Robert Jan Veldman

Ceramide: second messenger or modulator of membrane structure and dynamics?

The physiological role of ceramide formation in response to cell stimulation remains controversial. Here, we emphasize that ceramide is not a priori an apoptotic signalling molecule. Recent work points out that the conversion of sphingomyelin into ceramide can play a membrane structural (physical) role, with consequences for membrane microdomain function, membrane vesiculation, fusion/fission and vesicular trafficking. These processes contribute to cellular signalling. At the Golgi, ceramide takes part in a metabolic flux towards sphingomyelin, diacylglycerol and glycosphingolipids, which drives lipid raft formation and vesicular transport towards the plasma membrane. At the cell surface, receptor clustering in lipid rafts and the formation of endosomes can be facilitated by transient ceramide formation. Also, signalling towards mitochondria may involve glycosphingolipid-containing vesicles. Ceramide may affect the permeability of the mitochondrial outer membrane and the release of cytochrome c. In the effector phase of apoptosis, the breakdown of plasma membrane sphingomyelin to ceramide is a consequence of lipid scrambling, and may regulate apoptotic body formation. Thus ceramide formation serves many different functions at distinct locations in the cell. Given the limited capacity for spontaneous intracellular diffusion or membrane flip-flop of natural ceramide species, the topology and membrane sidedness of ceramide generation are crucial determinants of its impact on cell biology.

Intracellular Triggering of Fas Aggregation and Recruitment of Apoptotic Molecules into Fas-enriched Rafts in Selective Tumor Cell Apoptosis

We have discovered a new and specific cell-killing mechanism mediated by the selective uptake of the antitumor drug 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, Edelfosine) into lipid rafts of tumor cells, followed by its coaggregation with Fas death receptor (also known as APO-1 or CD95) and recruitment of apoptotic molecules into Fas-enriched rafts. Drug sensitivity was dependent on drug uptake and Fas expression, regardless of the presence of other major death receptors, such as tumor necrosis factor (TNF) receptor 1 or TNF-related apoptosis-inducing ligand R2/DR5 in the target cell. Drug microinjection experiments in Fas-deficient and Fas-transfected cells unable to incorporate exogenous ET-18-OCH3 demonstrated that Fas was intracellularly activated. Partial deletion of the Fas intracellular domain prevented apoptosis. Unlike normal lymphocytes, leukemic T cells incorporated ET-18-OCH3 into rafts coaggregating with Fas and underwent apoptosis. Fas-associated death domain protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid were recruited into rafts, linking Fas and mitochondrial signaling routes. Clustering of rafts was necessary but not sufficient for ET-18-OCH3–mediated cell death, with Fas being required as the apoptosis trigger. ET-18-OCH3–mediated apoptosis did not require sphingomyelinase activation. Normal cells, including human and rat hepatocytes, did not incorporate ET-18-OCH3 and were spared. This mechanism represents the first selective activation of Fas in tumor cells. Our data set a framework for the development of more targeted therapies leading to intracellular Fas activation and recruitment of downstream signaling molecules into Fas-enriched rafts.

Accumulation of glycosphingolipids in Niemann-Pick C disease disrupts endosomal transport.

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus but are mistargeted to lysosomes in sphingolipid storage disorders. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Niemann-Pick type C (NPC) disease is a disorder of intracellular transport where glycosphingolipids (GSLs) and cholesterol accumulate in endosomal compartments. The mechanisms of altered intracellular trafficking are not known but may involve the mistargeting and disrupted function of proteins associated with GSL membrane microdomains. Membrane microdomains were isolated by Triton X-100 and sucrose density gradient ultracentrifugation. High pressure liquid chromatography and mass spectrometric analysis of NPC1–/– mouse brain revealed large increases in GSL. Sphingosine was also found to be a component of membrane microdomains, and in NPC liver and spleen, large increases in cholesterol and sphingosine were found. GSL and cholesterol levels were increased in mutant NPC1-null Chinese hamster ovary cells as well as U18666A and progesterone induced NPC cell culture models. However, inhibition of GSL synthesis in NPC cells with N-butyldeoxygalactonojirimycin led to marked decreases in GSL but only small decreases in cholesterol levels. Both annexin 2 and 6, membrane-associated proteins that are important in endocytic trafficking, show distorted distributions in NPC cells. Altered BODIPY lactosylceramide targeting, decreased endocytic uptake of a fluid phase marker, and mistargeting of annexin 2 (phenotypes associated with NPC) are reversed by inhibition of GSL synthesis. It is suggested that accumulating GSL is part of a mislocalized membrane microdomain and is responsible for the deficit in endocytic trafficking found in NPC disease.

Elevation of glucosylceramide in multidrug-resistant cancer cells and accumulation in cytoplasmic droplets.

Multidrug‐resistant (MDR) cancer cells have been shown to have an accumulation of glucosylceramide (GlcCer). In this study, we aim at localizing, at subcellular level, where these lipids accumulate. Neutral lipids and phospholipid containing organelles have been identified using confocal fluorescence microscopy and microspectrofluorometry by monitoring the emission of the fluorescent probe Nile‐red. Data from confocal fluorescence microscopy analysis shows accumulation of neutral lipids in cytoplasmic droplets of MDR human carcinoma MCF7R cells. Microspectrofluorometric measurements show an increase of the gold‐yellow emission intensity in MCF7R cells, corresponding to neutral lipids. Similar observations were made in human MDR vincristine‐HL60 and doxorubicin‐KB selected cells. Total cellular glucosylceramide (GlcCer) measurements using [3H]‐palmitic acid and thin layer chromatography show a significant increase of GlcCer in MCF7R cells. Moreover, MCF7R cells treated with fluorescent GlcCer‐bodipy exhibit an accumulation of this lipid in cytoplasmic droplets. Treatment of MCF7R cells with 1‐phenyl‐2‐palmitoylamino‐3‐morpholino‐1‐propanolol (PPMP), a potent inhibitor of GlcCer synthase, attenuates the Nile‐red fluorescence emission emanating from these structures and reverses MDR. Moreover, Golgi compartments stained with fluorescent PPMP‐bodipy, show an increase in the Golgi compartments density. Treatment of MCF7R cells with cyclosporine A (CSA), tamoxifen (TMX) and 3′‐azido‐3′deoxythymidine (AZT) leads to the same effect observed in the presence of PPMP. Treatment of MCF7 and MCF7R with the β‐glucosidase inhibitor conduritol β‐epoxide (CBE) significantly increases resistance to daunorubicin only in MCF7R cells. These data demonstrate also that: (i) CSA, an inhibitor of MDR, has an additional target in addition to P‐glycoprotein; and (ii) TMX (used in breast cancer treatment and prevention) and AZT (used in the treatment of HIV) could have side effects by disturbing lipid metabolism and inhibiting many cellular functions required in normal cells.

Altered sphingolipid metabolism in multidrug-resistant ovarian cancer cells is due to uncoupling of glycolipid biosynthesis in the Golgi apparatus.

Multidrug‐resistant tumor cells display enhanced levels of glucosylceramide. In this study, we investigated how this relates to the overall sphingolipid composition of multidrug‐resistant ovarian carcinoma cells and which mechanisms are responsible for adapted sphingolipid metabolism. We found in multidrug‐resistant cells substantially lower levels of lactosylceramide and gangliosides in sharp contrast to glucosylceramide, galactosylceramide, and sphingomyelin levels. This indicates a block in the glycolipid biosynthetic pathway at the level of lactosylceramide formation, with concomitant accumulation of glucosylceramide. A series of observations exclude regulation at the enzyme level as the underlying mechanism. First, reduced lactosylceramide formation occurred only in intact resistant cells whereas cell‐free activity of lactosylceramide synthase was higher compared with the parental cells. Second, the level of lactosylceramide synthase gene expression was equal in both phenotypes. Third, glucosylceramide synthase (mRNA and protein) expression and activity were equal or lower in resistant cells. Based on the kinetics of sphingolipid metabolism, the observation that brefeldin A does not restore lactosylceramide synthesis, and altered localization of lactosylceramide synthase fused to green fluorescent protein, we conclude that lactosylceramide biosynthesis is highly uncoupled from glucosylceramide biosynthesis in the Golgi apparatus of resistant cells.

DIFFERENTIAL EXPRESSION OF SPHINGOLIPIDS IN MRP1 OVEREXPRESSING HT29 CELLS

We have obtained a novel multidrug resistant cell line, derived from HT29 G+ human colon carcinoma cells, by selection with gradually increasing concentrations of the anti‐mitotic, microtubule‐disrupting agent colchicine. This HT29col cell line displayed a 25‐fold increase in colchicine resistance and exhibited cross‐resistance to doxorubicin, VP16, vincristine and taxol. Immunoblotting, combined with RT‐PCR showed that the multidrug resistance phenotype was conferred by specific overexpression of the multidrug resistance protein 1. Confocal scanning laser microscopy revealed that multidrug resistance protein 1 specifically localized in the plasma membrane of HT29col cells. In a functional assay, using the fluorescent multidrug resistance protein 1 substrate 5‐carboxyfluorescein, an increased efflux activity of HT29col cells was measured, as compared to the wild‐type HT29 G+ cells. MK571, a specific inhibitor of multidrug resistance protein 1, blocked the 5‐carboxyfluorescein efflux, but only partially reversed resistance to colchicine, indicating that additional multidrug resistance mechanisms operate in HT29col cells. In conclusion, these results show for the first time overexpression of a functional multidrug resistance protein 1 under colchicine pressure, indicating that colchicine is not a P‐glycoprotein‐specific substrate. Colchicine‐induced overexpression of multidrug resistance protein 1 is accompanied by a changed sphingolipid composition, i.e., enhanced levels of glucosylceramide and galactosylceramide. In addition, ceramide, a lipid messenger molecule involved in apoptosis‐related signal transduction processes, was much more abundant in HT29col cells, which is indicative of a stress response. Int. J. Cancer 87:172–178, 2000.

Enriching lipid nanovesicles with short-chain glucosylceramide improves doxorubicin delivery and efficacy in solid tumors

For amphiphilic anticancer drugs, such as the anthracyclin doxorubicin (Dox), uptake by tumor cells involves slow diffusion across the plasma membrane, a limiting factor in clinical oncology. Previously, we discovered that preinsertion of short‐chain sphingolipids such as AŁoctanoyl‐glucosylceramide (GC) in the tumor cell membrane enhances cellular Dox uptake. In the present study, we apply this strategy in vitro and in vivo by coadministering GC and Dox in a lipid nanovesicle (LNV). GC enrichment of Dox‐LNVs strongly enhanced in vitro cyto‐toxicity toward B16 melanoma and A431 carcinoma, as evidenced by 6‐fold decreased IC50 values compared with Dox‐LNVs. This correlated with enhanced cellular Dox uptake observed by confocal microscopy. Intravital optical imaging in window chamber‐bearing mice with ortho‐topically implanted B16 melanoma demonstrated enhanced GC‐mediated Dox delivery to tumor cells. Treatment of nude mice bearing human A431 xenografts with 6 mg/kg GC‐Dox‐LNVs almost doubled the tumor growth delay compared with Dox‐LNVs. A second administration of 5 mg/kg after 3 d induced even 3‐fold delay in tumor growth, while no systemic toxicity was found. GC‐enriched Dox‐LNVs displayed superior in vitro and in vivo antitumor activity, without systemic toxicity. This new drug delivery concept, aiming at increased membrane permeability for amphiphilic drugs, provides an opportunity to improve cancer chemotherapy.—Van Lummel, M., van Blitterswijk, W. J., Vink, S. R., Veldman, R. J., van der Valk, M. A., Schipper, D., Dicheva, B. M., Eggermont, A. M. M., ten Hagen, T. L. M., Verheij, M., Koning, G. A. Enriching lipid nanovesicles with short‐chain glucosylcer‐amide improves doxorubicin delivery and efficacy in solid tumors. FASEB J. 25, 280–289 (2011). www.fasebj.org

The absence of functional glucosylceramide synthase does not sensitize melanoma cells for anticancer drugs

Conversion of ceramide, a putative mediator of anticancer drug‐induced apoptosis, into glucosylceramide, by the action of glucosylceramide synthase (GCS), has been implicated in drug resistance. Herein, we compared GM95 mouse melanoma cells deficient in GCS activity, with cells stably transfected with a vector encoding GCS (GM95/GCS). Enzymatic and metabolic analysis demonstrated that GM95/GCS cells expressed a fully functional enzyme, resulting in normal ceramide glycosylation. However, cytotoxicity assays, as well as caspase activation and cytochrome c release studies, did not reveal any difference between the two cell lines with respect to their sensitivity toward doxorubicin, vinblastine, paclitaxel, cytosine arabinoside, or short‐chain ceramide analogs. Administration of doxorubicin resulted in ceramide accumulation in both cell lines, with similar kinetics and amplitude. Although glucosylceramide formation was detected in doxorubicin‐treated GM95/GCS cells, metabolism of drug‐induced ceramide did not appear to be instrumental in cell survival. Furthermore, N‐(n‐butyl)deoxynojirimycin, a potent and non‐toxic GCS inhibitor, had no chemosensitizing effect on wild‐type melanoma cells. Altogether, both genetic and pharmacological alterations of the cellular ceramide glycosylation capacity failed to sensitize melanoma cells to anticancer drugs, therefore moderating the importance of ceramide glucosylation in drug‐resistance mechanisms.

N -hexanoyl-sphingomyelin potentiates in vitro doxorubicin cytotoxicity by enhancing its cellular influx

Anticancer drugs generally have intracellular targets, implicating transport over the plasma membrane. For amphiphilic agents, such as the anthracycline doxorubicin, this occurs by passive diffusion. We investigated whether exogenous membrane-permeable lipid analogues improve this drug influx. Combinations of drugs and lipid analogues were coadministered to cultured endothelial cells and various tumour cell lines, and subsequent drug accumulation in cells was quantified. We identified N-hexanoyl-sphingomyelin (SM) as a potent enhancer of drug uptake. Low micromolar amounts of this short-chain sphingolipid, being not toxic itself, enhanced the uptake of doxorubicin up to 300% and decreased its EC50 toxicity values seven- to 14-fold. N-hexanoyl SM acts at the level of the plasma membrane, but was found not incorporated in (isolated) lipid rafts, and artificial disruption or elimination of raft constituents did not affect its drug uptake-enhancing effect. Further, any mechanistic role of the endocytic machinery, membrane leakage or ABC-transporter-mediated efflux could be excluded. Finally, a correlation was established between the degree of drug lipophilicity, as defined by partitioning in a two-phase octanol–water system, and the susceptibility of the drug towards the uptake-enhancing effect of the sphingolipid. A clear optimum was found for amphiphilic drugs, such as doxorubicin, epirubicin and topotecan, indicating that N-hexanoyl-SM might act by modulating the average degree of plasma membrane lipophilicity, in turn facilitating transbilayer drug diffusion. The concept of short-chain sphingolipids as amphiphilic drug potentiators provides novel opportunities for improving drug delivery technologies.

Ara-C- and daunorubicin-induced recruitment of Lyn in sphingomyelinase-enriched membrane rafts

Induction of apoptosis by DNA‐damaging agents such as 1‐β‐D‐arabinofuranosylcytosine (Ara‐C) includes the activation of Lyn protein tyrosine kinase. We have previously established that Ara‐C‐induced activation of Lyn results in its binding to a neutral sphingomyelinase (SMase) and is requisite for its stimulation and the induction of apoptosis in U937 cells. However, the spacio‐temporal organization of these events is unclear. This study demonstrates that part of the total cellular SMase activity is sequestered in sphingomyelin‐enriched plasma membrane microdomains (rafts). Under Ara‐C and daunorubicin (DNR) treatment, Lyn is rapidly activated and translocated into rafts. The compartmentalization of Lyn (as well as neutral SMase activation and apoptosis) induced by these drugs was blocked by the tyrosine kinase inhibitor herbimycin A and raft disruption. In conclusion, this study establishes that DNA‐damaging agents such as Ara‐C and DNR rapidly induce Lyn activation and its translocation into membrane rafts. This in turn leads to neutral SMase activation and raft‐associated sphingomyelin hydrolysis with the concomitant generation of the proapoptotic lipid second messenger, ceramide. The apparent topological partitioning between DNA damage and apoptosis signaling (integrated into specialized plasma membrane domains) is discussed.