TOPICAL USE OF CsA: POSSIBLE ROLE OF CsA IN APOPTOSIS, P53 EXPRESSION, AND GLYCOSPHINGOLIPID PROFILE
RESUMO: Introduction :Cyclosporine A (CsA) is an immunossupressant agent which structure is a cyclic polypeptide composed of 11 amino acids. It is produced as a metabolite by the fungus Tolypocladium inflatum Gams.(SCHREIBER and GRABTREE, 1993). CsA prolongs survival of allogeneic transplants involving skin, heart, kidney, pancreas, bone marrow, small bowel and lungs. Although the exact mechanism of action of CsA is not completely understood, recently LALLY, HEALY and RYAN, 1999 proposed that CsA binds to cyclophilin in the cytoplasm of T cells and this complex binds to calcineurin inhibiting the transcription block of the IL-2 gene. Malignancy is a common and dreaded complication following organ transplantation. The high incidence of neoplasm and its aggressive progression associated with immunossupressive therapy seems to be directly related to the resulting impairment of the immune surveillance system of the recipients. Nevertheless, literature shows that CsA can induce phenotypic changes of non transformed cells by a cell autonomous mechanism (HOJO, MORIMOTO, MALUCCIO, ASANO, MORIMOTO, LAGMAN, SHIMBO, SUTHANTHIRAN, 1999) and, sometimes, cell cycle arrest or apoptosis (LALLY, HEALY, RYAN,1999). CsA treatment induces cell cycle arrest, this is coincident with p53 elevated levels (LALLY et al, 1999). It is known that the tumor suppressor protein p53 is induced following DNA injury and is crucial in this cellular response by trans-activation of a number of genes. Also, HORTELANO, LOPEZ-COLLAZO and BOSCA, 1999, showed a protective inhibition of apoptosis exerted by CsA in macrofages suggesting the contribution of those cells to innate immunity under conditions of immunossupression of the host. The mechanisms underlying these changes in cell cycle and apoptosis induced by CsA require further investigation. It is also well known that many of the molecules that cause cell-cycle arrest or apoptosis can induce generation of ceramides in these cells (DBAIBO, PUSHKAREVA, RACHID, ALTER, SMYTH, OBEID and HANNUN, 1998). Ceramides are found in glycosphingolipids, which comprise a large class of membrane lipids that contain sphingosine or related sphingoid bases, an amide-linked fatty acyl group and different sugar units. Ceramides are products of the hydrolysis of these glycosphingolipids and seems to be a second messenger that mediates the effects of TNF on cell growth and differentiation (PUSHKAREWA, OBEID and HANNUN, 1995). Recently, SWEENEY, SAKAKURA, SHIRAHAMA, MASAMUNE, OHTA, HAKOMORI and IGARASHI, 1996 studied the role of sphingosine and ceramide in the apoptosis and it was found that they are an integral part of the apoptosis pathway in human cancer cells hematopoietic and carcinoma origin. These findings led us to study the effects of topical CsA in mice skin, using the model described by NUNES and SALVADOR, 1996 of local immunessupression, for correlation among the effect of topical CsA, p53 and glycosphingolipids. Objective: The aim of this study was to investigate the effect of local immunossupression with topical CsA in mice skin observing the pattern of p53 and glycosphingolipids. Methods: Twenty female mice C57BL/6, 9-11 weeks old were distributed, randomly, into two groups with 10 animals each. In the GROUP I (GI), each animal received topical CsA (oil solution, SANDIMUN), 25µl, 14mg/kg, after hair extraction. In GROUP II (GII) each animal had just had the hair pluked out. After 24 hours of treatment. Animals of both group were submitted to anesthesia with diethyl ether, the dorsal skin was excised (2x3cm) and divided into three specimens (1x2cm) to study: i) apoptosis, by microscopy using hematoxilyn/eosin method (HE) (ref); ii) the pattern of p53 by immunohistochemistry using polyclonal antibodies anti-p53 using rat monoclonal antibody, and p53 was visualized using StreptABCComplex/HRP Duet, mouse/rabbit (ref); and iii) the profile of glycosphingolipids was analyzed after extraction of the tissues with a mixture of isopropanol/hexane/water (55:20:25; v/v/v), and analyzed by high perfomance thin layer chromatography (HPTLC), the plates were stained with orcinol or resorcinol. CsA blood level was measured. by fluorescence polarized immunoassay (ref.). Results: Neither GI nor GII presented significant alterations in skin architecture (microscopy, HE), all the skin layers were preserved. Also, it was observed in both groups some hyperkeratosis and a few apoptotic cells in epidermis or basal layer. By immunohistochemistry using rat monoclonal antibody/StreptABCComplex, it was not observed presence of p53 cells in GI or GII. On the other hand, glycosphingolipids extracted from tissues of GI presented quantitative as well as qualitative differences when compared to GII (CsA treated). In GI, it was observed presence of ceramide monohexosyl, globoside and H1 whereas in GII it was observed the presence of ceramide monohexosyl, globoside, H1, and four other bands with lower migration. HPTLC plates with resorcinol . Use of topical CsA in mice of GI showed the presence of 65ng/mL of blood. Conversely in mice of GII (control group) no CsA was detected in the blood . These results clearly indicate that the level of CsA in the blood of GI mice was 3-4 times lower than the immunosupressive levels.
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16 Mar 2001
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