Ghazi Terisha, Nagiah Savania, Tiloke Charlette, Sheik Abdul Naeem, Chuturgoon Anil A
Discipline of Medical Biochemistry and Chemical Pathology, School of Laboratory Medicine and Medical Science, College of Health Sciences, University of Kwa-Zulu Natal, Congella, Durban, 4013, South Africa.
J Cell Biochem. 2017 Nov;118(11):3866-3874. doi: 10.1002/jcb.26037. Epub 2017 May 19.
Fusaric acid (FA), a common fungal contaminant of maize, is known to mediate toxicity in plants and animals; however, its mechanism of action is unclear. p53 is a tumor suppressor protein that is activated in response to cellular stress. The function of p53 is regulated by post-translational modifications-ubiquitination, phosphorylation, and acetylation. This study investigated a possible mechanism of FA induced toxicity in the human hepatocellular carcinoma (HepG ) cell line. The effect of FA on DNA integrity and post-translational modifications of p53 were investigated. Methods included: (a) culture and treatment of HepG cells with FA (IC : 580.32 μM, 24 h); (b) comet assay (DNA damage); (c) Western blots (protein expression of p53, MDM2, p-Ser-15-p53, a-K382-p53, a-CBP (K1535)/p300 (K1499), HDAC1 and p-Ser-47-Sirt1); and (d) Hoechst 33342 assay (apoptosis analysis). FA caused DNA damage in HepG cells relative to the control (P < 0.0001). FA decreased the protein expression of p53 (0.24-fold, P = 0.0004) and increased the expression of p-Ser-15-p53 (12.74-fold, P = 0.0126) and a-K382-p53 (2.24-fold, P = 0.0096). This occurred despite the significant decrease in the histone acetyltransferase, a-CBP (K1535)/p300 (K1499) (0.42-fold, P = 0.0023) and increase in the histone deacetylase, p-Ser-47-Sirt1 (1.22-fold, P = 0.0020). The expression of MDM2, a negative regulator of p53, was elevated in the FA treatment compared to the control (1.83-fold, P < 0.0001). FA also inhibited cell proliferation and induced apoptosis in HepG cells as evidenced by the Hoechst assay. Together, these results indicate that FA is genotoxic and post-translationally modified p53 leading to HepG cell death. J. Cell. Biochem. 118: 3866-3874, 2017. © 2017 Wiley Periodicals, Inc.
腐马素（FA）是玉米常见的真菌污染物，已知其可介导对植物和动物的毒性作用；然而，其作用机制尚不清楚。p53是一种肿瘤抑制蛋白，在细胞应激反应中被激活。p53的功能受翻译后修饰（泛素化、磷酸化和乙酰化）的调控。本研究调查了FA诱导人肝癌（HepG）细胞系毒性的可能机制。研究了FA对DNA完整性和p53翻译后修饰的影响。方法包括：（a）用FA（IC：580.32μM，24小时）培养和处理HepG细胞；（b）彗星试验（DNA损伤）；（c）蛋白质免疫印迹法（p53、MDM2、p-Ser-15-p53、α-K382-p53、α-CBP（K1535）/p300（K1499）、HDAC1和p-Ser-47-Sirt1的蛋白表达）；以及（d）Hoechst 33342试验（凋亡分析）。与对照组相比，FA导致HepG细胞DNA损伤（P＜0.0001）。FA降低了p53的蛋白表达（0.24倍，P = 0.0004），增加了p-Ser-15-p53（12.74倍，P = 0.0126）和α-K382-p53（2.24倍，P = 0.0096）的表达。尽管组蛋白乙酰转移酶α-CBP（K1535）/p300（K1499）显著降低（0.42倍，P = 0.0023），组蛋白去乙酰化酶p-Ser-47-Sirt1增加（1.22倍，P = 0.0020），上述情况仍发生。与对照组相比，FA处理组中p53的负调节因子MDM2的表达升高（1.83倍，P＜0.0001）。Hoechst试验证明，FA还抑制HepG细胞增殖并诱导其凋亡。总之，这些结果表明，FA具有遗传毒性，并对p53进行翻译后修饰，导致HepG细胞死亡。《细胞生物化学杂志》118：3866 - 3874，2017年。©2017威利期刊公司
