RESUMO
Since cancer cells have different mitochondrial bioenergetic requirements than non-cancerous cells, therapeutic inhibition of its mitochondrial functionality continues to be an important target for anticancer drug discovery. In this study, a series of acylhydroquinones with different acyl-chain length, and their chlorinated derivatives, in the aromatic ring, synthesized by Fries rearrangement under microwave irradiation, were evaluated for their anticancer activity in two leukemia cell lines. Findings from the primary and secondary screening of the 18 acylhydroquinones, tested at 5 µM on acute promyelocytic leukemia HL-60 and acute lymphoblastic leukemia CEM cells lines, identified an acylchlorohydroquinone (12) with a highly selective anti-proliferative effect toward HL-60 cells. This compound induced S-phase arrest in the cell cycle progression of HL-60 cells with insignificant toxicity on leukemic CEM cells and non-cancerous Hs27 cells. In HL-60 leukemic cells, 12 triggered increased mitochondrial NADH oxidation, increased respiration in presence of oligomycin (state 4o), mitochondrial depolarization, and ROS production, suggesting an uncoupling of OXPHOS. This provoked a metabolic adaptation dependent on AMPK/ACC/autophagy axis, having the mitochondrial ß-oxidation a pro-survival role since the combination of 12 and etomoxir, a carnitine palmitoyl-transferase (CPT) inhibitor promoted extensive HL-60 cell death. Finally, 12-induced metabolic stress sensitized to HL-60 cells to cell death by the FDA-approved anti-leukemic drug ABT-199, a BH3 mimetic. Therefore, our results suggest that acylchlorohydroquinone is a promising scaffold in anti-promyelocytic leukemia drug research.
Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Hidroquinonas/química , Fosforilação Oxidativa/efeitos dos fármacos , Sulfonamidas/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Hidroquinonas/farmacologia , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Immunosenescence has been described as age-associated changes in the immune function which are thought to be responsible for the increased morbidity with age. Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in immune defense against tumor and microbial diseases. Interestingly, aging-related NK cell dysfunction is associated with features of aging such as tumor incidence, reduced vaccination efficacy, and short survival due to infection. It is known that NK cell effector functions are critically dependent on cytokines and metabolic activity. Our aim was to determine whether there is a difference in purified human NK cell function in response to high concentration of IL-2 between young and elder donors. Here, we report that the stimulation of human NK cells with IL-2 (2000â¯U/mL) enhance NK cell cytotoxic activity from both young and elderly donors. However, while NK cells from young people responded to IL-2 signaling by increasing mitochondrial mass and mitochondrial membrane potential, no increase in these mitochondrial functional parameters was seen in purified NK cells from elderly subjects. Moreover, as purified NK cells from the young exhibited an almost three-fold increase in PGC-1α expression after IL-2 (2000â¯U/mL) stimulation, PGC-1α expression was inhibited in purified NK cells from elders. Furthermore, this response upon PGC-1α expression after IL-2 stimulation promoted an increase in ROS production in NK cells from elderly humans, while no increase in ROS production was observed in NK cells of young donors. Our data show that IL-2 stimulates NK cell effector function through a signaling pathway which involves a PGC-1α-dependent mitochondrial function in young NK cells, however it seems that NK cells from older donors exhibit an altered IL-2 signaling which affects mitochondrial function associated with an increased production of ROS which could represent a feature of NK cell senescence.
Assuntos
Células Matadoras Naturais/metabolismo , Mitocôndrias/metabolismo , Biogênese de Organelas , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Interleucina-2/farmacologia , Células K562 , Células Matadoras Naturais/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Transdução de Sinais , Regulação para CimaRESUMO
Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2.
Assuntos
Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/fisiologia , Mitocôndrias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Oligomicinas/farmacologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genéticaRESUMO
Folic acid (FA) consumption at high levels has been associated with colon cancer risk. Several mechanisms have been proposed to explain this association. The Notch signal pathway has been implicated in the regulation of cellular proliferation. Our aim was to demonstrate that high concentrations of FA or its reduced form, 5-methyltetrahydrofolic acid (5-MTHF), increase colorectal carcinoma HT29 cell proliferation through an increase of Notch1 activation and to prove if the inhibition of Notch1 activation by gamma secretase inhibitor, reduce the effect of folic acid. HT29 cells were cultured in high (400 nM), low (20 nM), or 0 nM FA or 5-MTHF concentrations during 96 h with or without DAPT (gamma secretase inhibitor). Cell proliferation was determined by the methylthiazole tetrazolium method, and Notch1-intracellular domain (NICD) was analyzed by flow cytometry. HT29 cells exposed to 400 nM FA or 5-MTHF showed higher proliferation rate than those exposed to 20 nM of FA or 5-MTHF (P < 0.01) during 96 h. NICD expression increased at higher FA or 5-MTHF concentrations compared with lower concentrations (P < 0.01). This effect on proliferation was partially reversible when we blocked Notch1 activation with the inhibitor of γ-secretase (P < 0.05).These data suggest that high concentration of FA and 5-MTHF induce HT29 cell proliferation activating Notch1 pathway.
Assuntos
Proliferação de Células/efeitos dos fármacos , Ácido Fólico/farmacologia , Receptor Notch1/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Neoplasias do Colo/patologia , Receptor 1 de Folato/genética , Receptor 1 de Folato/metabolismo , Células HT29 , Humanos , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Transdução de Sinais , Tetra-Hidrofolatos/farmacologiaRESUMO
OBJECTIVES: Folate supplementation may be associated with an increased risk of developing several types of cancer and a derangement of immune function. Among the latter, Natural killer (NK) cells are involved in non-MHC-restricted natural immunity against malignant target cells. Abnormalities in NK cell number or function have been associated with a higher cancer risk. The aim of this study was to study in vitro the possible effect of different concentrations of 5-methyltetrahydrofolic acid (5-MTHF) or folic acid on NK cell cytotoxic function, and expression of the stimulatory and inhibitory receptors KIRDL4, KIRDL3, and NKG2D. METHODS: Volunteer-derived peripheral mononuclear cells (PBMC) and highly enriched NK cells (95% CD56+ CD16+) were grown in folic acid free-RPMI 1640, supplemented either with folic acid or 5-MTHF (15-100 nM) during 72 h to 96 h. RESULTS: No differences in the cytolytic activity of PBMC and enriched NK cells were observed. After 96 h of in vitro culture without folate or supplemented with FA or 5-MTHF (30 or 100 nM), there were no changes in the percentage of HPNK receptor-positive cells. CONCLUSIONS: Our data indicate that a high dose of 5-MTHF or folic acid does not influence NK cell function in vitro.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Ácido Fólico/farmacologia , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Neoplasias/imunologia , Receptores de Células Matadoras Naturais/metabolismo , Complexo Vitamínico B/farmacologia , Antígeno CD56/metabolismo , Suplementos Nutricionais , Feminino , Ácido Fólico/imunologia , Ácido Fólico/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Neoplasias/metabolismo , Receptores de IgG/metabolismo , Tetra-Hidrofolatos/imunologia , Tetra-Hidrofolatos/metabolismo , Tetra-Hidrofolatos/farmacologia , Complexo Vitamínico B/imunologia , Complexo Vitamínico B/metabolismoRESUMO
Regulatory T cells (Treg) are important in the development of immune tolerance under normal physiological conditions. However, in pathological situations such as cancer, Treg increases have been correlated with bad prognoses. Treg depletion can be achieved in vitro under several stimuli, including the activation of the purinergic P2X7 receptor. Our aim was to determine whether polymyxin B (PMB), which is a positive modulator of this receptor, could affect mice Treg depletion by ATP and related compounds. For that purpose, we evaluated by flow cytometry changes in Treg populations under several treatments with PMB and/or purinergic agonists and antagonists. We found that both ATP and NAD induce a dose-dependent decrease on the Treg CD4+ CD25+ population. PMB not only potentiated the effect of exogenous ATP and NAD, but also decreased the CD4+ CD25+ population when it was applied alone. While ATP mediated effects are related to the P2X7 receptor, PMB effects appear to be related to another mechanism. We conclude that PMB positively modulates the depletion of the CD4+ CD25+ population of Treg. Therefore PMB could constitute a non-canonical drug with potential use on Treg depletion and cancer treatment.
Assuntos
Antibacterianos/farmacologia , Depleção Linfocítica , Polimixina B/farmacologia , Agonistas Purinérgicos/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Sinergismo Farmacológico , Feminino , Imunofenotipagem , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Numerous investigations exist about the alterations that oxygen free radicals can provoke on biomolecules; these modifications can be prevented and/or reversed by different antioxidants agents. On the other hand, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), a stable nitrogen synthetic radical, is used to evaluate the antioxidant capacity of medicinal herbal products; however, the structural changes that this radical provoke on the herbal active principles are not clear yet. In this work, we compared the redox reactivity of oxygen free radicals and DPPH radical on phospholipids and protein thiol groups present in rat liver microsomes. Cu2+/ascorbate was used as generator system of oxygen free radical and as antioxidant, an extract of Buddleja globosa's leaves. Cu2+/ascorbate provoked microsomal lipid peroxidation, microsomal thiols oxidation and oxygen consumption; all of these phenomena were inhibited by B. globosa extract. On the other hand, DPPH was bleached in different extension by the herbal extract and phosphatidyl choline; beside, DPPH decreased microsomal thiols content, but this phenomenon were not prevented by the herbal extract. Furthermore, DPPH did not induce oxygen consumption and neither modified the oxygen consumption induced by Cu2+/ascorbate. Distinct redox mechanisms may explain the differences between the reactivity of DPPH and oxygen free radicals on biomolecules, which is discussed.
Assuntos
Oxidantes/farmacologia , Picratos/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Compostos de Bifenilo , Buddleja/química , Cobre/farmacologia , Flavonoides/análise , Flavonoides/farmacologia , Técnicas In Vitro , Indicadores e Reagentes , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Oxirredução , Consumo de Oxigênio/efeitos dos fármacos , Fenóis/análise , Fenóis/farmacologia , Fosfatidilcolinas/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Polifenóis , Ratos , Ratos Sprague-Dawley , Compostos de Sulfidrila/metabolismoRESUMO
BACKGROUND: Highly active antiretroviral therapy (HAART) in HIV/AIDS infection induces an important reduction of the viral load (VL) and an immune system reconstitution. CD4+ T lymphocyte count is the immunological measurement commonly used for the follow up of HIV/AIDS patients. AIM: To study prospectively the restoration of the innate immune system in patients with HIV/AIDS infection during their first year on HAART. PATIENTS AND METHODS: 25 naive HIV/AIDS patients, from San José Hospital and University of Chile Clinical Hospital, Santiago, Chile, were studied between years 2002-2003. Every 4 months after HAART initiation, CD3+, CD4+, CD8+ T lymphocytes and CD16/56+ natural killer (NK) cells were quantified by flow cytometry. NK cell cytotoxicity was measured using radioactive chrome liberation (Cr51). Tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) were measured in peripheral blood mononuclear cells and viral load was determined using Amplicor HIV-1 from Roche Diagnostics Systems. RESULTS: Thirteen of the 25 patients continued in the study. They were all males, average age 35 years old (23-50). At baseline average CD4+ count was 146 cells/microL (31-362) and average viral load was 82.000 copies/mL (4.000-290.000). A raise in CD3+, CD4+, CD8+, and CD16/56 cells was noted at months 9-12 of therapy. Viral load became undetectable in the same period. NK cell function was decreased at the beginning of the therapy (1-4 months), reaching its highest values at months 9-12. There was no significant change in IL-10. TNF-alpha increased in six patients during the study. CONCLUSIONS: In this group of patients, innate immunity was restored during HAART. These results should be confirmed in studies with a longer follow up period and also measuring cytokines such as MIP-1a, MIP-1ss and RANTES.
Assuntos
Terapia Antirretroviral de Alta Atividade , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV-1/imunologia , Imunidade Inata , Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Síndrome da Imunodeficiência Adquirida/imunologia , Adulto , Contagem de Linfócito CD4 , Seguimentos , Inibidores da Protease de HIV/uso terapêutico , HIV-1/efeitos dos fármacos , Humanos , Interleucina-10/sangue , Células Matadoras Naturais/efeitos da radiação , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Inibidores da Transcriptase Reversa/uso terapêutico , Fatores de Tempo , Fator de Necrose Tumoral alfa/sangue , Carga ViralRESUMO
A series of tricyclic hydroquinones, incorporating a carbonyl group in the ortho position relative to the phenol function, were tested as inhibitors of oxygen uptake against the TA3 mouse carcinoma cell line and its multidrug-resistant variant TA3-MTX-R. The title compound, which proved to be the most active one, also exhibited low micromolar dose-dependent growth inhibition of the human tumor U937 cell line (human monocytic leukemia). A tentative structure-activity relationship is proposed for these substances. A comparison between the cytotoxicities of the title compound and 4,4-dimethyl-5,8-dihydroxynaphthalene-1-one, with their activities as inhibitors of oxygen uptake by the TA3-MTX-R cell line, is presented. Also, the inhibition of oxygen uptake by 6-(4-methylpent-3-enyl)-1,4-naphthoquinone was determined and compared with its reported cytotoxicity toward P-388 (murine lymphocytic leukemia), A-549 (human lung carcinoma), HT-29 (human colon carcinoma), and MEL-28 (human melanoma) cells. The inhibition of oxygen uptake by TA3-MTX-R cells is useful as a quick test for preliminary screening of possible anticancer activity.
Assuntos
Antracenos/química , Antracenos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Naftalenos/química , Naftalenos/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Camundongos , Neoplasias/metabolismo , Relação Estrutura-AtividadeRESUMO
Peripheral blood mononuclear cell (PBMC) cytotoxicity against S. typhi (wild type or mutant strain TYT1231)-infected U937 cells was significantly higher than its lytic effect against noninfected cells (control) at the various effector-to-target cell ratio used (30:1, 50:1 and 70:1). Natural killer cell activity [expressed as % specific lysis (mean +/- SEM); 30:1 (25.4 +/- 3.6, 25.1 +/- 4.2 and 16.3 +/- 3.3); 50:1 (27.8 +/- 3.7, 26.7 +/- 4.5 and 20.9 +/- 2.9) and 70:1 ratio (33.2 +/- 5.9, 29.4 +/- 4.2 and 22.8 +/- 2.8), respectively] appeared to be dependent on such ratios and independent of the S strain studied. Most (80%) of individual samples tested showed at least a 20% specific lysis increase over their own control; essentially no changes or smaller increases in NKC activity were observed in all other samples. Similar results were obtained when using highly purified NKC (HPNKC) preparations as effector cells [NKC activity (mean +/- SEM); 5:1 (46.2 +/- 4.7, 43.2 +/- 5.0 and 25.2 +/- 2.3) and 10:1 effector-to-target cell ratio (49.3 +/- 4.9, 44.7 +/- 5.2 and 27.2 +/- 2.6, respectively)]. All individual samples tested showed at least a 20% specific lysis increase over their own control. These results show that S. typhi-infected U937 cells are a significantly better target for NKCs than control cells and indicate that intracellular bacteria survival capacity is not a critical factor for infected cells becoming a NKC target.