RESUMEN
The phenotype and severity of cancer cachexia differ among tumor types and metastatic site in individual patients. In this study, we evaluated if differences in tumor microenvironment would affect the development of cancer cachexia in a murine model, and demonstrated that body weight, adipose tissue and gastrocnemius muscle decreased in tumor-bearing mice. Interestingly, a reduction in heart weight was observed in the intraperitoneal tumor group but not in the subcutaneous group. We evaluated 23 circulating cytokines and members of the TGF-ß family, and found that levels of IL-6, TNF-α and activin A increased in both groups of tumor-bearing mice. Eotaxin and G-CSF levels in the intraperitoneal tumor group were higher than in the subcutaneous group. Atrogin 1 and MuRF1 mRNA expressions in the gastrocnemius muscle increased significantly in both groups of tumor-bearing mice, however, in the myocardium, expression of these mRNAs increased in the intraperitoneal group but not in subcutaneous group. Based on these results, we believe that differences in microenvironment where tumor cells develop can affect the progression and phenotype of cancer cachexia through alterations in various circulating factors derived from the tumor microenvironment.
Asunto(s)
Caquexia/patología , Músculo Esquelético/patología , Atrofia Muscular/patología , Activinas/sangre , Animales , Caquexia/sangre , Caquexia/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Interleucina-6/sangre , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas Musculares/genética , Atrofia Muscular/sangre , Atrofia Muscular/genética , Miocardio/patología , ARN Mensajero/genética , Proteínas Ligasas SKP Cullina F-box/genética , Factor de Crecimiento Transformador beta/sangre , Proteínas de Motivos Tripartitos , Microambiente Tumoral/genética , Factor de Necrosis Tumoral alfa/sangre , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Although treatment with an antibody against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) combined with multiple therapeutic interventions has been explored, the effect of combination therapy with CTLA-4 inhibition and adoptive T-cell therapy has not been determined. In the present study, our aim was to determine whether CTLA-4 inhibition, combined with adoptive transfer of T cells at different stages of differentiation, exhibits synergistic antitumor effects in a murine colon cancer model. Mice bearing subcutaneous tumors were administered adoptive T-cell transfer of CD62Lhigh or CD62Llow cells combined with an anti-CTLA-4 antibody (α-CTLA-4) or control immunoglobulin G. Subcutaneous tumors were harvested, and the antitumor effects and helper T-cell polarization were analyzed. CTLA-4 inhibition combined with CD62Lhigh cell administration showed the strongest antitumor effect. Combination therapy increased the number of CD3+ cells within the tumor. Moreover, CTLA-4 inhibition induced polarization of T cells infiltrating the tumor toward the T helper 1 lineage, and suppressed the frequency of regulatory T cells within the tumor, particularly in combination with CD62Lhigh T-cell transfer. This is the first report demonstrating that the efficacy of α-CTLA-4 and adoptive T-cell transfer combination therapy depends on the state of differentiation of the transferred T cells. Our data support the notion that a combination of α-CTLA-4 and adoptive T-cell transfer containing an abundance of naïve phenotype cells could potentially exert antitumor effects in a clinical setting.
Asunto(s)
Antineoplásicos/inmunología , Antígeno CTLA-4/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo/métodos , Animales , Antígenos CD/inmunología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
Epithelial-mesenchymal transition (EMT) plays a crucial role in cancer metastasis. In this study, we evaluated the effect of heat treatment on tumor growth factor-ß1 (TGF-ß1)-induced EMT in pancreatic cancer cells and tried to ascertain the mechanism related to any observed effects. Human pancreatic cancer cell lines (BxPC-3, PANC-1 and MIAPaCa-2) were stimulated by TGF-ß1, and evaluated for morphological changes using immunofluorescence and EMT-related factors (i.e., E-cadherin, Vimentin, Snail or ZEB-1) using RT-PCR. To examine the effect of heat on EMT, the cancer cells were heat-treated at 43°C for 1 h then stimulated with TGF-ß1. We then evaluated whether or not heat treatment changed the expression of EMT-related factors and cell migration and also whether Smad activation was inhibited in TGF-ß signaling. After being treated with TGF-ß1, pancreatic cancer cells resulted in EMT and cell migration was enhanced. Heat treatment inhibited TGF-ß1-induced changes in morphology, inhibited the expression of EMT-related factors, and attenuated TGF-ß1-induced migration in pancreatic cancer cells. Additionally, we observed that heat treatment blocked TGF-ß1-induced phosphorylation of Smad2 in PANC-1 cells. Our results suggest that heat treatment can suppress TGF-ß1-induced EMT and opens the possibility of a new therapeutic use of hyperthermia as a potential treatment for cancer metastasis.
RESUMEN
Epithelial-mesenchymal transition (EMT) is considered to be a crucial event in the development of cancer metastasis. Anoxia/reoxygenation (A/R) is known to occur in cancer tissues due to angiogenesis and changes in tissue pressure that occur during tumor growth. We investigated whether A/R induces EMT in the human colon cancer cell line HT-29. Colon cancer cells were exposed to anoxia (2 h) followed by reoxygenation (4-22 h) and evaluated for EMT changes using immunofluorescence and western blot analyses. We also investigated the expression of EMT-related transcription factors (Snail and ZEB1) using RT-PCR and evaluated the expression of NF-κB using ELISA. To determine whether NF-κB is involved in A/R-induced EMT, HT-29 cells were treated with proteasome inhibitors. Colon cancer cells exposed to A/R underwent EMT morphological changes; the cancer cells acquired a spindle-shaped phenotype. The expression of E-cadherin on the cell surface and the total amount of E-cadherin proteins were reduced after A/R. The expression of EMT-related transcription factors (Snail, ZEB1) was increased after A/R. Pretreatment with proteasome inhibitors significantly attenuated the downregulation of E-cadherin induced by A/R. These results indicate that A/R induces EMT in human colon cancer cells through an NF-κB-dependent transcriptional pathway.
Asunto(s)
Transición Epitelial-Mesenquimal , Oxígeno/fisiología , Antígenos CD , Cadherinas/genética , Cadherinas/metabolismo , Hipoxia de la Célula , Forma de la Célula , Neoplasias del Colon , Regulación Neoplásica de la Expresión Génica , Células HT29 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Subunidad p50 de NF-kappa B/metabolismo , Unión Proteica , Factores de Transcripción de la Familia Snail , Factor de Transcripción ReIA/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Vimentina/genética , Vimentina/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
A core challenge in administering immune-based treatments for cancer is the establishment of easily accessible immunological assays that can predict patients' clinical responses to immunotherapy. In this study, our aim was to predict the therapeutic effects of adoptive T-cell therapy in patients with advanced pancreatic cancer. To do this, we evaluated whole blood cytokine levels and peripheral regulatory T cells (Tregs) in 46 patients with unresectable or recurrent pancreatic cancer who received adoptive T-cell therapy at 2-week intervals. To test immune function, venous blood was obtained from patients before the start of therapy and 2 weeks after the 4th treatment. Whole blood interferon (IFN)-α levels (after stimulation with the Sendai virus) were evaluated, as well as the levels of 9 cytokines stimulated with phytohemagglutinin [interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12(p70), IL-13, tumor necrosis factor-α, IFN-γ, and granulocyte-monocyte colony-stimulating factor]. Peripheral Tregs were analyzed by flow cytometry. Using the obtained data, we then observed the relationship between these immunological parameters and clinical outcome of patients. We found that the whole blood production of IFN-γ, IL-2, IL-4, IL-5 and IL-13 significantly increased after adoptive T-cell therapy, whereas the number of peripheral Tregs did not change. Multivariate Cox proportional hazards analyses indicated that the number of peripheral Tregs before receiving adoptive T-cell therapy and the change in IFN-γ levels after adoptive T-cell therapy were independent variables predicting overall survival. The findings of this study indicate that the assay of whole blood IFN-γ production offers promise for evaluating the clinical response of patients to cancer immunotherapy.
Asunto(s)
Inmunoterapia Adoptiva , Interferón gamma/sangre , Neoplasias Pancreáticas/terapia , Linfocitos T/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Citocinas/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/mortalidad , Modelos de Riesgos ProporcionalesRESUMEN
We investigated the effects of anticancer agents on peripheral blood mononuclear cells for the purpose of providing data to support new translational chemoimmunotherapy regimens. Peripheral-blood mononuclear cells were treated with one of four anticancer agents (5-fluorouracil, irinotecan, cisplatin, and gemcitabine) for 2 h, after which cell viability was determined. For assessment of effects of each drug on proliferation and cytokine production, cells were stimulated with phytohemagglutinin for 48 h. As a result, the anticancer agents did not affect cell viability. Cell proliferation was unaffected by 5-fluorouracil and irinotecan but inhibited by cisplatin and gemcitabine. Treatment with gemcitabine enhanced the production of IFN-γ and decreased the number of regulatory T cells. gemcitabine treatment increased IFN-γ production among CD4 T cells but not among CD8 T cells. The results indicated that GEM had immunoregulatory properties that might support immune response against cancer. This finding has implications for designing chemoimmunotherapy strategies.
RESUMEN
Acetyl salicylic acid (ASA) is one of the most frequently prescribed medications for the secondary prevention of cardiovascular and cerebrovascular events. It has recently been reported to cause small intestinal mucosal injury at a considerably higher rate than previously believed. The aim of this study is to investigate the mechanism by which this occurs using an in vitro small intestine model focusing on the role of oxidative stress and cell permeability. Differentiated Caco-2 exhibits a phenotype similar to human small intestinal epithelium. We measured whether ASA induced the increase of differentiated Caco-2 permeability, the decrease of tight junction protein expression, the production of reactive oxygen species (ROS), and the expression of ROS-modified zonula occludens-1 (ZO-1) protein. In some experiments, Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP, a superoxide dismutase mimetic) was used. The nontoxic concentration of ASA decreased transepithelial electrical resistance and increased the flux of fluorescein isothiocyanate-conjugated dextran across Caco-2 in a time-dependent manner. The same concentration of ASA significantly decreased ZO-1 expression among TJ proteins as assessed by Western blot and immunocytochemistry and increased ROS production and the expression of oxidative stress-modified ZO-1 protein. However, MnTMPyP suppressed the ASA-induced increased intercellular permeability and the ASA-induced ROS-modified ZO-1 expression. Our findings indicate that ASA-induced ROS production can specifically modify the expression of ZO-1 protein and induce increased cell permeability, which may ultimately cause small intestinal mucosal injury.
Asunto(s)
Aspirina/farmacología , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Proteína de la Zonula Occludens-1/metabolismo , Línea Celular Tumoral , Humanos , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Estrés Oxidativo/fisiología , Permeabilidad , Especies Reactivas de Oxígeno/metabolismo , Uniones Estrechas/metabolismoRESUMEN
OBJECTIVE: To investigate the relation between functional impairments of cancer patients and circulating cytokines using a multiplex technique. DESIGN AND METHODS: 50 patients with cancer were assessed using the quality of life (QOL) questionnaire. 27 plasma cytokine levels were determined by using the Bio-Plex array system. The relation to QOL scores was assessed using Chi-square test for categorical variables and univariate linear regression analysis for cytokine levels. RESULTS: Multivariate analysis showed that interleukin-6 (IL-6) level is a significant independent determinant of physical (ß=-0.238, P=0.0126) and cognitive functioning (ß=-0.462, P=0.0006) and that vascular endothelial growth factor (VEGF) level is a significant independent determinant of emotional functioning (ß=-0.414, P=0.039). CONCLUSION: This study, in which 27 cytokines are simultaneously tested with cutting edge technology, demonstrates that plasma IL-6 and VEGF are significant independent determinants of functional impairments in patients with cancer.
Asunto(s)
Citocinas/sangre , Neoplasias/sangre , Neoplasias/psicología , Adulto , Anciano , Anciano de 80 o más Años , Cognición , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias/patología , Neoplasias/fisiopatología , Análisis de Regresión , Adulto JovenRESUMEN
Protection of the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs including acetylsalicylic acid is a critical issue in the field of gastroenterology. Polaprezinc an anti-ulcer drug, consisting of zinc and L-carnosine, provides gastric mucosal protection against various irritants. In this study, we investigated the protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of the RIE1 rat intestinal epithelial cell line. Confluent rat intestinal epithelial cells were incubated with 70 µM polaprezinc for 24 h, and then stimulated with or without 15 mM acetylsalicylic acid for a further 15 h. Subsequent cellular viability was quantified by fluorometric assay based on cell lysis and staining. Acetylsalicylic acid-induced cell death was also qualified by fluorescent microscopy of Hoechst33342 and propidium iodide. Heat shock proteins 70 protein expression after adding polaprezinc or acetylsalicylic acid was assessed by western blotting. To investigate the role of Heat shock protein 70, Heat shock protein 70-specific small interfering RNA was applied. Cell viability was quantified by fluorometric assay based on cell lysis and staining and apoptosis was analyzed by fluorescence-activated cell sorting. We found that acetylsalicylic acid significantly induced apoptosis of rat intestinal epithelial cells in a dose- and time-dependent manner. Polaprezinc significantly suppressed acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells at its late phase. At the same time, polaprezinc increased Heat shock protein 70 expressions of rat intestinal epithelial cells in a time-dependent manner. However, in Heat shock protein 70-silenced rat intestinal epithelial cells, polaprezinc could not suppress acetylsalicylic acid -induced apoptosis at its late phase. We conclude that polaprezinc-increased Heat shock protein 70 expression might be an important mechanism by which polaprezinc suppresses acetylsalicylic acid-induced small intestinal apoptosis, a hallmark of acetylsalicylic acid-induced enteropathy.
RESUMEN
We examined the effects of adoptive T-cell transfer (ACT) on the population of regulatory T cells (Tregs) in a mouse colorectal cancer transplant model. In an in vivo study, Treg populations in Balb/c mice colon26 transplant model after ACT were analyzed in peripheral blood, local lymph node, and tumor. In an in vitro study CD4+ cells were cultured in medium containing TGF-beta to induce Tregs. LAK cells were added or not in this Treg induction system. Treg induction after coculture with LAK was investigated. We also studied the role of IFN-gamma in the mechanism of Treg induction. Tregs in the draining lymph nodes and tumor were significantly suppressed by ACT. The induction of Tregs in vitro was inhibited by coculture with LAK cells. Furthermore, Tregs in the cultured cells were significantly inhibited by addition of exogenous IFN-gamma. Moreover, Tregs were increased by addition of IFN-gamma mAb. ACT may decrease Tregs in tumor-bearing hosts. One of the mechanisms is considered to be IFN-gamma inhibiting the induction of Tregs.
Asunto(s)
Adenocarcinoma/inmunología , Linfocitos T CD4-Positivos/trasplante , Neoplasias del Colon/inmunología , Modelos Animales de Enfermedad , Inmunoterapia , Células Asesinas Activadas por Linfocinas/trasplante , Linfocitos T Reguladores/inmunología , Adenocarcinoma/prevención & control , Traslado Adoptivo , Animales , Western Blotting , Linfocitos T CD4-Positivos/inmunología , Neoplasias del Colon/prevención & control , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Factores de Transcripción Forkhead , Técnicas para Inmunoenzimas , Interferón gamma/metabolismo , Células Asesinas Activadas por Linfocinas/inmunología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Linfocitos T Reguladores/citología , Factor de Crecimiento Transformador beta/farmacología , Células Tumorales CultivadasRESUMEN
It remains to be clarified whether adoptive cellular therapy (ACT) in patients with advanced cancer, in whom strong immunosuppression and immune-escape mechanisms are established, has the potential to alter cytokine secretion from blood cells and affect the number of regulatory T cells (Tregs). In this study, the secretion of cytokines from peripheral blood cells and the number of peripheral blood Tregs were analyzed before and after ACT. Blood samples were collected from 109 consecutive cancer patients who received ACT, which consisted of anti-CD3 stimulated lymphokine-activated killer cells. For testing immune function, venous blood was obtained from patients before the start of therapy and after they had received 4 cycles of ACT. Of the 109 patients, 76 received ACT four times or more. All 109 blood samples at baseline and 76 follow-up samples were available. The secretion ability of various cytokines from peripheral blood cells was measured, as well as the number of peripheral blood Tregs. We found that the secretion ability of interferon (IFN)-γ and tumor necrosis factor (TNF)-α was enhanced significantly after treatment, while the number of Tregs and the ratio of Treg to CD4 was significantly decreased. Overall survival in patients with increased IFN-γ and TNF-α secretion after ACT was significantly longer. These findings suggest a potential therapeutic role for ACT in cancer immunotherapy.
RESUMEN
BACKGROUND AND AIMS: Ecabet sodium (ES) is a gastric mucosal protective and ulcer-healing agent. Recently enema therapy with ES was found to be effective for the treatment of human ulcerative colitis as well as experimental colitis in an animal model. Whereas ES possesses potential as a novel treatment for ulcerative colitis, its precise mechanism of action remains to be elucidated. In this study, we investigated the therapeutic efficacy of ES in an experimental rat model of colitis, and evaluated the restitution of intestinal epithelial cells treated with ES in vitro. METHODS: Acute colitis was induced with trinitrobenzene sulfonic acid (TNBS) in male Wistar rats. Rats received intrarectal treatment with ES daily starting on day 7 and were sacrificed on day 14 after the administration of TNBS. The distal colon was removed to evaluate various parameters of inflammation. Moreover, wound-healing assays were used to determine the enhanced restitution of rat intestinal epithelial (RIE) cells treated with ES. RESULTS: Intracolonic administration of ES accelerated TNBS-induced ulcer healing. Increases in the wet weight of the colon after TNBS administration were significantly inhibited by ES treatment. The wound assay revealed ES enhancement of the migration of RIE cells migration through the phosphorylation of extracellular signal-regulated kinase. CONCLUSION: Daily administration of an ES enema promoted the healing of intestinal mucosal injury, in part by the enhanced restitution of intestinal epithelial cells via extracellular signal-regulated kinase activation. ES may thus represent a novel therapeutic approach for the treatment of inflammatory bowel disease.
Asunto(s)
Abietanos/farmacología , Antiulcerosos/farmacología , Movimiento Celular/efectos de los fármacos , Colitis Ulcerosa/tratamiento farmacológico , Colon/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Ácido Trinitrobencenosulfónico , Cicatrización de Heridas/efectos de los fármacos , Abietanos/administración & dosificación , Enfermedad Aguda , Administración Rectal , Animales , Antiulcerosos/administración & dosificación , Línea Celular , Colitis Ulcerosa/enzimología , Colitis Ulcerosa/patología , Colon/enzimología , Colon/patología , Modelos Animales de Enfermedad , Enema , Células Epiteliales/enzimología , Células Epiteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Masculino , Fosforilación , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Previously we have demonstrated that whole body hyperthermia (WBH) improves insulin resistance in diabetic mice. The aim of the present study was to perform a gene expression analysis of the liver and adipose tissue of obesity-induced insulin resistant diabetic mice (db/db mice) after WBH and to define the molecules that play the important role in improvement of insulin resistance by WBH. Male db/db mice were treated with WBH 3 times per week for 12 weeks. Total RNA was extracted from the liver and adipose tissue of db/db mice, and differences in the gene expression profiles among db/+ mice, untreated db/db mice, and WBH-treated db/db mice were investigated using a high-density DNA microarray. WBH directly targets liver and adipose tissue, resulting in modifications in NF-kappaB and IL-6 signalling pathways, as well as lipid metabolism. Although the mechanisms have not yet been completely investigated, we can conclude that WBH may provide a new therapeutic or preventive modality against type 2 diabetes mellitus and metabolic or insulin resistance syndrome through the modification of several signalling pathways.
Asunto(s)
Diabetes Mellitus Experimental/genética , Hipertermia Inducida , Tejido Adiposo/fisiología , Animales , Análisis por Conglomerados , Diabetes Mellitus Experimental/metabolismo , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Resistencia a la Insulina/genética , Hígado/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal/fisiologíaRESUMEN
Induction of heme oxygenase-1 (HO-1) expression has been associated with cytoprotective and anti-inflammatory actions of lansoprazole, a proton pump inhibitor, but the underlying molecular mechanisms remain largely unresolved. In this study, we investigate the role of transcriptional NF-E2-related factor 2 (Nrf2), its phosphorylation/activation, and oxidation of Kelch-like ECH-associating protein 1 (Keap1) in lansoprazole-induced HO-1 up-regulation using cultured gastric epithelial cells (rat gastric mucosal cell line, RGM-1). HO-1 expression of RGM-1 cells was markedly enhanced in a time- and dose-dependent manner by the treatment with lansoprazole, and this up-regulation of HO-1 contributed to the inhibition of chemokine production from stimulated RGM-1 cells. Transfection of Nrf2-siRNA suppressed the lansoprazole-induced HO-1. An electrophoretic mobility shift assay showed increases in the nuclear translocation and stress-response elements (StRE) binding activity of Nrf2 proteins in RGM-1 cells treated with lansoprazole. Furthermore, in RGM-1 cells transfected with HO-1 enhancer luciferase reporter plasmid containing mutant StRE, lansoprazole-induced HO-1 reporter gene activity was diminished. Lansoprazole promoted the phosphorylation of extracellular signal-regulated kinase (ERK), and lansoprazole-induced HO-1 up-regulation was suppressed by U0126, an ERK-specific inhibitor. Phosphorylated Nrf2 protein was detected in the phosphoprotein fraction purified by a Pro-Q Diamond Phosphoprotein Enrichment kit. Finally, an oxidative form of the Keap1 protein was detected in lansoprazole-treated RGM-1 cells by analyzing S-oxidized proteins using biotinylated cysteine as a molecular probe. These results indicate that lansoprazole up-regulates HO-1 expression in rat gastric epithelial cells, and the up-regulated HO-1 contributes to the anti-inflammatory effects of the drug. Phosphorylation of ERK and Nrf2, activation and nuclear translocation of Nrf2, and oxidation of Keap1 are all involved in the lansoprazole-induced HO-1 up-regulation.
Asunto(s)
2-Piridinilmetilsulfinilbencimidazoles/farmacología , Antiinflamatorios no Esteroideos/farmacología , Mucosa Gástrica/metabolismo , Hemo-Oxigenasa 1/biosíntesis , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas/metabolismo , Inhibidores de la Bomba de Protones/farmacología , Animales , Línea Celular , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/fisiología , Mucosa Gástrica/citología , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/enzimología , Insectos , Péptidos y Proteínas de Señalización Intracelular , Proteína 1 Asociada A ECH Tipo Kelch , Lansoprazol , Ratones , Factor 2 Relacionado con NF-E2/fisiología , Oxidación-Reducción/efectos de los fármacos , RatasRESUMEN
BACKGROUND AND AIM: It is reported that NF-kappaB is activated by chemotherapy in some cancer cell lines and NF-kappaB activation is one of the mechanisms by which tumors are induced to become resistant to chemotherapy. We reported that heat-treatment-induced heat shock protein 70 (Hsp70) could inhibit I-kappa-B kinase, resulting in the inhibition of NF-kappaB activation. Therefore, we speculated that activated NF-kappaB in a pancreatic cell line might be inhibited by heat treatment, resulting in the enhancement of gemcitabine-induced cytotoxicity. METHODS: We used the human pancreatic carcinoma cell lines AsPC-1 and MIAPaCa-2. Both cell lines were treated with various concentrations (0, 5, 10, 20, and 30 microM) of gemcitabine for 24 h. Heat treatment (43 degrees C, 1 h) was performed at various times relative to gemcitabine treatment. The effect of gemcitabine and heat treatment on cell survival was determined by WST-8 assay. The status of NF-kappaB in carcinoma cells exposed to gemcitabine was investigated by electrophoretic mobility shift assay and immunocytochemistry. We analyzed apoptosis and necrosis in AsPC-1 and MIAPaCa-2 cells by flow cytometry. Furthermore, the levels of Hsp70, cyclin D1, caspase-3, and vascular endothelial growth factor in each treatment group were detected by western blotting. RESULTS: (1) Significant cytotoxicity was observed with gemcitabine. (2) Gemcitabine activated NF-kappaB binding activity in both cell lines. (3) Heat treatment inhibited the gemcitabine-induced activation of NF-kappaB. (4) Heat treatment enhanced the cytotoxicity of gemcitabine, especially when heat treatment was performed 24 h before gemcitabine was given. (5) The levels of Hsp70 were increased by heat treatment. Gemcitabine did not affect the protein level of Hsp70. The levels of pro-caspase-3 were decreased by heat treatment combined with gemcitabine. CONCLUSIONS: Heat treatment inhibited gemcitabine-induced activation of NF-kappaB, resulting in the enhancement of the cytotoxicity of gemcitabine.
Asunto(s)
Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Desoxicitidina/análogos & derivados , Hipertermia Inducida , FN-kappa B/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Desoxicitidina/uso terapéutico , Humanos , GemcitabinaRESUMEN
BACKGROUND/AIMS: Protecting intestinal mucosa from nonsteroidal anti-inflammatory drugs is still an unsolved problem. It has been revealed that apoptosis in epithelial cells as a result of mitochondrial injury is an important pathogenesis in indomethacin-induced gastric mucosal injury. In this study, we revealed the effect of overexpressed heat-shock protein 70 (HSP70) in indomethacin-induced apoptosis and oxidative stress. METHODS: HSP70-overexpressing rat gastric mucosal cells (7018-RGM-1 cells) and control cells (pBK-CMV-12 cells) were used and treated with 0-500 microM of indomethacin for 24 h. Cell viability and cytotoxity were measured by a WST-8 assay and a lactate dehydrogenase release assay, respectively. Apoptosis was observed by fluorescence microscopy staining with Hoechst 33342 and propidium iodide. The expression of Bcl-2 family proteins, activation of caspase-3, and 4-hydroxy-2-nonenal (4-HNE)-modified proteins were assessed by Western blot analysis. RESULTS: Indomethacin caused apoptosis of gastric epithelial cells. The 7018-RGM-1 cells survived significantly after indomethacin treatment compared to the control cells. The increase in pro-apoptotic Bad proteins, the decrease in anti-apoptotic Bcl-2 proteins, and caspase activation were all suppressed in the 7018-RGM-1 cells. A lower level of indomethacin-induced 4-HNE-modification was detected in the 7018-RGM-1 cells than in the control cells. CONCLUSION: Overexpressed HSP70 may potentiate resistance to apoptosis and oxidative stress in indomethacin-induced gastric epithelial cell injury.
Asunto(s)
Antiinflamatorios no Esteroideos/efectos adversos , Células Epiteliales/efectos de los fármacos , Mucosa Gástrica/metabolismo , Proteínas HSP70 de Choque Térmico/biosíntesis , Indometacina/efectos adversos , Animales , Antiinflamatorios no Esteroideos/farmacología , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/metabolismo , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/fisiopatología , Indometacina/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , RatasRESUMEN
PURPOSE: We examined whether hyperthermia attenuated the metastatic potential of colon cancer through the induction of heat shock protein 70 (Hsp70). MATERIALS AND METHODS: Colon26 cells were separated into four groups: (1) no pretreatment, (2) hyperthermia at 42 degrees C for 1 hour, (3) pretreatment with geranylgeranylacetone (GGA) 10(-6) M for 2 hours, and (4) hyperthermia after GGA treatment. We measured cell viabilities and the contents of Hsp70. We assessed nuclear factor-kappa-B (NF-kappa-B) status with and without tumor necrosis factor-alpha (TNF-alpha) stimulation. For in vivo study, colon26 cells were injected via the tail vein or into a subcutaneous area of mice and the numbers of lung metastatic nodules or the volumes of subcutaneous tumors were assessed. Untreated cells were incubated with PKH26. Experimental metastasis models were then generated and used to assess the fixed cancer cells. RESULTS: Tumor development in the subcutaneous tumor models and cell viabilities were similar among the four groups. However, the GGA plus hyperthermia group had fewer lung metastatic nodules in the experimental lung metastasis model and higher Hsp70 induction than the other cell groups. The GGA plus hyperthermia pretreatment group also showed a lower number of fixed cells in lungs and lower activation of NF-kappa-B by TNF-alpha than the other cell groups. CONCLUSIONS: It is suggested the metastatic potential but not the proliferation potency of cancer cells is inhibited by the transient induction of Hsp70.
Asunto(s)
Neoplasias del Colon , Modelos Animales de Enfermedad , Diterpenos/uso terapéutico , Hipertermia Inducida , Metástasis de la Neoplasia , Animales , Línea Celular Tumoral , Supervivencia Celular , Neoplasias del Colon/patología , Neoplasias del Colon/terapia , Proteínas HSP70 de Choque Térmico/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Trasplante de NeoplasiasRESUMEN
BACKGROUND: The precise pathogenic mechanism of nonsteroidal antiinflammatory drug-induced small intestinal injury is still unknown. In the present study, we investigated the mechanism by which indomethacin induced mucosal injury by using an in vitro model of small intestine. METHODS: The colon cancer cell line Caco-2, exhibiting a small intestinal phenotype starting as a crypt cell and differentiating to a villous phenotype, and RIE, a rat intestinal epithelial cell line, were employed. Indomethacin was added to differentiated the Caco-2 and RIE monolayer, and cell death was quantified by MTT assay and LDH release in the cell culture supernatant. Indomethacin-induced cell death was also qualified by fluorescent probes under the fluorescent microscope. As a functional study, the permeability of the Caco-2 monolayer was assessed by measuring transepithelial electrical resistance (TEER) and the flux of FITC-conjugated dextran across the monolayer. Indomethacin-induced reactive oxygen species production in Caco-2 and RIE was evaluated by redoxsensitive fluorogenic probes using a fluorometer. In some experiments, antioxidants were used to clarify the role of reactive oxygen species on indomethacin-induced Caco-2 cell death. RESULTS: Indomethacin caused cell death (mainly apoptosis) of Caco-2 and RIE in a dose-and time-dependent manner that was correlated with increased permeability of the Caco-2 monolayer. Exposure of Caco-2 and RIE with indomethacin also resulted in a significant reactive oxygen species production that was inhibited by the pretreatment of these cells with antioxidants. CONCLUSIONS: Taken together, reactive oxygen species production is one of the mechanisms by which indomethacin induced small intestinal injury.
Asunto(s)
Antiinflamatorios no Esteroideos/toxicidad , Apoptosis/efectos de los fármacos , Indometacina/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antioxidantes/farmacología , Células CACO-2 , Diferenciación Celular , Línea Celular , Relación Dosis-Respuesta a Droga , Impedancia Eléctrica , Fluorometría , Humanos , Indometacina/administración & dosificación , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Permeabilidad/efectos de los fármacos , Ratas , Factores de TiempoRESUMEN
Astaxanthin (ASX) is a carotenoid that has potent protective effects on diabetic nephropathy in mice model of type 2 diabetes. In this study, we investigated the protective mechanism of ASX on the progression of diabetic nephropathy using an in vitro model of hyperglycemia, focusing on mesangial cells. Normal human mesangial cells (NHMCs) were cultured in the medium containing normal (5 mM) or high (25 mM) concentrations of D-glucose. Reactive oxygen species (ROS) production, the activation of nuclear transcription factors such as nuclear factor kappa B (NFkappaB) and activator protein-1 (AP-1), and the expression/production of transforming growth factor-beta 1 (TGFbeta(1)) and monocyte chemoattractant protein-1 (MCP-1) were evaluated in the presence or absence of ASX. High glucose (HG) exposure induced significant ROS production in mitochondria of NHMCs, which resulted in the activation of transcription factors, and subsequent expression/production of cytokines that plays an important role in the mesangial expansion, an important event in the pathogenesis of diabetic nephropathy. ASX significantly suppressed HG-induced ROS production, the activation of transcription factors, and cytokine expression/production by NHMCs. In addition, ASX accumulated in the mitochondria of NHMCs and reduced the production of ROS-modified proteins in mitochondria. ASX may prevent the progression of diabetic nephropathy mainly through ROS scavenging effect in mitochondria of mesangial cells and thus is expected to be very useful for the prevention of diabetic nephropathy.
Asunto(s)
Neuropatías Diabéticas/prevención & control , Hiperglucemia/metabolismo , Células Mesangiales/efectos de los fármacos , Línea Celular , Quimiocina CCL2/metabolismo , Neuropatías Diabéticas/etiología , Neuropatías Diabéticas/metabolismo , Humanos , Hiperglucemia/complicaciones , Células Mesangiales/metabolismo , Mitocondrias/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factor de Transcripción AP-1/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Xantófilas/farmacologíaRESUMEN
AIM: In this study, we examined the efficacy of whole body hyperthermia (WBH) on obesity-induced insulin resistance in diabetic mice. METHODS: Male db/db mice were treated with WBH 3 times per week for 12 weeks. The rectal temperature of mice reached 38.0 degrees C 5 min after heating, and was kept at 38.0 degrees C for 30 min. At the end of each week, tail snip glucose levels were determined under fasting conditions. The GLUT-4 gene expression of muscle tissue was analyzed by real-time PCR. RESULTS: (1) WBH-treated db/db mice showed a significant decrease in fasting blood glucose level as compared with untreated db/db mice (p < 0.01). (2) Plasma insulin levels in untreated db/db mice at the age of 10 weeks were significantly increased compared with those of db/+ mice (p < 0.0001). On the other hand, the reduction (31%) in insulin levels in WBH-treated mice indicated improved insulin sensitivity. (3) The ability of WBH to increase insulin sensitivity was further established in glucose tolerance tests and insulin tolerance tests. (4) Urine albumin of db/db mice significantly increased compared with those of db/+ mice at 18 weeks of age (p < 0.001). This increase in urinary albumin was significantly inhibited by WBH (p < 0.01). (5) WBH up-regulated the expression of GLUT4 mRNA in skeletal muscle. CONCLUSION: Although the mechanisms have not yet been completely investigated, WBH may provide a new therapeutic or preventive modality against obesity-related diseases such as T2DM and metabolic or insulin resistance syndrome.