Mechanism of action of probucol on cholesteryl ester transfer protein (CETP) mRNA in a Chinese hamster ovary cell line that had been stably transfected with a human CETP gene

Jiafu Ou , Keijiro Saku , Shiro Jimi , Takao Ohta , Bo Zhang , Henry J. Pownall , Yoko Shimada , Yoshio Tsujita , Kikuo Arakawa
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引用次数: 10

Abstract

Probucol, a widely used lipid-lowering agent, is associated with a significant reduction of plasma high density lipoprotein (HDL)-cholesterol levels. To examine the mechanism of probucol HDL-lowering and probucol’s effects on cholesteryl ester transfer protein (CETP) and cholesterol metabolism in cells, we used a Chinese hamster ovary (CHO) cell line that had been stably transfected with a human CETP gene (hCETP-CHO). After this cell line was incubated with various concentrations of probucol (5, 10 and 50 μM) for 24 h, mean intracellular probucol concentrations reached 0.47, 0.67, and 1.52 μg/mg cell protein, respectively. Northern blot analysis showed that cellular CETP mRNA was increased by probucol in a dose-dependent manner (137%, 162%, and 221% of the control, respectively). The specific CET activity in the culture medium, measured as the percentage of [3H]cholesterol oleate transferred from discoidal bilayer particles (which mimic HDL) to LDL, also increased in a dose-dependent manner. Intracellular total cholesterol levels were decreased to 87.5%, 74.9%, and 52.5% of the control, respectively. Probucol had no effects on HMG-CoA reductase activity or cholesterol synthesis from [14C]acetate in hCETP-CHO. However, 14C-incorporated cholesterol secretion into the culture medium from hCETP-CHO was increased to 181%, 256% and 354% of the control by 5, 10 and 50 μM probucol, respectively. We concluded that (1) treatment with probucol increased the CETP mRNA level and specific CET activity in the hCETP-CHO cell line, and (2) probucol promoted cholesterol efflux from hCETP-CHO, which resulted in a decrease in intracellular cholesterol levels.

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普罗布考对稳定转染人CETP基因的中国仓鼠卵巢细胞系胆固醇酯转移蛋白mRNA的作用机制
普罗布考是一种广泛使用的降脂剂,可显著降低血浆高密度脂蛋白(HDL)-胆固醇水平。为了研究普罗布考降低高密度脂蛋白的作用机制,以及普罗布考对细胞内胆固醇酯转移蛋白(CETP)和胆固醇代谢的影响,我们使用稳定转染人CETP基因(hCETP-CHO)的中国仓鼠卵巢(CHO)细胞系。不同浓度(5 μM、10 μM和50 μM)的普罗布考作用24 h后,普罗布考细胞内平均浓度分别达到0.47、0.67和1.52 μg/mg细胞蛋白。Northern blot分析显示普罗布考使细胞CETP mRNA呈剂量依赖性增加(分别为对照组的137%、162%和221%)。培养基中的特定CET活性,以[3H]油酸胆固醇从盘状双层颗粒(类似HDL)转移到LDL的百分比来测量,也以剂量依赖的方式增加。细胞内总胆固醇水平分别降至对照组的87.5%、74.9%和52.5%。普罗布考对hCETP-CHO中HMG-CoA还原酶活性和[14C]乙酸合成胆固醇没有影响。然而,hCETP-CHO培养基中的14C-incorporated胆固醇分泌量分别增加了5 μM、10 μM和50 μM普罗布考,分别达到对照的181%、256%和354%。我们得出结论:(1)普罗布考提高了hCETP-CHO细胞系中CETP mRNA水平和特异性CET活性;(2)普罗布考促进了hCETP-CHO的胆固醇外排,导致细胞内胆固醇水平降低。
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