| Objective:Esophageal cancer is the 7th most common cancer in the world. South area of Hebei Province China is one of the highest incidence regions of esophageal cancer in the world, with a mortality rate of more than 100 per 100,000 people per year. The treatment methods for esophageal cancer are esophagectomy,chemotheraphy and radiothera- phy. Several epidemiological studies indicate that NSAIDs use may reduce the risk of death from esophageal cancer. Thus, NSAIDs have been considered as a chemopreventive agent of esophageal cancer.The best known function of DSAIDs action is to block the enzyme cyclooxygenase, the rate limiting enzyme in the conversion of arachidonic acid to prostaglandins. Prosta-glandins, such as PGE2, can promote cell proliferation, inhibit apoptosis and immune response to malignant cells. There are two different isoforms of COX, COX-1 and COX-2. COX-1 is constitutively expressed in most mammalian tissue. COX-2 is induced by various agents such as growth factors and tumor promoters. It is demonstrated that COX-2 has been up-regulated in esophageal cancer tissue. The studies on cell line of esophageal squamous cell carcinomahas demonstrated that COX-2 inhibitor and other NSAIDs can stimulate proliferation and induce apoptosis of esophageal cancer cells expressing COX-2.In pathogenesis of asthma and inflammation, the downstream regulation of COX-2 by NF-κB has been demonstrated. However, this pathway has not been shown in any cancers. Moreover, the effects of NSAIDs on cancer in human being have not been elucidated. This study is going to elucidate the effects of Mobic, a selective inhibitor, on esophageal cancer to look at whether NSAIDs have a downstream effect of NF-κB on COX-2 in human esophageal cancer. Methods:1. Patients and clinical data:From March 2001 to September 2002, fifty-three patients with squamous cell carcinoma of the esophagus were randomised into test group and control group in the Third Department of Thoracic Surgery, the Fourth Hospital of Hebei Medical University. These included 40 males and 13 females, with a mean age of 57.3 years (range 35-75 years). No chemotheraphy and radiotheraphy and other treatments targeting at cancer were given to these patients before surgery. In the test group, a daily dose of 7.5mg of Mobic were administered for 10-14 days before surgery. In the control group, no NSAIDs were used. After esophagectomy was performed, some esophageal cancer tissues were sampled and immediately frozen in liquid nitrogen and stored in a -80℃ refrigerator or fixed in 70%ethanol and stored in a 4℃refrigerator before analysis. 2. Western blot :Cytoplasmic protein was extracted from tissue of esophageal cancer. Protein concentration was measured with Forlin-phenol method. Protein was separated by 10% SDS-PAGE and transferred to PVDF membranes. For immunodetection of COX-2 protein and IκB-α protein, the membranes were probed with COX-2 antibody (rabbit anti-human polyclonal IGg) at 1:50 or IκB-α antibody (rabbit anti-human polyclonal IGg) at 1:100, followed by incubation with preoxidase-conjugated secondary antibody (goat anti-rabbit IGg) at 1:1000, and at last stained with DAB. 3. Electrophoretic mobility shift assay (EMSA):Nuclear extract was extracted from tissue of esophageal cancer. Protein concentration was measured with Forlin-phenol method. Double-stranded deoxyooligonucleotides containing the NF-κB consensus recognition site (5-AGT TGA GGG GAC TTT CCC AGG C-3, 3-TCA ACT CCC CTG AAA GGG TCC G-5) were labeled with [γ32P]ATP and purified with ethanol precipitation. Gel shift assay was carried out. X-ray film was visualized by autoradiography at -72℃. 4. Reverse transcription-polymerase chain reaction (RT-PCR):Total RNA was extracted from tissue of esophageal cancer with a Trizol reagent. The RT-PCR was carried out using RT-PCR kit. The sequences of COX-2 were 5,-TTC AAA TGA GAT TGT GGG AAA ATT GCT-3,(forward primer) and 5,-GTG GCC ATC TCC TGC TCG AAG TC-3,(reverse), giving a 30... |
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