title: Cardiac glycoside ouabain induces autophagic cell death in non-small cell lung cancer cells via a JNK-dependent decrease of Bcl-2.
abstract: Cardiac glycosides are Na/K-ATPase inhibitors, clinically used for congestive heart failure and cardiac arrhythmias. Epidemiological studies have reported that patients on cardiac glycosides treatment are protected from some types of cancers. This evidence together with the demonstration that cardiac glycosides show selective cytotoxicity against cancer cells has raised new interest on the anticancer properties of these drugs. This study examines the mechanism involved in the anticancer effect of ouabain in non-small cell lung cancer cells lines (A549 and H1975). Ouabain inhibited cell proliferation and induced cell death in a concentration-dependent manner. Cell death was caspase-independent and showed classical patterns of autophagic cell death: conversion of LC3-I to LC3-II, increase of LC3 puncta and increase of autophagic flux. Moreover, cell death was completely blocked by the class III phosphatidylinositol-3 kinase inhibitor 3-methyladenine. Here we show that ouabain caused the reduction of Bcl-2 protein levels, with no change in the expression of the autophagic protein Beclin 1. Early signalling events of ouabain exposure were ERK1/2 and JNK activation, however only JNK inhibition with SP600125 or JNK knockdown by shRNA were able to prevent Bcl-2 decrease, conversion of LC3-I to LC3-II and cell death. We propose that JNK activation by ouabain leads to a decrease of Bcl-2 levels, resulting in disruption of the inhibitory interaction of Bcl-2 with Beclin 1, that promotes autophagy. These findings indicate that pharmacological modulation of autophagy by cardiac glycosides could be exploited for anticancer therapy.
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Largo Meneghetti 2, 35131 Padova, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Trenti
authors.affiliation: Department of Molecular Medicine, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Grumati
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Largo Meneghetti 2, 35131 Padova, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Cusinato
authors.affiliation: E. MEDEA Scientific Institute, c/o University of Padova, Department of Pharmaceutical and Pharmacological Sciences, Largo Meneghetti 2, 35131 Padova, Italy.
authors.forename: Genny
authors.initials: G
authors.lastname: Orso
authors.affiliation: Department of Molecular Medicine, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Bonaldo
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Largo Meneghetti 2, 35131 Padova, Italy. Electronic address: lucia.trevisi@unipd.it.
authors.forename: Lucia
authors.initials: L
authors.lastname: Trevisi
mesh_terms: D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*; D002301:Cardiac Glycosides / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D002301:Cardiac Glycosides; D019253:Proto-Oncogene Proteins c-bcl-2; D010042:Ouabain; D048031:JNK Mitogen-Activated Protein Kinases
keywords: Autophagy; Bcl-2; Cardiac glycosides; JNK; Non-small cell lung cancer