RESUMEN

ABSTRACT: The aim of this study was to investigate the expression of phosphatase of regenerating live-3 (PRL-3) in human stage III colorectal cancer (CRC) and to evaluate its correlation with metachronous liver metastasis (MLM) and prognosis.The retrospective cohort study included 116 stage III CRC primary tumors and 60 normal colorectal tissues. PRL-3 expression was measured by immunohistochemistry. We investigated the correlation of PRL-3 with clinicopathologic features by the chi-square test. The association of PRL-3 expression with MLM was assessed by binary logistic regression. Overall survival (OS) and disease-free survival (DFS) between patients with positive PRL-3 expression and those with negative PRL-3 expression were compared by the Kaplan-Meier method and Cox proportional hazards regression model.We found that 32.8% of stage III CRC primary tumors were PRL-3 positive, and 15.0% of normal colorectal epithelia showed high PRL-3 expression (P = .012). Seventeen tumors (47.2%) among 36 cases that developed MLM were PRL-3 positive, and only 21 tumors (26.3%) in the 80 cases that did not develop MLM had positive PRL-3 expression (P = .026). PRL-3 expression was associated with MLM (P = .028). Patients with positive expression of PRL-3 showed a significantly shorter OS (40.32 ±â€Š3.97 vs 53.96 ±â€Š2.77 months, P = .009) and DFS (34.97 ±â€Š4.30 vs 44.48 ±â€Š2.89 months, P = .036). A multivariate analysis indicated that PRL-3 expression was an independent unfavorable prognostic factor for OS (P = .007).Our study suggested that high PRL-3 expression is an independent risk factor for MLM and poor prognosis. PRL-3 is expected to be a promising biomarker for predicting the incidence of MLM and prognosis in patients with stage III CRC.

Asunto(s)

Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/mortalidad , Proteínas de Neoplasias/metabolismo , Neoplasias Primarias Secundarias/enzimología , Neoplasias Primarias Secundarias/mortalidad , Proteínas Tirosina Fosfatasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Colon/enzimología , Neoplasias Colorrectales/patología , Supervivencia sin Enfermedad , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Análisis Multivariante , Estadificación de Neoplasias , Neoplasias Primarias Secundarias/patología , Valor Predictivo de las Pruebas , Pronóstico , Modelos de Riesgos Proporcionales , Recto/enzimología , Estudios Retrospectivos , Factores de Riesgo

RESUMEN

BACKGROUND: Increased acetylcholinesterase (AChE) activity on frozen sections of rectal mucosal biopsies accurately diagnoses Hirschsprung disease (HD). But the quest for a biomarker in blood as a screening test prompts one to look for AChE in blood and study its role in HD diagnosis. AIM: To develop a low-cost reliable method to estimate the AChE activity in plasma and red blood cells (RBCs) in normal children (control) and study its role in HD (test). MATERIALS AND METHODS: Optimized method derived after modifying and standardizing known AChE assay protocols for blood were employed on 30 controls to define the AChE cut-off range, on 40 suspected HD cases to categorize them as HD/non-HD based on cut-off values and later compared with gold standard tissue AChE histochemistry of rectal mucosal biopsies. RESULTS: An optimal in-house modified methods of Ellman's was found best suited to analyze plasma AChE activity, method by Wilson and Henderson was optimal for extraction and AChE estimation in RBCs. AChE levels (controls) obtained were 1.03 ± 0.31 U/mL and 5.17 ± 1.52 U/mL in plasma and RBCs, respectively while the plasma AChE was 1.35 ± 0.84 U/mL (HD) and 1.62 ± 0.85 U/mL (non-HD) while RBC AChE was 4.29 ± 3.2 U/mL (HD) and 6.48 ± 4.31 U/mL (non-HD). Sensitivity was 66.67% and 55.56%, specificity was 22.73% and 45.45%, and an accuracy rate of 42.5% and 50% for plasma and RBC, respectively. CONCLUSIONS: Mutually exclusive AChE activity range identified for test blood samples overlapped with the normal and hence, not considered a diagnostic tool for HD.

Asunto(s)

Acetilcolinesterasa/análisis , Acetilcolinesterasa/sangre , Eritrocitos/enzimología , Enfermedad de Hirschsprung/sangre , Enfermedad de Hirschsprung/diagnóstico , Recto/enzimología , Acetilcolinesterasa/metabolismo , Biomarcadores/análisis , Biomarcadores/sangre , Biopsia , Niño , Preescolar , Método Doble Ciego , Tránsito Gastrointestinal/fisiología , Enfermedad de Hirschsprung/patología , Histocitoquímica/métodos , Humanos , India , Membrana Mucosa/enzimología , Estudios Prospectivos , Sensibilidad y Especificidad

RESUMEN

Increased COX-2 and decreased 15-hydroxyprostaglandin dehydrogenase (15-HPGD) expression promote prostaglandin-mediated inflammation and colorectal carcinogenesis. Experimental studies suggest that vitamin D and calcium may inhibit these pathways, but their effects on colorectal tissue COX-2 and 15-HPGD expression in humans are unknown. We tested the effects of supplemental vitamin D (1,000 IU/day) and/or calcium (1,200 mg/day) on COX-2 and 15-HPGD expression in the morphologically normal rectal mucosa from 62 paients with colorectal adenoma in a placebo-controlled chemoprevention trial. We measured biomarker expression using automated IHC and quantitative image analysis at baseline and 1-year follow-up, and assessed treatment effects using mixed linear models. The primary outcome was the COX-2/15-HPGD expression ratio, because these enzymes function as physiologic antagonists. After 1 year of treatment, the mean COX-2/15-HPGD expression ratio in full-length crypts proportionately decreased 47% in the vitamin D group (P = 0.001), 46% in the calcium group (P = 0.002), and 34% in the calcium + vitamin D group (P = 0.03), relative to the placebo group. Among individuals with the functional vitamin D-binding protein isoform DBP2 (GC rs4588*A), the COX-2/15-HPDG ratio decreased 70% (P = 0.0006), 75% (P = 0.0002), and 60% (P = 0.006) in the vitamin D, calcium, and combined supplementation groups, respectively, relative to placebo. These results show that vitamin D and calcium favorably modulate the balance of expression of COX-2 and 15-HPGD-biomarkers of inflammation that are strongly linked to colorectal carcinogenesis-in the normal-appearing colorectal mucosa of patients with colorectal adenoma (perhaps especially those with the DBP2 isoform). PREVENTION RELEVANCE: Supplemental calcium and vitamin D reduce indicators of cancer-promoting inflammation in normal colorectal tissue in humans, thus furthering our understanding of how they may help prevent colorectal cancer.

Asunto(s)

Adenoma/prevención & control , Carbonato de Calcio/administración & dosificación , Neoplasias Colorrectales/prevención & control , Mucosa Intestinal/inmunología , Vitamina D/administración & dosificación , Adenoma/inmunología , Adenoma/patología , Anciano , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Colon/efectos de los fármacos , Colon/enzimología , Colon/inmunología , Colon/patología , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Ciclooxigenasa 2/análisis , Ciclooxigenasa 2/metabolismo , Suplementos Dietéticos , Femenino , Estudios de Seguimiento , Humanos , Hidroxiprostaglandina Deshidrogenasas/análisis , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Inflamación/diagnóstico , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/patología , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Recto/efectos de los fármacos , Recto/enzimología , Recto/inmunología , Recto/patología , Resultado del Tratamiento

RESUMEN

OBJECTIVE: To study the distribution of nitric oxide synthase (NOS) isoforms and protein levels in human haemorrhoids and rectal tissue. METHODS: Protein expression of NOS1, NOS2 and NOS3 was compared between haemorrhoids (n=14) and normal rectal submucosa (n=6) using Western blot analysis. The localisation of all NOS isoforms to specific structures was determined by immunohistochemistry. RESULTS: Western blot analysis showed median (interquartile range) protein levels of all NOS isoforms were 1.5-2.4 times higher in haemorrhoids than rectal tissue; 121.4 (55.2-165.5) vs 50.0 (25.5-73.7) for NOS1 (p=0.020), 32.2 (23.8-140.6) vs 14.8 (9.6-34.0) for NOS2 (p=0.109), and 80.1 (62.0-139.5) vs 54.3 (48.7 -61.7) for NOS3 (p=0.015). Immunohistochemistry revealed a different distribution and location of all NOS isoforms in vascular and non-vascular structure of haemorrhoids and rectal tissues. The number of haemorrhoid specimens showing positive immunoreactivity of NOS in the vascular endothelium was significantly higher than that in rectal tissue for NOS1 (11/14 (79%) vs 1/6 (17%); p=0.018) and NOS3 (8/14 (57%) vs 0/6 (0%); p=0.042), but not for NOS2 (6/14 (43%) vs 4/6 (67%); p=0.63). CONCLUSION: Haemorrhoids have significantly higher protein levels of NOS1 and NOS3 than rectal tissue. The vascular endothelium of haemorrhoids also has significantly higher positive immunoreactivity of NOS1 and NOS3 than rectal tissue suggesting that blood vessels in haemorrhoids are exposed to higher NO concentrations than those of rectal tissue. Since haemorrhoids exhibit marked vascular dilatation and present with bleeding or swelling, a reduction in NOS - by applying NOS inhibitors - may potentially improve the symptoms of haemorrhoids.

Asunto(s)

Hemorroides/enzimología , Óxido Nítrico Sintasa de Tipo III/análisis , Óxido Nítrico Sintasa de Tipo II/análisis , Óxido Nítrico Sintasa de Tipo I/análisis , Recto/irrigación sanguínea , Recto/enzimología , Anciano , Estudios de Casos y Controles , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Femenino , Hemorroides/patología , Humanos , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Proyectos Piloto , Recto/patología

RESUMEN

Cold-acclimated insects defend ion and water transport function during cold exposure. We hypothesized that this is achieved via enhanced active transport. The Malpighian tubules and rectum are likely targets for such transport modifications, and recent transcriptomic studies indicate shifts in Na+-K+ ATPase (NKA) and V-ATPase expression in these tissues following cold acclimation. Here we quantify the effect of cold acclimation (one week at 12°C) on active transport in the ionoregulatory organs of adult Gryllus pennsylvanicus field crickets. We compared primary urine production of warm- and cold-acclimated crickets in excised Malpighian tubules via Ramsay assay at a range of temperatures between 4 and 25°C. We then compared NKA and V-ATPase activities in Malpighian tubule and rectal homogenates from warm- and cold-acclimated crickets via NADH-linked photometric assays. Malpighian tubules of cold-acclimated crickets excreted fluid at lower rates at all temperatures compared to warm-acclimated crickets. This reduction in Malpighian tubule excretion rates may be attributed to increased NKA activity that we observed for cold-acclimated crickets, but V-ATPase activity was unchanged. Cold acclimation had no effect on rectal NKA activity at either 21°C or 6°C, and did not modify rectal V-ATPase activity. Our results suggest that an overall reduction, rather than enhancement of active transport in the Malpighian tubules allows crickets to maintain hemolymph water balance during cold exposure, and increased Malpighian tubule NKA activity may help to defend and/or re-establish ion homeostasis.

Asunto(s)

Aclimatación , Frío , Gryllidae/fisiología , Transporte Iónico , Animales , Túbulos de Malpighi/enzimología , Túbulos de Malpighi/metabolismo , Recto/enzimología , Recto/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Transcriptoma , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo

RESUMEN

OBJECTIVES: Drug transporters affect antiretroviral therapy (ART) tissue disposition, but quantitative measures of drug transporter protein expression across preclinical species are not available. Our objective was to use proteomics to obtain absolute transporter concentrations and assess agreement with corresponding gene and immunometric protein data. DESIGN: In order to make interspecies comparisons, two humanized mouse [hu-HSC-Rag (n = 41); bone marrow-liver-thymus (n = 13)] and one primate [rhesus macaque (nonhuman primate, n = 12)] models were dosed to steady state with combination ART. Ileum and rectum were collected at necropsy and snap frozen for analysis. METHODS: Tissues were analyzed for gene (quantitative PCR) and protein [liquid chromatography-mass spectrometry (LC-MS) proteomics and western blot] expression and localization (immunohistochemistry) of ART efflux and uptake transporters. Drug concentrations were measured by LC-MS/MS. Multivariable regression was used to determine the ability of transporter data to predict tissue ART penetration. RESULTS: Analytical methods did not agree, with different trends observed for gene and protein expression. For example, quantitative PCR analysis showed a two-fold increase in permeability glycoprotein expression in nonhuman primates versus mice; however, proteomics showed a 200-fold difference in the opposite direction. Proteomics results were supported by immunohistochemistry staining showing extensive efflux transporter localization on the luminal surface of these tissues. ART tissue concentration was variable between species, and multivariable regression showed poor predictive power of transporter data. CONCLUSION: Lack of agreement between analytical techniques suggests that resources should be focused on generating downstream measures of protein expression to predict drug exposure. Taken together, these data inform the use of preclinical models for studying ART distribution and the design of targeted therapies for HIV eradication.

Asunto(s)

Antirretrovirales/administración & dosificación , Antirretrovirales/farmacocinética , Íleon/enzimología , Proteínas de Transporte de Membrana/análisis , Recto/enzimología , Animales , Western Blotting , Cromatografía Liquida , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Macaca mulatta , Espectrometría de Masas , Ratones SCID , Proteoma/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

In the absence of an effective vaccine, strategies to prevent HIV transmission are urgently needed. Condomless receptive anal intercourse represents a major route of transmission, and efforts are being made to develop strategies, in which potent anti-HIV drugs are formulated for topical application to the rectum before sex. 5P12-RANTES is a promising candidate for such a purpose. It is an analog of the human chemokine RANTES/CCL5, which potently blocks CCR5, the principal coreceptor used by HIV to enter and infect target cells. As a protein, 5P12-RANTES is potentially vulnerable to attack by proteases in the rectal environment. In this study, we tested the stability of 5P12-RANTES on exposure to rectal lavage samples obtained from healthy volunteers, using a sensitive HIV entry inhibition assay as an indicator of stability. Varying levels of inactivating activity toward 5P12-RANTES were detected across the different lavage samples. Analysis of even the most aggressive samples indicated that protease activity in the rectal environment is unlikely to impact on the anti-HIV activity of 5P12-RANTES when applied pericoitally at the envisaged clinical dose (1 mM). This study indicates that 5P12-RANTES has adequate stability for further development as an HIV prevention drug for rectal use.

Asunto(s)

Antiinfecciosos/metabolismo , Quimiocinas CC/metabolismo , Recto/enzimología , Estabilidad de Medicamentos , Voluntarios Sanos , Humanos , Hidrólisis

RESUMEN

Mycophenolic acid (MPA) is an important immunosuppressant broadly used in renal transplantation. However, the large inter-patient variability in mycophenolic acid (MPA) pharmacokinetics (PK) limits its use. We hypothesize that extrahepatic metabolism of MPA may have significant impact on MPA PK variability. Two intestinal UDP-glucuronosyltransferases 1A8 and 1A10 plays critical role in MPA metabolism. Both in silico and previous genome-wide analyses suggested that vitamin D (VD) may regulate intestinal UGT1A expression. We validated the VD response elements (VDREs) across the UGT1A locus with chromatin immunoprecipitation (ChIP) and luciferase reporter assays. The impact of 1-alpha,25-dihydroxyvitamin D3 (D3) on UGT1A8 and UGT1A10 transcription and on MPA glucuronidation was tested in human intestinal cell lines LS180, Caco-2 and HCT-116. The correlation between transcription levels of VD receptor (VDR) and the two UGT genes were examined in human normal colorectal tissue samples (n = 73). PK alterations of MPA following the parent drug, mycophenolate mofetil (MMF), and D3 treatment was assessed among renal transplant recipients (n = 10). Our ChIP assay validate three VDREs which were further demonstrated as transcriptional enhancers with the luciferase assays. D3 treatment significantly increased transcription of both UGT genes as well as MPA glucuronidation in cells. The VDR mRNA level was highly correlated with that of both UGT1A8 and UGT1A10 in human colorectal tissue. D3 treatment in patients led to about 40% reduction in both AUC0-12 and Cmax while over 70% elevation of total clearance of MPA. Our study suggested a significant regulatory role of VD on MPA metabolism and PK via modulating extrahepatic UGT activity.

Asunto(s)

Glucuronosiltransferasa/genética , Trasplante de Riñón , Hígado/metabolismo , Ácido Micofenólico/farmacocinética , Vitamina D/análogos & derivados , Células CACO-2 , Inmunoprecipitación de Cromatina , Clonación Molecular , Colon/efectos de los fármacos , Colon/enzimología , Regulación de la Expresión Génica/efectos de los fármacos , Glucurónidos/metabolismo , Glucuronosiltransferasa/metabolismo , Células HCT116 , Humanos , Luciferasas/metabolismo , Ácido Micofenólico/farmacología , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Recto/efectos de los fármacos , Recto/enzimología , Reproducibilidad de los Resultados , Elementos de Respuesta/genética , Transcripción Genética , Vitamina D/farmacología

RESUMEN

BACKGROUND: 5-aminosalicylic acid (5-ASA) is a classic anti-inflammatory drug for the treatment of ulcerative colitis. N-acetyltransferase (NAT) enzymes convert 5-ASA to its metabolite N-acetyl-5-ASA, and it is unresolved whether 5-ASA or N-acetyl-5-ASA is the effective therapeutic molecule. We previously demonstrated that colonic production of N-acetyl-5-ASA (NAT activity) is decreased in dextran sulfate sodium-induced colitis. Our hypothesis is that 5-ASA is the therapeutic molecule to improve colitis, with the corollary that altered NAT activity affects drug efficacy. Since varying clinical effectiveness of 5-ASA has been reported, we also ask if NAT activity varies with inflammation in pediatric or adult patients. METHODS: Acute colonic inflammation was induced in C57BL/6 NAT wild-type (WT) or knockout mice, using 3.5% dextran sulfate sodium (w/v) concurrent with 5-ASA treatment. Adult and pediatric rectosigmoid biopsies were collected from control or patients with ulcerative colitis. Tissue was analyzed for NAT and myeloperoxidase activity. RESULTS: Dextran sulfate sodium-induced colitis was of similar severity in both NAT WT and knockout mice, and NAT activity was significantly decreased in NAT WT mice. In the setting of colitis, 5-ASA significantly restored colon length and decreased myeloperoxidase activity in NAT knockout but not in WT mice. Myeloperoxidase activity negatively correlated with NAT activity in pediatric patients, but correlation was not observed in adult patients. CONCLUSIONS: Inflammation decreases NAT activity in the colon of mice and human pediatric patients. Decreased NAT activity enhances the therapeutic effect of 5-ASA in mice. A NAT activity assay could be useful to help predict the efficacy of 5-ASA therapy.

Asunto(s)

Antiinflamatorios no Esteroideos/uso terapéutico , Arilamina N-Acetiltransferasa/metabolismo , Colitis Ulcerosa/enzimología , Isoenzimas/metabolismo , Mesalamina/uso terapéutico , Peroxidasa/metabolismo , Adolescente , Adulto , Anciano , Animales , Antiinflamatorios no Esteroideos/metabolismo , Arilamina N-Acetiltransferasa/genética , Biopsia , Estudios de Casos y Controles , Niño , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/patología , Colon Sigmoide/enzimología , Colon Sigmoide/patología , Sulfato de Dextran , Femenino , Humanos , Isoenzimas/genética , Masculino , Mesalamina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Peroxidasa/efectos de los fármacos , Recto/enzimología , Recto/patología , Adulto Joven

RESUMEN

In humans, the liver is generally considered to be the major organ contributing to drug metabolism, but studies during the last years have suggested an important role of the extra-hepatic drug metabolism. The gastrointestinal tract (GI-tract) is the major path of entry for a wide variety of compounds including food, and orally administered drugs, but also compounds - with neither nutrient nor other functional value - such as carcinogens. These compounds are metabolized by a large number of enzymes, including the cytochrome P450 (CYP), the glutathione S-transferase (GST) family, the uridine 5'-diphospho- glucuronosyltransferase (UDP-glucuronosyltransferase - UGT) superfamily, alcohol-metabolizing enzymes, sulfotransferases, etc. These enzymes can either inactivate carcinogens or, in some cases, generate reactive species with higher reactivity compared to the original compound. Most data in this field of research originate from animal or in vitro studies, wherein human studies are limited. Here, we review the human studies, in particular the studies on the phenotypic expression of these enzymes in the colon and rectum to get an impression of the actual enzyme levels in this primary organ of exposure. The aim of this review is to give a summary of currently available data on the relation between the CYP, the GST and the UGT biotransformation system and colorectal cancer obtained from clinical and epidemiological studies in humans.

Asunto(s)

Colon/metabolismo , Neoplasias Colorrectales/metabolismo , Recto/metabolismo , Xenobióticos/farmacocinética , Arilamina N-Acetiltransferasa/genética , Arilamina N-Acetiltransferasa/metabolismo , Biotransformación , Colon/enzimología , Neoplasias Colorrectales/enzimología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Recto/enzimología , Sulfotransferasas/genética , Sulfotransferasas/metabolismo

RESUMEN

OBJECTIVES: The aim of this study was to analyze cell kinetics through expression and apoptosis of topoisomerase 2-α (TOP2A), p53, and c-erb2 in rectosigmoid endometriotic lesions and in healthy endometrial tissue and to establish correlations between such findings and clinical data in patients with rectosigmoid endometriosis. METHODS: Sixty patients with rectosigmoid endometriosis and 20 control women without endometriosis were included. Immunohistochemical assays were used to measure expression of TOP2A, p53, and c-erB-2. Apoptosis was quantified by directly counting the apoptotic bodies. FINDINGS: The number of lesions was positively correlated with expression of TOP2A in the lesion. There was also significant correlation between the lesions' size and number and cell turnover index. Apoptosis index (AI) was the same for endometriosis lesions and eutopic endometrium. Expression of TOP2A was significantly lower in the endometriosis group compared to the controls. CONCLUSIONS: Changes in cell proliferation but not in the AI in rectosigmoid endometriosis are indicative of an imbalance in cell kinetics that may lead to the development of the disease.

Asunto(s)

Antígenos de Neoplasias/análisis , Apoptosis , Proliferación Celular , Colon Sigmoide/patología , ADN-Topoisomerasas de Tipo II/análisis , Proteínas de Unión al ADN/análisis , Endometriosis/patología , Endometrio/patología , Recto/patología , Adulto , Estudios de Casos y Controles , Colon Sigmoide/enzimología , Estudios Transversales , Endometriosis/enzimología , Endometrio/enzimología , Femenino , Humanos , Inmunohistoquímica , Cinética , Persona de Mediana Edad , Proteínas de Unión a Poli-ADP-Ribosa , Estudios Prospectivos , Receptor ErbB-2/análisis , Recto/enzimología , Proteína p53 Supresora de Tumor/análisis

RESUMEN

Incidence of colorectal cancer (CRC) is growing worldwide. Pathogenetic mechanisms responsible for its onset and progression need further clarification. Colorectal adenomatous polyps are precancerous lesions with malignant potential dependent on histological architecture and grade of nuclear dysplasia. One of the factors conditioning CRC development are abnormalities in sphingolipid metabolism. The aim of this study was to assess the levels of sphingolipids in human colorectal adenomas. The control group (C, n = 12) consisted of patients with no colonic polyps. The examined group consisted of patients with prior diagnosed colonic polyps, qualified to endoscopic polypectomy. This group was further divided due to histological architecture into tubular adenomas group (TA, n = 10), tubulovillous adenomas with low-grade dysplasia (LGD-TVA, n = 10), and tubulovillous adenomas group with high-grade dysplasia (HGD-TVA, n = 11). In tissue samples, sphingolipd metabolite contents were measured using high performance liquid chromatography (HPLC). In cases of polypoid lesions with low malignancy potential (tubular adenomas), concentration of ceramide, which is characterized by proapoptotic and anti-proliferative properties, increases compared with control group (p < 0.05), whereas content of sphingosine-1-phosphate with anti-apoptotic and stimulating cellular proliferation properties is reduced in comparison with control group (p < 0.05). On the contrary, in cases of more advanced form of adenomatous polyps (tubulovillous adenomas with high-grade dysplasia), the ceramide level decreases compared with control group (p < 0.05) while sphingosine-1-phosphate concentration is elevated (p < 0.05). We found that concentrations of pro-apoptotic ceramide are decreased and pro-proliferative S1P levels are increased in polypoid lesions with high malignancy potential, and it was the opposite in those with low malignancy potential.

Asunto(s)

Adenoma/metabolismo , Colon/patología , Pólipos del Colon/metabolismo , Neoplasias Colorrectales/metabolismo , Recto/patología , Esfingolípidos/metabolismo , Adenoma/sangre , Adenoma/enzimología , Adenoma/patología , Adulto , Anciano , Colon/enzimología , Colon/metabolismo , Pólipos del Colon/sangre , Pólipos del Colon/enzimología , Pólipos del Colon/patología , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Femenino , Humanos , Lisofosfolípidos/análisis , Lisofosfolípidos/metabolismo , Masculino , Persona de Mediana Edad , Recto/enzimología , Recto/metabolismo , Esfingolípidos/análisis , Esfingosina/análogos & derivados , Esfingosina/análisis , Esfingosina/metabolismo

RESUMEN

Locally advanced rectal cancer (LARC) comprises heterogeneous tumors with predominant hypoxic components, a hallmark of the tumor microenvironment and determinant of resistance to cytotoxic therapies, local recurrence, and metastatic progression. A rational integration of molecularly targeted agents in established combined-modality treatment regimens may improve local and systemic disease control, but will require a clear definition of functional biomarkers for patient stratification. In a prospective study of LARC patients given neoadjuvant chemotherapy and radiation, we applied a kinase substrate array technology to analyze the patients' tumor biopsies sampled at the time of diagnosis, and observed that receptor tyrosine kinase activities integrated by high phosphatidylinositol 3-kinase signaling were correlated both with poor tumor response to the neoadjuvant treatment and adverse progression-free survival. Conceptually, the specific tumor signature of phosphatidylinositol 3-kinase signaling activity may point to actionable therapy targets in LARC patients with unfavorable disease features. Clinical trial registration number NCT00278694.

Asunto(s)

Terapia Neoadyuvante , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias del Recto/patología , Neoplasias del Recto/terapia , Recto/patología , Transducción de Señal , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Terapia Neoadyuvante/métodos , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/terapia , Neoplasias del Recto/enzimología , Neoplasias del Recto/radioterapia , Recto/enzimología , Recto/efectos de la radiación , Análisis de Supervivencia , Resultado del Tratamiento

RESUMEN

Phospholipid scramblase 1 involve in biological processes including phospholipid movement, proliferation, and apoptosis. Treatment with an antiphospholipid scramblase 1 antibody (NP1) has been demonstrated to inhibit cell proliferation in colorectal cancer. This study aimed to explore the role of NP1 treatment in the apoptosis of colorectal cancer cells. Results showed that NP1 treatment significantly increases the apoptosis of colorectal cancer cells via the activation of caspase 8, caspase 9, and caspase 3. Moreover, pretreatment with a caspase 8 inhibitor did not fully prevent the apoptotic effects of NP1. Taken together, these data indicate NP1 induces cell apoptosis primary through the intrinsic apoptotic pathway. NP1 may serve as a potential therapeutic agent.

Asunto(s)

Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/enzimología , Terapia Molecular Dirigida , Proteínas de Transferencia de Fosfolípidos/metabolismo , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Colon/efectos de los fármacos , Colon/enzimología , Colon/patología , Neoplasias Colorrectales/patología , Humanos , Proteínas de Transferencia de Fosfolípidos/química , Estructura Terciaria de Proteína/efectos de los fármacos , Recto/efectos de los fármacos , Recto/enzimología , Recto/patología

RESUMEN

Irritable bowel syndrome (IBS) is characterized by altered bowel habits, persistent pain and discomfort, and typically colorectal hypersensitivity. Linaclotide, a peripherally restricted 14 aa peptide approved for the treatment of IBS with constipation, relieves constipation and reduces IBS-associated pain in these patients presumably by activation of guanylate cyclase-C (GC-C), which stimulates production and release of cyclic guanosine monophosphate (cGMP) from intestinal epithelial cells. We investigated whether activation of GC-C by the endogenous agonist uroguanylin or the primary downstream effector of that activation, cGMP, directly modulates responses and sensitization of mechanosensitive colorectal primary afferents. The distal 2 cm of mouse colorectum with attached pelvic nerve was harvested and pinned flat mucosal side up for in vitro single-fiber recordings, and the encoding properties of mechanosensitive afferents (serosal, mucosal, muscular, and muscular-mucosal; M/M) to probing and circumferential stretch studied. Both cGMP (10-300 µM) and uroguanylin (1-1000 nM) applied directly to colorectal receptive endings significantly reduced responses of muscular and M/M afferents to stretch; serosal and mucosal afferents were not affected. Sensitized responses (i.e., increased responses to stretch) of muscular and M/M afferents were reversed by cGMP, returning responses to stretch to control. Blocking the transport of cGMP from colorectal epithelia by probenecid, a mechanism validated by studies in cultured intestinal T84 cells, abolished the inhibitory effect of uroguanylin on M/M afferents. These results suggest that GC-C agonists like linaclotide alleviate colorectal pain and hypersensitivity by dampening stretch-sensitive afferent mechanosensitivity and normalizing afferent sensitization.

Asunto(s)

Colon/enzimología , Guanilato Ciclasa/metabolismo , Mecanorreceptores/enzimología , Recto/enzimología , Vías Aferentes/enzimología , Animales , Línea Celular Tumoral , Colon/inervación , Activación Enzimática/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Recto/inervación

RESUMEN

BACKGROUND AND AIMS: 15-Hydroxprostaglandin dehydrogenase (15-PGDH) mediates a colon neoplasia suppressor pathway, acting through metabolic antagonism of cyclooxygenase-mediated colon carcinogenesis. To determine whether the colon tumor prevention activity of 15-PGDH acts as a constant or variable effect among individuals, we determined whether 15-PGDH levels remain stable over subsite and time in the human colon, determined the extent of differences in 15-PGDH levels between different individuals, and determined whether 15-PGDH modulation mediates any part of the anti-colon tumor effect of aspirin. METHODS: Using real-time PCR, we measured 15-PGDH mRNA to determine the correlation of 15-PGDH level in replicate colon biopsies, in biopsies from throughout the length of the colon, in repeat biopsies taken 4 months apart, and in paired biopsies of individuals taken before and after aspirin treatment, and by Western-blot for 15-PGDH protein in mice. RESULTS: Colonic 15-PGDH levels varied 4.4-fold across the human population. Within individuals, 15-PGDH levels proved highly reproducible (r=0.81 in duplicate biopsies) and stable along the length of the colon, with average 15-PGDH levels deviating by only 17% from rectum to cecum. An individual's 15-PGDH levels are also highly stable over time, with a median coefficient of variation over a 4-month interval of only 12%. Last, colonic 15-PGDH levels proved resistant to alteration by aspirin, with only a 10% difference in 15-PGDH levels measured before and after aspirin treatment. CONCLUSIONS: 15-PGDH levels vary across the population in a stable and reproducible manner, and are resistant to alteration by aspirin. 15-PGDH represents an independent target for modulation by candidate colon tumor chemopreventive agents.

Asunto(s)

Aspirina/uso terapéutico , Colon/enzimología , Neoplasias del Colon/prevención & control , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Animales , Aspirina/farmacología , Quimioprevención , Colon/efectos de los fármacos , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Femenino , Humanos , Masculino , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Recto/enzimología

RESUMEN

Phenolic compounds are naturally occurring, bioactive substances with marked antioxidant and anti-inflammatory potential. The flavonoid chrysin, found in high levels in honey bee propolis, inhibits the activity of enzymes involved in carcinogenesis. We have investigated the effect of chrysin on pre-neoplastic colorectal lesions (ACF, aberrant crypt foci) in a rat model of chemical carcinogenesis induced by 1,2-dimethylhydrazine (DMH). Female Wistar rats weighing 137.2 ± 24.3 g received weekly one subcutaneous injection of DMH (20 mg/kg) for 10 weeks. The animals were divided into five groups each with seven animals: Group 1, 0.9% saline; Group 2, DMH+0.9% saline; Group 3, DMH+chrysin (10 mg/kg); Group 4, DMH+chrysin (100 mg/kg); Group 5, DMH+chrysin (200 mg/kg). Groups 2 and 3 showed a significant increase in ACF number, nucleolus organizer regions per enterocyte nucleus and nitrite/nitrate serum levels compared with Group 1. Groups 4 and 5 presented a significant reduction in all these parameters compared with Group 2. The levels of antioxidant minerals (copper, magnesium, selenium, zinc) and the number of enteroendocrine and mucin-producing cells were significantly reduced in Groups 2 and 3 but were similar in Groups 4 and 5 compared with Group 1. Chrysin, at 100 mg/kg and 200 mg/kg, was effective in attenuating pathological colorectal remodeling, reducing the number of pre-neoplastic lesions in rats exposed to DMH. Some of these effects might be attributable to the recovery of antioxidant mineral levels, a reduction in systemic nitrosative stress and an inhibition of the cellular proliferation induced by this flavonoid.

Asunto(s)

Colon/efectos de los fármacos , Neoplasias Colorrectales/prevención & control , Flavonoides/farmacología , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/tratamiento farmacológico , Recto/efectos de los fármacos , 1,2-Dimetilhidrazina , Animales , Carcinógenos , Colon/enzimología , Colon/patología , Neoplasias Colorrectales/inducido químicamente , Femenino , Lesiones Precancerosas/patología , Ratas , Ratas Wistar , Recto/enzimología , Recto/patología

RESUMEN

The role of PKC and RhoA/ROCK pathways in the phasic activities in the rectal smooth muscles (RSM) in the basal state is not known. We examined this issue by determining the effects of PKC inhibitors (calphostin C and Gö-6850) and a ROCK inhibitor (Y-27632) on the slow-rate (~3/min) and fast-rate (~25/min) phasic activities. We also examined the corresponding signal transduction cascades and the PKC and ROCK enzymatic activities in the RSM in the basal state. PKC inhibition with calphostin C and Gö-6850 (10(-5) M) caused a significant decrease (~25%) in slow-rate (but not fast-rate) phasic activity (monitored by frequency and amplitude of contractions) of the RSM. Conversely, ROCK inhibition with Y-27632 (10(-5) M) caused a significant decrease not only in slow-rate, but also fast-rate, phasic activity caused by ROCK inhibition in the RSM. Western blot analysis revealed that the PKC inhibition-induced decrease in RSM phasic activity was associated with decreases in PKCα translocation, phosphorylated (Thr(38)) PKC-potentiated inhibitor (CPI-17), and phosphorylated (Thr(18)/Ser(19)) 20-kDa myosin regulatory light chain. Conversely, decreases in the phasic activity in the RSM by ROCK inhibition were accompanied by the additional decrease in phosphorylated (Thr(696)) myosin phosphatase target subunit 1. Data show that while PKC and RhoA/ROCK pathways play a significant role in slow-rate high-amplitude spontaneous phasic activity, only the RhoA/ROCK pathway primarily mediates fast-rate low-amplitude phasic activity, in the RSM. Such knowledge is important in the understanding of the pathophysiology of large intestinal motility disorders. Relative contributions of the PKC vs. the RhoA/ROCK pathway in the phasic activity remain to be determined.

Asunto(s)

Incontinencia Fecal/metabolismo , Músculo Liso/enzimología , Proteína Quinasa C/metabolismo , Recto/enzimología , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Amidas/farmacología , Animales , Carcinógenos/farmacología , Inhibidores Enzimáticos/farmacología , Incontinencia Fecal/fisiopatología , Indoles/farmacología , Maleimidas/farmacología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/fisiología , Naftalenos/farmacología , Forbol 12,13-Dibutirato/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteína Quinasa C/antagonistas & inhibidores , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Recto/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Quinasas Asociadas a rho/antagonistas & inhibidores

RESUMEN

BACKGROUND: During normal development in human and other placental mammals, the embryonic cloacal cavity separates along the axial longitudinal plane to give rise to the urethral system, ventrally, and the rectum, dorsally. Defects in cloacal development are very common and present clinically as a rectourethral fistula in about 1 in 5,000 live human births. Yet, the cellular mechanisms of cloacal septation remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We previously detected Bone morphogenetic protein 7 (Bmp7) expression in the urorectal mesenchyme (URM), and have shown that loss of Bmp7 function results in the arrest of cloacal septation. Here, we present evidence that cloacal partitioning is driven by Bmp7 signaling in the cloacal endoderm. We performed TUNEL and immunofluorescent analysis on cloacal sections from Bmp7 null and control littermate embryos. We found that loss of Bmp7 results in a dramatic decrease in the endoderm survival and a delay in differentiation. We used immunological methods to show that Bmp7 functions by activating the c-Jun N-terminal kinase (JNK) pathway. We carried out confocal and 3D imaging analysis of mitotic chromosome bundles to show that during normal septation cells in the cloacal endoderm divide predominantly in the apical-basal direction. Loss of Bmp7/JNK signaling results in randomization of mitotic angles in the cloacal endoderm. We also conducted immunohistochemical analysis of human fetal sections to show that BMP/phospho-SMAD and JNK pathways function in the human cloacal region similar as in the mouse. CONCLUSION/SIGNIFICANCE: Our results strongly indicate that Bmp7/JNK signaling regulates remodeling of the cloacal endoderm resulting in a topological separation of the urinary and digestive systems. Our study points to the importance of Bmp and JNK signaling in cloacal development and rectourethral malformations.

Asunto(s)

Tipificación del Cuerpo , Proteína Morfogenética Ósea 7/metabolismo , Polaridad Celular , Cloaca/citología , Cloaca/embriología , Animales , Proteína Morfogenética Ósea 7/deficiencia , Diferenciación Celular , División Celular , Proliferación Celular , Supervivencia Celular , Cloaca/enzimología , Endodermo/citología , Epitelio/embriología , Feto/embriología , Feto/enzimología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Modelos Biológicos , Recto/embriología , Recto/enzimología , Proteínas Smad/metabolismo , Uretra/embriología , Uretra/enzimología

RESUMEN

RAS proteins act as molecular switches between several homeostatic inputs and signal transduction pathways that regulate important cellular processes including cell growth, differentiation and survival. Activating mutations change the function of normal proto-oncogenic RAS proteins to oncogenic RAS proteins that trigger a wide range of downstream effectors altering expression of transcription factors that together stimulate cell proliferation and modulate apoptosis and differentiation. RAS genes are amongst the most frequently mutated genes in human cancers, in particular KRAS is mutated in 40-50% of colorectal cancers. Mutation of this gene has a significant impact on treatment management and patients' survival, particularly in relation to anti-EGFR therapy, which is only effective in KRAS wild-type cases. Here, we discuss the regulation of KRAS signalling in the colorectum, some of the post-transcriptional and post-translational modifications that control its activity, the mutations and other DNA alterations that are found in this tumour type and the implications that they have for disease progression and current drug treatments.

Asunto(s)

Colon , Neoplasias Colorrectales/enzimología , Recto , Transducción de Señal , Proteínas ras/metabolismo , Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Colon/enzimología , Colon/patología , Colon/fisiología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Humanos , MicroARNs/metabolismo , Mutación , Procesamiento Proteico-Postraduccional , Recto/enzimología , Recto/patología , Recto/fisiología , Proteínas ras/genética , Proteínas ras/inmunología