1,721,023 research outputs found
Relevance of the Salvage Pathway to N-Hexanoylsphingosine Metabolic Downregulation in Human Neurotumor Cells: Implications for Apoptosis
No full textN-Hexanoylsphingosine (C6-Cer) is currently being evaluated as an antineoplastic agent, after preclinical studies showing its property to reduce tumor growth. Herein it is reported that the cytotoxic effect of C6-Cer, as observed in CHP-100 neurotumor cells, impinges on its continuous uptake from the culture medium, ensuring maintainance of elevated steady-state intracellular levels, in the face of the rapid metabolic removal. C6-Cer metabolism not only does occur by direct glucosylation but is also relevantly driven by utilization via the sphingosine salvage pathway, leading to accumulation of natural ceramide that, in CHP-100 cells, has been demonstrated to lack apoptotic properties. Upon inhibition of glucosylceramide synthase by D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, previously shown to enhance C6-Cer cytotoxic activity, short-chain ceramide metabolism was partly redirected to the salvage pathway, likely attenuating the chemosensitizing effect of the above-mentioned compound. Elucidation of the metabolic machinery driving C6-Cer recycling via the salvage pathway might thus be relevant for optimization of its therapeutic utilization
Different contribution of phospholipid and triacylglycerol metabolism to esterification of free intracellular arachidonate: a study on SK-N-BE(2) human neuroblastoma cells.
No full textBrefeldin A Limits N-Hexanoylsphingosine-Induced Accumulation of Natural Ceramide via the Salvage Pathway by Enhancing Glucosylation
No full textCells actively metabolize exogenously administered N-hexanoylsphingosine (C6-Cer) to natural (i.e. long-chain) ceramide (LC-Cer) via the sphingosine (Sph) salvage pathway, namely via C6-Cer deacylation and Sph reacylation with a long-chain fatty acid. Based on the observation that the mycotoxin brefeldin A (BFA), a Golgi complex disassembler, impairs C6-Cer-evoked LC-Cer accumulation, it has been hypothesized that the integrity of the above-mentioned organelle might be necessary for C6-Cer processing via the salvage pathway and that BFA might block the phenomenon at the step short-chain ceramide deacylation. The present study shows that BFA indeed attenuates C6-Cer-evoked LC-Cer accumulation in human neurotumor CHP-100 cells: evidence is however provided that the phenomenon is not due to impaired synthesis of LC-Cer, but to its enhanced conversion to glucosylceramide. The possibility is discussed that this outcome might be a consequence of the BFA well-established property to induce the merging of the cis-Golgi region with endoplasmic reticulum, namely the compartments in which glucosylceramide synthase and ceramide synthases have been reported to reside
On the role of agonist-evoked Ca2+ mobilization in sustaining the ongoing phosphoinositide hydrolysis. A study on intact SK-N-BE(2) neuroblastoma cells subjected to muscarinic stimulation
No full textStimulation of SK-N-BE(2) cells with 1 mM carbachol (Cch) elicited phosphoinositide (PPI) hydrolysis and a rapid elevation of cytosolic Ca2+ concentration ([Ca2+]i) from 115 nM to about 500 nM, followed by a plateau around 200 nM. In myo [3H]inositol-labelled cells, Cch-evoked accumulation of [3H]inositol phosphate (IPs) was not affected when [Ca2+]i was clamped at resting by cell loading with 10 microM BAPTA/AM; under these conditions, maximal 1,4,5-inositol trisphosphate accumulation was not reduced either. When [Ca2+]i was clamped around 700 nM by cell treatment with 600 nM ionomycin, Cch-evoked [3H]IPs accumulation was enhanced by less than 20%, but it was impaired by a 30% and a 55% after [Ca2+]i reduction to about 70 nM and 35-50 nM, by cell loading with 20 microM or 40 microM BAPTA/AM, respectively. These results show that, in SK-N-BE(2) cells, Cch-activated PPI-specific phospholipase C is sensitive to [Ca2+]i but it already operates under suboptimal conditions at resting [Ca2+]i
Differential chemosensitizing effect of two glucosylceramide synthase inhibitors in hepatoma cells
No full textIt has been proposed that ceramide mediates anthracyclin-induced apoptosis and that drug resistance may arise due to upregulated removal of this active lipid through glucosylation. We report that HepG2 hepatoma cells displayed only a modest apoptotic response to doxorubicin treatment, accompanied by a substantial elevation of ceramide levels only at toxic drug concentrations. D,L-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1-propanol (PDMP) and D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP), used at concentrations causing a 90% inhibition of ceramide glucosylation, enhanced doxorubicin-elicited ceramide elevation, but only PDMP potentiated apoptosis. Exogenously administered ceramide had only a marginal apoptotic effect on HepG2 cells; moreover, even in this case, apoptosis was propagated by PDMP but not by PPPP. PDMP moderately inhibited P-glycoprotein activity only at the highest concentration tested, but its chemosensitizing effect was still outstanding at lower concentrations, at which P-gp inhibition was no longer observed. These results demonstrate that the chemosensitizing effect of PDMP is, at least partly, independent from its activity as a glucosylceramide synthase inhibitor. Moreover, P-glycoprotein inhibition is not central to the phenomenon. (C) 2001 Academic Press
Two glucosylceramide synthase inhibitors attenuate doxorubicin-induced p21Cip1/Waf1 upregulation in HepG2 cells, irrespective of their differential chemosensitizing properties
No full textWe have previously reported that HepG2 human hepatocarcinoma cells are sensitized to doxorubicin-induced apoptosis by the glucosylceramide synthase inhibitor D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) but not by the more specific inhibitor D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP). Herein we investigated whether the chemosensitizing action of PDMP impinged on any unspecific effect of this compound on doxorubicin-induced expression of p53 and/or P21(Cip1/Waf1), namely two proteins reported to modulate the apoptotic response to DNA-damaging agents, in a positive or negative fashion, respectively. We show that, in HepG2 cells, PDMP did not substantially affect doxorubicin-induced p53 upregulation, whereas drug-evoked upregulation of p21(Cip1/Waf1) was markedly attenuated. Although this outcome could be expected to account for the chemosensitizing effect of PDMP, impaired upregulation of p21(Cip1/Waf1), in the setting of unaltered p53 expression, was also observed in the case of PPPP. These results, while raising the possibility of a link between attenuation of drug-evoked p21(Cip1/Waf1) expression and redirection of (glyco)sphingolipid metabolism, show that, differently from other tumor systems, attenuation of doxorubicin-induced p21(Cip1/Waf1) expression is at least not sufficient to sensitize HepG2 cells to the apoptotic action of the drug. (c) 2005 Elsevier Inc. All rights reserved
Pertussis toxin effect on carbachol-elicited stimulation of phosphoinositide hydrolysis in human neuroblastoma [SK-N-BE(2)]
No full textCharbachol (Cch) dose-dependently elicited accumulation of [3H]inositol phosphates (IPs) in human neuroblastoma cells SK-N-BE(2) prelabelled with [3H]myo-inositol and stimulated in the presence of 10 mM Li+. Cell response was observed at agonist concentrations higher than 10-6 M and was nearly maximal at 10-3 M; Cch concentration eliciting half- maximal stimulation of [3H]IPs production was about 3.3 x 10-5 M. Pirenzepine (PZ) inhibited [3H]IPs production, as elicited by 1 mM CCh in the presence of 10 mM Li+, in the concentration range 10-8 -10-4 M, with half maximal effect at 2.5 x 10-7 M. Cell treatment with pertussis toxin (PTX) (100 ng/ml for 6 h) resulted in a 20% reduction of total [3H]IPs accumulation elicited by cell stimulation with 1 mM Cch. Results indicate that the human neuroblastoma cell line SK-N-BE(2) possesses a population of muscarinic receptors, highly sensitive to PZ and coupled to stimulation of phosphoinositide hydrolysis through a mechanism which is only partially sensitive to PTX
Muscarinic stimulation of SK-N-BE(2) human neuroblastoma cells elicits phosphoinositide and phosphatidylcholine hydrolysis: relationship to diacylglycerol and phosphatidic acid accumulation.
No full textMuscarinic stimulation of the human neuroblastoma cell line SK-N-BE(2) elicits hydrolysis of phosphoinositides and phosphatidylcholine (PtdCho) and produces a rapid and sustained elevation of diacylglycerol (DG) mass. PtdIns(4,5)P2 cleavage by phospholipase C (PLC) occurred immediately after carbachol (CCh) addition, and phosphoinositide hydrolysis was then sustained for at least 5 min. Cell stimulation, after extensive PtdCho labelling by long-term [H-3]choline administration, resulted in an enhanced release of [H-3]phosphocholine (PCho) into the external medium; enhanced [H-3]PCho release, which occurred with a 15 s delay with respect to CCh addition, was particularly pronounced within the first minute of stimulation and proved to be caused by PtdCho-specific PLC activation. In fact, when cells were exposed to [H-3]choline for a short period, to extensively label the intracellular PCho pool but not PtdCho, stimulation did not result in an enhanced release of [H-3]PCho into the medium. PtdCho-specific phospholipase D (PLD) activation was documented by the accumulation of [H-3]phosphatidylethanol in cells prelabelled with [H-3]myristic acid and stimulated in the presence of 1 % (v/v) ethanol: this metabolic pathway, however, proved to be a minor one leading to generation of phosphatidic acid (PtdOH) during cell stimulation, whereas DG production by the sequential action of PtdCho-specific PLD and PtdOH phosphohydrolase was not observed. Studies on cells which were double-labelled with [H-3]myristic acid and [C-14]arachidonic acid indicated that within 15 s of stimulation DG is uniquely derived from PtdIns(4,5)P2. whereas PtdCho is the major source at later times. Evidence is provided that rapid and selective conversion of phosphoinositide-derived DG into PtdOH may play an important role in determining the temporal accumulation profile of DG from the above-mentioned sources
Effects of pherphenazine on the metabolism of inositol phospholipids in SK-N_BE (2) human neuroblastoma cells
No full textAdministration of myo-[H-3]inositol to SK-N-BE(2) human neuroblastoma cells for 24 hr resulted in equilibrium labelling of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2), as well as in retention of a large intracellular pool of free myo-[H-3]inositol. Equilibrium labelling was no longer observed when cells were treated for 2 hr with 20 mu M perphenazine (PPZ) in label-free medium; under these conditions, myo-[H-3]inositol from the retained intracellular pool was incorporated into PI and PIP but not into PIP2. Analysis of water-soluble myo-[H-3]inositol derivatives and inositol 1,4,5-trisphosphate mass determination indicated that PPZ did not stimulate phosphoinositide hydrolysis by phospholipase C. These results indicate that PPZ raises PI and PIP levels, whereas it is ineffective in expanding the PIP2 pool. The latter effect is not due to a concomitant synthesis and hydrolysis of this lipid
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