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.

Tumor acidosis enhances cytotoxic effects and autophagy inhibition by salinomycin on cancer cell lines and cancer stem cells

“…SAL was reported to induce cell death via autophagy upregulation in some experimental models [45, 46]. However, it was recently reported that 2 μM SAL inhibits the autophagic flux in breast and hepatocellular carcinomas [43, 47]. In order to establish the activity of SAL on autophagic flux, we started our investigation by using HOS cells stably transfected with a GFP-LC3 vector, which allows the analysis of the autophagic flux by flow cytometry by monitoring the accumulation of GFP-LC3-positive autophagosomes in the presence of lysosomal inhibitors [48].…”

Geno- and cytotoxicity of salinomycin in human nasal mucosa and peripheral blood lymphocytes

“…Recently, salinomycin was demonstrated to be an mTOR C1 and Wnt/b-catenin signaling inhibitor by suppressing lipoprotein receptor-related protein-6 (LRP6) in breast and prostate cancer cells (Lu and Li, 2014). Another study showed an inhibition of autophagic activity by salinomycin in hepatocellular carcinoma (Klose et al, 2014). Additionally, salinomycin was introduced as a potent drug to overcome apoptosis resistance in several types of cancer (Lieke et al, 2012).…”

Salinomycin inhibits metastatic colorectal cancer growth and interferes with Wnt/β-catenin signaling in CD133+ human colorectal cancer cells

“…Sal was dissolved in dimethyl sulfoxide (DMSO) for in vitro analysis [16] or in corn oil for in vivo applications [17]. 5-FU was dissolved in phosphate buffered saline (PBS).…”

“…Cells were analyzed for apoptosis, late apoptosis or necrosis induction following exposure to either Sal or 5-FU alone or combined Sal and 5-FU for 24 h applying the AnnexinV apoptosis detection kit (BD Biosciences) according to the manufacturer’s instructions as previously described [16, 23]. AnnexinV positive cells were regarded as apoptotic cells; AnnexinV/PI positive cells were regarded as late apoptotic and PI positive cells were regarded as necrotic cells.…”