RESUMO
Through its classic ATP-dependent ion-pumping function, basolateral Na/K-ATPase (NKA) generates the Na+ gradient that drives apical Na+ reabsorption in the renal proximal tubule (RPT), primarily through the Na+ /H+ exchanger (NHE3). Accordingly, activation of NKA-mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical (NHE3) Na+ reabsorption. In contrast, activation of the more recently discovered NKA signaling function triggers cellular redistribution of RPT NKA and NHE3 and decreases Na+ reabsorption. We used gene targeting to test the respective contributions of NKA signaling and ion pumping to the overall regulation of RPT Na+ reabsorption. Knockdown of RPT NKA in cells and mice increased membrane NHE3 and Na+ /HCO3 - cotransporter (NBCe1A). Urine output and absolute Na+ excretion decreased by 65%, driven by increased RPT Na+ reabsorption (as indicated by decreased lithium clearance and unchanged glomerular filtration rate), and accompanied by elevated blood pressure. This hyper reabsorptive phenotype was rescued upon crossing with RPT NHE3-/- mice, confirming the importance of NKA/NHE3 coupling. Hence, NKA signaling exerts a tonic inhibition on Na+ reabsorption by regulating key apical and basolateral Na+ transporters. This action, lifted upon NKA genetic suppression, tonically counteracts NKA's ATP-driven function of basolateral Na+ reabsorption. Strikingly, NKA signaling is not only physiologically relevant but it also appears to be functionally dominant over NKA ion pumping in the control of RPT reabsorption.
Assuntos
Túbulos Renais , Sódio , Animais , Camundongos , Trocador 3 de Sódio-Hidrogênio , ATPase Trocadora de Sódio-Potássio , Trifosfato de AdenosinaRESUMO
Iron is a key element in cell function; however, its excess in iron overload conditions can be harmful through the generation of reactive oxygen species (ROS) and cell oxidative stress. Activity of Na,K-ATPase has been shown to be implicated in cellular iron uptake and iron modulates the Na,K-ATPase function from different tissues. In this study, we determined the effect of iron overload on Na,K-ATPase activity and established the role that isoforms and conformational states of this enzyme has on this effect. Total blood and membrane preparations from erythrocytes (ghost cells), as well as pig kidney and rat brain cortex, and enterocytes cells (Caco-2) were used. In E1-related subconformations, an enzyme activation effect by iron was observed, and in the E2-related subconformations enzyme inhibition was observed. The enzyme's kinetic parameters were significantly changed only in the Na+ curve in ghost cells. In contrast to Na,K-ATPase α2 and α3 isoforms, activation was not observed for the α1 isoform. In Caco-2 cells, which only contain Na,K-ATPase α1 isoform, the FeCl3 increased the intracellular storage of iron, catalase activity, the production of H2O2 and the expression levels of the α1 isoform. In contrast, iron did not affect lipid peroxidation, GSH content, superoxide dismutase and Na,K-ATPase activities. These results suggest that iron itself modulates Na,K-ATPase and that one or more E1-related subconformations seems to be determinant for the sensitivity of iron modulation through a mechanism in which the involvement of the Na, K-ATPase α3 isoform needs to be further investigated.
Assuntos
Trifosfato de Adenosina/metabolismo , Cloretos/química , Compostos Férricos/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células CACO-2 , Cloretos/metabolismo , Enterócitos/citologia , Enterócitos/metabolismo , Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Compostos Férricos/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ratos , ATPase Trocadora de Sódio-Potássio/genética , SuínosRESUMO
Changes in metabolism are mechanisms that are largely implicated in the development, progression, and metastasis of head and neck squamous cell carcinoma (HNSCC) and also in resistance to different anticancer therapies. Identification of biomarkers for differentiation between cancerous and normal epithelium, treatment design and prognosis remain a vital issue in the field of head and neck cancer. The present study analyzed the main biochemical changes that occur in HNSCC tumors by through mechanisms involving oxidative stress. The release of substances reactive to thiobarbituric acid was significantly lower in HNSCC tumor tissue as compared to healthy tissue. The assays related to the lipid profile assays showed changes in membrane biophysics of tumor cells due to an increase in total phospholipids and total cholesterol, as well as an increased activity and expression of the α1 subunit of Na, K-ATPase, which is fundamental in the process of carcinogenesis. The modulation of the antioxidant system was also affected, with a decrease in the catalytic activity of the enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), as well as a reduction of glutathione (GSH) content and an increase in H2O2 content. A reduction in catalase (CAT) activity was observed. The data presented here are in accordance with important findings described by us in a previous study, involving the same individuals, but with a focus on the damage generated in red blood cells, resulting from tumor installation. Therefore, it was possible to conclude that the biochemical alterations found in HNSCC cells are fundamental for transformation and maintenance of the tumor cell and once it is installed, it is also capable of generating injuries in the patients' red blood cells. Our data demonstrate that this could be a promising biomarker for HNSCC.
Assuntos
Neoplasias de Cabeça e Pescoço , Estresse Oxidativo , Adenosina Trifosfatases , Humanos , Peróxido de Hidrogênio , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Cardiac glycosides (CGs) are useful drugs to treat cardiac illnesses and have potent cytotoxic and anticancer effects in cultured cells and animal models. Their receptor is the Na+,K+ ATPase, but other plasma membrane proteins might bind CGs as well. Herein, we evaluated the short- and long-lasting cytotoxic effects of the natural cardenolide glucoevatromonoside (GEV) on non-small-cell lung cancer H460 cells. We also tested GEV effects on Na+,K+ -ATPase activity and membrane currents, alone or in combination with selected chemotherapy drugs. GEV reduced viability, migration, and invasion of H460 cells spheroids. It also induced cell cycle arrest and death and reduced the clonogenic survival and cumulative population doubling. GEV inhibited Na+,K+-ATPase activity on A549 and H460 cells and purified pig kidney cells membrane. However, it showed no activity on the human red blood cell plasma membrane. Additionally, GEV triggered a Cl-mediated conductance on H460 cells without affecting the transient voltage-gated sodium current. The administration of GEV in combination with the chemotherapeutic drugs paclitaxel (PAC), cisplatin (CIS), irinotecan (IRI), and etoposide (ETO) showed synergistic antiproliferative effects, especially when combined with GEV + CIS and GEV + PAC. Taken together, our results demonstrate that GEV is a potential drug for cancer therapy because it reduces lung cancer H460 cell viability, migration, and invasion. Our results also reveal a link between the Na+,K+-ATPase and Cl- ion channels.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas , Cardenolídeos/farmacologia , Neoplasias Pulmonares , Proteínas de Neoplasias/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Células A549 , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Citotoxinas/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologiaRESUMO
Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous and complex disease, both from a clinical and molecular point of view. The prolonged use of alcohol and tobacco, along with the release of tumor secretions can modulate blood cells, such as erythrocytes. Here, this study was conducted with 24 patients diagnosed with HNSCC and an equal number of healthy individuals are matched by age and gender. The levels of lipid peroxidation were measured using the individual plasma, while for lipid concentrations, identification and quantification Na, K-ATPase activity and osmotic fragility, the red blood cell concentrate were used. The release of TBARS was significantly higher in patients with HNSCC. The lipid profile assays demonstrated a rearrangement of the erythrocyte membrane due to a decrease in total phospholipids and phosphatidylethanolamine followed by an increase in total cholesterol and phosphatidylcholine. Na, K-ATPase activity also increased. Erythrocytes were more fragile in patients with HNSCC than in health individuals. Therefore, the membrane of erythrocytes were rearranged and Na, K-ATPase function altered in the HNSCC patients. Our findings suggests that the alcohol, tobacco and tumor secretion modulate in a specific manner that the erythrocytes membranes of these patients making this system a potential tool for HNSCC biomarker of tumor progression.
Assuntos
Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Biomarcadores , Estudos de Casos e Controles , Humanos , Peroxidação de Lipídeos , Lipídeos de Membrana/metabolismo , Fragilidade Osmótica , Estresse Oxidativo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Iron is an essential element for human life. However, it is a pro-oxidant agent capable of reacting with hydrogen peroxide. An iron overload can cause cellular changes, such as damage to the plasma membrane leading to cell death. Effects of iron overload in cellular biochemical processes include modulating membrane enzymes, such as the Na, K-ATPase, impairing the ionic transport and inducing irreversible damage to cellular homeostasis. To avoid such damage, cells have an antioxidant system that acts in an integrated manner to prevent oxidative stress. In addition, the cells contain proteins responsible for iron transport and storage, preventing its reaction with other substances during absorption. Moreover, iron is associated with cellular events coordinated by iron-responsive proteins (IRPs) that regulate several cellular functions, including a process of cell death called ferroptosis. This review will address the biochemical aspects of iron overload at the cellular level and its effects on important cellular structures.
Assuntos
Sobrecarga de Ferro , Humanos , Peróxido de Hidrogênio , Ferro , Estresse Oxidativo , Espécies Reativas de OxigênioRESUMO
Cardiotonic steroids (CTS) are agents traditionally known for their capacity to bind to the Na,K-ATPase (NKA), affecting the ion transport and the contraction of the heart. Natural CTS have been shown to also have effects on cell signaling pathways. With the goal of developing a new CTS derivative, we synthesized a new digoxin derivative, 21-benzylidene digoxin (21-BD). Previously, we have shown that this compound binds to NKA and has cytotoxic actions on cancer, but not on normal cells. Here, we further studied the mechanisms of actions of 21-BD. Working with HeLa cells, we found that 21-BD decreases the basal, as well as the insulin stimulated proliferation. 21-BD reduces phosphorylation of the epidermal growth factor receptor (EGFR) and extracellular-regulated kinase (ERK), which are involved in pathways that stimulate cell proliferation. In addition, 21-BD promotes apoptosis, which is mediated by the translocation of Bax from the cytosol to mitochondria and the release of mitochondrial cytochrome c to the cytosol. 21-BD also activated caspases-8, -9 and -3, and induced the cleavage of poly (ADP-ribose) polymerase-1 (PARP-1). Altogether, these results show that the new compound that we have synthesized exerts cytotoxic actions on HeLa cells by inhibition of cell proliferation and the activation of both the extrinsic and intrinsic apoptotic pathways. These results support the relevance of the cardiotonic steroid scaffold as modulators of cell signaling pathways and potential agents for their use in cancer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Digoxina/análogos & derivados , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Digoxina/química , Digoxina/farmacologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Conformação Molecular , Inibidores de Proteínas Quinases/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Recently, cardiotonic steroids (CTS) have been shown to lead to the activation of Na,K-ATPase at low concentrations in brain, promoting neuroprotection against ischemia. We report here the results of the use of digoxin and its semisynthetic derivatives BD-14, BD-15, and BD-16 against partial chemical ischemic induction followed by reperfusion in murine neuroblastoma cells neuro-2a (N2a). For chemical ischemic induction, sodium azide (5 mM) was used for 5 hours, and then reperfusion was induced for 24 hours. Na,K-ATPase activity and protein levels were analyzed in membrane preparation of N2a cells pretreated with the compounds (150 nM), in the controls and in induced chemical ischemia. In the Na,K-ATPase activity and protein levels assays, the steroids digoxin and BD-15 demonstrated a capacity to modulate the activity of the enzyme directly, increasing its levels of expression and activity. Oxidative parameters, such as superoxide dismutase (SOD) activity, lipid peroxidation (thiobarbituric acid reactive substance), glutathione peroxidase (GPx), glutathione (GSH) levels, hydrogen peroxide content, and the amount of free radicals (reactive oxygen species) during induced chemical ischemia were also evaluated. Regarding the redox state, lipid peroxidation, hydrogen peroxide content, and GPx activity, we have observed an increase in the chemical ischemic group, and a reduction in the groups treated with CTS. SOD activity increased in all treated groups when compared to control and GSH levels decreased when treated with sodium azide and did not change with CTS treatments. Regarding the lipid profile, we saw a decrease in the content of phospholipids and cholesterol in the chemical ischemic group, and an increase in the groups treated with CTS. In conclusion, the compounds used in this study demonstrate promising results, since they appear to promote neuroprotection in cells exposed to chemical ischemia.
Assuntos
Digoxina/farmacologia , Expressão Gênica/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Animais , Isquemia Encefálica/prevenção & controle , Células CACO-2 , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Digoxina/análogos & derivados , Digoxina/síntese química , Glutationa/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Modelos Biológicos , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/síntese química , Estresse Oxidativo/efeitos dos fármacos , Fosfolipídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Azida Sódica/antagonistas & inibidores , Azida Sódica/farmacologia , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Cardiac glycosides (CGs) are natural compounds widely used to treat several cardiac conditions and more recently have been recognized as potential antitumor agents. They are known as Na,K-ATPases ligands, which is a promising drug target in cancer. In this study, the short and long-lasting cytotoxic effects of the natural cardenolide digitoxigenin monodigitoxoside (DGX) were evaluated against two non-small cell lung cancer lines (A549 and H460 cells). It was found that DGX induced cytotoxic effects in both cells and the apoptotic effects were more pronounced on H460 cells. In long-term analysis, using the clonogenic and the cumulative population doubling (CPD) assays, DGX showed a reduction of cell survival, after 15days without re-treatment. To better understand DGX effects in A549 cells, several assays were conducted. In cell cycle analysis, DGX caused an arrest in S and G2/M phases. This compound also increased the number of cells in subG1 phase in a concentration- and time-dependent manner. The presence of ß-galactosidase positive cells, large nucleus and flattened cells indicated senescence. Additionally, DGX inhibited Na,K-ATPase activity in A549 cells, as well as in purified pig kidney and in human red blood cell membrane preparations, at nanomolar range. Moreover, results of molecular docking showed that DGX binds with high efficiency (-11.4Kcal/mol) to the Na,K-ATPase (PDB:4HYT). Taken together, our results highlight the potent effects of DGX both in A549 and H460 cells, and disclose its link with Na,K-ATPase inhibition.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Digitoxigenina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Células A549 , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Digitoxigenina/farmacologia , Humanos , Neoplasias Pulmonares/patologia , Simulação de Acoplamento Molecular , Suínos , Fatores de TempoRESUMO
Digoxin and other cardiotonic steroids (CTS) exert their effect by inhibiting Na,K-ATPase (NKA) activity. CTS bind to the various NKA isoforms that are expressed in different cell types, which gives CTS their narrow therapeutic index. We have synthesised a series of digoxin derivatives (γ-Benzylidene digoxin derivatives) with substitutions in the lactone ring (including non-oxygen and ether groups), to obtain CTS with better NKA isoform specificity. Some of these derivatives show some NKA isoform selective effects, with BD-3, BD-8, and BD-13 increasing NKA α2 activity, BD-5 inhibiting NKA α1 and NKA α3, BD-10 reducing NKA α1, but stimulating NKA α2 and α3; and BD-14, BD-15, and BD-16 enhancing NKA α3 activity. A molecular-docking approach favoured NKA isoform specific interactions for the compounds that supported their observed activity. These results show that BD compounds are a new type of CTS with the capacity to target NKA activity in an isoform-specific manner.
Assuntos
Compostos de Benzilideno/farmacologia , Digoxina/farmacologia , Simulação de Acoplamento Molecular , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Compostos de Benzilideno/síntese química , Compostos de Benzilideno/química , Células Cultivadas , Digoxina/síntese química , Digoxina/química , Relação Dose-Resposta a Droga , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Conformação Molecular , Células Sf9 , ATPase Trocadora de Sódio-Potássio/metabolismo , Spodoptera , Relação Estrutura-AtividadeRESUMO
Cisplatin and other platinum-containing drugs have played a crucial role in anticancer treatments for over 30 years. However, treatment with cisplatin may cause serious side effects, such as myelosuppression, nausea, ototoxicity, nephrotoxicity, and cell resistance processes. In addition, cardiotonic steroids, particularly digoxin, have recently been suggested to exert potent anticancer effects. Therefore, it is possible that the combined treatment of HeLa cells with cisplatin and digoxin can ameliorate the cytotoxic effects and decrease the side effects of cisplatin. In this study, we demonstrated that the interaction between cisplatin and digoxin had a synergistic effect on cervical cancer cells and a significantly positive cytotoxic and antiproliferative effect on this cell line compared to the control and single cisplatin treatments. Although a decrease in the Na,K-ATPase α1 subunit expression was observed in total extracts, its expression remains unchanged in the membrane, as does the Na,K-ATPase activity. The antiproliferative effect of the synergistic treatment appears to depend on Src kinase activation, indicating the possible involvement of the Scr-EGFR-ERK1/2 pathway in the antitumor effect. The inhibition of ERK1/2 provoked the same synergism with 1 µM cisplatin as that observed with 1 nM digoxin plus 1 µM cisplatin but not with 1 nM digoxin. Pretreatment with PP2 during combined treatment abolished the synergistic effect on the antiproliferative activity. Cisplatin and digoxin are already used in the clinical setting; therefore, this study opens possibilities for future clinical trials of combined treatments to improve treatment outcomes with a lower incidence of toxicity and side effects.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Digoxina/farmacologia , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Neoplasias do Colo do Útero/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológicoRESUMO
Cardiotonic steroids (CTS) are compounds which bind to the Na,K-ATPase, leading to its inhibition and in some cases initiating signaling cascades. Long utilized as a treatment for congestive heart disease, CTS have more recently been observed to inhibit proliferation and cause apoptosis in several cancer cell lines. A synthetic derivative of the CTS digoxin, called 21-benzylidene digoxin (21-BD), activates the Na,K-ATPase rather than cause its inhibition, as its parent compound does. Here, the mechanism behind the unique effects of 21-BD are further explored. In HeLa cancer cells, low (5µM) and high (50µM) doses of 21-BD activated and inhibited the Na,K-ATPase, respectively, without altering the membrane expression of the Na,K-ATPase. While digoxin did not affect HeLa membrane cholesterol or phospholipid content, 50µM 21-BD increased both lipids via a mechanism reliant on an intact cell. Afterwards, the direct action of 21-BD was evaluated on erythrocyte membranes; however, no effect was observed. As CTS may generate reactive oxygen species (ROS) which can affect plasma membrane fluidity and therefore Na,K-ATPase activity, several markers involved in ROS generation were analyzed such as, lipid peroxidation (TBARS), reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD). GSH content and catalase activity were unaffected by digoxin or 21-BD. Surprisingly, TBARS and SOD activity was decreased with digoxin and with 50µM 21-BD. Thus, 21-BD and digoxin altered components involved in ROS generation and inhibition in a similar fashion. This study suggests alterations to the Na,K-ATPase and membrane lipids by 21-BD is not reliant on ROS generation.
Assuntos
Digoxina/análogos & derivados , Digoxina/farmacologia , Lipídeos de Membrana/química , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/metabolismo , Colesterol/metabolismo , Células HeLa , Humanos , Fosfolipídeos/metabolismo , Subunidades Proteicas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Cardenolides are cardiac glycosides, mostly obtained from natural sources. They are well known for their inhibitory action on the Na,K-ATPase, an effect that regulates cardiovascular alterations such as congestive heart failure and atrial arrhythmias. In recent years, they have also sparked new interest in their anticancer potential. In the present study, the cytotoxic effects of the natural cardenolide convallatoxin (CON) were evaluated on non-small cell lung cancer (A549 cells). It was found that CON induced cytostatic and cytotoxic effects in A549 cells, showing essentially apoptotic cell death, as detected by annexin V-propidium iodide double-staining, as well as changes in cell form. In addition, it prompted cell cycle arrest in G2/M and reduced cyclin B1 expression. This compound also increased the number of cells in subG1 in a concentration- and time-dependent manner. At a long term, the reduction of cumulative population doubling was shown along with an increase of ß-galactosidase positive cells and larger nucleus, indicative of senescence. Subsequently, CON inhibited the Na,K-ATPase in A549 cells at nM concentrations. Interestingly, at the same concentrations, CON was unable to directly inhibit the Na,K-ATPase, either in pig kidney or in red blood cells. Additionally, results of docking calculations showed that CON binds with high efficiency to the Na,K-ATPase. Taken together, our data highlight the potent anticancer effects of CON in A549 cells, and their possible link with non-classical inhibition of Na,K-ATPase.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Estrofantinas/farmacologia , Células A549 , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , Rim/enzimologia , Neoplasias Pulmonares/tratamento farmacológico , Simulação de Acoplamento Molecular , ATPase Trocadora de Sódio-Potássio/química , SuínosRESUMO
Culex quinquefasciatus is the main vector of lymphatic filariasis and combating this insect is of great importance to public health. There are reports of insects that are resistant to the products currently used to control this vector, and therefore, the search for new products has increased. In the present study, we have evaluated the effects of fatty acid methyl esters (FAMEs) that showed larvicidal activity against C. quinquefasciatus, on glucose, total protein, and triacylglycerol contents and Na(+)/K(+)-ATPase activity in mosquito larvae. The exposure of the fourth instar larvae to the compounds caused a decrease in the total protein content and an increase in the activity of the Na(+)/K(+)-ATPase. Furthermore, the direct effect of FAMEs on cell membrane was assessed on purified pig kidney Na(+)/K(+)-ATPase membranes, erythrocyte ghost membranes, and larvae membrane preparation. No modifications on total phospholipids and cholesterol content were found after FAMEs 20 min treatment on larvae membrane preparation, but only 360 µg/mL FAME 2 was able to decrease total phospholipid of erythrocyte ghost membrane. Moreover, only 60 and 360 µg/mL FAME 3 caused an activation of purified Na(+)/K(+)-ATPase, that was an opposite effect of FAMEs treatment in larvae membrane preparation, and caused an inhibition of the pump activity. These data together suggest that maybe FAMEs can modulate the Na(+)/K(+)-ATPase on intact larvae for such mechanisms and not for a direct effect, one time that the direct effect of FAMEs in membrane preparation decreased the activity of Na(+)/K(+)-ATPase. The biochemical changes caused by the compounds were significant and may negatively influence the development and survival of C. quinquefasciatus larvae.
Assuntos
Culex/metabolismo , Ésteres , Ácidos Graxos/metabolismo , Larva/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Colesterol/metabolismo , Membrana Eritrocítica/metabolismo , Ésteres/química , Ácidos Graxos/química , Ácidos Graxos/farmacologia , Rim , Fosfolipídeos/metabolismo , SuínosRESUMO
Iron is an essential chemical element for human life. However, in some pathological conditions, such as hereditary hemochromatosis type 1 (HH1), iron overload induces the production of reactive oxygen species that may lead to lipid peroxidation and a change in the plasma-membrane lipid profile. In this study, we investigated whether iron overload interferes with the Na,K-ATPase activity of the plasma membrane by studying erythrocytes that were obtained from the whole blood of patients suffering from iron overload. Additionally, we treated erythrocytes of normal subjects with 0.8 mM H2O2 and 1 µM FeCl3 for 24 h. We then analyzed the lipid profile, lipid peroxidation and Na,K-ATPase activity of plasma membranes derived from these cells. Iron overload was more frequent in men (87.5%) than in women and was associated with an increase (446%) in lipid peroxidation, as indicated by the amount of the thiobarbituric acid reactive substances (TBARS) and an increase (327%) in the Na,K-ATPase activity in the plasma membrane of erythrocytes. Erythrocytes treated with 1 µM FeCl3 for 24 h showed an increase (132%) in the Na,K-ATPase activity but no change in the TBARS levels. Iron treatment also decreased the cholesterol and phospholipid content of the erythrocyte membranes and similar decreases were observed in iron overload patients. In contrast, erythrocytes treated with 0.8 mM H2O2 for 24 h showed no change in the measured parameters. These results indicate that erythrocytes from patients with iron overload exhibit higher Na,K-ATPase activity compared with normal subjects and that this effect is specifically associated with altered iron levels.
Assuntos
Membrana Eritrocítica/metabolismo , Sobrecarga de Ferro/enzimologia , Lipídeos/sangue , ATPase Trocadora de Sódio-Potássio/sangue , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/enzimologia , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Sobrecarga de Ferro/sangue , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Fatores SexuaisRESUMO
Cardiotonic steroids (CS), natural compounds with traditional use in cardiology, have been recently suggested to exert potent anticancer effects. However, the repertoire of molecules with Na,K-ATPase activity and anticancer properties is limited. This paper describes the synthesis of 6 new digoxin derivatives substituted (on the C17-butenolide) with γ-benzylidene group and their cytotoxic effect on human fibroblast (WI-26 VA4) and cancer (HeLa and RKO) cell lines as well as their effect on Na,K-ATPase activity and expression. As digoxin, compound BD-4 was almost 100-fold more potent than the other derivatives for cytotoxicity with the three types of cells used and was also the only one able to fully inhibit the Na,K-ATPase of HeLa cells after 24h treatment. No change in the Na,K-ATPase α1 isoform protein expression was detected. On the other hand it was 30-40 fold less potent for direct Na,K-ATPase inhibition, when compared to the most potent derivatives, BD-1 and BD-3, and digoxin. The data presented here demonstrated that the anticancer effect of digoxin derivatives substituted with γ-benzylidene were not related with their inhibition of Na,K-ATPase activity or alteration of its expression, suggesting that this classical molecular mechanism of CS is not involved in the cytotoxic effect of our derivatives.
Assuntos
Antineoplásicos/síntese química , Compostos de Benzilideno/química , Digoxina/análogos & derivados , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/toxicidade , Sítios de Ligação , Encéfalo/enzimologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Digoxina/síntese química , Digoxina/toxicidade , Células HeLa , Humanos , Rim/enzimologia , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Ratos , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
The Actinomyces sp has been linked to osteoradionecrosis of the jaw. The identification of these bacteria on histopathological findings confers a determining factor of poor prognosis and is related to difficulties of local control of bone necrosis and risk of recurrent infections. The treatment is complicated due to insidious growth pattern of Actinomyces sp. The methods for diagnosis and therapeutic approaches constitute challenges that underscore the need for understanding the risk factors for infection and knowledge about clinical conditions associated with this pathology. The objective of this study is to report two cases of cancer patients suffering from an osteoradionecrosis infection by Actinomyces sp, to describe approaches and discuss therapeutic options in the light of the current literature.
O Actinomyces sp tem sido associado a osteorradionecrose. A identificação desta bactéria confere um fator determinante de mau prognóstico e está relacionado a dificuldades do controle da necrose óssea local e a risco de infecções recorrentes. O tratamento é complicado devido ao padrão de crescimento insidioso do Actinomyces sp. Os métodos de diagnóstico e as abordagens terapêuticas constituem desafios que reforçam a necessidade de compreender os fatores de risco para a infecção e o conhecimento sobre as condições clínicas associadas a esta patologia. O objetivo deste trabalho é relatar dois casos clínicos de pacientes com câncer que sofrem infecção osteorradionecrose por Actinomyces sp, para descrever as abordagens e discutir as opções terapêuticas à luz da literatura atual.
Assuntos
Osteonecrose , Osteorradionecrose , Actinomyces , Actinomicose , Difosfonatos , Arcada OsseodentáriaRESUMO
Cardiotonic steroids are used to treat heart failure and arrhythmia and have promising anticancer effects. The prototypic cardiotonic steroid ouabain may also be a hormone that modulates epithelial cell adhesion. Cardiotonic steroids consist of a steroid nucleus and a lactone ring, and their biological effects depend on the binding to their receptor, Na,K-ATPase, through which, they inhibit Na+ and K+ ion transport and activate of several intracellular signaling pathways. In this study, we added a styrene group to the lactone ring of the cardiotonic steroid digoxin, to obtain 21-benzylidene digoxin (21-BD), and investigated the effects of this synthetic cardiotonic steroid in different cell models. Molecular modeling indicates that 21-BD binds to its target Na,K-ATPase with low affinity, adopting a different pharmacophoric conformation when bound to its receptor than digoxin. Accordingly, 21-DB, at relatively high µM amounts inhibits the activity of Na,K-ATPase α1, but not α2 and α3 isoforms. In addition, 21-BD targets other proteins outside the Na,K-ATPase, inhibiting the multidrug exporter Pdr5p. When used on whole cells at low µM concentrations, 21-BD produces several effects, including: 1) up-regulation of Na,K-ATPase expression and activity in HeLa and RKO cancer cells, which is not found for digoxin, 2) cell specific changes in cell viability, reducing it in HeLa and RKO cancer cells, but increasing it in normal epithelial MDCK cells, which is different from the response to digoxin, and 3) changes in cell-cell interaction, altering the molecular composition of tight junctions and elevating transepithelial electrical resistance of MDCK monolayers, an effect previously found for ouabain. These results indicate that modification of the lactone ring of digoxin provides new properties to the compound, and shows that the structural change introduced could be used for the design of cardiotonic steroid with novel functions.
Assuntos
Apoptose/efeitos dos fármacos , Digoxina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Junções Íntimas/efeitos dos fármacos , Animais , Cardenolídeos/metabolismo , Cardenolídeos/farmacologia , Linhagem Celular Tumoral , Digoxina/análogos & derivados , Digoxina/química , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Modelos Moleculares , Conformação Molecular , Neoplasias/genética , Neoplasias/metabolismo , Ratos , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
BACKGROUND: In developing countries, the access to red blood cell (RBC) irradiators is restricted. Thus, it is a common practice in blood banks to stock irradiated RBC units until they expire. The aim of this work is to elucidate the involvement of Na,K-ATPase in potassium leakage from prophylactically irradiated RBCs. METHODS: Whole blood was collected from healthy donors, and blood concentrates were irradiated with 25Gy of γ-radiation within 24h of collection. At days 3, 5, 7, 9, 11, 14 and 28 post-irradiation, fractions were removed and centrifuged and Na,K-ATPase activity from ghost membranes was determined. RESULTS: The inhibition of Na,K-ATPase activity in RBCs reached 12.6% by day 7 of storage and up to 50% by day 14 of storage. The addition of vitamin C prevented the irradiation-induced loss of Na,K-ATPase activity. The irradiation of RBCs provoked an increase in potassium plasma levels and a decrease in sodium plasma levels. The incubation of RBCs with ouabain did not change the sodium or potassium levels in the plasma, and the addition of vitamin C only partially prevented a decrease in sodium levels caused by irradiation. CONCLUSION: Because the inhibition of Na,K-ATPase by ouabain did not cause potassium accumulation in the plasma, we conclude that the irradiation-induced inhibition of the pump is not a key factor driving this effect.
Assuntos
Eritrócitos/metabolismo , Eritrócitos/efeitos da radiação , Potássio/sangue , ATPase Trocadora de Sódio-Potássio/metabolismo , Manejo de Espécimes/métodos , Inibidores Enzimáticos/farmacologia , Humanos , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidoresRESUMO
The aim of the present work was to evaluate the redox and oligomeric effects associated with the human hemoglobin of stored red blood cells that had been previously submitted to gamma radiation. Whole blood was collected from healthy donors and irradiated with 25 Gy of γ-radiation within 24 h of collection. At days 3, 5, 7, 9, 11, 14, and 28 postirradiation, fractions were removed and centrifuged, and the levels of methehemoglobin and oxyhemoglobin were measured. Hb was isolated to measure the denaturation and UV-vis spectra. The results from electrophoresis demonstrated that there was no fragmentation or cross-linking of the hemoglobin. However, ferrous center oxidation was identified as a very significant process. This mechanism is likely through an autoxidation process of the ferrous heme center, which has a maximal intensity between 5 and 7 days of storage. Interestingly, a subsequent reduction of the oxidized heme species was observed, and after 9 days of storage, the difference between the ferric species present in the control and irradiated samples was not representative. This interesting fact suggests a type of "protective action" by the blood to control the oxidative stress generated by the gamma irradiation. The UV-vis measurements demonstrated that the oxidized species was predominantly formed by hemichrome species (bis-histidine ferric heme species), which are usually associated with Heinz bodies. After 28 days of storage, evidence from the UV-vis measurements indicated that the oxidation of the irradiated sample was much higher than that observed in the control sample. These results demonstrate that despite the minimal polypeptide changes observed in the hemoglobin of stored red blood cells after gamma irradiation, the oxidation of the heme metallic center is not irrelevant and must be controlled to improve the hematological clinical procedures associated with the storage of red blood cells.