Ceramide: From Embryos to Tumors

Savtchouk, Iaroslav; Mattie, Floyd J.; Ollis, Anne A.

doi:10.1126/stke.3942007jc1

Cited by 11 publications

(6 citation statements)

References 19 publications

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“…Thus, inhibition of AC might restore the sensitivity of cancer cells to exogenous ceramides or treatment with chemotherapeutic agents known to produce ceramide (24). Current techniques for AC inhibition are based on using ceramide analogs as inhibitors or anti-AC small interfering RNA.…”

Section: Discussionmentioning

confidence: 99%

Autoproteolytic Cleavage and Activation of Human Acid Ceramidase

Shtraizent

Eliyahu

Park

et al. 2008

Journal of Biological Chemistry

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Herein we report the mechanism of human acid ceramidase (AC; N-acylsphingosine deacylase) cleavage and activation. A highly purified, recombinant human AC precursor underwent self-cleavage into ␣ and ␤ subunits, similar to other members of the N-terminal nucleophile hydrolase superfamily. This reaction proceeded with first order kinetics, characteristic of selfcleavage. AC self-cleavage occurred most rapidly at acidic pH, but also at neutral pH. Site-directed mutagenesis and expression studies demonstrated that Cys-143 was an essential nucleophile that was required at the cleavage site. Other amino acids participating in AC cleavage included Arg-159 and Asp-162. Mutations at these three amino acids prevented AC cleavage and activity, the latter assessed using BODIPY-conjugated ceramide. We propose the following mechanism for AC self-cleavage and activation. Asp-162 likely forms a hydrogen bond with Cys-143, initiating a conformational change that allows Arg-159 to act as a proton acceptor. This, in turn, facilitates an intermediate thioether bond between Cys-143 and Ile-142, the site of AC cleavage. Hydrolysis of this bond is catalyzed by water. Treatment of recombinant AC with the cysteine protease inhibitor, methyl methanethiosulfonate, inhibited both cleavage and enzymatic activity, further indicating that cysteine-mediated self-cleavage is required for ceramide hydrolysis. Human acid ceramidase (AC;3 N-acylsphingosine deacylase; EC 3.5.1.23) hydrolyzes the sphingolipid, ceramide, into sphingosine and free fatty acid. AC is considered a lysosomal enzyme since it has optimal in vitro activity at acidic pH, and most of the lipid storage in Farber disease patients (the genetic disorder resulting from the deficiency of this enzyme) occurs within late endosomes and/or lysosomes (1). It is therefore thought that the main function of AC is to participate in lysosomal membrane turnover. A low level, secreted form of AC also has been described (2-4), although its biological function remains unknown.In addition to its important housekeeping function in sphingolipid metabolism, AC participates in signal transduction pathways that regulate various physiological and pathological processes. Recently, it was shown that the AC gene (Asah1) is among the first genes expressed in newly formed mouse embryos, and its deficiency causes embryo death at the two-cell stage (5). In addition, many studies have reported the involvement of AC in complex diseases. For example, AC is overexpressed in several types of human cancer (prostate, head and neck squamous cell, etc.) (6, 7), and cancer therapy based on the inhibition of AC activity has recently been proposed (8, 9). Moreover, impaired ceramide metabolism has been implicated in the pathogenesis of diabetes, Alzheimer disease, atherosclerosis, thrombosis, and cardiomyocyte apoptosis (10 -14). Although the precise role of AC in these common diseases is unknown, the enzyme is likely to act as a rheostat controlling the levels of ceramide, sphingosine, and sphingosine-1-phosphate in cel...

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Section: Discussionmentioning

confidence: 99%

Autoproteolytic Cleavage and Activation of Human Acid Ceramidase

Shtraizent

Eliyahu

Park

et al. 2008

Journal of Biological Chemistry

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“…Ceramide may also form membrane channels for proteins like cytochrome C, and stabilize membrane rafts (42). The early embryo is resistant to apoptosis, owing to low levels of ceramide (43,44); however, there has been no measurement of ceramide during fertilization.…”

Section: Lipid Signaling Related To Fertilizationmentioning

confidence: 99%

“…The early embryo is resistant to ceramide-induced apoptosis (42), so ceramide presumably would have functions other than induction of apoptosis. Ceramide stabilizes membrane rafts (79) and alters PKC activity (59,80).…”

Section: Sm and Ceramidementioning

confidence: 99%

Lipid levels in sperm, eggs, and during fertilization in Xenopus laevis

Petcoff

Holland

Stith

2008

Journal of Lipid Research

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Critical developmental periods, such as fertilization, involve metabolic activation, membrane fusion events such as sperm-egg or plasma membrane-cortical granule merger, and production and hydrolysis of phospholipids. However, there has been no large-scale quantification of phospholipid changes during fertilization. Using an enzymatic assay, traditional FA analysis by TLC and gas chromatography, along with a new method of phospholipid measurement involving HPLC separation and evaporative light-scattering detection, we report lipid levels in eggs, sperm, and during fertilization in Xenopus laevis. Sperm were found to contain different amounts of phospholipids as compared with eggs. During fertilization, total phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, and phosphatidylserine decreased, and ceramide increased, whereas there was no change in phosphatidylcholine, cardiolipin, or phosphatidylethanolamine. FA analysis of phospholipids found numerous changes during fertilization. Because there is an increase in sn-1,2-diacylglycerol at fertilization, the FAs associated with this increase and the source of the increase in this neutral lipid were examined. Finally, activation of phospholipase C, phospholipase D, phospholipase A2, autotoxin, and sphingomyelinase at fertilization is discussed. Major goals of the LIPID Metabolites and Pathways Strategy (LIPID MAPS) are to "separate and detect all of the lipids in a specific cell," "to quantitate each of the lipid metabolites present and determine the changes in their levels," and to study lipid metabolic pathways during biological events (1). Characterizing changes in the lipid composition during defined biological events is part of the concept of "functional lipidomics" (2). These fundamental, descriptive studies will facilitate our understanding of how lipids regulate membrane fluidity and fusion, lipid rafts, the cytoskeleton, kinases, and protein binding to membranes (e.g., 3-5) to induce pleiotropic cellular effects.About 15 years ago, quantification of lipid mass changes during maturation of the oocyte to the fertilizable egg, and during fertilization of the egg by sperm, was begun in Xenopus laevis (6-9). Critical developmental events can be isolated in vitro with this model system, and large numbers of gametes can be obtained to provide sufficient amounts of second messengers and lipids. During fertilization, membrane bending and fusion are associated with the acrosome reaction of sperm (wherein the large sperm acrosome undergoes exocytosis upon interaction with layers that cover the egg), with sperm-egg merger, and with subsequent fertilization events such as cortical granule exocytosis (10, 11). Furthermore, both Xenopus and human eggs are arrested in meiosis II (12).Compared with oocytes, one disadvantage of studying eggs is that they have a short life, much less than the estimated time for lipid precursor incorporation to near equilibrium levels (6, 7). Thus, lipid analysis based on label incorporation or turnover is not reliable, and ...

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“…Ceramide, the central molecule of sphingolipid metabolism, generally mediates anti-proliferative responses, such as inhibition of cell growth, induction of apoptosis and/or modulation of senescence (Saddoughi et al , 2008). Several therapeutic agents that induce ceramide-dependent apoptosis in cancerous cells currently exist, and a number of enzymes involved in ceramide metabolism are beginning to be recognised as potential targets for cancer therapy (Savtchouk et al , 2007; Carpinteiro et al , 2008). Recent research has shown that cannabinoids induce ceramide accumulation in cancer cells, which is related to the pro-apoptotic effect of cannabinoids in cancer cells (Guzman et al , 2001; Velasco et al , 2005).…”

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confidence: 99%

Inhibition of human tumour prostate PC-3 cell growth by cannabinoids R(+)-Methanandamide and JWH-015: Involvement of CB2

et al. 2009

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Background:We have previously shown that cannabinoids induce growth inhibition and apoptosis in prostate cancer PC-3 cells, which express high levels of cannabinoid receptor types 1 and 2 (CB1 and CB2). In this study, we investigated the role of CB2 receptor in the anti-proliferative action of cannabinoids and the signal transduction triggered by receptor ligation.Methods:The human prostate cancer cell lines, namely PC-3, DU-145 and LNCaP, were used for this study. Cell proliferation was measured using MTT proliferation assay, [3H]-thymidine incorporation assay and cell-cycle study by flow cytometry. Ceramide quantification was performed using the DAG kinase method. The CB2 receptor was silenced with specific small interfering RNA, and was blocked pharmacologically with SR 144528. In vivo studies were conducted by the induction of prostate xenograft tumours in nude mice.Results:We found that the anandamide analogue, R(+)-Methanandamide (MET), as well as JWH-015, a synthetic CB2 agonist, exerted anti-proliferative effects in PC-3 cells. R(+)-Methanandamide- and JWH-015-induced cell death was rescued by treatment with the CB2 receptor antagonist, SR 144528. Downregulation of CB2 expression reversed the effects of JWH-015, confirming the involvement of CB2 in the pro-apoptotic effect of cannabinoids. Further analysing the mechanism of JWH-015-induced cell growth inhibition, we found that JWH-015 triggered a de novo synthesis of ceramide, which was involved in cannabinoid-induced cell death, insofar as blocking ceramide synthesis with Fumonisin B1 reduced cell death. Signalling pathways activated by JWH-015 included JNK (c-Jun N-terminal kinase) activation and Akt inhibition. In vivo treatment with JWH-015 caused a significant reduction in tumour growth in mice.Conclusions:This study defines the involvement of CB2-mediated signalling in the in vivo and in vitro growth inhibition of prostate cancer cells and suggests that CB2 agonists have potential therapeutic interest and deserve to be explored in the management of prostate cancer.

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Ceramide: From Embryos to Tumors

Cited by 11 publications

References 19 publications

Autoproteolytic Cleavage and Activation of Human Acid Ceramidase

Autoproteolytic Cleavage and Activation of Human Acid Ceramidase

Lipid levels in sperm, eggs, and during fertilization in Xenopus laevis

Inhibition of human tumour prostate PC-3 cell growth by cannabinoids R(+)-Methanandamide and JWH-015: Involvement of CB2

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