Preferential killing of multidrug-resistant KB cells by inhibitors of glucosylceramide synthase

This study has compared the preferential killing of three multidrug-resistant (MDR) KB cell lines, KB-C1, KB-A1 and KB-V1 by two inhibitors of glucosylceramide synthase, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP), to the killing produced by these compounds in the drug-sensitive cell line, KB-3-1. Both of the inhibitors caused much greater induction of apoptosis in each of the three MDR cell lines than in the drug-sensitive cell line, as judged by morphological assay and confirmed by poly-(ADP-ribose)-polymerase cleavage. The highest level of apoptosis was produced following 24-h exposure to 5 μM PPPP. This treatment produced 75.8 (± 7.1)%, 73.6 (± 9.8)% and 75.3 (± 6.4)% apoptotic cells in the three MDR cell lines respectively, compared to 19.0 (± 9.8)% in the drug-sensitive cell line. A reduction in glucosylceramide level following inhibitor treatment occurred in KB-3-1 cells as well as in the MDR cell lines, suggesting that the increased apoptotic response in the MDR cells reflected a different downstream response to changes in the levels of this lipid in these cells compared to that in the drug-sensitive cells. These results suggest that the manipulation of glucosylceramide levels may be a fruitful way of causing the preferential killing of MDR cells in vitro and possibly in vivo. © 1999 Cancer Research Campaig

HIF-driven SF3B1 induces KHK-C to enforce fructolysis and heart disease.

Fructose is a major component of dietary sugar and its overconsumption exacerbates key pathological features of metabolic syndrome. The central fructose-metabolising enzyme is ketohexokinase (KHK), which exists in two isoforms: KHK-A and KHK-C, generated through mutually exclusive alternative splicing of KHK pre-mRNAs. KHK-C displays superior affinity for fructose compared with KHK-A and is produced primarily in the liver, thus restricting fructose metabolism almost exclusively to this organ. Here we show that myocardial hypoxia actuates fructose metabolism in human and mouse models of pathological cardiac hypertrophy through hypoxia-inducible factor 1α (HIF1α) activation of SF3B1 and SF3B1-mediated splice switching of KHK-A to KHK-C. Heart-specific depletion of SF3B1 or genetic ablation of Khk, but not Khk-A alone, in mice, suppresses pathological stress-induced fructose metabolism, growth and contractile dysfunction, thus defining signalling components and molecular underpinnings of a fructose metabolism regulatory system crucial for pathological growth

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 μM 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. © 1999 Cancer Research Campaig

Treatment of Gaucher disease with an enzyme inhibitor

The hypothesis is offered predicting that Caucher 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 β-glucosidase, with exogenous β-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 β-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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45689/1/10719_2004_Article_BF00731489.pd

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, GbOse 3 Cer and GbOse 4 Cer. It also directly inhibited the activity of UDP-glucose:ceramide β1 → 4-glucosyltransferase (GlcT-1) and UDP-galactose: GlcCer β1 → 4 galactosyltransferase (GalT-2). In contrast, L -PDMP had opposite effects on the metabolic labelling of GlcCer, LacCer, and GbOse 3 Cer. The levels of GlcCer and LacCer were increased, while the labelling and level of GbOse 4 Cer 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 GbOse 4 Cer 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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45706/1/10719_2004_Article_BF00731481.pd

Mitochondrially targeted ceramide LCL-30 inhibits colorectal cancer in mice

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 reduced the proliferative activity and the growth rate of established tumours. Sphingolipid profiles in tumour tissue also showed increased levels of S1P. In summary, we present the first in vivo application of a long-chain pyridinium ceramide for the treatment of experimental metastatic colorectal cancer, together with its pharmacokinetic parameters. LCL-30 was an efficacious and safe agent. Future studies should identify an improved application route and effective partners for combination treatment

CD40L induces multidrug resistance to apoptosis in breast carcinoma and lymphoma cells through caspase independent and dependent pathways

BACKGROUND: CD40L was found to reduce doxorubicin-induced apoptosis in non Hodgkin's lymphoma cell lines through caspase-3 dependent mechanism. Whether this represents a general mechanism for other tumor types is unknown. METHODS: The resistance induced by CD40L against apoptosis induced by a panel of cytotoxic chemotherapeutic drugs in non Hodgkin's lymphoma and breast carcinoma cell lines was investigated. RESULTS: Doxorubicin, cisplatyl, etoposide, vinblastin and paclitaxel increased apoptosis in a dose-dependent manner in breast carcinoma as well as in non Hodgkin's lymphoma cell lines. Co-culture with irradiated L cells expressing CD40L significantly reduced the percentage of apoptotic cells in breast carcinoma and non Hodgkin's lymphoma cell lines treated with these drugs. In breast carcinoma cell lines, these 5 drugs induced an inconsistent increase of caspase-3/7 activity, while in non Hodgkin's lymphoma cell lines all 5 drugs increased caspase-3/7 activity up to 28-fold above baseline. Co-culture with CD40L L cells reduced (-39% to -89%) the activation of caspase-3/7 induced by these agents in all 5 non Hodgkin's lymphoma cell lines, but in none of the 2 breast carcinoma cell lines. Co culture with CD40L L cells also blocked the apoptosis induced by exogenous ceramides in breast carcinoma and non Hodgkin's lymphoma cell lines through a caspase-3-like, 8-like and 9-like dependent pathways. CONCLUSION: These results indicate that CD40L expressed on adjacent non tumoral cells induces multidrug resistance to cytotoxic agents and ceramides in both breast carcinoma and non Hodgkin's lymphoma cell lines, albeit through a caspase independent and dependent pathway respectively

Isofagomine In Vivo Effects in a Neuronopathic Gaucher Disease Mouse

The pharmacological chaperone, isofagomine (IFG), enhances acid β-glucosidase (GCase) function by altering folding, trafficking, and activity in wild-type and Gaucher disease fibroblasts. The in vivo effects of IFG on GCase activity, its substrate levels, and phenotype were evaluated using a neuronopathic Gaucher disease mouse model, 4L;C* (V394L/V394L + saposin C-/-) that has CNS accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) as well as progressive neurological deterioration. IFG administration to 4L;C* mice at 20 or 600 mg/kg/day resulted in life span extensions of 10 or 20 days, respectively, and increases in GCase activity and protein levels in the brain and visceral tissues. Cerebral cortical GC and GS levels showed no significant reductions with IFG treatment. Increases of GC or GS levels were detected in the visceral tissues of IFG treated (600 mg/kg/day) mice. The attenuations of brain proinflammatory responses in the treated mice were evidenced by reductions in astrogliosis and microglial cell activation, and decreased p38 phosphorylation and TNFα levels. Terminally, axonal degeneration was present in the brain and spinal cord from untreated and treated 4L;C* mice. These data demonstrate that IFG exerts in vivo effects by enhancing V394L GCase protein and activity levels, and in mediating suppression of proinflammation, which led to delayed onset of neurological disease and extension of the life span of 4L;C* mice. However, this was not correlated with a reduction in the accumulation of lipid substrates

C6-ceramide synergistically potentiates the anti-tumor effects of histone deacetylase inhibitors via AKT dephosphorylation and α-tubulin hyperacetylation both in vitro and in vivo

Histone deacetylase inhibitors (HDACIs) have shown promising anti-tumor effects for a variety of malignancies, however, many tumors are reportedly resistant to them. In this study, we made a novel discovery that co-administration of HDACIs (Trichostatin A (TSA) and others) and exogenous cell-permeable short-chain ceramide (C6) results in striking increase in cancer cell death and apoptosis in multiple cancer cells. These events are associated with perturbations in diverse cell signaling pathways, including inactivation of Akt/mTOR and increase in α-tubulin acetylation (both in vivo and in vitro). TSA interacts in a highly synergistic manner with C6-ceramide to disrupt HDAC6/protein phosphatase 1 (PP1)/tubulin complex, to induce α-tubulin hyperacetylation, and to release and activate PP1, which then leads to AKT dephosphorylation and eventually causes cancer cell death. Interestingly, TSA itself results in short-term ceramide accumulation, which as a result of metabolic (glycosylation) removal, does not result in evident increase of cancer cell death. However, adding C6-ceramide led to a very pronounced increase in ceramide level and marked increase in cell death. Importantly, the effective synergistic anti-tumor activity of TSA plus C6-ceramide is also seen in in vivo mice xenograft pancreatic and ovarian cancer models, indicating that this regimen (HDACI plus C6-ceramide) may represent a more effective form of therapy against pancreatic and ovarian carcinoma