Acid Ceramidase Inhibitors Research Articles

Discovery of a New Class of Highly Potent Inhibitors of Acid Ceramidase: Synthesis and Structure–Activity Relationship (SAR)

Acid ceramidase (AC) is an intracellular cysteine amidase that catalyzes the hydrolysis of the lipid messenger ceramide. By regulating ceramide levels in cells, AC may contribute to the regulation of cancer cell proliferation and senescence and to the response to cancer therapy. We recently identified the antitumoral agent carmofur (4a) as the first nanomolar inhibitor of intracellular AC activity (rat AC, IC50 = 0.029 μM). In the present work, we expanded our initial structure-activity relationship (SAR) studies around 4a by synthesizing and testing a series of 2,4-dioxopyrimidine-1-carboxamides. Our investigations provided a first elucidation of the structural features of uracil derivatives that are critical for AC inhibition and led us to identify the first single-digit nanomolar inhibitors of this enzyme. The present results confirm that substituted 2,4-dioxopyrimidine-1-carboxamides are a novel class of potent inhibitors of AC. Selected compounds of this class may represent useful probes to further characterize the functional roles of AC.

Abstract 3396: Improving the anti-AML efficacy of liposomal ceramide by targeting sphingolipid metabolism.

Abstract Novel approaches to enhancing the delivery, uptake, and therapeutic effect of sphingolipids are of interest for the treatment of neoplasms. We attempt to promote cellular ceramide accumulation in murine and human models of acute myelogenous leukemia (AML) by modifying ceramide metabolism with either the sphingosine kinase1 inhibitor safingol (Lip-Saf), or the acid ceramidase inhibitor LCL204, preventing ceramide glycosylation by inhibition of p-glycoprotein (P-GP) activity with tamoxifen (Lip-Tam), and supplementing cells with exogenous C6 ceramide (Lip-C6) via liposomal delivery. Ceramide is widely recognized as an apoptotic signal and blocking its catabolism to sphingosisne-1-phosphate has been shown to induce autophagy. Safingol and LCL204 both modify ceramide catabolism. Supplementation of tamoxifen has been shown to promote apoptosis via ceramide accumulation by inhibiting the activity of P-GP at the Golgi membrane, which can participate in ceramide glycosylation. C1498, HL-60, HL-60/VCR, and KG-1 leukemia cell line viability was quantified following treatment with Lip-C6, Lip-Saf, or a 2:1 combination to measure synergy. Likewise, cell line viability was evaluated using Lip-Tam and Lip-C6, independently and in a 1:1 combination to study synergy. C1498 viability was also quantified with increasing concentrations of LCL204. CalcuSyn software was used to analyze dose-response and synergistic therapeutic efficacy. Colony-forming capacity was assessed using a poor-prognosis AML patient sample treated with Lip-C6, Lip-Saf, or the combination of both. Autophagy and apoptosis were further evaluated using flow cytometry with primary AML samples and leukemia cell lines. A reduction in IC50 of the drugs used in combination was noted in several cell lines. Dramatic IC50 shifts were observed in KG-1, HL-60, and the vincristine-resistant HL-60/VCR cell lines after exposure to a combination of Lip-C6 and Lip-Saf in a 2:1 ratio. Flow cytometry confirmed Lip-Saf enhances autophagy, whereas Lip-C6 exerts an effect primarily through apoptosis in C1498 and primary AML samples. Therefore, the observed cell death synergism is likely due to induction of both autophagy and apoptosis. Additionally, it was shown that the anti-AML efficacy of Lip-Tam was dramatically enhanced in combination with Lip-C6, demonstrating that inhibition of ceramide glycosylation is also a valid combinatorial therapeutic approach. Lastly, inhibition of acid ceramidase via LCL204 demonstrated efficacy as a cytotoxic agent both in vitro and in vivo using C1498 cells alone or engrafted into C57BL/6J mice. These studies verify inhibition of ceramide metabolism as an effective strategy to enhance the therapeutic efficacy of ceramide in the treatment of AML. Citation Format: Timothy J. Brown, Aileen M. Garcia, Su-Fern Tan, Lindsey N. Kissinger, David J. Feith, Brian M. Barth, David F. Claxton. Improving the anti-AML efficacy of liposomal ceramide by targeting sphingolipid metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3396. doi:10.1158/1538-7445.AM2013-3396

Discovery of highly potent acid ceramidase inhibitors with in vitro tumor chemosensitizing activity

The expression of acid ceramidase (AC) – a cysteine amidase that hydrolyses the proapoptotic lipid ceramide – is abnormally high in several human tumors, which is suggestive of a role in chemoresistance. Available AC inhibitors lack, however, the potency and drug-likeness necessary to test this idea. Here we show that the antineoplastic drug carmofur, which is used in the clinic to treat colorectal cancers, is a potent AC inhibitor and that this property is essential to its anti-proliferative effects. Modifications in the chemical scaffold of carmofur yield new AC inhibitors that act synergistically with standard antitumoral drugs to prevent cancer cell proliferation. These findings identify AC as an unexpected target for carmofur, and suggest that this molecule can be used as starting point for the design of novel chemosensitizing agents.

Cystatin SA – a molecule with novel anticancer properties based on the inhibition of acid ceramidase

Cystatin SA – a molecule with novel anticancer properties based on the inhibition of acid ceramidase

Ceramide formation is involved in Lactobacillus acidophilus-induced IFN-beta response in dendritic cells

Event Abstract Back to Event Ceramide formation is involved in Lactobacillus acidophilus-induced IFN-beta response in dendritic cells Eva Fuglsang1*, Louise Henningsen1 and Hanne Frøkiær1 1 University of Copenhagen, Department of Veterinary Disease Biology, Denmark The sphingolipid ceramide is known to play a role in lipid raft fusion and receptor clustering in the plasma membrane (PM). Upon bacterial encounter, dendritic cells (DCs) endocytose the bacteria and initiate a bacteria-specific downstream signaling event. We hypothesized that conversion of sphingomyelin to ceramide by acid sphingomyelinase (ASMase) at the outer leaflet of the PM is a key event in endocytosis of gram-positive Lactobacillus acidophilus (L. acidophilus) and the subsequent induction of IFN-beta in DCs and, as the gram-negative Escherichia coli (E. coli) does not induce appreciable amounts of IFN-beta, the ASMase activity would affect endocytosis and the ensuing cytokine response of L. acidophilus and E. coli differently. SMase or an inhibitor of ASMase and acid ceramidase, chlorpromazine (CPZ), was added to DCs prior to stimulation with either of the bacteria. Endocytosis of fluorescent bacteria +/- FITC-dextran was measured by flow cytometry and gene expression and cytokine response of IFN-beta and IL-12 was measured by qPCR and ELISA, respectively. Addition of SMase increased the uptake of L. acidophilus and L. acidophilus-induced IL-12/IFN-beta but showed no effect on the uptake of E. coli though decreasing IL-12 induced by E. coli. SMase also showed to down-regulate Pam3CSK4-induced macropinocytosis of both bacteria. Addition of CPZ increased actin-dependent uptake of dextran and increased Il-12/Ifn-beta expression induced by L. acidophilus, thus further substantiating the key role of ceramide and thus, phagocytosis, in L. acidophilus-induced IL-12/IFN-beta. Keywords: Dendritic Cells, ceramide, Endocytosis, Lactobacillus acidophilus, Escherichia coli Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Fuglsang E, Henningsen L and Frøkiær H (2013). Ceramide formation is involved in Lactobacillus acidophilus-induced IFN-beta response in dendritic cells. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01028 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Miss. Eva Fuglsang, University of Copenhagen, Department of Veterinary Disease Biology, Frederiksberg, 1870, Denmark, efu@sund.ku.dk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Eva Fuglsang Louise Henningsen Hanne Frøkiær Google Eva Fuglsang Louise Henningsen Hanne Frøkiær Google Scholar Eva Fuglsang Louise Henningsen Hanne Frøkiær PubMed Eva Fuglsang Louise Henningsen Hanne Frøkiær Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

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

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. 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. 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. 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.

Ceramide–Antiestrogen Nanoliposomal Combinations—Novel Impact of Hormonal Therapy in Hormone-Insensitive Breast Cancer

Although the sphingolipid ceramide exhibits potent tumor suppressor effects, efforts to harness this have been hampered by poor solubility, uptake, bioavailability, and metabolic conversion. Therefore, identification of avenues to improve efficacy is necessary for development of ceramide-based therapies. In this study, we used mutant p53, triple-negative breast cancer (TNBC) cells, a type of breast cancer highly refractory to treatment, and cell-permeable nanoliposomal C6-ceramide in conjunction with the antiestrogen tamoxifen, which has been shown to be an effective modulator of ceramide metabolism. We show for the first time that nanoliposomal tamoxifen enhances nanoliposomal C6-ceramide cytotoxicity in cultured TNBC cells, a response that was accompanied by induction of cell-cycle arrest at G(1) and G(2), caspase-dependent induction of DNA fragmentation, and enhanced mitochondrial and lysosomal membrane permeability at 18 and 2 hours, respectively. Tamoxifen metabolites were also effective. Only tamoxifen promoted lysosomal membrane permeability. In addition, we show for the first time that tamoxifen inhibits acid ceramidase, as measured in intact cell assays; this effect was irreversible. Together, our findings show that tamoxifen magnifies the antiproliferative effects of C6-ceramide via combined targeting of cell-cycle traverse and lysosomal and mitochondrial integrity. We adduce that C6-ceramide-induced apoptosis is amplified by tamoxifen's impact on lysosomes and perhaps accompanying inhibition of acid ceramidase, which could result in decreased levels of sphingosine 1-phosphate. This drug regimen could serve as a promising therapy for chemoresistant and triple-negative types of breast cancer, and thus represents an indication for tamoxifen, irrespective of estrogen receptor status.

Novel amide- and sulfonamide-based aromatic ethanolamines: Effects of various substituents on the inhibition of acid and neutral ceramidases

In the present study we describe the design and synthesis of a series of amide- and sulfonamide-based compounds as inhibitor of recombinant acid and neutral ceramidases. Inhibition of ceramidases has been shown to induce apoptosis and to increase the efficacy of conventional chemotherapy in several cancer models. B-13, lead in vitro inhibitor of acid ceramidase has been recently shown to be virtually inactive towards lysosomal acid ceramidase in living cells at lower concentrations and for a shorter time of treatment, suggesting the development of more potent inhibitors. In this study, a detailed SAR investigation has been performed to understand the effect of different substituents on the phenyl ring of amide- and sulfonamide-based compounds that partially resemble the structure of well-known inhibitors such as B-13, D-e-MAPP as well as NOE. Our results suggest that the electronic effects of the substituents on phenyl ring in B-13 and D-e-MAPP analogues have negligible effects either in enhancing the inhibition potencies or for selectivity towards aCDase over nCDase. However, the hydrophobicity and the steric effects of longer alkyl chains (n-Pr, n-Bu or t-Bu groups) at the phenyl ring were found to be important for an enhanced selectivity towards aCDase over nCDase.

Ceramidases in Hematological Malignancies: Senseless or Neglected Target?

Ceramidases are ubiquitous amidohydrolases that catalyze the cleavage of ceramides into sphingosine and fatty acids. This reaction exerts a cytoprotective role in physiological conditions, while altered ceramidase activities favour a number of human diseases. Among these diseases, several reports point to important roles of ceramidases, mainly the acid ceramidase, in the initiation and progression of cancer, and the response of tumors to radio- or chemotherapy. Multiple reports confirm the interest of acid ceramidase inhibitors as anticancer drugs, either alone or in combination with other therapies. Sphingolipid metabolism plays a role in hematological malignancies and appears as an interesting target for therapeutic intervention. Although the use of ceramidase inhibitors in chemotherapy of hematologic cancers has not been widely investigated, a number of indirect evidence suggest that inhibition of specific ceramidases could potentiate the effect of drugs in clinical use to treat hematologic malignancies and may afford strategies to combat relapses. The arsenal of ceramidase inhibitors so far available is wide and hopefully, upcoming research will assess the feasibility of this approach.

Identification of Cystatin SA as a Novel Inhibitor of Acid Ceramidase

Autoproteolytic cleavage of the inactive acid ceramidase (AC) precursor into the active heterodimer exposes a free cysteine residue, leading us to study whether AC could be regulated by one or more members of the cystatin family. Co-expression of the full-length AC and cystatin SA (cysSA) cDNAs led to significant reduction of AC activity in the transfected cells. Expression of cysSA also inhibited endogenous AC activity in cells and increased ceramide. Conversely, cysSA siRNA expression led to elevated AC activity and reduction in ceramide. The effects of cysSA siRNA expression could be reversed by the addition of recombinant cysSA into the culture media. These results were consistent with detection of a physical interaction between AC and cysSA, assessed by co-immunoprecipitation and nickel-nitrilotriacetic acid affinity chromatography, and further supported by co-localization of the endogenous proteins using confocal microscopy. In vitro kinetic analysis of purified, recombinant AC and cysSA confirmed the transfection results and suggested a non-competitive type of inhibition with a K(i) in the low micromolar range. Processing of the AC precursor into the active form was not affected by cysSA expression, suggesting that it likely inhibits AC by allosteric interference. Computer modeling and expression studies identified several potential inhibitory domains in cysSA, including a small "AC-like" domain (identical to the AC cleavage site, TICT). Small peptides, synthesized with combinations of this and a "cystatin-like" domain (QXVXG), exhibited significant AC inhibition as well. Such peptide-based AC inhibitors could potentially be used to regulate AC activity in cancer cells that are known to overexpress this enzyme alone and in combination with conventional anti-cancer drugs.

C6-Ceramide and targeted inhibition of acid ceramidase induce synergistic decreases in breast cancer cell growth

The sphingolipid ceramide is known to play a central role in chemo- and radiation-induced cell death. Acid ceramidase (AC) hydrolyzes ceramide, and thus reduces intracellular levels of this proapoptotic lipid. The role of AC as a putative anticancer target is supported by reports of upregulation in prostate cancer and in some breast tumors. In this study, we determined whether the introduction of an AC inhibitor would enhance the apoptosis-inducing effects of C6-ceramide (C6-cer) in breast cancer cells. Cultured breast cancer cells were treated with DM102 [(2R,3Z)-N-(1-hydroxyoctadec-3-en-2-yl)pivalamide, C6-cer, or the combination. Cell viability and cytotoxic synergy were assessed. Activation of apoptotic pathways, generation of reactive oxygen species, and mitochondrial transmembrane potential were determined. DM102 was a more effective AC inhibitor than N-oleoylethanolamine (NOE) and (1R,2R)-2-N-(tetradecanoylamino)-1-(4'-nitrophenyl)-1,3-propandiol (B-13) in MDA-MB-231, MCF-7, and BT-474 cells. As single agents, C6-cer (IC(50) 5-10 μM) and DM102 (IC(50) 20 μM) were only moderately cytotoxic in MDA-MB-231, MCF-7, and SK-BR-3 cells. Co-administration, however, produced synergistic decreases in viability (combination index <0.5) in all cell lines. Apoptosis was confirmed in MDA-MB-231 cells by detection of caspase 3 cleavage and a >3-fold increase in caspase 3/7 activation, PARP cleavage, and a >70% increase in Annexin-V positive cells. C6-cer/DM102 increased ROS levels 4-fold in MDA-MB-231 cells, shifted the ratio of Bax:Bcl-2 to >9-fold that of control cells, and resulted in mitochondrial membrane depolarization. DM102 also increased the synthesis of (3)H-palmitate-labeled long-chain ceramides by 2-fold when C6-cer was present. These data support the effectiveness of targeting AC in combination with exogenous short-chain ceramide as an anticancer strategy, and warrant continued investigation into the utility of the C6-cer/DM102 drug duo in human breast cancer.

Abstract 5484: Targeted inhibition of acid ceramidase and C6-ceramide exposure induces synergistic killing in cancer cells

Abstract Introduction. In this study, we evaluated the anticancer activity of DM102 [(2R,3Z)-N-(1-hydroxyoctadec-3-en-2-yl)pivalamide], a novel inhibitor of the enzyme acid ceramidase (AC), a key regulator of ceramide metabolism, in combination with C6-ceramide (C6-cer) in human breast cancer cells. Ceramide, a bioactive sphingolipid and potent enhancer of apoptosis, is known to play a role in chemo- and radiation-induced cell death. AC tempers ceramide-induced apoptosis by hydrolysis, producing sphingosine and free fatty acid, thus reducing intracellular ceramide levels and providing substrate for sphingosine kinase, which converts sphingosine to sphingosine 1-phosphate. AC has emerged as a putative anticancer target following reports of upregulation in prostate cancer and in some breast tumors. In the present work, we determined whether the introduction of an AC inhibitor would enhance the apoptosis-inducing effects of C6-cer in breast cancer cells. Methods. Cultured breast cancer cells were pretreated with 10 μM DM102 before exposure to 2.5-10 μM C6-cer. Cell viability was assessed at 72 hr by 96-well colorimetric assays. Cytotoxic synergy was assessed by the Chou-Talalay method of combination index (CI) determined using CalcuSyn® software. Apoptosis was measured by Annexin V/propidium iodide staining using flow cytometry, and by caspase 3/7 activity. Generation of reactive oxygen species (ROS) was measured by flow cytometry. Autophagy was assessed by Western blot for LC3II activation. Lipid metabolic studies were performed using [3H]palmitic acid. Results. Treatment with DM102 (10 μM) for 24 hr increased levels of 3H-palmitic acid-labeled ceramide by 2-fold, compared to controls, in MDA-MB 231 breast cancer cells. As single agents in viability studies, both C6-cer (IC50 5-10 μM) and DM102 (IC50 20 μM) were only moderately cytotoxic in MDA-MB 231, MCF-7, and SKBr3 cells. Co-administration however, produced synergistic decreases in viability (CI &amp;lt;0.5) in all cell lines, at doses that were half the IC50 of either agent alone. Similar cytotoxic profiles were observed with C6-cer and the AC inhibitor B13, (1R,2R)-2-N-(tetradecanoylamino)-1-(4′-nitrophenyl)-1,3-propiandiol, suggesting that blocking AC activity was key to the synergistic effect. At 24 hr, combination C6-cer/DM102 increased ROS levels &amp;gt; 2-fold in MDA-MB 231 cells. This was accompanied by activation of the autophagy marker, LC3II and a decrease in phospho-Akt, a promoter of cell survival and negative regulator of autophagy. At 48 hr, caspase 3/7 activity increased &amp;gt; 2-fold, and by 72 hr there was a &amp;gt;70% increase in Annexin-V positive cells, compared to controls. Conclusions. These data support the efficacy of targeting AC in combination with exogenous short-chain ceramide as an anticancer strategy, and warrant further investigation into the mechanism of action of the C6 ceramide/DM102 drug duo. (Support: NIGMS77391) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5484. doi:10.1158/1538-7445.AM2011-5484

Inhibition of acid ceramidase by a 2‐substituted aminoethanol amide synergistically sensitizes prostate cancer cells to N‐(4‐hydroxyphenyl) retinamide

The purpose of this study was to determine whether the therapeutic efficacy of fenretinide (4-HPR), a ceramide-generating anticancer agent, could be enhanced in prostate cancer cells by inclusion of a novel synthetic acid ceramidase (AC) inhibitor, DM102, a pivaloylamide of a 2-substituted aminoethanol. In prostate cancer, AC plays a role in progression and resistance to chemotherapy. PC-3 and DU 145 hormone-refractory human prostate cancer cell lines were used. Cells were exposed to 4-HPR, DM102, and combinations; viability, apoptosis, cell migration, ceramide metabolism, and levels of reactive oxygen species (ROS) were assessed. Single agent 4-HPR and DM102 (2.5-10 µM) were weakly cytotoxic; however, combinations synergistically decreased cell viably to as low as 1.5% of control. N-oleoylethanolamine (NOE), a frequently employed AC inhibitor, was not effective in producing synergy. The 4-HPR/DM102 regimen enhanced caspase activity and increased [(3) H](dihydro)ceramide and ROS levels 6- and 30-fold over control, respectively. The antioxidant vitamin E, but not the de novo ceramide synthesis inhibitor myriocin, partially rescued cells from 4-HPR/DM102 cytotoxicity. The 4-HPR/DM102 combination also elicited synergistic cytotoxicity in DU 145 cells, another human hormone-refractory prostate cancer cell line. This study shows that 4-HPR cytotoxicity is enhanced in a synergistic fashion by inclusion of the AC inhibitor DM102, by a mechanism that enlists generation of ROS, and thus provides a system to raise 4-HPR therapeutic potential. The role of ceramide however in the cytotoxic response is not clear, as blocking ceramide generation failed to rescue PC-3 cells from 4-HPR/DM102 cytotoxicity.

Potent Inhibition of Acid Ceramidase by Novel B-13 Analogues

The lipid-signalling molecule ceramide is known to induce apoptosis in a variety of cell types. Inhibition of the lysosomal acid ceramidase can increase cellular ceramide levels and thus induce apoptosis. Indeed, inhibitors of acid ceramidase have been reported to induce cell death and to display potentiating effects to classical radio- or chemo therapy in a number of in vitro and in vivo cancer models. The most potent in vitro inhibitor of acid ceramidase, B-13, recently revealed to be virtually inactive towards lysosomal acid ceramidase in living cells. In contrast, a number of weakly basic B-13 analogues have been shown to accumulate in the acidic compartments of living cells and to efficiently inhibit lysosomal acid ceramidase. However, introduction of weakly basic groups at the ω-position of the fatty acid moiety of B-13 led to a significant reduction of potency towards acid ceramidase from cellular extracts. Herein, we report a novel B-13-derived scaffold for more effective inhibitors of acid ceramidase. Furthermore, we provide hints for an introduction of basic functional groups at an alternative site of the B-13 scaffold that do not interfere with acid ceramidase inhibition in vitro.

Activation of ceramide synthase 6 by celecoxib leads to a selective induction of C 16:0-ceramide

Ceramides serve as bioactive molecules with important roles in cell proliferation and apoptosis. Ceramides (Cer) with different N-acyl side chains (C 14:0-Cer–C 26:0-Cer) possess distinctive roles in cell signaling and are differentially expressed in HCT-116 colon cancer cells. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, exhibiting antiproliferative effects, activates the sphingolipid pathway. To elucidate the mechanism, HCT-116 cells were treated with 50 μM celecoxib leading to a significant increase of C 16:0-Cer. Interestingly, 50 μM celecoxib resulted in a 2.8-fold increase of ceramide synthase (CerS) activity as measured by a cell-based activity assay. siRNA against several CerSs revealed that CerS6 was predominantly responsible for the increase of C 16:0-Cer in HCT-116 cells. Moreover, the silencing of CerS6 partially protected HCT-116 cells from the toxic effects induced by celecoxib. Treatment of cells with celecoxib and fumonisin B1 (inhibitor of CerSs) or myriocin (inhibitor of l-serine palmitoyl transferase) or desipramine (inhibitor of acid sphingomyelinase and acid ceramidase) revealed that the increase of C 16:0-Cer results predominantly from activation of the salvage pathway. Using the nude mouse model we demonstrated that celecoxib induces also in vivo a significant increase of C 16:0-Cer in stomach, small intestine and tumor tissue. In conclusion, celecoxib causes a specific increase of C 16:0-Cer by activating CerS6 and the salvage pathway, which contribute to the toxic effects of celecoxib.

Acid Ceramidase Upregulation in Prostate Cancer Cells Confers Resistance to Radiation: AC Inhibition, a Potential Radiosensitizer

Radiation resistance in a subset of prostate tumors remains a challenge to prostate cancer radiotherapy. The current study on the effects of radiation on prostate cancer cells reveals that radiation programs an unpredicted resistance mechanism by upregulating acid ceramidase (AC). Irradiated cells demonstrated limited changes of ceramide levels while elevating levels of sphingosine and sphingosine-1-phosphate. By genetically downregulating AC with small interfering RNA (siRNA), we observed radiosensitization of cells using clonogenic and cytotoxicity assays. Conversely, AC overexpression further decreased sensitivity to radiation. We also observed that radiation-induced AC upregulation was sufficient to create cross-resistance to chemotherapy as demonstrated by decreased sensitivity to Taxol and C(6) ceramide compared to controls. Lower levels of caspase 3/7 activity were detected in cells pretreated with radiation, also indicating increased resistance. Finally, utilization of the small molecule AC inhibitor, LCL385, sensitized PPC-1 cells to radiation and significantly decreased tumor xenograft growth. These data suggest a new mechanism of cancer cell resistance to radiation, through upregulation of AC that is, in part, mediated by application of the therapy itself. An improved understanding of radiotherapy and the application of combination therapy achieved in this study offer new opportunities for the modulation of radiation effects in the treatment of cancer.

Synthesis and bioevaluation of ω- N-amino analogs of B13

Novel ω- N-amino analogs of B13 (Class E) were designed, synthesized and tested as inhibitors of acid ceramidase (ACDase) and potential anticancer agents deprived of unwanted lysosomal destabilization and ACDase proteolytic degradation properties of LCL204 [Szulc, Z. M.; Mayroo, N.; Bai, A.; Bielawski, J.; Liu, X.; Norris, J. S.; Hannun, Y. A.; Bielawska, A. Bioorg. Med. Chem. 2008, 16, 1015]. Representative analog LCL464, (1 R,2 R)-2- N-(12′- N, N-dimethylaminododecanoyl amino)-1-(4″-nitrophenyl)-1,3-propandiol, inhibited ACDase activity in vitro, with a similar potency as B13 but higher than LCL204. LCL464 caused an early inhibition of this enzyme at a cellular level corresponding to decrease of sphingosine and specific increase of C 14- and C 16-ceramide. LCL464 did not induce lysosomal destabilization nor degradation of ACDase, showed increased cell death demonstrating inherent anticancer activity in a wide range of different cancer cell lines, and induction of apoptosis via executioner caspases activation. LCL464 represents a novel structural lead as chemotherapeutic agent acting via the inhibition of ACDase.

Acid Sphingomyelinase Overexpression Enhances the Antineoplastic Effects of Irradiation In Vitro and In Vivo

Exposure of cells or animals to stress frequently induces acid sphingomyelinase (ASM)-mediated ceramide production that leads to cell death. Consistent with this, overexpression of ASM in subcutaneous B16-F10 mouse melanomas, in combination with irradiation, resulted in tumors that were up to 12-fold smaller than irradiated control melanomas. Similarly, when irradiated melanomas were pretreated with a single, peritumoral injection of recombinant ASM (rhASM), the tumors were up to threefold smaller. The in vivo effect of ASM was likely due to enhanced cell death of the tumor cells themselves, as well as the surrounding microvascular endothelial cells. In vitro, rhASM had little or no effect on the growth of tumor cells, even in combination with irradiation. However, when the culture media was acidified to mimic the acidic microenvironment of solid tumors, rhASM-mediated cell death was markedly enhanced when combined with irradiation. Microscopic analysis suggested that this was associated with an increase in autophagy. rhASM has been produced for the treatment of the lysosomal storage disorder, type B Niemann-Pick disease, and is currently being evaluated in a phase-1 clinical trial. Based on the data presented in this article, we propose that further investigation of this protein and gene as antineoplastic agents also is warranted.

Cytotoxicity and acid ceramidase inhibitory activity of 2-substituted aminoethanol amides

The acid ceramidase (AC) inhibitory activity of octanoylamides, p- tert-butylbenzamides and pivaloylamides of several 2-substituted aminoethanols is reported. All the aminoethanol amides bearing a hexadecyl substituent (C16), as well as ( S)- N-(1-(hexadecylthio)-3-hydroxypropan-2-yl)pivaloylamide (SC16-tb) were inhibitory in cell lysates overexpressing AC, while all other compounds were not inhibitors. Kinetic experiments with ( R, E)- N-(1-hydroxyoctadec-3-en-2-yl)pivaloylamide ( E-tb) and SC16-tb showed that inhibition was competitive, with K i values of 34 and 94.0 μM, respectively. None of the compounds inhibited neutral ceramidase. Compounds E-tb and E-c7 (the octanoylamide of the unsaturated base E), which elicited a dose–response inhibition with IC 50 values around 15 μM, were the only AC inhibitors in intact cells. Both compounds were toxic to A549 cells with LD 50 values nearly 40 μM. Flow cytometry studies with E-tb evidenced that this compound induced a concentration-dependent cell cycle arrest at G(1) and a 20–25% apoptosis/late apoptosis/necrosis after a 24-h incubation at 50 μM. In agreement with its activity as acidic ceramidase inhibitor, this effect was accompanied with an increase in the amounts of C14, C16 and C18 ceramides (LC–MS analyses), which suggested that these lipids may be responsible for the cytotoxic activity of E-tb.

Acid ceramidase inhibition: a novel target for cancer therapy

During the last decade, sphingolipid deregulation, namely the balance between the pro-apoptotic molecule ceramide and the anti-apoptotic sphingolipid sphingosine-1-phosphate, has emerged as an important factor in cancer pathology and resistance to therapy. Thus, our research has been focused on developing drugs that are able to restore normal sphingolipid balance, precisely through increasing the levels of ceramide and decreasing sphingosine-1-phosphate. Particularly, inhibition of the ceramide metabolizing enzyme acid ceramidase, whose over-expression in cancer cells has been implicated in resistance to treatment, is proving to be an efficient and promising strategy. In this review, we consider our recent work with acid ceramidase inhibitors, in combination with radiation or gene therapy as a sensitizer that enhance cancer therapy.