Dependence of aldehyde dehydrogenase-mediated oxazaphosphorine resistance on soluble thiols: importance of thiol interactions with the secondary metabolite acrolein.
Acrolein is a highly reactive and cytotoxic by-product released during activation of oxazaphosphorine (OAP) anticancer alkylating agents. Previously, we demonstrated that transfected human aldehyde dehydrogenase (ALDH, EC 1.2.1.3) isozymes (class 1 or 3) protect V79/SD1 cells from mafosfamide (MAF) cytotoxicity, but protection from 4-hydroperoxy-cyclophosphamide (4-hpCPA) was weaker. Acrolein, an ALDH inhibitor, may be detoxified by conjugation with the nucleophilic thiol 2-mercaptoethanesulfonate (MESNA), which is released from MAF but not from 4-hpCPA. We examined the effect of acrolein or acrolein/thiol conjugates on ALDH activity in vitro. We found that both ALDH isozymes were inhibited by acrolein, with IC50 values of 35 and 144 microM for ALDH-1 or ALDH-3, respectively. Both isozymes were partially protected by NAD+ cofactor, being at least five-fold more sensitive to acrolein if added before cofactor. In contrast, thiol conjugates of acrolein did not inhibit ALDH-3 activity, but were substrates only for ALDH-1. Further, acrolein was shown to be oxidized by ALDH-3, but not by ALDH-1. The effect of acrolein on ALDH-mediated resistance to OAP agents in intact cells was also examined. In control cells (without ALDH expression), acrolein and 4-hpCPA rapidly depleted intracellular GSH levels, whereas the effect of MAF was much less. Depletion of GSH by preincubation of V79/SD1 cells with a low concentration of acrolein (2 microM) before MAF exposure caused a two-fold reduction in ALDH-mediated resistance. Conversely, protection from 4-hpCPA cytotoxicity was enhanced by the addition of thiols (GSH, 2-mercaptoethanesulfonate, or N-acetylcysteine) during the drug exposure. These results suggest 1) that thiol content is an important determinant of the OAP resistance conferred by ALDH isoenzymes; and 2) a new mechanism whereby thiol modulation could increase the therapeutic index of OAP chemotherapy.
Biochemistry Department, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
K D
KD
Bunting
A J
AJ
Townsend
D000171:Acrolein / Q000378:metabolism / Q000494:pharmacology*; D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D018906:Antineoplastic Agents, Alkylating; D007527:Isoenzymes; D013438:Sulfhydryl Compounds; D000171:Acrolein; D000444:Aldehyde Dehydrogenase; D005978:Glutathione