PPG - Medicina (Hematologia)

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https://hdl.handle.net/11600/61354

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Navegando PPG - Medicina (Hematologia) por Orientador(es) "Kerbauy, Daniella Marcia Bahia [UNIFESP]"

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    Avaliação da expressão do gene twist1 no microambiente hipóxico da leucemia mieloide aguda
    (Universidade Federal de São Paulo (UNIFESP), 2015-11-25) Malafaia, Emilia Carolina Oliveira Brandao [UNIFESP]; Kerbauy, Daniella Marcia Bahia [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    TWIST1, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in mesodermal development and organogenesis. Overexpressed TWIST1 has been thoroughly related to epithelial-mesenchymal transition (EMT) in solid tumors and has been described as an emerging poor prognostic factor in hematological neoplasms. Many questions remain to be addressed concerning to the role of TWIST1 in acute myeloid leukemia (AML). The understanding of TWIST1 in leukemia cells and its interaction with microenvironment can offer new insights in regards to physiopathology and therapeutic targets for patients with AML. Objectives: to evaluate the role of stroma contact and hypoxia in TWIST1 expression in myeloid cell lines. 2) To evaluate the functional impact of overexpressing TWIST1 on KG1a and PL21 cells. 3) To evaluate TWIST1 expression in primary cells of AML patients. Methods: In order to mimic bone marrow microenvironment, myeloid cells were co-cultured with mesenchymal HS5 cell line and PO2 1% was established with Smart -Trak ? 2 (Sierra Instruments, Inc.) equipment. Quantitative mRNA was determined using TaqMan ? Universal Master Mix (Applied Biosystems, Foster City, CA) and 3-step standard cycling conditions with sequence-specific primer TWIST1 normalized to the expression of ?-actin. KG1a and PL21 cells were transduced with lentivirus vector carrying e-GFP ("enhanced green fluorescence protein") for stable expression of TWIST1. Transduced cells were sorted by FITC fluorochrome and then verified through western blot analysis with TWIST1 antibody. For quantification of apoptosis, cells were labeled with PE-conjugated antibody using annexin V?phycoerythrin and propidium iodide (BD Biosciences, USA). DAPI (4',6- diamidino-2-phenylindole dihydrochloride) was used to stain DNA and determine cell cycle information. Apoptosis and cell cycle were analyzed by FACS -Becton Dickinson Canto II (BD Biosciences). Statistical analysis was assessed with unpaired t test and Fisher?s exact test. Results: Hypoxia induced TWIST1 mRNA expression in OCIAML3, PL21, KG1a and ML1 cell lines (fold-increased 46.3, 29.8, 12.9 and 2.3 respectively). Cells expressing endogenous TWIST1 protein (OCIAML3 and ML1)! 88!showed resistance to apoptosis in a hypoxic microenvironment (normoxia versus hypoxia: OCI/AML3, 22.6 % vs 11.7% and ML1, 29.8% vs. 7.5%) in contrast, cells not expressing endogenous TWIST1 protein (KG1a and PL21) went to apoptosis in the same conditions. Thus, overexpressing TWIST1 in KG1a and PL21 induced apoptosis protection in hypoxia (KG1a unmodified vs. modified: 17.6 ± 6.3 vs. 2.8 ± 6.3, p=0.04; PL21 unmodified vs. modified: 26.9 ± 10.9 vs. 3.2 ± 0.6, p=0.04). We found increased TWIST1 mRNA levels in bone marrow samples of 18 AML patients (3,329 ± 0,64) compared with 6 healthy controls (1,25 ± 0,62) (p= 0.03). Patients in the highest tertile of TWIST1 expression did not show differences in prognostic and complete remission after treatment compared with patients in low and middle tertile. Conclusion: Our data suggest TWIST1 gene expression protects acute myeloid leukemia cells from apoptosis in a hypoxic microenvironment. Moreover, our results showed increased expression of TWIST1 in AML patients. Thus, TWIST1 is a potential gene involved in leukemogenesis and should be further explored to understand disease biology and potential therapeutic targets.
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    Novas Estratégias Terapêuticas Para O Linfoma De Células Do Manto: Estudos Dos Efeitos Da Halofuginona
    (Universidade Federal de São Paulo (UNIFESP), 2017-12-18) Santos, Melina Goncalves Dos [UNIFESP]; Kerbauy, Daniella Marcia Bahia [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Introduction: mantle cell lymphoma (MCL) is characterized by the translocation t(11;14)(q13;q32), which takes to cyclin D1 overexpression, signaling pathways alterations and cell cycle activation. MCL responds to many initial therapies, but remissions are reached shortly and patients become chemoresistants over time. Treatment changes towards more intensive immunochemotherapies allow prolonged remissions, but the therapy intensification is a problem to elderly patients during clinical practice. Therefore, new drugs that reduce resistance and provide lasting responses are necessary. Halofuginone is an antifungal commonly used in poultry and it has recognized antifibrotic properties and mode of action associated with the TGF-β signaling pathway. Objectives: 1 - evaluate effects of halofuginone on MCL cell lines, through in vitro dose-response and time-dependent experiments designed to determine cytotoxic effects, cell cycle alterations and apoptosis; 2 - develop a xenograft model of MCL in mice; 3 - evaluate in vivo effects of halofuginone in subcutaneous tumors of MCL developed in mice. Materials and Methods: for in vitro experiments, cytotoxicity assays were performed with MTT and cell cycle and apoptosis studies evaluated by flow cytometry. For drug efficacy studies in animals, an in vivo model of MCL cell line was performed. Ninety mice were used, divided between experiments of intrafemoral or subcutaneous MCL cell line injection. In the mice submitted to intrafemoral injection, a xenograft model of MCL was established. In the animals submitted to subcutaneous injection, drug efficacy studies were performed with intraperitoneal injections of halofuginone. Animals were then separated into groups: control or treated, for each MCL cell line, and the effect of halofuginone was measured by the decrease of subcutaneous tumor volume. Results: cytotoxicity assays showed that halofuginone has significant cytotoxic effects on MCL cell lines, with IC50 values ranging from 72 nM, for Mino cell line (considered halofuginone sensitive), to 142 nM, for HBL-2 cell line (considered halofuginone resistant). In cell cycle and apoptosis studies, halofuginone induces apoptosis and cell cycle arrest at G1, with concentrations of 50 ng/mL for 24 hours for Mino cells and 200 ng/mL for 48 hours for HBL-2 cells. Cytometry assays showed engraftment for both MCL cell lines demonstrated by the presence of these cells in the mice spleen and bone marrow. For animals inoculated subcutaneously with HBL-2, on day 10 after injection, significant decrease on tumor volume (p<0,05) was observed in treated group compared to control. For the mice inoculated subcutaneously with Mino cells, there was significant difference (p<0,05) between the averages on tumor volumes, observed on days 16, 21, 23, 26 and 30 after subcutaneous injection, in the treated group compared to control. The time for mice sacrifice was 28 days for the control group and 30.2 days for the treated group (p<0,05). Conclusions: the effects of halofuginone were demonstrated on MCL cell line, with emphasis on the greater sensitivity of Mino cells as opposed to greater resistance of HBL-2 cells to the effects of halofuginone. These findings support a better exploration of the effect of halofuginone on MCL.