RESUMEN
BACKGROUND: Previously, we showed that short-term inhibition of ß-catenin expression and reversal of aberrant ß-catenin subcellular localization by the selective COX-2 inhibitor celecoxib is associated with adenoma regression in the C57BL/6J Min/+ mouse. Conversly, long-term administration resulted in tumor resistance, leading us to investigate alternative methods for selective ß-catenin chemoprevention. In this study, we hypothesized that disruption of ß-catenin expression by EZN-3892, a selective locked nucleic acid (LNA)-based ß-catenin inhibitor, would counteract the tumorigenic effect of Apc loss in Min/+ adenomas while preserving normal intestinal function. MATERIALS AND METHODS: C57BL/6J Apc(+/+) wild-type (WT) and Min/+ mice were treated with the maximum tolerated dose (MTD) of EZN-3892 (30mg/kg). Drug effect on tumor numbers, ß-catenin protein expression, and nuclear ß-catenin localization were determined. RESULTS: Although the tumor phenotype and ß-catenin nuclear localization in Min/+ mice did not change following drug administration, we observed a decrease in ß-catenin expression levels in the mature intestinal tissue of treated Min/+ and WT mice, providing proof of principle regarding successful delivery of the LNA-based antisense vehicle. Higher doses of EZN-3892 resulted in fatal outcomes in Min/+ mice, likely due to ß-catenin ablation in the intestinal tissue and loss of function. CONCLUSIONS: Our data support the critical role of Wnt/ß-catenin signaling in maintaining intestinal homeostasis and highlight the challenges of effective drug delivery to target disease without permanent toxicity to normal cellular function.
Asunto(s)
Carcinogénesis , Neoplasias Intestinales/patología , Oligonucleótidos/farmacología , beta Catenina/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Neoplasias Intestinales/genética , Neoplasias Intestinales/metabolismo , Intestinos/efectos de los fármacos , Dosis Máxima Tolerada , Ratones , Ratones Endogámicos C57BL , beta Catenina/metabolismoRESUMEN
Adenomatous polyposis coli (APC)-regulated Wnt and transforming growth factor-ß (TGFß) signaling cooperate in the intestine to maintain normal enterocyte functions. Human clinical trials showed that estrogen [17ß-estradiol (E2)], the ligand of nuclear receptors estrogen receptor α (ERα) and ERß, inhibited colorectal cancer (CRC) in women. Consistent with this finding, we reported that E2, ERα and ERß suppressed intestinal tumorigenesis in the C57BL/6J-Min/+ (Min/+) mouse, a CRC model. Here, we extended our results with further comparisons of colon and small intestine from intact female Apc (+/+) (WT), Min/+ and ER-deficient Min/+ mice. In the colon of ER-deficient Min/+ mice, ER loss reduced TGFß signaling in crypt base cells as evidenced by minimal expression of the effectors Smad 2, 3 and 4 in these strains. We also found reduced expression of Indian hedgehog (Ihh), bone morphogenetic protein 4 and hepatocyte nuclear factor 3ß or FoxA2, factors needed for paracrine signaling between enterocytes and mesenchyme. In proximal colon, ER loss produced a >10-fold increased incidence of crypt fission, a marker for wound healing and tumor promotion. These data, combined with our previous work detailing the specific roles of E2, ERα and ERß in the colon, suggest that ER activity helps to maintain the intestinal stem cell (ISC) microenvironment by modulating epithelial-stromal crosstalk in ways that regulate cytokine, Wnt and Ihh availability in the extracellular matrix (ECM).
Asunto(s)
Colon/metabolismo , Colon/patología , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Factor Nuclear 3-beta del Hepatocito/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Focos de Criptas Aberrantes/metabolismo , Focos de Criptas Aberrantes/patología , Animales , Microambiente Celular , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/genética , Femenino , Proteínas Hedgehog/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Células de Paneth/metabolismo , Células de Paneth/patología , Transducción de Señal , Proteína Smad4/metabolismo , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
BACKGROUND: Optimal surgical and medical therapy for the treatment of desmoid tumors (DT) is still undefined. Partial response to tyrosine kinase inhibitors (TKI) has previously been described. Here, we examined the role of the tyrosine kinases c-Src and c-Kit in driving desmoid tumorigenesis. METHODS: Six consecutive DT and matched normal tissues were collected from the operating room. Tissues were embedded in paraffin for immunohistochemical analysis, and protein lysates were prepared for immunoblot and immunoprecipitation. RESULTS: We found increased levels of ß-catenin in five of six (83%) DT relative to matched normal tissue by immunoblot analysis. By immunohistochemistry, ß-catenin expression was also increased in DT and localized to the nucleus. In contrast, we observed variable levels of total and activated c-Src and c-Kit expression in DT compared with normal tissue. Finally, ß-catenin tyrosine phosphorylation (p-Y) among tumors was variably increased, despite the increased amount of total ß-catenin in tumors. CONCLUSIONS: Our results suggest that c-Src and c-Kit activity in DT is variable, consistent with the heterogeneous nature of this disease. Clinical response to TKI in DT may be via alternative mechanisms unrelated to c-Src or c-Kit activity. Further insight into DT biology will help identify novel drug regimens to limit the morbidity and mortality associated with this disease.
Asunto(s)
Fibromatosis Agresiva/enzimología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , beta Catenina/metabolismo , Adulto , Proteína Tirosina Quinasa CSK , Transformación Celular Neoplásica , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Fosforilación , Familia-src QuinasasRESUMEN
Desmoid tumors are nonmalignant neoplasms of mesenchymal origin that mainly contain fibroblast lineage cells. These tumors often occur in patients with familial adenomatous polyposis (FAP) coli who have germ line mutations in the APC gene. Given emerging data that has implicated multipotent mesencyhmal stromal cells (MSC) in the origin of mesenchymal tumors, we hypothesized that desmoid tumors may arise in patients with FAP after MSCs acquire somatic mutations during the proliferative phase of wound healing. To test this idea, we examined 16 desmoid tumors from FAP-associated and sporadic cases, finding that all 16 of 16 tumors expressed stem cell markers, whereas matching normal stromal tissues were uniformly negative. Desmoid tumors also contained a subclass of fibrocytes linked to wound healing, angiogenesis, and fibrosis. Using an MSC cell line derived from an FAP-associated desmoid tumor, we confirmed an expected loss in the expression of adenomatous polyposis coli (APC) and the transcriptional repressor BMI-1 while documenting the coexpression of markers for chondrocytes, adipocytes, and osteocytes. Together, our findings argue that desmoid tumors result from the growth of MSCs in a wound healing setting that is associated with deregulated Wnt signaling due to APC loss. The differentiation potential of these MSCs combined with expression of BMI-1, a transcriptional repressor downstream of Hedgehog and Notch signaling, suggests that desmoid tumors may respond to therapies targeting these pathways.
Asunto(s)
Fibromatosis Agresiva/etiología , Células Madre Mesenquimatosas/metabolismo , Mutación , Cicatrización de Heridas , Línea Celular Tumoral , Fibromatosis Agresiva/patología , Humanos , InmunohistoquímicaRESUMEN
Cyclooxygenase-2 (COX-2) inhibition prevents adenoma formation in humans and mouse models of colon cancer. The selective COX-2 inhibitor celecoxib reduces COX-2 and prostaglandin E(2) (PGE(2)) expression and adenomas in the intestine of Min/+ mice after treatment for several weeks, but prolonged treatment increases PGE(2) production, resulting in drug-resistant tumor formation and transforming growth factor beta (TGFbeta)-dependent intestinal fibrosis. In this study, we examined pathways that regulate COX-2 expression and suppress chronic intestinal inflammation. We show that NF-kappaB signaling was inhibited in the ileum of Min/+ mice receiving long-term treatment with celecoxib. This effect was associated with inhibition of TGFbeta-associated kinase-1 and IkappaB kinase alpha/beta activities and reduced expression of the Toll-like receptor (TLR) 2 and TLR4 that enhance colonic barrier function. Additionally, we observed reduced activities of protein kinases c-Jun NH(2)-terminal kinase 1 and protein kinase A and transcription factor cyclic AMP-responsive element binding protein, regulators of COX-2 expression, which cross-talk with NF-kappaB. In ileum subjected to long-term celecoxib treatment, we noted relatively higher expression of COX-2, vascular endothelial growth factor, and interleukin-1beta in Paneth cells, whereas NF-kappaB and COX-2 were more strongly expressed by an expanded population of stromal myofibroblasts. Our findings argue that celecoxib resistance is an acquired adaptation to changes in the crypt microenvironment that is associated with chronic intestinal inflammation and impaired acute wound-healing responsiveness.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Colitis/metabolismo , Neoplasias del Colon/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Ciclooxigenasa 2/metabolismo , FN-kappa B/metabolismo , Animales , Antígenos CD34/biosíntesis , Celecoxib , Transformación Celular Neoplásica/patología , Colitis/enzimología , Colitis/patología , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/patología , Neoplasias del Colon/prevención & control , Ciclooxigenasa 2/biosíntesis , Regulación hacia Abajo , Íleon/irrigación sanguínea , Íleon/enzimología , Íleon/metabolismo , Íleon/patología , Interleucina-1beta/biosíntesis , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Ratones Endogámicos C57BL , Pirazoles/farmacología , Sulfonamidas/farmacología , Receptores Toll-Like/metabolismoRESUMEN
BACKGROUND: Activation of the Wnt/beta-catenin signaling pathway is implicated in thyroid tumorigenesis, and up to 90% of papillary thyroid cancer (PTC) demonstrate aberrant expression of beta-catenin. Nonsteroidal antiinflammatory drugs reverse aberrant beta-catenin expression and localization in colon cancer. In this study, we tested the hypothesis that the nonsteroidal antiinflammatory drug sulindac would reverse aberrant beta-catenin activity in thyroid cancer cells. METHODS: beta-catenin protein levels were determined in thyroidectomy specimens from six consecutive patients and in three different thyroid cancer cells lines (8505-C, KTC-1, and TPC-1) by immunoblotting. Cells of 8505-C and KTC-1 harbor the BRAF(V600E) mutation, and TPC-1 has the RET/PTC rearrangement. All cell lines were treated with sulindac (100 microM for up to 72 hours). Protein levels of c-myc and cyclin D1 were detected by immunoblotting, and beta-catenin localization was determined by immunocytochemistry in the PTC cell lines. PCCL3 rat thyroid cells that conditionally overexpress either BRAF(V600E) or RET/PTC were also treated with sulindac. RESULTS: All PTC specimens and cell lines expressed high levels of beta-catenin protein and displayed aberrant nuclear and cytoplasmic localization of beta-catenin. Exposure to sulindac for 48 hours reduced beta-catenin expression in 8505-C and KTC-1 cells, but not in TPC-1 cells. Further, sulindac treatment reduced c-myc and cyclin D1 levels in 8505-C and KTC-1 cells, but had no effect in TPC-1 cells. Immunocytochemistry demonstrated that sulindac treatment redistributed beta-catenin from the nucleus to the membrane in 8505-C and KTC-1 cells. However, sulindac did not affect beta-catenin localization in TPC-1 cells. Finally, sulindac was effective in decreasing beta-catenin expression and cellular proliferation in BRAF(V600E)-overexpressing cells, but not in RET/PTC3-overexpressing cells. CONCLUSIONS: Taken together, our findings demonstrate that sulindac treatment reverses beta-catenin activity in 8505-C and KTC-1 cell lines with the BRAF(V600E), but not in TPC-1 cells with the RET/PTC mutation. Future studies should investigate the potential for beta-catenin-directed therapy for patients with advanced thyroid cancers.
Asunto(s)
Sulindac/farmacología , Neoplasias de la Tiroides/metabolismo , beta Catenina/genética , Adenocarcinoma Papilar/genética , Adenocarcinoma Papilar/metabolismo , Animales , Línea Celular Tumoral , Humanos , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Ratas , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/genética , beta Catenina/biosíntesisRESUMEN
Anti-inflammatory drugs prevent intestinal tumor formation, an activity related to their ability to inhibit inflammatory pathway signaling in the target tissue. We previously showed that treatment of Min/(+) mice with the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib induced rapid tumor regression; however, drug-resistant tumors appeared with long-term treatment. In this study, we investigated whole-tissue changes in inflammatory signaling by studying constituents of the tissue stroma and extracellular matrix. We found that celecoxib resistance was associated with changes in factors regulating autocrine transforming growth factor-beta (TGFbeta) signaling. Chronic drug treatment expanded the population of bone marrow-derived CD34(+) vimentin(+) alphaSMA(-) myofibroblast precursors and alphaSMA(+) vimentin(+) F4/80(-) myofibroblasts in the lamina propria and submucosa, providing a source of increased TGFbeta and COX-2 expression. Membrane constituents regulating TGFbeta availability, including syndecan-1 and heparanase-1, were also modified by chronic treatment in a manner promoting increased TGFbeta signaling. Finally, long-term celecoxib treatment induced tissue fibrosis, as indicated by increased expression of collagen, fibronectin, and laminin in the basement membrane. We conclude that chronic COX-2 inhibition alters TGFbeta signaling in the intestinal mucosa, producing conditions consistent with chronic inflammation.
Asunto(s)
Inhibidores de la Ciclooxigenasa 2/farmacología , Ciclooxigenasa 2/química , Matriz Extracelular/patología , Fibrosis/patología , Intestinos/efectos de los fármacos , Pirazoles/farmacología , Sulfonamidas/farmacología , Animales , Celecoxib , Ciclooxigenasa 2/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/enzimología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/patología , Fibrosis/inducido químicamente , Fibrosis/enzimología , Genes APC/fisiología , Immunoblotting , Técnicas para Inmunoenzimas , Neoplasias Intestinales , Intestinos/enzimología , Intestinos/patología , Ratones , Ratones Endogámicos C57BL , Polisacárido Liasas/metabolismo , Transducción de Señal , Células del Estroma/efectos de los fármacos , Células del Estroma/enzimología , Células del Estroma/patología , Sindecano-1/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Although most patients with papillary thyroid cancer (PTC) have favorable outcomes, some have advanced PTC that is refractory to external beam radiation and systemic chemotherapy. Galectin-3 (Gal-3) is a beta-galactoside-binding protein with antiapoptotic activity that is consistently overexpressed in PTC. The purpose of this study is to determine if Gal-3 inhibition promotes apoptosis, chemosensitivity, and radiosensitivity in PTC. PTC cell lines (8505-C and TPC-1) and human ex vivo PTC were treated with a highly specific small molecule inhibitor of Gal-3 (Td131_1). Apoptotic activity was determined by flow cytometric analysis as well as caspase-3 and PARP cleavage. The minimum inhibitory concentrations of Td131_1 and doxorubicin were determined, and their combined effects were measured to test for synergistic activity. The effects of Td131_1 on radiosensitivity were determined by a clonogenic assay. Td131_1 promoted apoptosis, improved radiosensitivity, and synergistically enhanced chemosensitivity to doxorubicin in PTC cell lines. In PTC ex vivo, Td131_1 treatment alone induced the cleavage of caspase-3 and PARP. Td131_1 and doxorubicin together activated apoptosis in PTC ex vivo to a greater degree than their combined individual effects. Td131_1 activated apoptosis and had synergistic activity with doxorubicin in PTC. We conclude that Gal-3 targeted therapy is a promising therapeutic strategy for advanced PTC that is refractory to surgery and radioactive iodine therapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Papilar/tratamiento farmacológico , Galectina 3/antagonistas & inhibidores , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Tioglicósidos/uso terapéutico , Neoplasias de la Tiroides/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Carcinoma Papilar/metabolismo , Carcinoma Papilar/radioterapia , Caspasa 3/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Sinergismo Farmacológico , Citometría de Flujo , Galectina 3/metabolismo , Humanos , Estructura Molecular , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/fisiología , Fármacos Sensibilizantes a Radiaciones/síntesis química , Ratas , Tiogalactósidos/síntesis química , Tiogalactósidos/uso terapéutico , Tioglicósidos/síntesis química , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/radioterapiaRESUMEN
Aberrant crypt foci (ACF) are the earliest visible neoplastic lesions in the colorectum. The natural history of these lesions and their role in the adenoma-carcinoma sequence are unknown. We studied ACF in a subset of patients randomized to placebo (n = 17), celecoxib (200 mg twice daily; n = 15), or celecoxib (400 mg twice daily; n = 13) in the Adenoma Prevention with Celecoxib (APC) trial. Magnification chromoendoscopy was done to identify, count, and biopsy ACF within the rectum at baseline and after 8 to 12 months of treatment. A total of 655 ACF were identified in 45 patients. We examined 70 of these ACF histologically, and all 70 were nondysplastic. Cohort characteristics and APC trial treatment results for substudy patients were similar to those of the overall APC trial. There was no significant modulation of ACF by celecoxib (versus placebo; P = 0.77). Immunohistochemical comparison of ACF with adjacent normal mucosa showed that ACF had an increased proliferative index as determined by Ki-67 (P < 0.0001), but lacked other features of neoplasia such as increased cyclooxygenase-2 expression and microvessel density, nuclear localization of beta-catenin, or decreased expression of the tumor suppressors SMAD4, Estrogen Receptor alpha, or MGMT. Only baseline SMAD4 expression in ACF correlated with posttreatment adenoma recurrence (independent of treatment arm; P = 0.01). The presence or number of nondysplastic ACF did not correlate with a higher risk of synchronous advanced or recurrent adenomas. Our overall results indicated that nondysplastic ACF were not accurate surrogate endpoint biomarkers of recurrent colorectal adenomas in the APC trial.
Asunto(s)
Adenoma/prevención & control , Mucosa Intestinal/patología , Neoplasias Intestinales/prevención & control , Lesiones Precancerosas/patología , Pirazoles/uso terapéutico , Sulfonamidas/uso terapéutico , Adenoma/diagnóstico , Adenoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Algoritmos , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Biomarcadores/análisis , Celecoxib , Inhibidores de la Ciclooxigenasa/administración & dosificación , Inhibidores de la Ciclooxigenasa/uso terapéutico , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Neoplasias Intestinales/diagnóstico , Neoplasias Intestinales/patología , Pólipos Intestinales/patología , Masculino , Persona de Mediana Edad , Placebos , Pirazoles/administración & dosificación , Sulfonamidas/administración & dosificaciónRESUMEN
Estrogen replacement therapy in postmenopausal women is associated with a reduction in colorectal cancer risk, potentially via interactions between 17beta-estradiol (E(2)) and the estrogen receptors (ER) alpha and beta. To study the role of E(2) in intestinal tumor inhibition, we separately crossed C57BL/6J-Min/+ (Min/+) mice with Eralpha(+/-) and Erbeta(+/-) mice to generate ER-deficient Min/+ progeny. We found an increased incidence of visible colon tumors and dysplastic microadenomas in ER-deficient Min/+ relative to Er(+/+)Min/+ controls. Small intestinal tumor numbers were unaffected. Invasive carcinomas were found only in Eralpha(+/-)Min/+ mice, suggesting that ERalpha plays additional non-cell autonomous roles that limit tumor progression. Histologic analyses of ER-deficient Min/+ colons, as well as colons from ovariectomized Min/+ mice (OvxMin/+) and E(2)-treated OvxMin/+ mice (OvxMin/+ +E(2)), revealed significant differences in crypt architecture, enterocyte proliferation, and goblet cell differentiation relative to Min/+ and Er(+/+)Apc(+/+) (wild-type) controls. The expression of ERalpha and ERbeta was regionally compartmentalized along the colonic crypt axis, suggesting functional antagonism. Our results indicate that ERalpha and ERbeta are inhibitory modifiers of Apc-dependent colon tumorigenesis. As a result, loss of E(2) and ER signaling in postmenopausal women may contribute to colorectal cancer development.
Asunto(s)
Neoplasias del Colon/patología , Receptor alfa de Estrógeno/fisiología , Receptor beta de Estrógeno/fisiología , Genes APC , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/genética , Neoplasias del Colon/genética , Enterocitos/citología , Enterocitos/efectos de los fármacos , Estrógenos/farmacología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos BiológicosRESUMEN
Selective cyclooxygenase-2 (COX-2) inhibitors are widely prescribed for severe arthritis and are currently under study in human chemoprevention trials. Recently, long-term use of these agents has come under scrutiny due to reports of treatment-associated cardiovascular toxicity. On short-term administration, the selective COX-2 inhibitor celecoxib inhibits adenoma growth in animal tumor models, including the C57BL/6J-Min/+ (Min/+) mouse. With uninterrupted long-term celecoxib administration, intestinal tumors in Min/+ mice initially regressed and then recurred to levels comparable with untreated controls. Celecoxib treatment initially suppressed COX-2 and prostaglandin E2 (PGE2) expression, but long-term use produced significantly higher levels of these molecules and reactivated PGE2-associated growth factor signaling pathways in tumor and normal tissues. These results indicate that COX-2 is an important chemoprevention target and that inhibition of this enzyme alters a paracrine enterocyte regulatory pathway. Chronic uninterrupted celecoxib treatment, however, induces untoward effects that enhance early progression events in intestinal tumorigenesis and may contribute to treatment toxicity.
Asunto(s)
Inhibidores de la Ciclooxigenasa 2/administración & dosificación , Neoplasias Intestinales/tratamiento farmacológico , Pirazoles/administración & dosificación , Sulfonamidas/administración & dosificación , Adenoma/tratamiento farmacológico , Adenoma/enzimología , Adenoma/metabolismo , Animales , Celecoxib , Dinoprostona/metabolismo , Esquema de Medicación , Neoplasias Intestinales/enzimología , Neoplasias Intestinales/metabolismo , Lipooxigenasa/biosíntesis , Ratones , Ratones Endogámicos C57BL , Receptores de Prostaglandina/biosíntesis , Transducción de Señal/efectos de los fármacosRESUMEN
The Min/+ mouse is a model for APC-dependent colorectal cancer (CRC). We showed that tumorigenesis in this animal was associated with decreased E-cadherin adhesion and increased epidermal growth factor receptor (Egfr) activity in the non-tumor intestinal mucosa. Here, we tested whether these abnormalities correlated with changes in the actin cytoskeleton due to increased Rho-ROCK signaling. We treated Apc+/+ (WT) littermate small intestine with EGTA, an inhibitor of E-cadherin, and with LPA, an RhoA activator; both induced effects on adhesion and kinase activity that mimicked the Min/+ phenotype. GTP-bound Rho was increased in Min/+ enterocytes relative to WT. Since RhoA activity is associated with actomyosin contractility, markers of this signaling cascade were assessed including phosphorylated myosin light chain (MLC), cofilin, Pyk2, Src, and MAPK kinases. The increased actomyosin contractility characterizing Min/+ intestinal tissue was suppressed by the ROCK inhibitor, Y27632, but was inducible in the WT by EGTA or LPA. Finally, ultrastructural imaging revealed changes consistent with actomyosin contractility in Min/+ enterocytes. Thus, the positive regulation of E-cadherin adhesion provided by Apc+ in vivo allows proper negative regulation of Egfr, Src, Pyk2, and MAPK, as well as RhoA activities.
Asunto(s)
Actomiosina/metabolismo , Cadherinas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/fisiología , Amidas/farmacología , Animales , Antiinflamatorios no Esteroideos/farmacología , Cadherinas/genética , Calcio/deficiencia , Miosinas Cardíacas/metabolismo , Celecoxib , Adhesión Celular/efectos de los fármacos , Ácido Egtácico/farmacología , Enterocitos/efectos de los fármacos , Enterocitos/metabolismo , Enterocitos/ultraestructura , Receptores ErbB/agonistas , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Intestino Delgado/efectos de los fármacos , Intestino Delgado/fisiología , Lisofosfolípidos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Electrónica de Transmisión , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Contracción Muscular/efectos de los fármacos , Relajantes Musculares Centrales/farmacología , Cadenas Ligeras de Miosina/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Pirazoles/farmacología , Piridinas/farmacología , Quinazolinas , Sulfonamidas/farmacología , Tirfostinos/farmacología , Regulación hacia Arriba , beta Catenina/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA/agonistasRESUMEN
The present study was designed to investigate the effects of two main constituents of green tea, (-)-epigallocatechin-3-gallate (EGCG) and caffeine, on intestinal tumorigenesis in Apc(min/+) mice, a recognized mouse model for human intestinal cancer, and to elucidate possible mechanisms involved in the inhibitory action of the active constituent. We found that p.o. administration of EGCG at doses of 0.08% or 0.16% in drinking fluid significantly decreased small intestinal tumor formation by 37% or 47%, respectively, whereas caffeine at a dose of 0.044% in drinking fluid had no inhibitory activity against intestinal tumorigenesis. In another experiment, small intestinal tumorigenesis was inhibited in a dose-dependent manner by p.o. administration of EGCG in a dose range of 0.02% to 0.32%. P.o. administration of EGCG resulted in increased levels of E-cadherin and decreased levels of nuclear beta-catenin, c-Myc, phospho-Akt, and phospho-extracellular signal-regulated kinase 1/2 (ERK1/2) in small intestinal tumors. Treatment of HT29 human colon cancer cells with EGCG (12.5 or 20 micromol/L at different times) also increased protein levels of E-cadherin by 27% to 58%, induced the translocation of beta-catenin from nucleus to cytoplasm and plasma membrane, and decreased c-Myc and cyclin D1 (20 micromol/L EGCG for 24 hours). These results indicate that EGCG effectively inhibited intestinal tumorigenesis in Apc(min/+) mice, possibly through the attenuation of the carcinogenic events, which include aberrant nuclear beta-catenin and activated Akt and ERK signaling.
Asunto(s)
Anticarcinógenos/farmacología , Catequina/análogos & derivados , Neoplasias Intestinales/prevención & control , Animales , Cadherinas/metabolismo , Cafeína/farmacología , Catequina/farmacología , Dinoprostona/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Genes APC , Células HT29 , Humanos , Neoplasias Intestinales/genética , Neoplasias Intestinales/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/patología , Leucotrieno B4/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfolipasas A/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , beta Catenina/metabolismoRESUMEN
Carnosol, a constituent of the herb, rosemary, has shown beneficial medicinal and antitumor effects. Using the C57BL/6J/Min/+ (Min/+) mouse, a model of colonic tumorigenesis, we found that dietary administration of 0.1% carnosol decreased intestinal tumor multiplicity by 46%. Previous studies showed that tumor formation in the Min/+ mouse was associated with alterations in the adherens junctions, including an increased expression of tyrosine-phosphorylated beta-catenin, dissociation of beta-catenin from E-cadherin, and strongly reduced amounts of E-cadherin located at lateral plasma membranes of histologically normal enterocytes. Here, we confirm these findings and show that treatment of Min/+ intestinal tissue with carnosol restored both E-cadherin and beta-catenin to these enterocyte membranes, yielding a phenotype similar to that of the Apc(+/+) wild-type (WT) littermate. Moreover, treatment of WT intestine with the phosphatase inhibitor, pervanadate, removed E-cadherin and beta-catenin from the lateral membranes of enterocytes, mimicking the appearance of the Min/+ tissue. Pretreatment of WT tissue with carnosol inhibited the pervanadate-inducible expression of tyrosine-phosphorylated beta-catenin. Thus, the Apc(Min) allele produces adhesion defects that involve up-regulated expression of tyrosine-phosphorylated proteins, including beta-catenin. Moreover, these data suggest that carnosol prevents Apc-associated intestinal tumorigenesis, potentially via its ability to enhance E-cadherin-mediated adhesion and suppress beta-catenin tyrosine phosphorylation.
Asunto(s)
Abietanos/farmacología , Adenoma/prevención & control , Neoplasias del Colon/prevención & control , Proteínas del Citoesqueleto/metabolismo , Fenantrenos/farmacología , Transactivadores/metabolismo , Animales , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Enterocitos/efectos de los fármacos , Enterocitos/metabolismo , Femenino , Intestino Delgado/citología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Rosmarinus/química , Tirosina/metabolismo , Vanadatos/antagonistas & inhibidores , Vanadatos/farmacología , beta CateninaRESUMEN
Epidemiological studies suggest that post-menopausal hormone replacement therapy (HRT) reduces colorectal cancer (CRC) incidence. Phytoestrogens, including the soy isoflavone genistein and coumestrol, are used by many women as alternatives to HRT. Previous studies showed that ovariectomy induced a 77% increase in intestinal adenoma number in the C57BL/6J-Min/+ (Min/+) mouse, an animal model of adenomatous polyposis coli (APC)-associated CRC. Replacement of estradiol (E(2)) in ovariectomized Min/+ mice reduced tumor number to baseline and up-regulated the expression of estrogen receptor beta (ERbeta). We hypothesized that the phytoestrogens genistein and coumestrol would inhibit intestinal tumorigenesis in ovariectomized Min/+ mice. Min/+ and Apc(+/+) (WT) mice were ovariectomized and assigned to either a control diet or treatment with E(2), genistein or coumestrol. Treatment of ovariectomized Min/+ (Min/+ OX) mice with genistein resulted in a non-significant reduction in tumor number. Min/+ OX mice treated with coumestrol had significantly fewer tumors than untreated Min/+ OX controls and the same number of tumors as non-ovariectomized Min/+ mice. Bromodeoxyuridine migration assays also demonstrated that treatment with E(2) or coumestrol improved enterocyte migration rate. Immunoprecipitation and immunohistochemistry analyses showed that impaired association of the adherens junction proteins E-cadherin and beta-catenin in Min/+ mice was improved by treatment with either E(2) or coumestrol. Immunoblot analyses also showed that expression of ERbeta was elevated in enterocytes of Min/+ OX mice treated with E(2) or coumestrol as compared with those of untreated Min/+ OX mice. In conclusion, both coumestrol and E(2) prevent intestinal tumorigenesis and ameliorate enterocyte migration and intercellular adhesion in the Apc(Min/+) mouse model of CRC.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Neoplasias Colorrectales/prevención & control , Cumestrol/farmacología , Modelos Animales de Enfermedad , Genes APC , Genisteína/farmacología , Intestinos/efectos de los fármacos , Fitoestrógenos/farmacología , Animales , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Inmunohistoquímica , Inmunoprecipitación , Mucosa Intestinal/metabolismo , Intestinos/patología , Ratones , Ratones Endogámicos C57BL , Ovariectomía , Receptores de Estrógenos/metabolismoRESUMEN
Overexpression of the epidermal growth factor receptor (EGFR) and its increased tyrosine kinase activity are implicated in colorectal cancer (CRC) development and malignant progression. The C57BL/6J-Min/+ (Min/+) mouse is a model for CRC and develops numerous intestinal adenomas. We analyzed the normal mucosa of Min/+ and Apc+/+ (WT) littermate mice together with Apc-null adenomas to gain insight into the roles of Egfr in these intestinal tissues. Protein analyses showed that Egfr activity was highest in the tumors, and also up-regulated in Min/+ relative to WT enterocytes. Expression of ubiquitylated Egfr (Egfr-Ub) was increased in Min/+ enterocytes and tumors. Tumors exhibited increased association of Egfr with clathrin heavy chain (CHC), Gab1, and p85alpha, the regulatory subunit of phosphoinositide 3-kinase (PI3K), and tumors also overexpressed c-Src, PDK1, and Akt. Immunohistochemistry for Akt-p-Ser473 revealed a low level of this active kinase in Min/+ and WT enterocytes and its strong presence in tumors. Prostaglandin E2 (PGE2) is a product of cyclooxygenase-2 (Cox-2) activity that is up-regulated in Min/+ tumors and transactivates Egfr. PGE2 expression was significantly higher in untreated Min/+ tumors and reduced by treatment with the Cox-2 inhibitor, celecoxib. Dietary administration of this NSAID also inhibited Egfr activity in tumors. Increased activation of the EGFR-PI3K-Akt signaling pathway in tumors relative to Apc+/+ and ApcMin/+ enterocytes provides potential opportunities for therapeutic interventions to differentially suppress tumor formation, promotion, progression, and/or recurrence.
Asunto(s)
Adenoma/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/deficiencia , Enterocitos/metabolismo , Receptores ErbB/metabolismo , Genes APC , Proteínas Adaptadoras Transductoras de Señales , Adenoma/patología , Alelos , Animales , Antiinflamatorios no Esteroideos/farmacología , Línea Celular , Clatrina/metabolismo , Neoplasias Colorrectales/metabolismo , Ciclooxigenasa 2 , Dinoprostona/metabolismo , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Células HeLa , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Isoenzimas/metabolismo , Células Jurkat , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Prostaglandina-Endoperóxido Sintasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Recurrencia , Transducción de Señal , Distribución Tisular , Activación Transcripcional , Tirosina/química , Ubiquitina/metabolismo , Regulación hacia Arriba , Vanadatos/farmacología , Familia-src Quinasas/metabolismoRESUMEN
Partial loss of function of adenomatous polyposis coli (APC) protein by truncation of its carboxy (C)-terminus is an early factor in the development of many sporadic colorectal cancers. In the C57BL/6J Min/+ (Min/+) mouse, an animal with a germline mutation of Apc, we found that APC truncation was associated with reduced enterocyte migration and loss of association and membrane expression of adherens junction proteins. We hypothesized that these defects were related to changes in cytoskeletal function resulting from truncation of the APC C-terminus, which contains microtubule binding regions, as well as putative sites for indirect actin binding. We investigated this further by determining whether APC truncation produced in vivo changes in actin cytoskeletal structure and microtubule stability. The actin cytoskeleton of histologically normal enterocytes from Min/+ mice was compared to that of Apc+/+ (wild-type) mice by confocal indirect immunofluorescence microscopy. We found a significant loss of actin localization at the apical plasma membrane in Min/+ enterocytes. In addition, immunoblotting revealed increased levels of both unstable Tyr-tubulin and alpha-tubulin turnover in Min/+ enterocytes, indicating an alteration in microtubule dynamics. These studies suggest that loss of actin localization and changes in microtubule dynamics may be dominant negative effects of truncated APC. These changes are consistent with the defects in enterocyte migration and junctional complex formation observed in the Min/+ model of early APC-associated colorectal tumorigenesis.