RESUMEN
Galectin-12 is a tissue-specific galectin that has been largely defined by its role in the regulation of adipocyte differentiation and lipogenesis. This study aimed to evaluate the role of galectin-12 in the differentiation and polarization of neutrophils within a model of acute myeloid leukemia HL-60 cells. All-trans retinoic acid and dimethyl sulfoxide were used to induce differentiation of HL-60 cells which led to the generation of two phenotypes of neutrophil-like cells with opposite changes in galectin-12 gene (LGALS12) expression and different functional responses to N-formyl- l-methionyl- l-leucyl- l-phenylalanine. These phenotypes showed significant differences of differentially expressed genes on a global scale based on bioinformatics analysis of available Gene Expression Omnibus (GEO) data sets. We also demonstrated that HL-60 cells could secrete and accumulate galectin-12 in cell culture medium under normal growth conditions. This secretion was found to be entirely inhibited upon neutrophilic differentiation and was accompanied by an increase in intracellular lipid droplet content and significant enrichment of 22 lipid gene ontology terms related to lipid metabolism in differentiated cells. These findings suggest that galectin-12 could serve as a marker of neutrophilic plasticity or polarization into different phenotypes and that galectin-12 secretion may be influenced by lipid droplet biogenesis.
Asunto(s)
Galectinas , Leucemia Promielocítica Aguda , Neutrófilos , Humanos , Diferenciación Celular , Galectinas/metabolismo , Galectinas/genética , Células HL-60 , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patología , Metabolismo de los Lípidos/genética , Neutrófilos/metabolismo , Fenotipo , Tretinoina/farmacologíaRESUMEN
The G protein-coupled receptors, GPR43 (free fatty acid receptor 2, FFA2) and GPR41 (free fatty acid receptor 3, FFA3), are activated by short-chain fatty acids produced under various conditions, including microbial fermentation of carbohydrates. Previous studies have implicated this receptor energy homeostasis and immune responses as well as in cell growth arrest and apoptosis. Here, we observed the expression of both receptors in human blood cells and a remarkable enhancement in leukemia cell lines (HL-60, U937, and THP-1 cells) during differentiation. A reporter assay revealed that GPR43 is coupled with Gαi and Gα12/13 and is constitutively active without any stimuli. Specific blockers of GPR43, GLPG0974 and CATPB function as inverse agonists because treatment with these compounds significantly reduces constitutive activity. In HL-60 cells, enhanced expression of GPR43 led to growth arrest through Gα12/13 . In addition, the blockage of GPR43 activity in these cells significantly impaired their adherent properties due to the reduction of adhesion molecules. We further revealed that enhanced GPR43 activity induces F-actin formation. However, the activity of GPR43 did not contribute to butyrate-induced apoptosis in differentiated HL-60 cells because of the ineffectiveness of the inverse agonist on cell death. Collectively, these results suggest that GPR43, which possesses constitutive activity, is crucial for growth arrest, followed by the proper differentiation of leukocytes.
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Ácidos Grasos Volátiles , Leucocitos , Receptores de Superficie Celular , Humanos , Ácidos Grasos Volátiles/metabolismo , Leucocitos/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Diferenciación Celular , Células HL-60RESUMEN
The A3 adenosine receptor (AR) is an important inflammatory and immunological target. However, the underlying mechanisms are not fully understood. Here, we report the gene regulation in HL-60 cells treated acutely with highly selective A3AR agonist MRS5698, positive allosteric modulator (PAM) LUF6000, or both. Both pro- and anti-inflammatory genes, such as IL-1a, IL-1ß, and NFκBIZ, are significantly upregulated. During our observations, LUF6000 alone produced a lesser effect, while the MRS5698 + LUF6000 group demonstrated generally greater effects than MRS5698 alone, consistent with allosteric enhancement. The number of genes up- and down-regulated are similar. Pathway analysis highlighted the critical involvement of signaling molecules, including IL-6 and IL-17. Important upstream regulators include IL-1a, IL-1ß, TNF-α, NF-κB, etc. PPAR, which modulates eicosanoid metabolism, was highly downregulated by the A3AR agonist. Considering previous pharmacological results and mathematical modeling, LUF6000's small enhancement of genetic upregulation suggested that MRS5698 is a nearly full agonist, which we demonstrated in both cAMP and calcium assays. The smaller effect of LUF6000 on MRS5698 in comparison to its effect on Cl-IB-MECA was shown in both HL-60 cells endogenously expressing the human (h) A3AR and in recombinant hA3AR-expressing CHO cells, consistent with its HL-60 cell genetic regulation patterns. In summary, by using both selective agonists and PAM, we identified genes that are closely relevant to immunity and inflammation to be regulated by A3AR in differentiated HL-60 cells, a cell model of neutrophil function. In addition, we demonstrated the previously uncharacterized allosteric signaling-enhancing effect of LUF6000 in cells endogenously expressing the hA3AR.
RESUMEN
Modifications in sphingolipid (SL) metabolism and mitochondrial bioenergetics are key factors implicated in cancer cell response to chemotherapy, including chemotherapy resistance. In the present work, we utilized acute myeloid leukemia (AML) cell lines, selected to be refractory to various chemotherapeutics, to explore the interplay between SL metabolism and mitochondrial biology supportive of multidrug resistance (MDR). In agreement with previous findings in cytarabine or daunorubicin resistant AML cells, relative to chemosensitive wildtype controls, HL-60 cells refractory to vincristine (HL60/VCR) presented with alterations in SL enzyme expression and lipidome composition. Such changes were typified by upregulated expression of various ceramide detoxifying enzymes, as well as corresponding shifts in ceramide, glucosylceramide, and sphingomyelin (SM) molecular species. With respect to mitochondria, despite consistent increases in both basal respiration and maximal respiratory capacity, direct interrogation of the oxidative phosphorylation (OXPHOS) system revealed intrinsic deficiencies in HL60/VCR, as well as across multiple MDR model systems. Based on the apparent requirement for augmented SL and mitochondrial flux to support the MDR phenotype, we explored a combinatorial therapeutic paradigm designed to target each pathway. Remarkably, despite minimal cytotoxicity in peripheral blood mononuclear cells (PBMC), co-targeting SL metabolism, and respiratory complex I (CI) induced synergistic cytotoxicity consistently across multiple MDR leukemia models. Together, these data underscore the intimate connection between cellular sphingolipids and mitochondrial metabolism and suggest that pharmacological intervention across both pathways may represent a novel treatment strategy against MDR.
Asunto(s)
Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Leucemia/metabolismo , Mitocondrias/metabolismo , Fosforilación Oxidativa , Esfingolípidos/metabolismo , Citarabina/farmacología , Daunorrubicina/farmacología , Células HL-60 , Humanos , Leucemia/patología , Mitocondrias/patología , Vincristina/farmacologíaRESUMEN
Leukemia is a type of disease in which hematopoietic stem cells proliferate clonally at the genetic level. We discovered previously by high-resolution mass spectrometry that diallyl disulfide (DADS), which is one of the effective ingredients of garlic, reduces the performance of RhoGDI2 from APL HL-60 cells. Although RhoGDI2 is oversubscribed in several cancer categories, the effect of RhoGDI2 in HL-60 cells has remained unexplained. We aimed to investigate the influence of RhoGDI2 on DADS-induced differentiation of HL-60 cells to elucidate the association among the effect of inhibition or over-expression of RhoGDI2 with HL-60 cell polarization, migration and invasion, which is important for establishing a novel generation of inducers to elicit leukemia cell polarization. Co-transfection with RhoGDI2-targeted miRNAs apparently decreases the malignant biological behavior of cells and upregulates cytopenias in DADS-treated HL-60 cell lines, which increases CD11b and decreases CD33 and mRNA levels of Rac1, PAK1 and LIMK1. Meanwhile, we generated HL-60 cell lines with high-expressing RhoGDI2. The proliferation, migration and invasion capacity of such cells were significantly increased by the treated with DADS, while the reduction capacity of the cells was decreased. There was a reduction in CD11b and an increase in CD33 production, as well as an increase in the mRNA levels of Rac1, PAK1 and LIMK1. It also confirmed that inhibition of RhoGDI2 attenuates the EMT cascade via the Rac1/Pak1/LIMK1 pathway, thereby inhibiting the malignant biological behavior of HL-60 cells. Thus, we considered that inhibition of RhoGDI2 expression might be a new therapeutic direction for the treatment of human promyelocytic leukemia. The anti-cancer property of DADS against HL-60 leukemia cells might be regulated by RhoGDI2 through the Rac1-Pak1-LIMK1 pathway, which provides new evidence for DADS as a clinical anti-cancer medicine.
Asunto(s)
Leucemia , Inhibidor beta de Disociación del Nucleótido Guanina rho , Humanos , Compuestos Alílicos/farmacología , Diferenciación Celular/efectos de los fármacos , Disulfuros/farmacología , Células HL-60/efectos de los fármacos , Células HL-60/metabolismo , Leucemia/metabolismo , Leucemia/patología , Quinasas Lim/genética , Quinasas Lim/metabolismo , Quinasas p21 Activadas/metabolismo , Quinasas p21 Activadas/farmacología , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/farmacología , Inhibidor beta de Disociación del Nucleótido Guanina rho/efectos de los fármacos , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , ARN Mensajero , Invasividad Neoplásica/genética , Invasividad Neoplásica/patologíaRESUMEN
Acetylshikonin (ASK) is a natural naphthoquinone derivative of traditional Chinese medicine Lithospermum erythrorhyzon. It has been reported that ASK has bactericidal, anti-inflammatory and antitumour effects. However, whether ASK induces apoptosis and autophagy in acute myeloid leukaemia (AML) cells and the underlying mechanism are still unclear. Here, we explored the roles of apoptosis and autophagy in ASK-induced cell death and the potential molecular mechanisms in human AML HL-60 cells. The results demonstrated that ASK remarkably inhibited the cell proliferation, viability and induced apoptosis in HL-60 cells through the mitochondrial pathway, and ASK promoted cell cycle arrest in the S-phase. In addition, the increased formation of autophagosomes, the turnover from light chain 3B (LC3B) I to LC3B II and decrease of P62 suggested the induction of autophagy by ASK. Furthermore, ASK significantly decreased PI3K, phospho-Akt and p-p70S6K expression, while enhanced phospho-AMP-activated protein kinase (AMPK) and phospho-liver kinase B1(LKB1) expression. The suppression of ASK-induced the conversion from LC3B I to LC3B II caused by the application of inhibitors of AMPK (compound C) demonstrated that ASK-induced autophagy depends on the LKB1/AMPK pathway. These data suggested that the autophagy induced by ASK were dependent on the activation of LKB1/AMPK signalling and suppression of PI3K/Akt/mTOR pathways. The cleavage of the apoptosis-related markers caspase-3 and caspase-9 and the activity of caspase-3 induced by ASK were markedly reduced by inhibitor of AMPK (compound C), an autophagy inhibitor 3-methyladenine (3-MA) and another autophagy inhibitor chloroquine (CQ). Taken together, our data reveal that ASK-induced HL-60 cell apoptosis is dependent on the activation of autophagy via the LKB1/AMPK and PI3K/Akt-regulated mTOR signalling pathways.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Proteínas Proto-Oncogénicas c-akt , Proteínas Quinasas Activadas por AMP/metabolismo , Antraquinonas , Apoptosis , Autofagia , Caspasa 3 , Proliferación Celular , Células HL-60 , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
To migrate, cells assume a polarized morphology, extending forward with a leading edge with their trailing edge retracting back toward the cell body. Both cell extension and retraction critically depend on the organization and dynamics of the actin cytoskeleton, and the small, monomeric GTPases Rac and Rho are important regulators of actin. Activation of Rac induces actin polymerization and cell extension, whereas activation of Rho enhances acto-myosin II contractility and cell retraction. To coordinate migration, these processes must be carefully regulated. The myosin Myo9b, a Rho GTPase-activating protein (GAP), negatively regulates Rho activity and deletion of Myo9b in leukocytes impairs cell migration through increased Rho activity. However, it is not known whether cell motility is regulated by global or local inhibition of Rho activity by Myo9b. Here, we addressed this question by using Myo9b-deficient macrophage-like cells that expressed different recombinant Myo9b constructs. We found that Myo9b accumulates in lamellipodial extensions generated by Rac-induced actin polymerization as a function of its motor activity. Deletion of Myo9b in HL-60-derived macrophages altered cell morphology and impaired cell migration. Reintroduction of Myo9b or Myo9b motor and GAP mutants revealed that local GAP activity rescues cell morphology and migration. In summary, Rac activation leads to actin polymerization and recruitment of Myo9b, which locally inhibits Rho activity to enhance directional cell migration.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Miosinas/metabolismo , Seudópodos/metabolismo , Movimiento Celular/fisiología , Células Cultivadas , Proteínas Activadoras de GTPasa/genética , Humanos , Miosinas/genéticaRESUMEN
Oncostatin M (OSM) is essential in a wide range of inflammatory responses, and most OSM is produced by neutrophils in respiratory diseases. While resveratrol (RES) is regarded as an anti-inflammatory agent in a variety of conditions, the mechanism of OSM inhibition by RES in neutrophils remains to be elucidated. In this study, we investigated whether RES could inhibit OSM production in neutrophil-like differentiated (d)HL-60 cells. The effects of RES were measured by means of an enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blotting. Increases in production and mRNA expression of OSM resulted from the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) in neutrophil-like dHL-60 cells; however, these increases were downregulated by RES treatment. Exposure to GM-CSF led to elevations of phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt, and nuclear factor (NF)-kB. Treatment with RES induced downregulation of the phosphorylated levels of PI3K, Akt, and NF-κB in neutrophil-like dHL-60 cells. These results suggest that RES could be applicable to prevent and/or treat inflammatory disorders through blockade of OSM.
RESUMEN
Protein kinase CK2 is a potential target for the discovery of anticancer drugs. Flavonoids are reported to be effective CK2 inhibitors. Herein, based on structural trimming of flavonoids, a series of chromone-2-aminothiazole derivatives (1a-d, 2a-g, 4a-j, 5a-k) were designed and synthesized by hybridizing the chromone skeleton with 2-aminothiazole scaffold. Among these compounds, compound 5i was the most effective CK2 inhibitor (IC50 = 0.08 µM) and possessed potent anti-proliferative activity against HL-60 tumor cells (IC50 = 0.25 µM). Cellular thermal shift assay (CESTA) confirmed that 5i directly bound to the CK2, and the possible binding mode of 5i toward CK2 was also simulated. Further studies showed that 5i induced the apoptosis of HL-60 cells and arrested the cell cycle. Finally, western-blot analysis showed that 5i could inhibit the downstream of CK2, including α-catenin/Akt pathway and PARP/Survivin pathway.
Asunto(s)
Antineoplásicos , Quinasa de la Caseína II , Antineoplásicos/química , Cromonas/farmacología , Flavonoides , Humanos , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
A cathepsin B (Cat B)-responsive optical nanoprobe is designed and prepared for report of HL60 differentiation into macrophage. A peptide sequence FRFK is linked to fluorescein (FITC) via the distant amino group of its lysine and N-terminated with acrylic acid (AA) to yield a molecular fluorescent probe AA-FRFK (FITC). The molecular probe is further embedded in poly(lactic-co-glycolic acid) (PLGA) to form a fluorescent nanoprobe AA-FRFK (FITC)@PLGA. The resultant optical nanoprobe is degradable by lysosomal Cat B, which is expressed in macrophages with a level of 5-10 times of that in HL60 cells. As a result, a significant decrease in fluorescence intensity is associated with the differentiation process of HL60 to macrophage and can be used as an indication of the differentiation process. The findings may pave a way toward the development of a universal in vitro labeling strategy of exogenous stem cells for report of in vivo cell differentiation by a dual-mode imaging modality involving optical imaging and magnetic resonance imaging.
Asunto(s)
Catepsina B , Macrófagos , Diferenciación Celular , Fluoresceína-5-Isotiocianato/química , Células HL-60 , HumanosRESUMEN
Multidrug resistance (MDR), having a multifactorial nature, is one of the major clinical problems causing the failure of anticancer therapy. The aim of this study was to examine the antitumour effects of selected pyridinium salts, 1-methyl-3-nitropyridine chloride (MNP) and 3,3,6,6,10-pentamethyl-3,4,6,7-tetrahydro-[1,8(2H,5H)-dion]acridine chloride (MDION), on sensitive leukaemia HL60 cells and resistant topoisomerase II-defective HL60/MX2 cells. Cell growth was determined by the MTT test. Intracellular ROS level was measured with the aid of 2',7'-DCF-DA. The cell cycle distribution was investigated by performing PI staining. DSB formation was examined using the γ-H2AX histone phosphorylation assay. The activity of caspase-3 and caspase-8 was measured with the use of the FLICA test. The assays for examining the lysosome membrane permeabilization were carried out with the aid of LysoTracker Green DND-26. Both studied compounds exerted very similar cytotoxic activities towards sensitive HL60 cells and their MDR counterparts. They modulated the cellular ROS level in a dose-dependent and time-dependent manner and significantly increased the percentage of sensitive HL60 and resistant HL60/MX2 cells with sub-diploid DNA (sub-G1 fraction). However, the induction of DSB formation was not a significant mechanism of action of these pyridinium salts in studied cells. Both examined compounds triggered caspase-3/caspase-8-dependent apoptosis of sensitive HL60 cells and their MDR counterparts. Additionally, the findings of the study indicate that lysosomes may also participate in the programmed death of HL60 as well as HL60/MX2 cells induced by MDION. The data obtained in this work showed that both examined pyridinium salts, MNP and MDION, are able to retain high antileukaemic effects against multidrug resistant topoisomerase II-defective HL60/MX2 cells.
Asunto(s)
Antineoplásicos/farmacología , ADN-Topoisomerasas de Tipo II , Leucemia , Apoptosis , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Cloruros/farmacología , ADN-Topoisomerasas de Tipo II/metabolismo , Resistencia a Antineoplásicos , Células HL-60 , Humanos , Proteínas de Resistencia a Mixovirus/metabolismo , Proteínas de Resistencia a Mixovirus/farmacología , Especies Reactivas de Oxígeno/metabolismo , Sales (Química)/metabolismo , Sales (Química)/farmacologíaRESUMEN
Chemoresistance causes cancer relapse and metastasis, thus remaining the major obstacle to cancer therapy. While some light has been shed on the underlying mechanisms, it is clear that chemoresistance is a multifaceted problem strictly interconnected with the high heterogeneity of neoplastic cells. We utilized two different human cell lines, i.e., LoVo colon cancer and promyelocytic leukemia HL60 cells sensitive and resistant to doxorubicin (DXR), largely used as a chemotherapeutic and frequently leading to chemoresistance. LoVo and HL60 resistant cells accumulate less reactive oxygen species by differently modulating the levels of some pro- and antioxidant proteins. Moreover, the content of intracellular magnesium, known to contribute to protect cells from oxidative stress, is increased in DXR-resistant LoVo through the upregulation of MagT1 and in DXR-resistant HL60 because of the overexpression of TRPM7. In addition, while no major differences in mitochondrial mass are observed in resistant HL60 and LoVo cells, fragmented mitochondria due to increased fission and decreased fusion are detected only in resistant LoVo cells. We conclude that DXR-resistant cells evolve adaptive mechanisms to survive DXR cytotoxicity by activating different molecular pathways.
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Neoplasias del Colon/tratamiento farmacológico , Doxorrubicina/farmacología , Leucemia Promielocítica Aguda/tratamiento farmacológico , Magnesio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Antibióticos Antineoplásicos/farmacología , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Resistencia a Antineoplásicos , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Canales Catiónicos TRPM/metabolismoRESUMEN
A small library of derivatives carrying a polycyclic scaffold recently identified by us as a new privileged structure in medicinal chemistry was designed and synthesized, aiming at obtaining potent MDR reverting agents also endowed with antitumor properties. In particular, as a follow-up of our previous studies, attention was focused on the role of the spacer connecting the polycyclic core with a properly selected nitrogen-containing group. A relevant increase in reverting potency was observed, going from the previously employed but-2-ynyl- to a pent-3-ynylamino moiety, as in compounds 3d and 3e, while the introduction of a triazole ring proved to differently impact on the activity of the compounds. The docking results supported the data obtained by biological tests, showing, for the most active compounds, the ability to establish specific bonds with P-glycoprotein. Moreover, a multifaceted anticancer profile and dual in vitro activity was observed for all compounds, showing both revertant and antitumor effects on leukemic cells. In this respect, 3c emerged as a "triple-target" agent, endowed with a relevant reverting potency, a considerable antiproliferative activity and a collateral sensitivity profile.
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Antracenos/farmacología , Antineoplásicos/farmacología , Hidrocarburos Aromáticos con Puentes/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Succinimidas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antracenos/síntesis química , Antracenos/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Hidrocarburos Aromáticos con Puentes/síntesis química , Hidrocarburos Aromáticos con Puentes/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Succinimidas/síntesis química , Succinimidas/metabolismoRESUMEN
Genistein (GEN) has been shown to induce apoptotic cell death in various human cancer cells. L-asparaginase (Asp), a clinical drug for leukemia, has been shown to induce cell apoptosis in leukemia cells. No available information concerning GEN combined with Asp increased the cell apoptosis compared to GEN or Asp treatment alone. The objective of this study is to evaluate the anti-leukemia activity of GEN combined with Asp on human leukemia HL-60 cells in vitro. The cell viability, the distribution of cell cycle, apoptotic cell death, and the level of ΔΨm were examined by flow cytometric assay. The expressions of apoptosis-associated proteins were measured by western blotting. GEN combined with Asp revealed a more significant decrease in total viable cells and induced a higher percentage of G2/M phase arrest, DNA damage, and cell apoptosis than that of GEN or Asp treatment only in HL-60 cells. Furthermore, the combined treatments (GEN and Asp) showed a higher decrease in the level of ΔΨm than that of GEN or Asp treatment only. These results indicated that GEN combined with Asp induced mitochondria dysfunction by disrupting the mitochondrial membrane potential. The results from western blotting demonstrated that the treatment of GEN combined with Asp showed a higher increase in the levels of Bax and Bak (pro-apoptotic proteins) and an active form of caspase-3 and a higher decrease in Bcl-2 (anti-apoptotic protein) than that of GEN or Asp treatment alone. GEN significantly enhances the efficiency of Asp on cytotoxic effects (the induction of apoptosis) in HL-60 cells.
Asunto(s)
Genisteína , Leucemia , Apoptosis , Asparaginasa , Genisteína/farmacología , Células HL-60 , HumanosRESUMEN
Kaempferol is a polyphenol found in a variety of plants. Kaempferol exerts antitumor properties by affecting proliferation and apoptosis of cancer cells. We investigated whether kaempferol and its glycoside derivatives-kaempferol 3-O-[(6-O-E-caffeoyl)-ß-D-glucopyranosyl-(1â2)]-ß-D-galactopyranoside-7-O-ß-D-glucuropyranoside (P2), kaempferol 3-O-[(6-O-E-p-coumaroyl)-ß-D-glucopyranosyl-(1â2)]-ß-D-galactopyranoside-7-O-ß-D-glucuropyranoside (P5) and kaempferol 3-O-[(6-O-E-feruloyl)-ß-D-glucopyranosyl-(1â2)]-ß-D-galactopyranoside-7-O-ß-D-glucuropyranoside (P7), isolated from aerial parts of Lens culinaris Medik.-affect the antitumor activity of etoposide in human promyelocytic leukemia (HL-60) cells. We analyzed the effect of kaempferol and its derivatives on cytotoxicity, DNA damage, apoptosis, cell cycle progression and free radicals induced by etoposide. We demonstrated that kaempferol increases the sensitivity of HL-60 cells to etoposide but does not affect apoptosis induced by this drug. Kaempferol also reduces the level of free radicals generated by etoposide. Unlike kaempferol, some of its derivatives reduce the apoptosis of HL-60 cells (P2 and P7) and increase the level of free radicals (P2 and P5) induced by etoposide. Our results indicate that kaempferol and its glycoside derivatives can modulate the activity of etoposide in HL-60 cells and affect its antitumor efficacy in this way. Kaempferol derivatives may have the opposite effect on the action of etoposide in HL-60 cells compared to kaempferol.
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Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Daño del ADN , Etopósido/toxicidad , Quempferoles/farmacología , Estrés Oxidativo/efectos de los fármacos , Antioxidantes/farmacología , Ensayo Cometa , ADN/efectos de los fármacos , Células HL-60 , Humanos , Lens (Planta)/químicaRESUMEN
Acute myeloid leukemia is characterized by uncontrolled clonal proliferation of abnormal myeloid progenitor cells. Despite recent advances in the treatment of this disease, the prognosis and overall long-term survival for patients remain poor, which drives the search for new chemotherapeutics and treatment strategies. Piceatannol, a polyphenolic compound present in grapes and wine, appears to be a promising chemotherapeutic agent in the treatment of leukemia. The aim of the present study was to examine whether piceatannol induces autophagy and/or apoptosis in HL-60 human acute myeloid leukemia cells and whether HL-60 cells are able to acquire resistance to piceatannol toxicity. We found that piceatannol at the IC90 concentration of 14 µM did not induce autophagy in HL-60 cells. However, it induced caspase-dependent apoptosis characterized by phosphatidylserine externalization, disruption of the mitochondrial membrane potential, caspase-3 activation, internucleosomal DNA fragmentation, PARP1 cleavage, chromatin condensation, and fragmentation of cell nuclei. Our findings also imply that HL-60 cells are able to acquire resistance to piceatannol toxicity via mechanisms related to MRP1 activity. Our results suggest that the use of piceatannol as a potential chemotherapeutic agent may be associated with the risk of multidrug resistance, warranting its use in combination with other chemotherapeutic agents.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Leucemia Mieloide Aguda/metabolismo , Resveratrol/análogos & derivados , Estilbenos/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/química , Autofagia/efectos de los fármacos , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Células HL-60 , Humanos , Concentración 50 Inhibidora , Leucemia Mieloide Aguda/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Estructura Molecular , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Estilbenos/químicaRESUMEN
N-acetylcysteine (NAC), often used as an antioxidant-scavenging reactive oxygen species (ROS) in vitro, was recently shown to increase the cytotoxicity of other compounds through ROS-dependent and ROS-independent mechanisms. In this study, NAC itself was found to induce extensive ROS production in human leukemia HL-60 and U937 cells. The cytotoxicity depends on ROS-modulating enzyme expression. In HL-60 cells, NAC activated NOX2 to produce superoxide (O2â¢-). Its subsequent conversion into H2O2 by superoxide dismutase 1 and 3 (SOD1, SOD3) and production of ClO- from H2O2 by myeloperoxidase (MPO) was necessary for cell death induction. While the addition of extracellular SOD potentiated NAC-induced cell death, extracellular catalase (CAT) prevented cell death in HL-60 cells. The MPO inhibitor partially reduced the number of dying HL-60 cells. In U937 cells, the weak cytotoxicity of NAC is probably caused by lower expression of NOX2, SOD1, SOD3, and by the absence of MOP expression. However, even here, the addition of extracellular SOD induced cell death in U937 cells, and this effect could be reversed by extracellular CAT. NAC-induced cell death exhibited predominantly apoptotic features in both cell lines. Conclusions: NAC itself can induce extensive production of O2â¢- in HL-60 and U937 cell lines. The fate of the cells then depends on the expression of enzymes that control the formation and conversion of ROS: NOX, SOD, and MPO. The mode of cell death in response to NAC treatment bears apoptotic and apoptotic-like features in both cell lines.
Asunto(s)
Acetilcisteína/farmacología , Leucemia/genética , NADPH Oxidasa 2/genética , Estrés Oxidativo/efectos de los fármacos , Peroxidasa/genética , Superóxido Dismutasa/genética , Catalasa/genética , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Células U937RESUMEN
Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HOâ) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.
Asunto(s)
Diferenciación Celular , Radicales Libres/metabolismo , Monocitos/citología , Monocitos/metabolismo , Proteínas/metabolismo , Acetofenonas/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Espectroscopía de Resonancia por Spin del Electrón , Células HL-60 , Humanos , Radical Hidroxilo , Monocitos/efectos de los fármacos , NADP/metabolismo , Coloración y Etiquetado , Acetato de Tetradecanoilforbol/farmacología , Células U937RESUMEN
Helicobacter pylori neutrophil-activating protein (HP-NAP)-induced production of reactive oxygen species (ROS) by neutrophils and monocytes is regulated by pertussis toxin (PTX)-sensitive G proteins, whereas HP-NAP-induced cytokine secretion by monocytes is mediated by Toll-like receptor 2 (TLR2). However, it is unclear whether TLR2 participates in HP-NAP-induced cytokine secretion by neutrophils. Here, all-trans retinoic acid (ATRA)-induced differentiated HL-60 cells were first employed as a neutrophil model to investigate the molecular mechanisms underlying neutrophil responses to HP-NAP. HP-NAP-induced ROS production in ATRA-induced differentiated HL-60 cells is mediated by the PTX-sensitive heterotrimeric G protein-dependent activation of extracellular signal-regulated kinase 1/2 and p38-mitogen-activated protein kinase, which is consistent with the findings reported for human neutrophils. Next, whether TLR2 participated in HP-NAP-induced secretion of interleukin-8 (IL-8) was investigated in neutrophils and ATRA-induced differentiated HL-60 cells. In both cells, TLR2 participated in HP-NAP-induced IL-8 secretion but not HP-NAP-induced ROS production. Interestingly, PTX-sensitive G proteins also contributed to the HP-NAP-induced secretion of IL-8 from neutrophils and the differentiated HL-60 cells. Our ELISA-based binding assay further revealed the competitive binding of Pam3CSK4, a TLR2 agonist, and HP-NAP to TLR2, which suggests the presence of specific and direct interactions between HP-NAP and TLR2. Thus, HP-NAP directly interacts with and activates TLR2 to induce IL-8 secretion in neutrophils and ATRA-induced differentiated HL-60 cells.
Asunto(s)
Proteínas Bacterianas/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Interleucina-8/metabolismo , Neutrófilos/metabolismo , Receptor Toll-Like 2/metabolismo , Tretinoina/metabolismo , Línea Celular Tumoral , Proteínas de Unión al GTP/metabolismo , Células HL-60 , Humanos , Monocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Oncostatin M (OSM) plays a role in various inflammatory reactions, and neutrophils are the main source of OSM in pulmonary diseases. However, there is no evidence showing the mechanism of OSM production in neutrophils. While dexamethasone (Dex) has been known to exert anti-inflammatory activity in various fields, the precise mechanisms of OSM downregulation by Dex in neutrophils remain to be determined. Here, we examined how OSM is produced in neutrophil-like differentiated HL-60 cells. Enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blot analysis were utilized to assess the potential of Dex. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation resulted in OSM elevation in neutrophil-like dHL-60 cells. OSM elevation induced by GM-CSF is regulated by phosphatidylinositol 3-kinase (PI3K)/Akt/nuclear factor (NF)-kB signal cascades. GM-CSF stimulation upregulated phosphorylated levels of PI3K or Akt or NF-κB in neutrophil-like dHL-60 cells. Treatment with Dex decreased OSM levels as well as the phosphorylated levels of PI3K or Akt or NF-κB in neutrophil-like dHL-60 cells. Our findings show the potential of Dex in the treatment of inflammatory diseases via blocking of OSM.