| Objective:Salivary adenoid cystic carcinoma (SACC) is one of the most common subtypes of malignant tumors occurring in the salivary gland. The characteristics of SACC are the invasion of adjacent tissue and hematogenous spread to the distant organs of the lung, bone and liver. The process of tumor cell invasion into the stromal tissue is closely associated with interactions between the tumor cells and the extracellular matrix (ECM). Altered expression and modification of ECM proteins in tumor cells plays a significant role in their invasion into surrounding normal tissues. MMP-2 can degrade the components of the ECM and basement membrane, destroy the structure of local tissues. The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene is expressed in a number of normal tissues, however it is not expressed in transformed and tumor-derived cells. RECK has been demonstrated to inhibit MMP activity through several mechanisms, including the direct inhibition of protease activity, theregulation of cellular release and possible sequestration at the cell surface. Therefore, we hypothesize that RECK is a novel suppressor gene for invasion and metastasis in SACC, and that it is potentially a good target for SACC therapy. DNA methylation changes were commonly found in human cancers, Hypomethylation of oncogenes can result in its aberrant activation, and hypermethylation of suppressor genes can lend to its silencing. Several methylation-regulated candidate genes have been identified in SACC, including Suprabasin (SBSN), Aquaporin 1 (AQP1), phosphatase and tensin homolog deleted on chromosome 10(PTEN), cyclin-dependent kinase inhibitors, RAS-associated domain family protein 1A (RASSF1) and death-associated protein kinase (DAPK), but the methylation of RECK in SACC had not yet been reported.The purpose of the present study was to examine the expression of RECK and MMP-2 in vitroand in vivo, and to analyze the correlation between RECK expression and MMP-2 expression in SACC. This study also aimed to clarify the roles of RECK and MMP-2 in the invasion and development of SACC and to explore their role in the prognosis of SACC and aimed to examine the methylation status of RECK gene and determined the 5-aza-dC effects of treatment on the methylation status and expression level of RECK gene in human adenoid cystic carcinoma cell lines, and expect to development of new strategies for cancer prevention and therapeutic interventionMethods:1.83 paraffin-embedded specimens were retrospectively recruited and immunohistochemical method was used to detect the expression of RECK and MMP-2 in SACC. Based on the score of immunochemistry, the correlation between the expression of RECK and various clinicopathologic parameters (age, sex, tumor size, clinical stage, histological grade and Perineural invasion) was investigated.2. Using the statistical software, SPSS 13.0 (SPSS, Inc., Chicago, IL, USA), to evaluate the association between RECK and MMP-2 expression。3. Using the the Kaplan-Meier method and log-rank test, to evaluate the the overall survival in the RECK-positive expression group to the RECK-negative expression group.4. Using the the univariate analysis and the multivariate analysis, to revealed which variables were significantly associated with the prognosis.5. Methylation-specific PCR, were used to investigate the methylation status of RECK gene, and to investigate the methylation status of RECKgene after treated with different dose of 5-aza-dC with different time in SACC cell lines.6. Western blot analysis and quantitative real-time PCR were used to investigate the RECK gene expression after treated with different dose of 5-aza-dC for different time in SACC cell lines.7. Transwell invasion assay were used to investigate the invasion ability of SACC cell lines after treated with different dose of 5-aza-dC for 72 hours.Results:1. Immunohistochemistry results showed that the percentage of RECK expression increased from the SACC, to the benign salivary tumors, to the normal cases; 25.3% in the SACC,48.1% in the benign salivary tumors and 87.5% in the normal salivary tissues. The difference in expression rates was considered statistically significant (P<0.05) The percentage of expression decreased from the SACC, to the benign salivary tumors, to the normal salivary tissues; 83.1% in the ACC,74.1% in the benign salivary tumors and 25.0% in the normal salivary tissues. The difference of expression rates was considered to be statistically significant (P<0.05).2. The MMP-2 expression rate was 45.5%(10/22) in tissues with positive (+and++) RECK expression. In tissues negative (-) for RECK expression, the MMP-2 expression rate was 95.2%(59/62). Pearson’s x2test revealed that there was a significant negative correlation between the expression of RECK and MMP-2 (x2, 38.202; P<0.0001).3. The average follow-up period was 54 months (range,10-120 months). In total,42 patients (50.6%) succumbed to SACC,5 patients (6.0%) succumbed during the follow-up period,4 patients (4.8%) succumbed to an unrelated cause and 32 patients (38.6%) remained alive on the day of the study. The survival curves demonstrated that the overall survival in the RECK-positive expression group was improved in comparison to the RECK-negative expression group (P=0.009), based on the use of the Kaplan-Meier method and log-rank test.4. The univariate analysis revealed that the following variables were significantly associated with the prognosis, includingRECK and MMP-2 expression, histological grade, TNM stage and perineural invasion, while the multivariate analysis revealed that RECK expression and histological grade also had an independent prognostic effect on the overall survival of the SACC patients.5. Promoter methylation was only found in ACC-M cell line, and treatment of ACC-M cell lines with 5-aza-dC partially reversed the hypermethylation status of the RECK gene by MSP.6.5-aza-dC significantly enhanced the expression level of mRNA and protein of RECK gene in ACC-M cell lines by Western blot analysis and quantitative real-time PCR。7.5-aza-dC significantly suppressed ACC-M cell invasive ability by transwell assay。Conclusions:The positive expression of RECK was observed in 21/83 (25.3%) of SACC cases, and RECK expression was significantly associated with the TNM stage, histological pattern and perineural invasion of. the present data indicates that RECK was discovered as a novel molecular biomarker and may actively be involved in the progression, invasion and metastasis of SACC. RECK may reduce tumor progression through functionally downregulating MMP-2 expression. Measurements of the expression of RECK and MMP-2 are valuable, not only to assess patient prognosis, but to also develop new strategies for cancer prevention and therapeutic intervention.our findings first show that 5-aza-dC could inhibit cancer cell invasion through reversal of hypermethylation status of RECK gene, which could be a promising therapeutic strategy for chemotherapeutic approaches in SACC treatment. Additional work is needed to find less side and more effective drug, and to find the mechanism underlying RECK hypermethylation in SACC... |
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