GSH Synthetic Analogue O-Methyl-L-Tyrosinylglutathione Regulates Nrf2-Mediated Expression of GCLc and GCLm

Mats Hansen,Riina Mahlapuu,Ursel Soomets,Rando Porosk,Ceslava Kairane,Kalle Kilk

doi:10.1155/2019/3841219

Abstract

A glutathione (L-γ-glutamyl-L-cysteinylglycine, GSH) analogue, UPF1 (O-methyl-L-tyrosinylglutathione), has been shown to increase intracellular concentration of total glutahione (tGSH) in K562 cells. The synthesis of GSH is a two-step process that requires the actions of two distinct enzymes:γ-glutamyl-cysteine ligase (GCL) and glutathione synthetase (GS). Transcription of the GCL is controlled by multiple different factors, among others the nuclear factor (erythroid-derived 2)-like 2 transcription factor (Nrf2), which under the oxidative stress translocates into nucleus, where it binds to the dedicated binding site—antioxidant response element (ARE). In the present study, we investigated if the observed increased concentration of intracellular tGSH is a result of activation of Nrf2 protein—a key transcription factor in the cellular antioxidant response. Two distinct cell lines, adherent human hepatocarcinoma cell line HepG2 and nonadherent human myelogenous cell line K562, were chosen to establish if the increased intracellular tGSH is a universal response to the UPF1 treatment. Western blot analysis demonstrated that, after 3 h, the catalytic subunit of GCL (GCLc) level in HepG2 cells was higher than the modifying subunit of GCL (GCLm), while in K562 cells no change was observed. After 24 h, the GCLc level was higher than GCLm in K562 cells but not in the HepG2 cell line. Reverse-transcriptase PCR experiment demonstrated that no statistically significant difference was found in GCLm or GCLc mRNA levels, while the expression of the mRNA of Nrf2 and GS was elevated in the K562 cell line. Our findings suggest that UPF1 displays unique properties of mobilizing cellular defence mechanisms against reactive oxygen species while it is previously been shown to act as potent antioxidant per se.

Highlights

Xenobiotics are removed from the organism in 3 distinct phases—functionalization, conjugation, and excretion [1].e first two are more conventionally called phase I and phase II of detoxification
Since we have previously demonstrated the upregulation of the intracellular levels of GSH in response to UPF1 treatment in K562 cells, the same cell line seemed an obvious candidate for the study
K562 and HepG2 cells were incubated with 0.1 mM UPF1 for 3 and 24 h

Summary

Introduction

Xenobiotics are removed from the organism in 3 distinct phases—functionalization, conjugation, and excretion [1]. The majority of conjugation reactions in phase II are mediated via different enzymes, such as the methylations, glucuronidations, and acetylations, the nonenzymatic conjugations of xenobiotics with glutathione (GSH) are amongst the very central events in the detoxification processes [5]. E expression of GCL is tightly regulated by the nuclear factor (erythroid-derived 2)-like 2 transcription factor (NFE2L2) or Nrf2 [13], which is released from its anchoring protein Keap and binds thereafter to the antioxidant response elements (ARE) during the oxidative stress [14]. Nrf regulates the expression of other phase II antioxidant molecules such as glutathione peroxidase, N-acetyltransferase, and glutathione S-transferase [18] and other enzymes related to the drug metabolism and disposition like aldehyde dehydrogenase, alcohol dehydrogenase, or multidrug resistance-associated protein [19]. In attempt to establish whether UPF1 could alter the intracellular GSH levels in other cell lines, the HepG2 was chosen, since the Nrf2-mediated increase in GSH levels in the same cell line was recently reported in response to the homocysteine treatment [23]

Materials and Methods

Results

Discussion