Role of Na/K-ATPase α1 caveolin-binding motif in adipogenesis.

Deficiencies in mice and in humans have brought to the fore the importance of the caveolar network in key aspects of adipocyte biology. The conserved N-terminal caveolin-binding motif (CBM) of the ubiquitous Na/K-ATPase (NKA) α1 isoform, which allows NKA/caveolin-1 (Cav1) interaction, influences NKA signaling and caveolar distribution. It has been shown to be critical for animal development and ontogenesis, as well as lineage-specific differentiation of human induced pluripotent stem cells (hiPSCs). However, its role in postnatal adipogenesis has not been fully examined. Using a genetic approach to alter CBM in hiPSC-derived adipocytes (iAdi-mCBM) and in mice (mCBM), we investigated the regulatory function of NKA CBM signaling in adipogenesis. Seahorse XF cell metabolism analyses revealed impaired glycolysis and decreased ATP synthesis-coupled respiration in iAdi-mCBM. These metabolic dysfunctions were accompanied by evidence of extensive remodeling of the extracellular matrix (ECM), including increased collagen staining, overexpression of ECM marker genes, and heightened TGF-ß signaling uncovered by RNAseq analysis. Rescue of mCBM by lentiviral delivery of WT NKA α1 or treatment of mCBM hiPSCs with the TGF-ß inhibitor SB431542 normalized ECM, suggesting that NKA CBM signaling integrity is required for adequate control of TGF-ß signaling and ECM stiffness during adipogenesis. The physiological impact was revealed in mCBM male mice with reduced fat mass accompanied by histological and transcriptional evidence of elevated adipose fibrosis and decreased adipocyte size. Based on these findings, we propose that the genetic alteration of the NKA/Cav1 regulatory path uncovered in human iAdi leads to lipodystrophy in mice.NEW & NOTEWORTHY A Na/K-ATPase α1 caveolin-binding motif regulates adipogenesis. Mutation of this binding motif in the mouse leads to reduced fat with increased extracellular matrix production and inflammation. RNA-seq analysis and pharmacological interventions in human iPSC-derived adipocytes revealed that TGF-ß signal, rather than Na/K-ATPase-mediated ion transport, is a key mediator of NKA regulation of adipogenesis.

The Na/K-ATPase α1/Src Signaling Axis Regulates Mitochondrial Metabolic Function and Redox Signaling in Human iPSC-Derived Cardiomyocytes.

Na/K-ATPase (NKA)-mediated regulation of Src kinase, which involves defined amino acid sequences of the NKA α1 polypeptide, has emerged as a novel regulatory mechanism of mitochondrial function in metazoans. Mitochondrial metabolism ensures adequate myocardial performance and adaptation to physiological demand. It is also a critical cellular determinant of cardiac repair and remodeling. To assess the impact of the proposed NKA/Src regulatory axis on cardiac mitochondrial metabolic function, we used a gene targeting approach in human cardiac myocytes. Human induced pluripotent stem cells (hiPSC) expressing an Src-signaling null mutant (A420P) form of the NKA α1 polypeptide were generated using CRISPR/Cas9-mediated genome editing. Total cellular Na/K-ATPase activity remained unchanged in A420P compared to the wild type (WT) hiPSC, but baseline phosphorylation levels of Src and ERK1/2 were drastically reduced. Both WT and A420P mutant hiPSC readily differentiated into cardiac myocytes (iCM), as evidenced by marker gene expression, spontaneous cell contraction, and subcellular striations. Total NKA α1-3 protein expression was comparable in WT and A420P iCM. However, live cell metabolism assessed functionally by Seahorse extracellular flux analysis revealed significant reductions in both basal and maximal rates of mitochondrial respiration, spare respiratory capacity, ATP production, and coupling efficiency. A significant reduction in ROS production was detected by fluorescence imaging in live cells, and confirmed by decreased cellular protein carbonylation levels in A420P iCM. Taken together, these data provide genetic evidence for a role of NKA α1/Src in the tonic stimulation of basal mitochondrial metabolism and ROS production in human cardiac myocytes. This signaling axis in cardiac myocytes may provide a new approach to counteract mitochondrial dysfunction in cardiometabolic diseases.

Evaluation of Cardiotonic Steroid Modulation of Cellular Cholesterol and Phospholipid.

We have previously shown that 21-benzylidene digoxin (21-BD) increases the total cholesterol and phospholipid content on the membrane of HeLa cells. Lipid modulation caused by cardiotonic steroids (CTS) is still unexplored. Therefore, the aim of the present study was to evaluate the cholesterol and phospholipid modulation of the cell membrane caused by ouabain and 21-BD and the possible involvement of the caveolae on this modulation. For this, one cell line containing caveolae (HeLa) and other not containing (Caco-2) were used. The modulation of the lipid profile was evaluated by total cholesterol and phospholipids measurements, and identification of membrane phospholipids by HPTLC. The cholesterol distribution was evaluated by filipin staining. The caveolin-1 expression was evaluated by Western Blotting. Ouabain had no effect on the total membrane lipid content in both cell lines. However, 21-BD increased total membrane phospholipid content and had no effect on the membrane cholesterol content in Caco-2 cells. CTS were not able to alter the specific phospholipids content. In the filipin experiments, 21-BD provoked a remarkable redistribution of cholesterol to the perinuclear region of HeLa cells. In Caco-2 cells, it was observed only a slight increase in cholesterol, especially as intracellular vesicles. The caveolin-1 expression was not altered by any of the compounds. Our data mainly show different effects of two cardiotonic steroids. Ouabain had no effect on the lipid profile of cells, whereas 21-BD causes important changes in cholesterol and phospholipid content. Therefore, the modulation of cholesterol content in the plasma membrane of HeLa cells is not correlated with the expression of caveolin-1.

Implications of Synthetic Modifications of the Cardiotonic Steroid Lactone Ring on Cytotoxicity.

Na,K-ATPase (NKA) and cardiotonic steroids (CTS) have shown potent cytotoxic and anticancer effects. Here, we have synthesized a series of CTS digoxin derivatives (γ-benzylidene) with substitutions in the lactone ring and evaluated the cytotoxicity caused by digoxin derivatives in tumor and non-tumor cells lines, as well as their effects on NKA. The cytotoxicity assay was determined in HeLa, A549, and WI-26 VA4 after they were treated for 48 h with increased concentrations of CTS. The effects of CTS on NKA activity and immunoblotting of α1 and ß1 isoforms were evaluated at IC50 concentrations in A549 cell membrane. NKA activity from mouse brain cortex was also measured. The majority of CTS exhibited low cytotoxicity in tumor and non-tumor cells, presenting IC50 values at micromolar concentrations, while digoxin showed cytotoxicity at nanomolar concentrations. BD-15 presented the lowest IC50 value (8 µM) in A549 and reduced its NKA activity in 28%. In contrast, BD-7 was the compound that most inhibited NKA (56% inhibition) and presented high IC50 value for A549. In mouse cortex, only BD-15 modulated the enzyme activity in a concentration-dependent inhibition curve. These results demonstrate that the cytotoxicity of these compounds is not related to NKA inhibition. The substitutions in the lactone ring of digoxin led to an increase in the cytotoxic concentration in tumor cells, which may not be interesting for cancer, but it has the advantage of increasing the therapeutic margin of these molecules when compared to classic CTS, and can be used safely in research for other diseases.

Avaliação da cadeia de frio do transporte de vacina: estudo transversal em Minas Gerais, Brasil / Evaluation of the cold chain of vaccine transportation: cross-sectional study in Minas Gerais, Brazil

Estudo transversal de abordagem avaliativa do grau de implantação da cadeia de frio durante o transporte de vacina do nível regional para municípios. Avaliamos 53 rotas de transporte de vacina de municípios de Minas Gerais. Para a coleta de dados, utilizamos um questionário estruturado e o monitoramento das temperaturas das caixas de vacina encaminhadas aos municípios através do data logger. O grau de implantação foi definido por meio de um sistema de escores, com pesos diferenciados para indicadores em cada dimensão avaliada. Os escores obtidos a partir da soma dos pontos dos indicadores foram transformados em percentuais, com referência à pontuação máxima possível. A seguir, foram definidas as categorias para o grau de qualidade: "adequado", "parcialmente adequado" e "não adequado". Os dados foram digitados no programa Epidata 3,0 e analisados no EPI-INFO 7.0, GraphPad Prism 5 e BioEstat 5.0, Excel. Realizamos análise univariada com cálculo das frequências, medidas de aferição e dispersão. A cadeia de frio durante o transporte de vacina não está adequadamente implantada na maioria dos municípios avaliados. Em vinte e dois municípios as vacinas foram expostas a temperaturas abaixo de +2°C, sendo que em seis destes a temperatura variou de 0°C a +0,5°C. O transporte de vacina apresenta falhas que podem comprometer a qualidade dos imunobiológicos.

Cross-sectional study aimed to evaluate the degree of cold chain implementation during vaccine transport from regions to municipalities. We evaluated 53 vaccine delivery routes in municipalities from the state of Minas Gerais. For data collection, we utilized a structured questionnaire and data logger in order to monitor the temperature inside the vaccine coldboxesthatwere distributed to thesemunicipalities through the data logger device. The degree of implementation was defined by means of a scoring system, with differentscores of different weights attributed to indicators for each rated dimension. The scores obtained from the sum of indicator points were transformed into percentages and referenced to the maximum possible score. The categories for quality grades were then defined as: "adequate", "partially adequate" and "not adequate". The data were entered in the Epidata 3.0 program and analyzed with EPI-INFO 7.0, GraphPad Prism 5 and BioEstat 5.0, Excel. We conducted univariate analysis to calculate frequencies,standardized measures and dispersion. Cold chain implementation during vaccine transport was found to be inadequate in the majority of the municipalities evaluated. In twenty-two municipalities, vaccines were exposed to temperatures below +2ºC and in six of these twenty-two,temperatures varied from 0oC to +0.5ºC. The vaccine transportation reveals shortcomings that could potentially compromise the immunobiologicals quality.

Cardiac Oxidative Signaling and Physiological Hypertrophy in the Na/K-ATPase α1s/sα2s/s Mouse Model of High Affinity for Cardiotonic Steroids.

The Na/K-ATPase is the specific receptor for cardiotonic steroids (CTS) such as ouabain and digoxin. At pharmacological concentrations used in the treatment of cardiac conditions, CTS inhibit the ion-pumping function of Na/K-ATPase. At much lower concentrations, in the range of those reported for endogenous CTS in the blood, they stimulate hypertrophic growth of cultured cardiac myocytes through initiation of a Na/K-ATPase-mediated and reactive oxygen species (ROS)-dependent signaling. To examine a possible effect of endogenous concentrations of CTS on cardiac structure and function in vivo, we compared mice expressing the naturally resistant Na/K-ATPase α1 and age-matched mice genetically engineered to express a mutated Na/K-ATPase α1 with high affinity for CTS. In this model, total cardiac Na/K-ATPase activity, α1, α2, and ß1 protein content remained unchanged, and the cardiac Na/K-ATPase dose-response curve to ouabain shifted to the left as expected. In males aged 3-6 months, increased α1 sensitivity to CTS resulted in a significant increase in cardiac carbonylated protein content, suggesting that ROS production was elevated. A moderate but significant increase of about 15% of the heart-weight-to-tibia-length ratio accompanied by an increase in the myocyte cross-sectional area was detected. Echocardiographic analyses did not reveal any change in cardiac function, and there was no fibrosis or re-expression of the fetal gene program. RNA sequencing analysis indicated that pathways related to energy metabolism were upregulated, while those related to extracellular matrix organization were downregulated. Consistent with a functional role of the latter, an angiotensin-II challenge that triggered fibrosis in the α1r/rα2s/s mouse failed to do so in the α1s/sα2s/s. Taken together, these results are indicative of a link between circulating CTS, Na/K-ATPase α1, ROS, and physiological cardiac hypertrophy in mice under baseline laboratory conditions.

The Na/K-ATPase α1/Src interaction regulates metabolic reserve and Western diet intolerance.

AIM: Highly prevalent diseases such as insulin resistance and heart failure are characterized by reduced metabolic flexibility and reserve. We tested whether Na/K-ATPase (NKA)-mediated regulation of Src kinase, which requires two NKA sequences specific to the α1 isoform, is a regulator of metabolic capacity that can be targeted pharmacologically. METHODS: Metabolic capacity was challenged functionally by Seahorse metabolic flux analyses and glucose deprivation in LLC-PK1-derived cells expressing Src binding rat NKA α1, non-Src-binding rat NKA α2 (the most abundant NKA isoform in the skeletal muscle), and Src binding gain-of-function mutant rat NKA α2. Mice with skeletal muscle-specific ablation of NKA α1 (skα1-/-) were generated using a MyoD:Cre-Lox approach and were subjected to treadmill testing and Western diet. C57/Bl6 mice were subjected to Western diet with or without pharmacological inhibition of NKA α1/Src modulation by treatment with pNaKtide, a cell-permeable peptide designed by mapping one of the sites of NKA α1/Src interaction. RESULTS: Metabolic studies in mutant cell lines revealed that the Src binding regions of NKA α1 are required to maintain metabolic reserve and flexibility. Skα1-/- mice had decreased exercise endurance and mitochondrial Complex I dysfunction. However, skα1-/- mice were resistant to Western diet-induced insulin resistance and glucose intolerance, a protection phenocopied by pharmacological inhibition of NKA α1-mediated Src regulation with pNaKtide. CONCLUSIONS: These results suggest that NKA α1/Src regulatory function may be targeted in metabolic diseases. Because Src regulatory capability by NKA α1 is exclusive to endotherms, it may link the aerobic scope hypothesis of endothermy evolution to metabolic dysfunction.

Índice fuzzy de qualidade de água para ambiente lótico - IQAFAL / IQA FAL - Fuzzy water quality index for lotic environments

RESUMO A divulgação de informações sobre qualidade das águas para um público não especialista é fundamental para subsidiar ações políticas e institucionais de gestão dos ambientes aquáticos. Para tanto, índices de qualidade de água têm sido propostos por serem capazes de sintetizar em um único valor ou categoria a informação, normalmente descrita a partir de um conjunto extenso de variáveis de qualidade de água. Este trabalho propõe um novo índice de qualidade de água, IQAFAL, baseado em lógica nebulosa, direcionado para o ambiente lótico, desenvolvido com a colaboração do quadro de especialistas da área de qualidade de água do Instituto Estadual do Ambiente (INEA). O índice proposto foi aplicado a dados de qualidade de água do Rio Paraíba do Sul, obtidos pelo INEA, nos anos de 2002 a 2009. Os resultados do IQAFAL mostraram que esse índice foi capaz de descrever a qualidade da água desse trecho do Rio Paraíba do Sul, correspondendo satisfatoriamente às avaliações de qualidade de água contidas nos relatórios disponíveis. Verificou-se também que com essa metodologia foi possível evitar que a influência de uma variável em condições críticas fosse atenuada pela influência das outras variáveis em condições favoráveis, produzindo um resultado impreciso no índice final.

ABSTRACT The dissemination of information on water quality for a non-specialist audience is essential to support political and institutional actions for the management of aquatic environments. Therefore, water quality indices have been proposed since they are able to synthesize into a single value or category information, usually described from an extensive set of water quality variables. This research proposes a new water quality index, based on fuzzy logic, aimed at lotic environments, developed through the collaboration of experts in water quality of the Rio de Janeiro Environmental Agency (Instituto Estadual do Ambiente - INEA). The proposed index was applied to water quality data from the Paraíba do Sul River, obtained by INEA, in the years 2002 to 2009. The results of IQAFAL showed that the index was able to synthesize the water quality of this stretch of the Paraíba do Sul, satisfactorily matching the assessments of the water quality assessments contained in the reports available. It was also noticed that with this methodology it was possible to avoid the attenuation of the influence of a variable in critical condition was attenuated by the influence of other variables in favorable conditions, producing an inaccurate result in the final index.

21-Benzylidene digoxin decreases proliferation by inhibiting the EGFR/ERK signaling pathway and induces apoptosis in HeLa cells.

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.

Iron overload: Effects on cellular biochemistry.

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.

Opioid prescription patterns among patients with fibromyalgia.

OBJECTIVES: To investigate opioid prescribing patterns among patients with fibromyalgia (FM) in terms of age, gender, race, type of opioids, and to examine changes in opioid prescription over the past 8 years compared to the US Food and Drug Administration (FDA)-approved FM medications. DESIGN: Retrospective review of data using the Healthcare Enterprise Repository for Ontological Narration database. The collected data were analyzed descriptively and a chi-square test for trend was used to analyze a possible linear relationship between the proportions of opioid and non-opioid users along the time. PARTICIPANTS: Patients with a diagnosis of FM who had received opioid prescriptions from January 1, 2010 to December 31, 2017, and FM patients who had received prescriptions of FDA-approved FM medications in the same period. MAIN OUTCOME MEASURE: Trends in opioid and non-opioid prescriptions in patients with FM. RESULTS: The opioid medications were prescribed more frequently in 2010 (40 percent) and 2011 (42 percent), but the percentages have decreased since 2012 and reached the lowest numbers in 2016 (27 percent). The chi-square test for trend shows that from 2010 to 2017 the prescriptions of opioids had a statistically significant (p < 0.0001) decrease. CONCLUSION: This study suggests that the frequency of prescribed opioids in FM patients has decreased since 2012. This decline could be attributed to (1) FDA monitoring programs, (2) national efforts to increase awareness of the addictive and harmful effects of opioids, and (3) the growing research on the efficacy of non-opioid therapies to treat chronic pain conditions including FM.

Iron overload impact on P-ATPases.

Iron is a chemical element that is active in the fundamental physiological processes for human life, but its burden can be toxic to the body, mainly because of the stimulation of membrane lipid peroxidation. For this reason, the action of iron on many ATPases has been studied, especially on P-ATPases, such as the Na+,K+-ATPase and the Ca2+-ATPase. On the Fe2+-ATPase activity, the free iron acts as an activator, decreasing the intracellular Fe2+ and playing a protection role for the cell. On the Ca2+-ATPase activity, the iron overload decreases the enzyme activity, raising the cytoplasmic Ca2+ and decreasing the sarco/endoplasmic reticulum and the Golgi apparatus Ca2+ concentrations, which could promote an enzyme oxidation, nitration, and fragmentation. However, the iron overload effect on the Na+,K+-ATPase may change according to the tissue expressions. On the renal cells, as well as on the brain and the heart, iron promotes an enzyme inactivation, whereas its effect on the erythrocytes seems to be the opposite, directly stimulating the ATPase activity, or stimulating it by signaling pathways involving ROS and PKC. Modulations in the ATPase activity may impair the ionic transportation, which is essential for cell viability maintenance, inducing irreversible damage to the cell homeostasis. Here, we will discuss about the iron overload effect on the P-ATPases, such as the Na+,K+-ATPase, the Ca2+-ATPase, and the Fe2+-ATPase.

Selectivity analyses of γ-benzylidene digoxin derivatives to different Na,K-ATPase α isoforms: a molecular docking approach.

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.

Differences of lipid membrane modulation and oxidative stress by digoxin and 21-benzylidene digoxin.

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.

Avaliação do Programa de Controle da Tuberculose: um estudo de caso / Evaluation of Tuberculosis Control Program: a case report

RESUMO A falta de integração entre os serviços que compõem a rede de atenção à saúde, além da incapacidade de muitos profissionais, são apontadas como causas da problemática para o controle da tuberculose no Brasil. Este estudo avaliou o grau de implantação do Programa de Controle de Tuberculose (PCT) em Divinópolis (MG). Trata-se de uma pesquisa avaliativa na perspectiva de análise de implantação. O PCT encontra-se parcialmente implantado. Como limitações, destacam-se a deficiência de gestão e planejamento, e a insuficiência de profissionais capacitados e insumos. Sugere-se a descentralização das ações do PCT e o aumento das ações de vigilância como possíveis soluções para a melhoria da assistência e da prevenção.

ABSTRACT The lack of integration between health care services network, in addition to the inefficiency of health professionals, are some of the identified problems for the control of Tuberculosis in Brazil. This study evaluated the level of implementation of the Tuberculosis Control Program (PCT) in Divinopolis (MG). The results signalize that PCT is partially implemented, due mostly to deficiencies related to management and planning, the insufficiency of trained professionals and insufficient resources. The decentralization of PCT actions and the enhancement of surveillance activities are signalized as possible solutions for the improvement of TB health care and prevention.

21-Benzylidene digoxin: a proapoptotic cardenolide of cancer cells that up-regulates Na,K-ATPase and epithelial tight junctions.

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.