Concurrent acetylation of FoxO1/3a and p53 due to sirtuins inhibition elicit Bim/PUMA mediated mitochondrial dysfunction and apoptosis in berberine-treated HepG2 cells

Toxicol Appl Pharmacol. 2016 Jan 15;291:70-83. doi: 10.1016/j.taap.2015.12.006. Epub 2015 Dec 19.

Abstract

Post-translational modifications i.e. phosphorylation and acetylation are pivotal requirements for proper functioning of eukaryotic proteins. The current study aimed to decode the impact of acetylation/deacetylation of non-histone targets i.e. FoxO1/3a and p53 of sirtuins (NAD(+) dependent enzymes with lysine deacetylase activity) in berberine treated human hepatoma cells. Berberine (100 μM) inhibited sirtuins significantly (P<0.05) at transcriptional level as well as at translational level. Combination of nicotinamide (sirtuin inhibitor) with berberine potentiated sirtuins inhibition and increased the expression of FoxO1/3a and phosphorylation of p53 tumor suppressor protein. As sirtuins deacetylate non-histone targets including FoxO1/3a and p53, berberine increased the acetylation load of FoxO1/3a and p53 proteins. Acetylated FoxO and p53 proteins transcriptionally activate BH3-only proteins Bim and PUMA (3.89 and 3.87 fold respectively, P<0.001), which are known as direct activator of pro-apoptotic Bcl-2 family protein Bax that culminated into mitochondria mediated activation of apoptotic cascade. Bim/PUMA knock-down showed no changes in sirtuins' expression while cytotoxicity induced by berberine and nicotinamide was curtailed up to 28.3% (P<0.001) and it restored pro/anti apoptotic protein ratio in HepG2 cells. Sirtuins inhibition was accompanied by decline in NAD(+)/NADH ratio, ATP generation, enhanced ROS production and decreased mitochondrial membrane potential. TEM analysis confirmed mitochondrial deterioration and cell damage. SRT-1720 (1-10 μM), a SIRT-1 activator, when pre-treated with berberine (25 μM), reversed sirtuins expression comparable to control and significantly restored the cell viability (P<0.05). Thus, our findings suggest that berberine mediated sirtuins inhibition resulting into FoxO1/3a and p53 acetylation followed by BH3-only protein Bim/PUMA activation may in part be responsible for mitochondria-mediated apoptosis.

Keywords: Berberine; Bim; FoxO; PUMA; SIRT-1; SIRT-3.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation / drug effects
  • Apoptosis / drug effects
  • Apoptosis / physiology
  • Apoptosis Regulatory Proteins / metabolism*
  • Bcl-2-Like Protein 11
  • Berberine / toxicity*
  • Dose-Response Relationship, Drug
  • Forkhead Box Protein O1
  • Forkhead Box Protein O3
  • Forkhead Transcription Factors / metabolism*
  • Genes, p53 / drug effects
  • Genes, p53 / physiology*
  • Hep G2 Cells
  • Humans
  • Membrane Proteins / metabolism*
  • Mitochondria / drug effects
  • Mitochondria / metabolism
  • Proto-Oncogene Proteins / metabolism*
  • Sirtuins / antagonists & inhibitors*
  • Sirtuins / metabolism

Substances

  • Apoptosis Regulatory Proteins
  • BBC3 protein, human
  • BCL2L11 protein, human
  • Bcl-2-Like Protein 11
  • FOXO1 protein, human
  • FOXO3 protein, human
  • Forkhead Box Protein O1
  • Forkhead Box Protein O3
  • Forkhead Transcription Factors
  • Membrane Proteins
  • Proto-Oncogene Proteins
  • Berberine
  • Sirtuins