RESUMEN
Population-based studies have established that long-term intake of non-steroidal anti-inflammatory drugs (NSAIDs), compounds that inhibit the enzymatic activity of cyclooxygenase (COX), reduces the relative risk for developing colorectal cancer. These studies led to the identification of a molecular target, COX-2, that is involved in tumour promotion during colorectal cancer progression. Recent studies in humans indicate that therapy with specific COX-2 inhibitors might be an effective approach to colorectal cancer prevention and treatment.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Neoplasias Colorrectales/prevención & control , Inhibidores de la Ciclooxigenasa/uso terapéutico , Isoenzimas/antagonistas & inhibidores , Animales , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/enzimología , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Modelos Animales de Enfermedad , Humanos , Isoenzimas/fisiología , Proteínas de la Membrana , Prostaglandina-Endoperóxido Sintasas/fisiología , Prostaglandinas/biosíntesis , Receptores de Prostaglandina E/fisiologíaRESUMEN
Quantifying tumor burden is important for following the natural history of orthotopic colon cancer and therapeutic efficacy. Bioluminescence imaging (BLI) is commonly used for such assessment and has both advantages and limitations. We compared BLI and magnetic resonance imaging (MRI) for quantifying orthotopic tumors in a mouse model of colon cancer. Among sequences tested, T2-based MRI imaging ranked best overall for colon cancer border delineation, contrast, and conspicuity. Longitudinal MRI detected tumor outside the colon, indistinguished by BLI. Colon tumor weights calculated from MRI in vivo correlated highly with tumor weights measured ex vivo whereas the BLI signal intensities correlated relatively poorly and this difference in correlations was highly significant. This suggests that MRI may more accurately assess tumor burden in longitudinal monitoring of orthotopic colon cancer in this model as well as in other models.
Asunto(s)
Neoplasias del Colon/diagnóstico por imagen , Mediciones Luminiscentes , Imagen por Resonancia Magnética , Animales , Células HCT116 , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de NeoplasiasRESUMEN
Cyclooxygenase-2 (COX-2; Ptgs2) acts as a tumor promoter in rodent models for colorectal cancer, but its precise role in carcinogenesis remains unclear. We evaluated the contribution of host-derived COX-1 and COX-2 in tumor growth using both genetic and pharmacological approaches. Lewis lung carcinoma (LLC) cells grow rapidly as solid tumors when implanted in C57BL/6 mice. We found that tumor growth was markedly attenuated in COX-2(-/-), but not COX-1(-/-) or wild-type mice. Treatment of wild-type C57BL/6 mice bearing LLC tumors with a selective COX-2 inhibitor also reduced tumor growth. A decrease in vascular density was observed in tumors grown in COX-2(-/-) mice when compared with those in wild-type mice. Because COX-2 is expressed in stromal fibroblasts of human and rodent colorectal carcinomas, we evaluated COX-2(-/-) mouse fibroblasts and found a 94% reduction in their ability to produce the proangiogenic factor, VEGF. Additionally, treatment of wild-type mouse fibroblasts with a selective COX-2 inhibitor reduced VEGF production by 92%.
Asunto(s)
Isoenzimas/fisiología , Neoplasias Experimentales/etiología , Prostaglandina-Endoperóxido Sintasas/fisiología , Animales , Western Blotting , Carcinoma Pulmonar de Lewis/patología , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Factores de Crecimiento Endotelial/biosíntesis , Linfocinas/biosíntesis , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/patología , Neovascularización Patológica/etiología , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Growth factors and tumor promoters have been shown to play a role in intestinal epithelial growth regulation and transformation. In this study, transforming growth factor-alpha (TGF alpha) and the tumor promoter, tetradecanoyl phorbol acetate (TPA), are shown to stimulate the production of eicosanoids by rat intestinal epithelial (RIE-1) cells in culture. A 4.5-kb mRNA, which hybridizes to the mouse cyclooxygenase-2 cDNA probe, is elevated 18-fold within 30 min after TGF alpha or TPA treatment. Stimulation of RIE-1 cells with TGF alpha leads to the increase of a protein (M(r) approximately 69,000), which binds a monospecific antibody to the mouse cyclooxygenase-2 protein. Dexamethasone markedly inhibits the increase of the 4.5-kb mRNA. Pretreatment of TGF alpha or TPA-stimulated RIE-1 cells with dexamethasone or cyclooxygenase inhibitors prevents the increase in eicosanoid production by these cells. Treatment of quiescent RIE-1 cells with TGF alpha stimulates mitogenesis. This mitogenic activity is blocked by pretreating the cells with dexamethasone or cyclooxygenase inhibitors. A mitogen-inducible cyclooxygenase gene is thus shown to be regulated by TGF alpha and TPA in rat intestinal epithelial cells. We suggest that products of an intestinal growth factor-inducible cyclooxygenase may play a role in the regulation of mitogenesis.
Asunto(s)
División Celular/efectos de los fármacos , Eicosanoides/biosíntesis , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Factor de Crecimiento Transformador alfa/farmacología , Animales , Línea Celular , ADN/biosíntesis , Sondas de ADN , Complejo IV de Transporte de Electrones/biosíntesis , Inducción Enzimática , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Indometacina/farmacología , Mucosa Intestinal/metabolismo , Cinética , Ratones , Microsomas/enzimología , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Ratas , Sulindac/análogos & derivados , Sulindac/farmacología , Timidina/metabolismo , TransfecciónRESUMEN
The kidney is a rich source of prostaglandins. These eicosanoids, formed by cyclooxygenase-dependent metabolism of arachidonic acid, are important physiologic mediators of renal glomerular hemodynamics and tubular sodium and water reabsorption. Two separate isoforms of cyclooxygenase (COX) have now been identified: constitutive COX-1, encoded by a 2.8-kb mRNA, and mitogen-activated COX-2, encoded by a 4.0-4.5-kb mRNA. COX-2 expression increases during development and inflammation, but, except for brain, constitutive expression is low. It has been generally accepted that physiologic renal production of prostaglandins is mediated by COX-1. However, in the absence of inflammation, low levels of COX-2 mRNA are also detectable in the kidney. To examine the role of COX-2 in the kidney and determine its intrarenal localization, we used a 1.3-kb cDNA probe specific for the 3' untranslated region of rat COX-2 and COX-2-specific antiserum. The COX-2-specific cDNA probe hybridized with a 4.4-kb transcript in total RNA from adult rat kidney. Immunoblots of microsomes isolated from kidney cortex and papilla indicated immunoreactive COX-2 in both locations. In situ hybridization and immunohistochemistry indicated that renal cortical COX-2 expression was localized to the macula densa of the juxtaglomerular apparatus and to adjacent epithelial cells of the cortical thick ascending limb of Henle. In addition, COX-2 immunoreactivity was detected in interstitial cells in the papilla. No COX-2 message or immunoreactive protein was detected in arterioles, glomeruli, or cortical or medullary collecting ducts. When animals were chronically sodium restricted, the level of COX-2 in the region of the macula densa increased threefold (from 0.86 +/- 0.08 to 2.52 +/- 0.43/mm2) and the total area of the COX-2 immunoreactive cells in cortex increased from 34 microns2/mm2 of cortex to 226 microns2/mm2 of cortex. The intrarenal distribution of COX-2 and its increased expression in response to sodium restriction suggest that in addition to its proposed role in inflammatory and growth responses, this enzyme may play an important role in the regulation of salt, volume, and blood pressure homeostasis.
Asunto(s)
Isoenzimas/aislamiento & purificación , Aparato Yuxtaglomerular/enzimología , Prostaglandina-Endoperóxido Sintasas/aislamiento & purificación , Sodio en la Dieta/farmacología , Sodio/deficiencia , Animales , Northern Blotting , Western Blotting , Hibridación in Situ , Isoenzimas/biosíntesis , Isoenzimas/genética , Aparato Yuxtaglomerular/anatomía & histología , Aparato Yuxtaglomerular/efectos de los fármacos , Aparato Yuxtaglomerular/metabolismo , Riñón/anatomía & histología , Riñón/efectos de los fármacos , Riñón/enzimología , Riñón/metabolismo , Masculino , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
A considerable amount of evidence collected from several different experimental systems indicates that cyclooxygenase-2 (COX-2) may play a role in colorectal tumorigenesis. Large epidemiologic studies have shown a 40-50% reduction in mortality from colorectal cancer in persons taking aspirin or other nonsteroidal antiinflammatory drugs on a regular basis. One property shared by all of these drugs is their ability to inhibit COX, a key enzyme in the conversion of arachidonic acid to prostaglandins. Two isoforms of COX have been characterized, COX-1 and COX-2. COX-2 is expressed at high levels in intestinal tumors in humans and rodents. In this study, we selected two transformed human colon cancer cell lines for studies on the role of COX-2 in intestinal tumorigenesis. We evaluated HCA-7 cells which express high levels of COX-2 protein constitutively and HCT-116 cells which lack COX-2 protein. Treatment of nude mice implanted with HCA-7 cells with a selective COX-2 inhibitor (SC-58125), reduced tumor formation by 85-90%. SC-58125 also inhibited colony formation of cultured HCA-7 cells. Conversely, SC-58125 had no effect on HCT-116 implants in nude mice or colony formation in culture. Here we provide evidence that there may be a direct link between inhibition of intestinal cancer growth and selective inhibition of the COX-2 pathway.
Asunto(s)
Neoplasias Colorrectales/enzimología , Inhibidores de la Ciclooxigenasa/farmacología , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Pirazoles/farmacología , Animales , Northern Blotting , Western Blotting , División Celular , Neoplasias Colorrectales/tratamiento farmacológico , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/uso terapéutico , Humanos , Isoenzimas/genética , Isoenzimas/inmunología , Proteínas de la Membrana , Ratones , Ratones Desnudos , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/inmunología , Prostaglandinas/inmunología , Prostaglandinas/metabolismo , Pirazoles/uso terapéutico , ARN/análisis , ARN/metabolismo , Trasplante Heterólogo , Células Tumorales CultivadasAsunto(s)
Poliposis Adenomatosa del Colon/prevención & control , Antineoplásicos/uso terapéutico , Compuestos Orgánicos , Sulindac/uso terapéutico , Aminoquinolinas , Compuestos de Anilina , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Inhibidores de la Ciclooxigenasa/uso terapéutico , Quimioterapia Combinada , Receptores ErbB/antagonistas & inhibidores , Ratones , Modelos Biológicos , Transducción de Señal/efectos de los fármacosRESUMEN
Cyclooxygenase (COX) 2 expression is regulated via the Ras signaling pathway, and induction of mutated Ras rapidly increases COX-2 levels in intestinal epithelial cells. Protein kinase B (Akt/PKB) is an important effector of Ras signaling and a critical component of Ras-mediated transformation. Here we investigate the role of Akt/PKB in K-Ras-mediated induction of COX-2. Rat intestinal epithelial cells (IEC-6) were transfected with an inducible K-RasVal12 cDNA (IEC-iK-Ras cells). Addition of 5 mM isopropyl-1-thio-beta-D-galactopyranoside induced the expression of K-RasVal12, followed by increased activity of extracellular signal-regulated kinase and Akt/PKB. COX-2 levels were dramatically increased after induction of K-RasVal12. Inhibition of MAPK/ERK kinase activity by PD 98059 completely blocked the K-Ras-mediated induction of COX-2, whereas inhibition of PI3K/Akt/PKB activity with LY 294002 or by expressing a dominant negative Akt (Akt-K179M) partially blocked the induction of COX-2 by K-Ras. Transient transfection of cells with phosphatidylinositol 3-kinase and Akt expression vectors revealed that PI3/Akt/PKB activity predominantly regulates the stability of COX-2 mRNA. Thus, Akt/PKB activity is involved in K-Ras-induced expression of COX-2 and stabilization of COX-2 mRNA largely depends on the activation of Akt/PKB.
Asunto(s)
Isoenzimas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/metabolismo , Proteínas ras/fisiología , Animales , Línea Celular , Ciclooxigenasa 2 , Activación Enzimática , Inducción Enzimática , Células Epiteliales/enzimología , Células Epiteliales/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Genes ras , Mucosa Intestinal/citología , Mucosa Intestinal/enzimología , Mucosa Intestinal/fisiología , Isoenzimas/genética , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Prostaglandina-Endoperóxido Sintasas/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-akt , ARN Mensajero/genética , Ratas , Transfección , Proteínas ras/genéticaRESUMEN
Colorectal cancer is the second leading cause of death from cancer in the United States. Continuous use of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to reduce the risk of colorectal cancer in humans by 40-50%. Patients with familial adenomatous polyposis who take NSAIDs, such as sulindac, undergo a regression of intestinal adenomas. Rodents exposed to carcinogens that cause colon cancer have a 50-60% reduction in the size and number of colonic tumors when treated continuously with NSAIDs. One common target for these drugs is prostaglandin endoperoxide synthase, also referred to as cyclooxygenase (COX). We and others have shown recently that COX-2 levels are increased dramatically in 85-90% of human colorectal adenocarcinomas and in 40-50% of colonic adenomas. We prepared intestinal epithelial cells that express the COX-2 gene permanently and found that they have altered adhesion properties and resist undergoing apoptosis. We report here that these cells also have a 3-fold increase in the duration of G1, lower levels of cyclin D1 protein, and a marked decrease in retinoblastoma kinase activity associated with cyclin-dependent kinase 4. The delay in G1 transit may relate to the resistance of these cells to undergo programmed cell death, which could affect their tumorigenic potential.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Anticarcinógenos/uso terapéutico , Aspirina/uso terapéutico , Neoplasias Colorrectales/prevención & control , Fase G1 , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Proteínas Proto-Oncogénicas , Adenoma/prevención & control , Animales , Línea Celular , Neoplasias del Colon/prevención & control , Neoplasias Colorrectales/mortalidad , Ciclina D1 , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/biosíntesis , Ciclinas/biosíntesis , ADN/biosíntesis , ADN/efectos de los fármacos , Epitelio , Humanos , Intestinos , Cinética , Proteínas Oncogénicas/biosíntesis , Sistemas de Lectura Abierta , Ratas , Proteínas Recombinantes/biosíntesis , Proteína de Retinoblastoma/biosíntesis , Factores de Tiempo , Transfección , Estados Unidos/epidemiologíaRESUMEN
Previously, we have shown that forced expression of prostaglandin endoperoxide synthase-2 [also called cyclooxygenase (COX) 2] leads to inhibition of programmed cell death in intestinal epithelial cells. More recently, we have demonstrated that growth of human colonic cancer xenografts is inhibited by treatment with a highly selective COX-2 inhibitor in tumors that express COX-2 (HCA-7) but not in those that lack COX-2 expression (HCT-116). To explore the biochemical mechanisms involved in these effects, we have evaluated the role of COX-2-derived eicosanoid products on programmed cell death in human colon cancer cells. Here we report that PGE2 treatment of human colon cancer cells leads to increased clonogenicity of HCA-7, but not HCT-116 cells. Treatment with a highly selective COX-2 inhibitor (SC-58125) decreases colony formation in monolayer culture and this growth inhibition was reversed by treatment with PGE2. Additionally, PGE2 inhibits programmed cell death caused by SC-58125 and induces Bcl-2 expression, but did not affect Bcl-x or Bax expression in human colon cancer (HCA-7) cells. Therefore, decreased cell death caused by PGE2 would enhance the tumorigenic potential of intestinal epithelial cells. Thus, these results may help to explain a component of the mechanism by which COX inhibitors prevent colorectal cancer in humans.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias del Colon/metabolismo , Dinoprostona/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Células Clonales/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Immunoblotting , Isoenzimas/metabolismo , Proteínas de la Membrana , Ratones , Ratones Desnudos , Neoplasias Experimentales/patología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Pirazoles/farmacología , Células Madre/efectos de los fármacos , Trasplante Heterólogo , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Nonsteroidal anti-inflammatory drugs have potential for use in the prevention and/or treatment of colorectal cancer. We have studied the cytotoxic effect of a specific COX-2 inhibitor, celecoxib, against LLC, HCA-7, and HCT-15 cells grown in cell culture and have compared these results with its effect on HCA-7 cells grown as xenografts in nude mice. "High-dose" celecoxib (>20 microM) reduced the viability of all three cell lines in vitro as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometric analysis demonstrated that this loss of viability was attributable to the induction of apoptosis. Significantly, concentrations of the drug <10 microM had no effect on cell viability in vitro. The cytotoxic effects of high-dose celecoxib were independent of COX-2 inhibition because similar effects were observed in cox-2 (+/+), cox-2 (+/-) and cox-2 (-/-) fibroblasts. A plasma concentration of 2.3+/-0.7 microM was achieved when celecoxib (1250 mg/kg of chow) was fed to animals ad libitum. Despite a lack of toxicity at 2-3 microM celecoxib in vitro, there was attenuation of HCA-7 xenograft growth in vivo. Celecoxib had no effect on apoptosis, cell division, or the epithelial architecture of the normal gut in treated mice. These results support the need for additional clinical evaluation of celecoxib for treatment and/or prevention of colorectal cancer in humans.
Asunto(s)
Anticarcinógenos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa/farmacología , Mucosa Intestinal/efectos de los fármacos , Sulfonamidas/farmacología , Animales , Anticarcinógenos/toxicidad , Apoptosis/efectos de los fármacos , Celecoxib , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Neoplasias Colorrectales/patología , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/toxicidad , Relación Dosis-Respuesta a Droga , Humanos , Mucosa Intestinal/citología , Isoenzimas/antagonistas & inhibidores , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Prostaglandina-Endoperóxido Sintasas , Pirazoles , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We postulated that increased expression of the cell cycle regulators cyclin D1 and cyclin-dependent kinase (Cdk) 4 may be involved in the development of intestinal adenomas associated with familial adenomatous polyposis (FAP). In the present study of multiple intestinal neoplasia (Min) mice and human FAP patients, the expression and distribution of cyclin D1, Cdk4, and cell proliferative activity (5-bromo-2'-deoxyuridine incorporation) in normal and adenomatous intestinal epithelium were investigated. Immunohistochemical analysis of Min mouse intestine revealed that cyclin D1 immunoreactivity in the intestinal epithelium was restricted to the adenomatous areas, with a significantly higher percentage of positively staining nuclei in high-grade dysplasia versus low-grade dysplasia (54.8 +/- 18.4% versus 34.6 +/- 16.9%, P = 0.016). Morphologically normal areas of intestinal epithelia were uniformly negative for cyclin D1 immunoreactivity. Cdk4 nuclear immunoreactivity was restricted to the crypt areas in morphologically normal small intestine and colon. Conversely, Cdk4 immunoreactivity was uniformly abundant in adenomatous areas regardless of the degree of dysplasia. Increased expression of cyclin D1 and Cdk4 in adenomas was accompanied by a significantly increased 5-bromo-2'-deoxyuridine incorporation rate in the same areas. Immunoblot analysis of lysates from surgical specimens revealed increased levels of cyclin D1 and Cdk4 in the majority of intestinal adenomas from human FAP patients in comparison to the adjacent grossly normal colonic mucosa. Our results indicate that overexpression of cyclin D1 and Cdk4 occurs in intestinal adenomas and is associated with increased cell proliferative activity in premalignant neoplastic cells. Increased cyclin D1 immunoreactivity is associated with more severe dysplasia. These data suggest that abnormal up-regulation of these important G1 cell cycle proteins is a relatively early event in intestinal carcinogenesis and that these changes may contribute to malignant progression within those lesions.
Asunto(s)
Poliposis Adenomatosa del Colon/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Neoplasias Intestinales/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Primarias Múltiples/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas , Poliposis Adenomatosa del Colon/patología , Animales , División Celular , Ciclina D1 , Quinasa 4 Dependiente de la Ciclina , Humanos , Neoplasias Intestinales/patología , Ratones , Ratones Endogámicos AKR , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias Primarias Múltiples/patología , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
Tumor cells often become resistant to the growth-inhibitory effects of transforming growth factor beta (TGF-beta). Recent studies have identified TGF-beta type II receptor (RII) mutations in a subset of cancers, including colon cancer. To evaluate the expression of TGF-beta RII in premalignant intestinal adenomas and the relationship with cell cycle regulation, we investigated the expression of TGF-beta RII, cyclin D1, and cyclin-dependent kinase 4 (Cdk4) in Min/+ mouse intestinal adenomas. Immunohistochemistry indicated that TGF-beta RII cytoplasmic immunoreactivity was undetectable in the proliferative crypt zones of the normal small intestinal and normal colonic epithelium but was abundant toward the villus tips of the normal small intestine and the lumenal third of the colonic glands. As was observed in the proliferating crypt zones, TGF-beta RII immunoreactivity was dramatically decreased or undetectable in all adenomas examined in comparison to the abundant levels in adjacent normal differentiated intestinal epithelium. TGF-beta RII mRNA was also reduced in the adenomas in comparison to normal mucosa as determined by reverse transcription-PCR. In an inverse distribution to TGF-beta RII, Cdk4 nuclear immunoreactivity was restricted to the crypt regions of the small and large intestine, whereas cyclin D1 immunoreactivity was uniformly absent in normal intestinal epithelium. For both cyclin D1 and Cdk4, protein and mRNA levels were increased in intestinal adenomas but not in normal intestinal epithelium as determined by immunohistochemistry, in situ hybridization, and reverse transcription-PCR. In summary, the lack of TGF-beta RII expression was associated with increased cyclin D1 and Cdk4 expression in Min/+ mouse intestinal adenomas. We hypothesize that the former may enable tumor cells to escape from the normal growth-constraining influence of TGF-beta, whereas the latter promotes inappropriate cell proliferation and adenoma progression.
Asunto(s)
Adenoma/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Neoplasias Intestinales/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteína de la Poliposis Adenomatosa del Colon , Animales , Ciclina D1 , Quinasa 4 Dependiente de la Ciclina , Proteínas del Citoesqueleto/genética , Hibridación in Situ , Mucosa Intestinal/metabolismo , Ratones , Ratones Mutantes , Proteínas Serina-Treonina Quinasas , ARN Mensajero/genética , ARN Neoplásico/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genéticaRESUMEN
15-Lipoxygenase (15-LOX)-2 is expressed in benign prostate secretory cells and benign prostate produces 15S-hydroxyeicosatetraenoic acid (15S-HETE) from exogenous arachidonic acid (AA). In contrast, 15S-LOX-2 and 15S-HETE formation are reduced in prostate carcinoma (Pca). The mechanisms whereby reduced 15-LOX-2 may contribute to Pca development or progression are not known. We investigated the expression of peroxisome proliferator-activated receptor (PPAR) gamma in benign and malignant prostate tissues and the ability of 15S-HETE to activate PPARgamma-dependent transcription and modulate proliferation of the Pca cell line PC3. In contrast to benign prostate and similar to most Pca tissues, 15-LOX-2 mRNA was not detected in PC3 cells, and they did not produce detectable 15-HETE from [14C]AA. By reverse transcription-PCR, PPARgamma mRNA was present in 18 of 18 benign and 9 of 9 tumor specimens. The PPARgamma ligand BRL 49653 and 15S-HETE caused a dose-dependent inhibition of PC3 proliferation in a 14-day soft agar colony-forming assay (IC50 of 3 and 30 microM, respectively). 15S-HETE (10 microM) caused greater inhibition than 10 microM 15R-HETE. At 3 days, BRL 49653 and 15S-HETE caused a slight increase in cells in G0-G1 and a corresponding decrease in cells in S phase. In PC3 cells transiently transfected with a luciferase reporter linked to a PPAR response element, 1 microM BRL 49653 and 10 microM 15S-HETE caused approximately threefold and greater than twofold induction of PPAR-dependent transcription, respectively. By quantitative real-time reverse transcription-PCR and Northern analysis, 3-day treatment with BRL 49653 and 15S-HETE caused a reduction of PPARgamma expression but a marked up-regulation of the PPAR response element containing adipocyte type fatty acid binding protein. These results support the hypothesis that 15-LOX-2-derived 15S-HETE may constitute an endogenous ligand for PPARgamma in the prostate and that loss of this pathway by reduced expression of 15-LOX-2 may contribute to increased proliferation and reduced differentiation in prostate carcinoma.
Asunto(s)
División Celular/efectos de los fármacos , Ácidos Hidroxieicosatetraenoicos/farmacología , Neoplasias de la Próstata/genética , Receptores Citoplasmáticos y Nucleares/genética , Tiazolidinedionas , Factores de Transcripción/genética , Agar/farmacología , Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/metabolismo , Northern Blotting , Catálisis , Medios de Cultivo/farmacología , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Luciferasas/efectos de los fármacos , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Neoplasias de la Próstata/patología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Rosiglitazona , Tiazoles/farmacología , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Most colorectal adenomas and carcinomas arise in the setting of chromosomal instability characterized by progressive loss of heterozygosity. In contrast, approximately 15-20% of colorectal neoplasms arise through a distinct genetic pathway characterized by microsatellite instability (MSI) associated with frequent loss of expression of one of the DNA mismatch repair enzymes, most often hMLH1 or hMSH2. These distinct genetic pathways are reflected by differences in tumor histopathology, distribution in the colon, prognosis, and dwell time required for progression from adenoma to carcinoma. To determine whether these two groups of tumors differ in their expression of cyclooxygenase-2 (COX-2), a putative chemopreventative target, immunostaining for this protein was performed in colorectal cancers categorized by the presence (n = 41) and absence (n = 66) of defective mismatch repair. Defective mismatch repair was defined by the presence of tumor microsatellite instability (MSI-H, > or =40% of markers demonstrating instability) and by the absence of protein expression for either hMLH1 or hMSH2. Overall, our results showed that low or absent COX-2 staining was significantly more common among tumors with defective mismatch repair (P = 0.001). Other features predictive of low COX-2 staining included marked tumor infiltrating lymphocytosis, and solid/cribiform or signet ring histological patterns. These observations indicate that colorectal cancers with molecular and phenotypic characteristics of defective DNA mismatch repair express lower levels of COX-2. The clinical implications of this biological distinction remain unknown but should be considered when assessing the efficacy of COX-2 inhibitors for chemoprevention in patients whose tumors may arise in the setting of defective DNA mismatch repair.
Asunto(s)
Neoplasias Colorrectales/enzimología , Reparación del ADN , Proteínas de Unión al ADN , Isoenzimas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Proteínas Adaptadoras Transductoras de Señales , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Portadoras , Neoplasias Colorrectales/genética , Ciclooxigenasa 2 , Femenino , Humanos , Inmunohistoquímica , Masculino , Proteínas de la Membrana , Repeticiones de Microsatélite , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS , Proteínas de Neoplasias/biosíntesis , Proteínas Nucleares , Estudios Prospectivos , Proteínas Proto-Oncogénicas/biosíntesisRESUMEN
Numerous reports suggest that use of nonsteroidal anti-inflammatory drugs (NSAIDs) decrease mortality from colorectal cancer. To better understand all of the mechanisms underlying this effect, the global pattern of gene expression in colon carcinoma cells following treatment with NS-398, a selective cyclo-oxygenase-2 inhibitor was evaluated. We utilized suppression subtractive hybridization combined with differential screening to identify genes whose expression was affected following treatment. Among the subtracted cDNA fragments confirmed as differentially expressed, there were two which are known to be involved in the regulation of cell adhesion (human FAT and proto-cadherin-7). We identified two other genes whose levels were decreased and these are known to be involved in the regulation of cell proliferation (cyclin K and p-100). We identified additional genes which are involved in different signaling pathways which regulate programmed cell death (Dynamin 2, Pdcd4 and LIP.1). These results provide evidence that some of the effects of NS-398 on carcinoma cells may be due to modulation of genes which regulate programmed cell death, cell proliferation and cell-cell communication. Additional studies are underway to determine the biological function of the novel genes that were identified.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Neoplasias del Colon/genética , Inhibidores de la Ciclooxigenasa/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Isoenzimas/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Nitrobencenos/farmacología , Proteínas de Unión al ARN , Sulfonamidas/farmacología , Proteínas Adaptadoras Transductoras de Señales , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis , Northern Blotting , Cadherinas/biosíntesis , Cadherinas/genética , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Cateninas , Adhesión Celular , Ciclinas/biosíntesis , Ciclinas/genética , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , ADN Complementario/genética , Dinamina I , Dinaminas , GTP Fosfohidrolasas/biosíntesis , GTP Fosfohidrolasas/genética , Perfilación de la Expresión Génica , Humanos , Proteínas de la Membrana , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , Prostaglandina-Endoperóxido Sintasas , Biosíntesis de Proteínas , Proteínas/genética , Protocadherinas , ARN Mensajero/genética , ARN Neoplásico/genética , Neoplasias del Recto/genética , Neoplasias del Recto/metabolismo , Neoplasias del Recto/patología , Transducción de Señal/genética , Especificidad por Sustrato , Técnica de Sustracción , Células Tumorales Cultivadas/efectos de los fármacos , Catenina deltaRESUMEN
The cyclooxygenase (COX) enzymes catalyze a key step in the conversion of arachidonate to PGH2, the immediate substrate for a series of cell specific prostaglandin and thromboxane synthases. Prostaglandins play critical roles in numerous biologic processes, including the regulation of immune function, kidney development, reproductive biology, and gastrointestinal integrity. There are two COX isoforms, which differ mainly in their pattern of expression. COX-1 is expressed in most tissues, whereas COX-2 usually is absent, but is induced by numerous physiologic stimuli. Surprisingly, disruption of Cox1 (Ptgs1) in the mouse did not result in gastrointestinal abnormalities. cox-2 (Ptgs2) null mice show reproductive anomalies and defects in kidney development. Epidemiologic, animal, and human data indicate that NSAIDs, inhibitors of cyclooxygenase, are chemopreventive for colon cancer. COX-2 is overexpressed in 50% of benign polyps and 80-85% of adenocarcinomas. Offspring from cox-2 null by Apcdelta716 matings exhibit an 86% reduction in polyp number when compared to offspring from control animals, thus providing genetic evidence that COX-2 contributes to tumor formation or growth. The in vivo mechanism by which COX-2 affects tumor growth has not been determined. It is possible that both tumor and stromally derived COX-2 could influence tumor angiogenesis and/ or immune function.
Asunto(s)
Desarrollo Embrionario y Fetal , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Anticarcinógenos/uso terapéutico , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Neoplasias del Colon/prevención & control , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/uso terapéutico , Humanos , Isoenzimas/deficiencia , Isoenzimas/genética , Proteínas de la Membrana , Ratones , Ratones Noqueados , Prostaglandina-Endoperóxido Sintasas/deficiencia , Prostaglandina-Endoperóxido Sintasas/genéticaRESUMEN
The precise role of TGF-beta in colorectal carcinogenesis is not clear. The purpose of this study was to determine the phenotypic alterations caused by chronic exposure to TGF-beta in non-transformed intestinal epithelial (RIE-1) cells. Growth of RIE-1 cells was inhibited by >75% following TGF-beta1 treatment for 7 days, after which the cells resumed a normal growth despite the presence of TGF-beta1. These 'TGF-beta-resistant' cells (RIE-Tr) were continuously exposed to TGF-beta for >50 days. Unlike the parental RIE cells, RIE-Tr cells lost contact inhibition, formed foci in culture, grew in soft agarose. RIE-Tr cells demonstrated TGF-beta-dependent invasive potential in an in vitro assay and were resistant to Matrigel and Na-butyrate-induced apoptosis. The RIE-Tr cells were also tumorigenic in nude mice. The transformed phenotype of RIE-Tr cells was associated with a 95% decrease in the level of the type II TGF-beta receptor (TbetaRII) protein, a 40-fold increase in cyclooxygenase-2 (COX-2) protein, and 5.9-fold increase in the production of prostacyclin. Most RIE-Tr subclones that expressed low levels of TbetaRII and high levels of COX-2 were tumorigenic. Those subclones that express abundant TbetaRII and low levels of COX-2 were not tumorigenic in nude mice. A selective COX-2 inhibitor inhibited RIE-Tr cell growth in culture and tumor growth in nude mice. The reduced expression of TbetaRII, increased expression of COX-2, and the ability to form colonies in Matrigel were all reversible upon withdrawal of exogenous TGF-beta1 for the RIE-Tr cells.
Asunto(s)
Transformación Celular Neoplásica/inducido químicamente , Regulación hacia Abajo , Células Epiteliales/efectos de los fármacos , Intestinos/efectos de los fármacos , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Animales , Apoptosis , Recuento de Células , División Celular/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Ciclooxigenasa 2 , Resistencia a Medicamentos , Inducción Enzimática , Células Epiteliales/metabolismo , Intestinos/citología , Fenotipo , Proteínas Serina-Treonina Quinasas , Ratas , Receptor Tipo II de Factor de Crecimiento Transformador betaRESUMEN
It has been proposed that Cyclooxygenase (COX)-2 inhibitors may be able to enhance the effects of chemotherapeutic or radiation treatment; however, currently few studies have been reported that define the radiation-enhancing effect of COX-2 inhibitors. We conducted in vitro radiation survival experiments using rat intestinal epithelial cells which were stably transfected with COX-2 cDNA in the sense (RIE-S) and antisense (RIE-AS) orientations to investigate the potential radiosensitizing effect of the selective COX-2 inhibitor, NS-398. Apoptosis was measured using 7-aminoactinomycin-D with flow cytometry to investigate underlying mechanisms for the effect of NS-398 on radiosensitivity. The same experiments were repeated with NCI-H460 human lung cancer cells, which express COX-2 constitutively, and HCT-116 human colon cancer cells, which lack COX-2 expression. In vivo tumor growth delay assays were also performed with tumors formed by H460 and HCT-116 cells. No difference was observed in the intrinsic radiation sensitivity of RIE-S and RIE-AS cells exposed to radiation alone. However, 150-400 microM of NS-398 enhanced radiosensitivity in a concentration-dependent manner in RIE-S cells with dose enhancement ratios of 1.2-1.9 at a surviving fraction of 0.25. However, this effect was not shown in RIE-AS cells. NS-398 enhanced radiosensitivity in H460 cells with a dose enhancement ratio of 1.8 but protected HCT-116 cells from the effects of radiation. Radiation-induced apoptosis was enhanced by NS-398 in RIE-S and H460 cells but not in RIE-AS and HCT-116 cells. Additionally, this radiation-enhancing effect in RIE-S cells seemed to be attributable to some mechanisms other than the reversal of radioresistance induced by COX-2. NS-398 (36 mg/kg) enhanced the effect of radiation on H460 tumors in vivo by an enhancement factor of 2.5; however, it did not enhance the radiosensitivity of HCT-116 tumors (enhancement factor = 1.04). These in vitro and in vivo results suggest that selective COX-2 inhibitors enhance the effect of radiation on tumors that express COX-2 but not on COX-2-lacking tumors. This effect may be attributable to enhancement of radiation-induced apoptosis. Thus, selective COX-2 inhibitors may have potential as radiosensitizers for treatment of human cancers.
Asunto(s)
Inhibidores de la Ciclooxigenasa/farmacología , Isoenzimas/antagonistas & inhibidores , Nitrobencenos/farmacología , Sulfonamidas/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Terapia Combinada , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/uso terapéutico , ADN sin Sentido/genética , Relación Dosis-Respuesta en la Radiación , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/efectos de la radiación , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de la radiación , Isoenzimas/genética , Proteínas de la Membrana , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Neoplasias Experimentales/radioterapia , Nitrobencenos/uso terapéutico , Prostaglandina-Endoperóxido Sintasas/genética , Ratas , Sulfonamidas/uso terapéutico , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Modulation of cyclooxygenase-2 (COX-2) mRNA stability plays an important role in the regulation of its expression by oncogenic Ras. Here, we evaluate COX-2 mRNA stability in response to treatment with two known endogenous promoters of gastrointestinal cancer, the bile acid (chenodeoxycholate; CD) and ceramide. Treatment with CD and ceramide resulted in a 10-fold increase in the level of COX-2 protein and a four-fold lengthening of the half-life of COX-2 mRNA. COX-2 mRNA stability was assessed by Northern blot analysis and by evaluating the AU-rich element located in the COX-2 3'-UTR. A known inhibitor of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK), PD98059, reversed the effects of CD or ceramide to stabilize COX-2 mRNA. Overexpression of a dominant-negative ERK-1 or ERK-2 protein also led to destabilization of COX-2 mRNA. Treatment with a p38 MAPK inhibitor, PD169316, or transfection with a dominant-negative p38 MAPK construct reversed the effect of CD or ceramide to stabilize COX-2 mRNA. Expression of a dominant-negative c-Jun N-terminal kinase (JNK) had no effect on COX-2 mRNA stability in cells treated with CD or ceramide. We conclude that posttranscriptional mechanisms play an important role in the regulation of COX-2 expression during carcinogenesis.