title: Concurrent acetylation of FoxO1/3a and p53 due to sirtuins inhibition elicit Bim/PUMA mediated mitochondrial dysfunction and apoptosis in berberine-treated HepG2 cells.
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.
authors.affiliation: Herbal Research Section, CSIR - Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow-226001, India; Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi -110062, India.
authors.forename: Shatrunajay
authors.initials: S
authors.lastname: Shukla
authors.affiliation: Herbal Research Section, CSIR - Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow-226001, India.
authors.forename: Ankita
authors.initials: A
authors.lastname: Sharma
authors.affiliation: Herbal Research Section, CSIR - Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow-226001, India; Academy of Scientific and Innovative Research, India.
authors.forename: Vivek Kumar
authors.initials: VK
authors.lastname: Pandey
authors.affiliation: Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi -110062, India.
authors.forename: Sheikh
authors.initials: S
authors.lastname: Raisuddin
authors.affiliation: Herbal Research Section, CSIR - Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow-226001, India; Academy of Scientific and Innovative Research, India. Electronic address: kakkarp59@gmail.com.
authors.forename: Poonam
authors.initials: P
authors.lastname: Kakkar
mesh_terms: D000107:Acetylation / Q000187:drug effects; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D001599:Berberine / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D000071161:Forkhead Box Protein O1; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D016158:Genes, p53 / Q000187:drug effects / Q000502:physiology*; D056945:Hep G2 Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D037761:Sirtuins / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; C085126:FOXO1 protein, human; C499517:FOXO3 protein, human; D000071161:Forkhead Box Protein O1; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D001599:Berberine; D037761:Sirtuins
keywords: Berberine; Bim; FoxO; PUMA; SIRT-1; SIRT-3