Inhibition of Membrane-Associated Catalase, Extracellular ROS/RNS Signaling and Aquaporin/H2O2-Mediated Intracellular Glutathione Depletion Cooperate during Apoptosis Induction in the Human Gastric Carcinoma Cell Line MKN-45.

Georg Bauer

doi:10.3390/antiox10101585

Abstract

The human gastric carcinoma cell line MKN-45 is a prototype of bona fide tumor cells, as it is protected from the NADPH oxidase-1 (NOX-1)-driven HOCl- and nitric oxide (NO)/peroxynitrite apoptosis-inducing signaling pathways by a membrane-associated catalase. The use of inhibitors/scavengers shows that inhibition of membrane-associated catalase is sufficient for the activation of NO/peroxynitrite or HOCl signaling. However, this signaling is not sufficient for apoptosis induction, as intracellular glutathione peroxidase/glutathione counteracts these signaling effects. Therefore, intrusion of extracellular tumor cell-derived H2O2 through aquaporins is required for the full apoptosis-inducing effect of extracellular reactive oxygen/nitrogen species. This secondary step in apoptosis induction can be prevented by inhibition of aquaporins, inhibition of NOX1 and decomposition of H2O2. Pretreatment with inhibitors of glutathione synthase or the cysteine-glutamine antiporter (xC transporter) abrogate the requirement for aquaporin/H2O2-mediated glutathione depletion, thus demonstrating that intracellular glutathione is the target of intruding H2O2. These data allow definition of mechanistic interactions between ROS/RNS signaling after inhibition of membrane-associated catalase, the sensitizing effects of aquaporins/H2O2 and the counteraction of the xC transporter/glutathione synthase system. Knowledge of these mechanistic interactions is required for the understanding of selective apoptosis induction in tumor cells through reestablishment of apoptosis-inducing ROS/RNS signaling.

Highlights

The demonstration of a steady state level of compound I of catalase in hemoglobinfree perfused rat liver by Sies and Chance [1] was the first proof for the generation ofH2 O2 under physiological conditions
There was still substantial protection against apoptosis induction at lower concentrations of glucose oxidase (GOX). These findings show (i) that an influx of H2 O2 into the cells was required for apoptosis induction under the conditions of this experiment, (ii) that membrane-associated catalase controls the effective H2 O2 level and (iii) that aquaporins control the influx of H2 O2
The data show that the concentration of exogenous, GOX-derived H2 O2 determines the time window in which aquaporins play their role in this specific experimental setting

Summary

Introduction

This finding opened the path for the elucidation of numerous physiological and pathophysiological roles of H2 O2 , as well as its control by enzymes such as catalase, peroxidase and others [2,3,4]. One classical mechanism for H2O2-mediated damaging effects is based on Fenton chemistry, a process in which an electron derived from ferrous iron causes decomposition of H2O2 into hydroxyl anions and highly reactive hydroxyl radicals [9]. As this reaction lacks sitespecificity, sufficiently high concentrations of H2O2 are needed to cause damage through this

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Discussion

Conclusion