School of Medicine Cancer Center, Texas Tech University Health Sciences Center, Lubbock, Texas, United States of America.
PLoS One. 2013 Sep 9;8(9):e74768. doi: 10.1371/journal.pone.0074768. eCollection 2013.
We previously reported that fenretinide (4-HPR) was cytotoxic to acute lymphoblastic leukemia (ALL) cell lines in vitro in association with increased levels of de novo synthesized dihydroceramides, the immediate precursors of ceramides. However, the cytotoxic potentials of native dihydroceramides have not been defined. Therefore, we determined the cytotoxic effects of increasing dihydroceramide levels via de novo synthesis in T-cell ALL cell lines and whether such cytotoxicity was dependent on an absolute increase in total dihydroceramide mass versus an increase of certain specific dihydroceramides. A novel method employing supplementation of individual fatty acids, sphinganine, and the dihydroceramide desaturase-1 (DES) inhibitor, GT-11, was used to increase de novo dihydroceramide synthesis and absolute levels of specific dihydroceramides and ceramides. Sphingolipidomic analyses of four T-cell ALL cell lines revealed strong positive correlations between cytotoxicity and levels of C22:0-dihydroceramide (ρ = 0.74-0.81, P ≤ 0.04) and C24:0-dihydroceramide (ρ = 0.84-0.90, P ≤ 0.004), but not between total or other individual dihydroceramides, ceramides, or sphingoid bases or phosphorylated derivatives. Selective increase of C22:0- and C24:0-dihydroceramide increased level and flux of autophagy marker, LC3B-II, and increased DNA fragmentation (TUNEL assay) in the absence of an increase of reactive oxygen species; pan-caspase inhibition blocked DNA fragmentation but not cell death. C22:0-fatty acid supplemented to 4-HPR treated cells further increased C22:0-dihydroceramide levels (P ≤ 0.001) and cytotoxicity (P ≤ 0.001). These data demonstrate that increases of specific dihydroceramides are cytotoxic to T-cell ALL cells by a caspase-independent, mixed cell death mechanism associated with increased autophagy and suggest that dihydroceramides may contribute to 4-HPR-induced cytotoxicity. The targeted increase of specific acyl chain dihydroceramides may constitute a novel anticancer approach.
我们之前报道过，芬维 A 酯（4-HPR）在体外对急性淋巴细胞白血病（ALL）细胞系具有细胞毒性，同时伴有从头合成的二氢神经酰胺水平升高，二氢神经酰胺是神经酰胺的直接前体。然而，天然二氢神经酰胺的细胞毒性潜力尚未确定。因此，我们确定了通过从头合成增加 T 细胞 ALL 细胞系中二氢神经酰胺水平的细胞毒性作用，以及这种细胞毒性是否依赖于总二氢神经酰胺质量的绝对增加与某些特定二氢神经酰胺的增加。采用补充单个脂肪酸、神经酰胺和二氢神经酰胺去饱和酶-1（DES）抑制剂 GT-11 的新方法来增加从头合成的二氢神经酰胺和特定二氢神经酰胺和神经酰胺的绝对水平。对四种 T 细胞 ALL 细胞系的神经鞘脂组学分析显示，细胞毒性与 C22:0-二氢神经酰胺（ρ=0.74-0.81，P≤0.04）和 C24:0-二氢神经酰胺（ρ=0.84-0.90，P≤0.004）水平之间存在强烈的正相关，但与总二氢神经酰胺或其他特定二氢神经酰胺、神经酰胺或神经鞘氨醇碱基或磷酸化衍生物之间没有相关性。选择性增加 C22:0-和 C24:0-二氢神经酰胺会增加自噬标记物 LC3B-II 的水平和通量，并增加 DNA 片段化（TUNEL 检测），而不会增加活性氧的产生；全半胱氨酸蛋白酶抑制剂阻断 DNA 片段化但不阻断细胞死亡。补充 C22:0-脂肪酸到 4-HPR 处理的细胞中进一步增加 C22:0-二氢神经酰胺水平（P≤0.001）和细胞毒性（P≤0.001）。这些数据表明，特定二氢神经酰胺的增加通过一种与自噬增加相关的 caspase 非依赖性混合细胞死亡机制对 T 细胞 ALL 细胞具有细胞毒性，并表明二氢神经酰胺可能有助于 4-HPR 诱导的细胞毒性。靶向增加特定酰基链二氢神经酰胺可能构成一种新的抗癌方法。
