title: 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 µM), 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 PP242-treatment 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 HCC-autophagy, leading to impaired recycling and accumulation of dysfunctional mitochondria with increased ROS-production all of which are associated with induction of apoptosis.
authors.affiliation: Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany; Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Klose
authors.affiliation: Department of Clinical Immunology and Rheumatology, Hannover Medical School, Hannover, Germany.
authors.forename: Metodi V
authors.initials: MV
authors.lastname: Stankov
authors.affiliation: Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.
authors.forename: Moritz
authors.initials: M
authors.lastname: Kleine
authors.affiliation: Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.
authors.forename: Wolf
authors.initials: W
authors.lastname: Ramackers
authors.affiliation: Department of Dermatology, Venereology and Allergology, Medical Faculty Mannheim, University Heidelberg, Mannheim, Germany.
authors.forename: Diana
authors.initials: D
authors.lastname: Panayotova-Dimitrova
authors.affiliation: Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Jäger
authors.affiliation: Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Klempnauer
authors.affiliation: Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Winkler
authors.affiliation: Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.
authors.forename: Hüseyin
authors.initials: H
authors.lastname: Bektas
authors.affiliation: Department of Clinical Immunology and Rheumatology, Hannover Medical School, Hannover, Germany; German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Hannover, Germany.
authors.forename: Georg M N
authors.initials: GM
authors.lastname: Behrens
authors.affiliation: Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany; German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Hannover, Germany.
authors.forename: Florian W R
authors.initials: FW
authors.lastname: Vondran
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects* / Q000235:genetics; D000071193:Autophagy-Related Protein 7; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003049:Coccidiostats / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D011714:Pyrans / Q000494:pharmacology*; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000235:genetics; D000095662:Polyether Polyketides
chemical_list: D000970:Antineoplastic Agents; D000071193:Autophagy-Related Protein 7; D003049:Coccidiostats; D049452:Green Fluorescent Proteins; D008869:Microtubule-Associated Proteins; D011714:Pyrans; D017382:Reactive Oxygen Species; D044764:Ubiquitin-Activating Enzymes; C477012:MAP1LC3A protein, human; C010327:salinomycin; D000095662:Polyether Polyketides; C509349:ATG7 protein, human