Differential apoptotic effect and metabolism of N-acetylsphingosine and N-hexanoylsphingosine in CHP-100 human neurotumor cells.

The cytotoxic effects of N-acetylsphingosine (C2-Cer) and N-hexanoylsphingosine (C6-Cer) were compared together with their specific intracellular accumulation profiles and metabolism in human CHP-100 neuroepithelioma cells. The two short-chain ceramides, administered in the culture medium at an equimolar concentration, evoked a differential apoptotic response, with C6-Cer showing markedly more cytotoxic than C2-Cer. Apoptosis, that was suppressed in both cases by inhibition of caspase-9, but not of caspase-8, associated with a higher intracellular accumulation of C6-Cer over C2-Cer, notwithstanding C6-Cer was actively metabolized by direct glucosylation or by conversion to natural ceramide via the sphingosine salvage pathway, whereas C2-Cer was apparently metabolically inhert.

Brefeldin A limits N-hexanoylsphingosine-induced accumulation of natural ceramide via the salvage pathway by enhancing glucosylation.

Cells 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.

Relevance of the salvage pathway to N-hexanoylsphingosine metabolic downregulation in human neurotumor cells: implications for apoptosis.

N-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.

P-Glycoprotein is not a key target for the chemosensitizing effect of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol in HepG2 cells exposed to doxorubicin.

Chemosensitization of HepG2 cells to doxorubicin by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol neither impinged on downregulation of P-glycoprotein expression nor on severe impairment of its activity. Moreover, differently from verapamil, a potent P-glycoprotein inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol chemosensitized HepG2 cells in a fashion that was insensitive to the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. At concentrations exceeding the one employed for chemosensitization, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol was by itself strongly toxic to HepG2 cells, and also this effect was insensitive to the pancaspase inhibitor.

Long-chain ceramide produced in response to N-hexanoylsphingosine does not induce apoptosis in CHP-100 cells.

It has been previously reported that treatment of CHP-100 human neuroepithelioma cells with N-hexanoylsphingosine (C6-Cer) induces intracellular accumulation of long-chain ceramide (LC-Cer) and apoptosis. Herein, we investigated the existence of any causal relationship between the two phenomena. We report that C6-Cer-evoked LC-Cer accumulation is potently attenuated by the ceramide synthase inhibitor fumonisin B1; however, fumonisin B1 neither affects the apoptotic response evoked by C6-Cer administration, nor is toxic by itself to CHP-100 cells.