Identification of Dihydroceramide Desaturase as a Direct in Vitro Target for Fenretinide

Abstract

The dihydroceramide desaturase (DES) enzyme is responsible for inserting the 4,5-trans-double bond to the sphingolipid backbone of dihydroceramide. We previously demonstrated that fenretinide (4-HPR) inhibited DES activity in SMS-KCNR neuroblastoma cells. In this study, we investigated whether 4-HPR acted directly on the enzyme in vitro. N-C8:0-D-erythrodihydroceramide (C 8 -dhCer) was used as a substrate to study the conversion of dihydroceramide into ceramide in vitro using rat liver microsomes, and the formation of tritiated water after the addition of the tritiated substrate was detected and used to measure DES activity. NADH served as a cofactor Sphingolipids are known to be modulators of various cell functions. They are not only components of cell membranes but also play a role in cell survival, apoptosis, senescence, and differentiation (1, 2). Ceramide, a central molecule in the metabolism of sphingolipids and glycosphingolipids, is involved in these regulatory cellular events. Intracellulary, ceramide is generated by different pathways. De novo synthesis of ceramide starts with condensation of L-serine with palmitoylCoA. Further reduction and subsequent N-acylation generates dihydroceramide. Ceramide is finally generated by introduction of the 4,5-double bond into dihydroceramide by dihydroceramide desaturase (DES) 2 (3). The DES enzyme was characterized previously, and an in vitro assay was developed to determine its activity (4). In subsequent studies, a family of sphingolipid ⌬4-desaturases (homologs of the Drosophila melanogaster degenerative spermatocyte gene 1 (des-1)) were identified via a bioinformatics approach (5). These proteins contain three His-containing consensus motifs that are characteristic of a group of membrane fatty acid desaturases. The human homolog of des-1 is now referred to as DEGS-1, although it was first cloned in 1997 and named as membrane lipid desaturase because its physiologic substrate was not determined at the time (6). DEGS-1 is the only dihydroceramide desaturase reported to be present in human cells, and its mouse homolog (mDES1) was shown to have desaturase activity (7). hDES2, the human homolog of the mouse DES2 (mDes2) gene, like mDES2 has dihydroceramide hydroxylase activity (8). Although mDES2 has been reported to have both desaturase and hydroxylase activity, no desaturase activity was detected in HEK 293 human embryonic kidney cells overexpressing hDES2 (8). In this work, we refer to enzyme as DES in experiments with rat liver microsomes and as DEGS-1 in experiments with human SMS-KCNR cells.We previously developed an assay to evaluate the in situ activity of DEGS-1 using cell-permeable dihydroceramidoids (dhCCPS analogs) (9). We showed in these studies that the

Inhibition of dihydroceramide desaturase activity by the sphingosine kinase inhibitor SKI II

“…Blockade of Des1 produces an increase in dihydroceramides (dhCers), which have emerged as bioactive lipids and the target of several drugs ( 34 ), including fenretinide. Several studies have shown that cells respond to fenretinide treatment with a robust production of dhCers (35)(36)(37)(38)(39)(40)(41)(42)(43)(44), and that this increase induces autophagy ( 45,46 ). Furthermore, experimental evidence exists on the connection between resistance to fenretinide and increased S1P production ( 38,44 ) due to an increased SK activity and SK1 mRNA and protein levels ( 38 ).…”

“…Kinetic studies showed that SKI II was a noncompetitive inhibitor with a K i value in the high nanomolar range. Other Des1 inhibitory compounds produced competitive (GT11, K i = 6 M) ( 74 ) or mixed-type inhibition (XM462, K i = 2 M) ( 47 ), while fenretinide functions as a competitive inhibitor ( K i = 8 M) at short incubation times and as an irreversible inhibitor at long incubation times ( 40 ). While GT11 and XM462 are structural analogs of the Des1 substrate, fenretinide and other Des1 inhibitors ( 34 ) are not.…”

“…Cell lysis was performed with [30][31][32][33][34][35][36][37][38][39][40] l of lysis buffer [150 mM NaCl, 1% Igepal-CA630, 50 mM TrisHCl (pH 8), 2 g/ml aprotinin, 5 g/ml leupeptin, and 1 mM PMSF] by three cycles of bath sonication (5 s)/rest on ice (10 s). Then samples were kept on ice for 30 min and centrifuged for 3 min at 10,000 rpm.…”

Cell density-dependent reduction of dihydroceramide desaturase activity in neuroblastoma cells

“…Recent studies using depletion or inhibition of dihydroceramide desaturase, which uses dihydroceramide as a substrate, changed the way we think about this concept ( 10,(18)(19)(20)(21)(22). Fenretinide and resveratrol treatments are reported to inhibit dihydroceramide desaturase and increase endogenous dihydroceramides, resulting in autophagy in cancer cells ( 10,20,22,23 ). Treatment of tumor cells with celecoxib, a COX2 inhibitor, has also been shown to increase some dihydroceramide species by inhibiting dihydroceramide desaturase.…”

Sphingolipid in Lung Cancer Pathogenesis and Therapy

“…These lipids are synthesized A Global Scientific Vision -Prevention, Diagnosis, and Treatment of Lung Cancerin an interconnected network of enzymes, which is centered on ceramide (Figure 1). There are three pathways that produce ceramide, de novo, sphingomyelin cycle, and the salvage pathways [14][15][16]. In the de novo pathway, ceramide synthesis is initiated by serine palmitoyltransferase which condenses serine and palmitate to form ketosphinganine, followed by reduction of the ketone group to dihydrosphingosine.…”