RESUMEN
L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.
Asunto(s)
Antineoplásicos/farmacología , Asparaginasa/farmacología , Productos Biológicos/farmacología , Fenómenos Inmunogenéticos/efectos de los fármacos , Lisosomas/inmunología , Péptido Hidrolasas/farmacología , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Asparaginasa/química , Asparaginasa/uso terapéutico , Productos Biológicos/química , Productos Biológicos/uso terapéutico , Bovinos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Pollos , Relación Dosis-Respuesta a Droga , Escherichia coli , Femenino , Caballos , Humanos , Fenómenos Inmunogenéticos/fisiología , Células Jurkat , Lisosomas/química , Ratones , Ratones Endogámicos BALB C , Péptido Hidrolasas/química , Péptido Hidrolasas/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Estructura Secundaria de ProteínaRESUMEN
Annexin A1 (AnxA1) modulates neutrophil life span and bone marrow/blood cell trafficking thorough activation of formyl-peptide receptors (FPRs). Here, we investigated the effect of exogenous AnxA1 on haematopoiesis in the mouse. Treatment of C57BL/6 mice with recombinant AnxA1 (rAnxA1) reduced the granulocyte-macrophage progenitor (GMP) population in the bone marrow, enhanced the number of mature granulocytes Gr-1+Mac-1+ in the bone marrow as well as peripheral granulocytic neutrophils and increased expression of mitotic cyclin B1 on hematopoietic stem cells (HSCs)/progenitor cells (Lin-Sca-1+c-Kit+: LSK). These effects were abolished by simultaneous treatment with Boc-2, an FPR pan-antagonist. In in vitro studies, rAnxA1 reduced both HSC (LSKCD90lowFLK-2-) and GMP populations while enhancing mature cells (Gr1+Mac1+). Moreover, rAnxA1 induced LSK cell proliferation (Ki67+), increasing the percentage of cells in the S/G2/M cell cycle phases and reducing Notch-1 expression. Simultaneous treatment with WRW4, a selective FPR2 antagonist, reversed the in vitro effects elicited by rAnxA1. Treatment of LSK cells with rAnxA1 led to phosphorylation of PCLγ2, PKC, RAS, MEK, and ERK1/2 with increased expression of NFAT2. In long-term bone marrow cultures, rAnxA1 did not alter the percentage of LSK cells but enhanced the Gr-1+Mac-1+ population; treatment with a PLC (U73122), but not with a PKC (GF109203), inhibitor reduced rAnxA1-induced phosphorylation of ERK1/2 and Elk1. Therefore, we identify here rAnxA1 as an inducer of HSC/progenitor cell differentiation, favouring differentiation of the myeloid/granulocytic lineage, via Ca2+/MAPK signalling transduction pathways.
RESUMEN
Due to the cytotoxic effect of antimicrobial peptides (AMP) against several microorganism and tumor cells has been proposed their association with the immune system. However, just a few reports have shown this relationship. In this study, mice were treated with gomesin, a ß-hairpin AMP that exhibit high cytotoxicity against bacterial and tumor cells. Different effects in the immune system were observed, such as, decrease of CD3+ in T lymphocytes (Control: 17.7±1.4%; Gomesin: 7.67±1.2%) and in hematopoietic progenitors and increase of hematopoietic stem cell (Control: 0.046±0.004%; Gomesin: 0.067±0.003%), B220+ B lymphocytes (Control: 38.63±1.5%; Gomesin: 47.83±0.48%), and Mac-1+F4/80+ macrophages (Control: 11.76±3.4%; Gomesin: 27.13±4.0%). Additionally, macrophage increase was accompanied by an increase of macrophage phagocytosis (Control 20.85±1.53; Gomesin 31.32±1 Geometric mean), interleukin 6 (Control: 47.24±1.9ng/mL; Gomesin: 138.68±33.68ng/mL) and monocyte chemoattractant protein-1 (Control: 0.872±0.093ng/mL; Gomesin: 1.83±0.067ng/mL). Thus, this report showed immunomodulatory activity of gomesin in the immune system of mice.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/inmunología , Diferenciación Celular/genética , Activación de Macrófagos/genética , Células Mieloides/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/administración & dosificación , Sistema Inmunológico/metabolismo , Inmunomodulación/genética , Activación de Macrófagos/inmunología , Ratones , Monocitos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Leptin is a cytokine that regulates food intake, energy expenditure and hematopoiesis. Based on the tridimensional structure of the human leptin molecule, six fragments have been synthesized, (Ac-Lep23-47-NH2, [LEP1]; Ac-Lep48-71-NH2, [LEP2]; Ac-Lep72-88-NH2, [LEP3]; Ac-Lep92-115-NH2, [LEP4], Ac-[Ser(117)]-Lep116-140-NH2, [LEP5] and Ac-Lep141-164-NH2, [LEP6]), and their effects on hematopoiesis were evaluated. The mice were treated with 1mg/kg LEP5 for 3 days. The mature and primitive hematopoietic populations were quantified. We observed that the mature populations from the bone marrow and spleen were not affected by LEP5. However, the peptide caused at least a two-fold increase in the number of hematopoietic stem cells, the most primitive population of the bone marrow. Additionally, the number of granulocyte/macrophage colony-forming units produced by bone marrow cells in methylcellulose also increased by 40% after treatment with LEP5, and the leptin receptor was activated. These results show that the leptin fragment LEP5 is a positive modulator of the in vivo expansion of hematopoietic stem cells.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Leptina/farmacología , Fragmentos de Péptidos/farmacología , Bazo/efectos de los fármacos , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Proliferación Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/síntesis química , Receptores de Leptina/agonistas , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Bazo/citología , Bazo/metabolismoRESUMEN
The reports regarding the mutual influence between the central nervous system and the immune system constitute a vast and somewhat controversial body of literature. Stress is known to disturb homeostasis, impairing immunological functions. In this study, we investigated the hematopoietic response of Chlorella vulgaris (CV)-treated mice exposed to single (SST) and repeated stress (RST). We observed a reduction in the numbers of hematopoietic progenitors (HP) in the bone marrow and long-term bone marrow cultures (LTBMC) using flow cytometry and a coinciding decrease in the number of granulocyte-macrophage colonies (CFU-GM) after treatment with both stressors, but SST caused a more profound suppression. We observed a proportional increase in the colony-stimulating activity (CSA) of the serum of animals subjected to SST or RST. In the bone marrow, SST and RST induced a decrease in both mature myeloid and lymphoid populations but did not affect pluripotent hematopoietic progenitors (Lin(-)Sca-1(+)c-kit(+), LSK), and again, a more profound suppression was observed after SST. We further quantified the levels of interleukin-1α (IL-1α) and interleukin-6 (IL-6) and the number of myeloid cells in LTBMC. Both SST and RST reduced the levels of these cytokines to similar degrees. The myeloid population was also reduced in LTBMC, and SST induced a more intense suppression. Importantly, CV treatment prevented the changes produced by SST and RST in all of the parameters evaluated. Together, our results suggest that CV treatment is an effective tool for the prophylaxis of myelosuppression caused by single or repeated stressors.
Asunto(s)
Chlorella vulgaris/química , Hematopoyesis/fisiología , Estrés Psicológico/tratamiento farmacológico , Estrés Psicológico/fisiopatología , Animales , Médula Ósea/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interleucina-1alfa/biosíntesis , Interleucina-6/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Células Mieloides/efectos de los fármacos , Células Madre/efectos de los fármacos , Estrés Psicológico/líquido cefalorraquídeoRESUMEN
Apoptosis induction is often associated with increased autophagy, indicating interplay between these two important cellular events in cell death and survival. In this study, the programmed cell death and autophagy induced by two nitrostyrene derivative compounds (NTS1 and NTS2) was studied using the tumorigenic Ehrlich ascitic tumor (EAT) cells. EAT cells were highly sensitive to NTS1 and NTS2 cytotoxicity in a dose-dependent manner. NTS1 and NTS2 IC(50) was less than 15.0µM post 12h incubation. Apoptosis was primarily induced by both compounds, as demonstrated by an increase in Annexin-V positive cells, concurrently with cytochrome c release from mitochondria to cytosol and caspase-3 activation. Although cytosolic Ca(2+) mobilization is involved in autophagy as well as apoptosis in response to cellular stress in many cancer cell types, from the two nitrostyrene derivative compounds studied, mainly NTS1 mobilized this ion and disparate autophagy in EAT cells. These results suggest that EAT induced cell death by NTS1 and NTS2 involved a Ca(2+)-dependent and a Ca(2+)-independent pathways, respectively. In accordance with these results, the treatment of EAT cells with 3 methyladenine (3-MA), an autophagy inhibitor; significantly increased the number of apoptotic cells after NTS1 treatment, suggesting that pharmacological modulation of autophagy augments the NTS1 efficacy. Thus, we denote the importance of studies involving autophagy and apoptosis during pre-clinical studies of new drugs with anticancer properties.
Asunto(s)
Apoptosis/fisiología , Autofagia/fisiología , Calcio/metabolismo , Inhibidores Enzimáticos/farmacología , Nitrocompuestos/farmacología , Estirenos/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/efectos de los fármacos , Carcinoma de Ehrlich , Caspasa 3/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Citosol/metabolismo , Relación Dosis-Respuesta a DrogaRESUMEN
Tocopherols promote or inhibit growth in different cell types. In the hematopoietic system, the radioprotective property of tocopherols is thought to act through the expansion of primitive hematopoietic cells. However, the mechanisms activated by tocopherols and which HPs are affected remain poorly understood. To better address these questions, mice were treated with α-tocopherol, and its effects were investigated in the BM microenvironment. α-Tocopherol induced increased proliferation in HSC/HP cells, leading to BM hyperplasia. In addition, differentiation to the granulocytic/monocytic lineage was enhanced by α-tocopherol treatment. α-Tocopherol treatment resulted in decreased basal phosphorylation of ERK1/2, PKC, and STAT-5 in HSC/HP cells. In contrast, α-tocopherol enhanced ERK1/2 activation in response to IL-3 stimulation in HSC/HP cells without altering the expression of IL-3Rs. Moreover, α-tocopherol-induced differentiation and ERK1/2 activation were abolished in mice pretreated with a MEK inhibitor (PD98059); however, pretreatment with PD98059 did not reduce the α-tocopherol-mediated increase in HSC/HP cells but instead, further enhanced their proliferation. Therefore, α-tocopherol induces expansion of HSC/HP cells by a nonidentified intracellular pathway and granulocytic/monocytic differentiation through ERK1/2 activation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , alfa-Tocoferol/farmacología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/patología , Diferenciación Celular/fisiología , Proliferación Celular/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Hiperplasia , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismoRESUMEN
Chlorella vulgaris (CV) was examined for its modulating effects on the reduction induced by lead (Pb) on the numbers of marrow hematopoietic stem cells (HSCs) (c-Kit(+)Lin(-)), granulocyte-macrophage progenitors (Gr1(+)Mac1(+)) and total bone marrow cellularity. In mice gavage-treated daily with 50mg/kg dose of CV for 10 days, concomitant to a continuous offering of 1300 ppm lead acetate in drinking water, the treatment with the algae recovered the significantly reduced numbers of these cell populations to control values. As CV may have a myelostimulating effect through the induction of cytokines, we evaluated its modulating effects on the production of IL-1α, TNF-α, IFN-γ, IL-10 and IL-6. Our results demonstrated that lead significantly impairs the production of IFN-γ, IL-1α and TNF-α and increases the production of IL-10 and IL-6 and that these effects are successfully modulated by the CV treatment. The activity of NK cells, reduced in Pb-exposed animals, was raised to levels higher than those of controls in the exposed group treated with CV. Treatment with the algae also stimulated the production of IFN-γ, IL-1α, TNF-α and NK cells activity in normal mice. In addition, zinc bone concentrations, reduced in lead-exposed mice, were partially, but significantly, reversed by the treatment with CV.
Asunto(s)
Células de la Médula Ósea/fisiología , Chlorella vulgaris , Citocinas/metabolismo , Plomo/toxicidad , Animales , Células de la Médula Ósea/efectos de los fármacos , Proliferación Celular , Citocinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Fitoterapia , ZincRESUMEN
Even though the involvement of intracellular Ca(2+) Ca(i)(2+) in hematopoiesis has been previously demonstrated, the relationship between Ca(i)(2+) signaling and cytokine-induced intracellular pathways remains poorly understood. Herein, the molecular mechanisms integrating Ca(2+) signaling with the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in primary murine and human hematopoietic stem/progenitor cells stimulated by IL-3 and GM-CSF were studied. Our results demonstrated that IL-3 and GM-CSF stimulation induced increased inositol 1,4,5-trisphosphate (IP(3) ) levels and Ca(i)(2+) release in murine and human hematopoietic stem/progenitor cells. In addition, Ca(i)(2+) signaling inhibitors, such as inositol 1,4,5-trisphosphate receptor antagonist (2-APB), PKC inhibitor (GF109203), and CaMKII inhibitor (KN-62), blocked phosphorylation of MEK activated by IL-3 and GM-CSF, suggesting the participation of Ca(2+) -dependent kinases in MEK activation. In addition, we identify phospholipase Cγ2 (PLCγ2) as a PLCγ responsible for the induction of Ca(2+) release by IL-3 and GM-CSF in hematopoietic stem/progenitor cells. Furthermore, the PLCγ inhibitor U73122 significantly reduced the numbers of granulocyte-macrophage colony-forming units after cytokine stimulation. Similar results were obtained in both murine and human hematopoietic stem/progenitor cells. Taken together, these data indicate a role for PLCγ2 and Ca(2+) signaling through the modulation of MEK in both murine and human hematopoietic stem/progenitor cells.
Asunto(s)
Señalización del Calcio , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Células Madre Hematopoyéticas/enzimología , Interleucina-3/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Fosfolipasa C gamma/metabolismo , Adulto , Animales , Biomarcadores/metabolismo , Señalización del Calcio/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Separación Celular , Células Cultivadas , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/inmunología , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Fosfolipasa C gamma/antagonistas & inhibidores , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteínas Recombinantes , Factores de Tiempo , Adulto JovenRESUMEN
Angiotensin I-converting enzyme (ACE), a common element of renin-angiotensin system (RAS) and kallikrein-kinin system (KKS), is involved in myelopoiesis modulation, mainly by cleaving the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP). Based on this finding and in our results showing B1 and B2 kinin receptors expression in murine bone marrow (BM) cells, we evaluated the ACE influence on myelopoiesis of kinin B1 receptor knockout mice (B1KO) using long-term bone marrow cultures (LTBMCs). Captopril and AcSDKP were used as controls. Enhanced ACE activity, expressed by non-hematopoietic cells (Ter-199(-) and CD45(-)), was observed in B1KO LTBMCs when compared to wild-type (WT) cells. ACE hyperfunction in B1KO cells was maintained when LTBMCs from B1KO mice were treated with captopril (1.0microM) or AcSDKP (1.0nM). Although no alterations were observed in ACE mRNA and protein levels under these culture conditions, 3.0nM of AcSDKP increased ACE mRNA levels in WT LTBMCs. No alteration in the number of GM-CFC was seen in B1KO mice compared to WT animals, even when the former were treated with AcSDKP (10microg/kg) or captopril (100mg/kg) for 4 consecutive days. Hematological data also revealed no differences between WT and B1KO mice under basal conditions. When the animals received 4 doses of lipopolysaccharide (LPS), a decreased number of blood cells was detected in B1KO mice in relation to WT. We also found a decreased percentage of Gr1(+)/Mac-1(+), Ter119(+), B220(+), CD3(+), and Lin(-)Sca1(+)c-Kit(+) (LSK) cells in the BM of B1KO mice compared to WT animals. Low AcSDKP levels were observed in BM cultures from B1KO in comparison to WT cultures. We conclude that ACE hyperfunction in B1KO mice resulted in faster hydrolysis of AcSDKP peptide, which in turn decreased in BM tissues allowing HSC to enter the S stage of the cell cycle.
Asunto(s)
Mielopoyesis/efectos de los fármacos , Oligopéptidos/farmacología , Peptidil-Dipeptidasa A/metabolismo , Receptor de Bradiquinina B1/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Captopril/farmacología , Granulocitos/citología , Granulocitos/efectos de los fármacos , Sistema Calicreína-Quinina , Lipopolisacáridos/toxicidad , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peptidil-Dipeptidasa A/genética , Receptor de Bradiquinina B1/deficiencia , Receptor de Bradiquinina B1/genética , Sistema Renina-AngiotensinaRESUMEN
Previous studies reported by our group have introduced a new antitumoural drug called Biphosphinic Palladacycle Complex (BPC). In this paper we show that BPC causes apoptosis in leukaemia cells (HL60 and Jurkat), but not in normal human lymphocytes. IC(50) values obtained for both cell lines using the MTT and trypan blue exclusion assays 5h after BPC treatment were lower than 8.0 microM. Using metachromatic fluorophore, acridine orange, we observed that BPC elicited lysosomal rupture of leukaemic cells. Furthermore, BPC triggered caspase-3 and caspase-6 activation and apoptosis in cell lines, inducing chromatin condensation, apoptotic bodies, and DNA fragmentation. Interestingly, the lysosomal cathepsin B inhibitor CA074 markedly decreased BPC-induced caspase-3 and caspase-6 activation as well as cell death. Lysosomal BPC-induced membrane destabilisation was not dependent on reactive oxygen species generation, which was consistent with the absence of cellular HL60 and Jurkat membrane lipid peroxidation. We conclude that, following BPC treatment, lysosomal membrane rupture precedes cell death and the apoptotic signalling pathway is initiated by the release of cathepsin B in the cytoplasm of leukaemia cells. As no toxic effects for human lymphocytes were observed, we suggest that BPC is more selective for transformed cells, mainly due to their exacerbated lysosome expression.
Asunto(s)
Antineoplásicos/farmacología , Leucemia/tratamiento farmacológico , Compuestos Organometálicos/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Humanos , Células Jurkat , Leucemia/enzimología , Leucemia/patología , Peroxidación de Lípido , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The cell death mechanism of cytotoxicity induced by the Biphosphinic Palladacycle Complex (BPC) was studied using a K562 leukaemia cell line. The IC50 values obtained for K562 cells post-72 h of BPC were less than 5.0 microM by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and trypan blue assays. Using the Acridine Orange vital staining combining fluorescence microscopy it was observed that the complex triggers apoptosis in K562 cells, inducing DNA fragmentation, as analysed through electrophoresis. Lysosomal-membrane permeabilization was also observed in K562 cells post-5 h of BPC, which suggests intralysosomal accumulation by proton-trapping, since its pKa value ranged from 5.1 to 6.5. Caspase-3, and -6 activity induced by BPC in K562 cells was prevented by the cathepsin-B inhibitor [N-(L-3-trans-propylcarbamoyl-oxirane-2-carbonyl)-L-isoleucyl-L-proline] (CA074). These events occurred in the presence of endogenous bcl-2 and bax expression. Acute toxicological studies demonstrated that BPC produces no lesions for liver and kidney fourteen-days after drug administration (100 mg/kg--i.p.). White and red blood cells of BPC-treated mice presented normal morphological characteristics. Taken together, these data suggest a novel lysosomal pathway for BPC-induced apoptosis, in which lysosomes are the primary target and cathepsin B acts as death mediator.