Cardiac glycosides are secondary plant metabolites, which are commonly used for treatment of heart failure and cardiac arrhythmias due to their positive ionotropic effect. On molecular basis, they are composed of a steroid skeleton and a sugar moiety. Their mechanism of action is mediated through an interaction with Na+/K+-ATPase, the inhibition of which also leads to apoptosis. Nowadays, cardiac glycosides are studies as potential therapeutics for cancer treatment. One of the most important members of cardiac glycosides is digitoxin.
The aim of this work was design, synthesis and biological evaluation of novel fluorescent digitoxin conjugates, possibly applied in theranostics, having selective cytotoxicity between cancer and primary cell lines. We prepared digitoxin derivatives conjugated to various BODIPY dyes by modification of the terminal digitoxose of digitoxin using copper-catalyzed click chemistry and studied their interactions with Na+/K+-ATPase (organism: wild boar) using molecular docking methods. Further, we evaluated their cytotoxicity in cancer (A549) and primary (MRC-5) cell lines after 72 h of treatment using WST-1 method. Fluorescent derivatives of digitoxin were less cytotoxic in comparison with digitoxin, which is satisfactory according to excessive digitoxin toxicity and numerous side effects when used in clinics. All tested compounds were more cytotoxic against the tested cancer cell line in comparison to the primary cell line. By means of fluorescence microscopy, we investigated intracellular localization of the digitoxin-BODIPY conjugates, which were detected in endoplasmic reticulum and lysosomes of A549, HEK 293T and MRC-5 cells. Last but not least, the effect of digitoxin-BODIPY on the cell cycle of A549 cancer cell line was studied using flow cytometry. We detected a decrease in the G0/G1 and, on the contrary, an increase in the S and G2/M phases. We also confirmed that digitoxin-BODIPY induced apoptosis as a predominant type of cell death in A549 cells. The results of this study could help us understand how the derivatization of a sugar moiety changes the behavior of digitoxin within biological compartments and also how to develop new compounds with better targeting toward cancer cells.
Financial support from specific university research (MSMT No 21-SVV/2019), by Martina Roeselová Foundation, Czech Republic and by projects OPPC CZ.2.16/3.1.00/24503 and NPU I LO1601.