Full text
| |
| Author(s): |
Patricia Aparecida de Assis
Total Authors: 1
|
| Document type: | Master's Dissertation |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2010-08-03 |
| Examining board members: |
Eduardo Magalhães Rego;
Roger Chammas;
Sergio Britto Garcia
|
| Advisor: | Eduardo Magalhães Rego |
| Abstract | |
Angiogenesis is the term used to describe the growth of new blood vessels from the existing ones. Several studies have demonstrated the microvascular density (MVD) as a prognostic factor in leukemia, particularly acute promyelocytic leukemia (APL). This subtype of leukemia corresponds to 20-25% of acute myeloid leukemia in Latin America and presents clinical, morphological and biological peculiar characteristics. The halofuginone (HF) originally described as an antifungal agent has ability to inhibit tumor growth and vessel formation in animal models of solid tumors. Our group demonstrated that HF inhibits the VEGF secretion and cell proliferation in APL cell lineages. Thus, this study aimed to determine the antiproliferative and antiangiogenic potential of HF in an experimental model of APL in vivo and evaluate the mechanisms underlying its action. First, the cell cycle analysis in NB4 cells treated with HF showed a significant decrease in cell proliferation (2.093 ± 0.304 vs. 41.21 ± 3.25), along with a significant increase in apoptosis (12.53 ± 1.53 vs . 21.95 ± 0.79, p = 0.0007). Through Real Time Array it was possible to identify two groups of apoptosis associated genes differentially expressed in HF treated cells: TNF, TNFRSF9, TNFTSF10B, CD40, FAS, CASP10, CASP8 and CASP3, suggesting that HF induces apoptosis by extrinsic pathway. In vivo analysis of HF was performed in irradiated NOD/SCID mice transplanted with murine PML-RAR leukemic cells. Mice treated with HF for 21 days after transplantation showed no molecular remission as determined by amplification of PML-RARA gene by PCR, however minor leukemic infiltration was observed compared to untreated mice (leukocytes: 4.2 ± 3.89 vs . 20.6 ± 21.9, p <0.0001), hemoglobin: 12.0 ± 1.40 vs. 9.6 ± 1.67, p <0.0001, and Platelets: 932.0 ± 122.5 vs. 552.0 ± 83.2, p <0.001 respectively) and a lower relative weight of spleen (0.006 vs. 0.012, p = 0.0415). Furthermore, the differential count and immunophenotyping of bone marrow showed a lower percentage of immature myeloid cells (16.88 ± 6.27 vs. 44.06 ± 27.06). The HF was also able to inhibit the SMAD2 phosphorylation and consequently block the TGF- pathway in NB4 cells. However, leukemic animals presented lower serum TGF- compared to the healthy and treated (475.58 vs. 1378.45/1146.82 pg / mL, p <0.0001), suggesting that the leukemic blasts produces this cytokines and the decrease in leukemic cells resulted in decreased serum levels of TGF-. The HF did not increase the survival of leukemic animals and elevated liver enzymes suggested that the treatment was hepatotoxic. Finally, regarding angiogenesis, gene expression analysis showed that the HF treatment inhibited the expression of VEGF and EGF and the study by immunohistochemistry of sections of bone marrow showed less VEGF expression (30 vs. 80%, p = 0 0227), but there was no decrease in the MVD. Taken together, these results showed that angiogenesis is an important therapeutic target in APL, and despite the toxicity, HF has antileukemic potential, for both the antiproliferative and proapoptotic effects, and also for its ability to inhibit the production of proangiogenic factors. (AU) | |