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Effect of Zebularine on Apoptotic Pathways in Hepatocellular Carcinoma Cell Lines
Masumeh Sanaei1, Fraidoon Kavoosi1
1Department of Anatomy, Research Center for Non Communicable Diseases, Jahrom University of Medical Sciences, Jahrom, Iran.
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.
Insights
Zebularine, a DNA methyltransferase inhibitor, effectively induces apoptosis and inhibits growth in hepatocellular carcinoma cell lines by activating both intrinsic and extrinsic pathways. This epigenetic therapy shows promise for liver cancer treatment.
Area of Science:
- Oncology
- Epigenetics
- Molecular Biology
Background:
- DNA methylation alterations are common epigenetic changes in human cancers.
- DNA methyltransferases (DNMTs) regulate gene promoter methylation.
- DNMT inhibitors (DNMTIs) exhibit therapeutic and apoptotic effects in various cancers.
Purpose of the Study:
- To evaluate zebularine's effects on intrinsic and extrinsic apoptotic pathways in hepatocellular carcinoma (HCC) cell lines.
- To assess zebularine's impact on DNMTs (1, 3a, 3b), p21, p53, cell viability, and apoptosis.
- To investigate zebularine's potential as a therapeutic agent for HCC.
Main Methods:
- Hepatocellular carcinoma cell lines (HCCLM3, MHCC97H, MHCC97L) were treated with zebularine.
- MTT assay was used to assess cell viability.
- Flow cytometry and real-time RT-PCR analyzed apoptosis and gene expression.
- Statistical analysis was performed using GraphPad Prism (P < 0.05).
Main Results:
- Zebularine significantly up-regulated pro-apoptotic genes (DR4, DR5, FAS, FAS-L, TRAIL, Bax, Bak, Bim, p21, p53).
- Zebularine significantly down-regulated DNMTs (1, 3a, 3b) and anti-apoptotic genes (Bcl-2, Bcl-xL, Mcl-1).
- Apoptosis induction varied among cell lines, with maximal effect in HCCLM3 and minimal in MHCC97L.
Conclusions:
- DNMT inhibitor zebularine induces apoptosis and inhibits cell growth in HCC cell lines.
- Zebularine acts through both intrinsic and extrinsic apoptotic pathways.
- These findings support zebularine's potential as a therapeutic strategy for HCC.
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