The tobacco-specific carcinogen NNK induces DNA methyltransferase 1 accumulation in Laryngeal carcinoma

Abstract

Over-expression of DNA methyltransferase 1 (DNMT1) correlates with hypermethylation of tumor suppressor genes (TSGs) in tobacco-induced cancers. The tobacco component nitrosamine 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK) increases protein levels of the DNMT1 in human lung cancer. However, the role of DNMT1 expression induced by NNK is not clear during laryngeal carcinogenesis. We investigated DNMT1 expression levels in 101 cases of human laryngeal carcinoma specimens and 54 cases clear surgical margin specimens by reverse transcription polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry (IHC) detection. Then, we analyzed the relationship between the DNMT1 expression and the smoking status of the patients with laryngeal carcinoma. Moreover, we investigated the effects of tobacco carcinogen NNK on DNMT1 expression in Hep-2 cells. We found that DNMT1 mRNA and protein expressions were up-regulated in laryngeal cancer tissues (p<0.05 and p<0.01, respectively). Among the 101 cases, DNMT1 protein from patients with heavier smoking habit had a significant trend of an increase with IHC scores (p<0.01). The overall survival rates of patients DNMT1-positive were significantly lower than those of patients DNMT1-negative (p<0.05). We observed that NNK increased DNMT1 protein levels, not mRNA levels, in cultured Hep-2 cells significantly in both dose- and time-dependent manner (p<0.05). These results supported the idea that NNK-induced DNMT1 expression may result from protein stabilization. Increased DNMT1 protein expression may play a critical role in the malignant progression of larynx.