RESUMO

ABSTRACT Introduction: Culturing bone marrow mesenchymal stem cells (BM-MSCs) is a key point in different fields of research, including tissue engineering and regenerative medicine and studies of the bone marrow microenvironment. However, isolating and expanding murine BM-MSCs in vitro has challenged researchers due to the low purity and yield of obtained cells. In this study, we aimed to evaluate five different protocols to culture murine BM-MSCs in vitro. Methods: All protocols were based on the adhesion capacity of BM-MSCs to the tissue culture plastic surface and varied in the types of plate, culture media, serum, additional supplementation and initial cell density. Flow cytometry analysis was used to investigate lineage purity after expansion. Results: The expression of CD45 and CD11b was detected in the cultures generated according to all protocols, indicating low purity with the presence of hematopoietic cells and macrophages. The cellular growth rate and morphology varied between the cultures performed according to each protocol. Cells cultured according to protocol 5 (8 × 107cells/plate, Roswell Park Memorial Institute (RPMI) culture medium during first passage and then Iscove's Modified Delbecco's Medium (IMDM) culture medium, both supplemented with 9% fetal bovine serum, 9% horse serum, 12μM L-glutamine) presented the best performance, with a satisfactory growth rate and spindle-shape morphology. Conclusion: Our results point out that the purity and satisfactory growth rate of murine BM-MSC cultures are not easily achieved and additional approaches must be tested for a proper cell expansion.

Assuntos

Animais , Masculino , Ratos , Células-Tronco Mesenquimais , Medula Óssea , Técnicas In Vitro , Técnicas de Cultura de Células , Camundongos

RESUMO

Acute myeloid leukemia (AML) is a highly heterogeneous hematological neoplasm with low survival rates. Thus, the investigation of new therapeutic targets is essential. The Rac subfamily of GTPase proteins has been shown to participate in the physiopathology of hematological malignancies. However, their expression and function in AML remain unclear. In this study, we evaluated Rac1, Rac2 and Rac3 gene expressions in AML and their impact on clinical outcomes. We further investigated the effects of the in vitro treatment with a Rac inhibitor (EHT-1864) on AML cell lines. Rac3 expression was increased in AML derived from myelodysplastic syndromes compared to healthy donors. Rac2 expression did not differ between AML patients and healthy donors, but de novo AML patients with higher Rac2 presented lower overall survival. Oncogenic pathway gene-sets related to AKT/mTOR were identified as associated with Rac1, Rac2 and Rac3 expressions. EHT-1864 treatment reduced the viability of OCI-AML3, KG1 and Kasumi-1 cells in a time and dose-dependent manner. In OCI-AML3 cells, treatment with EHT-1864 induced apoptosis, autophagy, and led to the accumulation of cells in the G1 phase of the cell cycle. These changes were concomitant with alterations in p53 and cyclins. Dowregulation of the PI3K/AKT/mTOR pathway was also observed. Interestingly, the combined treatment of EHT-1864 and low doses of daunorubicin enhanced OCI-AML3 cell apoptosis. In conclusion, Rac2 expression is a prognostic factor in AML and our preclinical results suggest that Rac inhibition may be an attractive mechanism to compose the antineoplastic strategy for this disease.

Assuntos

GTP Fosfo-Hidrolases , Leucemia Mieloide Aguda , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt , Serina-Treonina Quinases TOR

RESUMO

INTRODUCTION: Culturing bone marrow mesenchymal stem cells (BM-MSCs) is a key point in different fields of research, including tissue engineering and regenerative medicine and studies of the bone marrow microenvironment. However, isolating and expanding murine BM-MSCs in vitro has challenged researchers due to the low purity and yield of obtained cells. In this study, we aimed to evaluate five different protocols to culture murine BM-MSCs in vitro. METHODS: All protocols were based on the adhesion capacity of BM-MSCs to the tissue culture plastic surface and varied in the types of plate, culture media, serum, additional supplementation and initial cell density. Flow cytometry analysis was used to investigate lineage purity after expansion. RESULTS: The expression of CD45 and CD11b was detected in the cultures generated according to all protocols, indicating low purity with the presence of hematopoietic cells and macrophages. The cellular growth rate and morphology varied between the cultures performed according to each protocol. Cells cultured according to protocol 5 (8 × 107cells/plate, Roswell Park Memorial Institute (RPMI) culture medium during first passage and then Iscove's Modified Delbecco's Medium (IMDM) culture medium, both supplemented with 9% fetal bovine serum, 9% horse serum, 12µM L-glutamine) presented the best performance, with a satisfactory growth rate and spindle-shape morphology. CONCLUSION: Our results point out that the purity and satisfactory growth rate of murine BM-MSC cultures are not easily achieved and additional approaches must be tested for a proper cell expansion.

RESUMO

ARHGAP21 is a member of the RhoGAP family of proteins involved in cell growth, differentiation, and adhesion. We have previously shown that the heterozygous Arhgap21 knockout mouse model (Arhgap21+/-) presents several alterations in the hematopoietic compartment, including increased frequency of hematopoietic stem and progenitor cells (HSPC) with impaired adhesion in vitro, increased mobilization to peripheral blood, and decreased engraftment after bone marrow transplantation. Although these HSPC functions strongly depend on their interactions with the components of the bone marrow (BM) niche, the role of ARHGAP21 in the marrow microenvironment has not yet been explored. In this study, we investigated the composition and function of the BM microenvironment in Arhgap21+/- mice. The BM of Arhgap21+/- mice presented a significant increase in the frequency of phenotypic osteoblastic lineage cells, with no differences in the frequencies of multipotent stromal cells or endothelial cells when compared to the BM of wild type mice. Arhgap21+/- BM cells had increased capacity of generating osteogenic colony-forming units (CFU-OB) in vitro and higher levels of osteocalcin were detected in the Arhgap21+/- BM supernatant. Increased expression of Col1a1, Ocn and decreased expression of Trap1 were observed after osteogenic differentiation of Arhgap21+/- BM cells. In addition, Arhgap21+/- mice recipients of normal BM cells showed decreased leucocyte numbers during transplantation recovery. Our data suggest participation of ARHGAP21 in the balanced composition of the BM microenvironment through the regulation of osteogenic differentiation.

RESUMO

PURPOSE: Despite great advances that have been made in the understanding of the molecular complexity of acute myeloid leukemia (AML), very little has been translated into new therapies. Here, we set out to investigate the impact of cytoskeleton regulatory genes on clinical outcomes and their potential as therapeutic targets in AML. METHODS: Gene expression and clinical data were retrieved from The Cancer Genome Atlas (TCGA) AML study and used for survival and functional genomics analyses. For pharmacological tests, AML cells were exposed to ezrin (EZR) inhibitors and submitted to several cellular and molecular assays. RESULTS: High EZR expression was identified as an independent marker of worse outcomes in AML patients from the TCGA cohort (p < 0.05). Functional genomics analyses suggested that EZR contributes to responses to stimuli and signal transduction pathways in leukemia cells. EZR pharmacological inhibition with NSC305787 and NSC668394 reduced viability, proliferation, autonomous clonal growth, and cell cycle progression in AML cells (p < 0.05). NSC305787 had a greater potency and efficiency than NSC668394 in leukemia models. At the molecular level, EZR inhibitors reduced EZR, S6 ribosomal protein and 4EBP1 phosphorylation, and induced PARP1 cleavage in AML cells. NSC305787, but not NSC668394, favored a gene network involving cell cycle arrest and apoptosis in Kasumi 1 AML cells. CONCLUSIONS: From our data we conclude that EZR expression may serve as a prognostic factor in AML. Our preclinical findings indicate that ezrin inhibitors may be employed as a putative novel class of AML targeting drugs.

Assuntos

Biomarcadores Tumorais/genética , Proteínas do Citoesqueleto/genética , Citoesqueleto/metabolismo , Regulação Leucêmica da Expressão Gênica , Genes Reguladores/genética , Leucemia Mieloide/genética , Doença Aguda , Adamantano/análogos & derivados , Adamantano/farmacologia , Adulto , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/metabolismo , Intervalo Livre de Doença , Feminino , Células HL-60 , Humanos , Células K562 , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/metabolismo , Masculino , Fenóis/farmacologia , Prognóstico , Quinolinas/farmacologia , Quinolonas/farmacologia , Células THP-1 , Células U937

Assuntos

Plaquetas/fisiologia , Proteínas Ativadoras de GTPase/genética , Megacariócitos/citologia , Regulação para Cima , Animais , Plaquetas/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Células Cultivadas , Proteínas Ativadoras de GTPase/metabolismo , Inativação Gênica , Humanos , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Camundongos , Selectina-P/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Cultura Primária de Células , Trombina/farmacologia

RESUMO

RhoA and RhoC contribute to the regulation of glutamine metabolism, which is a crucial determinant of cell growth in some types of cancer. Here we investigated the participation of RhoA and RhoC in the response of prostate cancer cells to glutamine deprivation. We found that RhoA and RhoC activities were up- or downregulated by glutamine reduction in PC3 and LNCaP cell lines, which was concomitant to a reduction in cell number and proliferation. Stable overexpression of wild type RhoA or RhoC did not alter the sensitivity to glutamine deprivation. However, PC3 cells expressing dominant negative RhoAN19 or RhoCN19 mutants were more resistant to glutamine deprivation. Our results indicate that RhoA and RhoC activities could affect cancer treatments targeting the glutamine pathway.

Assuntos

Glutamina

RESUMO

Tissue factor (TF) is a procoagulant protein associated with increased risk of thrombotic events in antiphospholipid syndrome (t-APS). The mechanisms by which TF levels are increased in APS have not yet been established. The aim of this study was to investigate whether TF mRNA expression is associated with TF levels and thrombosis in APS. We compared levels of circulating TF and high sensitivity C-reactive protein (hs-CRP) between t-APS and controls (individuals without thrombosis). The association between TF mRNA expression, quantified by real time quantitative polymerase chain reaction, and t-APS was accessed using regression analysis. We included 41 controls and 42 t-APS patients, mean age was 41 years old (SD 14) in both groups. Hs-CRP and TF levels were higher in t-APS patients (mean hs-CRP levels 0.81 mg/dL [SD 1.88] and median TF levels 249.0 pg/mL [IQR 138.77-447.61]) as compared to controls (mean hs-CRP levels 0.24 mg/dL [SD 0.26] and median TF levels 113.0 pg/mL [IQR 81.17-161.53]; P = 0.02 and P < 0.0001, respectively). There was no correlation between TF mRNA expression and TF levels in t-APS (r - 0.209, P = 0.19). TF mRNA expression was not associated with t-APS (adjusted OR 1.16; 95%CI 0.72-1.87). Despite circulating TF levels being higher in patients with t-APS than in controls, TF mRNA expression was similar between groups. The results demonstrate that TF mRNA expression is not associated with levels of circulating TF and hypercoagulability in t-APS.

Assuntos

Síndrome Antifosfolipídica/genética , RNA Mensageiro/genética , Tromboplastina/genética , Trombose/genética , Adulto , Síndrome Antifosfolipídica/complicações , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Trombose/etiologia

RESUMO

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Chemotherapy with cytotoxic agents is the standard of care, but is associated with a high rate of adverse events. Elderly patients are frequently intolerant to such treatment, presenting a very poor prognosis. The hypomethylating agents (HMA) azacitidine or decitabine represent one of the main therapeutic alternatives for these patients. Isocitrate dehydrogenase inhibitors (IDH) constitute another therapeutic class with DNA methylation effects in AML. In this article, we review the use of first- and second-generation HMA and IDH inhibitors in AML. The data collected demonstrated that HMA are generally considered effective and safe for AML patients who are not eligible for standard chemotherapy. The combination of azacitidine or decitabine with venetoclax was recently approved by the US Food and Drug Administration (FDA) for older AML patients and those unfit for intense chemotherapy. IDH inhibitors also showed encouraging results for relapsed/refractory AML patients harboring an IDH mutation and received FDA approval. Therefore, recent studies have led to the emergence of new therapeutic options using HMA and IDH inhibitors for specific groups of AML patients, representing an important step in the treatment of this aggressive malignancy. New options should emerge from the ongoing studies in the coming years.

Assuntos

Antineoplásicos/uso terapêutico , Metilação de DNA/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Terapia de Alvo Molecular , Idoso , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Azacitidina/administração & dosagem , Azacitidina/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Ensaios Clínicos como Assunto , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Decitabina/administração & dosagem , Decitabina/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Metanálise como Assunto , Estudos Multicêntricos como Assunto , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Sulfonamidas/administração & dosagem , Resultado do Tratamento

RESUMO

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell-based disorders characterized by ineffective hematopoiesis, increased genomic instability and a tendency to progress toward acute myeloid leukemia (AML). MDS and AML cells present genetic and epigenetic abnormalities and, due to the heterogeneity of these molecular alterations, the current treatment options remain unsatisfactory. Hypomethylating agents (HMA), especially azacitidine, are the mainstay of treatment for high-risk MDS patients and HMA are used in treating elderly AML. The aim of this study was to investigate the potential role of the epigenetic reader bromodomain-containing protein-4 (BRD4) in MDS and AML patients. We identified the upregulation of the short variant BRD4 in MDS and AML patients, which was associated with a worse outcome of MDS. Furthermore, the inhibition of BRD4 in vitro with JQ1 or shRNA induced leukemia cell apoptosis, especially when combined to azacitidine, and triggered the activation of the DNA damage response pathway. JQ1 and AZD6738 (a specific ATR inhibitor) also synergized to induce apoptosis in leukemia cells. Our results indicate that the BRD4-dependent transcriptional program is a defective pathway in MDS and AML pathogenesis and its inhibition induces apoptosis of leukemia cells, which is enhanced in combination with HMA or an ATR inhibitor.