Apoptosis induced by N-hexanoylsphingosine in CHP-100 cells associates with accumulation of endogenous ceramide and is potentiated by inhibition of glucocerebroside synthesis

Spinedi, Angelo; Bartolomeo, Sabrina Di; Piacentini, Mauro

doi:10.1038/sj.cdd.4400428

Cited by 41 publications

(26 citation statements)

References 19 publications

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“…(e) C 6 -Ceramide was found to produce apoptosis in CHP-100 neuroepithelioma cells, an effect that was markedly potentiated by PDMP, a potent inhibitor of ceramide glucosylation [59]. At the concentration of PDMP used here, there was no effect on cell viability when administered alone.…”

Section: R I T E R I O N ( C ) : U S E O F T W O C E R a M I D E -Ementioning

confidence: 87%

Killing cancer cells by poly-drug elevation of ceramide levels. A hypothesis whose time has come?

Radin¹

2001

Eur J Biochem

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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.

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Section: R I T E R I O N ( C ) : U S E O F T W O C E R a M I D E -Ementioning

confidence: 87%

Killing cancer cells by poly-drug elevation of ceramide levels. A hypothesis whose time has come?

Radin¹

2001

Eur J Biochem

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“…We have previously reported that in unstimulated CHP-100 cells ceramide mass amounts to about 3 pmol/nmol of lipid phosphate and that changes of ceramide mass can reliably be monitored by radiometric analysis, after cell labeling to equilibrium with [ 14 C]-palmitate. 17 Therefore, [ 14 C]-palmitatelabeled cells were exposed for 24 h to different OA concentrations and radioactivity accumulation in ceramide monitored. Figure 3 shows that OA started to induce ceramide elevation at concentrations higher than 10 nM; at 50 ± 100 nM OA, when apoptosis was maximal, ceramide levels reached 230 ± 250% of those observed in untreated cells.…”

Section: Resultsmentioning

confidence: 99%

“…Ceramide and glucosylceramide separation was achieved by developing samples in n-hexane/diethylether/acetic acid (25 : 75 : 1, vol/vol) for full plate height, followed by development in the same direction in chloroform/methanol/0.7 N NH 4 OH (20 : 5 :0.5, vol/vol), up to 3 cm from the top, and again in the same direction in n/hexane/ diethylether/acetic acid (25 : 75 : 1, vol/vol) for full plate height. 17 Lipids were visualized under iodine and spots scraped off from the plates into counting vials for radioactivity determination. Radiometric estimation of changes in ceramide levels was confirmed by gas-chromatographic analysis, as previously reported.…”

Section: Cell Labeling With [mentioning

confidence: 99%

Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatase inhibitor okadaic acid

et al. 1999

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The protein phosphatase inhibitor okadaic acid (OA) dosedependently induced apoptosis in CHP-100 neuroepithelioma cells when administered for 24 h at concentrations ranging from 10 ± 100 nM. Apoptosis was largely, albeit not completely, dependent on cystein protease (caspase) activation. CPP32 processing and poly(ADP-ribose) polymerase (PARP) cleavage started to be observed only at 20 nM OA; moreover, the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk) (100 mM) had negligible effect on apoptosis induced by 10 nM OA, but rescued from death an increasing cell fraction as OA concentration was raised from 20 ± 100 nM. Cell treatment for 24 h with OA induced ceramide accumulation; the phenomenon started to be evident at 20 nM OA and reached its maximum at 50 ± 100 nM OA. In cells exposed to 50 nM OA, ceramide was already elevated by 5 h; at this time, however, PARP cleavage and apoptosis were not yet observed. Z-VAD.fmk (100 mM) had no effect on ceramide elevation induced by 50 nM OA within 5 h, but markedly reduced ceramide accumulation as the incubation was prolonged to 24 h. The latter phenomenon was accompanied by elevation of glucosylceramide levels, thus suggesting that a caspase-dependent reduction of glucosylceramide synthesis might contribute to late ceramide accumulation. Shortchain ceramide (30 mM) induced apoptosis in CHP-100 cells and its effect was additive with that evoked by OA (10 ± 20 nM). These results suggest that ceramide generation might be an important mechanism through which sustained protein phosphatase inhibition induces caspase activation and apoptosis in CHP-100 cells.

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“…In the current study, we increased the intracellular concentrations of ceramide via inhibition of GCS and examined the possibility of enhancing apoptosis in response to imatinib in both sensitive and resistant cells. GCS inhibitors have been found to raise cellular ceramide levels by blocking its conversion into GlcCer and to induce apoptosis (Nicholson et al 1999;Spinedi et al 1998). It has previously been demonstrated that PDMP sensitizes murine neuroblastoma cells to Taxol and Vincristine (Sietsma et al 2000).…”

Section: Discussionmentioning

confidence: 99%

Targeting glucosylceramide synthase sensitizes imatinib-resistant chronic myeloid leukemia cells via endogenous ceramide accumulation

Baran

Bielawski

Gündüz

et al. 2011

J Cancer Res Clin Oncol

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Purpose Drug resistance presents a major obstacle for the treatment of some patients with chronic myeloid leukemia (CML). Pro-apoptotic ceramide mediates imatinib-induced apoptosis, and metabolism of ceramide by glucosylceramide synthase (GCS) activity, converting ceramide to glucosyl ceramide, might contribute to imatinib resistance. In this study, we investigated the role of ceramide metabolism by GCS in the regulation of imatinib-induced apoptosis in drug-sensitive and drug-resistant K562 and K562/IMA-0.2 and K562/IMA-1 human CML cells, which exhibit about 2.3-and 19-fold imatinib resistance, respectively. Methods Cytotoxic eVects of PDMP and imatinib were determined by XTT cell proliferation assay. Expression levels of GCS were determined by RT-PCR and western blot. Intracellular ceramide levels were determined by LC-MS. Cell viability analyses was conducted by Trypan blue dye exclusion assay. Cell cycle and apoptosis analyses were examined by Xow cytometry. Results We Wrst showed that mRNA and protein levels of GCS are increased in drug-resistant K562/IMA as compared to sensitive K562 cells. Next, forced expression of GCS in sensitive K562 cells conferred resistance to imatinib-induced apoptosis. In reciprocal experiments, targeting GCS using its known inhibitor, PDMP, enhanced ceramide accumulation and increased cell death in response to imatinib in K562/IMA cells. Conclusion Our data suggest the involvement of GCS in resistance to imatinib-induced apoptosis, and that targeting GCS by PDMP increased imatinib-induced cell death in drug-sensitive and drug-resistant K562 cells via enhancing ceramide accumulation.

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Apoptosis induced by N-hexanoylsphingosine in CHP-100 cells associates with accumulation of endogenous ceramide and is potentiated by inhibition of glucocerebroside synthesis

Cited by 41 publications

References 19 publications

Killing cancer cells by poly-drug elevation of ceramide levels. A hypothesis whose time has come?

Killing cancer cells by poly-drug elevation of ceramide levels. A hypothesis whose time has come?

Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatase inhibitor okadaic acid

Targeting glucosylceramide synthase sensitizes imatinib-resistant chronic myeloid leukemia cells via endogenous ceramide accumulation

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