| Objective: Esophageal cancer is one of the most common malignant tumors in China, with a very obvious regional distribution. Due to the fact that early symptoms of esophageal cancer are not typical, as well as other reasons such as lacking routine health screening, esophageal cancer are very often found at late stage when diagnosed, leading to poor prognosis. Recent studies suggest that cyclooxygenase-2(COX-2) plays an important role in development of malignant tumors, which can promote tumor cell proliferation, inhibition of tumor cell apoptosis and promote tumor invasion and metastasis, etc. Studies using animal tumor model demonstrated that selective COX-2 inhibitors can inhibit tumor growth significantly, thus targeted treatment with COX-2 inhibitors becomes one of the promising option for esophageal cancer therapy.Nimesulide is a novel selective COX-2 inhibitor. Animal experiments confirmed that Nimesulide can inhibit tumor growth and metastasis, preventing tumor progression. In addition, when used in combination with chemotherapy and/or radiotherapy, it enhanced the efficacy of chemotherapy or radiation. Oxaliplatin is a third generation chemotherapeutic drug, it can crosslink DNA strands intra- and inter chain through generation of hydration derivatives, thereby inhibiting DNA replication and DNA synthesis.In this study, a variety of experimental methods are utilized to investigate the role of COX-2 in the progression of esophageal cancer. Both specimens from human esophageal cancer surgery and mice xenografts were used in this study. The synergistic effects of COX-2 inhibitor nimesulide and chemotherapy drug oxaliplatin on inhibition of esophageal xenograft tumor growth are also investigated.Methods:1 Expression of a COX-2 in esophageal cancer and its significanceFreshly dissected esophageal cancer specimens were collected from patients immediately after surgery(n=63). Specimens were fixed in 10% formalin, then embedded and micro-dissected. H&E stained slides were reviewed by two experienced histopathological physicians for diagnosis. Immunohistochemistry was used to detect the expression of COX-2 protein. Fresh samples were fixed in 70% ethanol and analyzed by flow cytometry(FCM) to quantify the expression of COX-2 in the specimens.2 Combinational effect of Nimesulide and Oxaliplatin on human esophageal xenografts growth in mice.Male C57BL/6 mice of 6-7 weeks old(~25-30g) were housed in IVC environment with sterile food and water, with ambient temperature and humidity control in the appropriate range. The human esophageal Eca-109 cancer cells were cultured DMEM/F12 medium supplemented with 10% fetal calf serum, penicillin and streptomycin(100U/ml) at 37 ℃with 5% carbon dioxide. Cells were passed at 70%-80% confluence. ECA cells at log phase were collected by digestion with 2.5g L-1 trypsin, washed with PBS then dispersed into single cell suspension. Cells were diluted with FBS free DMEM/F12 medium and plated for counting. Cell suspension was adjusted to 5x106/ml. Mouse skin on the right hip was disinfected and 1ml of the above cell suspension was injected to establish human esophageal carcinoma xenograft mouse model. In this assay, all 26 mice inoculated grew tumor. Tumor sizes were measured every 5 days. The two smallest tumors were removed from analysis when the average diameter of the tumor reached 4 mm. The 24 mice were randomly divided into four groups with 6 per each of the following groups: control group, Nimesulide group, Oxaliplatin group, Nimesulide/Oxaliplatin group. Nimesulide group of mice were given oral gavage nimesulide 20mg/ kg/time, administered once a day, while the same amount of sterile saline intraperitoneally; oxaliplatin group of mice were intraperitoneally injected with 10mg/kg/time of oxaliplatin/Mannitol, administered once every four days, while the same amount of sterile saline through oral gavage; Nimesulide/oxaliplatin mice were given oral gavage nimesulide, intraperitoneal injection of mannitol oxaliplatin, the same dosage as the alone group; control group of mice were given oral gavage sterile saline, the same amount of sterile saline intraperitoneally. The growth of mice was observed periodically and the tumor size including long diameter and short diameter was measured every 5 days. After 30 days, the mice were sacrificed and tumor tissue were removed, the tumor mass was weighed. COX-2 expression and apoptosis rate in xenograft tumor cells was quantified using flow cytometry(FCM) analysis.Result:1 COX-2 expression in esophageal carcinogenesis processImmunohistochemistry staining showed that COX-2 gene expression in esophageal cancer cell is mainly localized in the cytoplasm, visualized as positive brown granules stain in cytoplasm. In the cancer tissue, the expression rate is 90% while in the surrounding normal tissue, the expression is significantly lower(20%). FCM quantitative analysis of COX-2 expression showed FI value of 1.00±0.12 for COX-2 in normal esophageal squamous epithelium, while significantly increased expression of COX-2 in well- differentiated carcinoma highest with the FI value of 2.89±0.22. The decreased level of COX-2 expression in esophageal squamous carcinoma cells correlates with the degree of cancer tissue differentiation.2 Synergistic effect of Nimesulide and oxaliplatin on human esophageal cancer and the mechanisms for inhibition of tumor growth in miceTotal 26 mice were inoculated, and the tumor formation rate is 100% after 5-8 days oftumor cell inoculation. The mean tumor diameter reached 4mm after 13 days of tumor cell inoculation.After treatment, the volume of each group were as follows: control group: tumor volume 2047.42 ± 976.55mm3, tumor weight 1.41 ± 0.49g; Nimesulide group: tumor volume 1267.14 ± 476.38mm3, tumor weight 0.97 ± 0.51g; Oxaliplatin group: tumor volume 1054.28±102.82mm3, tumor weight of 0.89 ±0.20g; Nimesulide /Oxaliplatin combination therapy group: tumor volume 532.66±220.11mm3, tumor weight of 0.45±0.31 g. The tumor inhibition rate for Nimesulide group, oxaliplatin group and the inhibition rate nimesulide / oxaliplatin combination therapy group are 46.22%, 51.93% and 72.88% respectively. Differential display analysis of variance between groups of nimesulide group, oxaliplatin group and nimesulide / oxaliplatin combination group for tumor growth was significantly slower compared to the control group(P<0.05). Inhibitory effect of Nimesulide/oxaliplatin combination therapy group is significantly higher than Nimesulide group or Xxaliplatin group(P<0.05), while there is no difference between Nimesulide group and Oxaliplatin group(P>0.05).Quantification of xenograft tumor cell apoptosis using flow cytometry showed apoptosis rate of 12.12%±0.47%(control), 22.16%±5.24%(Nimesulide), 37.33%±7.25%(Oxaliplatin) and 47.26%±6.73%(Nimesulide/ Oxaliplatin), respectively. Statistic analysis showed significant difference of all three groups compared to control group(P<0.05). Nimesulide/oxaliplatin combinational group has significantly higher apoptosis rate than when using alone(P<0.05). There is no difference between Nimesulide and Oxaliplatin group in apoptosis(P>0.05).The expression of COX-2 protein in xenograft tumors were quantified using blow cytometry. Results showed FI value of 0.598 ± 0.090 for control group, 1.390±0.192 for Oxaliplatin group, and 0.791±0.121 for Nimesulide/Oxaliplatin group, respectively. Nimesulide and Nimesulide/ oxaliplatin groups revealed much lower COX-2 expression(P<0.05) compared to control group. Oxaliplatin group COX-2 protein expression level was higher than control group(P<0.05).Conclusion:1 Higher expression of COX-2 was found in esophageal tumor as well as tissue adjacent to tumor cells, indicating that COX-2 may play an important role in esophageal carcinogenesis. In esophageal squamous carcinoma cells, reduced COX-2 expression is found to correlates to lower esophageal tissue differentiation.2 Both Nimesulide and Oxaliplatin can inhibit tumor growth, there is synergistic effect when Nimesulide and Oxaliplatin were used together with enhanced inhibitory efficiency. Nimesulide can improve the effect of Oxaliplatin in tumor suppression, through induction of apoptosis of tumor cells.In summary, using specimens from esophageal cancer surgery and mice xenograft tumor cells, this study demonstrated that COX-2 plays an important role in esophageal carcinogenesis. In addition, the selective COX-2 inhibitors Nimesulide and Oxaliplatin, can be used to suppress tumor progression by inducing tumor cell apoptosis. When used together with Oxaliplatin, Nimesulide can improve tumor inhibition effect of Oxaliplatin. |
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