Infections and glycolipids.
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Anti-Bacterial Agents↗
Biomedical subjects
N S Radin
Publications and source records attributed to N S Radin.
Killing cancer cells by poly-drug elevation of ceramide levels: a hypothesis whose time has come?
Many papers have shown that sphingolipids control the balance in cells between growth and proliferation, and cell death by apoptosis. Sphingosine-1-phosphate (Sph1P) and glucosylceramide (GlcCer) induce proliferation processes, and ceramide (Cer), a metabolic intermediate between the two, induces apoptosis. In cancers, the balance seems to have come undone and it should be possible to kill the cells by enhancing the processes that lead to ceramide accumulation. The two control systems are intertwined, modulated by a variety of agents affecting the activities of the enzymes in Cer-GlcCer-Sph1P interdependence. It is proposed that successful cancer chemotherapy requires the use of many agents to elevate ceramide levels adequately. This review updates current knowledge of sphingolipid metabolism and some of the evidence showing that ceramide plays a causal role in apoptosis induction, as well as a chemotherapeutic agent.
Antineoplastic Agents↗
Apoptotic death by ceramide: will the real killer please stand up?
Many studies have shown that the addition of ceramide to an incubation medium, or procedures that lead to increased ceramide concentrations, can begin a process that leads to slowing of cell growth or apoptotic cell death. Only a few studies have examined the nature of the accumulating ceramide: is it composed of a fatty acid and sphinganine, or a fatty acid and sphingosine? Of the studies involving addition of ceramide to a cell culture, almost all have found that the sphingosine amide is active, not the sphinganine amide. Nearly all of these studies have utilized the rare form of ceramide, containing an acetyl group rather than the commonly found palmitoyl, stearoyl, or longer group. Acetyl sphingosine produces some unexpected effects with cells, the most striking being the formation of reactive oxygen species and mitochondrial damage. This mitochondrial damage appears to be an essential step in apoptosis. The possibility should be considered that the reactive oxygen species appear as the result of oxidation of the allylic alcohol group in unsaturated ceramides. This question is very relevant to a host of ceramide functions, particularly cancer chemotherapy and cell growth.
Apoptosis↗
Treating glucosphingolipid disorders by chemotherapy: use of approved drugs and over-the-counter remedies.
The accumulation of a glucosphingolipid (GSL) in individuals lacking an adequate level of hydrolase activity could be minimized by chemotherapeutic measures that slow the formation of the GSL and stimulate the defective hydrolase. By achieving a balance in the rates of formation and breakdown, one should be able to alleviate the symptoms of excess storage and achieve a satisfactory accommodation. While several drugs seem to be specifically suitable for this purpose, only one of these has been approved for human use. However, less effective drugs and over-the-counter substances are available for human use and may prove satisfactory for a few years until better ones are made available. The proposed materials and the evidence behind the recommendations are presented in this paper.
Animals↗
Chemotherapy by slowing glucosphingolipid synthesis.
The hypothesis offered here is that many different illnesses could be treated by slowing the synthesis of glucosphingolipids (GSLs) with a suitable inhibitor. In people with inadequate hydrolases for the GSLs (e.g. Gaucher's disease), the lipids accumulate to a pathological degree. It should be possible to eliminate the accumulation by slowing the synthesis of the GSLs to match the ability of the patient to degrade them. In people with cancer, the tumors secrete excessive amounts of GSLs, which block the ability of the immune system to attack the tumor cells. By blocking the synthesis of tumor GSLs, it should be possible to enable the patient to generate antibodies and activated T cells that can destroy the tumor. Tumors exhibiting multidrug resistance may do so by synthesizing GSLs even faster than usual. It should be possible to restore the sensitivity of the tumor to anti-cancer drugs by inhibiting their synthesis of GSLs. Metastasis of tumors also appears to require the formation of GSLs, so an inhibitor should help block tumor dissemination. Diabetics tend to have high levels of blood glucose, which acts to stimulate kidney growth via more rapid synthesis of GSLs. This pathological growth can be blocked by inhibiting the formation of kidney GSLs. Viruses, bacteria, and bacterial toxins have been found to bind to specific GSLs of human and animal cells. Presumably, this binding leads to the damaging process of infection. It should be possible to treat such infections by depleting the host's body of its GSLs.
Animals↗
Effects of the glucolipid synthase inhibitor, P4, on functional and phenotypic parameters of murine myeloma cells.
This study describes the effects of the glucolipid synthase inhibitor P4, (DL-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol ), on various functional and phenotypic parameters of 5T33 murine myeloma cells. Cell recovery was reduced by >85% following incubation of the cells for 3 days in the presence of 4 microM P4 (the IC50 concentration). Both cytostatic and cytotoxic inhibition was observed with tumour cell metabolic activity and clonogenic potential reduced to 42% and 14% of controls, respectively, and viability reduced to 52%. A dose-dependent increase in cells undergoing apoptosis (from 7% to 26%) was also found. P4 induced a decrease in the number of cells expressing H-2 Class I and CD44, and a large increase in cells expressing H-2 Class II and the IgG2b paraprotein. It did not affect surface expression of CD45 or CD54 (ICAM-1). Based on these alterations in tumour cell growth, adhesion molecule expression and potential immunogenicity, it is anticipated that P4 will provide a novel therapeutic approach for the treatment of multiple myeloma. In addition, given that essentially all tumours rely heavily on overexpressed or abnormal glucosphingolipids for growth, development and metastasis, glucolipid synthase inhibitors may prove to be universally effective anti-cancer agents.
Animals↗
Effect of an inhibitor of glucosylceramide synthesis on cultured human keratinocytes.
Glucosylceramide (GlcCer) is a major glycosphingolipid component of epidermis, which is thought to be related to the barrier function of skin permeability. However, the role of glycosphingolipids in keratinocyte growth and differentiation has not been fully clarified. It has been reported that D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of GlcCer synthase (EC 2.4.1.80), depletes cells of glycosphingolipids. This inhibitor has been used as a tool for elucidating their functions. In the present study, the effect of PDMP on cultured normal human keratinocytes was investigated. The cells were treated with various concentrations of PDMP. Forty-eight hours later cell growth, thymidine incorporation, and lipid content were studied. The cell growth and the incorporation of thymidine into cells were inhibited by PDMP in a dose dependent manner. The synthesis of GlcCer was strongly inhibited by PDMP treatment, whereas no significant changes in ceramide level were observed. We concluded that GlcCer in epidermis may play an important role in regulating epidermal growth and suggested that PDMP may be beneficial for treating proliferative skin disorders in the future.
Adult↗
A novel enzyme that catalyzes the esterification of N-acetylsphingosine. Metabolism of C2-ceramides.
A unique transacylase that catalyzes esterification of a short chain ceramide, N-acetylsphingosine, was found in Madin-Darby canine kidney cell and mouse tissue homogenates. It esterified the hydroxyl group at the carbon-1 position of the ceramide. The enzyme has a pH optimum of 4.2 and a Km of 9.4 microM for N-acetylsphingosine at pH 4.5. The transacylase activity is independent of free fatty acid or acyl-CoA and instead uses the 2-acyl group of phosphatidylethanolamine or phosphatidylcholine. The transacylase activity in the homogenate was present in the 100,000 x g supernatant, and the lipid extracted from the membranous fraction could function as a donor of the acyl group. When liposomes consisting of dioleoylphosphatidylcholine:1-palmitoyl-2-[14C]arachidonoyl-phosphati dylethanolamine:sulfatide (70:0.2:30) were incubated with the supernatant and N-acetylsphingosine, the formation of free arachidonic acid and O-arachidonoyl-N-acetylsphingosine was observed. The ratio of the two products depended on the concentration of ceramide; only the free acid was formed if the truncated ceramide was absent. Both deacylase and transacylase activities were inhibited 50-60% by 20 microM D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of several glucosphingolipid synthases. Neither activity was inhibited by nonadecyltetraenyl trifluoromethyl ketone, a potent inhibitor of cytosolic phospholipase A2. N-Acetyldihydrosphingosine and N-octanoylsphingosine were only 55 and 10%, respectively, as effective as N-acetylsphingosine as acyl acceptors. Oleoylsphingosine was only slightly reactive. An esterase that releases the truncated ceramide from its ester linkage appears to be membrane bound. Lecithin was less effective than phosphatidylethanolamine as an acyl donor in the transacylation. Madin-Darby canine kidney cell cultures treated with N-acetyl-[3-3H]sphingosine formed radioactive polar sphingolipids, long chain ceramide, free sphingosine, and O-acyl-N-acetylsphingosine. This suggests that the deacylation and transacylation reactions observed in vitro occur in growing cells as well.
Acyltransferases↗
Induction of glucosylceramide synthase by synthase inhibitors and ceramide.
Glucosylceramide (GlcCer) synthase acts on the sphingolipid, ceramide, to transer a glucose moiety from UDP-glc, thus forming the first member of a large family of glucosphingolipids. Two inhibitors of the enzyme, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) and N-butyldeoxynojirimycin (NBDN), have been found to induce an elevated level of the synthase in MDCK cells. In cells treated with 20 muM PDMP, then assayed for synthase activity under conditions in which the absorbed PDMP was partially diluted out, the assay showed that the enzyme's specific activity had risen considerably in only 1 h and reached a maximum of about three times the control activity within 6 h. Both cycloheximide and actinomycin D, inhibitors of translational and transcriptional protein synthesis, caused much of the synthase activity to disappear in 6 h, presumably because of normal catabolic destruction. However, simultaneous inclusion of PDMP or NBDN in the cell medium slowed the rate of synthase disappearance. L-Cycloserine, which blocked the synthesis of ceramide, nevertheless allowed PDMP to elevate the synthase activity. Thus the inductive effect appears to be due, in part at least, to resistance of the enzyme-inhibitor complex to the normal process of enzyme degradation. Two other inhibitors of GlcCer synthase, more active than PDMP, did not produce detectable induction because they could not be dissociated from the enzyme during the cell washing and diluting steps. Agents that produced a large increase in endogenous cell ceramide level (DL-erythro-PDMP,N-acetylsphingosine, and bacterial sphingomyelinase) also induced an elevated level of GlcCer synthase. The latter two agents did not protect the synthase from catabolism in the presence of cycloheximide. These findings suggest the existence of a second mechanism of enzyme induction, enhanced synthesis of the enzyme due to the increased availability of the enzyme's lipoidal substrate. The possibility is raised that events involving ceramide in cell signalling may be mediated in part by changes in glucosphingolipid levels.
1-Deoxynojirimycin↗
Studies of the action of ceramide-like substances (D- and L-PDMP) on sphingolipid glycosyltransferases and purified lactosylceramide synthase.
We have studied the effects of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and its L-enantiomer on glycosphingolipids in cultured normal human kidney proximal tubular cells. We found that D-PDMP exerted a concentration-dependent reduction in the metabolic labelling and cellular levels of glucosylceramide (GlcCer), lactosylceramide (LacCer), and the globo-series glycosphingolipids, GbOSe3Cer and GbOse4Cer. It also directly inhibited the activity of UDP-glucose:ceramide beta 1--> 4-glucosyltransferase (GlcT-1) and UDP-galactose: GlcCer beta 1-->4 galactosyltransferase (GalT-2). In contrast, L-PDMP had opposite effects on the metabolic labelling of GlcCer, LacCer, and GbOse3Cer. The levels of GlcCer and LacCer were increased, while the labelling and level of GbOse4Cer were strongly reduced. Purified GalT-2 from human kidney was inhibited by D-PDMP and stimulated by L-PDMP. It appears likely that the different glycosphingolipid glycosyltransferases possess similar binding sites for the ceramide moiety, which are blocked by binding to D-PDMP and, in the case of GbOse4Cer synthase, by L-PDMP as well. The stimulatory effects of L-PDMP on GlcCer and LacCer synthases may be the result of binding to a modulatory site on the glycosyltransferases; in intact cells, the enzyme-analog complex may afford protection against the normal catabolic inactivation of the enzymes.
Carbohydrate Conformation↗
Treatment of Gaucher disease with an enzyme inhibitor.
The hypothesis is offered predicting that Gaucher patients could be treated with a drug that slows the synthesis of glucosylceramide, the lipid that accumulates in this disorder. The present therapeutic approach involves augmenting the defective enzyme, glucosylceramide beta-glucosidase, with exogenous beta-glucosidase isolated from human tissue. This spectacularly expensive mode of treatment should be replaceable with a suitable enzyme inhibitor that simply slows formation of the lipid and matches the rate of synthesis with the rate of the defective, slowly working beta-glucosidase. Several drugs that possess this ability are available, the best known of which is 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a designer inhibitor that resembles the synthase's substrate and product. PDMP has been found to be effective in mice, rats, fish, and a wide variety of cultured cells. Its use, at suitable dosages, seems to be harmless, although long-term tests have not been made. The lack of suitable animal models of Gaucher disease has made it difficult to test the hypothesis adequately, but PDMP does rapidly lower the levels of glucosylceramide in normal animal tissues and the animals evidently do well with the lowered levels of glucosylceramide and its more complex glycolipid metabolites.
Animals↗
Glycosphingolipid synthesis and proliferation in a renal cell line grown in high glucose.
We evaluated the role of sphingolipids as potential mediators of the renal epithelial growth response to growth in high-glucose media. The mouse cortical tubule (MCT) cell line was studied under high-glucose (450 mg/dl) and normal glucose (100 mg/dl) conditions. In cells plated at low-density, high-glucose media stimulated cell proliferation as measured by DNA, protein, and cell number and [3H]thymidine incorporation with a corresponding increase in glucosylceramide (GlcCer). The GlcCer synthase inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), blocked the proliferative response to high glucose in association with a decrease in endogenous GlcCer and an increase in ceramide concentrations. Addition of N-acetylsphingosine, a short-chain homologue of natural ceramides, increased the levels of endogenous ceramides and inhibited proliferation. In contrast, the beta-glucosidase inhibitor conduritol B epoxide resulted in increased cell GlcCer but did not increase proliferation. We conclude that MCT cells proliferate when grown in the presence of high glucose. GlcCer levels increase and ceramide levels decrease in concert with the proliferative response. Pharmacologically increasing endogenous levels of ceramide inhibits the proliferative response to high glucose, but increasing endogenous levels of GlcCer does not stimulate proliferation.
Animals↗
Cell cycle arrest induced by an inhibitor of glucosylceramide synthase. Correlation with cyclin-dependent kinases.
In an attempt to define the basis for sphingolipid regulation of cell proliferation, we studied the effects of glucosylceramide (GlcCer) synthase inhibition by threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on NIH 3T3 cells overexpressing insulin-like growth factor-1 (IGF-1) receptor. PDMP treatment resulted in a time-dependent decrease in GlcCer levels and an increase in cellular ceramide levels. PDMP abolished serum and IGF-1-stimulated cell proliferation, as measured by a reduction in [3H]thymidine incorporation, protein, and DNA levels. However it did not affect IGF-1-mediated early signaling events, including receptor tyrosine kinase, MAP kinase, and phosphatidylinositol 3-kinase activities. Two-color flow cytometry with propidium iodide and 5-bromo-2'-deoxyuridine monophosphate labeling revealed an arrest of the cell cycle at G1/S and G2/M transitions in an asynchronous population of cells. These changes were time dependent, with maximal effects seen by 12-24 h. Removal of PDMP from the cell medium resulted in reversal of the cell cycle changes, with cells re-entering the S phase. The cell cycle arrest at the G1/S and G2/M transitions was confirmed in cells synchronized by pretreatment with nocodazole, aphidicolin, or hydroxyurea, and released from blockade in the presence of PDMP. A decrease in the activities of two cyclin-dependent kinases, p34cdc2 kinase and cdk2 kinase, was observed with PDMP treatment. When cell ceramide levels were increased by N-acetylsphingosine, comparable changes in the cell cycle distribution were seen. However, sphingomyelinase treatment was without effect. Therefore, it appears that ceramide mediates in part the inhibitory effect of GlcCer synthase inhibition on IGF-1-induced cell proliferation in 3T3 cells. The rapid production of decreased cyclin-dependent kinase activities by PDMP suggests that one of the crucial sites of action of the inhibitor lies in this area.
3T3 Cells↗
Structural and stereochemical studies of potent inhibitors of glucosylceramide synthase and tumor cell growth.
Analogs and homologs of PDMP were synthesized, based on its structure (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol). This compound had previously been found to block the synthesis of GlcCer (glucosylceramide). Increasing the acyl chain length from 10 to 16 carbon atoms greatly enhanced the efficacy of the enzyme inhibitor, as did the use of a less polar cyclic amine, especially a pyrrolidine instead of a morpholine ring. Replacement of the phenyl ring by a chain corresponding to sphingosine also yielded a strongly inhibitory material. By using a chiral synthetic route, we showed that the isomers active against GlcCer synthase had the R,R-(D-threo)-configuration. However, strong inhibition of the growth of human cancer cells in plastico was produced by both the threo and erythro racemic compounds, showing involvement of an additional factor (beyond simple depletion of cell glycosphingolipids by blockage of GlcCer synthesis). The growth arresting effects could be correlated with increases in cellular ceramide and diglyceride levels. The aliphatic pyrrolidino compound was strongly inhibitory toward the glucosyltransferase and produced almost complete depletion of glycolipids, but did not inhibit growth or cause an accumulation of ceramide. Attempts were made to see whether the differences in growth effects could be attributed to the influence of the inhibitors on related enzymes (ceramide and sphingomyelin synthase and ceramidase and sphingomyelinase). While some stimulation of enzyme activity was noted, particularly at high inhibitor concentrations (50 microM), these findings did not explain the differing effects of the different inhibitors. The best inhibitors of GlcCer synthase compared favorably in efficacy with some cancer chemotherapeutic drugs in current use when tested with a battery of human cancer cells.
Animals↗
Glucosylceramide in the nervous system--a mini-review.
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Animals↗
Epithelial glucosphingolipid expression as a determinant of bacterial adherence and cytokine production.
D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) is a structural analog of ceramide that inhibits glucosylation of this molecule and thus of glucosphingolipid (GSL) expression by living cells. In this study, we used PDMP to slow the synthesis of the globoseries of GSLs (globo-GSLs) (derived from the precursor Gal alpha 1-4Gal beta 1-4Glc-ceramide) by cultured human kidney and large intestinal epithelial cells. The aim was to deplete the cells of receptors for P-fimbriated Escherichia coli and to examine the effects on the bacterially induced cytokine response. The mammalian cells (A498, HT-29, and Caco2) were cultured in the presence of PDMP in order to deplete them of GSLs. The cells were then subjected to GSL analysis or used to test bacterial adherence and cytokine production. The globo-GSLs were identified by thin-layer chromatography. Bacterial adherence was quantitated by microscopy, and interleukin-6 secretion was quantitated by the B9 bioassay. The interaction of bacteria with the globo-GSLs was studied by using E. coli strains and recombinant clones expressing P fimbriae. E. coli strains expressing type 1 fimbriae binding to mannose-containing glycoproteins were used as controls. PDMP treatment was found to reduce the content of the globo-GSLs in mammalian cells and the adherence of P-fimbriated E. coli to these cells. In contrast, PDMP treatment had no effect on the adherence of type 1-fimbriated E. coli or their activation of cytokine production by A498 cells. P-fimbriated E. coli elicited an interleukin-6 response in the A498 cells; this response was reduced after treatment with PDMP. The results emphasize the role of GSLs as receptors for P-fimbriated E. coli and for the cytokine response elicited by attaching bacteria.
Bacterial Adhesion↗
Abnormalities of glycosphingolipid, sulfatide, and ceramide in the polycystic (cpk/cpk) mouse.
Polycystic kidney disease is a disorder marked by aberrant renal tubular epithelial cell proliferation and transport abnormalities. Sphingolipids are ubiquitous membrane components implicated in several cellular functions including cell membrane sorting, signaling, growth, ion transport, and adhesion. To investigate a potential pathogenic role for sphingolipids in cystic kidney disease, we studied the sphingolipid content and associated enzymatic activities of the kidneys from cpk/cpk mice and their phenotypically normal litter mates. The neutral glycolipids, including glucosylceramide and lactosylceramide, displayed a striking increase in 3-week-old cpk/cpk mice as did the acidic lipid, ganglioside GM3. However, a correspondingly significant decrease in sulfoglycolipid and ceramide concentration was observed in the cpk/cpk kidneys. Glucosylceramide synthase activity was higher in the kidneys of the cpk/cpk mice than in those of the controls. Kinetic analysis of the glucosylceramide synthase revealed the presence of an endogenous activator in the cystic kidney. A marked decrease in sulfotransferase activity was observed in both whole kidney homogenates and in microsomal preparations that was consistent with the decrement in sulfolipid content. The increase in GM3, glucosyl- and lactosylceramide may therefore be the result of impaired sulfolipid synthesis at the 3-week time point. While sulfolipid and glucosylceramide concentrations are not different at 1 and 2 weeks of age, ceramide concentrations in cystic kidneys are significantly reduced compared to kidneys from phenotypically normal mice. These results suggest that sphingolipids may play a potential role in the proliferative and transport abnormalities associated with cystic renal disease and the development of azotemia.
Animals↗
A role for glycosphingolipid accumulation in the renal hypertrophy of streptozotocin-induced diabetes mellitus.
Glucosylceramide (GlcCer) and related glycosphingolipids have been implicated as causal elements in both the growth of cells and in the regulation of hormonal signaling. We therefore studied whether the renal hypertrophy induced by diabetes was associated with enhanced synthesis of glycosphingolipids. 16 d after the induction of diabetes, increases in renal size and concentration of glucocerebroside and ganglioside GM3 were observed paralleling an increase in UDP-Glc concentration. GlcCer synthase and beta-glucosidase-specific activities were no different between control and diabetic kidneys. The apparent Km of the GlcCer synthase with respect to UDP-Glc was 250 microM and was unchanged in the diabetic kidneys. The observed concentrations of UDP-Glc were 149 and 237 microM in control and diabetic kidneys, respectively. The UDP-Glc level is thus rate limiting with regard to GlcCer synthesis. To determine whether the changes in glycolipid content were functionally significant, diabetic and control groups were treated with the GlcCer synthase inhibitor, D-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1- propanol, 2 wk after the induction of diabetes. Kidney weights in the diabetic rats treated with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol were no different than the control groups. Morphometric analysis of glomerular volumes paralleled changes in renal growth. Glycosphingolipid formation may therefore represent a significant pathway for glucose utilization in early diabetic nephropathy.
Animals↗