Abstract：
The oncogene and drug metabolism enzyme glutathione S-transferase P (GSTP) is also a GSH-dependent chaperone of signal transduction and transcriptional proteins with key role in liver carcinogenesis. In this study, we explored this role of GSTP in hepatocellular carcinoma (HCC) investigating the possible interaction of this protein with one of its transcription factor and metronome of the cancer cell redox, namely the nuclear factor erythroid 2-related factor 2 (Nrf2). Expression, cellular distribution, and function as glutathionylation factor of GSTP1-1 isoform were investigated in the mouse model of N-nitrosodiethylamine (DEN)-induced HCC and in vitro in human HCC cell lines. The physical and functional interaction of GSTP protein with Nrf2 and Keap1 were investigated by immunoprecipitation and gene manipulation experiments. GSTP protein increased its liver expression, enzymatic activity and nuclear levels during DEN-induced tumor development in mice; protein glutathionylation (PSSG) was increased in the tumor masses. Higher levels and a preferential nuclear localization of GSTP protein were also observed in HepG2 and Huh-7 hepatocarcinoma cells compared to HepaRG non-cancerous cells, along with increased basal and Ebselen-stimulated levels of free GSH and PSSG. GSTP activity inhibition with the GSH analogue EZT induced apoptotic cell death in HCC cells. Hepatic Nrf2 and c-Jun, two transcription factors involved in GSTP expression and GSH biosynthesis, were induced in DEN-HCC compared to control animals; the Nrf2 inhibitory proteins Keap1 and β-TrCP also increased and oligomeric forms of GSTP co-immunoprecipitated with both Nrf2 and Keap1. Nrf2 nuclear translocation and β-TrCP expression also increased in HCC cells, and GSTP transfection in HepaRG cells induced Nrf2 activation. In conclusion, GSTP expression and subcellular distribution are modified in HCC cells and apparently contribute to the GSH-dependent reprogramming of the cellular redox in this type of cancer directly influencing the transcriptional system Nrf2/Keap1.
摘要：
癌基因和2相解毒酶谷胱甘肽S-转移酶P（GSTP）是GSH依赖性信号转导和转录蛋白的伴侣，在肝癌发生中起关键作用。在这项研究中,我们探索了GSTP在肝细胞癌（HCC）中的作用，研究了该蛋白与其转录因子之一和癌细胞氧化还原节拍器的可能相互作用，即核因子红系2相关因子2（Nrf2）。表达式,细胞分布，在N-亚硝基二乙胺（DEN）诱导的HCC小鼠模型中以及在人HCC细胞系中的体外研究了GSTP1-1同工型作为谷胱甘肽酰化因子的功能。通过免疫沉淀和基因操作实验研究了GSTP-Nrf2/Keap1物理和功能相互作用。GSTP蛋白增加其肝脏表达，DEN诱导的小鼠肿瘤发生过程中的酶活性和核水平；肿瘤中的蛋白谷胱甘肽酰化（PSSG）增加。与HepaRG非癌细胞相比，在HepG2和Huh-7肝癌细胞中也观察到GSTP蛋白的更高水平和优先核定位。随着游离GSH和PSSG的基础和Ebselen刺激水平的增加。用GSH类似物EZT抑制GSTP活性诱导HCC细胞凋亡。肝Nrf2和c-Jun，参与GSTP表达和GSH生物合成的两个转录因子，与对照动物相比，在DEN-HCC中诱导；Nrf2抑制蛋白Keap1和β-TrCP也增加，GSTP与Nrf2和Keap1共免疫沉淀。Nrf2核转位和β-TrCP表达也在肝癌细胞中增加，而GSTP转染HepaRG细胞诱导Nrf2转录激活。总之,GSTP的表达和亚细胞分布可有助于直接影响Nrf2/Keap1系统的HCC细胞的GSH依赖性氧化还原重编程。
