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

Despite recent advances in the development of novel therapies against castration resistant prostate cancer, the advanced form of the disease remains a major treatment challenge. Aberrant sphingolipid signaling through sphingosine kinases and their product sphingosine-1-phosphate can promote proliferation, drug resistance, angiogenesis and inflammation. The sphingosine kinase 2 inhibitor ABC294640 is undergoing clinical testing in cancer patients, and in this study we investigated the effects this first-in-class inhibitor in castration resistant prostate cancer. In vitro, ABC294640 decreased prostate cancer cell viability as well as the expression of c-Myc and the androgen receptor while lysosomal acidification increased. ABC294640 also induced a greater than 3-fold increase in dihydroceramides that inversely correlated with inhibition of dihydroceramide desaturase (DEGS) activity. Expression of sphingosine kinase 2 was dispensable for the ABC294640-mediated increase in dihydroceramides. In vivo, ABC294640 diminished the growth rate of TRAMP-C2 xenografts in syngeneic hosts and elevated dihydroceramides within tumors as visualized by MALDI imaging mass spectroscopy. The plasma of ABC294640 treated mice contained significantly higher levels of C16- and C24:1-ceramides (but not dihydro-C16-ceramide) compared to vehicle treated mice. In summary, our results suggest that ABC294640 may reduce the proliferative capacity of castration resistant prostate cancer cells through both, inhibition of sphingosine kinase 2 and dihydroceramide desaturase, which provides a foundation for future exploration of this small molecule inhibitor for the treatment of advanced disease.

Oxidative stress was previously implicated in regulation of ceramide metabolism. Here, its effects on dihydroceramide desaturase were investigated. To stimulate oxidative stress HEK293, MCF7, A549, and SMS-KCNR cells were treated with hydrogen peroxide, menadione, or tert-butylhydroperoxide. In all cell lines, an increase in dihydroceramide was observed upon oxidative stress as measured by LC/MS. In contrast, total ceramide levels were relatively unchanged. Mechanistically, dihydroceramide desaturase activity was measured by an in-situ assay and was decreased in a time- and dose-dependent fashion. Interestingly, no detectable changes in the protein levels were observed, suggesting that oxidative stress does not induce degradation of dihydroceramide desaturase. In summary, oxidative stress leads to potent inhibition of dihydroceramide desaturase resulting in significant elevation in dihydroceramide levels in vivo.

Photodynamic therapy (PDT) is not always effective as an anticancer treatment, therefore, PDT is combined with other anticancer agents for improved efficacy. The combination of dasatinib and PDT with the silicone phthalocyanine photosensitizer Pc 4 was assessed for increased killing of SCCVII mouse squamous cell carcinoma cells, a preclinical model of head and neck squamous cell carcinoma, using apoptotic markers and colony formation as experimental end-points. Because each of these treatments regulates the metabolism of the sphingolipid ceramide, their effects on mRNA levels of ceramide synthase, a ceramide-producing enzyme, and the sphingolipid profile were determined. PDT + dasatinib induced an additive loss of clonogenicity. Unlike PDT alone or PDT + dasatinib, dasatinib induced zVAD-fmk-dependent cell killing. PDT or dasatinib-induced caspase-3 activation was potentiated after the combination. PDT alone induced mitochondrial depolarization, and the effect was inhibited after the combination. Annexin V+ and propidium iodide+ cells remained at control levels after treatments. In contrast to PDT alone, dasatinib induced upregulation of ceramide synthase 1 mRNA, and the effect was enhanced after the combination. Dasatinib induced a modest increase in C20:1-and C22-ceramide but had no effect on total ceramide levels. PDT increased the levels of 12 individual ceramides and total ceramides, and the addition of dasatinib did not affect these increases. PDT alone decreased substantially sphingosine levels and inhibited the activity of acid ceramidase, an enzyme that converts ceramide to sphingosine. The data suggest that PDT-induced increases in ceramide levels do not correlate with ceramide synthase mRNA levels but rather with inhibition of ceramidase. Cell killing was zVAD-fmk-sensitive after dasatinib but not after either PDT or the combination and enhanced cell killing after the combination correlated with potentiated caspase-3 activation and upregulation of ceramide synthase 1 mRNA but not the production of ceramide. The data imply potential significance of the combination for cancer treatment.

Studies were performed to further characterize the male-specific hepatic recombinant microsomal vitamin D 25-hydroxlase CYP2C11, expressed in baculovirus-infected insect cells, and determine whether it is also a vitamin D 24-hydroxylase. 25-and 24-hydroxylase activities were compared with those of 10 other recombinant hepatic microsomal cytochrome P-450 enzymes expressed in baculovirus-infected insect cells. Each of them 25-hydroxylated vitamin D 2, vitamin D3, 1␣-hydroxyvitamin D2 (1␣OHD2), and 1␣-hydroxyvitamin D3 (1␣OHD3). CYP2C11 had the greatest activity with these substrates, except vitamin D3, which had the same activity as four of the other enzymes. The descending order of 25-hydroxylation by CYP2C11 was 1␣OHD3 Ͼ 1␣OHD2 Ͼ vitamin D2 Ͼ vitamin D3. Each of the recombinant cytochrome P-450 enzymes 24-hydroxylated 1␣OHD2. CYP2C11 had the greatest activity. 24-Hydroxylation of 1␣OHD3 was very low, and there was none with vitamin D3. Only CYP2C11 24-hydroxylated vitamin D2. Structures of vitamin D metabolites, including 24-hydroxyvitamin D2, 1,24(S)-dihydroxyvitamin D2, and 1,24-dihydroxyvitamin D3, were confirmed by HPLC and gas chromatography retention times and characteristic mass spectrometric fragmentation patterns. In male rats, hypophysectomy significantly reduced body weight, liver weight, hepatic CYP2C11 mRNA expression, and 24-and 25-hydroxylation of 1␣OHD2. Expression of CYP2J3 and CYP2R1 mRNA did not change. In male rat hepatocytes, CYP2C11 mRNA expression and 24-and 25-hydroxylation were significantly reduced after culture for 24 h compared with uncultured cells. Expression of CYP2J3 and CYP2R1 either increased or did not change. It is concluded that CYP2C11 is a male-specific hepatic microsomal vitamin D 25-hydroxylase that hydroxylates vitamin D2, vitamin D3, 1␣OHD2, and 1␣OHD3. CYP2C11 is also a vitamin D 24-hydroxylase. (16,24,28). 25OHD and 1,25(OH) 2 D are further hydroxylated to calcitroic acid in the kidney and elsewhere by the rate-limiting enzyme 25OHD-24-hydroxylase (CYP24A1) (17,32,35). In rats, hepatic 25-hydroxylase activity is present in both mitochondria and microsomes. CYP27A1 is a mitochondrial vitamin D 25-hydroxylase that is involved in the alternative pathway for bile acid synthesis from cholesterol (5,12,34), and CYP2C11 and the recently identified CYP2J3 are microsomal vitamin D 25-hydroxylases of rats (35). CYP2C11 is expressed in male but not female rat livers, whereas CYP2J3 is expressed in both male and female rat livers (34). 24-Hydroxylation of vitamin D 2 and conversion of 24-hydroxyvitamin D 2 to biologically active 1,24(S)-dihydroxyvitamin D 2 [1,24(S)(OH) 2 D 2 ] by CYP27B1 represent an alternative pathway for vitamin D 2 metabolism (22). The purpose of the present studies is to characterize the substrate specificity of recombinant CYP2C11, to determine whether CYP2C11 is a vitamin D 24-hydroxylase, to compare its activity with that of other rat recombinant microsomal cytochrome P-450 enzymes, to confirm identity of the hydroxylation products, and to determine the...

Background/Aim Combining an anticancer agent fenretinide (HPR) or C6-pyridinium ceramide (LCL29) with Foscan-mediated photodynamic therapy (FoscanPDT) is expected to augment anticancer benefits of each substance. We showed that treatment with FoscanPDT+HPR enhanced accumulation of C16-dihydroceramide, and that fumonisin B1 (FB), an inhibitor of ceramide synthase, counteracted caspase-3 activation and colony-forming ability of head and neck squamous cell carcinoma (HNSCC) cells. Because cancer cells appear to be more susceptible to increased levels of the endoplasmic reticulum (ER) stress than normal cells, herein we tested the hypothesis that FoscanPDT combined with HPR or LCL29 induces FB-sensitive ER stress-associated apoptosis that affects cell survival. Materials and Methods Using an HNSCC cell line, we determined: cell survival by clonogenic assay, caspase-3 activity by spectrofluorometry, the expression of the ER markers BiP and CHOP by quantitative real-time polymerase chain reaction and western immunoblotting, and sphingolipid levels by mass spectrometry. Results Similar to HPR+FoscanPDT, LCL29+FoscanPDT induced enhanced loss of clonogenicity and caspase-3 activation, that were both inhibited by FB. Our additional pharmacological evidence showed that the enhanced loss of clonogenicity after the combined treatments was singlet oxygen-, ER stress- and apoptosis-dependent. The combined treatments induced enhanced, FB-sensitive, up-regulation of BiP and CHOP, as well as enhanced accumulation of sphingolipids. Conclusion Our data suggest that enhanced clonogenic cell killing after the combined treatments is dependent on oxidative- and ER-stress, apoptosis, and FB-sensitive sphingolipid production, and should help develop more effective mechanism-based therapeutic strategies.

This article is available online at http://www.jlr.orgIn the last two decades, sphingolipids and their precursors have been extensively studied for their cell signaling roles (recently reviewed in Ref. 1 ). Changes in sphingolipid levels are shown to play a role in regulating important cellular processes, such as cell proliferation, growth arrest, and cell death. Here we investigated the role of sphingolipids in cell-cycle arrest at high cell density, a phenomenon known as contact inhibition at saturation density ( 2 ). Density-dependent inhibition in cultured cells serves as a model to study the balance between cell proliferation and growth arrest in a multicellular organism. Cell-number control is important for maintenance of tissue homeostasis within multicellular organisms, and cell-cycle arrest is a critical aspect of that control mechanism ( 2, 3 ). Cell-cycle arrest at high saturation density is a complex process dependent on multiple factors, and earlier studies have shown that sphingolipids play a role in it. Gangliosides, GM 3 in particular, have been shown to promote growth inhibition in confl uent cells ( 2,4,5 ), and a recent study has revealed that abundance of another ganglioside, GM 1 , on the cellular membrane depends on the cell's population context ( 6 ). A more recent work showed that very long chain (VLC) ceramides (i.e., ceramides with fatty acid chain length C 22 -C 26 ) also play a role in cell-cycle arrest at contact inhibition in breast cancer cells ( 7 ).Changes in sphingolipids, resulting from various cellular insults or manipulation of the sphingolipid pathway, are often complex and include simultaneous changes of several sphingolipid species ( 1,8 ). Therefore, considering the interdependency of the sphingolipid pathway, we applied a metabolic approach to decipher the role of sphingolipids in density-dependent growth arrest in neuroblastoma cells. Neuroblastoma is the most common cancer in infants and understanding its biology is important for devising new therapeutic strategies.Abstract We applied a metabolic approach to investigate the role of sphingolipids in cell density-induced growth arrest in neuroblastoma cells. Our data revealed that sphingolipid metabolism in neuroblastoma cells signifi cantly differs depending on the cells' population context. At high cell density, cells exhibited G0/G1 cell-cycle arrest and reduced ceramide, monohexosylceramide, and sphingomyelin, whereas dihydroceramide was signifi cantly increased. In addition, our metabolic-labeling experiments showed that neuroblastoma cells at high cell density preferentially synthesized very long chain (VLC) sphingolipids and dramatically decreased synthesis of sphingosine-1-phosphate (S1P). Moreover, densely populated neuroblastoma cells showed increased message levels of both anabolic and catabolic enzymes of the sphingolipid pathway. Notably, our metabolic-labeling experiments indicated reduced dihydroceramide desaturase activity at confl uence, which was confi rmed by direct measurement of dihydroceramide desat...

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