DISCUSSION
Several studies have reported that individual differences in drug response cannot be fully explained by polymorphisms in genes encoding drug-metabolizing enzymes or transporters17, 18. Recently, epigenetic modifications, which regulate the expression of several enzymes and transporters involved in drug metabolism, have been recognised as important factors that affect individual differences in clinical drug response19. DNA methylation affects the expression ofCYP450 (CYP1A1 , CYP1A2 , CYP1B1 ,CYP2C19 , CYP2D6 , CYP2E1 , and CYP2W1 ), thus leading to significant individual differences in enzyme expression20-23. In addition, DNA methylation regulates the expression of ABCG2 and ABCB1 , which play a crucial role in determining the success or failure of cancer chemotherapy by mediating multi-drug resistance and individual differences in drug transport24, 25. As most studies on the methylation of genes encoding drug transporters have been carried out in the field of oncology, we investigated, for the first time, whether ABCB1 DNA methylation in donor livers affects tacrolimus plasma concentrations in liver transplant recipients by regulating its expression.
In this study, we analysed 15 donor liver samples carrying theCYP3A5*3/* 3 genotype using DNA methylation microarray technology, and we found ABCB1 methylation levels to be correlated with tacrolimus serum concentrations in liver transplantation patients. Based on the findings of previous studies carried out on ABCB1methylation and tacrolimus metabolism,2, 26, 27 we speculated that ABCB1 DNA methylation might be another key factor that affects tacrolimus metabolism by regulating ABCB1expression.
Previous studies have demonstrated that there exists no correlation between the frequency of ABCB1 gene polymorphisms and tacrolimus plasma concentrations following renal transplantation,4, 28, 29 and this is consistent with the findings of one of our previous studies [in publishing progress] . However, our studies found that there exist significant individual differences in tacrolimus plasma concentrations in liver transplant recipients who receive donor livers with theCYP3A5*3/*3 genotype; thus, for the first time, we evaluated the methylation status of 23 liver samples carrying the CYP3A5*3/*3genotype using a methylation microarray assay validated by pyrosequencing. Our findings showed that DNA methylation levels at threeABCB1 CpG sites (cg12501229, cg00634941, and cg05496710) in donor livers were significantly different between the high and low tacrolimus C0/D ratio groups following liver transplantation. In addition, in consonance with the findings of previous studies,30-32 ABCB1 mRNA levels in donor livers were found to be negatively correlated with its methylation levels.
To the best of our knowledge, no studies have been carried out on the effects of ABCB1 methylation, especially of its three CpG sites (cg12501229, cg00634941, and cg05496710), on tacrolimus metabolism. Therefore, in this study, the effects of ABCB1 methylation on tacrolimus metabolism were first evaluated using the methylation inhibitor, 5-Aza-2-DC. It was shown that in HepG2 cells, ABCB1 methylation levels at its three methylation sites significantly decreased following treatment with 5-Aza-2-DC. Moreover, tacrolimus intracellular concentrations significantly decreased following treatment with 5-Aza-2-DC. In addition, ABCB1mRNA and protein levels significantly increased following treatment with 5-Aza-2-DC. For clinical samples, the methylation levels of the cg12501229, cg00634941, and cg05496710 sites in the high C0/D group were all significantly lower than those in the low C0/D group, and ABCB1 mRNA expression was found to be positively correlated with tacrolimus C0/D ratio. These findings indicate that a decrease in DNA methylation could result in an increase in ABCB1 expression in donor livers, which would lead to an increase in tacrolimus excretion from liver cells and a consequent increase in tacrolimus plasma concentrations of the recipient.
This study had one limitation, namely, we did not construct a methylation-specific expression plasmid to determine which of the threeABCB1 methylation sites plays a leading role in the regulation of gene expression; however, further studies will be conducted on this in the future.