van der Luit Arnold H, Vink Stefan R, Klarenbeek Jeffrey B, Perrissoud Daniel, Solary Eric, Verheij Marcel, van Blitterswijk Wim J
Division of Cellular Biochemistry, the Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.
Mol Cancer Ther. 2007 Aug;6(8):2337-45. doi: 10.1158/1535-7163.MCT-07-0202.
Single-chain alkylphospholipids, unlike conventional chemotherapeutic drugs, act on cell membranes to induce apoptosis in tumor cells. We tested four different alkylphospholipids, i.e., edelfosine, perifosine, erucylphosphocholine, and compound D-21805, as inducers of apoptosis in the mouse lymphoma cell line S49. We compared their mechanism of cellular entry and their potency to induce apoptosis through inhibition of de novo biosynthesis of phosphatidylcholine at the endoplasmic reticulum. Alkylphospholipid potency closely correlated with the degree of phosphatidylcholine synthesis inhibition in the order edelfosine > D-21805 > erucylphosphocholine > perifosine. In all cases, exogenous lysophosphatidylcholine, an alternative source for cellular phosphatidylcholine production, could partly rescue cells from alkylphospholipid-induced apoptosis, suggesting that phosphatidylcholine biosynthesis is a direct target for apoptosis induction. Cellular uptake of each alkylphospholipid was dependent on lipid rafts because pretreatment of cells with the raft-disrupting agents, methyl-beta-cyclodextrin, filipin, or bacterial sphingomyelinase, reduced alkylphospholipid uptake and/or apoptosis induction and alleviated the inhibition of phosphatidylcholine synthesis. Uptake of all alkylphospholipids was inhibited by small interfering RNA (siRNA)-mediated blockage of sphingomyelin synthase (SMS1), which was previously shown to block raft-dependent endocytosis. Similar to edelfosine, perifosine accumulated in (isolated) lipid rafts independent on raft sphingomyelin content per se. However, perifosine was more susceptible than edelfosine to back-extraction by fatty acid-free serum albumin, suggesting a more peripheral location in the cell due to less effective internalization. Overall, our results suggest that lipid rafts are critical membrane portals for cellular entry of alkylphospholipids depending on SMS1 activity and, therefore, are potential targets for alkylphospholipid anticancer therapy.
与传统化疗药物不同，单链烷基磷脂作用于细胞膜以诱导肿瘤细胞凋亡。我们测试了四种不同的烷基磷脂，即依地福新、哌立福新、芥酰磷酰胆碱和化合物D - 21805，作为小鼠淋巴瘤细胞系S49中凋亡的诱导剂。我们比较了它们进入细胞的机制以及通过抑制内质网中磷脂酰胆碱的从头生物合成来诱导凋亡的效力。烷基磷脂的效力与磷脂酰胆碱合成抑制程度密切相关，顺序为依地福新>D - 21805>芥酰磷酰胆碱>哌立福新。在所有情况下，外源性溶血磷脂酰胆碱（细胞磷脂酰胆碱产生的替代来源）可部分挽救细胞免受烷基磷脂诱导的凋亡，这表明磷脂酰胆碱生物合成是凋亡诱导的直接靶点。每种烷基磷脂的细胞摄取依赖于脂筏，因为用破坏脂筏的试剂甲基 - β - 环糊精、制霉菌素或细菌鞘磷脂酶预处理细胞会降低烷基磷脂摄取和/或凋亡诱导，并减轻对磷脂酰胆碱合成的抑制。所有烷基磷脂的摄取均被小干扰RNA（siRNA）介导的鞘磷脂合酶（SMS1）阻断所抑制，先前已证明SMS1可阻断脂筏依赖性内吞作用。与依地福新类似，哌立福新在（分离的）脂筏中积累，与脂筏鞘磷脂含量本身无关。然而，哌立福新比依地福新更容易被无脂肪酸血清白蛋白反萃取，这表明由于内化效率较低，其在细胞中的位置更外围。总体而言，我们的结果表明脂筏是依赖于SMS1活性的烷基磷脂进入细胞的关键膜通道，因此是烷基磷脂抗癌治疗的潜在靶点。
