The
sphingolipid ceramide is known to play a central role in chemo- and radiation-induced cell death.
Acid ceramidase (AC) hydrolyzes
ceramide, and thus reduces intracellular levels of this proapoptotic
lipid. The role of AC as a putative anticancer target is supported by reports of upregulation in
prostate cancer and in some
breast tumors. In this study, we determined whether the introduction of an AC inhibitor would enhance the apoptosis-inducing effects of
C6-ceramide (C6-cer) in
breast cancer cells. Cultured
breast cancer cells were treated with
DM102 [(2R,3Z)-N-(1-hydroxyoctadec-3-en-2-yl)pivalamide, C6-cer, or the combination. Cell viability and cytotoxic synergy were assessed. Activation of apoptotic pathways, generation of
reactive oxygen species, and mitochondrial transmembrane potential were determined.
DM102 was a more effective AC inhibitor than
N-oleoylethanolamine (NOE) and (1R,2R)-2-N-(tetradecanoylamino)-1-(4'-nitrophenyl)-1,3-propandiol (B-13) in MDA-MB-231, MCF-7, and BT-474 cells. As single agents, C6-cer (IC(50) 5-10 μM) and
DM102 (IC(50) 20 μM) were only moderately cytotoxic in MDA-MB-231, MCF-7, and SK-BR-3 cells. Co-administration, however, produced synergistic decreases in viability (combination index <0.5) in all cell lines. Apoptosis was confirmed in MDA-MB-231 cells by detection of
caspase 3 cleavage and a >3-fold increase in
caspase 3/7 activation, PARP cleavage, and a >70% increase in
Annexin-V positive cells. C6-cer/
DM102 increased ROS levels 4-fold in MDA-MB-231 cells, shifted the ratio of Bax:Bcl-2 to >9-fold that of control cells, and resulted in mitochondrial membrane depolarization.
DM102 also increased the synthesis of (3)H-palmitate-labeled long-chain
ceramides by 2-fold when C6-cer was present. These data support the effectiveness of targeting AC in combination with exogenous short-chain
ceramide as an anticancer strategy, and warrant continued investigation into the utility of the C6-cer/
DM102 drug duo in human
breast cancer.