P-glycoprotein antagonists confer synergistic sensitivity to short-chain ceramide in human multidrug-resistant cancer cells.

P-glycoprotein (P-gp) antagonists inhibit ceramide metabolism at the juncture of glycosylation. The purpose of this study was to test whether targeting P-gp would be a viable alternative to targeting glucosylceramide synthase (GCS) for enhancing ceramide cytotoxicity. A2780 wild-type, and multidrug-resistant 2780AD and NCI/ADR-RES human ovarian cancer cell lines and the cell-permeable ceramide analog, C6-ceramide (C6-cer), were employed. Compared to P-gp-poor A2780 cells, P-gp-rich 2780AD cells converted 3.7-fold more C6-cer to nontoxic C6-glucosylceramide (C6-GC), whereas cell-free GCS activities were equal. 2780AD cells displayed resistance to C6-cer (10 µM) that was reversed by inclusion of the P-gp antagonist tamoxifen (5 µM) but not by inclusion of a GCS inhibitor. Co-administration of C6-cer and P-gp antagonists was also effective in NCI/ADR-RES cells. For example, C6-cer, VX-710 (Biricodar), and cyclosporin A (cyc A) exposure resulted in viabilities of ~90% of control; however, C6-cer/VX-710 and C6-cer/cyc A additions were synergistic and resulted in viabilities of 22% and 17%, respectively. Further, whereas C6-ceramide and cyc A imparted 1.5- and 0-fold increases in caspase 3/7 activity, the combination produced a 3.5-fold increase. Although the upstream elements of cell death have not been elucidated, the novel C6-ceramide/P-gp antagonist combination merits further study and assessment of clinical translational potential.

Glucosylceramide in humans.

Glucosylceramide has a unique and often ambiguous role in mammalian cells. Activation of glucosylceramide synthase, the enzyme that places a glucosyl moiety onto ceramide, is the first pathway-committed step to the production of more complex glycosphingolipids such as lactosylceramide and gangliosides. Alterations in the level of glucosylceramide are noted in cells and tissues in response to cardiovascular disease, diabetes, skin disorders and cancer. Overall, upregulation of glucosylceramide offers cellular protection and primes certain cells for proliferation. However, prolonged overabundance of glucosylceramide is detrimental, as seen in Gaucher disease in humans.

2-Chlorohexadecanal and 2-chlorohexadecanoic acid induce COX-2 expression in human coronary artery endothelial cells.

2-Chlorohexadecanal (2-ClHDA), a 16-carbon chain chlorinated fatty aldehyde that is produced by reactive chlorinating species attack of plasmalogens, is elevated in atherosclerotic plaques, infarcted myocardium, and activated leukocytes. We tested the hypothesis that 2-ClHDA and its metabolites, 2-chlorohexadecanoic acid (2-ClHA) and 2-chlorohexadecanol (2-ClHOH), induce COX-2 expression in human coronary artery endothelial cells (HCAEC). COX-2 protein expression increased in response to 2-ClHDA treatments at 8 and 20 h. 2-ClHA also increased COX-2 expression following an 8 h treatment. Quantitative PCR showed that 2-ClHDA treatment increased COX-2 mRNA over 8 h, while 2-ClHA treatment led to a modest increase by 1 h and those levels remained constant over 8 h. 2-ClHDA led to a significant increase in 6-keto-PGF(1alpha) release (a measure of PGI(2) release) by HCAEC. These data suggest that 2-ClHDA and its metabolite 2-ClHA, which are produced during leukocyte activation, may alter vascular endothelial cell function by upregulation of COX-2 expression.

Selective plasmenylcholine oxidation by hypochlorous acid: formation of lysophosphatidylcholine chlorohydrins.

The plasmalogen sn-1 vinyl ether bond is targeted by hypochlorous acid (HOCl) produced by activated phagocytes. In the present study, the attack of the plasmalogen sn-1 vinyl ether bond by HOCl is shown to be preferred compared to the attack of double bonds present in the sn-2 position aliphatic chain (sn-2 alkenes) of both plasmenylcholine and phosphatidylcholine. Lysophosphatidylcholine (LPC) is a product from the initial HOCl attack of plasmenylcholine and the sn-2 alkene bonds present in this LPC product are secondary targets of HOCl leading to the production of LPC-chlorohydrins (ClOH). The aliphatic ClOH was demonstrated in both the positive and negative ion mode using collisionally-activated dissociation (CAD) of the molecular ion of LPC-ClOH. Furthermore, HOCl treatment of endothelial cells led to the preferential attack of plasmalogens in comparison to that of diacyl choline glycerophospholipids. Taken together, plasmenylcholine is oxidized preferentially over phosphatidylcholine and leads to the production of LPC-ClOH.

Characterization of alkylacyl, alk-1-enylacyl and lyso subclasses of glycerophosphocholine by tandem quadrupole mass spectrometry with electrospray ionization.

Positive ion tandem quadrupole mass spectrometric methods for structural characterization of the subclasses of sn-glycero-3-phosphocholine (PC), including alkylacyl- and alk-1-enylacylphosphocholine and lysophosphatidylcholine (LPC), are described. Following collisionally activated dissociation, the [M + Li](+) ions generated by electrospray ionization yield abundant informative fragment ions that permit structural determination, and distinction of regioisomers among lysophosphatidylcholine can be easily achieved. In contrast, structurally informative ions arising from [M + H](+) or [M + Na](+) ions are less prominent. The most abundant ion observed in the product-ion spectra of the [M + Li](+) ions of plasmenyl- and plasmanyl-PC and of LPC arises from loss of N(CH(3))(3) ([M + Li - 59](+)). This feature permits their distinction from a product-ion spectrum arising from a diacylphosphatidylcholine, in which the [M + Li - 183](+) ion reflecting loss of phosphocholine is the most prominent. Examples for identification of various subclasses of PC in biological extracts by tandem mass spectrometry applying various constant neutral loss scannings are also shown.

Arterial cells and CNS sheath cells from Aplysia californica produce factors that enhance neurite outgrowth in co-cultured neurons.

Substrate-bound and soluble factors regulate neurite outgrowth and synapse formation during development, regeneration, and learning and memory. We report that sheath cells from CNS connectives and arterial cells from the anterior aorta of the sea slug, Aplysia californica, enhance neurite outgrowth from co-cultured Aplysia neurons. Sheath and arterial cell cultures contain several cell types, including fibrocytes, myocytes, and amoebocytes. When compared to controls (neurons with defined growth medium alone), the percentage of neurons with growth and the average neurite lengths are significantly enhanced by sheath and arterial cells at 48 h after plating of the neurons; these parameters are comparable to those of neurons cultured in medium containing hemolymph. Our results indicate that sheath cells produce substrate-bound factor(s) and arterial cells produce diffusible factor(s) that promote growth. These growth factors likely promote neuron survival and neurite outgrowth during neural plasticity exhibited in the adult CNS.

Potential role of acid ceramidase in conversion of cytostatic to cytotoxic end-point in pancreatic cancer cells.

PURPOSE: Acid ceramidase (AC) occupies an important place in the control of cancer cell proliferation. We tested the influence of AC inhibition on the effects of PSC 833, a P-glycoprotein antagonist with potent ceramide-generating capacity, to determine whether AC could be a therapeutic target in pancreatic cancer. METHODS: Ceramide metabolism was followed using (3)H-palmitate, and molecular species were determined by mass spectroscopy. Apoptosis was measured by DNA fragmentation, autophagy by acridine orange staining, and cell cycle was assessed by flow cytometry and RB phosphorylation. AC was measured in intact cells using fluorescent substrate. RESULTS: Exposure of human PANC-1 or MIA-PaCa-2 cells to PSC 833 promoted increases in de novo (dihydro)ceramides, (dihydro)glucosylceramides, and (dihydro)sphingomyelins, demarking ceramide generation and robust metabolism. Despite the multifold increases in (dihydro)ceramide levels, cells were refractory to PSC 833. However, PSC 833 produced a dose-dependent decrease in DNA synthesis and dose- and time-dependent decreases in RB phosphorylation, consistent with cell cycle arrest as demonstrated at G1. Cytostatic effects of PSC 833 were converted to cytotoxic end-point by acid ceramidase inhibition. Cytotoxicity was accompanied by formation of acridine orange-stained acidic vesicles and an increase in LC3 expression, indicative of autophagic response. Cell death was not reversed by preexposure to myriocin, which blocks PSC 833-induced ceramide generation. CONCLUSION: Although the role of ceramide in end-point cytotoxicity is unclear, our results suggest that acid ceramidase is a viable target in pancreatic cancer. We propose that AC inhibition will be effective in concert with other anticancer therapies.

Cytotoxic responses to N-(4-hydroxyphenyl)retinamide in human pancreatic cancer cells.

PURPOSE: Although fenretinide (4-HPR) has been studied in breast cancer and in neuroblastoma, little is known regarding its activity in pancreatic cancer, a neoplasm for which there are few therapeutic options. Since pancreatic cancer cells are susceptible to reactive oxygen species (ROS) and ceramide, two hallmarks of 4-HPR cytotoxicity, we investigated the effect of 4-HPR on human pancreatic cancer cells. METHODS: Human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 were treated with 4-HPR, followed by measurement of viability, proliferation, ROS and ceramide production, and Western blotting. RESULTS: At the measured IC(50) of 10 µM, 4-HPR led to a 44-68% reduction in [(3)H]thymidine incorporation, a >3-fold increase in de novo ceramide levels, a 2.7-fold increase in ROS, and minor increases in markers of apoptosis. 4-HPR induced a robust, sustained increase in LC3 II expression and enhanced formation of acridine orange-stained acidic vesicles that are markers of autophagy. In addition, sustained, dose-dependent increases in JNK and p38 phosphorylation and decreased ERK phosphorylation were observed following treatment. Pretreatment with vitamin E, a ROS scavenger, and 3-methyladenine, an autophagy inhibitor, individually led to decreased sensitivity to 4-HPR; however, the de novo ceramide inhibitor myriocin had no effect. CONCLUSIONS: These data show that 4-HPR triggers pancreatic cancer cell death by apoptosis and autophagy and that sensitivity appears to be mediated by ROS and not ceramide. This study is the first to characterize the response of human pancreatic cancer cells to 4-HPR and opens the door to investigations into this compound in pancreatic adenocarcinomas.

Metabolism of short-chain ceramide by human cancer cells--implications for therapeutic approaches.

Due to recent use of short-chain ceramides in preclinical studies, we characterized C6-ceramide metabolism in cancer cell lines and assessed metabolic junctures for enhancing efficacy. MDA-MB-231 breast cancer cells decreased the amount of C6-ceramide metabolized to C6-sphingomyelin (C6-SM) and increased the amount metabolized to C6-glucosylceramide (C6-GC) in response to increasing concentrations. A similar trend was seen in DU-145 (prostate cancer), PANC-1 (pancreatic cancer), and LoVo (colorectal cancer) cells. KG-1 leukemia cells favored C6-SM synthesis at low (0.6muM) and high-dose (12muM) C6-ceramide. Partnering C6-ceramide with tamoxifen, a P-glycoprotein antagonist that impedes ceramide glycosylation, was an effective regimen for enhancing cytotoxicity in cells. Experiments to assess the mechanism of cell death using KG-1 cells showed that tamoxifen inhibited synthesis of C6-GC and C6-SM from C6-ceramide by 80% and 50%, respectively, which was accompanied by enhanced apoptosis. Radiolabeling of KG-1 cells with [(3)H]palmitic acid produced a 2-fold increase in (3)H-long-chain ceramides when unlabeled C6-ceramide was added and a 9-fold increase when C6-ceramide and tamoxifen were added. The increase in (3)H-palmitate radiolabeling of long-chain ceramides was blocked by inclusion of a ceramide synthase inhibitor; however, inhibiting synthesis of long-chain ceramide did not rescue cells. These studies show that tamoxifen enhances the apoptotic effects of C6-ceramide. The proposed mechanism involves blocking short-chain ceramide anabolism to favor hydrolysis and generation of sphingosine. We propose that use of tamoxifen and other P-glycoprotein antagonists can be an effective means for enhancing cytotoxic potential of short-chain ceramides in the treatment of cancer.

Chromatographic methods for the analyses of 2-halofatty aldehydes and chlorohydrin molecular species of lysophosphatidylcholine.

Plasmalogens are targeted by hypohalous acids resulting in the production of 2-chlorofatty aldehydes, 2-bromofatty aldehydes and chlorohydrin species of lysophosphatidylcholine. These novel lipids may have important roles in the pathophysiological sequelae of cardiovascular diseases as well as serve as biomarkers of cardiovascular disease. Accordingly, the discovery of these new lipid species have required the development of techniques for their purification and quantification. Thin layer chromatography, high performance liquid chromatography (LC) and gas chromatography (GC) of these lipids and their derivatives have provided a battery of tools for their analyses. These lipids have been quantified using flame ionization detection (FID) and mass spectrometry (MS).

Endogenous neurotrophic factors enhance neurite growth by bag cell neurons of Aplysia.

Mechanisms that regulate neurite outgrowth are phylogenetically conserved, including the signaling molecules involved. Here, we describe neurotrophic effects on isolated bag cell neurons (BCNs) of substrate-bound growth factors endogenous to the sea slug Aplysia californica. Sheath cells dissociated from the pleural-visceral connectives of the Aplysia CNS and arterial cells dissociated from the anterior aorta enhance neurite outgrowth when compared to controls, i.e., BCNs grown in defined medium alone. In addition, the substrate remaining after sheath cells or arterial cells are killed significantly enhances growth, relative to all other conditions tested. For instance, primary neurites are more numerous and greater in length for BCNs cultured on substrate produced by arterial cells. These results suggest that sheath and arterial cells produce growth-promoting factors, some of which are found in the substrates produced by these cell types. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), we found that Aplysia collagen-like peptides are produced by dissociated arterial cells, and therefore likely contribute to the observed growth effects. Collagen-like peptides and other factors produced by sheath and arterial cells likely influence neurite growth in the Aplysia CNS during development, learning and memory, and regeneration after injury.

Myeloperoxidase-derived reactive chlorinating species from human monocytes target plasmalogens in low density lipoprotein.

A role for myeloperoxidase (MPO) in atherosclerosis has received considerable attention recently. To identify potential chlorinated lipid products in human low density lipoprotein (LDL), studies were designed to demonstrate that MPO-derived reactive chlorinating species (RCS) target the plasmalogen pool of LDL isolated from peripheral human blood in vitro. The vinyl ether bond of LDL plasmalogens was targeted by MPO-derived RCS, resulting in the release of the 16- and 18-carbon-containing alpha-chloro fatty aldehydes, 2-chlorohexadecanal and 2-chlorooctadecanal, respectively, from the plasmalogen glycerol backbone. Targeting of the LDL plasmalogen vinyl ether bond was dependent on the presence of MPO-derived RCS. Electrospray ionization mass spectrometric analysis of MPO-treated LDL demonstrated that a novel population of unsaturated lysophosphatidylcholine molecular species was produced by a phospholipase A2-independent mechanism. Unsaturated lysophosphatidylcholine molecular species elicited cyclic AMP response element binding protein phosphorylation in RAW 264.7 cells. Additionally, MPO-mediated targeting of both monocyte and LDL plasmalogen pools was demonstrated in phorbol myristate acetate-stimulated human monocytes, resulting in the production of both 2-chlorohexadecanal and 2-chlorooctadecanal. In contrast, alpha-chloro fatty aldehydes were not produced in phorbol myristate acetate-stimulated mouse monocytes. Collectively, the present studies demonstrate a novel MPO-specific mechanism that mediates the production of a novel group of unsaturated lysophosphatidylcholine molecular species and chlorinated aldehydes from both LDL and monocyte plasmalogen pools that may have important effects during inflammatory reactions mediated by monocytes, most notably atherosclerosis.