br Materials and methods br Results The frequency of
Materials and methods
Results The frequency of hypermethylation of RARbeta, RASSF1A, FHIT, GSTP1, MGMT, VHL and DAPK in patients with primary LSCC was assessed in both the tumor tissue samples and macroscopically and histologically normal mucosa from distant sites of the surgical specimen, Dehydroandrographolide sale and trachea. Table 2 shows the frequencies of gene methylation in clinical samples. Only VHL was not methylated in any of the tested samples while the highest methylation frequency was detected in case of DAPK. Interestingly, the frequency of gene methylation was overall only slightly higher in tumor samples than in epiglottis and trachea mucosa samples. No correlation was found between the hypermethylation of any of the analyzed genes and demographic, epidemiological or clinicopathological data with the exception of MGMT and RARbeta. The methylation of these genes in tumor samples was correlated with lymph node involvement (N0 vs. >N0), positively or negatively, respectively (Table 3). At least one gene was methylated in each tumor sample. On the other hand, only in a small fraction of normal mucosa samples no methylation was detected (Table 4). Nearly half of the tumor samples showed concurrent hypermethylation of three or four genes. The analysis of the convergence of gene methylation between tumor tissue samples and the corresponding normal mucosa of the same patient showed that the appearance of gene hypermethylation in the mucosa with lack of its methylation in the tumor occurred with low frequency (2.4–17.1%, mean 5.9%) with differences depending on gene analyzed and tissue origin (Table 5).
Discussion The essential role for the epigenetic defects in carcinogenesis is now well established. Aberrant methylation is a mechanism of inactivation of tumor suppressor genes as common as genetic mutations. The important difference is the reversibility of epigenetic modifications. Current data indicate that epigenetic abnormalities may take place at the earliest stages of carcinogenesis, and be responsible for the formation of cancer stem cells. Tumor-type specificity and an early appearance of aberrant DNA methylation make it an excellent cancer biomarker and the target for both chemoprevention and therapy.7, 24 The aim of this study was to assess the methylation profile of selected genes in primary laryngeal squamous cell carcinomas and paired normal mucosa samples. Our results show that gene hypermethylation is a very frequent event in LSCC patients. The highest methylation rate was found for DAPK and similar observations were reported in other studies.25, 26 On the other hand Dikshit et al. reported a significantly lower frequency of DAPK methylation. The frequency of hypermethylation of MGMT in our study was found to be higher than reported by Zhang et al. or Dikshit et al. However, a similar frequency of the methylation of MGMT, and also RASSF1A and GSTP1 was observed in the study of Park et al. To our knowledge, this is the first report assessing the frequency of methylation of RARbeta, FHIT or VHL in laryngeal cancers. The methylation of the analyzed genes may be of functional importance for laryngeal carcinogenesis. The loss of RASSF1A, RARbeta, DAPK and FHIT upon gene methylation may facilitate the acquirement of uncontrollable growth potential and apoptosis inhibition. The frequent methylation of MGMT may increase the sensitivity towards the mutagenic effects of DNA alkylating agents like cigarette smoke nitrosamines but on the other hand it can facilitate the cytotoxic effects of DNA alkylating chemotherapeutics in cancer patients. Recently, it has been suggested that the hypermethylation of DAPK correlates with lymph node metastasis in larynx carcinomas. Such a correlation was not found in our study. However, in the investigated group of patients lymph node involvement was significantly associated with the hypermethylation of MGMT and lack of hypermethylation of RARbeta. The correlation between MGMT methylation and lymph node metastasis was previously found in the case of gastric cancers, where it was also associated with a more frequent occurrence of K-ras mutation. It can be hypothesized that in laryngeal cancers the epigenetic loss of MGMT function may increase the mutation rate as a result of an impaired repair of DNA damage induced by cigarette smoke nitrosamines. This may facilitate the acquirement of an enhanced migration potential and invasiveness.