| With the recent advances in life science, it has long been recognized that both the epigenetic alterations and genetic alterations participate in the pathogenesis of cancer. DNA methylation is one of the most frequent epigenetic alterations. Pancreatic adenocarcinoma is one of the most common human cancers. Since the symptoms are unspecific, chemotherapy and radiotherapy have little effect , the patients have a poor prognosis. The precise pathogenesis of pancreatic adenocarcinoma remains unknown. The studies reveal that there are aberrant epigenetic alterations in pancreatic intraepithelial neoplasia, as well as in pancreatic adenocarcinoma.RAS proteins play important roles in several biological processes, including cellular proliferation, migration and apoptosis. Activated forms of RAS also can induce senescence and apoptosis by interacting with its negative effetors. RASSF (RAS-association domain family) are the most common RAS negative effectors that have been identified. They are thought to be function as tumor suppressor genes and are inactivated or downregulated in a variety of tumors.The RASSF2 gene encodes three different isoforms and only RASS2A has CpG islands. RASSF2A binds to K-RAS in a GTP-dependent manner. RASSF2A promotes apoptosis and cell cycle arrest as well as inhibits growth. RASSF2A inhibits the transcriptional activity of NF-κB. Genes regulated via the NF-kB pathway are downregulated by RASSF2A. These genes are involved in inflammatory responses, which promote tumor angiogenesis and invasion. RASSF2A also suppresses the RAS-Rho pathway.The promoter hypermethylation of RASSF2A is frequently observed in colorectal carcinomas and adenomas. RASSF2A is also methylated in other human cancers, including lung, gastric, breast, nasopharyngeal and endometrial cancers. However, the promoter hypermethylation of RASSF2A in pancreatic adenocarcinoma has not been reported.In the present study, RASSF2 gene expression was examined in 8 human pancreatic carcinoma cell lines and one normal pancreatic tissue by real-time PCR. To find out whether the inactivation of RASSF2 is related to its promoter hypermethylation in pancreatic adenocarcinoma, we examined the methylation status of RASSF2A promoter region CpG islands in 8 human pancreatic carcinoma cell lines and 41 cases of pancreatic ductal adenocarcinoma by methylation-specific PCR. 29 cases of matching non-neoplastic tissue were also investigated for methylation study. We also reviewed the clinicopathological data of the pancreatic ductal adenocarcinomas and analysed the correlations between methylation frequency and clinicopathological parameters. The pancreatic carcinoma cell lines BxPC-3 and AsPC-1 were treated with different concentration of 5-aza-2'-deoxycytidine (5-aza-dC) for 5 successive days. The total RNA was isolated and RASSF2 expression was determined in these two cell lines by real-time PCR after treatment with 5-aza-dC.We found that the expression of RASSF2 was downregulated in all human pancreatic carcinoma cell lines. Among 8 human pancreatic carcinoma cell lines, AsPC-1, BxPC-3 and PANC-1 had a weak expression of RASSF2. Aberrant hypermethylation of RASSF2A was detected in 8 of 8(100%) pancreatic carcinoma cell lines and they were completely methylated (only methylated alleles were detected). 9 of 41(22%) primary pancreatic ductal adenocarcinoma showed promoter hypermethylation, which were partially methylated (unmethylated and methylated alleles were both detected). Whereas none of the matched normal pancreatic tissue presented hypermethylation of RASSF2A (P< 0.01). Statistical analyses of the correlation between RASSF2A promoter hypermethylation and clinicopathological parameters demonstrated that there was no significant difference between methylated and unmethylated patients with regard to age, gender, histopathological grading and lymph node metastasis. The expression of RASSF2 could be restored by the methyltransferase inhibitor 5-aza-dC in pancreatic carcinoma cell lines, AsPC-1 and BxPC-3. An increase was shown at 3 days after 5-aza-dC treatment and a significant increase was observed in AsPC-1 cell lines. It confirmed the role played by promoter hypermethylation in the expression of RASSF2 in pancreatic carcinoma cell lines. Other epigenetic modifications, such as histone deacetyloation might be involved in the expression of RASSF2 in BxPC-3 cell lines.In conclusion, the expression of RASSF2 is downregulated in human pancreatic carcinoma cell lines. Promoter hypermethylation of RASSF2A is observed in pancreatic ductal adenocarcinoma, while not in normal pancreatic tissue. RASSF2A is inactivated in pancreatic adenocarcinoma by CpG island promoter hypermethylation, and may play a role in pancreatic carcinogenesis. |
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