Carrasco María P, Jiménez-López José M, Segovia Josefa L, Marco Carmen
Department of Biochemistry and Molecular Biology I, Faculty of Sciences, University of Granada, Avenida Fuentenueva s/n, Granada, Spain.
FEBS J. 2008 Apr;275(8):1675-86. doi: 10.1111/j.1742-4658.2008.06322.x. Epub 2008 Feb 27.
We have shown, in a previous publication, that nontoxic concentrations of hexadecylphosphocholine exert an antiproliferative effect on HepG2 cells. Hexadecylphosphocholine also interferes with the biosynthesis of cholesterol and phosphatidylcholine. We have now extended our studies to try to establish the molecular mechanism by which hexadecylphosphocholine disrupts cholesterol homeostasis. Using radiolabelled substrates we determined the effect of hexadecylphosphocholine on cholesterol synthesis, the destiny of cholesterol from low-density lipoprotein and the transport of cholesterol between the plasma membrane and the endoplasmic reticulum. Protein levels and gene expression of the main proteins involved in cholesterol homeostasis were analysed by western blotting and RT-PCR, respectively. HepG2 cells exposed to hexadecylphosphocholine showed an increase in cholesterol biosynthesis when acetate, but not mevalonate, was used as a substrate. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) and low-density lipoprotein receptor, as well as the corresponding mRNA expression, increased after 24 h of treatment with hexadecylphosphocholine. Cholesteryl linoleate in low-density lipoprotein uptake and further hydrolysis of these esters increased but the cholesterol esterification was reduced after 6 h of treatment with alkylphosphocholine. Cholesterol transport from the plasma membrane to the endoplasmic reticulum was impaired by hexadecylphosphocholine. In conclusion, hexadecylphosphocholine interfered with the transport of cholesterol from the cell surface to the endoplasmic reticulum, leading to a depletion of cholesterol in the endoplasmic reticulum and a deregulation of cholesterol biosynthesis. The accumulation of cholesterol within the cell and the reduction in phosphatidylcholine synthesis produces an alteration in the phosphatidylcholine/cholesterol ratio that may well be responsible for the antiproliferative activity exhibited by hexadecylphosphocholine in HepG2 cells.
我们在之前的一篇出版物中表明，无毒浓度的十六烷基磷胆碱对HepG2细胞具有抗增殖作用。十六烷基磷胆碱还会干扰胆固醇和磷脂酰胆碱的生物合成。我们现在扩展了研究，试图确定十六烷基磷胆碱破坏胆固醇稳态的分子机制。使用放射性标记的底物，我们确定了十六烷基磷胆碱对胆固醇合成、低密度脂蛋白中胆固醇的去向以及胆固醇在质膜和内质网之间运输的影响。分别通过蛋白质印迹法和逆转录聚合酶链反应分析了参与胆固醇稳态的主要蛋白质的水平和基因表达。当使用乙酸盐而非甲羟戊酸作为底物时，暴露于十六烷基磷胆碱的HepG2细胞显示胆固醇生物合成增加。用十六烷基磷胆碱处理24小时后，3-羟基-3-甲基戊二酰辅酶A还原酶（EC 1.1.1.34）和低密度脂蛋白受体的活性以及相应的mRNA表达增加。用烷基磷胆碱处理6小时后，低密度脂蛋白摄取中的胆固醇亚油酸酯以及这些酯的进一步水解增加，但胆固醇酯化减少。十六烷基磷胆碱损害了胆固醇从质膜向内质网的运输。总之，十六烷基磷胆碱干扰了胆固醇从细胞表面向内质网的运输，导致内质网中胆固醇的消耗和胆固醇生物合成的失调。细胞内胆固醇的积累和磷脂酰胆碱合成的减少导致磷脂酰胆碱/胆固醇比值的改变，这很可能是十六烷基磷胆碱在HepG2细胞中表现出抗增殖活性的原因。
