Shukla Shatrunajay, Sharma Ankita, Pandey Vivek Kumar, Raisuddin Sheikh, Kakkar Poonam
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.
Herbal Research Section, CSIR - Indian Institute of Toxicology Research, Post Box No. 80, Mahatma Gandhi Marg, Lucknow-226001, India.
Toxicol Appl Pharmacol. 2016 Jan 15;291:70-83. doi: 10.1016/j.taap.2015.12.006. Epub 2015 Dec 19.
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.
翻译后修饰，即磷酸化和乙酰化，是真核生物蛋白质正常功能的关键要求。当前研究旨在解读小檗碱处理的人肝癌细胞中，去乙酰化酶（具有赖氨酸脱乙酰酶活性的NAD⁺依赖性酶）对非组蛋白靶点即FoxO1/3a和p53进行乙酰化/去乙酰化的影响。小檗碱（100μM）在转录水平和翻译水平均显著抑制去乙酰化酶（P<0.05）。烟酰胺（去乙酰化酶抑制剂）与小檗碱联合使用增强了对去乙酰化酶的抑制作用，并增加了FoxO1/3a的表达以及肿瘤抑制蛋白p53的磷酸化。由于去乙酰化酶使包括FoxO1/3a和p53在内的非组蛋白靶点去乙酰化，小檗碱增加了FoxO1/3a和p53蛋白的乙酰化负荷。乙酰化的FoxO和p53蛋白转录激活仅含BH3结构域的蛋白Bim和PUMA（分别为3.89倍和3.87倍，P<0.001），它们是促凋亡Bcl-2家族蛋白Bax的直接激活剂，最终导致线粒体介导的凋亡级联反应激活。敲低Bim/PUMA后去乙酰化酶的表达没有变化，而小檗碱和烟酰胺诱导的细胞毒性降低了28.3%（P<0.001），并恢复了HepG2细胞中促凋亡/抗凋亡蛋白的比例。去乙酰化酶的抑制伴随着NAD⁺/NADH比值下降、ATP生成减少、活性氧生成增加以及线粒体膜电位降低。透射电镜分析证实了线粒体退化和细胞损伤。SRT-1720（1-10μM），一种SIRT-1激活剂，与小檗碱（25μM）预处理后，使去乙酰化酶的表达恢复至与对照相当的水平，并显著恢复了细胞活力（P<0.05）。因此，我们的研究结果表明，小檗碱介导的去乙酰化酶抑制导致FoxO1/3a和p53乙酰化，随后激活仅含BH3结构域的蛋白Bim/PUMA，这可能部分导致了线粒体介导的凋亡。
