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.

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.

RESUMO Glicosídeos cardíacos como a digoxina e digitoxina em doses terapêuticasPalavras chaves: glicosídeos cardíacos, digitálicos, câncer cervical, atividade Na+/K+-ATPase. INTRODUÇÃOA busca por novos compostos com ação antitumoral é uma parte importante no estudo para a cura do câncer. A Na + /K + -ATPase é uma proteína de membrana cuja principal função é atuar como regulador da homeostase iônica celular. Além disso, é um regulador do crescimento celular e da transcrição gênica. A inibição da Na + /K + -ATPase está sendo utilizada como . Novos compostos sintetizados a partir da digoxina ainda não possuem nenhuma caracterização biológica de efeitos na Na + /K + -ATPase e nem seu potencial efeito antitproliferativo em células tumorais. O objetivo do trabalho foi verificar o efeito citotóxico de novos compostos glicosídeos cardíacos em células de câncer de colo uterino (HeLa) e cólon intestinal retal (RKO), correlacionando o efeito antitumoral com a atividade da Na + / K + -ATPase. MATERIAL E MÉTODOS Cultura de células e tratamento com os glicosídeos cardíacosAs células de colo uterino (HeLa) e cólon intestinal retal (RKO) foram cultivadas a 37ºC e atmosfera de 5% de CO 2 em meio de cultura RPMI (Sigma) complementado com 10% de soro fetal bovino (Hyclone) em atmosfera de 5% de CO 2 . Para os experimentos de proliferação celular, foram adicionadas 22,5x10 5 células em cada garrafa de cultivo, nas quais foi possível observar uma multiplicação e aderência celular satisfatória. As células foram tratadas com digoxina a 150nM durante 24h de exposição e com os glicosídeos cardíacos DGB2, DGB3 e DGB4 nas concentrações de 150nM e 10µM. A exposição de DGB2 às células foi de 1h e 24h, e DGB3 e DGB4 foram de 24h.

Recently, new data began to consider cardiac glycoside as a potential antitumor drug. We synthesized 21‐benzylidene digoxin (21‐BD) through an aldol condensation of digoxin lactone ring. Molecular docking was performed to assess the main interactions between cardiac glycosides and Na,K‐ATPase, demonstrating a more stable binding energy for 21‐BD, compared with digoxin. Therefore, we used HeLa and RKO cancer cell lines after their exposure to cardiac glycosides to asses the cell viability as to cytotoxicity and proliferative ability. On HeLa cells, digoxin presented an IC50 = 1,47 μM, whereas 21‐BD presented an IC50 = 56,16 μM. Nevertheless, on RKO cells, digoxin presented an IC50 = 0,46 μM and 21‐BD presented an IC50 = 54,36 μM. Cometa assay showed DNA damage upon 20 μM of both cardiac glycosides. The treatment of cells with 21‐BD for 48h caused an increase of Na,K‐ATPase activity, whereas digoxin caused a decrease on enzyme activity. However, 21‐BD did not cause inhibition of Na,K‐ATPase of a membrane preparation from rat cerebral hemisphere. In attempt to avoid the 21‐BD effect though estrogen receptor, the pre‐treatment with tamoxifen did not interrupted the DGB1 effect. Thus, these data demonstrate that this new glycoside has a different mechanism of action then digoxin, and it can be an important tool to study the enzyme.Supported by: FAPEMIG, CNPq and CAPES