Role of Reduced Glutathione Efflux in Apoptosis of Immortalized Human Keratinocytes Induced by UVA

Abstract

We have investigated the role played by GSH efflux in apoptosis of human HaCaT keratinocytes induced by UVA irradiation. UVA irradiation of HaCaT cells caused a rapid rise in GSH efflux across the intact cell membrane, followed by an increase in apoptosis. GSH efflux was stimulated by glucose and was reduced by the addition of exogenous GSH and intracellular GSH depletion by buthionine sulfoximine, suggesting that GSH transport is active and is influenced by the GSH concentration gradient across the cell membrane. Verapamil and cyclosporin A, blockers of the multidrug resistanceassociated protein, decreased UVA-induced GSH efflux. GSH efflux occurred within 2 h of UVA irradiation, suggesting that the stimulation of GSH efflux is due to an increase in the activity of pre-existing multidrug resistance-associated protein transporter carrier. Although inhibition of GSH efflux did not affect caspase activation and DNA fragmentation, it delayed the gradual increase in plasma membrane permeability and reduced phosphatidylserine translocation in HaCaT cells. It is therefore likely that upon UVA irradiation, GSH efflux increased the intracellular oxidative stress without intervention of reactive oxygen species, thus resulting in more phosphatidylserine externalization and membrane rearrangement. These provide targets for macrophage recognition and phagocytosis and thus minimize the potential to invoke inflammation or neoplastic transformation.

Nitrogen monoxide (NO)-mediated iron release from cells is linked to NO-induced glutathione efflux via multidrug resistance-associated protein 1

“…This hypothesis is supported by studies where M s induced marked Fe release from tumor targets (1), an effect mediated by NO (2). GSH efflux is a key signal mediating apoptosis (37), and it is well known that cell Fe mobilization by chelators results in antitumor activity (38). Hence, the dual action of NO leading to Fe and GSH release may play a role in cytotoxicity of M s against tumors.…”

Chronic UVA irradiation of human HaCaT keratinocytes induces malignant transformation associated with acquired apoptotic resistance

“…Apoptosis was determined by Annexin-V/propidium iodide (PI) staining followed by flow cytometry, as described previously (He et al, 2003b). DNA fragmentation was determined as described previously (He et al, 2003a) as was caspase activity (He et al, 2003b).…”

“…DNA fragmentation was determined as described previously (He et al, 2003a) as was caspase activity (He et al, 2003b).…”

Cleavage of epidermal growth factor receptor by caspase during apoptosis is independent of its internalization

“…Apoptosis was determined by Annexin-V/propidium iodide (PI) staining followed by flow cytometry, as described previously (He et al, 2003b). DNA fragmentation was determined as described previously (He et al, 2003a).…”