Asiatic acid uncouples respiration in isolated mouse liver mitochondria and induces HepG2 cells death

Abstract

Asiatic acid, one of the triterpenoid components isolated from Centella asiatica, has received increasing attention due to a wide variety of biological activities. To date, little is known about its mechanisms of action. Here we examined the cytotoxic effect of asiatic acid on HepG2 cells and elucidated some of the underlying mechanisms. Asiatic acid induced rapid cell death, as well as mitochondrial membrane potential (MMP) dissipation, ATP depletion and cytochrome c release from mitochondria to the cytosol in HepG2 cells. In mitochondria isolated from mouse liver, asiatic acid treatment significantly stimulated the succinate-supported state 4 respiration rate, dissipated the MMP, increased Ca(2+) release from Ca(2+)-loaded mitochondria, decreased ATP content and promoted cytochrome c release, indicating the uncoupling effect of asiatic acid. Hydrogen peroxide (H2O2) produced by succinate-supported mitochondrial respiration was also significantly inhibited by asiatic acid. In addition, asiatic acid inhibited Ca(2+)-induced mitochondrial swelling but did not induce mitochondrial swelling in hyposmotic potassium acetate medium which suggested that asiatic acid may not act as a protonophoric uncoupler. Inhibition of uncoupling proteins (UCPs) or blockade of adenine nucleotide transporter (ANT) attenuated the effect of asiatic acid on MMP dissipation, Ca(2+) release, mitochondrial respiration and HepG2 cell death. When combined inhibition of UCPs and ANT, asiatic acid-mediated uncoupling effect was noticeably alleviated. These results suggested that both UCPs and ANT partially contribute to the uncoupling properties of asiatic acid. In conclusion, asiatic acid is a novel mitochondrial uncoupler and this property is potentially involved in its toxicity on HepG2 cells.

Keywords: 6-ketocholestanol (pubchemcid: 102008); Antimycin a (pubchemcid: 12550); Asiatic acid; Asiatic acid (pubchemcid: 119034); Carboxyatractyloside (pubchemcid: 36403); Cyclosporine a (pubchemcid: 5284373); Fccp (pubchemcid: 3330); Guanosine 5′-diphosphate (pubchemcid: 8977); HepG2 cell death; Mitochondrial respiration; Oligomycin (pubchemcid: 5281899); Propanolol (pubchemcid: 4946); Reactive oxygen species; Rhodamine 123 (pubchemcid: 65217); Trypan blue (pubchemcid: 6364561); Uncoupler; Valinomycin (pubchemcid: 3000706).