Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation

Possa SS, Charafeddine HT, Righetti RF, da Silva PA, Almeida-Reis R, Saraiva-Romanholo BM, Perini A, Prado CM, Leick-Maldonado EA, Martins MA, Tiberio ID. Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation. Am J Physiol Lung Cell Mol Physiol 303: L939-L952, 2012. First published September 21, 2012; doi:10.1152/ajplung.00034.2012.-Several studies have demonstrated the importance of Rho-kinase in the modulation of smooth muscle contraction, airway hyperresponsiveness, and inflammation. However, the effects of repeated treatment with a specific inhibitor of this pathway have not been previously investigated. We evaluated the effects of repeated treatment with Y-27632, a highly selective Rho-kinase inhibitor, on airway hyperresponsiveness, oxidative stress activation, extracellular matrix remodeling, eosinophilic inflammation, and cytokine expression in an animal model of chronic airway inflammation. Guinea pigs were subjected to seven ovalbumin or saline exposures. the treatment with Y-27632 (1 mM) started at the fifth inhalation. Seventy-two hours after the seventh inhalation, the animals' pulmonary mechanics were evaluated, and exhaled nitric oxide (E-NO) was collected. the lungs were removed, and histological analysis was performed using morphometry. Treatment with Y-27632 in sensitized animals reduced E-NO concentrations, maximal responses of resistance, elastance of the respiratory system, eosinophil counts, collagen and elastic fiber contents, the numbers of cells positive for IL-2, IL-4, IL-5, IL-13, inducible nitric oxide synthase, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, transforming growth factor-beta, NF-kappa B, IFN-gamma, and 8-iso-prostaglandin F2 alpha contents compared with the untreated group (P < 0.05). We observed positive correlations among the functional responses and inflammation, remodeling, and oxidative stress pathway activation markers evaluated. in conclusion, Rho-kinase pathway activation contributes to the potentiation of the hyperresponsiveness, inflammation, the extracellular matrix remodeling process, and oxidative stress activation. These results suggest that Rho-kinase inhibitors represent potential pharmacological tools for the control of asthma.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ São Paulo, Sch Med, Dept Med, BR-01246903 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, São Paulo, BrazilWeb of Scienc

The Mitochondrial Genome Is a “Genetic Sanctuary” during the Oncogenic Process

Since Otto Warburg linked mitochondrial physiology and oncogenesis in the 1930s, a number of studies have focused on the analysis of the genetic basis for the presence of aerobic glycolysis in cancer cells. However, little or no evidence exists today to indicate that mtDNA mutations are directly responsible for the initiation of tumor onset. Based on a model of gliomagenesis in the mouse, we aimed to explore whether or not mtDNA mutations are associated with the initiation of tumor formation, maintenance and aggressiveness. We reproduced the different molecular events that lead from tumor initiation to progression in the mouse glioma. In human gliomas, most of the genetic alterations that have been previously identified result in the aberrant activation of different signaling pathways and deregulation of the cell cycle. Our data indicates that mitochondrial dysfunction is associated with reactive oxygen species (ROS) generation, leading to increased nuclear DNA (nDNA) mutagenesis, but maintaining the integrity of the mitochondrial genome. In addition, mutational stability has been observed in entire mtDNA of human gliomas; this is in full agreement with the results obtained in the cancer mouse model. We use this model as a paradigm of oncogenic transformation due to the fact that mutations commonly found in gliomas appear to be the most common molecular alterations leading to tumor development in most types of human cancer. Our results indicate that the mtDNA genome is kept by the cell as a “genetic sanctuary” during tumor development in the mouse and humans. This is compatible with the hypothesis that the mtDNA molecule plays an essential role in the control of the cellular adaptive survival response to tumor-induced oxidative stress. The integrity of mtDNA seems to be a necessary element for responding to the increased ROS production associated with the oncogenic process

Retinoblastoma Loss Modulates DNA Damage Response Favoring Tumor Progression

Senescence is one of the main barriers against tumor progression. Oncogenic signals in primary cells result in oncogene-induced senescence (OIS), crucial for protection against cancer development. It has been described in premalignant lesions that OIS requires DNA damage response (DDR) activation, safeguard of the integrity of the genome. Here we demonstrate how the cellular mechanisms involved in oncogenic transformation in a model of glioma uncouple OIS and DDR. We use this tumor type as a paradigm of oncogenic transformation. In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb) inactivation respectively. In this scenario, the absence of pRb confers a proliferative advantage and activates DDR to a greater extent in a DNA lesion-independent fashion than cells that express only HRasV12. Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation. Thus, Rb loss acts as a switch mediating the transition between premalignant lesions and cancer through DDR modulation. These findings may have important implications for the understanding the biology of gliomas and anticipate a new target, Wip1 phosphatase, for novel therapeutic strategies

lmmunohistochemical demonstration of neuronal and astrocytic markers and oncofoetal antigens in retinoblastomas

General opinion is that retinoblastomas, though not everyone agrees with that view. Some authors suggest that retinoblastomas are derived from a primitive retinal cell able to differentiate into both neuronal and glial cell lines. The aim of tlie present work was to study immunohistocheniically tlie expression of neuronal and astrocytic markers in retinoblastomas and at the same time the presence of tlie oncofoetal antigens carcinocnihryoiiic antigen (CEA) and alpha Focto Protein (AFP). since patients with retinoblastomas often show high oncofoctal antigen in serum levels. For this purpose we cnnployed the strcptavidin-biotin inimunopcroxidase technique in 13 cases of retinobla5toma to evaluate the presence ancl distribution of neuron-specific enolase (NSE). neurofilament protein (NF), glial fihrillary acidic piutein (GFAP), S-l00 protein. CEA and AFP. All 13 tumours \tudicd stained for NSE. Seven of them showed GFAP- and S-l00 positive per ivasc~~lgalri al cells as well as cells distributed randomly in the tuniour that were interpreted as non tumour cells. All 13 retinohlustomas lacked detectable NF, CEA, and AFP. These results \ ~ ~ p p o rtth e idea that retinoblastomas are neuronal tumour\. although retinal glial cells may become incorporated in the tumour and proliferate in response to the t u ~ ~ i o

Immunohistochemical evaluation of EGFR expression in lip squamous cell carcinoma. Correlation with clinicopathological characteristics

Background: The majority of lip cancer is the squamous cell carcinoma (SCC) type that exhibits clinical and biological characteristics intermediate between skin and oral SCC. The aim of this study was to assess the impact of epidermal growth factor receptor (EGFR) expression on prognosis of lip squamous cell carcinoma (LSCC) and to relate it with clinicopathological features. The role of EGFR expression as a possible therapeutic target was also discussed. Methods: A series of 55 patients with LSCC was analyzed. EGFR expression was determined by standardized immunohistochemistry (pharmDx assay) and evaluated by both manual and automated image analysis (ACIS III). The Kappa statistic test was used to evaluate the concordance of manual and automated scores. EGFR results were correlated with clinicopathologic characteristics. Statistical differences between proportions were determined by the chi-squared test (with linear-by-linear correction where appropriate). The Mann-Whitney and the Kruskal-Wallis test were employed for comparison of continuous variables. Results: Correlation between manual and automated score was obtained in 50/55 cases (90.9%). EGFR expression was absent or weak in 14 cases (25.5%); borderline (2+) in 20 cases (36.4%) and positive (3+) in 21 cases (38.2%). Significant relationships were found between EGFR expression and tumour ulceration (p=0.022) and tumour thickness (p=0,002) and width (p=0.021). Conclusions: Our results revealed EGFR high expression in LSCC and its relationship with bad prognosis criteria (tumour size and ulceration)

Effects of Rho-kinase inhibition in lung tissue with chronic inflammation

We evaluated whether Rho-kinase inhibition (Y-27632) modulated distal lung responsiveness, inflammation, extracellular matrix remodeling and oxidative stress activation in guinea pigs (GPs) with chronic allergic inflammation. GPs were submitted to inhalation of ovalbumin (OVA-2x/week/4 weeks). From the 5th inhalation on, the Rho-kinase inhibitor group animals were submitted to Y-27632 inhalation 10 min before each inhalation of OVA. Seventy-two hours after the seventh inhalation, the oscillatory mechanics of the distal lung strips were assessed under the baseline condition and after the ovalbumin challenge. Subsequently, the lung slices were submitted to morphometry. Rho-kinase inhibition in the ovalbumin-exposed animals attenuated distal lung elastance and resistance, eosinophils, IL-2, IL-4, IL-5, IL-13, TIMP-1, MMP-9, TGF-beta, IFN-gamma, NF-kappa B and iNOS-positive cells and the volume fraction of 8-iso-PGF2 alpha, elastic, collagen and actin in alveolar walls compared with the OVA group (P < 0.05). Rho-kinase inhibition contributed to the control of distal lung responsiveness, eosinophilic and Th1/Th2 responses and extracellular matrix remodeling in an animal model of chronic allergic inflammation. (C) 2014 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ São Paulo, Sch Med, Dept Med, BR-01246903 São Paulo, BrazilUniv Fed Rio de Janeiro, Ilha Fundao, Carlos Chagas Filho Inst Biophys, Lab Pulm Invest, Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, BR-09972270 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, BR-09972270 São Paulo, BrazilWeb of Scienc