Alteraciones en la proliferación celular y muerte celular programada tipo apoptosis en muestras de carcinoma de células escamosas de mucosa oral

56 p.El carcinoma de células escamosas de la cavidad oral (CCECO) representa el 90% de las neoplasias malignas bucales. La carcinogénesis oral se ha vinculado a daños genéticos no letales, especialmente en secuencias de genes supresores de tumores y oncogenes. Estas mutaciones y otros mecanismos permiten a las células proliferar a un ritmo que supera la muerte celular, especialmente la de tipo apoptosis. Este desbalance se debe tanto al aumento en la proliferación como a una disminución de la muerte celular. Así, la desregulación de la muerte celular tipo apoptosis también juega un rol fundamental en el proceso de carcinogénesis.El objetivo del presente estudio fue analizar la posible desregulación de proliferación tumoral (determinada mediante la expresión de marcadores de proliferación tumoral) y muerte celular tipo apoptosis en el CCECO. Se estudiaron 5 muestras correspondientes a biopsias incisionales de CCECO obtenidas de la Unidad de Diagnostico Oral e Histopatología de las Clínicas Odontológicas de la Facultad de Ciencias de la Salud, Universidad de Talca. Éstas fueron sometidas a análisis inmunohistoquímico para el antígeno de proliferación celular nuclear (PCNA) y el gen supresor de tumores p53. Las muestras fueron analizadas adicionalmente con el método histoquímico de detección de Regiones Organizadoras de Nucléolos (Técnica AgNOR). La posible muerte celular tipo apoptosis fue determinada mediante el método de TUNEL (TdT-mediated dUTP Nick-End Labeling). Los resultados de nuestro estudio confirman que el CCECO presenta una desregulación de procesos celulares básicos, específicamente: 1) Aumento de la proliferación celular 2) Presencia de genes supresores de tumores mutados (p53) 3) Alteración en el patrón de maduración epitelial, manifestado por una alteración en la expresión normal de células TUNEL+ en el epitelio./ABSTRACT:Oral squamous cell carcinoma (OSCC) represents the 90% of the oral malignant neoplasms. The oral carcinogenesis has been related to non-lethal genetic damage, especially in tumor-supressor genes and oncogenes. These mutations and other deregulated mechanisms results in an increased cellular proliferation rate that overtake the cell death rate, the latter one is additionally decreased. Therefore, cell death deregulation, specially apoptotic cell death, plays a fundamental role in the oral carcinogenesis.The principal aim of the present study was to analyze the possible deregulation of cellular proliferation (determined by proliferation markers) and the apoptotic cell death in OSCC. We analyzed five samples of incisional biobsies of OSCC from the “Unidad de Diagnostico Oral e Histopatología de las Clínicas Odontológicas de la Facultad de Ciencias de la Salud, Universidad de Talca”. Immunohistochemistry for the proliferating celular nuclear antigen (PCNA) and the p53 tumor suppressor gene was performed. Additionally, nucleolar organizer regions (AgNORs) were analyzed histochemically and DNA fragmentation was determined by the TUNEL (TdT-mediated dUTP Nick- End Labeling) method.Our results confirm that OSCC present deregulation of basic cellular processes, specifically: 1) Increase in cell proliferation 2) Mutation of tumor suppressor genes 3) Alteration in normal epithelial differentiation, evidenced by altered expression of TUNEL+ cell

Determinación del efecto quimiopreventivo de quercetina en un modelo de carcinogénesis oral en ratones CF-1 expuestos a 4-nitroquinolina 1- oxido

104 p.El cáncer es un problema de salud pública a nivel mundial, afectando por igual a países desarrollados, como a los en vías en desarrollo. El cáncer orofaríngeo es una de las neoplasias más prevalentes (5,7%), afectando anualmente aproximadamente a 400.000 personas. En particular el carcinoma de células escamosas de la cavidad oral (CCECO) representa el 80% de estas neoplasias. El tratamiento de este tipo de cáncer es sumamente costoso y radical, lo que conlleva graves secuelas para el paciente durante toda su vida. La detección temprana de esta neoplasia es la mejor alternativa para impedir estas consecuencias.Quercetina es uno de los flavonoides más comunes y ampliamente distribuido en frutas y hortalizas. Es un potente antioxidante, que podría inducir respuestas celulares que contribuyan a disminuir la severidad de las neoplasias, convirtiéndolo en un candidato a ser un agente quimiopreventivo. Sin embargo, en la literatura se han reportado que la quercetina podría tener efectos oxidantes in vitro y que las dosis necesarias para obtener efectos antioxidantes in vivo son difícilmente aplicables.El objetivo de este estudio fue determinar el posible efecto quimiopreventivo de quercetina en CCECO inducido por 4-NQO. Para esto se utilizó un modelo de carcinogénesis químico murino determinando el efecto de quercetina sobre las lesiones preneoplásicas y neoplásicas, nivel de lipoperoxidación y glutatión reducido en sangre de ratones tratados. Además, se evaluó el potencial efecto tóxico del compuesto en cultivos celulares de fibroblastos pulmonares (MCR-5).Quercetina a bajas concentraciones previene la disminución de la viabilidad celular causada por 4-NQO, pero no mejora la sobrevida, ni la incidencia, ni la severidad de las lesiones preneoplásicas y de CCECO de ratones tratados con 4-NQO. Además mantiene bajos los niveles de GSH sanguíneo en ratones expuestos a 4-NQO y quercetina. Se concluye que Quercetina no presenta un efecto quimiopreventivo sobre las lesiones en la mucosa lingual de ratones sometidos a carcinogénesis experimental inducida por 4-NQO./ ABSTRACT: Cancer is a worldwide public health problem, affecting both developed and developing countries. Oropharyngeal cancer is one of the most prevalent carcinomas (5.7%), affecting approximately 400000 individuals annually. Squamous cell carcinoma of the oral cavity (SCCOC) represents 80% of these tumors. Treatment of this type of cancer is extremely expensive and radical, and has serious consequences for the patient throughout their lives. Early detection of this neoplasm is the best alternative to prevent these consequences. Quercetin is one of the most common and widely distributed flavonoids in fruits and vegetables. It is a powerful antioxidant which could induce cellular responses that might contribute to reduce the severity of tumors, making it a candidate as a chemopreventive agent. However, the scientific reports indicates that Quercetin may have oxidant effects in vitro and that the doses required for in vivo antioxidant effects are hardly applicable.The objetive of this study was to determine the potential chemopreventive effect of Quercetin over 4-NQO induced SCCOC in mice. We determined the effect of Quercetin on preneoplastic and neoplastic lesions, lipoperoxidation levels and reduced glutathione levels in the blood of treated animals. Additionally, the potential toxic effects on lung fibroblast cell cultures (MCR-5) was assessed. Quercetin at low concentrations prevent the decrease of cell viability caused by 4-NQO, but does not improve survival, nor the incidence or severity of preneoplastic lesions and SCCOC in mice treated with 4-NQO. Quercetin also maintains low blood GSH levels in CF-1 mice exposed to 4-NQO and quercetin. We conclude, that Quercetin has no chemopreventive effect on lesions in the lingual mucosa in mice subjected to experimental carcinogenesis with 4-NQO

Expresion de cambios histopatologicos reaccionales, pre-neoplasicos y/o neoplasicos en el epitelio de la mucosa lingual de ratones cf-1 expuestos a 4-nitroquinolina-1-oxido.

77 p.INTRODUCCIÓN: El cáncer oral es una enfermedad que afecta a 400.000 personas cada año. El CCECO representa aproximadamente el 90% de los casos. El desarrollo de modelos de carcinogénesis experimental en animales juega un papel fundamental para su estudio y el desarrollo de terapias. OBJETIVO: Inducir cambios histopatológicos reaccionales, pre-neoplásicos y/o neoplásicos en el epitelio de la mucosa lingual de ratones CF-1 al exponerlos a 4- nitroquinolina-1-oxido (4-NQO). MATERIALES Y MÉTODOS: Se aplicó una solución de 4NQO más propilenglicol diluido en el agua de bebida a 20 ratones, a una concentración de 100 μg/ml y se mantuvo un grupo control de igual tamaño solo con agua y propilenglicol. Esta sustancia se aplicó por un periodo de 16 semanas, después del cual se suspendió, esperando hasta las 28 semanas para realizar la biopsia de la lengua. Se analizó la presentación de lesiones clínicas (leucoplasia, eritroplasia y lesiones mixtas). Se procesó la totalidad de la lengua para estudio histopatológico de hematoxilina-eosina convencional, buscando la presencia de focos de lesiones reaccionales, pre-neoplásicos y neoplásicos. RESULTADOS: Se encontró un total de 58 lesiones macroscópicamente detectables, localizadas principalmente en la zona dorsal-medial de la lengua, de apariencia leucoplásica, de tamaños < a 1 mm hasta 3 mm, y de forma sésil. En el estudio histopatológico, las lesiones de mayor incidencia, en orden decreciente, fueron las hiperqueratosis, la hiperplasia, el carcinoma invasor y la displasia severa o CA in situ. CONCLUSIÓN: El 4-nitroquinolina 1-óxido es capaz de inducir cambios histopatológicos reaccionales, pre-neoplásicos y neoplásicos en el epitelio de la mucosa lingual de ratones CF-1 en forma similar a otros modelos animales ya sea de otras especies o cepa

Infection and invasion mechanisms of Trypanosoma cruzi in the congenital transmission of chagas' disease: A proposal

Chagas' disease is produced by the haemophlagelated protozoan Trypanosoma cruzi and transmitted by haematophages insects such as Triatoma infestans (vinchuca). Due to vector control, congenital transmission gains importance and is responsible for the presence and expansion of this disease in non-endemic areas. The mechanisms of congenital infection are uncertain. It has been suggested that the parasite reaches the fetus through the bloodstream by crossing the placental barrier, and that congenital Chagas' disease is the result of complex interactions between the immune response, placental factors, and the parasite's characteristics. We review the cellular and molecular mechanisms of infection and invasion of the parasite and how immune and placental factors may modulate this process. Finally, we propose a possible model for the vertical transmission of Chagaś disease

Curcumin rescues breast cells from epithelial‑mesenchymal transition and invasion induced by anti‑miR‑34a

Breast cancer is the most prevalent type of cancer among women worldwide and it is characterized by a high morbidity. Curcumin is a naturally occurring compound derived from the rhizome of Curcuma longa and is known to have antioxidant and anticarcinogenic properties. Emerging evidence has indicated that microRNAs (miRNAs or miRs) function as oncogenes or tumor suppressor genes to control invasion and migration. The aim of this study was to evaluate the effects of curcumin on genes implicated in epithelial-mesenchymal transition (EMT) and to examine the involvement of Rho-A in the migration and invasion of MCF-10F and MDA-MB-231 breast cell lines. Furthermore, to the best of our knowledge, this is the first study to examine the effects of curcumin on Rho-A and on genes involved in EMT, such as Axl, Slug and CD24 in order to determine whether the compound is able to prevent migration and invasion by targeting miRNAs as a regulator of such genes. Specifically, we focused on miR-34a which acts as a tumor suppressor gene in human breast cell lines. The present study demonstrated that the Axl, Slug and CD24 genes were implicated in EMT, and Rho-A was also involved in the migration and invasion of MCF-10F and MDA-MB-231 cell lines. Curcumin also acted upon the miRNA as a regulator of genes implicated in EMT and upon Rho-A as well, affecting the migration and invasion of the cells. This occurred independently of their estrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (HER2) receptors in the non-malignant MCF-10F and malignant MDA-MB-231 breast cell lines, which are both negative for such receptors.Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) CONICYT FONDECYT 1120006 1161219 3190744 UTA-MINEDUC UTA1117 Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) CONICYT FONDAP 1513001

Chagas disease: Present status of pathogenic mechanisms and chemotherapy

There are approximately 7.8 million people in Latin America, including Chile, who suffer from Chagas disease and another 28 million who are at risk of contracting it. Chagas is caused by the flagellate protozoan Trypanosoma cruzi. It is a chronic disease, where 20%-30% of infected individuals develop severe cardiopathy, with heart failure and potentially fatal arrhythmias. Currently, Chagas disease treatment is more effective in the acute phase, but does not always produce complete parasite eradication during indeterminate and chronic phases. At present, only nifurtimox or benznidazole have been proven to be superior to new drugs being tested. Therefore, it is necessary to find alternative approaches to treatment of chronic Chagas. The current treatment may be rendered more effective by increasing the activity of anti-Chagasic drugs or by modifying the host's immune response. We have previously shown that glutathione synthesis inhibition increases nifurtimox and benznidazole activity

Early detection in saliva of polypeptides associated to isoproterenol-induced mouse parotid hypertrophy

Chronic administration of isoproterenol (IPR) results in a marked hypertrophy and in the induction of a group of putative proline-rich polypeptides in the mouse parotid glands. Some of these polypeptides (pps C-G) have been considered as molecular markers of the parotid gland enlargement. Given the secretory character of polypeptides C-G, the polypeptide composition of mouse saliva was used to monitor the IPR-induced salivary gland hypertrophy. Whole saliva was collected after an oral administration of pilocarpine (PIL). Under those conditions, PIL provoked a massive salivary secretion both in normal control mice and during the whole course of the IPR-induced gland enlargement. Striking changes in the polypeptide composition of saliva obtained from chronically IPR-stimulated animals were observed. Those changes consisted basically in the appearance and progressive increase in concentration of parotid polypeptides C-G and in the progressive diminution in concentration of a couple of no

Role of placental barrier integrity in infection by Trypanosoma cruzi

American trypanosomiasis has long been a neglected disease endemic in LatinAmerica, but congenital transmission has now spread Chagas disease to cause a global health problem. As the early stages of the infection of placental tissue and the vertical transmission by Trypanosoma cruzi are still not well understood, it is important to investigate the relevance of the first structure of the placental barrier in chorionic villi infection by T. cruzi during the initial stage of the infection. Explants of human chorionic villi from healthy pregnant women at term were denuded of their syncytiotrophoblast and co-cultured for 3 h, 24 h and 96 h with 800,000 trypomastigotes (simulating acute infection). T. cruzi infected cells were identified by immunohistochemistry for cytokeratin-7 (+cytotrophoblast) and CD68 (+macrophages), and the infection was quantified. In placental tissue, the parasite load was analyzed by qPCR and microscopy, and the motile trypomastigotes were quantified in culture supernatant. In denuded chorionic villous, the total area occupied by the parasite (451.23 mu m(2),1.33%) and parasite load (RQ: 87) was significantly higher (p < 0.05) than in the entire villous (control) (5.98 mu m(2), 0.016%) (RQ:1) and with smaller concentration of nitric oxide. Stromal non-macrophage cells were infected as well as cytotrophoblasts and some macrophages, but with significant differences being observed. The parasite quantity in the culture supernatant was significantly higher (p <0.05) in denuded culture explants from 96 h of culture. Although the human complete chorionic villi limited the infection, the detachment of the first structure of the placenta barrier (syncytiotrophoblast) increased both the infection of the villous stroma and the living trypomastigotes in the culture supernatant. Therefore structural and functional alterations to chorionic villi placental barrier reduce placental defenses and may contribute to the vertical transmission of ChagasSECyT-UNLaR SECyT-UNC MINCyT-Cordoba UNVM FONCyT-PICT-2012-1061 PICT-V-2015-007

DNA repair BER pathway inhibition increases cell death caused by oxidative DNA damage in Trypanosoma cruzi

Trypanosoma cruzi, a parasitic protozoan, is the etiological agent of Chagas disease, an endemic and neglected pathology in Latin America. It presents a life cycle that involves a hematophagous insect and man as well as domestic and wild mammals. The parasitic infection is not eliminated by the immune system of mammals; thus, the vertebrate host serves as a parasite reservoir. Additionally, chronic processes leading to dysfunction of the cardiac and digestive systems are observed. To establish a chronic infection some parasites should resist the oxidative damage to its DNA exerted by oxygen and nitrogen free radicals (ROS/RNS) generated in host cells. Till date there are no reports directly showing oxidative DNA damage and repair in T. cruzi. We establish that ROS/RNS generate nuclear and kinetoplastid DNA damage in T. cruzi that may be partially repaired by the parasite. Furthermore, we determined that both oxidative agents diminish T. cruzi cell viability. This effect is significant

Comparative ex vivo infection with Trypanosoma cruzi and Toxoplasma gondii of human, canine and ovine placenta: Analysis of tissue damage and infection efficiency

Trypanosoma cruzi, the causative agent of Chagas disease, and Toxoplasma gondii, which is responsible for Toxoplasmosis, are two parasites that cause significant protozoan zoonoses and consequently important economic losses in human, companion animals and livestock. For the congenital transmission to occur, both parasites must cross the barrier present in the mammalian placenta, which differs between species. Particularly, hemochorial, endotheliochorial and epitheliochorial placental barriers are present, respectively, in human, dog and sheep. The type of placental barrier has been associated with the probability of transmission of pathogens. In this study, we used experimental placental ex vivo infection models of T. cruzi and T. gondii in the above-mentioned mammals in order to study tissue alterations and to compare infection efficiency. Here, we infected placental term explants from human, dog and sheep and analyzed tissue damage by standard histological and histochemical methods. Comparative infection efficiency was determined by quantitative PCR. Both parasites are able to infect the different placental explants; however, more T. gondii parasites were detected, and T. gondii causes a more severe tissue damage in human and canine explants than T. cruzi. The histopathological changes observed in ovine placenta explants were similar in presence of both parasites. We conclude that the infection efficiency of T. gondii is higher, compared to T. cruzi, during the ex vivo infection of human, canine and ovine placental explants. In addition, the ex vivo infection of mammalian placental explants constitutes an interesting experimental approach to study part of the infection mechanisms as well as host responses during congenital infection of both parasites.ERANET-LAC ERANET17/HLH-0142 UREDES URC024/16 Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) CONICYT FONDECYT 1190341 1170126 318045