Abstract
Increased generation of dihydrosphingosine (DHS), a bioactive sphingolipid, has been implicated in the cytotoxicity of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in tumor cells. However, how 4-HPR increases DHS remains unclear. Here we demonstrate that 4-HPR increases the expression of ACER2, which catalyzes the hydrolysis of dihydroceramides to generate DHS, and that ACER2 up-regulation plays a key role in mediating the 4-HPR-induced generation of DHS as well as the cytotoxicity of 4-HPR in tumor cells. Treatment with 4-HPR induced the accumulation of dihydroceramides (DHCs) in tumor cells by inhibiting dihydroceramide desaturase (DES) activity, which catalyzes the conversion of DHCs to ceramides. Treatment with 4-HPR also increased ACER2 expression through a retinoic acid receptor-independent and caspase-dependent manner. Overexpression of ACER2 augmented the 4-HPR-induced generation of DHS as well as 4-HPR cytotoxicity, and 4-HPR-induced death in tumor cells, whereas knocking down ACER2 had the opposite effects. ACER2 overexpression, along with treatment with GT11, another DES inhibitor, markedly increased cellular DHS, leading to tumor cell death, whereas ACER2 overexpression or GT11 treatment alone failed to do so, suggesting that both ACER2 up-regulation and DES inhibition are necessary and sufficient to mediate 4-HPR-induced DHS accumulation, cytotoxicity, and death in tumor cells. Taken together, these results suggest that up-regulation of the ACER2/DHS pathway mediates the cytotoxicity of 4-HPR in tumor cells and that up-regulating or activating ACER2 may improve the anti-cancer activity of 4-HRR and other DHC-inducing agents.
Highlights
Increased generation of dihydrosphingosine (DHS), a bioactive sphingolipid, has been implicated in the cytotoxicity of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) in tumor cells
C–F, alkaline ceramidase 1 (ACER1)-TET-ON (C), alkaline ceramidase 2 (ACER2)-TET-ON (D), acid ceramidase (AC)-TET-ON (E), or neutral ceramidase (NC)-TET-ON (F) cells grown in the presence of ET or TET were treated with DMSO or 4-HPR (10 M) for 6 h before DHS and DHS1P were analyzed by ESI/MS/MS
C, ACER2-TET-ON cells grown in the presence of ET or TET (10 ng/ml) were treated with 4-HPR (10 M) or DMSO for 48 h before the percentage of sub-G1 cells was determined by Fluorescence-activated Cell Sorting (FACS) analysis
Summary
Reagents—The anti-ACER2 antibody was previously raised against a peptide located at the carboxyl terminus of ACER2 in our laboratory [17]. ACER2 Promoter Cloning and Activity Assays—The 1882base pair DNA fragment upstream of the ACER2 translation initiation site (ATG) was amplified by PCR from human genomic DNA, which was isolated from the whole blood of disease-free sources (Clontech) using the primers 5Ј-cggggtaccatggaatctcgctctgtcaccc-3Ј (forward) and 5Ј-cccaagcttggccactccggggcattggagcag-3Ј (reverse) This DNA sequence was cloned into a promoter reporter vector pGL3 (Promega) to generate an ACER2 promoter construct, pGL3-ACER2P. HPLC Analysis for SPH and DHS—The amount of SPH and DHS in enzymatic reactions or lipid extracts of cells was determined by high performance liquid chromatography (HPLC) after its derivatization with O-phthalaldehyde as described [23]. Cells were harvested after being washed with ice-cold 25 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl. Fifty l of a mixture (1 M) of internal sphingolipid standards including C17SPH, C17SPH-1-phosphate, D-e-C16-ceramide (d17:1/16:0), and D-e-18-ceramide (d17:1/18:0) was added to each cell pellet sample before lipid extraction with 4 ml of the ethyl acetate/isopropyl alcohol/water (60/30/10%;v/v) solvent system. Values of p Ͻ 0.05 were considered significant and are marked with an asterisk (*)
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