Association Of Celecoxib With Methylation Of CpG Island Of P16and CDH1Genes In Esophageal Squamous Cell Carcinoma

Objective: Esophageal cancer is a common malignant tumor, and has ahigh mortality rate. It has an obvious regional difference in incidence ofesophageal cancer in China, the central and southern Hebei province, northernpart of Henan province and Taihang mountain region is of the highestincidence area of esophageal cancer in the world. Squamous cell carcinoma isthe major tissue type of esophageal cancer in these areas. The occurrence ofesophageal cancer is due to long-term synergies of the activation of oncogenesand inactivation of tumor suppressor genes. Gene methylation is a kind ofabnormal genetic changes, and plays an important role in the treatment ofcancer. DNA methylation is reversible under certain conditions, and thedemethylation drugs can make the methylation of DNA to demethylated andactivate the tumor suppressor gene.Currently known P16and CDH1gene are important tumor suppressorgenes, of which P16participates the regulation of cell cycle located in the9p21chromosome, plays a key role in the negative regulation of the G1/Srestriction point in the cell cycle, and finally regulates the proliferation of cell.E-cadherin which is encoded by CDH1is part of the selective cell recognitionand adhesion surface glycoprotein family. Studies have shown that theinactivation of CDH1occurs frequently in epithelium-derived tumors.Celecoxib is one of non-steroidal anti-inflammatory drugs, which isproven to inhibit methylation. In this study, RT-PCR and MSP wereperformed to evaluate the mRNA expression and the methylation of P16andCDH1in13cases of ESCC to investigate possible anticancer mechanisms ofcelecoxib.Methods：1The experimental group included13patients with esophageal squamous cell carcinoma who intook400mg0f celecoxib (twice a day for10-15days before surgery. Fresh cancer tissue was taken for investigation inthese patients. The control group included19cases patients with ESCC whodid not take any NSAIDs and fresh cancer tissue was also taken for analysis inthese patients.2MSP was used to detect their methylation of CpG island of p16andCDH1in esophageal squamous cell carcinoma from patients who did not useNSAIDs.3MSP and RT-PCR were used to detect the methylation of CpG islandand mRNA expression level of P16and CDH1gene, in order to reveal theimpact of celecoxib on the methylation of CpG island of p16and CDH1geneof esophageal squamous cell carcinoma.4The purpose of detecting the rates of apoptosis of the esophagealsquamous carcinoma cell before and after taking celecoxib by the technologyof flow cytometry, was to confirm the relationship between the demethylationaffection of celecoxib with the apoptosis of cancer cells.Results:1Methylation of CpG island of p16The percentage of methylation of the CpG island in the P16genepromoter region among cases and the controls were69%(9/13) and74%(14/19), respectively before taking celecoxib (χ~2=0.073, P=0.787, P>0.05).After taking celecoxib, this figure was44%(4/9) in cases co-paired to7%(1/14) in controls (χ~2=4.286, P=0.038, P <0.05).RT-PCR results showed that the extent of P16mRNA expression inesophageal squamous cell carcinoma before and after taking celecoxib were0.3333±0.05966and0.6460±0.14309, respectively. Statistical analysisshowed that P16mRNA expression in squamous cell carcinoma of theesophagus was significantly higher in patients used celecoxib than those whodid not use the drug (t=-10.785, P=0.000<0.05).2CDH1gene promoter region CpG island methylation status Gene promoter region CpG island methylation was54%(7/13) inpatients before use of celecoxib, and57%(11/19) in controls (χ~2=0.050, P=0.823>0.05). After use of celecoxib, this figure was43%(3/7) in patients and0%(0/11) in controls who never used NSAIDs (χ~2=5.343, P=0.021<0.05).RT-PCR results showed that the extent of P16mRNA expression inesophageal squamous cell carcinoma was0.3171±0.04582before use ofcelecoxib and0.6164±0.13150after use of the drug (t=-11.515,P=0.000<0.05). Statistical analysis showed that the extent of CDH1mRNAexpression in squamous cell carcinoma of the esophagus was higher inpatients who used celecoxib than those who did not used the drug (t=-11.515,P=0.000<0.05).3The results of flow cytometryIn experimental group, the apoptosis rate of esophageal squamouscarcinoma cell before and after taking celecoxib were12.32%±6.46%and19.18%±6.09%, respectively. The difference is subject to normal distribution(W=0.950, P=0.73>0.1). Statistical analysis showed that the apoptosis rateof esophageal squamous carcinoma cell after taking celecoxib wassignificantly higher than before taking celecoxib, and the difference wasstatistically significant between the two (t=-11.507, P=0.000).Conclusions:1. The methylation rate of P16and CDH1in control group is obviouslyhigher than that in experimental group. Celecoxib can reduce the methylationrate of P16and CDH1in ESCC.2. The expression level of P16mRNA and CDH1mRNA after takingcelecoxib in the ESCC is higher than the level before useing this drug.Celecoxib can strengthen the expression level of P16mRNA and CDH1mRNAof the ESCC.3Celecoxib can promote the apoptosis of cancer cells, which may beassociated with the demethylation and elevated expression of P16and CDH1gene in esophageal squamous cell carcinoma.