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.
Highlights
Salinomycin (Sal) is a polyether antibiotic widely-used as anticoccidial in poultry [1] and dietary supplement in ruminants’ and pigs’ breed [2,3] due to its antimicrobial activity
Our data support the notion that Sal exerts a pro-apoptotic effect in hepatocellular carcinoma (HCC) cells by inhibition of autophagic flux which leads to accumulation of dysfunctional mitochondria and increased ROSformation
To dissect the precise mode of action of Sal we focused on the molecular mechanism of Sal in HCC
Summary
Salinomycin (Sal) is a polyether antibiotic widely-used as anticoccidial in poultry [1] and dietary supplement in ruminants’ and pigs’ breed [2,3] due to its antimicrobial activity. The potential of Sal as an anti-cancer agent has been elucidated [4]. The precise mode of action of Sal as an anti-cancer agent remains unclear. Several mechanisms such as blocking Wingless-type (Wnt)/b-catenin pathway [12] or activation of the conventional caspase driven apoptotic pathways [13] have been proposed. A new mechanism responsible for the anti-cancer effect of Sal involving drug-mediated alteration of cancer cell autophagic activity was suggested. Depending on the experimental system, either inhibition [14] or activation [15,16,17] by Sal of this cellular degradation pathway have been reported
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