Clinical significance of epigenetic silencing and re-expression of O6-methylguanine-DNA methyltransferase using epigenetic agents in laryngeal carcinoma.

Jing Yang,Zhao-Wei Gu,Ming-Zhu Jin,Xin-Bing Zhu,Wen-Yue Ji,Li-Xia He

doi:10.3892/ol.2014.2662

Jing Yang, Zhao-Wei Gu + Show 4 more

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https://doi.org/10.3892/ol.2014.2662

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Abstract

The aim of the present study was to investigate the association between O6-methylguanine-DNA methyltransferase (MGMT) gene expression levels, and DNA methylation status and histone modifications in laryngeal squamous cell carcinoma (LSCC). Chromatin immunoprecipitation, methylation-specific polymerase chain reaction (PCR), and reverse transcription-quantitative PCR were performed to analyze histone modifications, DNA methylation status and mRNA expression levels in the promoter region of the MGMT gene in laryngeal carcinoma HEp-2 cells, as well as in 50 paired healthy and LSCC tissue samples. The present study demonstrated that treatment of HEp-2 cells with 5-aza-2′-deoxycytidine (Aza), a DNA methyltransferase inhibitor, significantly upregulated MGMT mRNA expression levels, reduced MGMT DNA methylation, reduced MGMT histone H3 lysine 9 (H3K9) di-methylation, and increased MGMT histone H3 lysine 4 di-methylation without a significant change in H3K9 acetylation. Trichostatin A (TSA), a histone deacetylase inhibitor, marginally upregulated MGMT mRNA expression levels without affecting the DNA methylation status, or H3K9 or H3K4 di-methylation, however, TSA treatment caused a significant increase in H3K9 acetylation. Furthermore, Aza and TSA combination treatment produced a synergistic effect. In the LSCC samples, the rate of DNA methylation in the MGMT gene was 54%, compared with 24% in the healthy control group (P<0.05). Therefore, data from the present study indicates that MGMT may serve as a novel therapeutic target in the treatment of LSCC.

Highlights

Laryngeal cancer is a common malignancy in otolaryngology, accounting for 1‐5% of all cases of cancer, worldwide
H3K9 acetylation was significantly increased at methylguanine‐DNA methyltransferase (MGMT) promoter regions following treatment with Trichostatin A (TSA) (0.470±0.001 vs. 0.612±0.003; P0.05)
The present study identified that the DNA methylation status of MGMT exhibited no correlation with the age or gender of the patient, the degree of tumor differentiation, the tumor T stage or lymph node metastasis in laryngeal squamous cell carcinoma (LSCC) tissues (P>0.05; Table I)

Summary

Introduction

Laryngeal cancer is a common malignancy in otolaryngology, accounting for 1‐5% of all cases of cancer, worldwide It is the eleventh most common type of cancer, accounts for 35.4% of cases of head and neck cancer, and is the third most common type of head and neck malignancy, worldwide [1]. Numerous carcinogens target O6‐guanine, the loss of MGMT gene expression results in the accumulation of unrepaired DNA damage and subsequent tumor development. MGMT is transcriptionally downregulated via the hypermethylation of CpG islands in its promoter region [2,3]. Histone modification is closely associated with the DNA methylation status of a gene and is key for gene regulation. DNA hypermethylation in the promoter CpG islands of tumor suppressor genes (TSGs) inhibits transcriptional initiation and results in permanent gene silencing, a key process in carcinogenesis [4,5,6]. Studies investigating the interaction between DNA methylation status and various histone modifications are currently ongoing

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