C6-Ceramide and targeted inhibition of acid ceramidase induce synergistic decreases in breast cancer cell growth

Abstract

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.

Acid Ceramidase in Melanoma

“…Compounds that increase cellular ceramide levels, such as oleoylethanolamide and B13, synergize with antitumoral agents to decrease viability of glioblastoma (29), hepatoma (30), colon (31), and prostate cancer cells (32). Similar synergistic effects have been reported in human breast cancer cells with the AC inhibitor DM102, when this compound is combined with the cell-permeating ceramide analog C6 (33), and in colon cancer cells with substituted 2,4-dioxopyrimidine-1-carboxamides, such as ARN398 (12,35). Evidence indicates that the same approach may be used to slow down or stop proliferation in melanoma cells (36 -39).…”

Construction of Conditional Acid Ceramidase Knockout Mice andin vivoEffects on Oocyte Development and Fertility

“…AC and its role in ceramide-mediated apoptosis has been the subject of numerous recent studies (see for reviews [22, 23]), and inhibitors of this enzyme are currently being developed for cancer therapy e.g., [24, 25]. …”

Sphingolipid biology and its role in cancer development and therapy

“…PDMP has also been used on human colon cancer and breast cell lines. It was shown to increase ceramide levels and stimulate autophagy in these cell lines [16].…”