Pro-death and pro-survival properties of ouabain in U937 lymphoma derived cells

Cuozzo et al. Journal of Experimental & Clinical Cancer Research 2012, 31:95 http://www.jeccr.com/content/31/1/95 RESEARCH Open Access Pro-death and pro-survival properties of ouabain in U937 lymphoma derived cells Francesca Cuozzo, Marisa Raciti, Laura Bertelli, Rosanna Parente and Livia Di Renzo* Abstract Background: Epidemiological studies revealed significantly lower mortality rates in cancer patients receiving cardiac glycosides, which turned on interest in the anticancer properties of these drugs. However, cardiac glycosides have also been shown to stimulate cell growth in several cell types. In the present investigation we analyzed the pro-death and pro-survival properties of ouabain in the human lymphoma derived cell line U937. Methods: ROS, intracellular Ca++, cell cycle were evaluated by loading the cells with fluorescent probes under cytofluorimetry. Cell counts and evaluation of trypan blue-excluding cells were performed under optic microscope. Protein detection was done by specific antibodies after protein separation from cellular lysates by SDS-PAGE and transfer blot. Results: High doses of ouabain cause ROS generation, elevation of [Ca++]i and death of lymphoma derived U937 cells. Lower doses of OUA activate a survival pathway in which plays a role the Na+/Ca++-exchanger (NCX), active in the Ca++ influx mode rather than in the Ca++ efflux mode. Also p38 MAPK plays a pro-survival role. However, the activation of this MAPK does not appear to depend on NCX. Conclusion: This investigation shows that the cardiac glycoside OUA is cytotoxic also for the lymphoma derived cell line U937 and that can activate a survival pathway in which are involved NCX and p38 MAPK. These molecules can represent potential targets of combined therapy. Keywords: Ouabain, Ca++, NCX, p38 MAPK, Cell death, Cell survival, Lymphoma Background The Na+/K+ ATPase catalyzes the electrogenic exchange of three intracellular Na+ ions for two extracellular K+ ions using for this transport energy that is released from the hydrolysis of ATP. In this way Na+/K+ ATPase plays an important role in the regulation of intracellular Na+ and K+ concentrations and in the maintenance of electrical membrane potential, cell volume, and Na+-coupled transport of amino acids, glucose, nucleotides, and other compounds with low molecular mass [1-3]. Ouabain (OUA) is a cardiac glycoside that has been used for long time for the treatment of cardiac insufficiency. OUA by binding to the α-subunit of Na+/K+ ATPase inhibits it. The inhibition of the Na+/K+ ATPase, reducing the sodium gradient, leads to increased cytosolic [Ca++] probably by impairing the activity of the * Correspondence: Department of Experimental Medicine, University of Rome “La Sapienza”, Viale Regina Elena 324, Rome 00161, Italy Na+/Ca++-exchanger (NCX) [4-9]. NCX is one of the main pathways for intracellular Ca++ clearance [9] and the inhibition of the Na+/K+ ATPase by cardiac glycosides, causing the inversion of the Na+/K+ gradient, leads to impairment of the NCX activity, contributing to accumulation of Ca++ [4-9]. Results from epidemiological studies showed significantly lower mortality rates in cancer patients receiving cardiac glycosides, which turned on interest in the antineoplastic properties of these drugs [10]. In various cancer cell lines, including prostate cancer cells or breast tumor cells, cardiac glycosides induce apoptosis [11-16]. These glycosides are considered to be cytotoxic for tumors because malignant cells express high levels of Na+/K+ ATPase α-isoforms, which are inhibited by them [17]. However, cardiac glycosides induce complex signaling cascades that lead to a variety of effects including the induction of proliferation on vascular smooth muscle cells [18], lymphocytes [19], prostate © 2012 Cuozzo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Cuozzo et al. Journal of Experimental & Clinical Cancer Research 2012, 31:95 http://www.jeccr.com/content/31/1/95 cells [20] and HeLa cells [21]. It appears that cardiac glycosides affect multiple signaling pathways, suggesting that their anti-cancer effect may be multifactorial and context dependent. To clarify the pro-survival or prodeath properties of OUA in the lymphoma derived U937 cells, we set out to investigate how high doses and low doses of the drug affect these parameters. Interestingly, by this means we detected that high doses of OUA are cytotoxic also for U937 cells, while low doses of OUA cause a rise of cytoplasmic Ca++ through NCX which appears to counter cell death. We detected also the activation and the pro-survival role of p38 MAPK upon OUA treatment, which appears to be NCX independent. Methods Reagents RPMI 1640, fetal calf serum, l-glutamine, penicillinstreptomycin, phosphate buffered saline (PBS), ouabain, monensin, tunicamycin and antibodies anti β-actin were from Sigma-Aldrich (St. Louis, MO, USA). Anisomycin, SB203580 and PD98059 were from Calbiochem (Inalco, Milan, Italy). KB-R7943 was from Tocris (Cookson Inc., Ellisville, MO, USA). Antibodies anti phospho-p38 and anti p38 were from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase (HRP)-conjugated antiimmunoglobulin antibodies, enhanced chemiluminescence (ECL) reagents and Hyperfilm-ECL film were from Amersham (Arlington Heights, IL, USA). Protein standards for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and nitrocellulose membranes were from Bio-Rad (Segrate, Milan, Italy). The membrane permeant CDCF-DA and FLUO-3-AM were from Molecular Probes (SIC, Rome, Italy), and other reagents were of the highest purity and purchased from Bio-Rad or Sigma. Cell viability and growth U937 cells, derived from the pleural effusion of a patient with histiocytic lymphoma [22], were grown in complete medium (RPMI-1640 medium supplemented with 1.0% sodium pyruvate, 5% FCS, 2 μM glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin) at 37°C, in fully humidified atmosphere 95% room air/5% CO2. Cells were resuspended three times a week in fresh complete medium as 3×105/ml. Cell growth was evaluated by hemocytometry counts of cells excluding Trypan blue (0.04% Trypan blue in PBS, w/v), and viability was assessed by calculating alive (trypan blue-excluding) cells as percentage of all cells counted. Cells used in every experiment were ≥93% viable and taken from cultures in exponential growth. They were washed once and resuspended in complete medium, 1×106/ml, and transferred to 24-well microplates. They were then treated with Page 2 of 8 inhibitors or vehicles, incubated for 30 min, and susequently exposed to test agents or, again, to vehicles. At the end of each experiment, the cells were gently mixed and aliquots were taken (...truncated)