Department of Experimental Therapeutics, John Wayne Cancer Institute, Santa Monica, CA, USA.
Breast Cancer Res Treat. 2012 Jun;133(2):447-58. doi: 10.1007/s10549-011-1768-8. Epub 2011 Sep 21.
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.
神经酰胺是一种已知在化疗和放疗诱导的细胞死亡中起核心作用的鞘脂。酸性神经酰胺酶（AC）水解神经酰胺，从而降低这种促凋亡脂质的细胞内水平。AC 作为一种潜在的抗癌靶点的作用得到了以下报告的支持：前列腺癌和一些乳腺癌中上调。在这项研究中，我们确定了引入 AC 抑制剂是否会增强 C6-神经酰胺（C6-cer）在乳腺癌细胞中的诱导凋亡作用。培养的乳腺癌细胞用 DM102[（2R，3Z）-N-（1-羟基十八-3-烯-2-基）戊酰胺、C6-cer 或两者的组合处理。评估细胞活力和细胞毒性协同作用。测定凋亡途径的激活、活性氧的产生和线粒体跨膜电位。DM102 是 MDA-MB-231、MCF-7 和 BT-474 细胞中比 N-油酰乙醇胺（NOE）和（1R，2R）-2-N-（十四烷酰氨基）-1-（4'-硝基苯基）-1,3-丙二醇（B-13）更有效的 AC 抑制剂。作为单一药物，C6-cer（IC505-10 μM）和 DM102（IC5020 μM）在 MDA-MB-231、MCF-7 和 SK-BR-3 细胞中仅具有中度细胞毒性。然而，联合给药可使所有细胞系的活力协同降低（组合指数<0.5）。在 MDA-MB-231 细胞中，通过检测半胱天冬酶 3 切割和半胱天冬酶 3/7 激活增加 3 倍以上、PARP 切割和 Annexin-V 阳性细胞增加 70%以上，证实了细胞凋亡。C6-cer/DM102 使 MDA-MB-231 细胞中的 ROS 水平增加 4 倍，使 Bax：Bcl-2 的比值增加到对照细胞的>9 倍，并导致线粒体膜去极化。当存在 C6-cer 时，DM102 还使（3）H-棕榈酸标记的长链神经酰胺的合成增加 2 倍。这些数据支持靶向 AC 与外源性短链神经酰胺联合作为抗癌策略的有效性，并证明在人类乳腺癌中继续研究 C6-cer/DM102 药物双药的效用是合理的。
