Yy. Liu et al., Uncoupling ceramide glycosylation by transfection of glucosylceramide synthase antisense reverses adriamycin resistance, J BIOL CHEM, 275(10), 2000, pp. 7138-7143
Previous work from our laboratory demonstrated that increased competence to
glycosylate ceramide conferred adriamycin resistance in MCF-7 breast cance
r 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 w
ith glucosylceramide synthase (GCS), the enzyme that converts ceramide to g
lucosylceramide, With this, we hypothesized that a decrease in cellular cer
amide 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 G
CS antisense (asGCS), using a pcDNA 3.1/his A vector and developed a new ce
ll 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/mu g protein, p <
0.001) and mere 28-fold more sensitive to adriamycin (EC50, 0.44 +/- 0.01 v
ersus 12.4 +/- 0.7 mu M, p < 0.0001). GCS antisense transfected cells were
also 2.4-fold more sensitive to C-6-ceramide compared with parental cells (
EC50 = 4.0 +/- 0.03 versus 9.6 +/- 0.5 mu M, p < 0.0005). Under adriamycin
stress, GCS antisense transfected cells compared with parental cells displa
yed time- and dose-dependent increases in endogenous ceramide and dramatica
lly higher levels of apoptotic effector, caspase-3. Western blotting showed
that adriamycin sensitivity, introduced by asGCS gene transfection, was in
dependent of P-glycoprotein and Bcl-2 expression. In summary, this work sho
ws that transfection of GCS antisense tempers the expression of native GCS
and restores cell sensitivity to adriamycin. Therefore, limiting the potent
ial to glycosylate ceramide, which is an apoptotic signal in chemotherapy a
nd radiotherapy, provides a promising approach to combat drug resistance.