ABSTRACT
Title
Ouabain at nanomolar concentrations is citostatic and induces programmed cell death in two different cancer cell lines
Authors
A. Trenti
PhD School in Pharmacological Sciences
Dept of Pharmacology and Anaesthesiology, University of Padova, Padova, Italy
Abstract
The cardiac glycoside ouabain binds to Na/K-ATPase and activates signalling cascades resulting in changes of cell proliferation, differentiation or apoptosis in a cell-specific manner (1). Accordingly, we have previously showed that ouabain has an antiapoptotic effect on human endothelial cells through the activation of phosphoinositide-3 kinase (PI-3K) and ERK (2). Recently cardiac glycosides have been studied as potential anticancer agents. A beneficial effect of cardiac glycosides in cancer treatment were drawn from epidemiological data: death rate and cancer recurrence is lower in women with breast cancer treated with digitalis. Moreover, there is a reduced incidence of leukemia/lymphoma and kidney/urinary tract tumors in subjects with elevated plasmatic concentrations of digitoxin (3).
The effect of ouabain (1-100 nM) was studied in two cancer cell lines: Jurkat (human T cell lymphoblast-like cell line) and A549 (human lung adenocarcinoma epithelial cell line). Cell viability was determined by methyl thiazolyl tetrazolium (MTT) assay. Proliferation was measured by cell counting and clonogenic assay. Apoptosis was ascertained by flow cytometric analysis of annexin V/propidium iodide binding, analysis of caspase-3 activation and analysis of DNA fragmentation. Levels of antiapoptotic Bcl-2 protein was determined by western blotting. In both cell lines ouabain induced a decrease in cell proliferation at low concentrations and cell death at concentrations over 50nM. Decrease of Bcl-2 protein expression was an early event. In Jurkat, cell death was preceded by a decrease of mitochondrial potential and an increase of caspase-3 activity. DNA laddering of ouabain-treated Jurkat was characteristic of caspase-dependent apoptosis. Instead, in A549 cells no changes in mitochondrial potential and caspase-3 activity were found and a smear pattern of DNA fragmentation was observed after ouabain treatment. Pretreatment with the pan-caspase inhibitor z-VAD-fmk prevented cell death only in Jurkat. In A549, the cytotoxic effect of ouabain was completely blocked by treatment with an inhibitor of autophagy such as 3-methyladenine or NH4Cl suggesting that autophagic cell death is involved. These findings indicate that ouabain, at the same concentrations that induce antiapoptotic and proliferative effects in normal cells, blocks cell proliferation and promotes programmed cell death in cancer cells. Depending on the cell type ouabain activates type I (apoptosis) or type two (autophagy) cell death.
The effect of ouabain (1-100 nM) was studied in two cancer cell lines: Jurkat (human T cell lymphoblast-like cell line) and A549 (human lung adenocarcinoma epithelial cell line). Cell viability was determined by methyl thiazolyl tetrazolium (MTT) assay. Proliferation was measured by cell counting and clonogenic assay. Apoptosis was ascertained by flow cytometric analysis of annexin V/propidium iodide binding, analysis of caspase-3 activation and analysis of DNA fragmentation. Levels of antiapoptotic Bcl-2 protein was determined by western blotting. In both cell lines ouabain induced a decrease in cell proliferation at low concentrations and cell death at concentrations over 50nM. Decrease of Bcl-2 protein expression was an early event. In Jurkat, cell death was preceded by a decrease of mitochondrial potential and an increase of caspase-3 activity. DNA laddering of ouabain-treated Jurkat was characteristic of caspase-dependent apoptosis. Instead, in A549 cells no changes in mitochondrial potential and caspase-3 activity were found and a smear pattern of DNA fragmentation was observed after ouabain treatment. Pretreatment with the pan-caspase inhibitor z-VAD-fmk prevented cell death only in Jurkat. In A549, the cytotoxic effect of ouabain was completely blocked by treatment with an inhibitor of autophagy such as 3-methyladenine or NH4Cl suggesting that autophagic cell death is involved. These findings indicate that ouabain, at the same concentrations that induce antiapoptotic and proliferative effects in normal cells, blocks cell proliferation and promotes programmed cell death in cancer cells. Depending on the cell type ouabain activates type I (apoptosis) or type two (autophagy) cell death.