RESUMEN
Calpain-2 levels are higher in colorectal tumors resistant to chemotherapy and previous work showed calpain-2 inhibitor therapy reduced inflammation-driven colorectal cancer, but direct effects of the inhibitor on colon cancer cells themselves were not demonstrated. In the present study, five human colon cancer cell lines were directly treated with a calpain-2 inhibitor and results showed increased cell death in 4 of 5 cell lines and decreased anchorage-independent growth for all cell five lines. When tested for levels of calpain-2, three cell lines exhibited increasing levels of this enzyme: HCT15 (low), HCC2998 (medium), and HCT116 (significantly higher). This was consistent with gel shift assays showing that calpain-2 inhibitor reduced of NF-κB nuclear translocation most effectively in HCT116 cells. Ability of calpain-2 inhibitor to impede tumor progression in vivo was evaluated using intrarectal transplant of luciferase-expressing cells for these three cell lines. Results showed that calpain-2 inhibitor therapy reduced tumor growth and increased survival only in mice injected with HCT116 cells. These data suggest calpain-2 inhibitor treatment may be most effective on colorectal tumors expressing highest levels of calpain-2.
Asunto(s)
Antineoplásicos/farmacología , Calpaína/antagonistas & inhibidores , Neoplasias del Colon/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Calpaína/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Neoplasias del Colon/mortalidad , Neoplasias del Colon/patología , Progresión de la Enfermedad , Regulación hacia Abajo , Células HCT116 , Humanos , Masculino , Ratones , Ratones Desnudos , FN-kappa B/metabolismo , Análisis de Supervivencia , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: An important role has emerged for calpain enzymes in regulating inflammation with one isoform, calpain-2, particularly important for macrophage activation. The goal of this study was to determine the therapeutic potential of a synthetic calpain-2 inhibitor, zLLY-CH2F, for colitis and inflammation-associated colorectal cancer. METHODS: Mice were then subjected to the azoxymethane/dextran sulfate sodium model of colitis and colitis-associated cancer incorporating intervention with daily injections of 0.75 mg/kg calpain-2 inhibitor beginning after the first signs of colitis. RESULTS: Calpain-2 inhibitor treatment alleviated weight loss and bloody diarrhea, and reduced inflammatory infiltration into colon tissues and inflammatory cytokine mRNA. Calpain-2 inhibitor intervention also reduced total colitis-associated cancer tumor volume by up to 70% in vehicle control mice and decreased cancer pathology scores of blinded histological colon tissue analyses. Mechanistic investigations showed that calpain-2 inhibition during macrophage activation reduced inhibitor of kappa beta (IκB) degradation and nuclear factor kappa beta (NFκB) nuclear localization as well as secretion of specific inflammatory cytokines. In addition, calpain-2 inhibitor treatment of CT26.WT mouse and HT-29 human colorectal cancer cells decreased proliferation and reduced IκB degradation and NFκB translocation. CONCLUSIONS: Overall, these findings suggest that intervention with a calpain-2 inhibitor may reduce colitis and colitis-associated cancer through a two-hit process of limiting macrophage activation and inhibiting growth of the colorectal cancer cells themselves.
Asunto(s)
Colitis/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Activación de Macrófagos/efectos de los fármacos , Oligopéptidos/farmacología , Animales , Azoximetano , Proliferación Celular , Colitis/inducido químicamente , Colitis/patología , Neoplasias del Colon/etiología , Neoplasias del Colon/patología , Citocinas/metabolismo , Sulfato de Dextran , Modelos Animales de Enfermedad , Células HT29/efectos de los fármacos , Humanos , Proteínas I-kappa B/efectos de los fármacos , Proteínas I-kappa B/genética , Inflamación/complicaciones , Inflamación/patología , Inyecciones Intraperitoneales , Ratones , FN-kappa B/efectos de los fármacos , FN-kappa B/genética , Translocación GenéticaRESUMEN
Calpain enzymes proteolytically modulate cellular function and have been implicated in inflammatory diseases. In this study, we found that calpain levels did not differ between intestinal tissues from inflammatory bowel disease (IBD) patients and healthy controls, but IBD tissues showed increased levels of the endogenous calpain inhibitor, calpastatin (CAST). To investigate the role of CAST in the immune system during IBD, mice were x-ray irradiated, reconstituted with either CAST-knockout (KO) or wild-type (WT) bone marrow, and subjected to dextran sulfate sodium-induced colitis. CAST-KO recipients with induced colitis exhibited more severe weight loss, bloody diarrhea, and anemia compared with WT controls. Histological evaluation of colons from KO recipients with colitis revealed increased inflammatory pathology. Macrophages purified from the colons of KO recipients had higher IL-6, TNF-α, and IFN-γ mRNA levels compared with WT controls. Mechanistic investigations using small interfering RNA and KO bone marrow to generate CAST-deficient macrophages showed that CAST deficiency during activation with bacterial pathogen associated molecular patterns, including heat-killed Enterococcus faecalis or CpG DNA, led to increased IκB cleavage, NF-κB nuclear localization, and IL-6 and TNF-α secretion. Thus, CAST plays a central role in regulating macrophage activation and limiting pathology during inflammatory disorders like IBD.
Asunto(s)
Proteínas de Unión al Calcio/farmacología , Colitis/inmunología , Colitis/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Animales , Colitis/genética , Colitis/patología , Inhibidores de Cisteína Proteinasa/farmacología , Citocinas/biosíntesis , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Humanos , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Masculino , Ratones , Ratones Noqueados , Transporte de Proteínas , Transducción de SeñalRESUMEN
Selenium (Se) is thought to confer cardioprotective effects through the actions of antioxidant selenoprotein enzymes that directly limit levels of ROS such as hydrogen peroxide (H(2)O(2)) or that reverse oxidative damage to lipids and proteins. To determine how the selenoproteome responds to myocardial hypertrophy, two mouse models were employed: triidothyronine (T3)- or isoproterenol (ISO)-treatment. After 7days of T3- and ISO-treatment, cardiac stress was demonstrated by increased H(2)O(2) and caspase-3 activity. Neither treatment produced significant increases in phospholipid peroxidation or TUNEL-positive cells, suggesting that antioxidant systems were protecting the cardiomyocytes from damage. Many selenoprotein mRNAs were induced by T3- and ISO-treatment, with levels of methionine sulfoxide reductase 1 (MsrB1, also called SelR) mRNA showing the largest increases. MsrB enzymatic activity was also elevated in both models of cardiac stress, while glutathione peroxidase (GPx) activity and thioredoxin reductase (Trxrd) activity were moderately and nonsignificantly increased, respectively. Western blot assays revealed a marked increase in MsrB1 and moderate increases in GPx3, GPx4, and Trxrd1, particularly in T3-treated hearts. Thus, the main response of the selenoproteome during hypertrophy does not involve increased GPx1, but increased GPx3 for reducing extracellular H(2)O(2) and increased GPx4, Trxrd1, and MsrB1 for minimizing intracellular oxidative damage.
Asunto(s)
Antioxidantes/metabolismo , Miocardio/metabolismo , Miocardio/patología , Selenoproteínas/metabolismo , Animales , Hipertrofia/inducido químicamente , Hipertrofia/metabolismo , Hipertrofia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , ARN Mensajero/genética , Selenoproteínas/genética , Regulación hacia ArribaRESUMEN
Selenoprotein K (Sel K) is a selenium-containing protein for which no function has been identified. We found that Sel K is an endoplasmic reticulum transmembrane protein expressed at relatively high levels in immune cells and is regulated by dietary selenium. Sel K(-/-) mice were generated and found to be similar to wild-type controls regarding growth and fertility. Immune system development was not affected by Sel K deletion, but specific immune cell defects were found in Sel K(-/-) mice. Receptor-mediated Ca(2+) flux was decreased in T cells, neutrophils, and macrophages from Sel K(-/-) mice compared with controls. Ca(2+)-dependent functions including T cell proliferation, T cell and neutrophil migration, and Fcγ receptor-mediated oxidative burst in macrophages were decreased in cells from Sel K(-/-) mice compared with that in cells from controls. West Nile virus infections were performed, and Sel K(-/-) mice exhibited decreased viral clearance in the periphery and increased viral titers in brain. Furthermore, West Nile virus-infected Sel K(-/-) mice demonstrated significantly lower survival (2 of 23; 8.7%) compared with that of wild-type controls (10 of 26; 38.5%). These results establish Sel K as an endoplasmic reticulum-membrane protein important for promoting effective Ca(2+) flux during immune cell activation and provide insight into molecular mechanisms by which dietary selenium enhances immune responses.