RESUMEN
BACKGROUND: Reports indicate that vascular endothelial growth factor receptor type 3 (VEGFR3) regulates cellular functions such as invasion, proliferation, and chemo-resistance. However, the exact function of the VEGFR3 signaling axis in prostate epithelial cells is poorly characterized. METHODS: The goal of this study was to evaluate whether TGFbeta1 in combination with VEGFD can promote pre-malignant invasive activities of intermediate basal cells (IBC-10a) isolated from human prostate cancer (Gleason score 6). RESULTS: hTERT immortalized IBC-10a cells normally grew as confluent "cobblestoned" monolayers, but treatment with TGFbeta1 (10 ng/ml for 2-6 hr) dissociated the cell-cell junctions and induced VEGFR3 translocation to the cell surface. This event was not inhibited by 10 microM cycloheximide or puromycin, indicating transcription and protein synthesis were not required. We further discovered that TGFbeta1 in combination with VEGFD induced a significant increase in the invasive activity of IBC-10a cells (>26% and 53% after 24 and 48 hr, respectively) in modified Boyden Chamber assays. TGFbetaRII receptor antibodies specifically blocked TGFbeta1 induction of VEGFR3 translocation to the cell surface and blocked VEGFD-induced invasion. Zymograms revealed that TGFbeta1 (and not VEGFR3) stimulated the secretion of MMP-2 and MMP-9, presumably to promote cell invasion. The cell invasion assays confirmed that antibodies specific for TGFbetaII receptor, MMP-2 and MMP-9 and VEGFR3, independently blocked TGFbeta1-induced invasion. CONCLUSIONS: For the first time, we have demonstrated the mechanism by which TGFbeta1 stimulates VEGFD/VEGFR3 receptor axis activation leading to increased cell migration and invasion by primary intermediate basal cell cultures.
Asunto(s)
Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Factor de Crecimiento Transformador beta1/metabolismo , Factor D de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Línea Celular Transformada , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Humanos , Uniones Intercelulares/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Próstata/metabolismo , Próstata/patología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta1/farmacología , Células Tumorales Cultivadas , Factor D de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
BACKGROUND: The CD133(hi) sub-population of prostate epithelial cells has been demonstrated to possess tumor-initiating capacity consistent with that of the cancer stem cell theory. However, the involvement of oncogenes such as c-myc has not been fully elucidated in the CD133(hi) sub-population. METHODS: We have isolated primary prostate cell strains (IBC-10a) and immortalized them by transfection with hTERT. The in vitro and in vivo tumorigenic capacity of isolated CD133(hi) and CD133(lo) cells was evaluated with respect to c-myc expression using specific sense and anti-sense oligonucleotides. RESULTS: Freshly immortalized cells consisted of <3.3% CD133(hi)/CD24(hi) sub-population (SP). "Prostaspheres" generated from single CD133(hi) cells in the presence of EGF consisted of approximately 10% CD133(hi) SPs in 12-21 day cultures. A single Prostasphere generated from single CD133(hi) cells (6-10 cell stage at day 6 injected i.t.) produced dysplastic lesions in NOD-SCID mice (n = 4/5). Treatment of Prostaspheres from CD133(hi) SPs in vitro with c-myc or cyclin D1 anti-sense oligonucleotides totally blocked colony forming ability and growth. Furthermore, treatment of fully formed, 6-day Prostaspheres for 48 hr with c-myc anti-sense significantly reduced c-myc expression and their ability to generate lesions in NOD-SCIDs (n = 10 Prostaspheres injected i.t./mouse). CONCLUSIONS: These data demonstrate for the first time that a single CD133(hi) cell is competent to generate Prostaspheres in vitro and that CD133(hi) Prostaspheres require c-myc to grow and form dysplastic lesions in vivo.
Asunto(s)
Antígenos CD/biosíntesis , ADN sin Sentido/genética , Glicoproteínas/biosíntesis , Células Madre Neoplásicas/patología , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-myc/genética , Antígeno AC133 , Animales , Western Blotting , ADN sin Sentido/administración & dosificación , Citometría de Flujo , Humanos , Cariotipificación , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Péptidos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Telomerasa/genética , TransfecciónRESUMEN
We have previously reported that interleukin 10 (IL-10) signalling stimulated activation of a specific enhancer element, termed HTE-1, to promote tissue inhibitor of matrix metalloproteinase1 (TIMP-1) expression in human bone metastatic PC-3 subclone (PC-3 ML) cells. Recently, we have identified an IL-10 responsive signal molecule, termed IL-10E1, which binds the HTE-1 element and cloned the gene encoding for the 22 kDa protein. In this paper, we have examined the mechanism of IL-10/IL-10 receptor signalling in two distinct human prostate cell lines, a 'normal' prostate epithelial cell line, termed NPTX-1532 and highly metastatic PC-3 ML tumour cells. Signalling cascade studies revealed that IL-10 stimulated tyrosine phosphorylation of JAK1 and TYK2 receptor kinases and tyrosine phosphorylation of IL-10E1. Phosphorylation, triggered IL-10E1's rapid translocation to the nucleus by 10-30 min. Deletion analysis combined with transient transfection experiments revealed that the n-terminal domain (approximately 74 a.a.) of the IL-10E1 protein, the nt-nls peptide, was stimulated by IL-10 to translocate to the nucleus and induce TIMP-1 expression. Site-directed mutagenesis further showed that phosphorylation of two tyrosine moieties (Y57 and Y62) of the nt-nls peptide was required for IL-10 activation of signalling and TIMP-1 expression. The data demonstrate, for the first time, that IL-10 receptor signalling of TIMP-1 expression is regulated by tyrosine phosphorylation of a novel gene, IL-10E1, in human prostate cells.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Interleucina-10/farmacología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Inhibidores de Proteasas/análisis , Receptores de Interleucina/fisiología , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Secuencia de Aminoácidos , Humanos , Masculino , Datos de Secuencia Molecular , Fosforilación , Receptores de Interleucina-10 , Transducción de Señal , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
We previously reported that a novel promoter enhancer element "human tissue inhibitors of metalloproteinases 1 (TIMP-1) enhancer" (HTE) and a novel transacting protein "cysteine rich transcription factor" (CRTF) induced TIMP-1 synthesis in prostate cancer cells 2xN.I.PC-3. In the present study, to clarify the significance of CRTF in gastrointestinal cancers we measured the binding activity of CRTF to HTE using an electrophoretic mobility shift assay (EMSA), and the TIMP-1 concentration by ELISA after various stimulation of six cancer cell lines (KE-3, TE-9, MKN-28, MKN-45, KM12SM, SW620). In three cell lines (KE-3, MKN-45, SW620), both the binding activity of CRTF and TIMP-1 concentration significantly increased after IL-10 stimulation. Fetal bovine serum (FBS) did not affect the binding activity of CRTF, whereas FBS induced TIMP-1 synthesis in all cell lines. In KE-3 esophageal cancer cells and SW620 colon cancer cells, both the binding activity of CRTF and TIMP-1 concentration increased in the presence of a conditioned medium (CM) of fibroblasts which was isolated from human colon cancer tissues, but did not increase in MKN-45 cells. Moreover, in the fibroblasts, both the binding activity of CRTF and the TIMP-1 concentration increased in the presence of CM from KM12SM, SW620, and TE-9 cancer cell lines. These results suggested that IL-I0, and unknown factors in addition to IL-10, induced TIMP-1 synthesis via an increase in the binding activity of CRTF in gastrointestinal cancers, and that interaction between cancer cells and fibroblasts may play an important role in TIMP-1 synthesis through a signal transduction pathway consisting of CRTF phosphorylation and HTE activation.
Asunto(s)
Neoplasias Gastrointestinales/genética , Proteínas Nucleares/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/genética , Factores de Transcripción/metabolismo , Células Cultivadas , Neoplasias del Colon/genética , Medios de Cultivo Condicionados , Elementos de Facilitación Genéticos , Neoplasias Esofágicas/genética , Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1/farmacología , Masculino , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Inhibidor Tisular de Metaloproteinasa-1/análisis , Células Tumorales CultivadasRESUMEN
Breast cancer is a classic hormone-dependent malignant disease that is influenced by estrogen. However, the molecular links between estrogen and cell proliferation in healthy and malignant breast tissue are complex and as yet not well understood. The selective estrogen receptor modulators (SERMs), which are competitive inhibitors of estrogen binding at estrogen receptors alpha and beta, have become important weapons in the prevention and treatment of breast cancer. These agents also offer opportunities for the elucidation of the multiple molecular mechanisms by which estrogen affects cell proliferation. Each SERM-estrogen receptor complex has a unique structure that influences its activity in different body tissues. Unraveling the links between SERM structure and function not only may shed light on the signaling pathways that connect estrogen to cell proliferation but also may allow the design of new agents specifically targeted to affect certain events along these pathways.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/prevención & control , Moduladores Selectivos de los Receptores de Estrógeno/uso terapéutico , Resistencia a Antineoplásicos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Clorhidrato de Raloxifeno/farmacología , Clorhidrato de Raloxifeno/uso terapéutico , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/fisiología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Tamoxifeno/uso terapéuticoRESUMEN
Changes in the native vasculature of the prostate gland associated with prostate adenocarcinoma have not been well characterized. Eighty-nine whole mounts of entirely submitted radical prostatectomies were reviewed. Thirty prostates with a minimum of five native arteries surrounded by carcinoma with corresponding control arteries were found and included in this study. The number of nuclei in the media of native arteries was recorded per 0.138 mm2 using a 40x objective. The number of nuclei in vessels embedded in carcinoma (n = 204) was increased when compared with controls (26.37 versus 20.58 mean nuclei per 0.138 mm2; P < .001). Pathologic Stage T3 carcinomas contained vessels that were more cellular than stage T2 (P < .001). Vessels embedded in Gleason Grade 4 showed more cellularity than arteries embedded in Gleason Grade 3 (P < .002). Increased media cellularity of native prostate vessels encased in carcinoma is a histologic feature of higher grade/stage prostate carcinoma and provides positive indicator of advanced prostate cancer.
Asunto(s)
Adenocarcinoma/irrigación sanguínea , Neovascularización Patológica/patología , Neoplasias de la Próstata/irrigación sanguínea , Adenocarcinoma/patología , Adenocarcinoma/cirugía , Anciano , Arterias/patología , Recuento de Células , Núcleo Celular/patología , Progresión de la Enfermedad , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Masculino , Persona de Mediana Edad , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Túnica Media/citologíaRESUMEN
Cultures from high grade prostatic intraepithelial neoplasia (HGPIN) have been established and immortalized by HPV-18 infection. The cultures were identified as PIN by Western blotting with anti-cytokeratin (34betaE12) and prostate specific antigen (PSA) antibodies. We examined the growth capabilities of the cultures in the presence of TGF-beta1, activin-A, follistatin (FS), androgens (DHEA, DHT) and several cytokines (IL-10, IL-2, IL-4). IL-10, FS, and DHT stimulated cell proliferation and colony forming ability, while the other cytokines and growth factors had no discernable effect. In addition, DHT and to a lesser extent IL-10 both stimulated PSA production. Activin-A blocked IL-10, FS, and DHT stimulated growth and PSA production. We interpret the data to mean that IL-10 induction of FS secretion (and FS binding of activin A) restores the normal growth capabilities of HGPIN cultures.
Asunto(s)
Dihidrotestosterona/farmacología , Glicoproteínas/farmacología , Sustancias de Crecimiento/farmacología , Inhibinas/farmacología , Interleucina-10/farmacología , Papillomaviridae , Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/patología , Activinas , Adyuvantes Inmunológicos/farmacología , Andrógenos/fisiología , Animales , Western Blotting , Adhesión Celular/fisiología , División Celular/efectos de los fármacos , Línea Celular Transformada , Transformación Celular Viral , Ensayo de Unidades Formadoras de Colonias , Deshidroepiandrosterona/farmacología , Ensayo de Inmunoadsorción Enzimática , Folistatina , Humanos , Luz , Masculino , Ratones , Ratones SCID , Microscopía , Neoplasia Intraepitelial Prostática/virología , Neoplasias de la Próstata/virología , Células Tumorales CultivadasRESUMEN
Transfection of primary human prostate tumor cells (i.e., HPCA-10a, 10b, 10c, and 10d lines) with the transforming growth factor (TGF)-beta1 gene stimulated anchorage-independent growth and promoted tumor growth, angiogenesis, and metastasis after orthotopic implantation in severe combined immunodeficiency mice. In contrast, interleukin (IL)-10 transfected cells or cells cotransfected with these two genes exhibited reduced growth rates and significantly reduced angiogenesis and metastasis after 8, 12, and 16 weeks. Enzyme-linked immunosandwich assays confirmed that the respective tumors expressed elevated levels of TGF-beta1 and IL-10 in vivo. ELISAs further showed that TGF-beta1 expression induced matrix metalloproteinases-2 (MMP-2) expression, whereas IL-10 down-regulated MMP-2 expression while up regulating TIMP-1 in the transfected cells. Also, tumor factor VIII levels correlated with TGF-beta1 and MMP-2 expression and inversely with IL-10 and TIMP-1 levels. More importantly, mouse survival was zero after 4-6 months in mice bearing TGF-beta1- and MMP-2-expressing tumors and increased significantly in mice implanted with IL-10- and TIMP-1-expressing tumors (i.e., to >80% survival). Analysis of the metastatic lesions showed that they expressed TGF-beta1 and MMP-2 but barely detectable levels of IL-10 or TIMP-1, suggesting that IL-10 and TIMP-1 might normally block tumor growth, angiogenesis, and metastasis.
Asunto(s)
Interleucina-10/genética , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/terapia , Factor de Crecimiento Transformador beta/genética , Animales , Ensayo de Unidades Formadoras de Colonias , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Gelatinasas/metabolismo , Terapia Genética , Humanos , Inmunohistoquímica , Interleucina-10/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz , Metaloendopeptidasas/metabolismo , Ratones , Ratones SCID , Metástasis de la Neoplasia/prevención & control , Trasplante de Neoplasias , Neovascularización Patológica , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Análisis de Supervivencia , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Transfección , Factor de Crecimiento Transformador beta/metabolismo , Trasplante Heterólogo , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
In in vitro angiogenesis assays, aggregates of human papilloma virus (HPV)-18-immortalized primary human prostate cancer cells (HPCA-5aHPV-18 or HPCA-10aHPV-18 cells) induced human bone marrow endothelial cells (HBMCE-1 cells) to form microvessels in three-dimensional collagen I gels after 1-2 days incubation at 37 degrees C. The microvessels aligned perpendicular to the tumor aggregates and abutted on the edges of the aggregates. The number and length of the microvessels increased significantly from day 1 to 2 (i.e., by approximately 30%). ELISAs showed that the HPCA-5aHPV-18 cells normally secreted low levels of tissue inhibitor of metalloproteinase (TIMP)-2, matrix metalloproteinase (MMP)-2, and MMP-9 but relatively high levels of TIMP-1. In contrast, HPCA-10aHPV-18 cells secreted high levels of MMP-2 and MMP-9 (>40 pg/microg protein) but low levels of TIMP-1 and TIMP-2 (<5 pg/microg protein). Interleukin 10 (IL-10) (15 ng/ml) induced TIMP-1 production (>15 pg/microg protein) but reduced MMP-2 and MMP-9 secretion (<5 pg/microg protein) by the HPCA-5aHPV-18 and HPCA-10aHPV-18 cells. IL-10 (15 ng/ml) and MMP-9/MMP-2 antibodies all blocked induction of microvessel formation in the coculture experiments. In contrast, IL-10 receptor antibodies and TIMP-1 antibodies countered IL-10's effects and promoted angiogenesis. The data demonstrated that IL-10 stimulation of TIMP-1 and inhibition of MMP-2 and MMP-9 secretion by prostate tumor cells can control induction of angiogenesis in vitro.
Asunto(s)
Colagenasas/metabolismo , Gelatinasas/metabolismo , Interleucina-10/farmacología , Metaloendopeptidasas/metabolismo , Neovascularización Patológica/enzimología , Neoplasias de la Próstata/irrigación sanguínea , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Anticuerpos/farmacología , Células de la Médula Ósea/fisiología , Técnicas de Cocultivo , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Gelatinasas/antagonistas & inhibidores , Humanos , Masculino , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Metaloendopeptidasas/antagonistas & inhibidores , Neovascularización Patológica/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/virología , Células Tumorales CultivadasRESUMEN
Upregulation of vascular endothelial growth factor (VEGF) expression induced by hypoxia is crucial event leading to neovascularization. Cyclooxygenase-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, has been demonstrated to be induced by hypoxia and play role in angiogenesis and metastasis. To investigate the potential effect of COX-2 on hypoxia-induced VEGF expression in prostate cancer. We examined the relationship between COX-2 expression and VEGF induction in response to cobalt chloride (CoCl2)-simulated hypoxia in three human prostate cancer cell lines with differing biological phenotypes. Northern blotting and ELISA revealed that all three tested cell lines constitutively expressed VEGF mRNA, and secreted VEGF protein to different degrees (LNCaP > PC-3 > PC3ML). However, these cell lines differed in the ability to produce VEGF in the presence of CoCl2-simulated hypoxia. CoCl2 treatment resulted in 40% and 75% increases in VEGF mRNA, and 50% and 95% in protein secretion by LNCaP and PC-3 cell lines, respectively. In contrast, PC-3ML cell line, a PC-3 subline with highly invasive, metastatic phenotype, exhibits a dramatic upregulation of VEGF, 5.6-fold in mRNA and 6.3-fold in protein secretion after treatment with CoCl2. The upregulation of VEGF in PC-3ML cells is accompanied by a persistent induction of COX-2 mRNA (6.5-fold) and protein (5-fold). Whereas COX-2 expression is only transiently induced in PC-3 cells and not affected by CoCl2 in LNCaP cells. Moreover, the increases in VEGF mRNA and protein secretion induced by CoCl2 in PC-3ML cells were significantly suppressed following exposure to NS398, a selective COX-2 inhibitor. Finally, the effect of COX-2 inhibition on CoCl2-induced VEGF production was reversed by the treatment with exogenous PGE2. Our data demonstrate that VEGF induction by cobalt chloride-simulated hypoxia is maintained by a concomitant, persistent induction of COX-2 expression and sustained elevation of PGE2 synthesis in a human metastatic prostate cancer cell line, and suggest that COX-2 activity, reflected by PGE2 production, is involved in hypoxia-induced VEGF expression, and thus, modulates prostatic tumor angiogenesis.
Asunto(s)
Cobalto/farmacología , Factores de Crecimiento Endotelial/biosíntesis , Isoenzimas/biosíntesis , Isoenzimas/farmacología , Linfocinas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/farmacología , Neoplasias de la Próstata/metabolismo , Andrógenos/fisiología , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/biosíntesis , Dinoprostona/farmacología , Interacciones Farmacológicas , Inducción Enzimática/efectos de los fármacos , Humanos , Masculino , Proteínas de la Membrana , Metástasis de la Neoplasia , Neoplasias Hormono-Dependientes/enzimología , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Nitrobencenos/farmacología , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Estimulación Química , Sulfonamidas/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
We have identified an IL-10 inducible enhancer (HTE) (5'-CACGATGACTCATCACTGTTGAAAGACA-3') (-864 to -836 bp) and associated silencer element (HTS) (5'-CCACTGGCCCATCGTATAT-3') (-1284 to -1266 bp) in the 5' promoter region of the human tissue inhibitor of metalloproteinase-1 (TIMP-1) gene. Chloramphenicol acetyl transferase (CAT), electrophoretic migration shift assays (EMSAs), and DNase footprinting revealed that IL-10 (15 ng/ml for 1-2 h) induced the HTE enhancer. In comparison, phorbol ester stimulated the HTS silencer and blocked IL-10's effects in a dose-dependent, orientation- and position-independent fashion, suggesting that HTS is a true silencer element. EMSAs combined with deletion and mutation analysis of the HTE and HTS elements confirmed these observations. Finally, Northern blot, Western blot, immunoprecipitation, and ELISA analysis showed that IL-10 (15 ng/ml) induced TIMP-1 expression (approximately 10-fold by 18 h), whereas PMA (100 ng/ml) inhibited the stimulatory effects of IL-10 on TIMP-1 expression. The data indicate that HTE and HTS function as positive and negative regulatory elements that control human TIMP-1 expression.
Asunto(s)
ADN de Neoplasias/genética , Genes Reguladores , Interleucina-10/farmacología , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Inhibidor Tisular de Metaloproteinasa-1/genética , Secuencia de Bases , Carcinógenos/farmacología , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/metabolismo , Clonación Molecular , ADN de Neoplasias/metabolismo , Desoxirribonucleasa I/análisis , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-6/farmacología , Masculino , Datos de Secuencia Molecular , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
We have previously identified (M. Wang et al., Oncol. Res., in press, 1998) an enhancer element [human tissue inhibitor of metalloproteinase-1 enhancer-1 (HTE)] for the human tissue inhibitor of metalloproteinase-1 promoter that binds a novel zinc finger, cysteine-rich transcription factor (CRTF). In this study, we have used electrophoretic mobility shift assays to examine the relative level of expression of CRTF, jun/fos, and IFN-gamma responsive signal transducer activators of transcription (STATs) that bind specific HTE, activator protein, and IFN-gamma (Fcy and interferon regulatory factor) response motifs in tumor lines and human prostate tissue [i.e., normal (n = 3); benign prostatic hyperplasia (BPH; n = 12); high grade prostate intraepithelial neoplasia (PIN; n = 10); and prostate cancer adenocarcinoma (PCA; n = 61) plus seminal vesicle (n = 10) tissues]. The data showed that CRTF was overexpressed in PCA (Gleason's score, 10>8>6>5>4) compared with BPH, PIN, seminal vesicle, and normal tissues. To a much lesser degree, jun/fos and STAT 1 were also elevated in PCA compared to BPH, PIN, and normal tissues. In addition, blinded studies showed that CRTF and jun/fos were present at low levels in organ-confined specimens but at significantly elevated levels (P < 0.001) in samples exhibiting capsular penetration and localized spread, which indicated that CRTF and perhaps jun/fos were markers for cancer progression.
Asunto(s)
Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Factores de Transcripción/biosíntesis , Sitios de Unión , Cisteína/metabolismo , Sondas de ADN , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/biosíntesis , Progresión de la Enfermedad , Humanos , Masculino , Estadificación de Neoplasias , Pronóstico , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Factor de Transcripción STAT1 , Sensibilidad y Especificidad , Transactivadores/biosíntesis , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
We have permanently transfected human prostate PC-3 ML tumor cells and examined the influence of interleukin 10 (IL-10) production on tumor growth and metastasis following orthotopic implantation in the prostate gland of severe combined immunodeficient mice. Measurements of tumor volume after 5, 8, and 12 weeks indicated that tumor volume was negatively correlated with the amount of IL-10 production. Likewise, the extent of metastasis was inversely related to the amount of IL-10 produced. Following i.v. injection, the IL-10-expressing clones also failed to metastasize to the bone marrow. Controls showed that PC-3 ML and PC-3 ML mock clones grew rapidly and metastasized when implanted orthotopically or injected i.v. via the tail vein. Mouse survival curves showed that all of the mice injected orthotopically with the PC-3 ML clones died by about 14-16 weeks, whereas the PC-3 ML-IL10a or PC-3 ML-ILl0b clones induced only 10-20% death after 23-24 weeks. Likewise, survival studies showed a high death rate by approximately 30 days with PC-3 ML mock cells but <10% death by 12 weeks with the IL-10-transfected clones injected i.v. via the tail vein. The data strongly suggest that IL-10 production blocks tumor growth and metastasis in severe combined immunodeficient mice.
Asunto(s)
Interleucina-10/genética , Interleucina-10/fisiología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Animales , División Celular/fisiología , Células Clonales , Ensayo de Inmunoadsorción Enzimática , Humanos , Inyecciones Intravenosas , Interleucina-10/biosíntesis , Masculino , Ratones , Ratones SCID , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias de la Próstata/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
We have previously shown that alendronate can prevent human PC-3 ML tumor cell metastasis to the bone (Wang and Stearns, 1991, Differentiation, 48, 115-25). In this paper, ELISAs and Western blots showed that TGF-beta1 stimulated the secretion of a 72 kDa matrix metalloproteinase 2 (MMP-2) to enhance the solubilization of radiolabeled collagen 1 by metastatic human prostate PC-3 ML cells. A potent bisphosphonate compound, alendronate, inhibited MMP-2 secretion to block solubilization of collagen 1. Alendronate failed to inhibit MMP-2 activity directly, but instead appeared to block cellular secretion of MMP-2. Alendronate failed to inhibit secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2; the inhibitor of MMP-2) and the decrease in collagen 1 solubilization observed may occur, in part, from changes in the molar stoichiometry of TIMP-2 to MMP-2. We conclude that alendronate may be a potent inhibitor of bone resorption based on its ability to block MMP-2 secretion by tumor cells.
Asunto(s)
Alendronato/farmacología , Colágeno/metabolismo , Neoplasias de la Próstata/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Antineoplásicos/farmacología , Neoplasias Óseas/enzimología , Neoplasias Óseas/prevención & control , Neoplasias Óseas/secundario , Colágeno/antagonistas & inhibidores , Gelatinasas/antagonistas & inhibidores , Gelatinasas/metabolismo , Humanos , Hidroxiprolina/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/metabolismo , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Factor de Crecimiento Transformador beta/farmacología , Tritio , Células Tumorales CultivadasRESUMEN
We have previously shown that alendronate, a potent bisphosphonate compound, can prevent human PC-3 ML tumor cell metastasis to the bone (Stearns and Stearns, 1996, Oncol Res, 8, 69-75). In this paper, tumor cells were injected into the bone medullary cavity of SCID mice femurs both in vivo and following isolation in vitro. ELISAs showed that the amount of collagen I released in the bone marrow (i.e. in in vitro experiments) and the blood plasma (i.e. in in vivo experiments) was a function of the time of incubation or the number of cells injected in the femurs. ELISAs also showed that the levels of matrix metalloproteinase (MMP-2 and MMP-9) secreted in the bone medullary cavity of the femurs directly correlated with the extent of collagen 1 release. In vitro experiments carried out with 'live' and 'devitalized bone' yielded similar results suggesting that the tumor cells (not the osteoclasts) were primarily responsible for the bone solubilization observed. Alendronate pretreatment of the SCID mice (0.1 mg/kg biweekly for 3 weeks) (or the tumor cells) blocked both MMP production by the tumor cells (and the osteoclasts) and collagen I release, providing direct evidence that alendronate might be utilized to prevent bone destruction by metastatic tumor cells. Zymography indicated that MMP-2 activation might be responsible for bone solubilization. In addition, the data suggest that the plasma levels of collagen I might be a marker of bone metastasis and osteolysis.
Asunto(s)
Alendronato/farmacología , Neoplasias Óseas/enzimología , Colágeno/antagonistas & inhibidores , Gelatinasas/antagonistas & inhibidores , Inhibidores de la Metaloproteinasa de la Matriz , Metaloendopeptidasas/antagonistas & inhibidores , Neoplasias de la Próstata/enzimología , Animales , Neoplasias Óseas/metabolismo , Colágeno/sangre , Colágeno/metabolismo , Colagenasas/metabolismo , Ensayo de Inmunoadsorción Enzimática/normas , Gelatina , Gelatinasas/metabolismo , Humanos , Masculino , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Metaloendopeptidasas/metabolismo , Ratones , Ratones SCID , Neoplasias de la Próstata/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Células Tumorales CultivadasRESUMEN
The expression of MMP-2, MMP-9, TIMP-1, TIMP-2, and the urokinase receptor were examined in fetal and normal prostate tissues, benign prostatic hyperplasia and prostate cancer (n = 117). In situ hybridization with digoxigenin-labeled oligonucleotide probes demonstrated that TIMP-1 and TIMP-2 were expressed at elevated levels in the stroma of Gleason sum 5 tissues, whereas MMP-2 and MMP-9 were expressed at relatively low levels. In higher Gleason sum tissues (GS 8-10), TIMP-1 and TIMP-2 were not expressed, whereas MMP-2 and MMP-9 were intensely expressed. Furthermore, TIMP-1 and TIMP-2 expression was high in organ-confined specimens (OC, n = 43), somewhat lower in specimens with capsular penetration (CP, n = 29), and low or negative in samples with surgical margin/seminal vesicle (M/SV, n = 17) and lymph node (LN, n = 13) involvement. In contrast, MMP-2 and MMP-9 expression was low in the OC tissues; and noticeably higher in CP, M/SV, and LN specimens. Finally, correlation of TIMP and MMP expression with GS and pathological stage versus cure rate further revealed that a high percentage of organ-confined, GS 5 specimens expressing TIMP and little MMP were cured. In comparison, few of the GS 7-10 patients with capsular penetration and expressing MMP and little TIMP were cured. The data suggest that TIMP-1 (and TIMP-2) and MMP-2 (and MMP-9) are independent predictors of outcome.
Asunto(s)
Colagenasas/genética , Gelatinasas/genética , Glicoproteínas/genética , Hibridación in Situ , Metaloendopeptidasas/genética , Neoplasias de la Próstata/metabolismo , Proteínas/genética , Colagenasas/análisis , Estudios de Seguimiento , Gelatinasas/análisis , Glicoproteínas/análisis , Humanos , Masculino , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Metaloendopeptidasas/análisis , Estadificación de Neoplasias , Neoplasias de la Próstata/patología , Proteínas/análisis , ARN Mensajero/análisis , Inhibidor Tisular de Metaloproteinasa-2 , Inhibidores Tisulares de MetaloproteinasasRESUMEN
The molecular mechanism by which IL-10 inhibits metastases was examined using a SCID mouse model. Human PC-3 ML subclones normally metastasize to the lumbar vertebrae (approximately 70% mice injected, n = 14/20) following intravenous injection in severe combined immunodeficient (SCID) mice. IL-10 treatment of the PC-3 ML cells (15 ng/ml for 36 h) and the SCID mice (0.03 mg/kg/day for 30 days) reduced the number of metastases to 5% of the mice (n = 1/20). More importantly, following discontinuation of IL-10 treatment on day 30, the mice remained tumor-free and mouse survival rates increased dramatically (from < 30% in untreated mice) to about 85% in IL-10-treated mice. IL-10 did not appear to alter the growth rates or colony-forming ability of the PC-3 ML cells in vitro. Likewise, the growth of subcutaneous tumors and established bone marrow metastases was not inhibited by IL-10 treatment of the SCID mice. However IL-10 may inhibit the production of matrix metalloproteases (MMP) and prevent the establishment of metastasis. We therefore examined the influence of IL-10 on PC-3 ML production of MMP-2/MMP-9 and the tissue inhibitors of metalloproteinases (TIMP-1/2). Enzyme-linked immunosandwich assays (ELISAs) revealed that IL-10 (15 ng/ml for 36 h) treatment of the PC-3 ML cells down-regulated MMP-2 and MMP-9 while up-regulating TIMP-1 (not TIMP-2) expression. Likewise, IL-10-treated mice exhibited similar changes in TIMP-1 and MMP-2/MMP-9 expression. The IL-10 effects were blocked by IL-10 receptor antibodies. In comparison to IL-10, IL-4 failed to influence metastasis or the expression of TIMP-1, TIMP-2, MMP-2 and MMP-9 by PC-3 ML cells. We suggest that IL-10-regulated increases in the molar ratio of TIMP-1/MMP-9 and TIMP-2/MMP-2 might inhibit processes critical to the establishment of bone marrow metastasis.
Asunto(s)
Neoplasias Óseas/secundario , Colagenasas/efectos de los fármacos , Gelatinasas/efectos de los fármacos , Interleucina-10/farmacología , Metaloendopeptidasas/efectos de los fármacos , Proteínas de Neoplasias/efectos de los fármacos , Neoplasias de la Próstata/patología , Inhibidor Tisular de Metaloproteinasa-1/efectos de los fármacos , Animales , Colagenasas/metabolismo , Gelatinasas/metabolismo , Humanos , Masculino , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Metaloendopeptidasas/metabolismo , Ratones , Ratones SCID , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/metabolismo , Análisis de Supervivencia , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND: The objective of this study was to evaluate the expression of platelet-derived growth factor (PDGF A, PDGF B) and their receptors (PDGF alpha, PDGF beta) in prostatic intraepithelial neoplasia (PIN) and adenocarcinoma. METHODS: Peroxidase-antiperoxidase immunoperoxidase labeling was used to detect the extent of antibody labeling in 29 different high grade PIN specimens. RESULTS: PDGF A and PDGF alpha were uniformly expressed in glandular epithelial and stromal cells, whereas PDGF B was not expressed by either tissue. However, PDGF beta was lightly expressed in a uniform manner by both the glandular epithelial and stromal cells. CONCLUSIONS: The data suggest that an autocrine loop may exist in which the epithelial cells of high-grade PIN express PDGF AA and PDGF alpha to modulate growth of precancerous lesions.
Asunto(s)
Carcinoma in Situ/química , Factor de Crecimiento Derivado de Plaquetas/análisis , Neoplasias de la Próstata/química , Proteínas Proto-Oncogénicas/análisis , Receptores del Factor de Crecimiento Derivado de Plaquetas/análisis , Humanos , Procesamiento de Imagen Asistido por Computador , Técnicas para Inmunoenzimas , Masculino , Proteínas Proto-Oncogénicas c-sis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas , Receptor beta de Factor de Crecimiento Derivado de PlaquetasRESUMEN
We have utilized in situ hybridization and immunocytochemistry techniques to examine the expression of both alpha 2-macroglobulin (alpha 2-MR) and the 39 kDa receptor-associated protein (RAP) in 8 benign (BPH) and 34 malignant human prostate tissues, including 4 metastases. The levels of alpha 2-MR mRNA expression (but not RAP) increased significantly in high Gleason score carcinomas ( > 8) and in metastatic lesions, suggesting that alpha 2-MR expression is associated with advanced cancer. Semi-quantitative analysis with computer-assisted system analysis (CASA) confirmed this interpretation. This is the first report of alpha 2-MR expression being associated with advanced prostate cancer.