Delta-like 3 is silenced by methylation and induces apoptosis in human hepatocellular carcinoma

Maemura,Kentaro; Yoshikawa,Hirohide; Yokoyama,Kazutake; Ueno,Teruo; Kurose,Hitomi; Uchiyama,Kazuhisa; Otsuki,Yoshinori

doi:10.3892/ijo.2013.1778

Delta-like 3 is silenced by methylation and induces apoptosis in human hepatocellular carcinoma

Authors:
- Kentaro Maemura
- Hirohide Yoshikawa
- Kazutake Yokoyama
- Teruo Ueno
- Hitomi Kurose
- Kazuhisa Uchiyama
- Yoshinori Otsuki
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Affiliations: Department of Anatomy and Cell Biology, Osaka Medical College, Takatsuki, Osaka, Japan, Department of Internal Medicine, Sassa General Hospital, Nishi-tokyo, Tokyo, Japan, Central Research Unit, Osaka Medical College, Takatsuki, Osaka, Japan, Department of General and Gastroenterological Surgery, Osaka Medical College, Takatsuki, Osaka, Japan
Published online on: January 17, 2013 https://doi.org/10.3892/ijo.2013.1778
Pages: 817-822
Copyright: © Maemura et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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Abstract

The genetic and epigenetic events of hepatocarcinogenesis are relatively poorly understood. By analyzing genes from human hepatocellular carcinoma (HCC) with restriction landmark genomic scanning, several aberrantly methylated genes, including Delta-like 3 (DLL3), have been isolated. In this study, we investigated the function of DLL3 in hepatocarcinogenesis. Methylation of the DLL3 gene in HCC cell lines was investigated with methylation-specific PCR and expression of DLL3 mRNA in HCC cells was examined by RT-PCR. Reactivation of DLL3 expression by treatment with a demethylating agent was examined in methylation-silenced HuH2 cells. Human DLL3 cDNA was cloned and DLL3 function was examined by restoring DLL3 expression in HuH2 cells. The effects of DLL3 on cell growth were evaluated by colony formation assay. Induction of cell death by overexpression of DLL3 was examined by flow cytometric assay using Annexin V and PI. Apoptotic cells were detected by TUNEL staining and the amount of single-stranded DNA was measured by ELISA. As a result, the promoter region of the DLL3 gene was methylated in four of ten HCC cell lines. This aberrant methylation correlated well with the suppression of RNA expression and a demethylating agent reactivated DLL3 expression in methylation-silenced HCC cells. Interestingly, the restoration of DLL3 in the methylation-silenced HuH2 cells led to growth suppression on colony formation assay. Flow cytometric assay with Annexin V and PI showed that this growth suppression by DLL3 expression is associated with the induction of apoptosis. Furthermore, these apoptotic effects were confirmed by TUNEL staining and measurement of single-stranded DNA. These results suggest that DLL3 was silenced by methylation in human HCC and that it negatively regulates the growth of HCC cells.

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