Cardiac glycoside ouabain induces autophagic cell death in non-small cell lung cancer cells via a JNK-dependent
Annalisa Trenti1, Paolo Grumati2, Federico Cusinato1
1Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Largo Meneghetti 2, 35131 Padova, Italy.
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.
Insights
Cardiac glycosides like ouabain induce cancer cell death through autophagy, not apoptosis. This mechanism involves JNK activation, leading to reduced Bcl-2 and promoting autophagy, suggesting potential anticancer therapies.
Area of Science:
- Pharmacology
- Cancer Biology
- Cellular Biology
Background:
- Cardiac glycosides, such as ouabain, are Na/K-ATPase inhibitors used for heart conditions.
- Epidemiological and in vitro studies suggest cardiac glycosides possess anticancer properties.
- Ouabain demonstrates selective cytotoxicity against cancer cells, warranting mechanistic investigation.
Purpose of the Study:
- To investigate the anticancer mechanism of ouabain in non-small cell lung cancer (NSCLC) cell lines.
- To elucidate the signaling pathways and cellular processes involved in ouabain-induced cancer cell death.
Main Methods:
- Cell proliferation and death assays in A549 and H1975 NSCLC cell lines.
- Analysis of apoptosis markers (caspase-independent) and autophagic markers (LC3-I/II conversion, LC3 puncta, autophagic flux).
- Inhibition studies using 3-methyladenine (PI3K inhibitor), SP600125 (JNK inhibitor), and JNK knockdown (shRNA).
- Western blot analysis for Bcl-2 and Beclin 1 protein levels.
- Assessment of ERK1/2 and JNK signaling pathway activation.
Main Results:
- Ouabain inhibited NSCLC cell proliferation and induced cell death in a concentration-dependent manner.
- Cell death was caspase-independent and exhibited characteristics of autophagic cell death, confirmed by increased LC3-II, LC3 puncta, and autophagic flux.
- Autophagic cell death was blocked by 3-methyladenine.
- Ouabain reduced Bcl-2 protein levels but did not alter Beclin 1 expression.
- Ouabain activated ERK1/2 and JNK pathways; JNK inhibition or knockdown prevented Bcl-2 reduction, LC3 conversion, and cell death.
Conclusions:
- Ouabain induces autophagic cell death in NSCLC cells via JNK activation.
- JNK activation by ouabain leads to decreased Bcl-2, disrupting its interaction with Beclin 1 and promoting autophagy.
- Pharmacological modulation of autophagy using cardiac glycosides represents a potential anticancer therapeutic strategy.
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