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.