DNA Methyltransferase Inhibitor Zebularine Inhibits Human Hepatic Carcinoma Cells Proliferation and Induces Apoptosis

Abstract

Hepatocellular carcinoma is one of the most common cancers worldwide. During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. In this study, we explore the effect and possible mechanism of action of zebularine on hepatocellular carcinoma cell line HepG2. We demonstrate that zebularine exhibits antitumor activity on HepG2 cells by inhibiting cell proliferation and inducing apoptosis, however, it has little effect on DNA methylation in HepG2 cells. On the other hand, zebularine treatment downregulated CDK2 and the phosphorylation of retinoblastoma protein (Rb), and upregulated p21WAF/CIP1 and p53. We also found that zebularine treatment upregulated the phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). These results suggest that the p44/42 MAPK pathway plays a role in zebularine-induced cell-cycle arrest by regulating the activity of p21WAF/CIP1 and Rb. Furthermore, although the proapoptotic protein Bax levels were not affected, the antiapoptotic protein Bcl-2 level was downregulated with zebularine treatment. In addition, the data in the present study indicate that inhibition of the double-stranded RNA-dependent protein kinase (PKR) is involved in inducing apoptosis with zebularine. These results suggest a novel mechanism of zebularine-induced cell growth arrest and apoptosis via a DNA methylation-independent pathway in hepatocellular carcinoma.

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

“…In this study, we found that neither 5-aza-dC nor RG108 treatment could affect PKR phosphorylation or PKR protein level (Fig. 7A,B), while it is known that zebularine treatment downregulates PKR phosphorylation and PKR protein level in HepG2 cells22. These findings indicate that 5-aza-dC and RG108 affect DNMT1 activity but not PKR.…”

“…In our previous study22, we reported that zebularine treatment downregulated the expression of DNMT1 protein in HepG2 cells, implying that zebularine can upregulate the expression levels of CYP genes through suppressing the expression of DNMT1. To examine the action mechanism by which zebularine upregulates the expression levels of CYP genes, we knocked down DNMT1 gene expression in HepG2 cells and examined the expression levels of CYP genes.…”

“…reported that cell functions including albumin secretion, urea production and the expression of CYP1A1 and CYP2B1 were suppressed in cultured primary hepatocytes while the cell cycle was progressing, suggesting that cell cycle inhibition may be crucial to the maintenance of primary hepatocytes’ functions during culture32. In our previous report, we found that zebularine inhibited cell proliferation at least in part through the suppression of PKR in HepG2 cells22. Taken together, the available evidence suggests that PKR might trigger the promotion of cell proliferation through the NF-κB or p38 MAPK pathways, resulting in inhibition of CYP gene expression in HepG2 cells.…”

“…In order to investigate these possible mechanism(s), we assessed the effect of PKR inhibition on CYP gene expression, since our previous study had revealed that zebularine inhibits not only DNMT1 but also PKR in HepG2 cells22. We treated HepG2 cells with a PKR inhibitor for 72 h and investigated the expression levels of CYP genes.…”

Therapeutic Response to Paroxetine in Major Depressive Disorder Predicted by DNA Methylation

“…This calculated value, known as β, ranges from 0 (completely unmethylated) to 1 (fully methylated). Nakamura et al [17], Yuen et al [18], and Córdova-Palomera et al [19] proposed that a difference in methylation between 2 groups is significant if it is >10%. We adopted this assumption, setting a threshold at 10%, i.e., a difference in β between BR and WR (Δβ) >10% (|Δβ| >0.1) was defined as a significant difference.…”