Papers by Jacek Bielawski
The Journal of Lipid Research, 2011
Mammalian ceramide synthases 1 to 6 (CerS1-6) generate Cer in an acyl-CoA-dependent manner, and e... more Mammalian ceramide synthases 1 to 6 (CerS1-6) generate Cer in an acyl-CoA-dependent manner, and expression of individual CerS has been shown to enhance the synthesis of ceramides with particular acyl chain lengths. However, the contribution of each CerS to steady-state levels of specific Cer species has not been evaluated. We investigated the knockdown of individual CerS in the MCF-7 human breast adenocarcinoma cell line by using small-interfering RNA (siRNA). We found that siRNA-induced downregulation of each CerS resulted in counter-regulation of nontargeted CerS. Additionally, each CerS knockdown produced unique effects on the levels of multiple sphingolipid species. For example, downregulation of CerS2 decreased very long-chain Cer but increased levels of CerS4, CerS5, and CerS6 expression and upregulated long-chain and medium-long-chain sphingolipids. Conversely, CerS6 knockdown decreased C16:0-Cer but increased CerS5 expression and caused non-C16:0 sphingolipids to be upregulated. Knockdown of individual CerS failed to decrease total sphingolipids or upregulate sphingoid bases. Treatment with siRNAs targeting combined CerS, CerS2, CerS5, and CerS6, did not change overall Cer or sphingomyelin mass but caused upregulation of dihydroceramide and hexosyl-ceramide and promoted endoplasmic reticulum stress. These data suggest that sphingolipid metabolism is robustly regulated by both redundancy in CerS-mediated Cer synthesis and counter-regulation of CerS expression.
The FASEB Journal, 2015
Peripheral neuropathy is a major dose-limiting side effect of paclitaxel and cisplatin chemothera... more Peripheral neuropathy is a major dose-limiting side effect of paclitaxel and cisplatin chemotherapy. In the current study, we tested the involvement of a novel class of neurotoxic sphingolipids, the 1-deoxysphingolipids. 1-Deoxysphingolipids are produced when the enzyme serine palmitoyltransferase uses l-alanine instead of l-serine as its amino acid substrate. We tested whether treatment of cells with paclitaxel (250 nM, 1 µM) and cisplatin (250 nM, 1 µM) would result in elevated cellular levels of 1-deoxysphingolipids. Our results revealed that paclitaxel, but not cisplatin treatment, caused a dose-dependent elevation of 1-deoxysphingolipids levels and an increase in the message and activity of serine palmitoyltransferase (P < 0.05). We also tested whether there is an association between peripheral neuropathy symptoms [evaluated by the European Organization for Research and Treatment of Cancer (EORTC) QLQ-chemotherapy-induced peripheral neuropathy-20 (CIPN20) instrument] and the 1-deoxysphingolipid plasma levels (measured by mass spectrometry) in 27 patients with breast cancer who were treated with paclitaxel chemotherapy. Our results showed that there was an association between the incidence and severity of neuropathy and the levels of very-long-chain 1-deoxyceramides such as C24 (P < 0.05), with the strongest association being with motor neuropathy (P < 0.001). Our data from cells and from patients with breast cancer suggest that 1-deoxysphingolipids, the very-long-chain in particular, play a role as molecular intermediates of paclitaxel-induced peripheral neuropathy.-Kramer, R., Bielawski, J., Kistner-Griffin, E., Othman, A., Alecu, I., Ernst, D., Kornhauser, D., Hornemann, T., Spassieva, S. Neurotoxic 1-deoxysphingolipids and paclitaxel-induced peripheral neuropathy.
Methods in Enzymology
In recent years, sphingolipid metabolites ceramide, sphingosine, and sphingosine-1-phosphate have... more In recent years, sphingolipid metabolites ceramide, sphingosine, and sphingosine-1-phosphate have emerged as important second messengers in addition to their role as precursors of biomembrane components. The investigation of these sphingolipid metabolites requires the development of new, more sensitive methods for assaying the enzymes involved in their production. This chapter describes the utilization of mass spectrometry technology in combination with nonnaturally occurring C(17) sphingoid bases in the in vitro assays of two of the enzymes of the sphingolipid pathway, ceramide synthase and sphingosine kinase. These new in vitro methods provide high sensitivity and extreme accuracy even when crude extracts are used as enzyme sources.
Mutants of tumor suppressor p53 not only lose the activity in genome stabilizing and in tumor sup... more Mutants of tumor suppressor p53 not only lose the activity in genome stabilizing and in tumor suppression, but 23 also exhibit oncogenic function in cancer cells. Most efforts in restoring p53 biological activity focus on either al-24 tering mutant-protein conformation or introducing an exogenous p53 gene into cells to eliminate p53-mutant 25 cancer cells. Being different from these, we report that ceramide can restore the expression of wild-type p53 26 and induce p53-dependent apoptosis in deletion-mutant cancer cells. We show that endogenous long-carbon 27 chain ceramide species (C 16 -to C 24 -ceramides) and exogenous C 6 -ceramide, rather than other sphingolipids, re-28 store wild-type mRNA (intact exon-5), phosphorylated protein (Ser15 in exon-5) of p53, and p53-responsive 29 proteins, including p21 and Bax, in ovarian cancer cells, which predominantly express a deleted exon-5 of p53 30 mutant before treatments. Consequently, the restored p53 sensitizes these p53-mutant cancer cells to DNA 31 damage-induced growth arrest and apoptosis. Furthermore, we elucidate that ceramide activates protein 32 phosphatase-1, and then the dephosphorylated serine/arginine-rich splicing-factor 1 (SRSF1) is translocated to 33 the nucleus, thus promoting pre-mRNA splicing preferentially to wild-type p53 expression. These findings dis-34 close an unrecognized mechanism that pre-mRNA splicing dysfunction can result in p53 deletion-mutants. Cer-35 amide through SRSF1 restores wild-type p53 expression versus deletion-mutant and leads cancer cells to 36 apoptosis. This suggests that heterozygous deletion-mutants of p53 can be restored in posttranscriptional level 37 by using epigenetic approaches. 38 39 40 41 42 43 65 develop consistently effective approaches targeting p53 mutants, great-66 er promise would seem to rest in the possibility of regulating the ex-67 pression of wild-type p53 versus mutants in cancer cells, which are 68 mostly heterozygous for the p53 gene. 69 Ceramide is the central metabolite of sphingolipids, and has myriad 70 effects on cell function, including cell growth arrest, senescence, Biochimica et Biophysica Acta xxx (2014) xxx-xxx
A recent finding showed that ceramide and sphingosine-1-phosphate (S1P) become exposed on the sur... more A recent finding showed that ceramide and sphingosine-1-phosphate (S1P) become exposed on the surface of cells treated by photodynamic therapy (PDT) and acquire the capacity to act as danger-associated molecular patterns (DAMPs). To explore this further, the present study examined whether ceramide and S1P can be released from PDT-treated cells and investigated changes in the levels of these sphingolipids in tumor-associated macrophages (TAMs) left in contact with PDT-treated tumor cells. Mass spectroscopy-based analysis detected increased levels of C16-ceramide and dihydroC16-ceramide in media supernatants from SCCVII cells collected three hours after they were treated by PDT, compared to untreated cell supernatants. While no release of S1P was detected, elevated levels of its precursor sphingosine were found in the supernatants of PDT-treated cells. The co-incubation of TAMs-containing primary cultures derived from mouse SCCVII tumors with PDT-treated SCCVII cells was followed by c...
International journal of oncology, 2015
Because photodynamic therapy (PDT) alone is not always effective as an anticancer treatment, PDT ... more Because photodynamic therapy (PDT) alone is not always effective as an anticancer treatment, PDT is combined with other anticancer agents for improved efficacy. The clinically-relevant fenretinide [N-(4-hydroxyphenyl) retinamide; 4HPR], was combined with the silicon phthalocyanine photosensitizer Pc4-mediated PDT to test for their potential to enhance killing of SCC17B cells, a clinically-relevant model of human head and neck squamous cell carcinoma. Because each of these treatments induces apoptosis and regulates the de novo sphingolipid (SL) biosynthesis pathway, the role of ceramide synthase, the pathway-associated enzyme, in PDT+4HPR-induced apoptotic cell death was determined using the ceramide synthase inhibitor fumonisin B1 (FB). PDT+4HPR enhanced loss of clonogenicity. zVAD-fmk, a pan-caspase inhibitor, and FB, protected cells from death post-PDT+4HPR. In contrast, the anti-apoptotic protein Bcl2 inhibitor ABT199 enhanced cell killing after PDT+4HPR. Combining PDT with 4HPR ...
Journal of photochemistry and photobiology. B, Biology, 2015
Combining photodynamic therapy (PDT) with another anticancer treatment modality is an important s... more Combining photodynamic therapy (PDT) with another anticancer treatment modality is an important strategy for improved efficacy. PDT with Pc4, a silicon phthalocyanine photosensitizer, was combined with C6-pyridinium ceramide (LCL29) to determine their potential to promote death of SCC17B human head and neck squamous cell carcinoma cells. PDT+LCL29-induced enhanced cell death was inhibited by zVAD-fmk, a pan-caspase inhibitor, and fumonisin B1 (FB), a ceramide synthase inhibitor. Quantitative confocal microscopy showed that combining PDT with LCL29 enhanced FB-sensitive ceramide accumulation in the mitochondria. Furthermore, PDT+LCL29 induced enhanced FB-sensitive redistribution of cytochrome c and caspase-3 activation. Overall, the data indicate that PDT+LCL29 enhanced cell death via FB-sensitive, mitochondrial ceramide accumulation and apoptosis.
Prostaglandins & other lipid mediators, 2008
Ceramide has been implicated in regulatory processes vital for cell survival under different stre... more Ceramide has been implicated in regulatory processes vital for cell survival under different stressors, most notably hypoxia. Little has been done to investigate the contributions of the different ceramide species to the regulation of cell survival. This study aims to highlight the patterns of variation in total ceramide and its species in the growing and hypoxic mouse heart. Mus musculus mice were placed in a hypoxic environment at birth. Control animals remained in room air. The hearts were extracted at different time points: 1 day, 1 week, 4 weeks, and 8 weeks. The total ceramide content and the amounts of component species were assayed by a modified diacylglycerol kinase assay and high-performance liquid chromatography-tandem mass spectroscopy, respectively. Data was collected from both ventricles in hypoxic and control conditions. There was significant polycythemia in the hypoxic versus control animals with a nearly twofold increase in hematocrit levels. Hypoxic right ventricle...
British journal of cancer, Jan 15, 2008
The sphingolipid ceramide is intimately involved in the growth, differentiation, senescence, and ... more The sphingolipid ceramide is intimately involved in the growth, differentiation, senescence, and death of normal and cancerous cells. Mitochondria are increasingly appreciated to play a key role in ceramide-induced cell death. Recent work showed the C16-pyridinium ceramide analogue LCL-30 to induce cell death in vitro by mitochondrial targeting. The aim of the current study was to translate these results to an in vivo model. We found that LCL-30 accumulated in mitochondria in the murine colorectal cancer cell line CT-26 and reduced cellular ATP content, leading to dose- and time-dependent cytotoxicity. Although the mitochondrial levels of sphingosine-1-phosphate (S1P) became elevated, transcription levels of ceramide-metabolising enzymes were not affected. In mice, LCL-30 was rapidly absorbed from the peritoneal cavity and cleared from the circulation within 24 h, but local peritoneal toxicity was dose-limiting. In a model of subcutaneous tumour inoculation, LCL-30 significantly red...
Bioorganic & medicinal chemistry, Jan 15, 2014
Acid ceramidase (ACDase) is being recognized as a therapeutic target for cancer. B13 represents a... more Acid ceramidase (ACDase) is being recognized as a therapeutic target for cancer. B13 represents a moderate inhibitor of ACDase. The present study concentrates on the lysosomal targeting of B13 via its N,N-dimethylglycine (DMG) esters (DMG-B13 prodrugs). Novel analogs, the isomeric mono-DMG-B13, LCL522 (3-O-DMG-B13·HCl) and LCL596 (1-O-DMG-B13·HCl) and di-DMG-B13, LCL521 (1,3-O, O-DMG-B13·2HCl) conjugates, were designed and synthesized through N,N-dimethyl glycine (DMG) esterification of the hydroxyl groups of B13. In MCF7 cells, DMG-B13 prodrugs were efficiently metabolized to B13. The early inhibitory effect of DMG-B13 prodrugs on cellular ceramidases was ACDase specific by their lysosomal targeting. The corresponding dramatic decrease of cellular Sph (80-97% Control/1h) by DMG-B13 prodrugs was mainly from the inhibition of the lysosomal ACDase.
Fatty acid 2-hydroxylase (FA2H) is responsible for the synthesis of myelin galactolipids containi... more Fatty acid 2-hydroxylase (FA2H) is responsible for the synthesis of myelin galactolipids containing hydroxy fatty acid (hFA) as the N-acyl chain. Mutations in the FA2H gene cause leukodystrophy, spastic paraplegia, and neurodegeneration with brain iron accumulation. Using the Cre-lox system, we developed two types of mouse mutants, Fa2h 2/2 mice (Fa2h deleted in all cells by germline deletion) and Fa2h flox/flox Cnp1-Cre mice (Fa2h deleted only in oligodendrocytes and Schwann cells). We found significant demyelination, profound axonal loss, and abnormally enlarged axons in the CNS of Fa2h 2/2 mice at 12 months of age, while structure and function of peripheral nerves were largely unaffected. Fa2h 2/2 mice also exhibited histological and functional disruption in the cerebellum at 12 months of age. In a time course study, significant deterioration of cerebellar function was first detected at 7 months of age. Further behavioral assessments in water T-maze and Morris water maze tasks revealed significant deficits in spatial learning and memory at 4 months of age. These data suggest that various regions of the CNS are functionally compromised in young adult Fa2h 2/2 mice. The cerebellar deficits in 12-month-old Fa2h flox/flox Cnp1-Cre mice were indistinguishable from Fa2h 2/2 mice, indicating that these phenotypes likely stem from the lack of myelin hFA-galactolipids. In contrast, Fa2h flox/flox Cnp1-Cre mice did not show reduced performance in water maze tasks, indicating that oligodendrocytes are not involved in the learning and memory deficits found in Fa2h 2/2 mice. These findings provide the first evidence that FA2H has an important function outside of oligodendrocytes in the CNS. V
The international journal of biochemistry & cell biology, 2012
Crohn's disease is a chronic inflammatory condition largely affecting the terminal ileum and ... more Crohn's disease is a chronic inflammatory condition largely affecting the terminal ileum and large bowel. A contributing cause is the failure of an adequate acute inflammatory response as a result of impaired secretion of pro-inflammatory cytokines by macrophages. This defective secretion arises from aberrant vesicle trafficking, misdirecting the cytokines to lysosomal degradation. Aberrant intestinal permeability is also well-established in Crohn's disease. Both the disordered vesicle trafficking and increased bowel permeability could result from abnormal lipid composition. We thus measured the sphingo- and phospholipid composition of macrophages, using mass spectrometry and stable isotope labelling approaches. Stimulation of macrophages with heat-killed Escherichia coli resulted in three main changes; a significant reduction in the amount of individual ceramide species, an altered composition of phosphatidylcholine, and an increased rate of phosphatidylcholine synthesis in...
Archives of Biochemistry and Biophysics, 2003
In this study, we examined the role of endogenous ceramide in the inhibition of telomerase and in... more In this study, we examined the role of endogenous ceramide in the inhibition of telomerase and induction of morphologic differentiation in response to all-trans-retinoic acid (ATRA) in the SK-N-SH and SK-N-AS human neuroblastoma cell lines. The results showed that ATRA inhibited the activity of telomerase significantly in a time- and dose-dependent manner, as determined by telomere repeat amplification protocol (TRAP).
Faseb Journal, 2006
Sphingosine-1-phosphate (S1P), a sphin- golipid metabolite, promotes cell proliferation and survi... more Sphingosine-1-phosphate (S1P), a sphin- golipid metabolite, promotes cell proliferation and survival whereas its precursor, sphingosine, has the opposite effects. However, much remains unknown about their regulation. Here we identify a novel human ceramidase (haCER2) that regulates the levels of both sphingosine and S1P by controlling the hydrolysis of ceramides. haCER2 is localized to the Golgi complex and is highly expressed
The accelerated cell death 11 (acd11) mutant of Arabidopsis provides a genetic model for studying... more The accelerated cell death 11 (acd11) mutant of Arabidopsis provides a genetic model for studying immune response activation and localized cellular suicide that halt pathogen spread during infection in plants. Here, we elucidate ACD11 structure and function and show that acd11 disruption dramatically alters the in vivo balance of sphingolipid mediators that regulate eukaryotic-programmed cell death. In acd11 mutants, normally low ceramide-1phosphate (C1P) levels become elevated, but the relatively abundant cell death inducer phytoceramide rises acutely. ACD11 exhibits selective intermembrane transfer of C1P and phyto-C1P. Crystal structures establish C1P binding via a surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively ensheaths lipid chains via a cleft-like gating mechanism. Point mutation mapping confirms functional involvement of binding site residues. A p helix (p bulge) near the lipid binding cleft distinguishes apo-ACD11 from other GLTP folds. The global two-layer, a-helically dominated, ''sandwich'' topology displaying C1P-selective binding identifies ACD11 as the plant prototype of a GLTP fold subfamily.
Biochimica Et Biophysica Acta-biomembranes, 2007
The Saccharomyces cerevisiae inositol sphingolipid phospholipase C (Isc1p), a homolog of mammalia... more The Saccharomyces cerevisiae inositol sphingolipid phospholipase C (Isc1p), a homolog of mammalian neutral sphingomyelinases, hydrolyzes complex sphingolipids to produce ceramide in vitro. Epitope-tagged Isc1p associates with the mitochondria in the post-diauxic phase of yeast growth. In this report, the mitochondrial localization of Isc1p and its role in regulating sphingolipid metabolism were investigated. First, endogenous Isc1p activity was enriched in highly
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2014
Mutants of tumor suppressor p53 not only lose the activity in genome stabilizing and in tumor sup... more Mutants of tumor suppressor p53 not only lose the activity in genome stabilizing and in tumor suppression, but 23 also exhibit oncogenic function in cancer cells. Most efforts in restoring p53 biological activity focus on either al-24 tering mutant-protein conformation or introducing an exogenous p53 gene into cells to eliminate p53-mutant 25 cancer cells. Being different from these, we report that ceramide can restore the expression of wild-type p53 26 and induce p53-dependent apoptosis in deletion-mutant cancer cells. We show that endogenous long-carbon 27 chain ceramide species (C 16 -to C 24 -ceramides) and exogenous C 6 -ceramide, rather than other sphingolipids, re-28 store wild-type mRNA (intact exon-5), phosphorylated protein (Ser15 in exon-5) of p53, and p53-responsive 29 proteins, including p21 and Bax, in ovarian cancer cells, which predominantly express a deleted exon-5 of p53 30 mutant before treatments. Consequently, the restored p53 sensitizes these p53-mutant cancer cells to DNA 31 damage-induced growth arrest and apoptosis. Furthermore, we elucidate that ceramide activates protein 32 phosphatase-1, and then the dephosphorylated serine/arginine-rich splicing-factor 1 (SRSF1) is translocated to 33 the nucleus, thus promoting pre-mRNA splicing preferentially to wild-type p53 expression. These findings dis-34 close an unrecognized mechanism that pre-mRNA splicing dysfunction can result in p53 deletion-mutants. Cer-35 amide through SRSF1 restores wild-type p53 expression versus deletion-mutant and leads cancer cells to 36 apoptosis. This suggests that heterozygous deletion-mutants of p53 can be restored in posttranscriptional level 37 by using epigenetic approaches. 38 39 40 41 42 43 65 develop consistently effective approaches targeting p53 mutants, great-66 er promise would seem to rest in the possibility of regulating the ex-67 pression of wild-type p53 versus mutants in cancer cells, which are 68 mostly heterozygous for the p53 gene. 69 Ceramide is the central metabolite of sphingolipids, and has myriad 70 effects on cell function, including cell growth arrest, senescence, Biochimica et Biophysica Acta xxx (2014) xxx-xxx
Molecular Therapy, 2006
The potential anti-tumor agent Apoptin activates apoptosis in many human cancers and transformed ... more The potential anti-tumor agent Apoptin activates apoptosis in many human cancers and transformed cell lines, but is believed to be less potent in primary cells. Although caspase 3 is activated during apoptin-induced apoptosis, the mechanism of tumor cell killing remains elusive. We now show that apoptin-mediated cell death involves modulation of the sphingomyelin-ceramide pathway. Treating cells with Ad-GFPApoptin resulted in increased ceramide accumulation and enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin. Using confocal microscopy, ASMase, normally present in the endosomal/lysosomal compartment, was observed to translocate to the cell's periphery. Cotreatment of Ad-GFPApoptin-infected cells with the ASMase inhibitor desipramine (2.5 MM) attenuated (30%; P b 0.01) apoptin-induced cell death. Apoptin was also able to induce a significant decline in sphingosine content by inhibition of ceramide deacylation through down-regulation of acid ceramidase at the protein level. Supporting the role of ceramide in apoptin action, treatment of cells with the combination of an exogenous cell-permeable ceramide analog (C6-ceramide) and AdQGFPApoptin infection yielded a significant increase ( P b 0.01) in apoptosis over either treatment modality alone. Together, these data suggest that apoptin modulates ceramide/sphingolipid metabolism as part of its mechanism of action.
Journal of Clinical Investigation, 2007
Uterine decidualization, a process that occurs in response to embryo implantation, is critical fo... more Uterine decidualization, a process that occurs in response to embryo implantation, is critical for embryonic survival and thus is a key event for successful pregnancy. Here we show that the sphingolipid metabolic pathway is highly activated in the deciduum during pregnancy and disturbance of the pathway by disruption of sphingosine kinase (Sphk) genes causes defective decidualization with severely compromised uterine blood vessels, leading to early pregnancy loss. Sphk-deficient female mice (Sphk1 -/-Sphk2 +/-) exhibited both an enormous accumulation of dihydrosphingosine and sphingosine and a reduction in phosphatidylethanolamine levels in pregnant uteri. These mice also revealed increased cell death in decidual cells, decreased cell proliferation in undifferentiated stromal cells, and massive breakage of decidual blood vessels, leading to uterine hemorrhage and early embryonic lethality. Thus, sphingolipid metabolism regulates proper uterine decidualization and blood vessel stability. Our findings also suggest that disturbance in sphingolipid metabolism may be considered as a cause of pregnancy loss in humans.
Prostaglandins & Other Lipid Mediators, 2008
The tumor suppressor protein p53 and the putative lipid tumor suppressor ceramide play pivotal ro... more The tumor suppressor protein p53 and the putative lipid tumor suppressor ceramide play pivotal roles in inducing cell cycle arrest or in driving the cell towards apoptosis. Previously we had shown that, in a p53-dependent model of cell death, ceramide accumulated in a p53-dependent manner [Dbaibo GS, Pushkareva MY, Rachid RA, Alter N, Smyth MJ, Obeid LM, Hannun YA. J Clin Invest 1998;102:329-339]. In the current study, we investigated the biochemical pathways by which ceramide accumulated following p53 up-regulation. In both Molt-4 LXSN leukemia cells exposed to ␥-irradiation and in EB-1 colon cancer cells treated with ZnCl 2 , p53 up-regulation led to de novo ceramide synthesis with predominance of N-palmitoylsphingosine (C16-ceramide) synthesis. The activation of the de novo pathway was not associated with increased activity of the key enzyme serine palmitoyltransferase (SPT) but rather with the increased activity of ceramide synthase. Furthermore, transcriptional up-regulation of the palmitoylspecific Lass5 ceramide synthase gene was observed in Molt-4 but not in EB-1 cells. The SPT inhibitor ISP-1 or the ceramide synthase inhibitor fumonisin B1 led to substantial inhibition of ceramide accumulation in response to p53 up-regulation. Other biochemical pathways of ceramide generation such as sphingomyelinase activation were examined and found unlikely to contribute to p53-dependent ceramide formation. These studies indicate that p53 specifically drives de novo ceramide synthesis by activation of a ceramide synthase that favors the synthesis of N-palmitoylsphingosine.
Uploads
Papers by Jacek Bielawski