Fetal bovine serum and human constitutive androstane receptor: evidence for activation of the SV23 splice variant by artemisinin, artemether, and arteether in a serum-free cell culture system.
The naturally occurring SV23 splice variant of human constitutive androstane receptor (hCAR-SV23) is activated by di-(2-ethylhexyl)phthalate (DEHP), which is detected as a contaminant in fetal bovine serum (FBS). In our initial experiment, we compared the effect of dialyzed FBS, charcoal-stripped, dextran-treated FBS (CS-FBS), and regular FBS on the basal activity and ligand-activation of hCAR-SV23 in a cell-based reporter gene assay. In transfected HepG2 cells cultured in medium supplemented with 10% FBS, basal hCAR-SV23 activity varied with the type of FBS (regular>dialyzed>CS). DEHP increased hCAR-SV23 activity when 10% CS-FBS, but not regular FBS or dialyzed FBS, was used. With increasing concentrations (1-10%) of regular FBS or CS-FBS, hCAR-SV23 basal activity increased, whereas in DEHP-treated cells, hCAR-SV23 activity remained similar (regular FBS) or slightly increased (CS-FBS). Subsequent experiments identified a serum-free culture condition to detect DEHP activation of hCAR-SV23. Under this condition, artemisinin, artemether, and arteether increased hCAR-SV23 activity, whereas they decreased it in cells cultured in medium supplemented with 10% regular FBS. By comparison, FBS increased the basal activity of the wild-type isoform of hCAR (hCAR-WT), whereas it did not affect the basal activity of the SV24 splice variant (hCAR-SV24) or ligand activation of hCAR-SV24 and hCAR-WT by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). The use of serum-free culture condition was suitable for detecting CITCO activation of hCAR-WT and hCAR-SV24. In conclusion, FBS leads to erroneous classification of pharmacological ligands of hCAR-SV23 in cell-based assays, but investigations on functional ligands of hCAR isoforms can be conducted in serum-free culture condition.
Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada.
Aik Jiang
AJ
Lau
Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada. Electronic address: thomas.chang@ubc.ca.
Thomas K H
TK
Chang
D000077549:Artemether; D037621:Artemisinins / Q000494:pharmacology*; D001681:Biological Assay / Q000379:methods*; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D056945:Hep G2 Cells; D006801:Humans; D008024:Ligands; D020033:Protein Isoforms; D011944:Receptors, Androgen / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D044967:Serum / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
C410798:AR protein, human; D037621:Artemisinins; D008024:Ligands; D020033:Protein Isoforms; D011944:Receptors, Androgen; C031327:artemisinin; D000077549:Artemether; C055141:artemotil
Arteether; Artemether; Artemisinin; Constitutive androstane receptor; Di-(2-ethylhexyl)phthalate; Fetal bovine serum