Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Multidrug-resistant cancer cells display elevated levels of glucosylceramide (Lavie, Y., Cao, H. T., Volner, A., Lucci, A., Han, T. Y., Geffen, V., Giuliano, A. E., and Cabot, M. C. (1997) J. Biol. Chem. 272, 1682-1687). In this study, we have introduced glucosylceramide synthase (GCS) into wild type MCF-7 breast cancer cells using a retroviral tetracycline-on expression system, and we developed a cell line, MCF-7/GCS. MCF-7/GCS cells expressed an 11-fold higher level of GCS activity compared with the parental cell line. Interestingly, the transfected cells demonstrated strong resistance to adriamycin and to ceramide, whereas both agents were highly cytotoxic to MCF-7 cells. The EC50 values of adriamycin and ceramide were 11-fold (p < 0.0005) and 5-fold (p < 0.005) higher, respectively, in MCF-7/GCS cells compared with MCF-7 cells. Ceramide resistance displayed by MCF-7/GCS cells closely paralleled the activity of expressed GCS with a correlation coefficient of 0.99. In turn, cellular resistance and GCS activity were dependent upon the concentration of the expression mediator doxycycline. Adriamycin resistance in MCF-7/GCS cells was related to the hyperglycosylation of ceramide and was not related to shifts in the levels of either P-glycoprotein or Bcl-2. This work demonstrates that overexpression of GCS, which catalyzes ceramide glycosylation, induces resistance to adriamycin and ceramide in MCF-7 breast cancer cells.
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PMID:Expression of glucosylceramide synthase, converting ceramide to glucosylceramide, confers adriamycin resistance in human breast cancer cells. 987 62

Previous work from our laboratory demonstrated that increased competence to glycosylate ceramide conferred adriamycin resistance in MCF-7 breast cancer cells (Liu, Y. Y., Han, T. Y., Giuliano, A. E. , and M. C. Cabot. (1999) J. Biol. Chem. 274, 1140-1146). This was achieved by cellular transfection with glucosylceramide synthase (GCS), the enzyme that converts ceramide to glucosylceramide. With this, we hypothesized that a decrease in cellular ceramide glycosylation would result in heightened drug sensitivity and reverse adriamycin resistance. To down-regulate ceramide glycosylation potential, we transfected adriamycin-resistant breast cancer cells (MCF-7-AdrR) with GCS antisense (asGCS), using a pcDNA 3.1/his A vector and developed a new cell line, MCF-7-AdrR/asGCS. Reverse transcription-polymerase chain reaction assay and Western blot analysis revealed marked decreases in both GCS mRNA and protein in MCF-7-AdrR/asGCS cells compared with the MCF-7-AdrR parental cells. MCF-7-AdrR/asGCS cells exhibited 30% less GCS activity by in vitro enzyme assay (19.7 +/- 1.1 versus 27.4 +/- 2.3 pmol GC/h/microg protein, p < 0.001) and were 28-fold more sensitive to adriamycin (EC(50), 0.44 +/- 0.01 versus 12.4 +/- 0.7 microM, p < 0. 0001). GCS antisense transfected cells were also 2.4-fold more sensitive to C(6)-ceramide compared with parental cells (EC(50) = 4. 0 +/- 0.03 versus 9.6 +/- 0.5 microM, p < 0.0005). Under adriamycin stress, GCS antisense transfected cells compared with parental cells displayed time- and dose-dependent increases in endogenous ceramide and dramatically higher levels of apoptotic effector, caspase-3. Western blotting showed that adriamycin sensitivity, introduced by asGCS gene transfection, was independent of P-glycoprotein and Bcl-2 expression. In summary, this work shows that transfection of GCS antisense tempers the expression of native GCS and restores cell sensitivity to adriamycin. Therefore, limiting the potential to glycosylate ceramide, which is an apoptotic signal in chemotherapy and radiotherapy, provides a promising approach to combat drug resistance.
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PMID:Uncoupling ceramide glycosylation by transfection of glucosylceramide synthase antisense reverses adriamycin resistance. 1070 81

Ceramide glycosylation, through glucosylceramide synthase (GCS), allows cellular escape from ceramide-induced programmed cell death. This glycosylation event confers cancer cell resistance to cytotoxic anticancer agents [Liu, Y. Y., Han, T. Y., Giuliano, A. E., and M. C. Cabot. (1999) J. Biol. Chem. 274, 1140-1146]. We previously found that glucosylceramide, the glycosylated form of ceramide, accumulates in adriamycin-resistant breast carcinoma cells, in vinblastine-resistant epithelioid carcinoma cells, and in tumor specimens from patients showing poor response to chemotherapy. Here we show that multidrug resistance can be increased over baseline and then totally reversed in human breast cancer cells by GCS gene targeting. In adriamycin-resistant MCF-7-AdrR cells, transfection of GCS upgraded multidrug resistance, whereas transfection of GCS antisense markedly restored cellular sensitivity to anthracyclines, Vinca alkaloids, taxanes, and other anticancer drugs. Sensitivity to the various drugs by GCS antisense transfection increased 7- to 240-fold and was consistent with the resumption of ceramide-caspase-apoptotic signaling. GCS targeting had little influence on cellular sensitivity to either 5-FU or cisplatin, nor did it modify P-glycoprotein expression or rhodamine-123 efflux. GCS antisense transfection did enhance rhodamine-123 uptake compared with parent MCF-7-AdrR cells. This study reveals that GCS is a novel mechanism of multidrug resistance and positions GCS antisense as an innovative force to overcome multidrug resistance in cancer chemotherapy.
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PMID:Ceramide glycosylation potentiates cellular multidrug resistance. 1125 90

As a strategy to enhance tumor cell sensitivity to vincristine, we tested whether modulation of sphingolipid metabolism would alter vincristine cytotoxicity since this is linked to accumulation of the intermediate metabolite, ceramide. We blocked ceramide metabolism in a series of variably vincristine-resistant cell lines derived from CCRF-CEM leukemia cells using an inhibitor of glucosylceramide synthase, DL-threo-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP). PPPP alone (1.0 microM), while nearly completely blocking glucosylceramide synthesis, was not toxic and did not increase cellular ceramide levels. Vincristine alone was toxic, caused apoptosis or programmed cell death (PCD) and caused an elevation in ceramide levels. Strikingly, the combination of PPPP and vincristine resulted in a further increase, over that of vincristine alone, of (i) cellular ceramide concentration, (ii) cytotoxicity associated with PCD and (iii) G2/M cell-cycle arrest. PPPP had no effect on P-glycoprotein expression or function. We conclude that vincristine cytotoxicity occurs in part through a ceramide-dependent mechanism, resulting in both G2/M block as well as PCD, and that the blockade of glucosylceramide synthase, in itself not toxic, causes augmented accumulation of ceramide resulting from vincristine exposure, which in turn maximizes ceramide-dependent, vincristine-induced cytotoxicity. Inhibition of glucosylceramide synthesis may be a means of circumventing drug resistance by enhancing signaling through a cell-death pathway.
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PMID:Glucosylceramide synthase inhibition enhances vincristine-induced cytotoxicity. 1139 32

It 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-decanoylamino-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.
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PMID:Differential chemosensitizing effect of two glucosylceramide synthase inhibitors in hepatoma cells. 1159 84

Alterations in metabolism of ceramide (Cer) to the noncytotoxic metabolite glucosylceramide have been implicated in the multidrug resistance (MDR) phenomenon. This observation has been made with tumor cells that also overexpress P-glycoprotein (Pgp), raising the possibility that Pgp plays a role in regulating Cer metabolism. We investigated the effect of the glucosylceramide synthase inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on the chemosensitivity of two wild-type and multidrug-resistant human breast tumor cell lines. Subtoxic concentrations of PDMP sensitized drug-selected MCF7/AdrR and Pgp-overexpressing MDA435/LCC6MDR1 (MDR1 gene-transfected) cell lines to Taxol and vincristine but did not alter the chemosensitivity of the wild-type cells. Evaluation of Taxol uptake indicated that the effect of PDMP was not due to membrane permeability alterations because anticancer drug accumulation was unaffected by PDMP. Whereas both multidrug-resistant cell lines overexpress Pgp, only the MCF7/AdrR cell line overexpresses the glucosylceramide synthase enzyme. This difference enabled us to distinguish between sensitization effects associated with Cer metabolism versus Pgp-mediated transport. Interestingly, when Pgp function was blocked, the PDMP effect was reduced 3-fold in MCF7/AdrR cells and was no longer observed in the MDA435/LCC6MDR1 cells. These observations imply that Cer metabolism and apoptosis effects are regulated not only by enzymes that convert Cer to nontoxic metabolites but also by Pgp-mediated transport. Given the intracellular distribution patterns of Pgp, we propose that this effect is related to glucosylceramide translocation across the Golgi bilayer. We have applied this model to the situation of Cer metabolism-based chemosensitization and demonstrate that MDR modulation strategies aimed primarily at altering drug transport mechanisms can influence other MDR mechanisms such as glycosphingolipid metabolism. This work highlights the relationship between drug transport and Cer metabolism in the context of chemosensitization and cautions against making oversimplified assumptions that these mechanisms act independently.
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PMID:P-glycoprotein modulates ceramide-mediated sensitivity of human breast cancer cells to tubulin-binding anticancer drugs. 1246 15

High glucosylceramide synthase (GCS) activity is one factor contributing to multidrug resistance (MDR) in breast cancer. Enforced GCS overexpression has been shown to disrupt ceramide-induced apoptosis and to confer resistance to doxorubicin. To examine whether GCS is a target for cancer therapy, we have designed and tested the effects of antisense oligodeoxyribonucleotides (ODNs) to GCS on gene expression and chemosensitivity in multidrug-resistant cancer cells. Here, we demonstrate that antisense GCS (asGCS) ODN-7 blocked cellular GCS expression and selectively increased the cytotoxicity of anticancer agents. Pretreatment with asGCS ODN-7 increased doxorubicin sensitivity by 17-fold in MCF-7-AdrR (doxorubicin-resistant) breast cancer cells and by 10-fold in A2780-AD (doxorubicin-resistant) ovarian cancer cells. In MCF-7 drug-sensitive breast cancer cells, asGCS ODN-7 only increased doxorubicin sensitivity by 3-fold, and it did not influence doxorubicin cytotoxicity in normal human mammary epithelial cells. asGCS ODN-7 was shown to be more efficient in reversing drug resistance than either the GCS chemical inhibitor d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or the P-glycoprotein blocking agents verapamil and cyclosporin A. Experiments defining drug transport and lipid metabolism parameters showed that asGCS ODN-7 overcomes drug resistance mainly by enhancing drug uptake and ceramide-induced apoptosis. This study demonstrates that a 20-mer asGCS oligonucleotide effectively reverses MDR in human cancer cells.
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PMID:Oligonucleotides blocking glucosylceramide synthase expression selectively reverse drug resistance in cancer cells. 1496 19

Previously we have described a novel multidrug-resistant cell line, HT29(col), which displayed over expression of the multidrug-resistance protein 1 (MRP1) and an altered sphingolipid composition, including enhanced levels of glucosylceramide (GlcCer; Kok JW, Veldman RJ, Klappe K, Koning H, Filipeanu C, Muller M. Int J Cancer 2000;87:172-8). In our study, long-term screening revealed that, during colchicine-induced acquisition of multidrug resistance in a new HT29(col) cell line, increases in GlcCer occurred concomitantly with upregulation of MRP1 expression. Both MRP1 and GlcCer were found enriched in Lubrol-insoluble membrane domains. The expression of MRP1 and GlcCer were tightly correlated, as indicated also by a reversal of both at the later stage of colchicine consolidation. Resistance to colchicine was determined by MRP1, while glucosylceramide synthase (GCS) did not contribute: 1). Resistance was fully inhibited by MK571. 2). GCS expression and activity were not upregulated in HT29(col) cells. 3). Inhibition of GCS did not affect MRP1-mediated efflux function or sensitivity to colchicine. Instead, overall sphingolipid metabolism was upregulated through an increased rate of ceramide biosynthesis. In conclusion, upregulation of MRP1 occurs in concert with upregulation of GlcCer during multidrug-resistance acquisition, and both are enriched in rafts. The increased GlcCer pool does not directly modulate MRP1 function and cell survival.
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PMID:MRP1 and glucosylceramide are coordinately over expressed and enriched in rafts during multidrug resistance acquisition in colon cancer cells. 1512 83

Resistance to natural product chemotherapy drugs is a major obstacle to successful cancer treatment. This type of resistance is often acquired in response to drug exposure; however, the mechanisms of this adverse reaction are complex and elusive. Here, we have studied acquired resistance to Adriamycin, Vinca alkaloids, and etoposide in MCF-7 breast cancer cells, KB-3-1 epidermoid carcinoma cells, and other cancer cell lines to determine if there is an association between expression of glucosylceramide synthase, the enzyme catalyzing ceramide glycosylation to glucosylceramide, and the multidrug-resistant (MDR) phenotype. This work shows that glucosylceramide levels increase concomitantly with increased drug resistance in the KB-3-1 vinblastine-resistant sublines KB-V.01, KB-V.1, and KB-V1 (listed in order of increasing MDR). The levels of glucosylceramide synthase mRNA, glucosylceramide synthase protein, and P-glycoprotein (P-gp) also increased in parallel. Increased glucosylceramide levels were also present in Adriamycin-resistant KB-3-1 sublines KB-A.05 and KB-A1. In breast cancer, detailed analysis of MCF-7 wild-type and MCF-7-AdrR cells (Adriamycin-resistant) demonstrated enhanced glucosylceramide synthase message and protein, P-gp message and protein, and high levels of glucosylceramide in resistant cells. Similar results were seen in vincristine-resistant leukemia, etoposide-resistant melanoma, and Adriamycin-resistant colon cancer cell lines. Cell-free glucosylceramide synthase activity was higher in lysates obtained from drug-resistant cells. Lastly, glucosylceramide synthase promoter activity was 15-fold higher in MCF-7-AdrR compared with MCF-7 cells. We conclude that selection pressure for resistance to natural product chemotherapy drugs selects for enhanced ceramide metabolism through glucosylceramide synthase in addition to enhanced P-gp expression. A possible connection between glucosylceramide synthase and P-gp in drug resistance biology is suggested.
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PMID:Overexpression of glucosylceramide synthase and P-glycoprotein in cancer cells selected for resistance to natural product chemotherapy. 1514 Oct 21

The multidrug-resistant cancer cell lines NCI/AdR(RES) and MES-SA/DX-5 have higher glycolipid levels and higher P-glycoprotein expression than the chemosensitive cell lines MCF7-wt and MES-SA. Inhibiting glycolipid biosynthesis by blocking glucosylceramide synthase has been proposed to reverse drug resistance in MDR cells by causing an increased accumulation of proapoptotic ceramide during treatment of cells with cytotoxic drugs. We treated both multidrug-resistant cell lines with the glucosylceramide synthase inhibitors PDMP (d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol), C9DGJ (N-nonyl-deoxygalactonojirimycin) or C4DGJ (N-butyl-deoxygalactonojirimycin). PDMP achieved a significant reversal of drug resistance in agreement with previous reports. However, the N-alkylated iminosugars C9DGJ and C4DGJ, which are more selective glucosylceramide synthase inhibitors than PDMP, failed to cause any reversal of drug resistance despite depleting glycolipids to the same extent as PDMP. Our results suggest that (a) inhibition of glucosylceramide synthase does not reverse multidrug resistance and (b) the chemosensitization achieved by PDMP cannot be caused by inhibition of glucosylceramide synthase alone.
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PMID:Inhibition of glucosylceramide synthase does not reverse drug resistance in cancer cells. 1526 8


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