Zebularine upregulates expression of CYP genes through inhibition of DNMT1 and PKR in HepG2 cells

Abstract

Drug-induced hepatotoxicity is one of the major reasons cited for drug withdrawal. Therefore, it is of extreme importance to detect human hepatotoxic candidates as early as possible during the drug development process. In this study, we aimed to enhance hepatocyte functions such as CYP gene expression in HepG2 cells, one of the most extensively used cell lines in evaluating hepatotoxicity of chemicals and drugs. We found that zebularine, a potent inhibitor of DNA methylation, remarkably upregulates the expression of CYP genes in HepG2 cells. In addition, we revealed that the upregulation of CYP gene expression by zebularine was mediated through the inhibition of both DNA methyltransferase 1 (DNMT1) and double-stranded RNA-dependent protein kinase (PKR). Furthermore, HepG2 cells treated with zebularine were more sensitive than control cells to drug toxicity. Taken together, our results show that zebularine may make HepG2 cells high-functioning and thus could be useful for evaluating the hepatotoxicity of chemicals and drugs speedily and accurately in in-vitro systems. The finding that zebularine upregulates CYP gene expression through DNMT1 and PKR modulation sheds light on the mechanisms controlling hepatocyte function and thus may aid in the development of new in-vitro systems using high-functioning hepatocytes.

Figure 1. The effect of zebularine on CYP gene expression in HepG2 cells.

(A–I) HepG2 cells were exposed to zebularine for 72 h. Expression levels of CYP1A1 (A), 1A2 (B), 2A6 (C), 2B6 (D), 2C9 (E), 2C19 (F), 2D6 (G), 2E1 (H) and 3A4 (I) were examined by qRT-PCR. The comparative threshold cycle (Ct) method was used to determine the relative ratio of expression for each gene, which was corrected against HPRT1. Each data point represents the mean ± SEM of the values obtained from three independent experiments. The expression level measured after exposure to 0 mM zebularine was considered to be 1.0 expression. *p < 0.05, compared to 0 mM.

Figure 2. The effect of 5-aza-dC on CYP gene expression in HepG2 cells.

(A–I) HepG2 cells were exposed to 5-aza-dC for 72 h. Expression levels of CYP1A1 (A), 1A2 (B), 2A6 (C), 2B6 (D), 2C9 (E), 2C19 (F), 2D6 (G), 2E1 (H) and 3A4 (I) were examined by qRT-PCR. The comparative threshold cycle (Ct) method was used to determine the relative ratio of expression for each gene, which was corrected against HPRT1. Each data point represents the mean ± SEM of the values obtained from three independent experiments. For each gene, the expression level detected in cells treated with 0 mM 5-aza-dC was considered to be 1.0 expression. *p < 0.05, compared to 0 mM.

Figure 3. The effect of RG108 on CYP gene expression in HepG2 cells.

(A–I) HepG2 cells were exposed to RG108 for 72 h. Expression levels of CYP1A1 (A), 1A2 (B), 2A6 (C), 2B6 (D), 2C9 (E), 2C19 (F), 2D6 (G), 2E1 (H) and 3A4 (I) were examined by qRT-PCR. The comparative threshold cycle (Ct) method was used to determine the relative ratio of expression for each gene, which was corrected against HPRT1. Each data point represents the mean ± SEM of the values obtained from three independent experiments. For each gene, the expression level detected in cells treated with 0 mM RG108 was considered to be 1.0 expression. *p < 0.05, compared to 0 mM.

Figure 4. The effect of knockdown of DNMT1 on CYP gene expression in HepG2 cells.

(A) HepG2 cells were transfected with siRNA against DNMT1 or negative control. After 72 h transfection, the cells were harvested and western blot analysis was performed to measure the DNMT1 protein level. GAPDH was used as a loading control. (B) Expression levels of CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 in HepG2 cells transfected with siRNA against DNMT1 were examined by qRT-PCR. The comparative threshold cycle (Ct) method was used to determine the relative ratio of expression for each gene, which was corrected against HPRT1. Each data point represents the mean ± SEM of the values obtained from three independent experiments. The expression level of HepG2 transfected with negative control was considered to be 1.0 expression. *p < 0.05, compared to control HepG2 cells.

Figure 5. The effect of PKR inhibitor on CYP gene expression in HepG2 cells.

(A–I) HepG2 cells were exposed to PKR inhibitor for 72 h. Expression levels of CYP1A1 (A), 1A2 (B), 2A6 (C), 2B6 (D), 2C9 (E), 2C19 (F), 2D6 (G), 2E1 (H) and 3A4 (I) were examined by qRT-PCR. The comparative threshold cycle (Ct) method was used to determine the relative ratio of expression for each gene, which was corrected with HPRT1. Each data point represents the mean ± SEM of the values obtained from three independent experiments. The expression level measured after exposure to 0 μM PKR inhibitor was considered to be 1.0 expression. *p < 0.05, compared to 0 μM.

Figure 6. The effect of combined DNMT1 and PKR inhibition on CYP gene expression in HepG2 cells.

(A) shDNMT1 was stably transfected into HepG2 cells and shDNMT1-HepG2-2, -5 and -6 cells showed downregulation of DNMT1 protein level. (B) Expression levels of CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 were examined in shDNMT1-HepG2-2, -5 and -6 cells by qRT-PCR. The expression level observed in parent cells was considered to be 1.0 expression. (C–E) shDNMT1-HepG2-2 cells (C), shDNMT1-HepG2-5 cells (D) and shDNMT1-HepG2-6 cells (E) were exposed to 1000 μM PKR inhibitor for 72 h, and expression levels of CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 were examined by qRT-PCR. The expression level of parent HepG2 cells without PKR inhibitor treatment was considered to be 1.0 expression. The comparative threshold cycle (Ct) method was used to determine the relative ratio of expression for each gene, which was corrected against HPRT1. Each data point represents the mean ± SEM of the values obtained from three independent experiments. *p < 0.05, compared to parent HepG2 cells.

Figure 7. The effect of the combination of either 5-aza-dC or RG108 and PKR inhibitor on CYP gene expression in HepG2 cells.

(A,B) The phosphorylation and expression of PKR after 5-aza-dC (A) or RG108 (B) treatment for 72 h at different concentrations. After treatment, the cells were harvested and western blot analysis was performed to detect the phosphorylated and total PKR protein levels. GAPDH was used as a loading control. A typical image is shown and the experiment was performed independently three times under each condition. (C,D) Expression levels of CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 were examined in HepG2 cells exposed to a combination of PKR inhibitor and either 5-aza-dC (C) or RG108 (D). After 72 h exposure, expression levels of CYPs were examined by qRT-PCR. The comparative threshold cycle (Ct) method was used to determine the relative ratio of expression for each gene, which was corrected against HPRT1. Each data point represents the mean ± SEM of the values obtained from three independent experiments. The expression levels measured in control HepG2 cells were considered to be 1.0 expression. *p < 0.05, compared to control HepG2 cells.

Figure 8. The effect of zebularine treatment on evaluation of drug toxicity.

(A,B) Comparison of cell viability with and without zebularine. After pretreatment with zebularine for 72 h, cells were exposed to acetaminophen (A) or aflatoxin B1 (B) for the next 72 h and then examined for cell viability. Each data point represents the mean ± SEM of the values obtained from three independent experiments. Zebularine-treated and untreated cell cultures exposed to 0 mM acetaminophen or 0 μM aflatoxin B1 were considered to be 100% viable. *p < 0.05, compared to the untreated (-zebularine) group. (C) Analysis of CYP2E1 and CYP3A4 protein levels after exposure to zebularine. After 72 h zebularine exposure, the cells were harvested and western blot analysis was performed to measure the CYP2E1 or 3A4 protein level. GAPDH was used as a loading control.