RESUMO
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
Recent findings have demonstrated new therapeutic functions of cardiotonic steroids, a process that is termed drug repositioning. Despite the confirmed anti-inflammatory effects of cardiotonic steroids, their clinical use has been discouraged due to toxicity related to inhibition of the Na+/K+ ATPase. A novel synthetic compound derived from digoxin, 21benzylidene digoxin (21BD), does not inhibit this enzyme. Herein, we evaluated the anti-inflammatory and antinociceptive effects and acute toxicity of 21BD. Murine (Swiss mice) models of paw oedema induced by carrageenan, acetic acid-induced abdominal writhing, and formalin and acute toxicity tests were used. Oral administration of 21BD (0.3â¯mg/kg) showed a significant and prolonged inhibition of paw oedema. Histological analysis demonstrated a reduction in inflammatory cells and expression of inducible nitric oxide synthase (iNOS) in footpads 6â¯h after administration of carrageenan. 21BD (0.3â¯mg/kg) also reduced the levels of tumour necrosis factor (TNF)-α 2 and 4â¯h after carrageenan. 21BD demonstrated antinociceptive activity, inhibiting abdominal writhes at all tested doses. However, in the formalin test, 21BD did not present antinociceptive activity. In the acute toxicity test, 21BD did not cause symptoms of toxicity or mortality. The present study demonstrated, for the first time, that 21BD is safe and exhibits a marked anti-inflammatory activity in acute local inflammation. This effect might be a consequence of its ability to inhibit the release of the PMN leucocyte-derived mediators, including TNF-α, and iNOS expression as well as its inhibitory effect on oedema and PMN leucocyte infiltration.
Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Digoxina/análogos & derivados , Analgésicos/química , Animais , Anti-Inflamatórios/química , Carragenina/toxicidade , Digoxina/administração & dosagem , Digoxina/química , Digoxina/farmacologia , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Edema/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Indometacina/farmacologia , Masculino , Camundongos , Estrutura Molecular , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Testes de Toxicidade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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
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
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.