Effects of Cholesterol on Autophagy and Apoptosis in HepG2 cells

Abstract

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent liver diseases. Recent studies have shown that cholesterol is accumulated in liver during the progression of NAFLD. Thus, increasing attention has been drawn to the role of cholesterol in the pathogenesis of the disease. Autophagy, a cellular degradative pathway, is an important self-defense mechanism to maintain cellular homeostasis in response to various stresses including lipid stimulation. In this context, this study was undertaken to determine cholesterol lipotoxicity in hepatocytes using cholesterol overloaded HepG2 cells. Precisely, whether dysregulated cholesterol homeostasis alters autophagic activity and contributes to apoptosis was investigated. Methods: Human hepatocarcinoma cell line, HepG2 cells were treated with cholesterol with either 25 or 50 µg/mL of concentrations, which are considered to deposit cholesterol as much as the amount found in human and experimental NAFLD model. Additional treatment using 10µM chloroquine was conducted in some experiments to inhibit lysosomal activity and degradation of autophagosome. Intracellular cholesterol level was measured by enzymatic colorimetric methods and the cell viability was determined through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The relative levels of several autophagy or apoptosis related proteins were analyzed by Western blotting. Production of reactive oxygen species (ROS) was assessed by observing emission of green fluorescence following oxidation of 2,7-dichlorofluorescein diacetate. Lipid peroxide levels were determined by measuring cellular thiobarbituric acid reactive substances (TBARS) levels. Results: Intracellular total cholesterol level increased in cells treated with 50 µg/mL cholesterol after 24 hours compared with vehicle treated cells. MTT assays showed dose-dependent cholesterol toxicity and microscopic observations showed reduction of cell populations compared with vehicle control cells when 25 or 50 µg/mL of cholesterol were treated. Detection of cleavage of non-erythroid spectrin and caspase-3 revealed that cholesterol treatment prominently induced the apoptosis in HepG2 cells. Meanwhile, regarding autophagy pathway, treatment of cholesterol induced the increases of LC3-II and p62 protein levels dose-dependently. Beclin-1 remained unchanged. Moreover, in a LC3-II turnover assay, cells treated with chloroquine plus cholesterol showed less LC3-II degradation than those with chloroquine alone. Cholesterol also resulted in concomitant increase of ubiquitinated protein levels. Thus, these data collectively indicated that cholesterol treatment reduced autophagic flux. To clarify whether defects in autophagy is the primary cause of cholesterol induced caspase-3 activation, cell viability was measured using MTT assay after chloroquine or/and cholesterol treatment. As a result, 10 µM chloroquine treatment induced 10.7% viability loss while 50 µg/mL cholesterol treatment 44.6% viability loss, despite similar level of autophagy inhibition. Thus, it can be inferred that blockage of autophagic flux is not enough to cause systemic cell death, unless other toxic mechanisms are involved. Production of ROS and induction of lipid peroxides were observed after 50 µg/mL of cholesterol were treated, suggesting cholesterol accumulation induced oxidative stress. Activation of c-Jun N-terminal kinase (JNK) was also detected following the cholesterol loading. Furthermore, mitochondrial dysfunction which is strongly associated with oxidative stress was confirmed by detection of cleavage of caspase-9. Increased phosphorylation level of adenosine monophosphate-activated kinase (AMPK) was also observed, which indicates occurrence of ATP depletion. Conclusion: These findings demonstrate that excessive hepatic cholesterol induces blockage of autophagic flux, oxidative stress and the subsequent mitochondrial dysfunction. Although reduced autophagic activity per se was insufficient to cause cholesterol induced hepatocyte apoptosis entirely, it seems to aggravate oxidative stress due to attenuated removal of dysfunctional mitochondria. Overall, the present study demonstrates that cholesterol, besides free fatty acids, may contribute to lipotoxicity occurring in the progression of NAFLD.