Signalling pathways involved in the apoptotic action of ouabain in two different cancer cell lines

Cardiac glycosides, like ouabain, are specific inhibitors of plasma membrane Na+/K+-ATPase, enzyme responsible for translocating Na+and K+ ions across cell membrane using ATP as energy source. Recent findings suggest for Na+/K+-ATPase a role as signal transducer, involved in the control of cell proliferation and growth or in the protection against apoptotic stimuli. In fact, the binding of ouabain to Na+/K+-ATPase (at concentrations that do not inhibit the pump activity) triggers a complex signaling cascade that is initiated by interacting with neighboring membrane proteins and organized cytosolic cascades of signaling molecules. These signaling complexes send messages to intracellular organelles via the activation of the protein tyrosine kinase Src, transactivation of epidermal growth factor receptor (EGFR) by Src, activation of Ras and the extracellular signal-regulated kinase (ERK). Also a ROS dependent c-Jun N-terminal kinase (JNK) activation has been shown to be involved in the pathways activated by ouabain (1). In agreement with these findings, we have previously showed that ouabain has an antiapoptotic effect on HUVEC through the activation of phosphoinositide-3 kinase (PI3K) and ERK (2). On the other hand, several studies have suggested that cardiac glycosides may have an anticancer utilization. Evidences of the antineoplastic potential of cardiac glycosides have been obtained with in vitro studies, but the most relevant evidences of the beneficial effects of cardiac glycosides in cancer treatment were drawn from epidemiological data. The death rate and cancer recurrence turned out to be lower in women with breast cancer treated with digitalis than in non-treated patients. Moreover, it was observed a reduced incidence of leukemia/lymphoma and kidney/urinary tract tumours in subjects with elevated plasmatic concentrations of digitoxin (3). The aim of my doctoral work is to characterize the effect of nM concentrations of ouabain on two cancer cell lines: Jurkat (immortalized cell line of T lymphocytes) and A549 (carcinomic human alveolar basal epithelial cells) with particular attention to the signaling pathway involved. Cell treatment with ouabain (1-100 nM) for 24 h induced a concentration-dependent decrease in cell viability measured by the MTT reduction assay both in Jurkat and A549 cells. The decrease in cell viability at 100 nM was 67.0±2.2 e 70.0±2.1 in Jurkat and A549, respectively. Incubation of both cell lines with 100 nM ouabain for 24 h induced a significant raise in the number of apoptotic cells, as indicated by flow cytometric analysis of annexin V/propidium iodide binding: ouabain increased the proportion of annexin V positive (apoptotic) cells from 7.0±0.7% to 33.0±6.0% in Jurkat cells and from 7.0±0.6% to 22.0±2.0% in A549 cells. However, an increase in caspase-3 activation induced by ouabain treatment was observed only in Jurkat cells. In order to clarify the signaling pathways involved in the mechanism of action of ouabain, the role of ERK, Src kinase and JNK was investigated. A transient increase in ERK1/2 phosphorylation (determined by Western blotting analysis) was observed in Jurkat cells treated with 100 nM ouabain for 30 min. Nevertheless, incubation of the cells with a specific MEK inhibitor, PD98059 (25 μM) or U0126 (10 μM) did not abolish the apoptotic effect of ouabain, as shown by MTT test and flow cytometric analysis of annexin V/PI binding. Similar results were obtained with A549 cells. Furthermore, treatment of both tumour cell lines with the inhibitor of JNK, SP600125, did not affect the apoptotic action of ouabain. To ascertain whether the activation of Src kinase is required for the ouabain-effect on cancer cells we used the Src-kinase inhibitor PP2 (50 μM). This compound did not affect the ouabain-induced apoptosis in both Jurkat and A549 cells. Our results show that nM concentrations of ouabain are pro-apoptotic for Jurkat and A549 cells through a mechanism that does not involve the classical pathways of signal transduction activated by ouabain in non-tumoral cells. References Schoner W et al. (2007) Am J Physiol Cell Physiol 293:509 Trevisi L et al. (2004) Biochem Biophys Res Commun 321:716 Newman RA et al. (2008) Mol Interv 8:36