P46 INHIBITION OF AUTOPHAGIC FLUX BY SALINOMYCIN RESULTS IN ANTI-CANCER EFFECT IN HEPATOCELLULAR CARCINOMA CELLS

Abstract

Salinomycin raised hope to be effective in anti-cancer therapies due to its capability to overcome apoptosis-resistance in several types of cancer cells. Recently, its effectiveness against human hepatocellular carcinoma (HCC) cells both in vitro and in vivo was demonstrated. However, the mechanism of action remained unclear. Latest studies implicated interference with the degradation pathway of autophagy. This study aimed to determine the impact of Salinomycin on HCC-autophagy and whether primary human hepatocytes (PHH) likewise are affected. Following exposure of HCC cell lines HepG2 and Huh7 to varying concentrations of Salinomycin (0–10 mM), comprehensive analysis of autophagic activity using western-blotting and flow-cytometry was performed. Drug effects were analyzed in the settings of autophagy stimulation by starvation or PP242treatment and correlated with cell viability, proliferation, apoptosis induction, mitochondrial mass accumulation and reactive oxygen species (ROS) formation. Impact on apoptosis induction and cell function of PHH was analyzed. Constitutive and stimulated autophagic activities both were effectively suppressed in HCC by Salinomycin. This inhibition was associated with dysfunctional mitochondria accumulation, increased apoptosis and decreased proliferation and cell viability. Effects of Salinomycin were dose and time dependent and could readily be replicated by pharmacological and genetic inhibition of HCC-autophagy alone. Salinomycin exposure to PHH resulted in transient impairment of synthesis function and cell viability without apoptosis induction. In conclusion, our data suggest that Salinomycin suppresses late stages of HCCautophagy, leading to impaired recycling and accumulation of dysfunctional mitochondria with increased ROS-production all of which are associated with induction of apoptosis. Citation: Klose J, Stankov MV, Kleine M, Ramackers W, Panayotova-Dimitrova D, et al. (2014) Inhibition of Autophagic Flux by Salinomycin Results in Anti-Cancer Effect in Hepatocellular Carcinoma Cells. PLoS ONE 9(5): e95970. doi:10.1371/journal.pone.0095970 Editor: Manlio Vinciguerra, University College London, United Kingdom Received January 10, 2014; Accepted April 1, 2014; Published May 9, 2014 Copyright: 2014 Klose et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by Heidelberger Stiftung Chirurgie and Gesellschaft der Freunde fur Chirurgie (JK), KFO 250 (GB, MVS), Rebirth EXC 62/1 (GB), and Else Kroner-Fresenius-Stiftung (FWRV; 2010_A49). The authors acknowledge financial support by Deutsche Forschungsgemeinschaft and Ruprecht-KarlsUniversitat Heidelberg within the funding programme Open Access Publishing. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Johannes.Klose@med.uni-heidelberg.de . These authors contributed equally to this work.