopy and, as prostate is an androgen controlled gland, AR expression was measured by RT-PCR and Western blot. Cd altered the prostatic lipid profile. Triglycerides (TG) and esterified cholesterol (EC) decreased, free cholesterol (FC) and phospholipids (PL) increased and total cholesterol (TC) did not change. FAS, MDH and IDH activities did not vary but G6PDH decreased significantly in Cd group. Regarding TG synthesis, DGAT-1 decreased while GPAT increased and FAS, LPL and DGAT-2 remained unchanged. Regarding beta oxidation, CPT-1 increased while ACO expression decreased in Cd group. In the PL pathway, CT expression was increased. All these results would justify the decrease of TG in Cd group when compared to control. In the cholesterol metabolic pathway, HMGCoAR and SREBP-2 increased. PPAR-alpha increased but PPAR-gamma did not change. Regarding fatty acid transporters, FAT/CD36 decreased, while E-FABP increased. AR mRNA and protein expression decreased. Ultrastructural analysis showed a decrease in lipid droplets and signs of cellular damage in the Cd group. Cadmium exposure induces important changes in prostatic lipid profile and metabolism, confirmed by the morphology analyses, which also showed signs of cellular damage. These results could be important to further understanding the complex mechanism of cadmium toxicity in prostate and in the development of better treatments for people and animals exposed to the heavy metal.

Estrogen-induced upregulation of AR expression and enhancement of AR nuclear translocation in mouse fallopian tubes in vivo

Female mice lacking AR display alterations in ovarian and uterine function. However, the biology of AR in the fallopian tube is not fully understood. To gain an insight into potential roles of AR in this tissue, we demonstrated that eCG treatment increased AR expression in a time-dependent manner and subsequent treatment with hCG decreased AR expression in mouse fallopian tubes. This expression pattern was positively associated with 17beta-estradiol and testosterone levels in vivo. Immunohistochemical analysis of fallopian tube epithelial cells revealed that nuclear localization of AR increased in parallel with decreased AR in the cytoplasm following eCG treatment. Moreover, we found that treatment with flutamide upregulated AR expression in immature mice in association with a decrease in serum testosterone levels, whereas the same treatment resulted in downregulation of AR expression in gonadotropin-stimulated mice with concomitant decreases in serum 17beta-estradiol concentrations, suggesting that androgen differs from estrogen in the regulation of AR expression. Furthermore, we demonstrated that DES increased both AR protein expression and nuclear location over a 48-h time course. DHT had rapid effects, with induction of AR expression and translocation at 6 h after injection, but unlike DES it had prolonged efficacy. In addition, we provided direct in vivo evidence that nuclear protein interaction between AR and p21(Cip1), a previously reported AR-regulated gene, was enhanced by gonadotropin stimulation. To our knowledge, this study provides the first demonstration to illustrate that estrogen as a principal regulator may contribute to regulate and activate AR in the fallopian tubes in vivo.

Hinokitiol, a metal chelator derived from natural plants, suppresses cell growth and disrupts androgen receptor signaling in prostate carcinoma cell lines

Hinokitiol (β-thujaplicin), a troplone-related compound found in the heartwood of cupressaceous plants, strongly inhibits the proliferation of a broad range of tumor cell lines. This is the first report to demonstrate that hinokitiol, a metal chelator derived from natural plants, suppresses cell growth and disrupts AR signaling in prostate carcinoma cell lines. Our present studies indicate that hinokitiol suppresses androgen/AR-mediated cell growth and androgen-stimulated DNA synthesis by [ 3H]thymidine incorporation in a dose- and time-dependent manner. Hinokitiol simultaneously suppresses the intracellular and secreted PSA levels, a marker for the progression of prostate cancer. Hinokitiol significantly represses the AR mRNA and protein expression in a dose- and time-dependent manner. Additionally, the ligand-binding assay shows that hinokitiol blocks binding of the synthetic androgen [ 3H]R1881 to AR in LNCaP cells. These findings collectively suggest that hinokitiol is potentially effective against prostate cancer in vitro, and thus it might become a novel chemopreventive or chemotherapeutic agent for prostate cancer.

Celastrol, a Triterpene Extracted from the Chinese “Thunder of God Vine,” Is a Potent Proteasome Inhibitor and Suppresses Human Prostate Cancer Growth in Nude Mice

Interest in the use of traditional medicines for cancer prevention and treatment is increasing. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as novel anticancer drugs. Celastrol, an active compound extracted from the root bark of the Chinese medicine "Thunder of God Vine" (Tripterygium wilfordii Hook F.), was used for years as a natural remedy for inflammatory conditions. Although Celastrol has been shown to induce leukemia cell apoptosis, the molecular target involved has not been identified. Furthermore, whether Celastrol has antitumor activity in vivo has never been conclusively shown. Here, we report, for the first time, that Celastrol potently and preferentially inhibits the chymotrypsin-like activity of a purified 20S proteasome (IC(50) = 2.5 micromol/L) and human prostate cancer cellular 26S proteasome (at 1-5 micromol/L). Inhibition of the proteasome activity by Celastrol in PC-3 (androgen receptor- or AR-negative) or LNCaP (AR-positive) cells results in the accumulation of ubiquitinated proteins and three natural proteasome substrates (IkappaB-alpha, Bax, and p27), accompanied by suppression of AR protein expression (in LNCaP cells) and induction of apoptosis. Treatment of PC-3 tumor-bearing nude mice with Celastrol (1-3 mg/kg/d, i.p., 1-31 days) resulted in significant inhibition (65-93%) of the tumor growth. Multiple assays using the animal tumor tissue samples from both early and end time points showed in vivo inhibition of the proteasomal activity and induction of apoptosis after Celastrol treatment. Our results show that Celastrol is a natural proteasome inhibitor that has a great potential for cancer prevention and treatment.

Induction of Adenosine A1Receptor Expression by Pertussis Toxin via an Adenosine 5′-Diphosphate Ribosylation-Independent Pathway

Pertussis toxin ADP ribosylates G(i) and G(o) transducing proteins and functionally uncouples adenosine A(1) receptor (A(1)AR) from its effectors. We hypothesized that this loss in receptor coupling could lead to de novo A(1)AR synthesis by the cell in a futile attempt to re-establish normal receptor function. To test this hypothesis, we used hamster ductus deferens tumor (DDT(1) MF-2) cells, a cell culture model for studying A(1)AR, and showed that pertussis toxin (100 ng/ml) produced a time-dependent loss in A(1)AR-G(i) interaction and abolished A(1)AR activation of extracellular signal-regulated kinase 1/2. Interestingly, pertussis toxin increased the expression of A(1)AR, as measured by real-time polymerase chain reaction, immunocytochemistry, and [(3)H]cyclopentyl-1,3-dipropylxanthine (DPCPX) binding, suggesting a compensatory response to G(i) protein inactivation. DDT(1) MF-2 cells exposed to pertussis toxin demonstrated nuclear factor kappaB (NF-kappaB) activation within 30 min of exposure, a time point that preceded the loss of function of the A(1)AR. Inhibition of NF-kappaB attenuated the increase in A(1)AR induced by pertussis toxin. Cells exposed to B-oligomer subunit of pertussis toxin, devoid of significant ADP ribosyltransferase activity, showed increased A(1)AR protein expression, preceded by activation of NF-kappaB. B-Oligomer increased intracellular Ca(2+) in DDT(1) MF-2 cells. Chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or inhibition of protein kinase C (PKC) with bisindolylmaleimide hydrochloride reduced the activation of NF-kappaB and [(3)H]DPCPX binding. We conclude that pertussis toxin promotes de novo A(1)AR synthesis by activating NF-kappaB through an ADP ribosylation-independent mechanism involving intracellular Ca(2+) release and PKC activation.

Evidence for the Presence of α1 Adrenoceptor Subtypes in the Human Ureter

Several studies proposed a role for α1 adrenoceptors (α1 ARs) in the ureteral physiology, indicating that they are present in the ureter; however, few studies have been made to identify α1 AR subtypes present in this area. Thus, this study was carried out to characterize the α1 AR subtype gene and protein expression in proximal, medial and distal region of the human ureter. Methods Molecular characterization of α1 AR subtypes was done by semi-quantitative RT-PCR. The α1 AR protein expression was studied by saturation binding curves using the α1 AR antagonist 125I-HEAT. Analysis of data was performed using the GraphPad PRISM 4 software. Results Analysis of saturation binding curves revealed an heterogeneous distribution of α1 AR binding sites; the Bmax for the distal ureter was indeed 52.5 ±5.4 fmol/mg prot, while a lower similar density of α1 ARs was demonstrated in the medial (25.2 ±1.7 fmol/mg prot) and proximal (23.4 ±0.4 fmol/mg prot) ureters. Molecular characterization of α1 AR subtypes indicated that each receptor was present, although with differences in term of the amount expressed. Conclusions Human ureter was endowed with each α1 AR subtype, although α1D and α1A ARs were prevalent over α1B ARs. Radioligand binding results revealed that there were no significant differences in the Kd between ureteral regions, while an heterogeneous distribution of α1 AR binding sites was detected, with the highest density of α1 ARs in the distal ureter and a lower similar density in the medial and proximal ureter.

Alpha1 Adrenoceptor Subtypes in Human Urinary Bladder: Sex and Regional Comparison

The aim of this work is to report a detailed study of the presence of α1 AR subtypes in human urinary bladder areas involved in the micturition (i.e. detrusor, trigone and neck), investigating whether or not there are differences between sexes. Methods Urinary bladder specimens were obtained as discarded tissue after cystectomy for vesical carcinoma (17 men and 14 women). Molecular characterization of α1 AR subtypes was done by semi-quantitative RT-PCR. The α1 AR protein expression was studied by saturation binding curves using the α1 AR antagonist 125I-HEAT and by western blot. Analysis of data was performed using the GraphPad PRISM 4 software. Results Results obtained indicated that α1 AR proteins were detectable in each bladder area. In both sexes, the detrusor and the neck expressed similar levels of α1 ARs: respectively, detrusor: 14.6 ± 1.2 in men and 13.1 ± 1.1 fmol/mg prot in women; neck: 16.9±3.2 in men and 17.5 ± 4.1 fmol/mg prot in women. In the trigone, significantly higher α1 ARs were found in women compared to men (20.6 ± 1.1 vs 11.7 ± 0.7 fmol/mg prot). Subtype analysis indicated that in women, each area was endowed with mRNA and protein encoding for each α1 AR subtype. The men detrusor expressed α1A and α1D ARs, while in the trigone and the neck, each subtype was present. Conclusions A selective distribution of α1 ARs did exist between sexes and between the different areas involved in micturition, supporting evidence which indicates a role of different α1 AR subtypes in the pathophysiology of the lower urinary tract symptoms.

Inhibition of p53 function diminishes androgen receptor-mediated signaling in prostate cancer cell lines.

Current therapy for advanced prostate cancer is mainly based on androgen deprivation, although most patients relapse to androgen-insensitive disease. Several mechanisms contributing to androgen-independent growth including alterations in the structure or expression of the androgen receptor (AR) and its cofactors have been identified. Recent evidence suggests that p53 is involved in androgen signaling. The analysis of the effect of p53 on androgen signaling was performed in 22Rv1 and LNCaP prostate cancer cells that express both p53 and AR. The overexpression of p53 diminished the androgenic response in both cell lines in a reporter gene assay. Conversely, the inhibition of p53 by three different p53 inhibitors, Pifithrin-1alpha (PFT-1alpha), an inhibitor of p53-dependent transactivation; MDM2, a regulator of p53 expression; and a dominant-negative N-terminally truncated p53 gene also reduced transactivation of androgen-dependent reporter genes. The inactivation of p53 by PFT-1alpha decreased AR-protein expression in both 22Rv1 and LNCaP cells. Our findings confirm that the overexpression of wild-type p53 decreases androgen function, whereas p53 expression at physiological levels stabilizes AR signaling. Thus, our findings suggest that there is a balance of AR and p53 expression during the androgen-dependent growth of prostate cancer, which is obliterated during further progression of the disease.

Alteration of α1A-Adrenoceptor Gene Expression in the Prostate of Streptozotocin-Induced Diabetic Rats

Diabetes-associated alterations in prostate α_1A-adrenoceptor (α_1A-AR) gene expression were studied using a streptozotocin (STZ) induced diabetic rat model. Male Wistar rats were divided into four groups: group I animals were vehicle-treated normal rats; group II consisted of vehicle-treated, STZ-diabetic rats; group III represented insulin-treated, STZ-diabetic rats (0.5 IU/kg three times daily for 4 days), and group IV animals were phlorizin-treated, STZ-diabetic rats (1 mg/kg three times daily for 4 days). The expression of mRNA that encoded protein of α_1A-AR in the rat prostate was studied using reverse transcription combined with polymerase chain reaction. The α_1A-AR protein expression in the prostate was studied by Western blotting analysis with a polyclonal antiserum. A 2.51 ± 0.21-fold increase in the mRNA level of α_1A-AR was observed in the prostate of diabetic rats (n = 8, p < 0.05). Similarly, there was a 2.23 ± 0.10-fold increase in the α_1A-AR protein level (n = 8, p < 0.05). Both insulin and phlorizin treatments restored the normal levels of mRNA and protein expression. In conclusion, the gene expression of α_1A-AR is increased in the prostate of diabetic rats. Hyperglycemia plays a major role in this alteration.

The antiestrogen ICI 182,780 decreases the expression of estrogen receptor-alpha but has no effect on estrogen receptor-beta and androgen receptor in rat efferent ductules

BackgroundThe antiestrogen ICI 182,780 has been used successfully as an alternative experimental model for the study of estrogen action in the rodent adult male reproductive tract. Although ICI 182,780 causes severe alterations in testicular and efferent ductule morphology and function, the effects on the expression of estrogen and androgen receptors in the male have not been shown.MethodsIn the present study, adult male rats were treated with ICI 182,780 for 7 to 150 days, to evaluate the time-response effects of the treatment on the pattern of ERα, ERβ and AR protein expression in the efferent ductules. The receptors were localized using immunohistochemistry.ResultsERα, ERβ and AR have distinct cellular distribution in the testis and efferent ductules. Staining for ERα is nearly opposite of that for ERβ, as ERα shows an increase in staining intensity from proximal to distal efferent ductules, whereas ERβ shows the reverse. Androgen receptor follows that of ERα. ICI 182,780 caused a gradual but dramatic decrease in ERα expression in the testis and efferent ductules, but no change in ERβ and AR expression.ConclusionsThe differential response of ERα and ERβ proteins to ICI 182,780 indicates that these receptors are regulated by different mechanisms in the male reproductive tract.

Involvement of nuclear factor-kappa B, Bax and Bcl-2 in induction of cell cycle arrest and apoptosis by apigenin in human prostate carcinoma cells

Apigenin, a common dietary flavonoid abundantly present in fruits and vegetables, may have the potential for prevention and therapy for prostate cancer. Here, we report for the first time that apigenin inhibits the growth of androgen-responsive human prostate carcinoma LNCaP cells and provide molecular understanding of this effect. The cell growth inhibition achieved by apigenin treatment resulted in a significant decrease in AR protein expression along with a decrease in intracellular and secreted forms of PSA. These effects were also observed in DHT-stimulated cells. Further, apigenin treatment of LNCaP cells resulted in G1 arrest in cell cycle progression which was associated with a marked decrease in the protein expression of cyclin D1, D2 and E and their activating partner cdk2, 4 and 6 with concomitant induction of WAF1/p21 and KIP1/p27. The induction of WAF1/p21 appears to be transcriptionally upregulated and is p53 dependent. In addition, apigenin inhibited the hyperphosphorylation of the pRb protein in these cells. Apigenin treatment also resulted in induction of apoptosis as determined by DNA fragmentation, PARP cleavage, fluorescence microscopy and flow cytometry. These effects were found to correlate with a shift in Bax/Bcl-2 ratio more towards apoptosis. Apigenin treatment also resulted in down-modulation of the constitutive expression of NF-kappaB/p65. Taken together, these findings suggest that apigenin has strong potential for development as an agent for prevention against prostate cancer.

The Developmental and Physiological Consequences of Disrupting Genes Encoding β1 and β2 Adrenoceptors

Publisher Summary The role that individual β AR subtypes play in specific physiological processes has been traditionally defined using subtype-specific ligands. Unfortunately, many of these ligands either possess poor selectivity or are used at concentrations in vivo that can lead to occupation of nonspecific subtypes. The advent of gene disruption techniques in the mouse now enables to selectively delete or alter cloned genes as a means of identifying the specific function(s) of their gene products. To better understand β AR subtype-specific functions in the context of either the whole animal or isolated organs and cells, the genes encoding both β 1 - and β 2 ARs have been disrupted. Gene targeting vectors containing β 1 AR or β 2 AR gene sequences interrupted or partially replaced by a bacterial neomycin resistance gene cassette were flanked by a viral thymidine kinase gene cassette. A standard positive-negative selection strategy (G418 + gancyclovir) has been used to isolate R1 embryonic stem (ES) cells, having undergone homologous recombination at β 1 AR or β 2 AR loci. The pharmacological profile of β 1 AR and β 2 AR knockouts is consistent with the loss of these specific receptor subtypes. When the nonspecific β AR antagonist [ 125 I]cyanopindolol is used in competition binding studies, excess unlabeled CGP 20712A ( β 1 AR-specific antagonist) reveals a selective loss of specific high-affinity sites in β 1 AR knockout heart or lung membranes while excess unlabeled ICI 118,551 ( β 2 AR-specific antagonist) reveals a selective loss of specific high-affinity sites in β 2 AR knockout lung membranes. These results provide further evidence that the β 1 AR and β 2 AR gene disruptions have abolished β AR protein expression. The physiological impact of knocking out either β 1 - or β 2 ARs has been studied in instrumented animals and in isolated tissues.

Detection and location of amphiregulin and Cripto-1 expression in the developing postnatal mouse mammary gland.

Amphiregulin (Ar) and Cripto-1 (Cr-1) are growth promoting peptides that share amino acid sequence homology with epidermal growth factor (EGF). The present study examined Ar and Cr-1 mRNA and protein expression during various stages of C57BL/6 mouse mammary morphogenesis. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect transcripts for Ar and Cr-1 at all stages of mammary development. Immunocytochemical (ICC) localization demonstrated that in virgin 4-week to mature 12-week-old mouse fourth inguinal mammary gland, Ar and Cr-1 are expressed in the stromal cells, luminal epithelial cells, and myoepithelial cells of the branching ducts. Ar, and to lesser extent Cr-1, were also found in the epithelial cap cells and in the luminal epithelial cells of the advancing terminal end bud (TEB) from virgin 4-week and 6-week-old mice. Western blot analysis demonstrated that both Ar (28 and 26 kDa) and Cr-1 (90, 67, 56, and 21 kDa) proteins are expressed in virgin, 13.5 day midpregnant and in the 14 day lactating mammary gland. In addition, Ar and Cr-1 are associated with developing alveolar structures as determined by ICC. These results imply that together with EGF and transforming growth factor alpha (TGF alpha), Ar and Cr-1 may play salient roles as modifiers in the morphogenesis and differentiation of the mammary gland.