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Objective: Lung cancer is the most common malignancy in the world, significantly increased morbidity and mortality of lung cancer in China in recent years. Typical early symptoms of lung cancer, the lack of health awareness and other reasons, the clinical diagnosis more than the development of advanced lung cancer has been transferred, so the prognosis is poor. Tumor invasion and metastasis is a complex process of multiple genes, and the interaction between these genes has become a hot research. Recent research results show that the cyclooxygenase -2 (cyclooxygenase-2, COX-2) have an important role in the process of tumor formation, which not only promote tumor cell proliferation, inhibition of apoptosis of tumor cells with tumor invasion and metastasis. Vascular endothelial growth factor-C (vascular endothelial growth factor-C, VEGF-C) is a member of the VEGF family, expressed in breast cancer, cervical cancer, colon cancer, lung cancer, prostate cancer and other tumor cells. VEGF-C secreted by tumor cells through an autocrine mechanism to its receptor - vascular endothelial growth factor receptor -3 (vascular endothelial growth factor receptor-2, VEGFR-3) binding regulation lymphangiogenesis most important signaling pathway. Survivin (Survivin) is the inhibitor of apoptosis protein (inhibitor of Apoptosis Proteins IAP) gene family, which inhibit apoptosis, involved in cell cycle regulation is found the strongest apoptosis inhibitory factor, and tumor , development and prognosis. beta-catenin (beta-catenin)-mediated cell adhesion, also has the function of signal transduction, abnormal expression can promote tumor metastasis. Studies have shown that COX-2 and VEGF-C, VEGFR-3, survivin, beta-catenin expression collaborative upward trend. Nimesulide (nimesulide) is a selective COX-2 inhibitors, Animal experiments confirmed that nimesulide can not only prevent the occurrence of tumors, can also inhibit tumor growth and metastasis, and has enhanced the role of chemotherapy and radiotherapy. Oxaliplatin (oxaliplatin) is a third generation platinum broad-spectrum anticancer drugs produce alkylated conjugates role in DNA, forming a chain and inter-chain crosslinking, thereby inhibiting DNA synthesis and replication. In this study, through the establishment of subcutaneously into nude mice xenograft model of lung cancer observed COX-2 inhibitor nimesulide combined with chemotherapy drug oxaliplatin tumor growth, as well as on COX-2, VEGF-C, VEGFR-3, Survivin, beta -catenin expression, to further explore the molecular mechanisms of tumor metastasis, and the synergistic effect of the combination of two drugs that may arise. Methods: 4 to 5-week-old BALB / c male nude mice 26, weighing 20 to 24 g, IVC sterile feed and sterilized pure water feeding, breeding environment specific pathogen-free, ambient temperature, humidity appropriate . DMEM medium containing 10% fetal calf serum, 100U/ml penicillin and 100U/ml streptomycin at 37 ° C containing 5? 2 incubator A549 human lung cancer cells, cells adherent growth to 70% to 80 % confluence with 0.25% trypsin digestion and passage. Take A549 cells in logarithmic growth phase were digested with 0.25% trypsin solution, PBS washed with serum-free DMEM medium was diluted by haemocytometer counts, cell density was adjusted to 1 × 107/ml. Nude skin disinfection after subcutaneous injection of the cell suspension 0.2ml, 1ml syringe in the right axilla established lung cancer xenografts in nude mice. 25 nude mice tumors, vernier caliper to measure the long diameter of the tumor nodules (a), short axis (b) by the formula V = ab2 / 2, to estimate tumor approximate volume. The average diameter of approximately 4 mm to be transplanted tumor, removed the tumor smallest nude mice were randomly divided into control group, nimesulide group, oxaliplatin group, nimesulide / oxaliplatin combination group, (n = 6). Oxaliplatin nude mice by intraperitoneal injection of oxaliplatin Mannitol Injection dose to 10mg/kg / time, administered once every four days, while oral administration of sterile distilled water; nimesulide group by gavage to drug, dose 20mg/kg/d were intraperitoneally injected with normal saline; the nimesulide / oxaliplatin combined with nimesulide group was given orally and oxaliplatin intraperitoneal injection, the amount of the two drugs alone the same group; the control group was treated with sterile distilled water, intraperitoneal injection of normal saline. Observed growth in nude mice, tumor volume was measured every 5 days. Administered 30 days later, the mice were sacrificed, cut transplanted tumor tissue, tumor tissue was detected by immunohistochemical method in COX-2, VEGF-C, VEGFR-3, Survivin, beta-catenin protein expression, Beijing University of Aeronautics and Astronautics true color pathological image analysis The system calculates the average integral optical density value. Quantitative real-time PCR analysis of tumor tissue in COX-2, VEGF-C, VEGFR-3, Survivin, the beta-cateninmRNA of expression. Results: The nude mice 26, 25 nude mice, tumor formation rate of 96.2%, the time of tumor cell seeding after 5 to 8 days. 12 days after the planting of the tumor cells, tumor diameter average of 4 mm. After the end of treatment, the tumor volume were: control group 1498.83 ± 429.11 mm3; nimesulide group 857.37 ± 113.73 mm3; oxaliplatin group 748.43 ± 42.22 mm3; combination group 442.53 ± 103.81 mm3. Nimesulide group, oxaliplatin group and the combined treatment group tumor inhibition rates were 44.34%, 51.60% and 73.00%, respectively. Analysis of variance between groups comparison showed that nimesulide group, oxaliplatin group and the combined treatment group tumor growth was slow compared with the control group (P lt; 0.05) Tumor inhibition rate of the combined treatment group compared with the nimesulide group and oxaliplatin tumor inhibition rate was significantly increased (P lt; 0.05). Nimesulide group and oxaliplatin group tumor growth inhibition was no significant difference (P gt; 0.05) COX-2, VEGF-C, VEGFR-3, Survivin, β-catenin protein immunohistochemical staining slice image analysis system integrated optical density measurement, analysis of variance showed that compared with the control group, the nimesulide group COX -2, VEGF-C, VEGFR-3, survivin, beta-catenin protein expression was significantly decreased (respectively p lt; 0.05); compared with the control group the the oxaliplatin group of COX-2, VEGF-C, VEGFR- 3 protein levels (respectively p lt; 0.05), and Survivin expression of beta-catenin protein levels decreased (respectively p lt; 0.05); compared with the control group, the combined treatment group, COX-2, VEGF-C, VEGFR- 3, survivin, beta-catenin protein expression was significantly lower (P LT; 0.05) Fluorescence quantitative RT-PCR method for the determination of COX-2, VEGF-C, VEGFR-3, Survivin, β-cateninmRNA expression, analysis of variance results show that compared with the control group, the nimesulide group COX-2, VEGF-C, VEGFR -3, Survivin, β-cateninmRNA expression was significantly decreased (respectively p lt; 0.05); compared with the control group the the oxaliplatin group of COX-2, VEGF-C and VEGFR-3 mRNA expression levels increased (P lt ; 0.05), and Survivin expression levels of beta-cateninmRNA lower (respectively p lt; 0.05); Compared with the control group, the the combination group COX-2, VEGF-C, VEGFR-3, Survivin, beta-cateninmRNA expression were significantly lower (P LT; 0.05). Conclusion: Nimesulide alone or combined with oxaliplatin significantly inhibited the growth of human lung cancer xenografts in nude mice and COX-2, VEGF-C, VEGFR-3, survivin, beta-catenin expression. Oxaliplatin can significantly inhibit the growth of human lung cancer xenografts in nude mice and Survivin, beta-catenin expression. Nimesulide with oxaliplatin in combination to improve the antitumor effects of oxaliplatin.
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