Expression of Ki-67 and P16 INK4a in chemically-induced perioral squamous cell carcinomas in mice / Expressão KI-67 e P16 INK4a em carcinomas espinocelulares periorais quimicamente induzidos em camundongos

ABSTRACT Objective: to evaluate the influence of Ki-67 and P16INK4a proteins immunohistochemical expressions on the clinical and morphological parameters of perioral squamous cell carcinoma induced with 9,10-dimethyl-1,2-benzanthracene (DMBA) in mice. Methods: we topically induced the lesions in the oral commissure of ten Swiss mice for 20 weeks, determining the time to tumors onset and the average tumor volume up to 26 weeks. In histopathological analysis, the variables studied were histological malignancy grade and the immunohistochemical expression of Ki-67 and P16INK4a proteins. The correlation between variables was determined by application of the Spearman correlation test. Results: the mean time to onset of perioral lesions was 21.1 ± 2.13 weeks; mean tumor volume was 555.91 ± 205.52 mm3. Of the induced tumors, 80% were classified as low score and 20% high score. There was diffuse positivity for Ki-67 in 100% of lesions - Proliferation Index (PI) of 50.1 ± 18.0. There was a strong direct correlation between Ki-67 immunoreactivity and tumor volume (R = 0.702) and a low correlation with the malignancy score (R = 0.486). The P16INK4a protein expression was heterogeneous, showing a weak correlation with tumor volume (R = 0.334). There was no correlation between the immunohistochemical expression of the two proteins studied. Conclusion: in an experimental model of DMBA-induced perioral carcinogenesis, tumor progression was associated with the tumor proliferative fraction (Ki-67 positive cells) and with tumor histological grading, but not with P16INK4a expression.

RESUMO Objetivo: avaliar a influência da expressão imuno-histoquímica das proteínas Ki-67 e p16INK4a sobre parâmetros clínico-morfológicos em carcinomas espinocelulares periorais quimicamente induzidos com 9,10-dimetil-1,2-benzantraceno (DMBA) em modelo murino. Métodos: as lesões foram induzidas topicamente na comissura labial de dez camundongos Swiss durante 20 semanas, sendo determinado o momento de surgimento dos tumores e volume tumoral médio até 26 semanas. Na análise histopatológica, as variáveis estudadas foram gradação histológica de malignidade tumoral e expressão imuno-histoquímica das proteínas Ki-67 e p16INK4a. A correlação entre as variáveis estudadas foi determinada pela aplicação do teste de correlação de Spearman. Resultados: o tempo médio de surgimento das lesões periorais foi 21,1±2,13 semanas. Volume tumoral médio foi de 555,91±205,52mm3. Dos tumores produzidos, 80% foram classificados como de baixo escore e 20%, alto escore. Evidenciou-se positividade difusa para Ki-67 em 100% das lesões - índice de marcação (PI) de 50,1±18,0. Verificou-se correlação direta forte entre a imunoexpressão do Ki-67 e o volume tumoral (R=0,702) e fraca correlação com o escore de malignidade (R=0,486). A expressão da proteína p16INK4a foi heterogênea, mostrando fraca correlação com o volume tumoral (R=0,334). Não houve correlação entre a expressão imuno-histoquímica das duas proteínas estudadas. Conclusão: Em modelo experimental de carcinogênese perioral DMBA-induzida, a progressão tumoral está associada à fração proliferativa do tumor (células ki-67 positivas) e com a gradação histológica tumoral, porém não com a expressão da p16INK4a.

Sheep gastrointestinal helminthiasis in the Sertão region of Paraíba State, Northeastern Brazil: prevalence and risk factors / Helmintoses gastrintestinais de ovinos no Sertão do Estado da Paraíba, Nordeste do Brasil: prevalência e fatores de riscos

In this study, we aimed to establish the prevalence and risk factors relating to gastrointestinal helminthiasis, and to characterize the sanitary management practiced among sheep herds in the Sertão region of the state of Paraíba, northeastern Brazil, based on factors that condition the ways of controlling these parasites in these herds. The research was carried out between April and July 2012. We visited 54 farms, where fecal and blood samples were individually collected from 465 animals. On each farm, a questionnaire was applied to gather information on variables relating to potential risk factors. The prevalence of sheep gastrointestinal helminthiasis in the region was 75.9%. At least one animal tested positive for this helminthiasis on 53 (98.1%) of the 54 farms evaluated. The eggs per gram of feces (EPG) analysis showed the following infection burdens: 51.8% with mild infection, 27.1% moderate infection, 9.9% heavy infection and 11.2% fatal infection. Among the sheep farms visited, anthelmintics were used on 81.5% (p <0.05). The most relevant risk factor in this study was the farm area, because it defines the area available for grazing animals. Properties with many animals and little pasture area, which are the most abundant type in the Sertão region of Paraíba, tend to have high prevalence of gastrointestinal helminthiasis, because the animals are more prone to reinfection. The Sertão region of Paraíba presents high prevalence of gastrointestinal helminthiasis among sheep, and the farm area is the most relevant risk factor for the development of these parasites.

Objetivou-se determinar a prevalência e os fatores de risco para as helmintoses gastrintestinais, caracterizando o manejo sanitário sob fatores condicionantes das formas de controle dessas parasitoses em rebanhos de ovinos da região do Sertão da Paraíba. A pesquisa foi desenvolvida no período de abril a julho de 2012. Foram visitadas propriedades, utilizando-se 465 animais, sendo coletadas individualmente amostras de fezes e sangue durante as visitas. Em cada propriedade, foi aplicado questionário para a coleta de informações acerca de variáveis que atuariam como possíveis fatores de risco. Observou-se que a prevalência das helmintoses gastrintestinais de ovinos na região do Sertão da Paraíba foi de 75,9%. Pelo menos um animal foi positivo para essas helmintoses, em 53 (98,1%) das 54 propriedades avaliadas. A análise de OPG (Ovos Por Gramas de Fezes) demonstrou que 51,8% dos animais apresentaram infecção leve, 27,1% infecção moderada, 9,9% infecção pesada e 11,2% infecção fatal. A utilização de anti-helmínticos ocorreu em 81,5% das propriedades (p <0,05). O fator de risco mais relevante neste estudo foi a área da propriedade, porque delimita a área de pastejo do animal. Propriedades com muitos animais e pouca área de pastejo, que são as mais abundantes no Sertão da Paraíba, tendem a apresentar alta prevalência de helmintoses gastrintestinais, pois os animais estão mais propensos à reinfecção. A região do Sertão da Paraíba apresenta uma elevada prevalência de helmintoses gastrintestinais em ovinos, e a área das propriedades é o fator de risco mais relevante para o desenvolvimento dessas parasitoses.

Prostate-specific membrane antigen can promote in vivo osseous metastasis of prostate cancer cells in mice

Reports remain insufficient on whether and how prostate-specific membrane antigen (PSMA) can influence in vivo osseous metastasis of prostate cancer (PCa). In the present study, the authors induced stable expression of PSMA in mouse PCa cell line RM-1. In vivo osseous metastasis was induced in 37 6-week-old female C57BL/6 mice weighing 22.45 ± 0.456 g. RM-1 cells were actively injected into the femoral bone cavity, leading to bilateral dissymmetry of bone density in the femoral bone. Tumor cells were also detected in bone tissue by pathological examination. The impact on bone density was demonstrated by the significant difference between animals injected with RM-PSMA cells (0.0738 ± 0.0185 g/cm²) and animals injected with RM-empty plasmid cells (0.0895 ± 0.0241 g/cm²). The lytic bone lesion of the RM-PSMA group (68.4%) was higher than that of the control group (27.8%). Immunohistochemistry showed that the expression of both vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) was distinctly higher in the RM-PSMA group than in the control group, while ELISA and Western blot assay indicated that VEGF and MMP-9 were higher in the RM-PSMA group compared to the control group (in vitro). Thus, the present study proposed and then confirmed for the first time that PSMA can promote in vivo osseous metastasis of PCa by increasing sclerotic destruction of PCa cells. Further analyses also suggested that PSMA functions positively on the invasive ability of RM-1 by increasing the expression of MMP-9 and VEGF by osseous metastases in vivo.

Epigallocatechin-3-gallate inhibits paracrine and autocrine hepatocyte growth factor/scatter factor-induced tumor cell migration and invasion

Aberrant activation of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, is involved in the development and progression of many human cancers. In the cell-based screening assay, (-)epigallocatechin-3-gallate (EGCG) inhibited HGF/SF-Met signaling as indicated by its inhibitory activity on HGF/SF-induced cell scattering and uPA activation (IC50 = 15.8 microg/ml). Further analysis revealed that EGCG at low doses specifically inhibited HGF/SF-induced tyrosine phosphorylation of Met but not epidermal growth factor (EGF)-induced phosphorylation of EGF receptor (EGFR). On the other hand, high-dose EGCG decreased both Met and EGFR proteins. We also found that EGCG did not act on the intracellular portion of Met receptor tyrosine kinase, i.e., it inhibited InlB-dependent activation of Met but not NGF-induced activation of Trk-Met hybrid receptor. This inhibition decreased HGF-induced migration and invasion by parental or HGF/SF-transfected B16F10 melanoma cells in vitro in either a paracrine or autocrine manner. Furthermore, EGCG inhibited the invasion/metastasis of HGF/SF-transfected B16F10 melanoma cells in mice. Our data suggest the possible use of EGCG in human cancers associated with dysregulated paracrine or autocrine HGF/SF-Met signaling.

Effects of Benzo(a)pyrene on the Expression of Heat Shock Proteins, Pro-inflammatory Cytokines and Antioxidant Enzymes in Hepatic Tumors Induced by Rat Hepatoma N1-S1 Cells

Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) that is easily introduced to humans via consumption of grilled or smoked meat. BaP causes harmful oxidative effects on cell development, growth and survival through an increase in membrane lipid peroxidation, oxidative DNA damage and mutagenesis. Therefore, the present study was conducted to evaluate the synergistic effects of BaP on oxidative stress in hepatic tumors. In this study, we established a hepatic tumor model by injecting rat hepatoma N1-S1 cells into healthy rats. Changes in the abundance of heat shock proteins (HSPs), antioxidant enzymes and pro-inflammatory cytokines were then investigated by western blot analysis. In addition, we examined changes in oxidative stress levels. Injection of N1-S1 cells or concomitant injection of BaP and N1-S1 cells resulted in the formation of hepatic tumors at the injection site. Evaluation of rat plasma reveals that hepatic tumors induced by BaP and N1-S1 cells expresses higher levels of Hsp27, superoxide dismutase (SOD), and tumor necrosis factor-alpha (TNF-alpha) when compared to those induced by N1-S1 cells only. The collective results of this study suggest that BaP exerts synergistic effects on the expression of HSP, cytokines and antioxidant enzymes in hepatic tumors induced by rat hepatoma N1-S1 cells.

Decrease in intestinal endocrine cells in Balb/c mice with CT-26 carcinoma cells

The density of intestinal endocrine cells, in Balb/c mice with colon 26 (CT-26) carcinoma cells, were examined immunohistochemically at 28 days after implantation. After CT-26 cell administration there was a significant decrease in most of the intestinal endocrine cells (p < 0.01) compared with the control group. The significant quantitative changes in the intestinal endocrine cell density might contribute to the development of the gastrointestinal symptoms commonly encountered in cancer patients.

Oxidative stress and experimental carcinogenesis.

The focus of this review is to provide state-of-the-art knowledge on the involvement of oxygen free radicals (OFR) in carcinogenesis with a particular reference to skin model system as the process of cancer development is best understood in this organ. However, a brief description of the role of OFR in other organs is also provided. The term OFR refers to forms of oxygen exhibiting high reactivity and having at least one unpaired electron. The role of OFR in different stages of carcinogenesis such as initiation, promotion and progression is described. Out of many mechanisms described for the chemical initiation of tumorigenesis, a number of them may involve free radicals in the cascade of reactions. Evidences that support the involvement of free radicals in tumor promotion include (i) a number of free radical-generating compounds are found to be tumor promoters in various animal model systems, (ii) ROS generating systems can mimic the biochemical action of tumor promoters, (iii) some tumor promoters stimulate the production of ROS, (iv) tumor promoters modulate the cellular antioxidant defense systems, and (v) free radical scavengers, detoxifiers and antioxidants inhibit the process of tumor promotion. The role of ROS in the progression stage of carcinogenesis is evident from the fact that a number of different free radical generating compounds enhance the malignant conversion of benign papillomas into carcinoma and their effectiveness may be related to the type of radicals produced into the biological system.

4-Epidoxorubicin activity in experimental malignant tumors

A literatura tem indicado a atividade positiva da doxorrubicina sobre adenocarcinomas gastrointestinais, seja isoladamente, seja em associacao com outros quimioterapicos. Permanece, porem, a critica representada pela toxicidade da droga, o que tem levado a pesquisa de derivados mais toleraveis e igualmente atuantes; e o caso da 4-epidoxorrubicina (4-EPI). E estudada a atividade da 4-EPI sobre o tumor maligno experimental denominado carcinossarcoma de Walker 256. Para tanto, foi observado o comportamento de 48 ratos Wistar adultos dividos aleatoriamente em cinco grupos: grupo T (seis ratos), com animais que receberam implante subcutaneo de celulas do tumor de Walker na regiao do flanco; grupo TE (11 ratos), em que os animais recebiam o implante tumoral, tal como descrito e simultaneamente a injecao endovenosa de 1 mg de 4-EPI na veia caudal; grupo T6E (oito ratos), em que os animais recebiam a 4-EPI, na dose referida, 6 dias apos o implante tumoral; grupo E (11 ratos) apenas injetados com a 4-EPI, sempre por via endovenosa; grupo C (12 ratos), que servia de controle para o comportamento dos demais grupos. Os animais de todos os grupos eram mantidos em gaiolas metabolicas individuais, recebendo dieta padronizada. Diariamente eram registrados: quantidade de dieta ingerida; peso corporeo;...

Development of the effect of an indifferentiated tumour extract on sinusoid littoral cells of young growing liver

El extracto de un tumor indiferenciado de crecimiento rápido, inyectado a las 1600/00 horas (hora del día/horas postinyección) inhibe la actividad mitótica de las células litorales del hígado joven en crecimiento durante el período circadiano dle día siguiente. El extracto inhibe también el aumento en el número de células litorales (SLOC/1000 cells) que sólo alcanza los valores de los animales inyectados con solución fisiológica, 12 horas después, a las 0200/30 horas. En los días subsiguientes la actividad mitótica a las 1400/46, 1400/70 y 1400/94 horas, no muestra ningún cambio. Los valores del número de células litorales por 1000 células son significativamente menores en ratones inyectados con extracto a las 1400/46, 1400/70 y 1400/94 horas. La inyección del extracto a las 0700/00 horas no muestra ningún efecto cuando el control de la actividad mitótica se hace a las 1400/07 horas. El efecto sobre la actividad mitótica se explicaría por la acción del extracto en la última parte de G1 o en G1-S (o en ambas). En cuanto al efecto sobre el número de células litorales por 1000 células podría deberse a una acción sobre el comportamiento migratorio de los monocitos, que aparecerían en el hígado como células de Kupffer o sobre producción en médula ósea (o en ambos efectos)