Arsenic trioxide inhibits DNA methyltransferase and restores methylation-silenced genes in human liver cancer cells

doi:10.1016/j.humpath.2005.10.013

Human Pathology

Volume 37, Issue 3, March 2006, Pages 298-311

https://doi.org/10.1016/j.humpath.2005.10.013 Get rights and content

Summary

In the present study, we investigated methylation status of the CpG islands of some major tumor suppressor genes both in human hepatocellular carcinoma and liver cancer cell lines and examined whether demethylation by arsenic trioxide (As₂O₃) could restore their expression in the cell lines. HepG2 and Huh-7 cells were treated with 2 to 10 μmol/L of As₂O₃ and/or 1 μmol/L of 5-aza-2′-deoxycytidine for 24, 48, and 72 hours. The methylation status of the CpG island around the promoter regions of p16^INK4a, RASSF1A, E cadherin, and GSTP1 was detected by a methylation-specific polymerase chain reaction (MSP). The messenger RNA (mRNA) and protein levels of these genes were determined by quantitative real-time reverse transcriptase–polymerase chain reaction, Western blot, and immunohistochemical analyses. The DNA methyltransferase (DNMT) mRNA levels and enzyme activity were also examined. The hypermethylated status of the promoter regions of p16^INK4a, RASSF1A, E cadherin, and GSTP1 was observed in 10 (40%), 14 (56%), 6 (24%), and 12 (48%) of 25 patients with hepatocellular carcinoma, respectively. CpG methylation of the p16^INK4a, RASSF1A, E cadherin, and GSTP1 genes was correlated to the reduction of mRNA levels in the cell lines, and mRNA expression of these 4 genes were indeed restored by low concentrations (2-6 μmol/L) of As₂O₃ through demethylation, as well as 1 μmol/L of 5-aza-2′-deoxycytidine. Western blot and immunohistochemical analyses confirmed that each protein was markedly enhanced after treatment with a low concentration of As₂O₃. In contrast, As₂O₃ at a high concentration (10 μmol/L) damaged cell membranes and remarkably suppressed these 4 protein levels. As₂O₃ decreased the mRNA expression of DNMT 1 and also dose-dependently inhibited DNMT activity. In conclusion, a low concentration of As₂O₃ induces CpG island demethylation of tumor suppressor genes by inhibition of DNMT and reactivates the partially/fully silenced genes in liver cancer cells.

Introduction

Inactivation of tumor suppressor genes is central to the development of all common forms of human cancer [1]. This inactivation often results from epigenetic silencing associated with promoter hypermethylation rather than intragenic mutations, and some genes are frequently inactivated by these reversible epigenetic events [2], [3]. Transcriptional silencing by CpG island methylation is a prevalent mechanism of tumor suppressor gene dysfunction in cancers [4].

Hepatocellular carcinoma (HCC), the major type of primary liver cancer, is one of the most common malignancies worldwide. Inactivation of the tumor suppressor genes p16^INK4a and Ras association domain family 1A (RASSF1A) caused by high frequency of hypermethylation of the 5′ CpG island of the genes in HCC has been reported [5], [6], [7], [8], [9]. In HCC, decreased E-cadherin expression was closely associated with loss of heterozygosity at the E-cadherin locus and methylation of CpG islands in the promoter region [10], [11]. Hypermethylation changes of GSTP1, a gene encoding the π-class glutathione S-transferase, have also been detected in many human cancers, including HCC [12], [13]. Therefore, inactivation of p16^INK4a, RASSF1A, E cadherin, and GSTP1 caused by hypermethylation of the CpG island around the promoter areas of the genes is one of the most important mechanisms of hepatocellular carcinogenesis.

The prototypic DNA methyltransferase 1 (DNMT1) is responsible for most of the methylation of the human genome and is necessary and sufficient for maintaining global methylation and aberrant CpG island methylation in human cancer cells [14], [15]. A well-known DNMT inhibitor, 5-aza-2′-deoxycytidine (5-aza-CdR), is thought to incorporate itself into the cellular DNA where it acts as a noncompetitive inhibitor of the DNMT1 and demethylates the hypermethylated CpG island of the tumor suppressor genes [16].

Recently, arsenic trioxide (As₂O₃) has been found to be effective for the treatment of malignant hematopoietic diseases. The mechanisms have been well discussed and are mostly considered to be induction of apoptosis and inhibition of proliferation [17], [18]. Arsenic has also been shown to induce DNA hypomethylation by continuous methyl depletion through consuming S-adenosyl-methionine (SAM) [19], [20]. Because SAM is an essential cofactor for a variety of methyltransferases in the cell, including DNMT, which are responsible for the methylation of DNA [21], we hypothesize that As₂O₃ may affect the expression of tumor suppressor genes by interfering with DNA methylation patterns. However, to the best of our knowledge, there have been no reports about the effects of As₂O₃ on demethylation and restoration of tumor suppressor genes.

In the present study, we investigated the status of CpG island methylation of p16^INK4a, RASSF1A, E cadherin, and GSTP1 in both HCC tissues and liver cancer cell lines and examined whether demethylation of CpG methylation around the promoter region of these tumor suppressor genes could restore their expression in 2 different liver cancer cell lines after treatment with As₂O₃.

Section snippets

Cell lines and reagents

Human liver cancer cell lines HepG2 (hepatoblastoma-derived) [22] and Huh-7 (HCC-derived) [23] were obtained from the RIKEN Cell Bank (Ibaraki, Japan). Cells were routinely cultured in Dulbecco modified Eagle medium with 10% heat-inactivated FBS (JRH Biosciences, Lenexa, Kan), 100 μg/mL streptomycin, and 100 U/mL penicillin and incubated at 37°C under 5% CO₂ in a humidified atmosphere. As₂O₃ (Sigma, St Louis, Mo) was dissolved in small amounts of 1.0 mol/L NaOH, then diluted to 10 mmol/L with

CpG methylation and immunohistochemistry of p16^INK4a, RASSF1A, E cadherin, and GSTP1 in HCC samples

We examined the methylation status of the CpG islands of p16^INK4a, RASSF1A, E cadherin, and GSTP1 genes in 25 HCC samples. The hypermethylated status of the promoter regions of p16^INK4a, RASSF1A, E cadherin, and GSTP1 was observed in 10 (40%), 14 (56%), 12 (48%), and 6 (24%) of 25 patients with HCC, respectively. All the clinicopathologic and molecular parameters are shown in Table 3. We did not find significant association between CpG hypermethylation of the genes and the following

Discussion

As₂O₃ is known to be effective for the treatment of acute promyelocytic leukemia. The mechanisms have been mostly considered to be induction of apoptosis and inhibition of proliferation [17], [18]. In the present study, we are the first to demonstrate that As₂O₃ induces demethylation of the CpG island of tumor suppressor genes, p16^INK4a, RASSF1A, E cadherin, and GSTP1, and restores its expression in the liver cancer cell lines, HepG2 and Huh-7. It is an important finding that the anticancer

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^☆: Grant Support: This study was supported in part by Grants-in-aid for Scientific Research (Young Scientists B, no. 14780433) from the Japan Society for the Promotion of Science (to Dr Cui).

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Original contributionArsenic trioxide inhibits DNA methyltransferase and restores methylation-silenced genes in human liver cancer cells☆

Summary

Introduction

Section snippets

Cell lines and reagents

CpG methylation and immunohistochemistry of p16INK4a, RASSF1A, E cadherin, and GSTP1 in HCC samples

Discussion

Cell

Am J Pathol

Hepatology

Hepatology

Am J Pathol

J Biol Chem

J Biol Chem

Hum Pathol

Blood

The history of cancer epigenetics

Nat Rev Cancer

Gene silencing in cancer in association with promoter hypermethylation

N Engl J Med

Epigenetic gene silencing in cancer

J Clin Invest

p16 is a major inactivation target in hepatocellular carcinoma

Cancer

Frequent epigenetic inactivation of the RASSF1A gene in hepatocellular carcinoma

Oncogene

Intensive hypermethylation of the CpG island of Ras association domain family 1A in hepatitis B virus–associated hepatocellular carcinomas

Clin Cancer Res

Inactivation of glutathione S-transferase P1 gene by promoter hypermethylation in human neoplasia

Cancer Res

DNMT1 and DNMT3b cooperate to silence genes in human cancer cells

Nature

Original contribution
Arsenic trioxide inhibits DNA methyltransferase and restores methylation-silenced genes in human liver cancer cells☆

CpG methylation and immunohistochemistry of p16^INK4a, RASSF1A, E cadherin, and GSTP1 in HCC samples