RESUMO
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome, which generates the oncogene BCR-ABL1. Protease-activated receptor 1 (PAR1) is involved in tumor progression and angiogenesis. We have previously reported that PAR1 expression is elevated in human leukemias that display a more aggressive clinical behavior, including the blast crisis of CML. In this study, we analyzed the crosstalk between the oncoprotein BCR-ABL and PAR1 in CML. Leukemic cell lines transfected with the BCR-ABL1 oncogene showed significantly higher expression levels of PAR1 compared with that of wild-type counterparts. This phenomenon was reversed by treatment with tyrosine kinase inhibitors (TKIs). Conversely, treatment with the PAR1 antagonist SCH79797 inhibited BCR-ABL expression. The PAR1 antagonist induced apoptosis in a dose- and time-dependent manner. Higher vascular endothelial growth factor (VEGF) levels were observed in cells transfected with BCR-ABL1 than in their wild-type counterparts. VEGF expression was strongly inhibited after treatment with either TKIs or the PAR1 antagonist. Finally, we evaluated PAR1 expression in CML patients who were either in the blast or chronic phases and had either received TKI treatment or no treatment. A significant decrease in PAR1 expression was observed in treatment-responsive patients, as opposed to a significant increase in PAR1 expression levels in treatment-resistant patients. Patients classified as high risk according to the Sokal index showed higher PAR1 expression levels. Our results demonstrate the crosstalk between BCR-ABL and PAR1. These data may offer important insight into the development of new therapeutic strategies for CML.
Assuntos
Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Inibidores de Proteínas Quinases/farmacologia , Receptor PAR-1/genética , Adulto , Idoso , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Estudos de Casos e Controles , Linhagem Celular Tumoral , Cromonas/farmacologia , Progressão da Doença , Relação Dose-Resposta a Droga , Feminino , Flavonoides/farmacologia , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Pessoa de Meia-Idade , Morfolinas/farmacologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Células Mieloides/patologia , Cromossomo Filadélfia , Pirimidinas/farmacologia , Pirróis/farmacologia , Quinazolinas/farmacologia , Receptor PAR-1/metabolismo , Transdução de Sinais , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Although hematopoietic stem cell (HSC) therapy for hematological diseases can lead to a good outcome from the clinical point of view, the limited number of ideal donors, the comorbidity of patients and the increasing number of elderly patients may limit the application of this therapy. HSCs can be generated from induced pluripotent stem cells (iPSCs), which requires the understanding of the bone marrow and liver niches components and function in vivo iPSCs have been extensively applied in several studies involving disease models, drug screening and cellular replacement therapies. However, the somatic reprogramming by transcription factors is a low-efficiency process. Moreover, the reprogramming process is also regulated by microRNAs (miRNAs), which modulate the expression of the transcription factors OCT-4 (also known as POU5F1), SOX-2, KLF-4 and MYC, leading somatic cells to a pluripotent state. In this Review, we present an overview of the challenges of cell reprogramming protocols with regard to HSC generation from iPSCs, and highlight the potential role of miRNAs in cell reprogramming and in the differentiation of induced pluripotent stem cells.
Assuntos
Diferenciação Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Reprogramação Celular/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fígado/citologia , Fígado/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética , Nicho de Células-Tronco/genética , Fatores de Transcrição/genéticaRESUMO
Snake venoms are complex mixtures of organic and inorganic compounds, including proteins belonging to the protease (serine and metalloproteinases), oxidase (L-amino acid oxidases), and phospholipase (especially phospholipases A2) enzyme classes. These toxins account for the serious deleterious effects of snake envenomations, such as tissue necrosis, neurotoxicity, and hemorrhage. In addition to their toxic effects, snake venom toxins have served as important tools for investigating the mechanisms underlying envenomation and discovering new pharmacologically active compounds with immunotherapeutic potential. In this sense, the present review discusses the new findings and therapeutic perspectives in the immune modulating potential of enzymatic toxins from snake venoms belonging to the classes metalloproteinase, serine protease, L-amino acid oxidase, and phospholipase A2.
Assuntos
Enzimas/química , Enzimas/metabolismo , Venenos de Serpentes/química , Venenos de Serpentes/enzimologia , Toxinas Biológicas/química , Toxinas Biológicas/metabolismo , Animais , Enzimas/imunologia , Humanos , Imunomodulação , Mordeduras de Serpentes/imunologia , Mordeduras de Serpentes/metabolismo , Mordeduras de Serpentes/patologia , Mordeduras de Serpentes/terapia , Venenos de Serpentes/imunologia , Venenos de Serpentes/uso terapêutico , Toxinas Biológicas/imunologiaRESUMO
Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the BCR-ABL1 tyrosine kinase (TK). The development of TK inhibitors (TKIs) revolutionized the treatment of CML patients. However, TKIs are not effective to those at advanced phases when amplified BCR-ABL1 levels and increased genomic instability lead to secondary oncogenic modifications. Wiskott-Aldrich syndrome protein (WASP) is an important regulator of signaling transduction in hematopoietic cells and was shown to be an endogenous inhibitor of the c-ABL TK. Here, we show that the expression of WASP decreases with the progression of CML, inversely correlates with the expression of BCR-ABL1 and is particularly low in blast crisis. Enforced expression of BCR-ABL1 negatively regulates the expression of WASP. Decreased expression of WASP is partially due to DNA methylation of the proximal WASP promoter. Importantly, lower levels of WASP in CML advanced phase patients correlate with poorer overall survival (OS) and is associated with TKI response. Interestingly, enforced expression of WASP in BCR-ABL1-positive K562 cells increases the susceptibility to apoptosis induced by TRAIL or chemotherapeutic drugs and negatively modulates BCR-ABL1-induced tumorigenesis in vitro and in vivo. Taken together, our data reveal a novel molecular mechanism that operates in BCR-ABL1-induced tumorigenesis that can be used to develop new strategies to help TKI-resistant, CML patients in blast crisis (BC).
Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Azacitidina/uso terapêutico , Carcinogênese/genética , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Proteínas de Fusão bcr-abl/biossíntese , Humanos , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/biossíntese , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
A new l-amino acid oxidase (LAAO) from Bothrops jararacussu venom (BjussuLAAO-II) was isolated by using a three-step chromatographic procedure based on molecular exclusion, hydrophobicity, and affinity. BjussuLAAO-II is an acidic enzyme with pI=3.9 and molecular mass=60.36kDa that represents 0.3% of the venom proteins and exhibits high enzymatic activity (4884.53U/mg/mim). We determined part of the primary sequence of BjussuLAAO-II by identifying 96 amino acids, from which 34 compose the N-terminal of the enzyme (ADDRNPLEECFRETDYEEFLEIARNGLSDTDNPK). Multiple alignment of the partial BjussuLAAO-II sequence with LAAOs deposited in the NCBI database revealed high similarity (95-97%) with other LAAOs isolated from Bothrops snake venoms. BjussuLAAO-II exerted a strong antiprotozoal effect against Leishmania amazonensis (IC50=4.56µg/mL) and Trypanosoma cruzi (IC50=4.85µg/mL). This toxin also induced cytotoxicity (IC50=1.80µg/mL) and apoptosis in MCF7 cells (a human breast adenocarcinoma cell line) by activating the intrinsic and extrinsic apoptosis pathways, but were not cytotoxic towards MCF10A cells (a non-tumorigenic human breast epithelial cell line). The results reported herein add important knowledge to the field of Toxinology, especially for the development of new therapeutic agents.
Assuntos
Antiprotozoários/isolamento & purificação , Antiprotozoários/farmacologia , Apoptose/efeitos dos fármacos , Bothrops , Venenos de Crotalídeos/enzimologia , L-Aminoácido Oxidase/isolamento & purificação , L-Aminoácido Oxidase/farmacologia , Sequência de Aminoácidos , Animais , Antiprotozoários/química , Humanos , L-Aminoácido Oxidase/química , Células MCF-7RESUMO
L-amino acid oxidases from snake venoms have been described to possess various biological functions. In this study, we investigated the inflammatory responses induced in vivo and in vitro by CR-LAAO, an L-amino acid oxidase isolated from Calloselasma rhodostoma venom, and its antitumor potential. CR-LAAO induced acute inflammatory responses in vivo, with recruitment of neutrophils and release of IL-6, IL-1ß, LTB4 and PGE2. In vitro, IL-6 and IL-1ß production by peritoneal macrophages stimulated with CR-LAAO was dependent of the activation of the Toll-like receptors TLR2 and TLR4. In addition, CR-LAAO promoted apoptosis of HL-60 and HepG2 tumor cells mediated by the release of hydrogen peroxide and activation of immune cells, resulting in oxidative stress and production of IL-6 and IL-1ß that triggered a series of events, such as activation of caspase 8, 9 and 3, and the expression of the pro-apoptotic gene BAX. We also observed that CR-LAAO modulated the cell cycle of these tumor cells, promoting delay in the G0/G1 and S phases. Taken together, our results suggest that CR-LAAO could serve as a potential tool for the development of novel immunotherapeutic strategies against cancer, since this toxin promoted apoptosis of tumor cells and also activated immune cells against them.
Assuntos
L-Aminoácido Oxidase/metabolismo , Venenos de Serpentes/enzimologia , Viperidae/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Citocinas/metabolismo , Humanos , Imunoterapia , Mediadores da Inflamação/metabolismo , L-Aminoácido Oxidase/imunologia , L-Aminoácido Oxidase/farmacologia , L-Aminoácido Oxidase/uso terapêutico , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/terapia , Infiltração de Neutrófilos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Venenos de Serpentes/imunologia , Venenos de Serpentes/farmacologia , Venenos de Serpentes/uso terapêutico , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
Background Tityus serrulatus scorpion venom (TsV) contains toxins that act on K + and Na + channels and account for the venoms toxic effects. TsV can activate murine peritoneal macrophages, but its effects on human lymphocytes have been poorly investigated. Considering that lymphocytes may play an important role in envenomation, we assessed whether TsV affects the expression of phenotypic (CD3, CD4, and CD8) and activation (CD69, CD25, and HLA-DR) markers, cell proliferation, and cytokine production in peripheral blood mononuclear cells. Methods Cytotoxicity of TsV was evaluated via the MTT assay. Cell proliferation, expression of phenotypic and activation markers, and release of cytokines were assessed using flow cytometry, after treatment with non-cytotoxic concentrations of TsV. The combined use of carboxyfluorescein diacetate succinimidyl ester and monoclonal antibodies against phenotypic and activation markers enabled us to simultaneously assess cell proliferation extent and cell activation status, and to discriminate among cell subpopulations. Results TsV at concentrations of 25 to 100 g/mL were not cytotoxic towards peripheral blood mononuclear cells. TsV did not induce significant changes in lymphocyte subpopulations or in the expression of activation markers on CD4 + and CD8 + T cells. TsV inhibited the phytohemagglutinin-stimulated lymphocyte proliferation, particularly in the CD8 + CD25 + T lymphocyte subset. TsV alone, at 50 and 100 g/mL, did not induce peripheral blood mononuclear cell proliferation, but elicited the production and release of IL-6, a proinflammatory cytokine that plays an important role in innate and adaptive immune responses. Conclusions TsV is a potential source of molecules with immunomodulatory action on human T lymphocytes.(AU)
Assuntos
Animais , Venenos de Escorpião , Animais Peçonhentos , Imunomodulação/efeitos dos fármacos , Linfócitos T/efeitos dos fármacosRESUMO
BACKGROUND: Tityus serrulatus scorpion venom (TsV) contains toxins that act on K(+) and Na(+) channels and account for the venom's toxic effects. TsV can activate murine peritoneal macrophages, but its effects on human lymphocytes have been poorly investigated. Considering that lymphocytes may play an important role in envenomation, we assessed whether TsV affects the expression of phenotypic (CD3, CD4, and CD8) and activation (CD69, CD25, and HLA-DR) markers, cell proliferation, and cytokine production in peripheral blood mononuclear cells. METHODS: Cytotoxicity of TsV was evaluated via the MTT assay. Cell proliferation, expression of phenotypic and activation markers, and release of cytokines were assessed using flow cytometry, after treatment with non-cytotoxic concentrations of TsV. The combined use of carboxyfluorescein diacetate succinimidyl ester and monoclonal antibodies against phenotypic and activation markers enabled us to simultaneously assess cell proliferation extent and cell activation status, and to discriminate among cell subpopulations. RESULTS: TsV at concentrations of 25 to 100 µg/mL were not cytotoxic towards peripheral blood mononuclear cells. TsV did not induce significant changes in lymphocyte subpopulations or in the expression of activation markers on CD4(+) and CD8(+) T cells. TsV inhibited the phytohemagglutinin-stimulated lymphocyte proliferation, particularly in the CD8(+) CD25(+) T lymphocyte subset. TsV alone, at 50 and 100 µg/mL, did not induce peripheral blood mononuclear cell proliferation, but elicited the production and release of IL-6, a proinflammatory cytokine that plays an important role in innate and adaptive immune responses. CONCLUSIONS: TsV is a potential source of molecules with immunomodulatory action on human T lymphocytes.
RESUMO
Background Tityus serrulatus scorpion venom (TsV) contains toxins that act on K + and Na + channels and account for the venoms toxic effects. TsV can activate murine peritoneal macrophages, but its effects on human lymphocytes have been poorly investigated. Considering that lymphocytes may play an important role in envenomation, we assessed whether TsV affects the expression of phenotypic (CD3, CD4, and CD8) and activation (CD69, CD25, and HLA-DR) markers, cell proliferation, and cytokine production in peripheral blood mononuclear cells. Methods Cytotoxicity of TsV was evaluated via the MTT assay. Cell proliferation, expression of phenotypic and activation markers, and release of cytokines were assessed using flow cytometry, after treatment with non-cytotoxic concentrations of TsV. The combined use of carboxyfluorescein diacetate succinimidyl ester and monoclonal antibodies against phenotypic and activation markers enabled us to simultaneously assess cell proliferation extent and cell activation status, and to discriminate among cell subpopulations. Results TsV at concentrations of 25 to 100 g/mL were not cytotoxic towards peripheral blood mononuclear cells. TsV did not induce significant changes in lymphocyte subpopulations or in the expression of activation markers on CD4 + and CD8 + T cells. TsV inhibited the phytohemagglutinin-stimulated lymphocyte proliferation, particularly in the CD8 + CD25 + T lymphocyte subset. TsV alone, at 50 and 100 g/mL, did not induce peripheral blood mononuclear cell proliferation, but elicited the production and release of IL-6, a proinflammatory cytokine that plays an important role in innate and adaptive immune responses. Conclusions TsV is a potential source of molecules with immunomodulatory action on human T lymphocytes.
Assuntos
Animais , Animais Peçonhentos , Imunomodulação/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Venenos de EscorpiãoRESUMO
Background Tityus serrulatus scorpion venom (TsV) contains toxins that act on K + and Na + channels and account for the venom's toxic effects. TsV can activate murine peritoneal macrophages, but its effects on human lymphocytes have been poorly investigated. Considering that lymphocytes may play an important role in envenomation, we assessed whether TsV affects the expression of phenotypic (CD3, CD4, and CD8) and activation (CD69, CD25, and HLA-DR) markers, cell proliferation, and cytokine production in peripheral blood mononuclear cells. Methods Cytotoxicity of TsV was evaluated via the MTT assay. Cell proliferation, expression of phenotypic and activation markers, and release of cytokines were assessed using flow cytometry, after treatment with non-cytotoxic concentrations of TsV. The combined use of carboxyfluorescein diacetate succinimidyl ester and monoclonal antibodies against phenotypic and activation markers enabled us to simultaneously assess cell proliferation extent and cell activation status, and to discriminate among cell subpopulations. Results TsV at concentrations of 25 to 100 μg/mL were not cytotoxic towards peripheral blood mononuclear cells. TsV did not induce significant changes in lymphocyte subpopulations or in the expression of activation markers on CD4 + and CD8 + T cells. TsV inhibited the phytohemagglutinin-stimulated lymphocyte proliferation, particularly in the CD8 + CD25 + T lymphocyte subset. TsV alone, at 50 and 100 μg/mL, did not induce peripheral blood mononuclear cell proliferation, but elicited the production and release of IL-6, a proinflammatory cytokine that plays an important role in innate and adaptive immune responses. Conclusions TsV is a potential source of molecules with immunomodulatory action on human T lymphocytes.(AU)