Subcellular distribution and Nrf2/Keap1-interacting properties of Glutathione S-transferase P in hepatocellular carcinoma.

The oncogene and phase 2 detoxification enzyme glutathione S-transferase P (GSTP) is 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 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 GSTP-Nrf2/Keap1 physical and functional interaction was 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 GSTP co-immunoprecipitated with both Nrf2 and Keap1. Nrf2 nuclear translocation and β−TrCP expression also increased in HCC cells, whereas the GSTP transfection in HepaRG cells induced Nrf2 transcriptional activation.In conclusion, GSTP expression and subcellular distribution can contribute to the GSH-dependent redox reprogramming of HCC cells directly influencing the Nrf2/Keap1 system.