Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

Efeitos da senescência no folheto intergeniculado do tálamo de ratos: análises morfológicas e neuroquímicas

The circadian timing system (CTS), in rodents, consists of interconnected neural structures such as the suprachiasmatic nucleus (SCN) of the hypothalamus, Intergeniculate Leaflet (IGL) of the thalamus, synchronous pathways and behavioral effectors. The SCN has been described as the major circadian pacemaker in several species of mammals, while the IGL appears to be involved in integration of photic and non-photic clues relaying them to SCN. The CTS allows an ordered internal temporal organization to the organism, providing the proper execution of physiological and behavioral mechanisms, which brings homeostasis. However, this stability is disrupted with aging process causing numerous pathological disorders, ranging from simple loss of physiological functions to decreases in cognitive performance. Therefore, is fundamental understanding the effects of senescence in this system. In this context, is proposed in this study to check if there are changes in IGL cytoarchitecture, neurochemical and retinal afferent markers with aging and their possible morpho-functional implications. To achieve this goal wistar rats were divided into 3 groups: young (3 months); Middle Age (13 months); Old (23 months). They were submitted to paraformaldhyde (4%) transcardiac perfusion to tissue fixation. Then, they had their brain removed and sectioned in 30 µm slices, which every sixth section were collected. This sections were processed by nissl method and immunostaining for GFAP, GAD, ENK, NPY and CTb in order to analyze the IGL features. It was observed a cell loss in middle age and old animals at Nissl, NPY and CTb stains. In addition, it was shown a increase in GFAP in middle aged animals compared to young and old ones. No differences were found in other neurochemichal stains. These data suggests IGL loss retinal afferents and neurons, in special the NPY-IR ones, likely having a compensatory gliogenesis. This supports the correlations between the CTS functional deficits and an anatomical deterioration of its components with the aging process.

Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway