Induction of Apoptosis in HepaRG Cell Line by Aloe-Emodin through Generation of Reactive Oxygen Species and the Mitochondrial Pathway

Cell Physiol Biochem. 2017;42(2):685-696. doi: 10.1159/000477886. Epub 2017 Jun 15.

Abstract

Background/aims: Aloe-emodin (1,8-dihydroxy-3-hydroxymethyl-anthraquinone), an anthraquinone active compounds, is isolated from some traditional medicinal plants such as Rheum palmatum L. and Cassia occidentalis, which induce hepatotoxicity in rats. The aim of this study was to determine potential cytotoxic effects of aloe-emodin on HepaRG cells and to define the underlying mechanism.

Methods: MTT was used to evaluate cell viability. Apoptotic cell death was analyzed via Annexin V-FITC/PI double staining. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were determined by flow cytometry, while the expression of apoptosis-related proteins was determined by Western blot analysis.

Results: Treatment with aloe-emodin significantly reduced cell viability and induced apoptosis in HepaRG cells in a dose- and time-dependent manner. It provoked ROS generation and depolarization of MMP in HepaRG cells when compared with controls. Aloe-emodin dose-dependently increased release of mitochondrial cytochrome c, and levels of Fas, p53, p21, Bax/Bcl-2 ratio, as well as activation of caspase-3, caspase-8, caspase-9, and subsequent cleavage of poly(ADP-ribose)polymerase (PARP). It also induced S-phase cell cycle arrest by increasing the expression of p21 and cyclin E proteins while significantly decreasing the expression of cyclin A and CDK2.

Conclusion: These results suggest that aloe-emodin inhibits cell proliferation and induces apoptosis in HepaRG cells, most probably through a mechanism involving both Fas death pathway and the mitochondrial pathway by generation of ROS. These findings underscore the need for risk assessment of human exposure to aloe-emodin.

Keywords: Aloe-emodin; Apoptosis; HepaRG cells; Hepatotoxicity; ROS.

MeSH terms

  • Animals
  • Anthraquinones / administration & dosage*
  • Apoptosis / drug effects*
  • Apoptosis Regulatory Proteins / biosynthesis
  • Caspase 3 / biosynthesis
  • Caspase 8 / biosynthesis
  • Caspase 9 / biosynthesis
  • Cell Cycle Checkpoints / drug effects
  • Cell Proliferation / drug effects*
  • Cell Survival / drug effects
  • Gene Expression Regulation / drug effects
  • Humans
  • Metabolic Networks and Pathways / drug effects
  • Mitochondria / drug effects
  • Mitochondria / metabolism*
  • Mitochondria / pathology
  • Rats
  • Reactive Oxygen Species / metabolism

Substances

  • Anthraquinones
  • Apoptosis Regulatory Proteins
  • Reactive Oxygen Species
  • aloe emodin
  • Caspase 3
  • Caspase 8
  • Caspase 9