RESUMEN
Understanding regional variations of soil heavy metals and their anthropogenic influence are very important for environmental planning. In this study, 286 surface soil samples were collected in Fuyang county, and the 'total' metals for copper (Cu), zinc (Zn), lead (Pb), cadmium (Cd) and nickel (Ni) were measured in 2005. Statistic analysis showed that Cu, Zn, Pb and Cd had been added by exterior factors, and Ni was mainly controlled by natural factors. The combination of multivariate statistical and geostatistical analysis successfully grouped three groups (Cu, Zn and Pb; Cd; and Ni) of heavy metals from different sources. Through pollution evaluation, it was found that 15.76% of the study area for Cu, Zn and Pb, and 46.14% for Cd suffered from moderate or severe pollution. Further spatial analysis identified the limestone mining activities, paper mills, cement factory and metallurgic activities were the main sources for the concentration of Cu, Zn, Pb and Cd in soils, and soil Ni was mainly determined by the parent materials.
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Monitoreo del Ambiente , Metales Pesados/análisis , Contaminantes del Suelo/análisis , Suelo/análisis , China , Análisis Multivariante , Medición de RiesgoRESUMEN
p66Shc is shown to negatively regulate the life span in mice through reactive oxygen species (ROS) production. Recent reports, however, revealed that p66Shc protein level is significantly elevated in several human cancer tissues and growth-stimulated carcinoma cells, suggesting a mitogenic and carcinogenic role for p66Shc. In this communication, we demonstrate for the first time that p66Shc mediates androgenic growth signals in androgen-sensitive human prostate cancer cells through mitochondrial ROS production. Growth stimulation of prostate cancer cells with 5alpha-dihydrotestosterone (DHT) is accompanied by increased p66Shc level and ROS production, which is abolished by antioxidant treatments. However, antioxidant treatments do not affect the transcriptional activity of androgen receptor (AR) as observed by its inability to block DHT-induced prostate-specific antigen expression, an AR-dependent correlate of prostate cancer progression. Elevated expression of p66Shc by cDNA transfection increases the basal cell proliferation and, thus, reduces additional DHT-induced cell proliferation. Furthermore, DHT increases the translocation of p66Shc into mitochondria and its interaction with cytochrome c. Conversely, both redox-negative p66Shc mutant (W134F), which is deficient in cytochrome c interaction, and p66Shc small interfering RNA decrease DHT-induced cell proliferation. These results collectively reveal a novel role for p66Shc-ROS pathway in androgen-induced prostate cancer cell proliferation and, thus, may play a role in early prostate carcinogenesis.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Andrógenos/farmacología , Proliferación Celular/efectos de los fármacos , Mitocondrias/metabolismo , Neoplasias de la Próstata/patología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Citocromos c/metabolismo , Progresión de la Enfermedad , Humanos , Masculino , Mitocondrias/fisiología , Modelos Biológicos , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Oxidación-Reducción , Neoplasias de la Próstata/metabolismo , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/farmacología , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal/fisiología , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Testosterona/análogos & derivados , Testosterona/farmacología , Células Tumorales CultivadasRESUMEN
Aberrant regulation in the adhesive ability of cancer cells is closely associated with their metastatic activity. In this study, we examine the role of ErbB-2 in regulating the adhesive ability of androgen receptor (AR)-positive human prostate cancer (PCa) cells, the major cell population of PCa. Utilizing different LNCaP and MDA PCa2b cells as model systems, we found that ErbB-2 activity was correlated with PYK2 activity and adhesive ability in those cells. Increased ErbB-2 expression or activity in LNCaP C-33 cells enhanced PYK2 activation and cell adhesion, while the high PYK2 activity and the rapid adhesion of LNCaP C-81 cells were decreased by diminishing ErbB-2 expression or activity. Knockdown studies revealed the predominant role of ErbB-2 in regulating LNCaP C-81 cell adhesion. Coimmunoprecipitation showed that C-81 cells had increased interaction between ErbB-2 and PYK2. Elevated ErbB-2 activity in LNCaP cells correlated with increased ERK/MAPK activity and enhanced adhesive ability, which were abolished by the expression of K457A-PYK2 mutant or the treatment of PD98059, a MEK inhibitor. In summary, our data suggested that ErbB-2, via PYK2-ERK/MAPK, upregulates the adhesive ability of AR-positive human PCa cells.
Asunto(s)
Adhesión Celular/fisiología , Quinasa 2 de Adhesión Focal/metabolismo , Receptor ErbB-2/genética , Receptores Androgénicos/fisiología , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Compuestos Orgánicos/farmacología , Neoplasias de la Próstata , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
PURPOSE: Understanding cell proliferation regulation in hormone refractory prostate cancer may provide answers for novel solutions. Protein tyrosine phosphatases have been thought to have key roles in regulating cell proliferation and be involved in oncogenesis, although to our knowledge their functional roles in human prostate cancer remain unknown. Human prostatic acid phosphatase (PAcP), a major phosphatase in prostate epithelium, has been shown to function as a neutral protein tyrosine phosphatase in these cells. We evaluated the biological significance of cellular prostatic acid phosphatase expression in human prostate cancer cells. MATERIALS AND METHODS: Immunohistochemical testing of human prostate cancer archival specimens was done to evaluate the expression of cellular PAcP. Immunoprecipitation and immunoblotting were performed to determine cellular PAcP and SH2 domain-bearing tyrosine phosphatase-1 levels as well as tyrosine phosphorylation of c-ErbB-2/neu in different human prostate cancer cells. The biological behavior of LNCaP derivative sublines was characterized in vitro and in vivo by soft agar analysis and xenograft animal inoculation. RESULTS: Immunohistochemical staining of human prostate clearly showed that cellular levels of PAcP significantly decreases in prostate cancer cells (p <0.001). The results of biochemical characterization revealed that the cellular level of PAcP but not SHP-1, another differentiation associated protein tyrosine phosphatase, consistently correlated negatively with the growth of several human prostate cancer cell lines. Reintroducing cellular PAcP activity in prostate cancer cells by PAcP complementary DNA transfection resulted in decreased tyrosine phosphorylation of c-ErbB-2/neu, decreased proliferation rates in culture as well as decreased anchorage independent growth in soft agar. The xenograft animal model demonstrated that a higher tumor growth rate as well as larger size is associated with a lower level of cellular PAcP. CONCLUSIONS: Cellular PAcP can down-regulate prostate cancer cell growth, at least partially by dephosphorylating c-ErbB-2/neu. Therefore, decreased cellular PAcP expression in cancer cells may be involved in prostate cancer progression.
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Neoplasias de la Próstata/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Receptor ErbB-2/metabolismo , Fosfatasa Ácida , Andrógenos/farmacología , Animales , Western Blotting , Regulación hacia Abajo , Femenino , Humanos , Inmunohistoquímica , Masculino , Ratones , Fosforilación , Próstata/citología , Próstata/enzimología , Proteína Fosfatasa 1 , Trasplante HeterólogoRESUMEN
The stimulated secretion of prostatic acid phosphatase (PAcP) has been known to be a hallmark of androgen action on human prostate epithelial cells for the last five decades. The molecular mechanism of androgen action on PAcP secretion, however, has remained mostly unknown. We investigated the molecular mechanism that promotes PAcP secretion in LNCaP human prostate carcinoma cells which express PAcP and are androgen-responsive. Treatment with 12-o-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase C (PKC) activator, resulted in an increased secretion of PAcP in a dose- and time-dependent fashion. 4Alpha-phorbol, a biologically inactive isomer of TPA, had no effect. This TPA stimulation of PAcP secretion was observed 2 h after exposure, while TPA did not have a significant effect on the mRNA level even with a 6 h treatment. A23187 calcium ionophore, known to mobilize cellular calcium which is a co-factor of PKC, also activated PAcP secretion. This TPA stimulation of PAcP secretion was more potent than the conventional stimulating agent 5alpha-dihydrotestosterone (DHT) at the same concentration of 50 nM. Furthermore, the action of TPA and DHT on PAcP secretion was blocked by five different PKC inhibitors. Results also showed that DHT, as well as TPA, could rapidly modulate PKC activity. Therefore, PKC can regulate PAcP secretion, and may also be involved in DHT action on PAcP secretion.
Asunto(s)
Neoplasias de la Próstata/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Fosfatasa Ácida , Dihidrotestosterona/farmacología , Humanos , Masculino , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/metabolismo , Acetato de Tetradecanoilforbol/farmacocinética , Células Tumorales CultivadasRESUMEN
Androgens regulate the expression of both human prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA), two major prostate epithelium-specific differentiation antigens. Due to the important role of these two enzymes as prostate epithelium differentiation markers, we investigated their regulation of expression at the mRNA level in LNCaP human prostate carcinoma cells. Interestingly, phenol red, a pH indicator in the culture medium, promoted cell growth. To eliminate this non-specific effect, a phenol red-free, steroid-reduced medium was utilized. When high-density cells were grown in that medium, 5alpha-dihydrotestosterone (DHT) suppressed PAcP but stimulated PSA. However, tumor promoter phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) functioned as a potent inhibitor of both PAcP and PSA expression. Prolonged treatment with DHT as well as TPA resulted in a similar down-regulation of protein kinase C and cellular PAcP activities. Thus, the levels of PAcP and PSA mRNA are differentially regulated by androgens in LNCaP cells.
Asunto(s)
Fosfatasa Ácida/efectos de los fármacos , Antígeno Prostático Específico/efectos de los fármacos , Próstata/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , ARN Mensajero/efectos de los fármacos , Fosfatasa Ácida/metabolismo , Andrógenos/farmacología , Carcinógenos/farmacología , División Celular/efectos de los fármacos , Dihidrotestosterona/farmacología , Humanos , Masculino , Fenolsulfonftaleína/farmacología , Próstata/metabolismo , Antígeno Prostático Específico/metabolismo , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Receptor protein tyrosine phosphatase alpha (RPTPalpha) is a transmembrane protein phosphatase, and has been proposed to be involved in the differentiation of the neuronal system. In the present study, we demonstrated the expression of RPTPalpha mRNA in several normal human tissues. We further investigated the regulation of expression of RPTPalpha mRNA in epithelial cells utilizing three commercially available human prostate cancer cell lines LNCaP, PC-3 and DU145. This is because these cells exhibit different levels of differentiation, defined by the expression of a tissue-specific differentiation antigen, prostatic acid phosphatase (PAcP), and their androgen sensitivity. LNCaP cells express PAcP and are androgen-sensitive cells, while PC-3 and DU145 cells do not express PAcP and are androgen-insensitive cells. Northern blot analyses revealed that, in LNCaP cells, fetal bovine serum (FBS) and 5alpha-dihydrotestosterone (DHT) down-regulates RPTPalpha mRNA expression, similar to the effect on PAcP. Contrarily, FBS up-regulated the RPTPalpha mRNA level in PC-3 and DU145 cells. In LNCaP cells, sodium butyrate inhibited cell growth and up-regulated RPTPalpha as well as PAcP mRNA expression. Although, sodium butyrate also inhibited the growth of PC-3 and DU145 cells, the level of RPTPalpha mRNA was decreased in PC-3, while increased in DU145 cells. Thus, data taken together indicate that the expression of RPTPalpha is apparently regulated by a similar mechanism to that of PAcP in LNCaP cells.
Asunto(s)
Fosfatasa Ácida/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Superficie Celular/metabolismo , Fosfatasa Ácida/genética , Animales , Northern Blotting , Butiratos/farmacología , Carcinógenos/farmacología , Bovinos , Dihidrotestosterona/farmacología , Proteínas Fetales/farmacología , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores , Receptores de Superficie Celular/genética , Albúmina Sérica Bovina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Cell lines expressing the human metabotropic glutamate receptor subtype 5a (hmGluR5a) and hmGluR1b were used as targets in an automated high-throughput screening (HTS) system that measures changes in intracellular Ca2+ ([Ca2+]i) using fluorescence detection. This functional screen was used to identify the mGluR5-selective antagonist, SIB-1757 [6-methyl-2-(phenylazo)-3-pyridinol], which inhibited the glutamate-induced [Ca2+]i responses at hmGluR5 with an IC50 of 0.37 microM compared with an IC50 of >100 microM at hmGluR1. Schild analysis demonstrated a noncompetitive mechanism of inhibition. Pharmacophore mapping was used to identify an additional compound, SIB-1893 [(E)-2-methyl-6-(2-phenylethenyl)pyridine], which was also shown to block glutamate-induced increases in [Ca2+]i at hmGluR5 with an IC50 of 0.29 microM compared with an IC50 of >100 microM at hmGluR1. SIB-1757 and SIB-1893 showed little or no activity when tested for agonist and antagonist activity at the other recombinant human mGluR subtypes, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and N-methyl-D-aspartate receptors. In rat neonatal brain slices, SIB-1757 and SIB-1893 inhibited (S)-3,5-dihydroxyphenylglycine (DHPG)-evoked inositol phosphate accumulation in hippocampus and striatum by 60% to 80%, with a potency similar to that observed on recombinant mGluR5. However, in the cerebellum, a brain region with low mGluR5 expression, SIB-1757 failed to inhibit DHPG-evoked inositol phosphate accumulation. In cultured rat cortical neurons, SIB-1757 and SIB-1893 largely inhibited DHPG-evoked [Ca2+]i signals, revealing a population of neurons that were less sensitive to SIB-1757 and SIB-1893. This is the first description of highly selective, noncompetitive mGluR5 antagonists. These compounds will be useful tools in evaluating the role of mGluR5 in normal physiology and in animal models of disease.
Asunto(s)
Antagonistas de Aminoácidos Excitadores/farmacología , Fenazopiridina/análogos & derivados , Piridinas/farmacología , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Animales , Animales Recién Nacidos , Unión Competitiva , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células CHO , Calcio/metabolismo , Células Cultivadas , Cricetinae , Antagonistas de Aminoácidos Excitadores/química , Humanos , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Fenazopiridina/química , Fenazopiridina/farmacología , Piridinas/química , Ratas , Ratas Sprague-Dawley , Receptor del Glutamato Metabotropico 5 , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadAsunto(s)
Expresión Génica , Receptores de N-Metil-D-Aspartato/genética , Animales , Células Cultivadas , Humanos , MamíferosRESUMEN
cDNAs encoding four isoforms of the human NMDA receptor (NMDAR) NMDAR2C (hNR2C-1, -2, -3, and -4) have been isolated and characterized. The overall identity of the deduced amino acid sequences of human and rat NR2C-1 is 89.0%. The sequences of the rat and human carboxyl termini (Gly925-Val1,236) are encoded by different exons and are only 71.5% homologous. In situ hybridization in human brain revealed the expression of the NR2C mRNA in the pontine reticular formation and lack of expression in substantia nigra pars compacta in contrast to the distribution pattern observed previously in rodent brain. The pharmacological properties of hNR1A/2C were determined by measuring agonist-induced inward currents in Xenopus oocytes and compared with those of other human NMDAR subtypes. Glycine, glutamate, and NMDA each discriminated between hNR1A/2C-1 and at least one of hNR1A/2A, hNR1A/2B, or hNR1A/2D subtypes. Among the antagonists tested, CGS 19755 did not significantly discriminate between any of the four subtypes, whereas 5,7-dichlorokynurenic acid distinguished between hNR1A/2C and hNR1A/2D. Immunoblot analysis of membranes isolated from HEK293 cells transiently transfected with cDNAs encoding hNR1A and each of the four NR2C isoforms indicated the formation of heteromeric complexes between hNR1A and all four hNR2C isoforms. HEK293 cells expressing hNR1A/ 2C-3 or hNR1A/2C-4 did not display agonist responses. In contrast, we observed an agonist-induced elevation of intracellular free calcium and whole-cell currents in cells expressing hNR1A/2C-1 or hNR1A/2C-2. There were no detectable differences in the macroscopic biophysical properties of hNR1A/2C-1 or hNR1A/2C-2.
Asunto(s)
Encéfalo/metabolismo , Genoma Humano , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcio/metabolismo , Línea Celular , Clonación Molecular , ADN Complementario/genética , Humanos , Isomerismo , Ratones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Oocitos/metabolismo , Ratas , Receptores de N-Metil-D-Aspartato/fisiología , Proteínas Recombinantes , Ribonucleasas , Distribución Tisular , XenopusRESUMEN
Androgen plays a critical role in regulating the growth and differentiation of normal prostate epithelia, as well as the initial growth of prostate cancer cells. Nevertheless, prostate carcinomas eventually become androgen-unresponsive, and the cancer is refractory to hormonal therapy. To gain insight into the mechanism involved in this hormone-refractory phenomenon, we have examined the potential role of the androgen receptor (AR) in that process. We have investigated the expression of AR and two prostate-specific androgen-responsive antigens, prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA), for the functional activity of AR in LNCaP and PC-3 human prostate carcinoma cells. Our results are as follows. (i) Clone 33 LNCaP cells express AR, PAcP, and PSA, and cell growth is stimulated by 5alpha-dihydrotestosterone (DHT). Stimulation of cell growth correlates with decreased cellular PAcP activity. (ii) In clone 81 LNCaP cells, the expression of PAcP decreases with a concurrent decrease in the degree of androgen stimulation of cell growth, whereas the expression of PSA mRNA level is up-regulated by DHT, as in clone 33 cells. Conversely, in PAcP cDNA-transfected clone 81 cells, an additional expression of cellular PAcP correlates with an increased stimulation by androgen, higher than the corresponding control cells. (iii) PC-3 cells express a low level of functional AR with no detectable PAcP or PSA, and the growth of PC-3 cells is not affected by DHT treatment. Nevertheless, in two PAcP cDNA-transfected PC-3 sublines, the expression of exogenous cellular PAcP correlates with androgen stimulation. This androgen stimulation of cell growth concurs with an increased tyrosine phosphorylation of a phosphoprotein of 185 kDa. In summary, the data indicate that the expression of AR alone is not sufficient for androgen stimulation of cell growth. Furthermore, in AR-expressing prostate cancer cells, the expression of cellular PAcP correlates with androgen stimulation of cell proliferation.
Asunto(s)
Fosfatasa Ácida/metabolismo , Andrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias de la Próstata/enzimología , Receptores Androgénicos/fisiología , División Celular/efectos de los fármacos , Humanos , Masculino , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Fosfotirosina/análisis , Antígeno Prostático Específico/metabolismo , ARN Mensajero/metabolismo , Testosterona/análogos & derivados , Testosterona/farmacología , Transfección/genética , Células Tumorales Cultivadas , Regulación hacia Arriba/fisiologíaRESUMEN
We isolated and characterized a cDNA encoding the human metabotropic glutamate receptor subtype 1b (hmGluR1b). In situ hybridization studies in human brain regions revealed a higher distribution of mGluR1 mRNA in the dentate gyrus of the hippocampus, the substantia nigra pars compacta and the Purkinje cell layer of the cerebellum compared to other regions studied. We established stable expression of recombinant hmGluR1b in L(tk-) mouse fibroblast and Chinese hamster ovary (CHO-dhfr-) cells. In both expression systems, agonist activation of hmGluR1b stimulated inositol phosphate (InsP) formation and elevation of the cytosolic free calcium ([Ca2+]i), and both responses were blocked by (S)-MCPG. The rank order of potency for agonists was quisqualate > glutamate > (1S,3R)-ACPD in both expression systems. Comparison of the agonist profiles of hmGluR1b and hmGluR5a, both stably expressed in L(tk-) cells, indicated the same rank order of potency (quisqualate > glutamate > or = (RS)-3,5-DHPG > or = (1S,3R)-ACPD), but each of the four agonists were more potent on hmGluR5a than on hmGluR1b. In antagonist studies, (S)-MCPG inhibited the agonist-induced InsP formation and elevation of [Ca2+]i in both hmGluR1b- and hmGluR5a-expressing cells. (S)-4CPG and (S)-4C3HPG both inhibited agonist responses only in hmGluR1b-expressing cells. However, in hmGluR5a-expressing cells the antagonist activity of (S)-4CPG and (S)-4C3HPG was dependent on the agonist used in the study, since they inhibited responses to glutamate but not to quisqualate. Stable cell lines expressing specific subtypes of human mGluRs represent valuable tools for the study of the mechanism of action of mGluRs at the molecular and cellular level and as screening targets for identification of subtype-selective agonists or antagonists.
Asunto(s)
Clonación Molecular , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Encéfalo/metabolismo , Células CHO , Línea Celular , Cricetinae , Relación Dosis-Respuesta a Droga , Expresión Génica , Humanos , Hibridación in Situ , Ratones , Ácido Quiscuálico/farmacología , ARN Mensajero/metabolismoRESUMEN
Mifepristone, also known as RU 486, is a 19-norsteroid derivative. Currently, mifepristone is being tested in clinical trials on meningioma and breast cancer. In this study we analyzed whether mifepristone could inhibit the growth of human prostate cancer cells including androgen-insensitive (PC-3 and DU145) and androgen-sensitive (LNCaP) cell lines. At 1-nM concentration, mifepristone exhibited a marginal stimulatory action on LN-CaP and PC-3 cells. Nevertheless, a dose-dependent growth inhibition on those same cell lines was observed at concentrations of 1 microM and 10 microM. Twenty-day exposure to the clinically achievable concentration of 1 microM mifepristone resulted in consistent inhibition of all three cell lines studied. Furthermore, this in vitro growth inhibition was reflected in an in vivo nude mouse system. Mifepristone at the dosage of 4 mg/100 g body weight completely suppressed the growth of PC-3 tumors for 21 days, although this was followed by a growth rate similar to that of the control tumor. To understand the possible mechanism of mifepristone inhibition, PC-3 cells were exposed to mifepristone in comparison with dexamethasone (Dex), progesterone, and 5 alpha-dihydrotestosterone (DHT), each at 1-microM concentration. The results demonstrated that while both DHT and Dex alone had essentially no effect on cell growth, progesterone alone resulted in a 20% growth inhibition, while mifepristone had more than 60% inhibition with a 16-day exposure. At an equal concentration, the degree of growth inhibition of PC-3 cells by mifepristone or progesterone was partially diminished by simultaneous exposure to Dex. In conclusion, our results demonstrated that the growth of androgen-insensitive prostate cancer cells can be directly inhibited by mifepristone in cultures. This in vitro inhibition is reflected in xenografted tumors.
Asunto(s)
Mifepristona/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Animales , División Celular/efectos de los fármacos , Dexametasona/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Mifepristona/uso terapéutico , Progesterona/farmacología , Neoplasias de la Próstata/patología , Receptores de Esteroides/análisis , Células Tumorales CultivadasRESUMEN
The expression of prostatic acid phosphatase (PAcP) in three human prostate carcinoma cell lines including LNCaP, DU 145 and PC-3, was studied to explore its potential role as a marker in the progression of prostate cancer. Although Southern blot analysis suggested the presence of PAcP gene in all three prostate carcinoma cell lines, the Northern blot analysis and the reverse transcriptase-polymerase chain reaction (RT-PCR) assay showed that PAcP mRNA can be detected only in LNCaP cells. As one of the major differences between LNCaP cells and PC-3 as well as DU 145 cells is the androgen-sensitivity of LNCaP cells, we then focused on the influence of PAcP expression by the presence of androgen receptor (AR) in human AR cDNA-transfected PC-3 cells and high passages of LNCaP cells. The results demonstrated that the transfection of human AR cDNA into PC-3 cells did not have any detectable effect on the expression of PAcP. Further, in LNCaP cells, while the level of PAcP mRNA diminished upon passage, the AR mRNA level remained approximately the same. Together, these data suggested that the differential expression of PAcP in different prostate carcinoma cells including high passages of LNCaP cells may occur at the transcriptional level and may have little linkage to the expression of AR.
Asunto(s)
Fosfatasa Ácida/genética , Regulación Neoplásica de la Expresión Génica , Próstata/enzimología , Neoplasias de la Próstata/enzimología , Secuencia de Bases , ADN Complementario/genética , Humanos , Masculino , Datos de Secuencia Molecular , Neoplasias de la Próstata/genética , ARN Mensajero/metabolismo , Receptores Androgénicos/genética , Transfección , Células Tumorales CultivadasRESUMEN
Prostatic acid phosphatase (PAcP) is a prostate epithelium-specific differentiation antigen. It has been demonstrated that human PAcP exhibits endogenous protein tyrosine phosphatase (PYP) activity, and that it represents the major PYP activity in normal prostate cells. Thus, it has been postulated that cellular PAcP may play a role in the tyrosine phosphorylation-mediated signal transduction. In this paper, we used LNCaP human prostate carcinoma cells, which express the endogenous PAcP, to study changes in cellular PAcP activity during cell growth. Our results demonstrated that PAcP activity increased when the cells reached confluence. Stimulation of cell growth by fresh culture medium or 5 alpha-dihydrotestosterone (DHT), a classical stimulator of prostate epithelial growth, resulted in a decline in PAcP activity. Moreover, transfection of PC-3 cells, which do not express PAcP, with a PAcP-expressing vector led to diminished cellular growth rate. These data established an inverse relationship between the cellular level of PAcP and the cell growth rate, suggesting that PAcP may be involved in regulating the growth of human prostate carcinoma cells.
Asunto(s)
Fosfatasa Ácida/análisis , Próstata/enzimología , Neoplasias de la Próstata/patología , Fosfatasa Ácida/genética , Fosfatasa Ácida/inmunología , División Celular , Humanos , Masculino , ARN Mensajero/análisis , Células Tumorales CultivadasRESUMEN
Human prostatic acid phosphatase (PAcP) is a prostate epithelium-specific differentiation antigen and its expression has been proposed to be regulated by androgens. Since cellular PAcP may function as a protein tyrosine phosphatase, we investigated the regulatory mechanism of its expression at different growth stages in LNCaP cells, the only cultured human prostate carcinoma cells that express an endogenous PAcP. Cells were plated at different densities to represent different stages of cellular growth for quantitating the expression of PAcP. In 4-d subconfluent cells, the cellular PAcP activity and protein level increased following the seeded cell density, consistent with mRNA levels. By day 7, all cultures had an approximately equal amount of total cellular proteins, indicating that cell growth approached to confluence, except the one that was plated at the lowest density. The cellular PAcP activity per cell was increased and corresponded to its protein level as observed in 4-d cultures. However, in 7-d cultured cells, although the PAcP protein level increased, its mRNA level declined. This increased PAcP protein level despite the decreased message was in part due to a prolonged half-life of the protein. Further, androgen effect on the PAcP mRNA level was also shown to be a cell density-dependent phenomenon. In low-density cultured cells, the PAcP mRNA level was elevated approximately 100% by 5 alpha-dihydrotestosterone (DHT) stimulation. However, in high-density confluent cells the mRNA level was slightly decreased by DHT treatment. Further, treatments with various growth stimulators resulted in various degrees of inhibition on PAcP mRNA levels. In conclusion, the data indicate that the cellular level of PAcP activity is associated with the cell density/confluence of LNCaP cells. Further, cell density could modulate androgen effect on PAcP expression at the mRNA level.