Effect of Zebularine on Apoptotic Pathways in Hepatocellular Carcinoma Cell Lines

BACKGROUND: The alteration of DNA cytosine methylation is one of the most common epigenetic changes that can play a significant role in human cancers. The enzymes involved in DNA methylation of promoter regions of the genes are DNA methyltransferases (DNMTs). The therapeutic activities and apoptotic effects of DNA methyltransferase inhibitors (DNMTIs) have been reported in various cancers. This study was assigned to assess the effect of zebularine on intrinsic and extrinsic pathways, DNAT 1, 3a, and 3b, p21, and p53, viability, and apoptosis in hepatocellular carcinoma (HCC) cell lines. METHODS: Hepatocellular carcinoma cell lines (HCCLM3, MHCC97H, and MHCC97L) were purchased from the National Cell Bank of Iran, Pasteur Institute, treated with zebularine, and the MTT assay was performed. Then, flow cytometry assay and real-time RT-PCR analysis were performed with zebularine. Statistical comparisons between groups were made using GraphPad Prism software version 8.0. A significant difference was considered as P < 0.05. RESULTS: Zebularine up-regulated DR4, DR5, FAS, FAS-L, TRAIL, Bax, Bak, Bim, p21WAF/CIP1 (p21), and p53 and down-regulated DNMTs (DNAT 1, 3a, and 3b), Bcl-2, Bcl-xL, and Mcl-1, significantly resulting in apoptosis induction in HCC cell lines. Maximal and minimal apoptosis was seen in HCCLM3 and MHCC97L cell lines, respectively. CONCLUSIONS: Our findings indicated that DNMTI zebularine can induce apoptosis and inhibit cell growth through both pathways (extrinsic and intrinsic) in HCC cell lines HCCLM3, MHCC97H, and MHCC97L.