Digitoxin inhibits ICC cell properties via the NF­κB/ST6GAL1 signaling pathway.

Intrahepatic cholangiocarcinoma (ICC) is a type of liver cancer associated with poor prognosis and increased mortality; the limited treatment strategy highlights the urgent need for investigation. Traditional Chinese Medicine (TCM), used alone or in combination with other treatments, can enhance therapeutic efficacy, improve life quality of patients and extend overall survival. In total, two rounds of screening of a TCM library of 2,538 active compounds were conducted using a Cell Counting Kit­8 assay and ICC cell lines. Cell proliferation and migration abilities were assessed through colony formation, 5­ethynyl­2'­deoxyuridine, would healing and Transwell assays. The impact of digitoxin (DT) on signaling pathways was initially investigated using RNA sequencing and further validated using reverse transcription­quantitative PCR, western blotting, lectin blotting and flow cytometry. ICC cells stably overexpressing ST6 ß­galactoside α­2,6­sialyltransferase 1 (ST6GAL1) were generated through lentiviral transfection. It was shown that DT emerged as a highly effective anti­ICC candidate from two rounds high­throughput library screening. DT could inhibit the proliferation and migration of ICC cells by suppressing NF­κB activation and reducing nuclear phosphorylated­NF­κB levels, along with diminishing ST6GAL1 mRNA and protein expression. The aforementioned biological effects and signal pathways of DT could be counteracted by overexpressing ST6GAL1 in ICC cells. In conclusion, DT suppressed ICC cell proliferation and migration by targeting the NF­κB/ST6GAL1 signaling axis. The findings of the present study indicated the promising therapeutic effects of DT in managing ICC, offering new avenues for treatment strategies.

Generation of affinity maps for thiazolidinediones with human serum albumin using affinity microcolumns. I. Studies of effects by glycation on multisite drug binding.

Human serum albumin (HSA) is known to undergo modifications by glucose during diabetes. This process produces glycated HSA that can have altered binding to some drugs. In this study, high-performance affinity microcolumns and competition studies were used to see how glycation affects the binding by two thiazolidinedione-class drugs (i.e., pioglitazone and rosiglitazone) at specific regions of HSA. These regions included Sudlow sites I and II, the tamoxifen and digitoxin sites, and a drug-binding site located in subdomain IB. At Sudlow site II, the association equilibrium constants (or binding constants) for pioglitazone and rosiglitazone with normal HSA were 1.7 × 105 M-1 and 2.0 × 105 M-1 at pH 7.4 and 37 °C, with values that changed by up to 5.7-fold for glycated HSA. Sudlow site I of normal HSA had binding constants for pioglitazone and rosiglitazone of 3.4 × 105 M-1 and 4.6 × 105 M-1, with these values changing by up to 1.5-fold for glycated HSA. Rosiglitazone was found to also bind a second region that had a positive allosteric effect on Sudlow site I for all the tested preparations of HSA (binding affinity, 1.1-3.2 × 105 M-1; coupling constant for Sudlow site I, 1.20-1.34). Both drugs had a strong positive allosteric effect on the tamoxifen site of HSA (coupling constants, 13.7-19.9 for pioglitazone and 3.7-11.5 for rosiglitazone). Rosiglitazone also had weak interactions at a site in subdomain IB, with a binding constant of 1.4 × 103 M-1 for normal HSA and a value that was altered by up to 6.8-fold with glycated HSA. Neither of the tested drugs had any significant binding at the digitoxin site. The results were used to produce affinity maps that described binding by these thiazolidinediones with HSA and the effects of glycation on these interactions during diabetes.

The antiangiogenic effect of digitoxin is dependent on a ROS-elicited RhoA/ROCK pathway activation.

We previously showed that digitoxin inhibits angiogenesis and cancer cell proliferation and migration and these effects were associated to protein tyrosine kinase 2 (FAK) inhibition. Considering the interactions between FAK and Rho GTPases regulating cell cytoskeleton and movement, we investigated the involvement of RhoA and Rac1 in the antiangiogenic effect of digitoxin. Phalloidin staining of human umbilical vein endothelial cells (HUVECs) showed the formation of stress fibers in cells treated with 10 nM digitoxin. By Rhotekin- and Pak1- pull down assays, detecting the GTP-bound form of GTPases, we observed that digitoxin (10-25 nM) induced sustained (0.5-6 h) RhoA activation with no effect on Rac1. Furthermore, inhibition of HUVEC migration and capillary-like tube formation by digitoxin was counteracted by hindering RhoA-ROCK axis with RhoA silencing or Y-27632 treatment. Digitoxin did not decrease p190RhoGAP phosphorylation at Tyr1105 (a site targeted by FAK), suggesting that RhoA activation was independent from FAK inhibition. Because increasing evidence points to a redox regulation of RhoA, we measured intracellular ROS and found that digitoxin treatment enhanced ROS levels in a concentration-dependent manner (1-25 nM). Notably, the flavoprotein inhibitor DPI or the pan-NADPH oxidase (NOX) inhibitor VAS-2870 antagonized both ROS increase and RhoA activation by digitoxin. Our results provide evidence that inhibition of HUVEC migration and tube formation by digitoxin is dependent on ROS production by endothelial NOX, which leads to the activation of RhoA/ROCK pathway. Digitoxin effects on proteins regulating cytoskeletal organization and cell motility could have a wider impact on cancer progression, beyond the antiangiogenic activity.

[Cloning and functional analysis of 3ß-hydroxysteroid dehydrogenase gene involved in biosynthesis of digitoxin].

Digitoxin, an important secondary metabolite of Digitalis purpurea, is a commonly used cardiotonic in clinical practice. 3ß-Hydroxysteroid dehydrogenase(3ßHSD) is a key enzyme involved in the biosynthesis of digitoxin. It belongs to the short-chain dehydrogenase/reductase(SDR) family, playing a role in the biosynthesis of cardiac glycosides by oxidizing and isomerizing the precursor sterol. In this study, two 3ßHSD genes were cloned from D. purpurea. The results showed that the open reading frame(ORF) of Dp3ßHSD1 was 780 bp, encoding 259 amino acid residues. The ORF of Dp3ßHSD2 was 774 bp and encoded 257 residues. Dp3ßHSD1/2 had the cofactor binding site TGxxxA/GxG and the catalytic site YxxxK. In vitro experiments confirmed that Dp3ßHSD1/2 catalyzed the generation of progesterone from pregnenolone, and Dp3ßHSD1 had stronger catalytic capacity than Dp3ßHSD2. The expression level of Dp3ßHSD1 was much higher than that of Dp3ßHSD2 in leaves, and digitoxin was only accumulated in leaves. The results implied that Dp3ßHSD1 played a role in the dehydrogenation of pregnenolone to produce progesterone in the biosynthesis of digitoxin. This study provides a reference for further exploring the biosynthetic pathway of cardiac glycosides in D. purpurea.

Broad spectrum post-entry inhibitors of coronavirus replication: Cardiotonic steroids and monensin.

A small molecule screen identified several cardiotonic steroids (digitoxin and ouabain) and the ionophore monensin as potent inhibitors of HCoV-229E, HCoV-OC43, and SARS-CoV-2 replication with EC50s in the low nM range. Subsequent tests confirmed antiviral activity in primary cell models including human nasal epithelial cells and lung organoids. Addition of digitoxin, ouabain, or monensin strongly reduced viral gene expression as measured by both viral protein and RNA accumulation. Furthermore, the compounds acted post virus entry. While the antiviral activity of digitoxin was dependent upon activation of the MEK and JNK signaling pathways but not signaling through GPCRs, the antiviral effect of monensin was reversed upon inhibition of several signaling pathways. Together, the data demonstrates the potent anti-coronavirus properties of two classes of FDA approved drugs that function by altering the properties of the infected cell, rendering it unable to support virus replication.

Estimulación de cardenólidos en brotes de Digitalis purpurea L. cultivados in vitro mediante elicitores / Stimulation of cardenolides production in Digitalis purpurea L. shoot cultures by elicitors addition

Digitalis purpurea L. es una de las principales fuentes de cardenólidos tales como digoxina y digitoxina. Estos fármacos son ampliamente usados en la disfunción cardíaca y para regular las arritmias del corazón. El presente trabajo se realizó con el objetivo de estudiar el efecto de tres elicitores en el cultivo de brotes de Digitalis purpurea var. Roter Berggold para incrementar la producción in vitro de cardenólidos. La elicitación es una estrategia para incrementar la producción de biomasa y metabolitos secundarios en el cultivo in vitro. Los elicitores evaluados fueron ChitoPlant (0,001; 0,01; 0,1 g.L-1); SilioPlant (0,01; 0,1; 1,0 g.L-1) y Jasmonato de metilo (60, 80 y 100 µM), descritos por primera vez para el incremento de cardenólidos. Se demostró que la elicitación es una estrategia viable para el incremento de cardenólidos en brotes de D. purpurea. El ChitoPlant®, redujo la altura sin afectación en el resto de las variables morfológicas evaluadas. Además indujo un incremento significativo en el contenido de cardenólidos. El SilioPlant® (0,01 g.L-1) no provocó afectaciones en la biomasa e incrementó significativamente la síntesis de cardenólidos en los brotes en 3,6 y 6,9 veces el contenido de digoxina y digitoxina respectivamente. La elicitación con el jasmonato de metilo provocó una reducción de la biomasa. Los contenidos de digoxina y digitoxina se incrementaron ligera y significativamente con 80 y 100 µM de jasmonato de metilo respectivamente. El mejor resultado integral se obtuvo con 0,01 g.L-1 de SilioPlant, el cual indujo la mayor producción neta de cardenólidos por frasco de cultivo (4,72 µg digoxina y 88,27 µg digitoxina).

Digitalis purpurea L. is one of the main sources of cardenolides such as digitoxin and digoxin. These drugs are widely used to strengthen cardiac diffusion and to regulate heart rhythm. The aim of this study was to evaluate the influence of three elicitors on shoots of Digitalis purpurea var. Roter Berggold in semisolid media in order to increase cardenolides biosynthesis. Elicitation is a strategy to increase biomass and secondary metabolites production on in vitro cultures. The elicitors evaluated were ChitoPlant (0,001; 0,01; 0,1 g.L-1); SilioPlant (0,01; 0,1; 1,0 g.L-1) and Methyl jasmonate (60, 80, 100 µM), which are reported here to induce cardenolide production for first time. Elicitation resulted an effective strategy to increase cardenolide production on D. purpurea shoot cultures. ChitoPlant induced a decrease in shoots length, but had no effect on the rest of morphological parameters evaluated. As well, ChitoPlant increased cardenolide content. SilioPlant (0,01 g.L-1) did not affect biomass production and at the same time, increased in 3,6-fold and 6,9-fold digoxin and digitoxin content respectively. Elicitation with Methyl jasmonate resulted in decreased biomass production. Digoxin and digitoxin content was slight and significantly increased by Methyl jasmonate 80 and 100 µM respectively. The best integral result was reached with 0,01 g.L-1 of SilioPlant, which induced the highest net yields per culture flask (4,72 µg of digoxin and 88,27 µg of digitoxin).

Digitoxina: superdosagem em medicamento manipulado / Digitoxin: overdose in pharmaceutical compounded preparation

Este relato de caso aborda medicamento manipulado em farmácia magistral, que apresentou teor da substância ativa Digitoxina de 565% em relação ao declarado em sua rotulagem. A solicitação da análise do medicamento veio acompanhada de informe do médico e da autoridade sanitária, na qual o paciente apresentou quadro de intoxicação grave após uso. Este episódio ilustra a dificuldade da garantia da qualidade em estabelecimento de farmácias magistrais, no processo da manipulação de medicamentos de fármacos com índice terapêutico estreito como neste caso dos digitálicos

Emergências cardiovasculares. Parte 1: intoxicaçäo digitálica / Cardiovascular emergencies. Part 1: Digitalis intoxication

Os autores mostram a relevância da intoxicaçäo digitálica dentre as emergências cardiológicas. Realçam a utilidade dos anticorpos monoclonais na intoxicaçäo suicida e o uso do sulfato de magnésio

Captopril no tratamento de pacientes com insuficiência cardíaca congestiva: avaliaçäo cicloergométrica / Captopril in the Treatment of Patients With Congestive Heart Failure. Evaluation in Bicycle Ergometric Test

Objetivo - Analisar a capacidade física de pacientes com insuficiência cardíaca congestiva (ICC), classes I e II da New York Heart Association (NYHA), submetidos à cicloergometria: 1) sob tratamento convencional com digital e diuréticos; 2) com um inibidor da enzima de conversão da angiotensina captopril, associado ao tratamento convencional; 3) usando captopril associado a digital e diurético. Métodos - Estudo randomizado e duplo cego utilizando 20 pacientes com ICC (I e II), submetidos ao teste cicloergométrico em diferentes fases terapêuticas. A carga inicial foi de 5 watts e aumentada progressivamente até o aparecimento de sintomas limitantes. Resultados - A introdução do captopril no esquema convencional de tratamento da ICC ou associado a digital e diurético promove significante aumento na duração do exercício físico, no consumo de oxigênio e na carga total desenvolvida, nos pacientes submetidos ao teste cicloergométrico. Conclusão - Nas formas iniciais de ICC, captopril melhorou a capacidade física dos pacientes em relação ao tratamento convencional e a terapêutica diurética pode ser substituída pelo inibidor da enzima de conversão da angiotensina com igual eficácia

Purpose - To analyze the physical performance of the patients with congestive heart failure (CHF), grades I and II of the New York Heart Association (NYHA), submitted to ergometric test: 1) under conventional treatment with digitalis and diuretic; 2) with an angiotensin converting enzyme inhibitor, captopril, associate with conventional treatment; 3) using captopril associated with digitalis or diuretic. Methods - A randomized double blind study was performed in 20 patients with CHF (I and II - NYHA) submitted to ergometric test in differents therapeutic phases. The initial workload was 5 watts and load was increased until the appearance of limiting symptoms. Results - The introduction of captopril to the conventional treatment for CFH or associated with digitalis or diuretic promotes significant increase in the duration of the physical exercise, in the oxygen consumption and in the total workload during the ergometric test. Conclusion - In the initial forms of CHF, captopril provides better physical performance when compared with conventional treatment and the diuretic treatment can be changed for the angiotensin converting enzyme inhibitor with equal efficacy

Determinaçäo das concentraçöes séricas, do período de meia-vida e das alteraçöes eletrocardiográficas na aplicaçäo de digitoxina em cäes sadios / Determination of serum concentrations after digitoxin administration in healthy dogs, for the half-life and electrocardiographic change periods

As concentraçöes séricas (CS) de digitoxina foram medidas através do método ELISA, após o uso de diferentes doses únicas, aplicadas por via i.v. ou oral, em cäes sadios. Determinaram-se, também, os níveis séricos desse glicosídio na fase platô de um tratamento por via oral e na fase de eliminaçäo, após a suspensäo da terapia. A CS média no platô foi de 20,7 ng/mlñ5,36. A meia-vida sérica do fármaco, calculada a partir das concentraçöes platô, foi de 8,2 horas. Os eletrocardiogramas realizadas durante o tratamento com digitoxina mostraram como única alteraçäo em relaçäo aos ECG controle um prolongamento do intervalo PQ, sendo que um dos animais tratados apresentou bloqueio atrioventricular de 1§ grau (PQ>0,14 seg.) associado a CS de digitoxina de 22,5 ng/ml

Identificaçäo cromatográfica de glicosídeos cardiotônicos em comprimidos e injetáveis / Chromatographic identifications of cardiac glycosides in tablets and injections

Os princípios ativos de comprimidos e injetáveis contendo glicosídeos cardiotônicos (digitoxina, digoxina e lanatosídeo C) foram identificados por Cromatografia em Camada Delgada

Intoxicaçäo digitálica: análise de 216 casos do HSE do Rio de Janeiro / Digitalis intoxication: review of 216 cases at the HSE of Rio de Janeiro

Foram revisados os prontuários de 216 pacientes do HSE do Rio de Janeiro que apresentavam intoxicaçäo digitálica e os resultados obtidos comparados com a literatura. Diversos parâmetros foram analisados, ressaltando-se que os mesmos foram correlacionados às drogas usadas. Houve maior incidência em pacientes do sexo masculino (81,93%), agrupados na faixa etária de 61 a 70 anos (34,72%). A insuficiência cardíaca estava presente em 67,12% dos pacientes. A droga mais utilizada foi digoxina (77,7%). As principais manifestaçöes clínicas foram: vômitos (53,24%), náuseas (52,77%) e descompensaçäo da insuficiência cardíaca (50%). A arritmia mais freqüêntemente encontrada foi extra-sístole ventricular multifocal (25,88%). Houve boa resposta à terapêutica conservadora em 86,11% dos pacientes, sendo que 2,78% evoluíram para o óbito

Acao antiarritmica da metoclopamida (Plasil) em intoxicacao digitalica experimental. Comparacao com a difenil-hidantoina. / Anti-arrhythmia effect of metoclopramide (Plasil) in experimental digitalis intoxication.Comparison with diphenylhydantoin

Foi estudada em caes a acao antiarritmica de metoclopramida (Plasil) em arritmias experimentais causadas por doses toxicas de desacetil-lanatosideo C e de digitoxina Foi realizada comparacao entre a metoclopramida e a fenil-hidantoina em arritmias causadas pela digitoxina. Os resultados mostram que a metoclopramida antagonizou com eficacia as arritmias causadas pelo desacetil-lanatosideo C e reverteu temporariamente, na maioria das experienicas, aquelas decorrentes da administracao de digitoxinas. Ja a definil-hidantoina nao exerceu acao antiarritmica na maioria dos caes estudados