Inhibition of reductase systems by 2-AAPA modulates peroxiredoxin oxidation and mitochondrial function in A172...

Thiol homeostasis has a critical role in the maintenance of proper cellular functions and survival, being coordinated by the action of several reductive enzymes, including glutathione (GSH)/glutathione reductase (GR) and thioredoxin (Trx)/thioredoxin reductase (TrxR) systems. Here, we investigated the effects of the GR inhibitor 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) on the activity of thiol reductases (GR and TrxR), redox balance and mitochondrial function of A172 glioblastoma cells. 2-AAPA inhibited cell GR (IC₅₀=6.7μM) and TrxR (IC₅₀=8.7μM). A significant decrease in the cellular ability to decompose cumene hydroperoxide was observed and associated to a greater susceptibility to this peroxide. The redox state of peroxiredoxins (Prx1, Prx2 and Prx3) was markedly shifted to dimer 30min after treatment with 100μM 2-AAPA, an event preceding 2-AAPA-induced decrease in cell viability. Furthermore, mitochondrial function was also severely impaired, leading to a decrease in the respiratory control ratio, reserve capacity, and ATP synthesis-coupled respiration, as well as an increase in mitochondrial membrane potential. Our results indicate that inhibition of GR and TrxR activities, disruption of the ability to detoxify peroxides, increased oxidation of Prxs, as well as compromised mitochondrial function represent early events mediating 2-AAPA toxicity to A172 glioblastoma cells.