Retinoic Acid Down-Regulates Aldehyde Dehydrogenase and Increases Cytotoxicity of 4-Hydroperoxycyclophosphamide and Acetaldehyde

Abstract

Multiple prior studies have identified aldehyde dehydrogenases (ALDH) that are capable of oxidizing retinal to retinoic acid. In this study, we test the hypothesis that the accumulation of intracellular retinoic acid may lead to the suppression of ALDH expression and thus increase cytotoxicity to 4-hydroperoxycyclophosphamide (4-HC) in vitro. Mainly A549, but also other lung cancer cell lines, were used in our experiments, with the former having high levels of two ALDH isozymes expressed. Dose-response and time-course experiments were performed by incubating the cells with all-trans retinoic acid (ATRA) as well as other commercially available retinoids. The results show that incubation of A549 cells with any of the retinoids at pharmacologic doses for Ն48 h results in a significant decrease in ALDH-1A1 and ALDH-3A1 enzyme activity and protein levels but not the corresponding mRNAs. Such a decrease in ALDH activity was seen in all cell lines tested and results in a significant increase in toxicity of 4-HC and acetaldehyde, both of which are substrates for the enzymes. Prior incubation with ATRA also results in increased cytotoxicity, although to a lesser degree, of phenylketophosphamide and melphalan, neither of which is a substrate for ALDHs. These results suggest a post-translational mechanism through which retinoids decrease both ALDH expression, which results in increased cytotoxicity of 4-HC and acetaldehyde, although other previously described effects of these retinoids may contribute to the slight increase in cytotoxicity seen with other chemotherapy agents. These results may have clinical implications in regard to the use of retinoids in lung cancer prevention and treatment.Aldehyde dehydrogenases (ALDH) are a group of enzymes that catalyze the conversion of a broad range of aldehydes to the corresponding acid via a NAD ϩ -dependent irreversible reaction. Two of these enzymes, cytosolic ALDH-1A1 and ALDH-3A1, have been found to be responsible for drug resistance in various tumor types against the antineoplastic drugs collectively known as oxazaphosphorines, which include cyclophosphamide and its active metabolites (Hilton, 1984;Manthey et al., 1990;Sreerama and Sladek, 1993;von Eitzen et al., 1994;Yoshida et al., 1998). Cyclophosphamide is a prodrug that requires cytochrome P450 hydroxylation for activation. Prior to the release of the active alkylating metabolite phosphoramide mustard, cyclophosphamide passes through an aldehyde intermediate, aldophosphamide. ALDH oxidizes aldophosphamide to the inactive metabolite carboxyphosphamide (Manthey et al., 1990). Several inhibitors of ALDH activity have been used to demonstrate the reversal of this drug resistance mechanism. We and others (Bunting et al., 1994;Bunting and Townsend, 1996;Magni et al., 1996;Moreb et al., 1996Moreb et al., , 1998 have successfully shown that overexpression of ALDH-1A1 or ALDH-3A1 in cell lines and normal hematopoietic progenitors results in a significant increase in the resistance to the active metabolites of cyclophospha...

Identification of Human Aldehyde Dehydrogenase 1 Family Member A1 as a Novel CD8+ T-Cell–Defined Tumor Antigen in Squamous Cell Carcinoma of the Head and Neck

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