Effects Of Celecoxib On The Methylation Status, MRNA Expression Of P14^ARF, DAP-K And TIMP-3gene Promotor And Apoptosis In Squamous Cell Carcinoma Of The Esophagus

Objective: Three pathways have been found to activate tumor suppressorgenes, including gene mutations, chromosomal variation promoter DNAmethylation. The promoter region of DNA methylation is a chemicalmodification process, mediated by the enzyme and it belongs to the epigeneticareas. Some studies have found that the expression of some tumor suppressorgenes, such as P14^ARF, DAP-K and TIMP-3have a close association with thedevelopment and metastasis of esophageal cancer. The gene promotermethylation leads to inactivation of tumor suppressor genes, and function loss,along with significantly increased chance of suffering from cancer.In recent years, NSAIDS （non-steroidal anti-inflammatory drugs） such asaspirin, indomethacin, celecoxib can reduce the risk of esophagealadenocarcinoma and squamous cell carcinoma. Now, it has become one of thehottest areas of cancer prevention. Epidemiological studies have shown thatnon-steroidal anti-inflammatory drugs can reduce the mortality rate ofesophageal cancer. However, little information is available on the impact ofesophageal methylation by NSAIDs. This study will observe the methylationstatus of P14^ARF, DAP-K and TIMP-3gene etc in esophageal cancer and theimpact of P14^ARF, DAP-K and TIMP-3gene methylation in ESCC bycelecoxib.Methods: Specimens: Thirteen patients who underwent esophagectomyfor squamous cell carcinoma were recruited in the Fourth Hospital of HebeiMedical University from June2012to January2013. There were9males and4females, with an average age of60.9years old. All of them werepathologically confirmed esophageal squamous cell carcinoma pre-andpostoperatively. There were no history of radiotherapy and chemotherapy, long-term use of aspirin and other non-steroidal anti-inflammatory drugs.Experimental Group: celecoxib was orally administered in a dose of400mg,bid for10-15days before surgery. Endoscopically biopsied esophageal cancertissues were taken from all recruited patients before celecoxib was administed.Cancer tissues were taken in the operation for the inveatigation. Control group:Nineteen patients who underwent esophagectomy for squamous cellcarcinoma were recruited. There were no history of radiotherapy andchemotherapy, long-term use of aspirin and other non-steroidalanti-inflammatory drugs. Both the preoperative and postoperative specimensshould be immediately immersed in later solution, and quickly be transferredto a-80℃refrigerator storage to spare. The specimens of the control groupand the experimental group must be grinded on ice, and the extracted DNAand RNA were placed in a-80℃refrigerator to prevent degradation.Methylation status of DNA were detected by methylation-specific PCR （MSP）technology and mRNA expression was detected by RT-PCR. Detect theapoptosis rate of before and after celecoxib administration in experimentalgroup. SPSS software package was used for statistical analysis of theexperimental data.Rusults:1The relationship between P14^ARF, DAP-K and the TIMP-3genepromoter methylation status and the clinical and pathological characteristicsThe methylation rates of P14^ARF, DAP-K and TIMP-3gene promotor inESCC were38.4%,46.1%and30.8%, respectively. There is no relationshipbetween the methylation status of P14^ARFand TIMP-3gene and the patient’sage and gender （P>0.05）. Meanwhile, methylation frequencies of P14^ARFandTIMP-3gene in lymph node metastasis group was significantly higher thanthat in non lymph node metastasis group （P <0.05）. DAP-K gene methylationstatus was not associated with age, gender and lymph node metastasis（P>0.05）.2The methylation status of P14^ARF, DAP-K and TIMP-3gene promoterin experimental group and control group P14^ARFgene promoter methylation rate was38.5%（5/13） beforecelecoxib administration in experimental group, while that in control groupwas47.4%（9/19）.The unmethylation rate was60%（3/5）after celecoxibadministration, while that in control group was0%（0/9）, through Fisher’sexact statistics, χ²=6.382，P=0.027<0.05, the difference was statisticallysignificant. DAP-K gene promoter methylation rate was46.1%（6/13），beforecelecoxib administration in experimental group, while that in control groupwas57.9%（11/19）. The unmethylation rate was50%（3/6）after celecoxibadministration, while that in control group was0%（0/11）, through Fisher’sexact statistics, χ²=6.286，P=0.029<0.05, the difference was statisticallysignificant. TIMP-3gene promoter methylation rate was30.8%（4/13） beforecelecoxib administration in experimental group, while that in control groupwas36.8%（7/19）. The unmethylation rate was50%（2/4）after celecoxibadministration, while that in control group was0%（0/9）, through Fisher’sexact statistics, χ²=3.889，P=0.049<0.05, the difference was statisticallysignificant.3The comparison of mRNA expression of P14^ARF, DAP-K and TIMP-3before and after celecoxib treatement administrationAfter celecoxib administration, P14^ARFgene mRNA relative IOD value（0.56±0.046） was significantly higher than that （0.37±0.051） before celecoxibadministration, the difference was statistically significant （t=12.290P=0.000）.After celecoxib administration, DAP-K gene mRNA relative IOD value（0.53±0.117） was significantly higher than that （0.37±0.051） before celecoxibadministration, and the difference was statistically significant （t=4.455P=0.011）. After celecoxib administration, the TIMP-3gene mRNA relativeIOD value （0.50±0.095） was significantly higher than that （0.38±0.049） beforecelecoxib administration and the difference was statistically significant（t=4.600P=0.019）.4The apoptosis status before and after celecoxib administrationThe overall apoptosis rate was12.32±6.46%before celecoxibadministration, while that after celecoxib administration was19.18±6.09%. The difference was statistically significant （t=-11.507，P=0.000<0.05）.Conclusion:1Celecoxib can reduce the degree of methylation of P14^ARF, DAP-K andTIMP-3gene promoter methylation.2Celecoxib can improve the expression of P14^ARF, DAP-K and TIMP-3gene in esophageal squamous cell carcinoma.3The methylation status of DAP-K gene promoter is not associated withage, gender and lymph node metastasis. The methylation status of P14^ARFandTIMP-3gene promoter is not associated with age and gender, but lymph nodemetastasis. It is suggested that methylation of DAP-K gene promoter is a riskfactor for lymoph node metastasis.4Celecoxib can induce apoptosis in esophageal squamous cellcarcinoma.