PCS 103 Genotoxicity Lab, Division of Toxicology and Experimental Medicine, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India; Molecular Biprospection Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Near Kukrail Picnic Spot Road, Lucknow 226015, India.
PCS 103 Genotoxicity Lab, Division of Toxicology and Experimental Medicine, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India.
Life Sci. 2018 Apr 15;199:23-33. doi: 10.1016/j.lfs.2018.02.037. Epub 2018 Feb 27.
Long-term treatment of Isoniazid (INH) in tuberculosis (TB) patients can lead to anti-tuberculosis drug-induced hepatotoxicity. To understand the mechanism of hepatotoxicity, an attempt has been made to elucidate the role of Nrf2, a transcription factor induced by oxidative stress, in INH induced apoptosis liver cancer cell lines.
Cytotoxicity was evaluated by MTT assay. Apoptosis and reactive oxygen species (ROS) generation was performed by flow cytometry. mRNA and protein expression of various genes involves in INH induced toxicity was evaluated via Real-time PCR and western blot analysis respectively. Differential protein expression was performed by two-dimensional gel electrophoresis followed by identification using MALDI TOF/TOF.
INH induced ROS and apoptosis in HepG2 as well as THLE-2 cells. Nuclear damage was also observed by INH treatment in HepG2 cells. Expression of apoptotic (Cytochrome C and Caspase 9) and antioxidative (Keap1 and Nrf2) genes were observed to increase. INH induced PKCδ phosphorylation and released Nrf2 from its inhibitor Keap1 in the cytoplasm of HepG2 cells. However, over-expression of Nrf2 did not affect nuclear Nrf2 protein level as well as its downstream target NQO1. Nrf2 importer, Karyopherin β1 level was observed to decrease in HepG2 as well as THLE-2 cells following INH treatment.
These findings suggest that INH prevented Nrf2 translocation into the nucleus by inhibiting its importer Karyopherin β1. Therefore Nrf2 might not able to bind ARE sequences from inducing antioxidative response for protecting the cells undergoing apoptosis.
长期使用异烟肼（INH）治疗结核病（TB）患者可能导致抗结核药物诱导的肝毒性。为了了解肝毒性的机制，我们试图阐明转录因子 Nrf2 在 INH 诱导的肝癌细胞系凋亡中的作用。
通过 MTT 测定评估细胞毒性。通过流式细胞术评估细胞凋亡和活性氧（ROS）的产生。通过实时 PCR 和 Western blot 分析分别评估涉及 INH 诱导毒性的各种基因的 mRNA 和蛋白表达。通过二维凝胶电泳进行差异蛋白表达，然后使用 MALDI TOF/TOF 进行鉴定。
INH 诱导 HepG2 和 THLE-2 细胞中的 ROS 和细胞凋亡。在 HepG2 细胞中还观察到 INH 处理引起的核损伤。观察到凋亡（细胞色素 C 和 Caspase 9）和抗氧化（Keap1 和 Nrf2）基因的表达增加。INH 诱导 PKCδ 磷酸化，并将 Nrf2 从 HepG2 细胞的细胞质中的抑制剂 Keap1 中释放出来。然而，Nrf2 的过表达并未影响核 Nrf2 蛋白水平及其下游靶标 NQO1。INH 处理后，观察到 HepG2 和 THLE-2 细胞中的 Nrf2 导入蛋白 Karyopherin β1 水平降低。
这些发现表明，INH 通过抑制其导入蛋白 Karyopherin β1 来阻止 Nrf2 向核内易位。因此，Nrf2 可能无法结合 ARE 序列，从而无法诱导抗氧化反应来保护正在经历凋亡的细胞。
