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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 8, 2003 as doi:10.1096/fj.02-1053fje. |
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* INSERM U.466, Laboratoire de Biochimie, Institut Louis Bugnard, CHU Rangueil, 31059 Toulouse Cedex 9, France;
Department of Membrane Cell Biology, Faculty of Medical Sciences, University of Groningen, 9713 AV Groningen, The Netherlands; and
Division of Experimental Therapeutics, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M1, Canada
3Correspondence: *INSERM U.466, Laboratoire de Biochimie, Institut Louis Bugnard, CHU Rangueil, 1 avenue Jean Poulhès, TSA 50032, 31059 Toulouse Cedex 9, France. E-mail: levade{at}toulouse.inserm.fr
SPECIFIC AIMS
Conversion of ceramide, a putative mediator of anticancer drug-induced apoptosis, into glucosylceramide (GlcCer) by action of the enzyme glucosylceramide synthase (GCS) has been implicated in drug resistance of cancer cells. We investigated the putative involvement of GCS in chemotherapeutic drug resistance regulation by comparing the sensitivity of mutant GM95 mouse melanoma cells, which are completely deficient in GlcCer owing to a defect in GCS, with GM95 cells stably expressing GCS (GM95/GCS) toward several common chemotherapeutic drugs.
PRINCIPAL FINDINGS
1. Transfected GM95 cells express a functional GCS
We restored the ceramide glucosylation capacity of GM95 cells by transfection with the pcDNA3.1 vector encoding human GCS. Using NBD-C6-ceramide as a substrate, in vitro enzyme measurements revealed that GM95/GCS lysates indeed exhibited GCS activity that was completely absent from the control-transfected GM95 cells. The difference in GCS activity was reflected by clear differences in the metabolism of NBD-C6-ceramide by living cells. Whereas formation of NBD-C6-GlcCer did not occur in GM95 cells, this product was clearly detected in GM95/GCS cells. Metabolic studies using [3H]palmitic acid as precursor of the de novo sphingolipid biosynthesis also showed that tritiated GlcCer and ganglioside GM3 were clearly detected in GM95/GCS cells but completely absent from GM95 cells. These data confirm that GM95 cells do not express functional GCS and that transfection with a human GCS cDNA fully restores their ceramide glucosylation capacity.
2. GCS does not affect cell viability and apoptosis induction upon drug exposure
GM95 and GM95/GCS cells were tested for their sensitivity toward several commonly used chemotherapeutic drugs. With the MTT viability assay, no differences were observed for doxorubicin, vinblastine, cytosine arabinoside, or paclitaxel (Fig. 1
). When the sensitivity of GM95 cells for either one of the above drugs, colchicine, or etoposide was compared with that of the parental B16 melanoma line from which GM95 cells were derived, no differences were observed either (data not shown). To further investigate this, we focused our studies on the anthracycline doxorubicin. This drug induced apoptosis both in GM95 and GM95/GCS cells, with similar kinetics and extent of doxorubicin-induced (caspase-3-like) effector caspase activation as measured by Ac-DEVD-AMC hydrolysis capacity (Fig. 2
, A). No difference in the cytosolic release of cytochrome c, another hallmark of apoptosis, was detected between GM95 and GM95/GCS cells (Fig. 2B
). When the nuclear morphology of doxorubicin-treated cells was examined by DAPI staining, typical DNA condensation and nuclear fragmentation were observed in GM95 and GM95/GCS cells (Fig. 2C
). Thus, GCS-corrected GM95 cells exhibited an identical sensitivity toward cytotoxic drugs compared with GCS-deficient GM95 cells, both in terms of overall viability and apoptosis induction.
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3. Doxorubicin treatment results in ceramide accumulation via de novo synthesis
Monitoring of cellular lipid levels after radiolabeling with [3H]palmitic acid showed an increase in ceramide content upon administration of doxorubicin that was detectable within several hours after drug addition and continued up to final levels of 170–190% over untreated cells. Notably, doxorubicin-induced ceramide formation occurred with the same amplitude and kinetics in GCS-deficient and GCS-containing cells. In both cell lines, however, the level of sphingomyelin remained remarkably stable during the process of doxorubicin-induced cell death, suggesting that sphingomyelin was not the metabolic source of the long-term increase seen in ceramide. Indeed, preincubation of GM95 cells with fumonisin B1, a mycotoxin inhibitor of ceramide synthase, markedly inhibited doxorubicin-triggered cell death, in agreement with previously published observations underscoring the role of de novo ceramide synthesis in anthracycline-induced cell death. As expected, GlcCer was not formed in (doxorubicin-treated) GCS-deficient cells. Although GlcCer formation was detected in doxorubicin-treated GM95/GCS cells, metabolism of drug-induced ceramide did not appear to be instrumental in cell survival.
4. Short-chain ceramide induces cell death in GM95 and GM95/GCS cells
Doxorubicin and other anthracyclines induce the generation of ceramide in mouse melanoma and other cell types. Since ceramide has been described as a potential lipid mediator of apoptosis, the effect of short-chain ceramide analogs was tested on GM95 and GM95/GCS cells. C6-ceramide, but not C6-dihydroceramide, induced cell death in a dose-dependent way with LC50 values of 
10 µM for both cell lines, suggesting that no differences exist between the two cell lines as to the effect of ceramide on cell signaling and functions.
5. Inhibition of GCS does not chemosensitize B16 melanoma cells
In addition to the genetically modified GM95 cell system, experiments were performed in which we pharmacologically inhibited GCS activity. We used the B16 parental melanoma cell line and N-(n-butyl)deoxynojirimycin, a potent and nontoxic GCS inhibitor. This compound strongly inhibited GCS activity of B16 cells yet had no chemosensitizing effect toward doxorubicin or vinblastine, confirming the results obtained with the genetically modified melanoma cells.
6. Drug transporter proteins are present in GM95 cells
ABC transporter proteins such as P-glycoprotein (Pgp) and multidrug resistance-related protein (MRP) are able to translocate fluorescent and other short-chain analogs of GlcCer over the plasma membrane, suggesting that the chemoprotective effect of GCS activity, and hence enhanced GlcCer levels, is somehow dependent on the presence of drug transporter proteins. Because most studies that indicated a role for GCS in multidrug resistance were performed on breast cancer cells that express high amounts of Pgp, we looked for the expression of drug transporter proteins in GM95 and GM95/GCS cells. Pgp and MRP1, two of the most common drug transporters, were detectable in both cell lines. Accumulation of doxorubicin proceeded with similar kinetics in the two cell lines. This indicates that doxorubicin efflux mechanisms are present and active to the same extent in both cell lines.
CONCLUSIONS
Doxorubicin and short-chain ceramide analogs both induced cell death of GM95 and GM95/GCS cells. Since doxorubicin also promoted an increase in cellular ceramide levels, it is likely that, in analogy to other cell lines, drug-induced apoptosis of melanoma cells involves (at least partly) ceramide production. By using both genetic and pharmacological models, we have demonstrated that GCS failed to sensitize melanoma cells to anticancer drugs (Fig. 3
). These findings moderate the importance of ceramide glucosylation in drug resistance mechanisms. This is in apparent contrast to several previous reports on tumor cells exhibiting abundant levels of drug transporter proteins. However, our data with GM95 cells indicate that the (in)ability of GCS to confer anticancer drug resistance is not directly correlated with the expression of ABC transporters. Our findings are consistent with the observations that constitutive overexpression of GCS in leukemic Jurkat T cells did not alter apoptosis sensitivity to anticancer drugs or other apoptosis-inducing stimuli and that expression of a GCS antisense in human neuroepithelioma cells did not increase apoptosis in response to anticancer drugs. This suggests that the cell sensitivity to chemotherapeutic drugs is not generally dictated by the level of GCS expression.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1053fje; to cite this article, use FASEB J. (April 8, 2003) 10.1096/fj.02-1053fje 
2 Present address: The Netherlands Cancer Institute, Division of Cellular Biochemistry, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
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