RESUMEN
One of the main problems of present-day oncology is the ability of neoplastic cells to develop different mechanisms of resistance to chemotherapeutic agent. A natural compound oleanolic acid (OA) was found to be active against many types of neoplastic cells. This paper examines the influence of eight semisynthetic oleanolic acid derivatives on drug-sensitive human acute promyelocytic leukemia cell line HL-60 and its multidrug resistant subline ABCC1 overexpressing HL-60/AR. Viability inhibition, proapoptotic activity, as well as influence on the ABCC1 gene expression level, ability to inhibit the transport function of multidrug resistance associated protein 1 (ABCC1) and to alter its level by the tested compounds, were evaluated. The most potent compounds were DIOXOL (methyl 3,11-dioxoolean-12-en-28-oate) and HIMOXOL (methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate). DIOXOL was most efficient in inducing apoptosis of HL-60 cells. It activated both intrinsic and extrinsic pathways of apoptotic cell death. Proapoptotic properties of DIOXOL were probably related to the significant decrease of p65 NFκB level and inhibition of its translocation to the nucleus. In turn, HIMOXOL was the most potent compound against resistant HL-60/AR cells. It inhibited ABCC1 transport function (short time response) and decreased the level of ABCC1 protein (long time response) as a result of reduction of ABCC1 expression.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Ácido Oleanólico/análogos & derivados , Fragmentación del ADN/efectos de los fármacos , Resistencia a Múltiples Medicamentos , Células HL-60/efectos de los fármacos , Células HL-60/enzimología , Humanos , Leucemia Promielocítica Aguda/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Terapia Molecular Dirigida/métodos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Ácido Oleanólico/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
BACKGROUND: Acute myeloid leukemia (AML) recurrence is largely a result of multidrug resistance (MDR). We aimed to examine the role of 14-3-3ζ in AML chemosensitivity using HL-60 and vincristine-resistant HL-60/VCR cells. METHODS: The effects of 14-3-3ζ siRNA on the growth and cell cycle progression of HL-60 and HL-60/VCR cells were determined. The effect of 14-3-3ζ siRNA on topotecan (TPT)-induced apoptosis was evaluated by several assays. RESULTS: Compared to HL-60 cells, HL-60/VCR cells had increased 14-3-3ζ mRNA and protein expression. Increased mdr-1 mRNA as well as mdr-1, Bcl-2 and Mcl-1 protein expression were observed in HL-60/VCR cells. In both HL-60 and HL-60/VCR cells, 14-3-3ζ was observed in the cytoplasm and nuclear compartments. 14-3-3ζ siRNA significantly reduced HL-60 and HL-60/VCR cell growth after 48 h and increased the proportion of cells in the G0/G1 phase. Moreover, 14-3-3ζ siRNA significantly increased the sensitivity of both HL-60 and HL-60/VCR cells to TPT, possibly through the inhibition of Bcl-2, Mcl-1 and mdr-1 protein expression. CONCLUSIONS: Silencing of 14-3-3ζ increased the sensitivity of both sensitive and resistant HL-60 cells to TPT-induced apoptosis, possibly through altering the expression of apoptosis-associated proteins, suggesting that it may be a potential target for MDR AML.
Asunto(s)
Proteínas 14-3-3/fisiología , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Regulación Leucémica de la Expresión Génica , Células HL-60/enzimología , Proteínas de Neoplasias/fisiología , Proteínas 14-3-3/antagonistas & inhibidores , Proteínas 14-3-3/biosíntesis , Proteínas 14-3-3/genética , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/fisiología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Perfilación de la Expresión Génica , Células HL-60/efectos de los fármacos , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/biosíntesis , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , ARN Interferente Pequeño/farmacología , Fracciones Subcelulares/metabolismo , Inhibidores de Topoisomerasa I/farmacología , Topotecan/farmacología , Vincristina/farmacologíaRESUMEN
BACKGROUND: We recently reported that the MeOH extract from bulbs of Odontioda Marie Noel 'Velano' exhibited diverse biological activities but most of the activity was concentrated into the EtOAc layer separated by sequential organic solvent extractions. In the present study, the EtOAc layer was subjected to silica-gel column chromatography for further separation into five fractions, and the cytotoxicity and apoptosis-inducing activity of the fractions against human normal oral and tumor cells was further investigated. MATERIALS AND METHODS: Cytotoxic activity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The 50% cytotoxic concentration (CC(50)) was determined by the dose-response curve. Tumor specificity (TS) was determined by the ratio of the mean CC(50) for normal cells to the one of tumor cell lines. DNA fragmentation was assayed by agarose gel electrophoresis, caspase-3/-7 activation was monitored by cleavage of substrates either spectrophotometrically or by western blot analysis. RESULTS: Among five fractions, the most hydrophobic fraction (Fr. 1) showed the highest cytotoxicity against all cell lines tested, followed by Fr. 2 >Fr. 3 >Fr. 4 >Fr. 5, in order of increasing polarity. Fr. 2 had the highest tumor-specificity, followed by Fr. 3, Fr. 4, Fr. 1 and Fr. 5. Fr. 1 induced caspase-3 activation more potently in promyelocytic leukemia HL-60 cells, than in oral squamous cell carcinoma (OSCC) HSC-2 cells, whereas it did not induce internucleosomal DNA fragmentation in either of these cell lines. CONCLUSION: The present study suggests that hydrophobic substances in the EtOAc extract of Odontioda Marie Noel 'Velano' exhibit tumor-specific cytotoxicity without inducing apoptosis in the HSC-2 OSCC cell line.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Carcinoma de Células Escamosas/patología , Neoplasias de la Boca/patología , Orchidaceae/química , Extractos Vegetales/farmacología , Acetatos , Antineoplásicos Fitogénicos/aislamiento & purificación , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Cromatografía en Gel , Fragmentación del ADN/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Células HL-60/efectos de los fármacos , Células HL-60/enzimología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Raíces de Plantas/química , SolventesRESUMEN
BACKGROUND: We recently reported that the MeOH extract of aerial parts and root of Rhinacanthus nasutus showed diverse biological activity, with most activity being concentrated into the EtOAc layer separated by sequential organic solvent extractions. In the present study, the EtOAc extracts were further separated by silica-gel column chromatography into five fractions (Frs. 1-5), and their cytotoxicity and apoptosis-inducing activity investigated. MATERIALS AND METHODS: Cytotoxic activity was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method. The 50% cytotoxic concentration (CC(50)) was determined from the dose-response curve. Tumor specificity (TS) was determined by the ratio of the mean CC(50) for normal cells to the one for tumor cell lines. DNA fragmentation was assayed by agarose gel electrophoresis. Caspase-3/-7 activation was monitored by cleavage of substrates either spectrophotometrically or by western blot analysis. RESULTS: Among five fractions of the EtOAc extract, Fr. 1, eluted with CHCl(3)-MeOH (50:1), showed the highest tumor specificity (TS=3.3) as compared with other fractions eluted at higher concentrations of MeOH in CHCl(3) (TS=1.0-2.8). Fr. 1 did not induce internucleosomal DNA fragmentation or induced only marginal level of caspase-3 activity in either human promyelocytic leukemia HL-60 cells and human oral squamous cell carcinoma (OSCC) cell lines HSC-2. CONCLUSION: The present study suggests that hydrophobic substances of EtOAc extract show tumor specific cytotoxicity by inducing little or no apoptosis.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Carcinoma de Células Escamosas/patología , Magnoliopsida/química , Neoplasias de la Boca/patología , Extractos Vegetales/farmacología , Acetatos , Antineoplásicos Fitogénicos/aislamiento & purificación , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Cromatografía en Gel , Fragmentación del ADN/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Células HL-60/efectos de los fármacos , Células HL-60/enzimología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Raíces de Plantas/química , Tallos de la Planta/química , SolventesRESUMEN
δ-Elemene, an antitumor component, is a chemical compound isolated from Curcuma wenyujin, a Chinese traditional herb. We examined whether δ-elemene could inhibit cell growth and cell cycle progression and induce apoptosis in human leukemia HL-60 cells. The results demonstrated that δ-elemene induces significant apoptosis of HL-60 cells, as shown by MTT assay, annexin V (AnV) binding of externalized phosphatidylserine (PS), and the mitochondrial probe JC-1 using flow cytometry. HL-60 cells treated with δ-elemene showed high percentages in the early apoptotic and late apoptoctic/necrotic stages, as well as caspase-3 activation of HL-60 cells. By monitoring the changes in cell cycle profiles, we confirmed that δ-elemene could interfere with the cell cycle in the G2/M phase and induce apoptosis in HL-60 cells in a time-dependent manner. Caspase-3 plays a direct role in proteolytic cleavage of the cellular proteins responsible for progression to apoptosis. Therefore we examined apoptosis in HL-60 cells after exposure to δ-elemene and measured caspase-3 activities with or without Z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk, a broad-spectrum caspase inhibitor) pretreatment using flow cytometric analysis. The results showed that δ-elemene could induce caspase-3 activation as detected by the decrease in δ-elemene-induced caspase-3 activities after treatment with z-VAD-fmk. In the present study, δ-elemene activated typical caspase-dependent apoptosis in HL-60 cells, as demonstrated by an inhibitory effect of z-VAD-fmk on this cell death. During δ-elemene-induced apoptosis, cytochrome c and apoptosis-inducing factor were released into the cytosol and BAX was translocated from the cytosol to mitochondria. However, these were not prevented by z-VAD-fmk. In conclusion, our study demonstrated that δ-elemene could induce G2/M cell cycle transition and trigger apoptosis through a caspase-3-dependent pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasa 3/fisiología , Células HL-60/enzimología , Células HL-60/patología , Sesquiterpenos/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Caspasa 3/metabolismo , División Celular/efectos de los fármacos , Línea Celular Tumoral , Curcuma/química , Citocromos c/metabolismo , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Fase G2/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/metabolismo , Sesquiterpenos/antagonistas & inhibidores , Sesquiterpenos/aislamiento & purificación , Factores de Tiempo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
It has long been observed that cancer cells rely more on glycolysis to generate ATP and actively use certain glycolytic metabolic intermediates for biosynthesis. Hexokinase II (HKII) is a key glycolytic enzyme that plays a role in the regulation of the mitochondria-initiated apoptotic cell death. As a potent inhibitor of hexokinase, 3-bromopyruvate (3-BrPA) is known to inhibit cancer cell energy metabolism and trigger cell death, supposedly through depletion of cellular ATP. The current study showed that 3-BrPA caused a covalent modification of HKII protein and directly triggered its dissociation from mitochondria, leading to a specific release of apoptosis-inducing factor (AIF) from the mitochondria to cytosol and eventual cell death. Co-immunoprecipitation revealed a physical interaction between HKII and AIF. Using a competitive peptide of HKII, we showed that the dissociation of hexokinase II from mitochondria alone could cause apoptotic cell death, especially in the mitochondria-deficient rho(0) cells that highly express HKII. Interestingly, the dissociation of HKII itself did not directly affect the mitochondrial membrane potential, ROS generation, and oxidative phosphorylation. Our study suggests that the physical association between HKII and AIF is important for the normal localization of AIF in the mitochondria, and disruption of this protein complex by 3-BrPA leads to their release from the mitochondria and eventual cell death.
Asunto(s)
Hexoquinasa/metabolismo , Mitocondrias/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Piruvatos/farmacología , Apoptosis , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glucólisis , Células HL-60/efectos de los fármacos , Células HL-60/enzimología , Hexoquinasa/genética , Humanos , Linfoma/enzimología , Linfoma/patología , Mitocondrias/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacosRESUMEN
During the last years remission rates of more than 72% for arsenic(III)-oxide (As(2)O(3)) treatment in relapsed or refractory acute promyelocytic leukemia have been published. As(2)O(3) is under clinical investigation for therapy of leukemia and solid tumors. Due to the chemical affinity of arsenic and antimony, we analyzed the potency of antimony(III)-oxide (Sb(2)O(3)) to exert As(2)O(3)-like effects. Based on the same molar concentrations, lower efficacy in apoptosis induction and caspase-independent decrease of mitochondrial membrane potential was observed for Sb(2)O(3). No difference in sensitivity to As(2)O(3) or Sb(2)O(3) was detected in CEM cells when compared to their multiple drug resistant derivatives. Apoptosis was induced by combining sub-apoptotic concentrations of Sb(2)O(3) or As(2)O(3) with sub-apoptotic concentrations of DL: -buthionine-[S,R]-sulfoximine (BSO). Other modulators of the cellular redox system showed this effect to a lower extent and enhancement was not consistent for the different cell lines tested. Caspase inhibitors protected cell lines from Sb(2)O(3)- and As(2)O(3)-induced apoptosis. When BSO was added, the inhibitors lost their protective ability. The ability of modulators of the cellular redox system in clinically applicable concentrations to enhance the apoptotic effects of the two oxides in a synergistic way may be helpful to reduce their toxicity by optimizing their dose.
Asunto(s)
Antimonio/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Arsenicales/farmacología , Glutatión/fisiología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucemia de Células T/patología , Óxidos/farmacología , Trióxido de Arsénico , Butionina Sulfoximina/farmacología , Inhibidores de Caspasas , Caspasas/metabolismo , Línea Celular Tumoral/citología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Células HL-60/citología , Células HL-60/efectos de los fármacos , Células HL-60/enzimología , Humanos , Células K562/citología , Células K562/efectos de los fármacos , Células K562/enzimología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Oxidación-ReducciónRESUMEN
AIMS: The ability of concentrated supernatants from Lactobacillus plantarum to produce a disruption of plasma membrane in eukaryotic and prokaryotic cells has been examined. METHODS AND RESULTS: A strain of Lact. plantarum (tolerant to acid and bile salts and resistant to several antibiotics) was used. It inhibited the growth of pathogenic Escherichia coli and L. monocytogenes. Supernatants from Lact. plantarum were concentrated by centrifugation. Either E. coli or HL-60 cells (a human promyelocytic cell line) were treated in the presence of the concentrated supernatants. The effect of concentrated supernatants from Lact. plantarum on E. coli growth demonstrated a bacteriostatic activity and a loss of cell viability measured by sytox green staining. Concentrated supernatants were capable of disturbing plasma membrane in E. coli and of promoting a cytotoxic and lyctic action on HL-60 cells and on human erythrocytes, respectively. CONCLUSIONS: These results suggest that Lact. plantarum release an effective compound responsible for an important effect in the disruption of E. coli plasma membrane and for a cytototoxic activity on promyelocytic leukaemia cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first in vitro study about the antimicrobial and biological activities of concentrated supernatants from Lact. plantarum.
Asunto(s)
Antibiosis/fisiología , Escherichia coli/efectos de los fármacos , Células HL-60/efectos de los fármacos , Lactobacillus plantarum/fisiología , Probióticos/farmacología , Membrana Celular/efectos de los fármacos , Recuento de Colonia Microbiana , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Células HL-60/enzimología , Humanos , Hidroliasas/metabolismoRESUMEN
Two-electron reduction of quinones catalyzed by NAD(P)H:quinone oxidoreductase (NQO1) protects cells against oxidative stress and toxic quinones. In fact, low level of NQO1 activity is often associated with increased risk of developing different types of tumours and with toxic effects linked to environmental quinones. In a previous report we analyzed the relationship between the oxidative stress induced by UV radiation and CoQ10 content in Burkitt's lymphoma cell lines compared to HL-60. The basal content of CoQ10 in Raji cells was slightly higher compared to HL-60. Moreover, after irradiation or ubiquinone supplementation in the medium, reduced CoQ10 levels were higher in Raji and Daudi cells compared to HL-60. In the present work, in order to inquire if NQO1 plays a role in the CoQ reducing capacity observed in the lymphoblastoid cell lines, we analyzed the transcription and translation products of this gene in Raji and Daudi cells, compared to cell lines possessing low and high NQO1 activity. The amount of transcripts of this gene in lymphoblastoid cells was comparable to that observed in HL-60 cells (low activity), as well as the level of two alternatively spliced mRNAs; one of which is described for the first time in this work. From the genotype analysis of polymorphisms C609T and C465T we observed that HL-60, Raji and Daudi cells were all heterozygous. Furthermore, NQO1 enzyme activity and protein synthesis in the cytosol of Raji and Daudi cells were undetectable. Therefore in Burkitt's lymphoma cell lines the NQO1 gene is not efficiently translated and this effect is not related to (C609T) polymorphism. Further studies will be necessary to find the enzyme responsible for CoQ10 reducing activity observed in lymphoma cell lines. On the other hand, this result suggests a careful re-evaluation of data concerning loss of NQO1 activity and polymorphisms in tumour cells.
Asunto(s)
Linfoma de Burkitt/enzimología , NAD(P)H Deshidrogenasa (Quinona)/genética , Empalme Alternativo , Línea Celular Tumoral , Células HL-60/enzimología , Humanos , Células K562 , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Polimorfismo Genético , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
Introducción. El colesterol es necesario para la proliferación y la correcta progresión del ciclo celular. La inhibición sostenida de la biosíntesis de colesterol inhibe la citocinesis y da lugar a la aparición de células poliploides, efectos que pueden implicar la participación de las vías de estrés. Objetivo. Estudiar el efecto de la inhibición terminal de la biosíntesis de colesterol sobre la vía de p38 MAPK y su papel en la progresión del ciclocelular. Metodología. La inhibición de la biosíntesis de colesterol en las células promielocíticas humanasHL-60 se llevó a cabo con SKF 104976. En determinados casos, junto a este inhibidor, se inhibieron las vías de p38 MAPK y ERK1/2empleando SB 203580 y PD 98059,respectivamente. El ciclo celular se estudió por marcaje con yoduro de propidio e incorporación de bromodesoxiuridina y análisis por citometría de flujo, y la expresión de proteínas se analizó por Western blot. Resultados. La inhibición de la biosíntesis de colesterol produjo la acumulación de células enG2/M y una activación transitoria de p38 MAPK, efectos que fueron revertidos por las lipoproteínas de baja densidad (LDL), demostrando que se debían a la deficiencia de colesterol. En aquellas condiciones, la adición de SB 203580 aceleró la replicación del ADN acompañada de un aumento de la poliploidía. Contrariamente, la adición de PD98059 inhibió la síntesis de ADN. Ni la inhibición de p38 MAPK ni la de ERK impidieron la división de las células previamente tratadas con SKF104976 tras suplementarlas con LDL, ni de las células previamente sincronizadas en prometafase con nocodazol. Conclusiones. La inhibición de la biosíntesis de colesterol activa la vía de p38 MAPK a fin de impedir la poliploidía pero no tiene efecto sobre la culminación de la mitosis (AU)
Introduction. Cells require cholesterol for proliferation and correct progression of the cell cycle. In the absence of cholesterol, the cells fail tounder go cytokines is and polynucleated cells are generated. These effects could be mediated by stress signal transduction pathways. Objective. To study the effects of cholesterol biosynthesis inhibition on the activity of p38MAPK and to evaluate the role of this pathway on cell cycle progression. Methodology. Human leukemia cells (HL-60)were incubated in the presence of SKF 104976, an inhibitor of cholesterol biosynthesis. In some cases inhibitors of p38 MAPK and ERK1/2, namely SB203580 and PD 98059, were added to the medium. Cell cycle progression was studied by flow citometry, both DNA content and bromodeoxyuridine incorporation into DNA, and protein expression of p38 MAPK was analyzed by western blot. Results. Inhibition of cholesterol biosynthesis led to accumulation of cells in G2/M and a transient activation of p38 MAPK. These effects were reversed by supplementing the medium with LDL. The addition of SB 203580 accelerated DNA replication, which was accompanied by an increase of polyploidy. By contrast, the addition of PD98059 inhibited DNA synthesis. Lastly, neither the inhibition of p38 MAPK nor ERK affected the division of cells treated with the cholesterol biosynthesis inhibitor following LDL provision, or mitosis completion from metaphase of cells previously synchronized with nocodazole. Conclusions. Cholesterol deficiency induces p38MAPK pathway activation in order to prevent polyploidy, but has no effect on mitosis completion (AU)
Asunto(s)
Anticolesterolemiantes , Colesterol/análisis , Proteínas Adaptadoras del Transporte Vesicular/análisis , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Activación Enzimática , División Celular/fisiología , Colesterol/deficiencia , Inhibidores Enzimáticos/farmacología , Células HL-60/enzimología , Células HL-60/metabolismo , Electroforesis/métodos , Western Blotting/métodosRESUMEN
CHR-2797 is a novel metalloenzyme inhibitor that is converted into a pharmacologically active acid product (CHR-79888) inside cells. CHR-79888 is a potent inhibitor of a number of intracellular aminopeptidases, including leucine aminopeptidase. CHR-2797 exerts antiproliferative effects against a range of tumor cell lines in vitro and in vivo and shows selectivity for transformed over nontransformed cells. Its antiproliferative effects are at least 300 times more potent than the prototypical aminopeptidase inhibitor, bestatin. However, the mechanism by which inhibition of these enzymes leads to proliferative changes is not understood. Gene expression microarrays were used to profile changes in mRNA expression levels in the human promyelocytic leukemia cell line HL-60 treated with CHR-2797. This analysis showed that CHR-2797 treatment induced a transcriptional response indicative of amino acid depletion, the amino acid deprivation response, which involves up-regulation of amino acid synthetic genes, transporters, and tRNA synthetases. These changes were confirmed in other leukemic cell lines sensitive to the antiproliferative effects of CHR-2797. Furthermore, CHR-2797 treatment inhibited phosphorylation of mTOR substrates and reduced protein synthesis in HL-60 cells, both also indicative of amino acid depletion. Treatment with CHR-2797 led to an increase in the concentration of intracellular small peptides, the substrates of aminopeptidases. It is suggested that aminopeptidase inhibitors, such as CHR-2797 and bestatin, deplete sensitive tumor cells of amino acids by blocking protein recycling, and this generates an antiproliferative effect. CHR-2797 is orally bioavailable and currently undergoing phase II clinical investigation in the treatment of myeloid leukemia.
Asunto(s)
Aminoácidos/metabolismo , Aminopeptidasas/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glicina/análogos & derivados , Ácidos Hidroxámicos/farmacología , Aminopeptidasas/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Electroforesis en Gel de Poliacrilamida , Factor 2 Eucariótico de Iniciación/metabolismo , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Glicina/farmacología , Células HL-60/efectos de los fármacos , Células HL-60/enzimología , Células HL-60/patología , Humanos , Immunoblotting , Leucina/análogos & derivados , Leucina/farmacología , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Fragmentos de Péptidos/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Fosforilación/efectos de los fármacos , Proteínas Quinasas/metabolismo , Inhibidores de la Síntesis de la Proteína , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina-Treonina Quinasas TOR , Tiofenos/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Using three different differentiation agents (1alpha, 25 dihydroxyvitamin D3, all-trans-retinoic acid, and Am80), down-regulation of telomerase activity was found to be a common response during the monocytic or granulocytic differentiation of human acute myeloblastic leukemia cell line 60 (HL60) cells. Rapid down-regulation of telomerase transcription occurred during early differentiation of HL60 cells prior to G(1) arrest. Akt kinase activity was suppressed after 6 h of differentiation along with inhibition of telomerase activity, and the extent of the suppression that occurred while maintaining telomerase protein expression suggested the post-translational regulation of telomerase activity. Recombinant Akt dose-dependently increased telomerase activity, and telomerase was inhibited at the transcriptional and post-translational levels by LY294002, suggesting that PI-3K/Akt is one of the key signaling proteins involved in telomerase regulation. Each of the three differentiation agents caused a significant increase of signaling proteins (including Akt) at 3 days after the initiation of differentiation. Changes of acetyl-histone H4, which regulates transcription of the telomerase gene, were observed before the activation of Akt. This finding suggests that epigenetic control of telomerase transcription occurs before activation of Akt during the late stage of differentiation. These results indicate that telomerase activity is regulated by at least two mechanisms during granulocytic and monocytic differentiation, with one mechanism being transcriptional and the other being post-translational.
Asunto(s)
Diferenciación Celular/fisiología , Células HL-60/enzimología , Telomerasa/genética , Calcitriol/farmacología , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Cromatina/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HL-60/efectos de los fármacos , Células HL-60/patología , Humanos , Telomerasa/metabolismo , Tretinoina/farmacologíaRESUMEN
OBJECTIVE: The roles of phosphatidylinositol 3 (PI3K) and mitogen-activated protein kinases (MAPK) have been widely studied in terms of the differentiation process induced by several drugs (phorbol ester, vitamin D-3, retinoic acid, etc.), but their exact functions in leukemic cells' phenotype and their potential therapeutic role remain incompletely clarified. MATERIALS AND METHODS: In order to investigate this query, leukemia cells were cultured in presence of kinase inhibitors (KIs). Proliferation, apoptosis, and differentiation were analyzed at the cellular and molecular levels, using flow cytometry and reverse transcriptase quantitative polymerase chain reaction. RESULTS: SB203580, a P38 MAPK inhibitor, had no effect on cell proliferation, whereas LY294002, a PI3K inhibitor, and PD098059, a selective inhibitor of mitogen-activated extracellular regulated kinase (MEK) phosphorylation, arrested cells in G(0)/G(1). However, LY294002 and PD098059 acted using different mechanisms: LY294002 decreased the expression of phosphorylated S6RP, whereas PD098059 increased P21/waf1 antigen expression. SP600125, an inhibitor of N-terminal c-jun kinases, arrested cells in G(2) and induced an endoreplicative process. SP600125 increased p21 at both the mRNA and protein levels. G(2) blockage is dependent on the PI3K pathway and the endoreplicative process is dependent on the PI3K and extracellular regulated kinase (ERK) pathways and mRNA synthesis. On the other hand, PD098059 potentiated the apoptotic process induced by either SP600125 or LY294002. Modulation of the expression of CD11, CD15, CD18, and CD54 was cell-dependent. CONCLUSION: Our results suggest that KIs modulate proliferation of leukemia cells and that the MEK/ERK inhibitor, PD098059, in combination with either SP600125 or LY294002, could have clinical value.
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Leucemia/enzimología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Antracenos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Cromonas/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Flavonoides/farmacología , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Células HL-60/efectos de los fármacos , Células HL-60/enzimología , Humanos , Imidazoles/farmacología , Leucemia/tratamiento farmacológico , Leucemia/patología , Monocitos/efectos de los fármacos , Monocitos/enzimología , Morfolinas/farmacología , Piridinas/farmacología , Células U937/efectos de los fármacos , Células U937/enzimologíaRESUMEN
The ends of human chromosomes are protected from the degradation associated with cell division by 15-20 kb long segments of hexameric repeats of 5'-TTAGGG-3' termed telomeres. In normal cells telomeres lose up to 300 bp of DNA per cell division that ultimately leads to senescence; however, most cancer cells bypass this lifespan restriction through the expression of telomerase. hTERT, the catalytic subunit essential for the proper function of telomerase, has been shown to be expressed in approximately 90% of all cancers. In this study we investigated the hTERT inhibiting effects of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea catechins, in MCF-7 breast cancers cells and HL60 promyelocytic leukemia cells. Exposure to EGCG reduced cellular proliferation and induced apoptosis in both MCF-7 and HL60 cells in vitro, although hTERT mRNA expression was decreased only in MCF-7 cells when treated with EGCG. Furthermore, down-regulation of hTERT gene expression in MCF-7 cells appeared to be largely due to epigenetic alterations. Treatment of MCF-7 cells with EGCG resulted in a time-dependent decrease in hTERT promoter methylation and ablated histone H3 Lys9 acetylation. In conjunction with demethylation, further analysis showed an increase in hTERT repressor E2F-1 binding at the promoter. From these findings, we propose that EGCG is effective in causing cell death in both MCF-7 and HL60 cancer cell lines and may work through different pathways involving both anti-oxidant effects and epigenetic modulation.
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Catequina/análogos & derivados , Metilación de ADN/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Telomerasa/antagonistas & inhibidores , Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Catequina/farmacología , División Celular/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Factor de Transcripción E2F1/metabolismo , Femenino , Células HL-60/efectos de los fármacos , Células HL-60/enzimología , Histonas/metabolismo , Humanos , Lisina/metabolismo , Masculino , Metilación/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Telomerasa/genéticaRESUMEN
The expression of delta 5 desaturase (D5D), delta 6 desaturase (D6D) and delta 9 desaturase (D9D) was determined by RT-PCR in the human promyelocytic cell line HL60. During 72 h of culture with 10% FBS, D5D and D6D were upregulated 5 to 6-fold, whereas D9D approximately doubled. The addition of fatty acids (FAs) to the culture medium suppressed upregulation of all desaturases. N-3 and n-6 FA appeared to be more effective than n-9 or saturated FA. When FAs were added after 72 h, further upregulation during the next 24 h was suppressed for nearly all desaturases and FAs tested, except for D5D when oleic acid (OA) or stearic acid (SA) was added. In cells cultured with restricted amounts of FBS, desaturase expression increased with decreasing concentrations of FBS. Cellular FA content decreased by 60% in the neutral lipid fraction, whereas that of the phospholipid fraction decreased by 10% during 72 h of culture. The largest decrease occurred in the sum of n-3 and n-6 FA of the neutral lipid fraction, which was reduced by 83%, whereas the content of these FAs in the phospholipid fraction decreased by 32%. The results indicate that when the supply of FA to HL60 cells is limited, the intracellular content of n-3 and n-6 FA decreases and this leads to upregulation of the desaturases, particularly D5D and D6D. Since HL60 cells resemble human leukocytes, the results suggest that desaturase expression in leukocytes may be exploited as a biomarker for FA status.
Asunto(s)
Grasas Insaturadas en la Dieta/sangre , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos Insaturados/sangre , Regulación Enzimológica de la Expresión Génica , Células HL-60/enzimología , Biomarcadores/sangre , Línea Celular , delta-5 Desaturasa de Ácido Graso , Grasas Insaturadas en la Dieta/farmacología , Ácido Graso Desaturasas/efectos de los fármacos , Ácidos Grasos Esenciales/sangre , Ácidos Grasos Esenciales/deficiencia , Ácidos Grasos Esenciales/genética , Ácidos Grasos Insaturados/deficiencia , Ácidos Grasos Insaturados/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HL-60/efectos de los fármacos , Humanos , Linoleoil-CoA Desaturasa/efectos de los fármacos , Linoleoil-CoA Desaturasa/metabolismo , ARN Mensajero/análisis , ARN Mensajero/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estearoil-CoA Desaturasa/efectos de los fármacos , Estearoil-CoA Desaturasa/metabolismoRESUMEN
Methyl methanesulphonate (MMS) is a DNA damaging agent, which induces oxidative stress, ATP depletion, and consequently, cell death, in HL-60 and K562 cells. The cell death induced by MMS predominantly exhibited the morphological and biochemical hallmarks of necrosis. A minor population of dying cells exhibited apoptotic hallmarks, especially in K562 cell cultures. Cyclosporin A (CsA) was used to modulate the MMS-induced cell death. Our results indicated that CsA did not prevent cells from dying, but changed the mode of death from necrotic to apoptotic. Surprisingly, CsA enhanced oxidative stress and increased the overall number of dead cells. Based on these results, we conclude that the modulatory effect of CsA on MMS-induced cell death might arise from an interference by CsA with mitochondrial metabolism, rather than from inhibition of the MMS efflux mediated by P-glycoprotein.
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Ciclosporina/toxicidad , Células HL-60/efectos de los fármacos , Inmunosupresores/toxicidad , Células K562/efectos de los fármacos , Metilmetanosulfonato/toxicidad , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Apoptosis/efectos de los fármacos , Caspasa 3/biosíntesis , Supervivencia Celular/efectos de los fármacos , Daño del ADN , ADN de Neoplasias/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Células HL-60/enzimología , Células HL-60/patología , Humanos , Células K562/enzimología , Células K562/patología , Necrosis/inducido químicamente , Estrés Oxidativo/efectos de los fármacos , ARN Mensajero/metabolismo , ARN Neoplásico/análisisRESUMEN
Gambogic acid (GA) is the major active ingredient of gamboge secreted from a Chinese traditional medicine Garcinia hanburryi possessing potent anti-tumor activity. N-(2-ethoxyethyl)gambogamide (NG-18), a derivative of GA, also efficiently inhibits proliferation of cultured human tumor cells. The inhibition effect of NG-18 is associated with its ability to induce apoptosis. In the present study, NG-18 markedly induced leukemia HL-60 cells apoptosis, and the extrinsic and intrinsic apoptosis pathways were activated almost at the same time. NG-18-induced tumor cell apoptosis was associated with up-regulation of pro-apoptotic Bcl-2 family member Bax, and downregulation of anti-apoptotic protein Bcl-2. The NG-18-induced apoptosis was blocked completely by a pan-caspase inhibitor Z-VAD-FMK, indicating that caspases were functionally and actively involved in this process. The specific inhibition of caspase-8 activity using Z-IETD-FMK significantly blocked NG-18-induced apoptosis. In contrast, inhibition of other initiator caspases, caspase-2 or -9, using Z-VDVAD-FMK or Z-LEHD-FMK respectively had no effect on NG-18-induced apoptosis. Altogether, our data demonstrated that NG-18-induced apoptosis was dependent on caspases and caspase-8 acted as a key executor in the event.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 8/metabolismo , Proliferación Celular/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Garcinia/química , Xantonas/farmacología , Western Blotting , Citocromos c/metabolismo , Citometría de Flujo , Células HL-60/efectos de los fármacos , Células HL-60/enzimología , Células HL-60/patología , Humanos , Estructura Molecular , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Xantonas/química , Proteína bcl-X/metabolismoRESUMEN
Anti-cancer and anti-angiogenesis effects of green tea catechins have been demonstrated. It has been found that chemical modification of tea catechins improves their biological activities. We examined the chemical modification of epicatechin enhanced anti-cancer and anti-angiogenic effects. Epicatechin conjugated with fatty acid (acyl-catechin) strongly inhibited DNA polymerase activity, HL-60 cancer cell growth and angiogenesis. Epicatechin conjugated with palmitic acid ((2R,3R)-3',4',5,7-tetrahydroxyflavan-3-yl hexadecanoate, epicatechin-C16) was the strongest inhibitor in DNA polymerase alpha, beta, lambda and angiogenesis assays. Epicatechin-C16 also suppressed human endothelial cell (HUVEC) tube formation on reconstituted basement membrane, suggesting that it affected not only DNA polymerase activity but also the signal transduction pathways needed for the tube formation in HUVECs. These results suggest that acylation of epicatechin is an effective chemical modification to improve the anti-cancer activity of epicatechin.
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Inhibidores de la Angiogénesis/farmacología , Catequina/farmacología , Inhibidores Enzimáticos/farmacología , Células HL-60/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico , Ácido Palmítico/farmacología , Acilación , Animales , Capilares/citología , Capilares/efectos de los fármacos , Capilares/crecimiento & desarrollo , Catequina/análogos & derivados , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/crecimiento & desarrollo , Inhibidores Enzimáticos/química , Células HL-60/enzimología , Humanos , Masculino , Estructura Molecular , Ratas , Ratas WistarRESUMEN
Tightly controlled proteolysis is a defining feature of apoptosis and caspases are critical in this regard. Significant roles for non-caspase proteases in cell death have been highlighted. Staurosporine causes a rapid induction of apoptosis in virtually all mammalian cell types. Numerous studies demonstrate that staurosporine can activate cell death under caspase-inhibiting circumstances. The aim of this study was to investigate the proteolytic mechanisms responsible for cell death under these conditions. To that end, we show that inhibitors of serine proteases can delay cell death in one such system. Furthermore, through profiling of proteolytic activation, we demonstrate, for the first time, that staurosporine activates a chymotrypsin-like serine protease-dependent cell death in HL-60 cells independently, but in parallel with the caspase controlled systems. Features of the serine protease-mediated system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease and DNA degradation. We also demonstrate a staurosporine-induced activation of a putative 16 kDa chymotrypsin-like protein during apoptosis.
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Apoptosis/efectos de los fármacos , Serina Endopeptidasas/fisiología , Inhibidores de Serina Proteinasa/farmacología , Western Blotting , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Quimasas , Células HL-60/enzimología , Células HL-60/patología , Humanos , Serina Endopeptidasas/metabolismo , Estaurosporina/farmacología , Fracciones SubcelularesRESUMEN
In a previous study, we reported an antileukaemic activity of auranofin (AF), demonstrating its dual effects: on the induction of apoptotic cell death and its synergistic action with retinoic acid on cell differentiation. In this study, we investigated the downstream signalling events of AF-induced apoptosis to determine the molecular mechanisms of AF activity. Treatment of HL-60 cells with AF induced apoptosis in a concentration- and time-dependent manner. Western blot analysis showed that AF-induced apoptosis was accompanied by the activation of caspase-8, caspase-9, and caspase-3, and the release of cytochrome c from the mitochondria. The phosphorylation and kinase activities of p38 mitogen-activated protein kinase (p38 MAPK) increased gradually until 12 h after AF (2 microM) treatment, and p38 MAPK was also activated concentration-dependently. Pretreatment with SB203580, a specific inhibitor of p38 MAPK, significantly blocked DNA fragmentation and the cleavage of procaspase-8, procaspase-3, and poly-ADP-ribose polymerase (PARP), whereas SB203580 alone had no effect. Reactive oxygen species (ROS) were also detected within 1 h after AF treatment, and the antioxidant N-acetyl-L-cysteine (NAC) effectively protected the cells from apoptosis by inhibiting the phosphorylation of p38 MAPK and the activation of caspases. These results suggest that ROS generation and the subsequent activation of p38 MAPK are essential for the proapoptotic effects of AF in human promyelocytic leukaemia HL-60 cells.