TY - JOUR
T1 - Uncoupling ceramide glycosylation by transfection of glucosylceramide synthase antisense reverses adriamycin resistance
AU - Liu, Yong Yu
AU - Han, Tie Yan
AU - Giuliano, Armando E.
AU - Hansen, Nora
AU - Cabot, Myles C.
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 2000/3/10
Y1 - 2000/3/10
N2 - 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/μg protein, p < 0.001) and were 28-fold more sensitive to adriamycin (EC50, 0.44 ± 0.01 versus 12.4 ± 0.7 μM, p < 0.0001). GCS antisense transfected cells were also 2.4-fold more sensitive to C6-ceramide compared with parental cells (EC50 = 4.0 ± 0.03 versus 9.6 ± 0.5 μM, 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.
AB - 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/μg protein, p < 0.001) and were 28-fold more sensitive to adriamycin (EC50, 0.44 ± 0.01 versus 12.4 ± 0.7 μM, p < 0.0001). GCS antisense transfected cells were also 2.4-fold more sensitive to C6-ceramide compared with parental cells (EC50 = 4.0 ± 0.03 versus 9.6 ± 0.5 μM, 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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U2 - 10.1074/jbc.275.10.7138
DO - 10.1074/jbc.275.10.7138
M3 - Article
C2 - 10702281
AN - SCOPUS:0034629305
SN - 0021-9258
VL - 275
SP - 7138
EP - 7143
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 10
ER -