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.
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 microM) for 24 h, mean intracellular probucol concentrations reached 0.47, 0.67, and 1.52 microg/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 microM 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.
Ou
J
J
Department of Internal Medicine, Fukuoka University School of Medicine, 45-1-7 Nanakuma Jonanku, Fukuoka 814-80, Japan.
Saku
K
K
Jimi
S
S
Ohta
T
T
Zhang
B
B
Pownall
H J
HJ
Shimada
Y
Y
Tsujita
Y
Y
Arakawa
K
K
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D016466:CHO Cells; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000096:biosynthesis; D053480:Cholesterol Ester Transfer Proteins; D006224:Cricetinae; D006023:Glycoproteins; D000960:Hypolipidemic Agents / Q000494:pharmacology*; D011341:Probucol / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis*; D014162:Transfection
D000975:Antioxidants; C506642:CETP protein, human; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D006023:Glycoproteins; D000960:Hypolipidemic Agents; D012333:RNA, Messenger; D002784:Cholesterol; D011341:Probucol