Decreased ABCB1 mRNA expression induced by atorvastatin results from enhanced mRNA degradation in HepG2 cells.
The mechanisms underlying atorvastatin supression of ABCB1 gene expression, at transcriptional and post-transcriptional levels of ABCB1 gene in HepG2 (human hepatocellular carcinoma) cells were investigated. Quantitative real-time PCR was used to measure mRNA levels, as well as to estimate the half-life of ABCB1 mRNA. Western blotting analysis was performed in order to measure protein levels of ABCB1. Electrophoretic mobility shift assay (EMSA) was used to evaluate interactions between protein(s) and ABCB1 promoter region. Exposure to atorvastatin for 24h resulted in a dose-dependent decrease of ABCB1 mRNA and protein levels, which was not abolished by addition of farnesyl or geranylgeranyl pyrophosphate. After removing fetal bovine serum from the media, however, ABCB1 expression was decreased by 2-fold in either HepG2 cells treated and non-treated with atorvastatin. Addition of cholesterol to serum free media abolished this latter effect on ABCB1 mRNA levels. In EMSA using a 5'-end-labeled 241bp ABCB1 promoter DNA fragment (-198 to +43) as probe, the binding of the proteins to the probe was reduced by NF-Y, but not changed by NFkappaB, AP-1, and SP1. However, the NF-Y binding activity was similar in control and atorvastatin-treated cells. mRNA stability studies revealed that ABCB1 mRNA degradation was increased in 1, 10 and 20muM atorvastatin-treated versus control cells (half-lives of 2h versus 7h). Therefore, evidence is provided that decreased mRNA stability by atorvastatin treatment may explain the decrease in ABCB1 transcript levels.
Rodrigues
Alice Cristina
AC
Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes, 580, Sao Paulo, SP 05508-900, Brazil. alice-rodrigues@usp.br
Curi
Rui
R
Hirata
Mario Hiroyuki
MH
Hirata
Rosario Dominguez Crespo
RD
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics; D000069059:Atorvastatin; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002784:Cholesterol / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D015536:Down-Regulation / Q000187:drug effects; D024202:Electrophoretic Mobility Shift Assay; D006207:Half-Life; D006538:Heptanoic Acids / Q000494:pharmacology*; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology*; D011758:Pyrroles / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016895:Culture Media, Serum-Free; D006538:Heptanoic Acids; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D011758:Pyrroles; D012333:RNA, Messenger; D014157:Transcription Factors; D002784:Cholesterol; D000069059:Atorvastatin