RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The Asháninkas, indigenous people of Peru, use cat's claw (Uncaria tomentosa) to restore health. Uncaria tomentosa has antioxidant activity and works as an agent to repair DNA damage. It causes different effects on cell proliferation depending on the cell type involved; specifically, it can stimulate the proliferation of myeloid progenitors and cause apoptosis of neoplastic cells. Neutropenia is the most common collateral effect of chemotherapy. For patients undergoing cancer treatment, the administration of a drug that stimulates the proliferation of healthy hematopoietic tissue cells is very desirable. It is important to assess the acute effects of Uncaria tomentosa on granulocyte-macrophage colony-forming cells (CFU-GM) and in the recovery of neutrophils after chemotherapy-induced neutropenia, by establishing the correlation with filgrastim (rhG-CSF) treatment to evaluate its possible use in clinical oncology. MATERIALS AND METHODS: The in vivo assay was performed in ifosfamide-treated mice receiving oral doses of 5 and 15 mg of Uncaria tomentosa and intraperitoneal doses of 3 and 9 µg of filgrastim, respectively, for four days. Colony-forming cell (CFC) assays were performed with human hematopoietic stem/precursor cells (hHSPCs) obtained from umbilical cord blood (UCB). RESULTS: Bioassays showed that treatment with Uncaria tomentosa significantly increased the neutrophil count, and a potency of 85.2% was calculated in relation to filgrastim at the corresponding doses tested. An in vitro CFC assay showed an increase in CFU-GM size and mixed colonies (CFU-GEMM) size at the final concentrations of 100 and 200 µg extract/mL. CONCLUSIONS: At the tested doses, Uncaria tomentosa had a positive effect on myeloid progenitor number and is promising for use with chemotherapy to minimize the adverse effects of this treatment. These results support the belief of the Asháninkas, who have classified Uncaria tomentosa as a 'powerful plant'.
Asunto(s)
Uña de Gato , Proliferación Celular/efectos de los fármacos , Células Progenitoras Mieloides/efectos de los fármacos , Neutropenia/tratamiento farmacológico , Neutrófilos/efectos de los fármacos , Extractos Vegetales/farmacología , Administración Oral , Animales , Uña de Gato/química , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sangre Fetal/citología , Filgrastim , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Células Progenitoras de Granulocitos y Macrófagos/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Ifosfamida , Inyecciones Intraperitoneales , Masculino , Medicina Tradicional , Ratones , Ratones Endogámicos BALB C , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patología , Neutropenia/inducido químicamente , Neutropenia/metabolismo , Neutropenia/patología , Neutrófilos/metabolismo , Neutrófilos/patología , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/administración & dosificación , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Plantas Medicinales , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/administración & dosificaciónRESUMEN
A new benzophenone O-glucoside neoannulatophenonoside (1) together with the known pinocembrin-7-O-glucoside were isolated from the aerial parts of Hyperium annulatum Moris (Guttiferae). The former was identified as 3',5',6-trihydroxy-4-methoxybenzophenone-2-O-beta-D-glucopyranoside by means of chemical and physical evidence. The cytoprotective effects of the new compound together with the previously isolated from this species hypericophenonoside (2), annulatophenone (3), annulatophenonoside (4), acetylannulatophenonoside (5) and 1,3,7-trihydroxyxanthone (6) were evaluated in a model of epirubicin-induced cellular toxicity in K-562 cells. While the benzophenone O-glycosides 1, 2, 4 and 5 exerted substantial cytoprotective effects against the epirubicin cytotoxicity in K-562 cells the aglycones 3 and 6 lacked any significant cytoprotective activity. Biochemical investigations aimed at evaluating the free-radical scavenging activity of the tested compounds as well as their effects on the cellular glutathione stores were carried out as well, aiming at unravelling the mechanisms of cytoprotection. Finally, the ability of 1, 4 and 5 to ameliorate epirubicin-induced anticlonogenic effects on bone marrow cells colony forming units, in vitro were also evaluated. Taken together, the experimental data indicate that the benzophenone glycosides isolated from H. annulatum have a substantial cytoprotective potential against the toxic effects induced by epirubicin and necessitates further detailed pharmacological evaluation of these compounds as possible chemoprotective/radioprotective agents.
Asunto(s)
Benzofenonas/farmacología , Citoprotección/efectos de los fármacos , Epirrubicina/farmacología , Depuradores de Radicales Libres/farmacología , Hypericum/química , Xantonas/farmacología , Animales , Benzofenonas/química , Benzofenonas/aislamiento & purificación , Supervivencia Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/aislamiento & purificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Células K562 , Ratones , Estructura Molecular , Células Progenitoras Mieloides/efectos de los fármacos , Células Progenitoras Mieloides/metabolismo , Componentes Aéreos de las Plantas/química , Proteínas Recombinantes , Relación Estructura-Actividad , Xantonas/química , Xantonas/aislamiento & purificaciónRESUMEN
STI571 is the most innovative drug for the cure of Chronic Myeloid Leukemia. It inhibits, in fact, the disease causative event, the p210 bcr-abl tyrosine kinase, and addresses clonal myeloid progenitors to apoptotic death. Here, we demonstrated that STI571 also induces growth arrest by activating the Chk2-Cdc25A-Cdk2 axis, a pathway complementary to p53 in the activation of G(1)/S cell cycle checkpoint. In vitro exposure to STI571 of 32D murine myeloid progenitor cell clones transducing a temperature-sensitive p210 bcr-abl construct was associated with Chk2 phosphorylation and activation, Cdc25A degradation and persistent Cdk2 inhibitory phosphorylation, preventing, in turn, cell transition to and progression throughout the S phase of cell cycle. Chk2 and Cdc25A are both components of a complex network that integrates signals involved in regulated cell cycle progression, DNA repair and cell decision between life or death. Chk2 gene mutations or decreased expression, leading to its protein loss of function on Cdc25A target, and Cdc25A overexpression have been linked to poor prognosis of human cancers. In CML, they might further enhance the proliferative advantage and genomic instability of clonal myeloid progenitors featuring a class of poor prognosis patients eventually resistant to STI571.
Asunto(s)
Antineoplásicos/farmacología , Fase G1/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/fisiología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Piperazinas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Pirimidinas/farmacología , Fase S/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Benzamidas , Línea Celular , Quinasa de Punto de Control 2 , Quinasas Ciclina-Dependientes/metabolismo , ADN/metabolismo , Proteínas de Fusión bcr-abl , Regulación Leucémica de la Expresión Génica/genética , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Ratones , Células Progenitoras Mieloides/metabolismo , Proteínas de Neoplasias/genética , Fosforilación/efectos de los fármacos , Piperazinas/uso terapéutico , Proteínas Tirosina Quinasas/genética , Pirimidinas/uso terapéutico , Fosfatasas cdc25/metabolismoRESUMEN
The t(10;11)(p12;q23) chromosomal translocation in human acute myeloid leukemia results in the fusion of the MLL and AF10 genes. The latter codes for a novel leucine zipper protein, one of many MLL fusion partners of unknown function. In this report, we demonstrate that retroviral-mediated transduction of an MLL-AF10 complementary DNA into primary murine myeloid progenitors enhanced their clonogenic potential in serial replating assays and led to their efficient immortalization at a primitive stage of myeloid differentiation. Furthermore, MLL-AF10-transduced cells rapidly induced acute myeloid leukemia in syngeneic or severe combined immunodeficiency recipient mice. Structure/function analysis showed that a highly conserved 82-amino acid portion of AF10, comprising 2 adjacent alpha-helical domains, was sufficient for immortalizing activity when fused to MLL. Neither helical domain alone mediated immortalization, and deletion of the 29-amino acid leucine zipper within this region completely abrogated transforming activity. Similarly, the minimal oncogenic domain of AF10 exhibited transcriptional activation properties when fused to the MLL or GAL4 DNA-binding domains, while neither helical domain alone did. However, transcriptional activation per se was not sufficient because a second activation domain of AF10 was neither required nor competent for transformation. The requirement for alpha-helical transcriptional effector domains is similar to the oncogenic contributions of unrelated MLL partners ENL and ELL, suggesting a general mechanism of myeloid leukemogenesis by a subset of MLL fusion proteins, possibly through specific recruitment of the transcriptional machinery.
Asunto(s)
Transformación Celular Neoplásica , Leucemia Mieloide/etiología , Células Progenitoras Mieloides/metabolismo , Proteínas de Fusión Oncogénica/fisiología , Proto-Oncogenes , Factores de Transcripción/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular , Células Cultivadas , Secuencia Conservada , Proteínas de Unión al ADN/genética , N-Metiltransferasa de Histona-Lisina , Leucina Zippers , Leucemia Mieloide/patología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Datos de Secuencia Molecular , Proteína de la Leucemia Mieloide-Linfoide , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Estructura Terciaria de Proteína , Alineación de Secuencia , Factores de Transcripción/química , Activación Transcripcional , Transducción GenéticaRESUMEN
The growth and survival of committed hematopoietic progenitors is dependent upon cytokine signaling. However, serum is also required for optimal growth of these progenitors in culture ex vivo. Here we report that serum withdrawal leads to myeloid progenitor cell apoptosis. Although serum deprivation-induced cell death has many hallmarks typical of apoptosis, these cell deaths were not inhibited by hemopoietins, survival factors such as IGF-I, or treatment with a broad-spectrum caspase inhibitor. Rather, apoptosis due to serum withdrawal was associated with damage to mitochondria. Surprisingly the serum factor required for myeloid cell survival was identified as iron, and loss of iron led to marked reductions in ATP production. Furthermore, supplementing serum-deprived myeloid cells with bound or free iron promoted cell survival and prevented mitochondrial damage. Therefore, serum suppresses hematopoietic cell apoptosis by providing an obligate source of iron and iron homeostasis is critical for proper myeloid cell metabolism and survival.
Asunto(s)
Apoptosis/fisiología , Deficiencias de Hierro , Hierro/metabolismo , Células Progenitoras Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Medio de Cultivo Libre de Suero/química , Medio de Cultivo Libre de Suero/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Interleucina-3/química , Interleucina-3/farmacología , Hierro/farmacología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/farmacologíaRESUMEN
The effects of deregulated Raf activation on the growth and differentiation of hematopoietic cells were investigated. The cytokine-dependent murine myeloid FDC-P1 and human erythroleukemic TF-1 cell lines were transformed to grow in response to deregulated Raf expression in the absence of exogenous cytokines. The conditionally active Raf proteins were regulated by beta-estradiol as cDNAs containing the Raf catalytic, but lacking negative-regulatory domains, were ligated to the hormone binding domain of the estrogen receptor (deltaRaf:ER). Continuous deltaRaf expression prevented apoptosis in the absence of exogenous cytokines and altered the morphology of the FD/deltaRaf:ER cells as they grew in large aggregated masses (>100 cells) whereas the parental cytokine-dependent FDC-P1 cells grew in smaller grape-like clusters (< 10 cells). FD/deltaRaf-1:ER cells growing in response to Raf activation displayed decreased levels of the Mac-2 and Mac-3 molecules on their cell surface. In contrast, when these cells were cultured in IL-3, higher levels of these adhesion molecules were detected. Expression of activated Raf oncoproteins also abrogated cytokine dependency and prevented apoptosis of TF-1 cells. Moreover, the differentiation status of these Raf-responsive cells was more immature upon Raf activation as culture with the differentiation-inducing agent phorbol 12 myristate 13-acetate (PMA) and beta-estradiol resulted in decreased levels of the CD11b and CD18 integrin molecules on the cell surface. In contrast when the Raf-responsive cells were induced to differentiate with PMA and GM-CSF, in the absence of deltaRaf:ER activation, increased levels of the CD11b and CD18 molecules were detected. Retinoic acid (RA) inhibited 3H-thymidine incorporation in response to GM-CSF. Interestingly, Raf activation counterbalanced the inhibition of DNA synthesis caused by RA but not PMA. Thus deregulated Raf expression can alter cytokine dependency, integrin expression and the stage of differentiation. These Raf-responsive cell lines will be useful in elucidating the roles of the MAP kinase cascade on hematopoietic cell differentiation and malignant transformation.