Therapeutic concentrations of digitoxin inhibit endothelial focal adhesion kinase and angiogenesis induced by different growth factors

Background and Purpose: Cardiac glycosides are Na⁺/K⁺-ATPases inhibitors used to treat congestive heart failure and cardiac arrhythmias. Epidemiological studies indicate that patients on digitalis therapy are more protected from cancer. Evidence of a selective cytotoxicity against cancer cells has suggested their potential use as anticancer drugs. The effect on angiogenesis of clinically used cardiac glycosides has not been extensively explored. Experimental Approach: We studied the effect of digoxin, digitoxin and ouabain on early events of the angiogenic process in HUVECs. We determined HUVEC viability, proliferation, migration and differentiation into capillary tube-like structures. We also tested drug activity using an in vivo angiogenesis model. Activation of protein tyrosine kinase 2 (FAK) and signalling proteins associated with the Na⁺/K⁺-ATPase signalosome was determined by Western blotting. Key Results: Digitoxin and ouabain (1–100 nM) inhibited HUVEC migration, concentration-dependently, without affecting cell viability, while digoxin induced apoptosis at the same concentrations. Digitoxin antagonized growth factor-induced migration and tubularization at concentrations (1–25 nM) within its plasma therapeutic range. The anti-angiogenic effect of digitoxin was confirmed also by in vivo studies. Digitoxin induced Src, Akt and ERK1/2 phosphorylation but did not affect FAK autophosphorylation at Tyr³⁹⁷. However, it significantly inhibited growth factor-induced FAK phosphorylation at Tyr^576/577. Conclusions and Implications: Therapeutic concentrations of digitoxin inhibited angiogenesis and FAK activation by several pro-angiogenic stimuli. These novel findings suggest a potential repositioning of digitoxin as a broad-spectrum anti-angiogenic drug for diseases where pathological angiogenesis is involved.