Effects of 5-Aza-2-DC on ABCB1 expression in HepG2 cells
First, the optimal concentration and administration time for 5-Aza-2-DC
were determined (Figure 4A-C ). Then, based on findings in
literature, 10 μM 5-Aza-2-DC was used to investigate the effects ofABCB1 methylation status on its expression in HepG2 cells. We
found that after treatment with 10 μM 5-Aza-2-DC for 24, 48 and 72 h,ABCB1 mRNA and protein expression levels significantly increased
at 72 h (Figure 4D-F ). Therefore, 10 μM 5-Aza-2-DC and 72 h
were selected as the optimal concentration and treatment duration,
respectively, for the determination of the effects of 5-Aza-2-DC on the
methylation levels of the three ABCB1 CpG sites (cg12501229,
cg00634941, and cg05496710).
Effects of
5-Aza-2-DC on ABCB1
methylation levels and tacrolimus metabolism in HepG2 cells
A pyrophosphorylation assay was performed to investigate the effects of
5-Aza-2-DC on ABCB1 methylation. As shown in Figure 5 ,
we found that the methylation levels of the ABCB1 sites in the
5-Aza-2-DC-treated group (10 μM) were significantly lower (P< 0.001) than those in the untreated group (0 μM). To
determine the effects of 5-Aza-2-DC on tacrolimus metabolism,
intracellular tacrolimus concentrations were determined by UHPLC-MS/MS.
We found the tacrolimus contents of 5-Aza-2-DC-treated cells to be
significantly lower than those of untreated cells at 0, 4, and 6 h
following the removal of tacrolimus-containing medium (P< 0.05). At 12 h, although there was a slight increase in
intracellular tacrolimus concentrations, its levels in the
5-Aza-2-DC-treated group were still significantly lower than those in
the untreated group (0 μM), and this may be due to its intracellular
metabolism in a dynamic equilibrium scenario (Figure 6 ).