Mechanism Of Tumor Growth Inhibition Of Celecoxib And The Synergistic Effect With Oxaliplatin In Lung Cancer Xenograft In Nude Mice

Objective: In recent years there is a growing body of evidence suggesting a correla-tion between NSAIDs use and lower incidence of cancer. Epidemiological studies showed that the rate of mortality from colorectal cancer in individuals taking NSAIDs was 40–50% lower than that in nonusers. The NSAIDs act by blocking cyclooxygenase(COX)-1 and COX-2. Thus inhibiting the conversion of arachidonic acid to thromboxane and prostaglandins. While COX-1 is ex-pressed in platelets and the gastric mucosa. COX-2 is specifically induced in sites of inflammation and neoplasia. COX-2 is frequently overexpressed in the cells of various tumors. Thus, cell proliferation and immune functions may be controlled in part by endogenous prostaglandin synthesis, a process that is inhibited by NSAIDs. However, the mechanism by which NSAIDs prevent cancer and induce apoptosis in neoplastic cells is not yet clear. Apoptosis is a tightly regulated process involving changes in the expression or activities of distinct genes. Dozens of such genes involved in the induction or inhibition of apoptosis have been cloned and analyzed.Survivin, one of the targets of the Wnt-signaling pathway counteracts cell death and controls mitotic progression. It is undetectable in normal adult tis-sues and becomes prominently expressed in nearly all human cancers. NSAIDs are able to downregulate survivin by inhibition of the Wntsignaling pathway as demonstrated in vitro. In vivo study was not found.COX-2 is an enzyme that can be inhibited with available medications, it has a major influence on cancer biology and applicable to many tumor types, it is involved in many aspects of carcinogenesis. A proposed mechanism for the antitumor effect of COX-2 inhibitors was that they inhibit the growth of newly formed blood vessels, COX-2-derived prostanoids were shown to modulate cytokine synthesis, to influence cell proliferation and apoptosis, and to modulate the nuclear translocation and function of tumor suppressor gene products. But it is naive to expect COX-2 inhibitor alone can have a deadly impact on the cancer process in which multiple aberrations are involved. Therefore, a combined use of COX-2 specific inhibitors and chemotherapy might result in a combination of cytotoxic and antiangiogenic effects or, pos-sibly, overcome multi-drug resistance, and enhance anti-cancer efficacy.Given that the mechanisms of action of celecoxib, an inhibitor of COX-2, and oxaliplatin, a chemotherapeutic agent, are different, combination of the two drugs could increase antitumor efficacy. This study is designed to evalu-ate the effects of different doses of celecoxib, or in combination with ox-aliplatin, on tumor growth, survivin expression and angiogenesis and in lung cancer xenograft in nude mice. At the third part we detect the expression of FEZ1 and Survivin in small cell lung carcinoma (SCLC) andⅢgraded squamous cell carcinoma (SCC), and provide a theoretical basis for clinical treatment.Method:1 Immunohistochemical and flow cytometry method were used to de-tect the expression of FEZ1, Survivin in SCLC and SCC; and to detect apop-tosis ratio and cell proliferation index in normal lung tissue, SCLC and SCC.2 Xenograft animal model of human lung cancer were established by injecting A549 cells into BALB/c nude mice subcutaneously. Then the mice were randomly divided into 4 groups:control group, celecoxib group one(25 mg.kg-1.d-1),celecoxib group two(50 mg.kg-1.d-1),celecoxib group three(100 mg.kg-1.d-1). celecoxib was administered respectively. Tumour vol-umes were measured every week. The mice were sacrificed at day 42 after treatment and tumor tissues were used to detect COX-2, VEGF and survivin, and microvessel density(MVD) by immunohistochemistry and RT-PCR.3 Xenograft animal model of human lung cancer were established by injecting A549 cells into BALB/c nude mice subcutaneously. Then the mice were randomly divided into 4 groups:control group, celecoxib-treated group, oxaliplatin-treated group and celecoxib combined with oxaliplatin-treated group. Medecine were administered respectively. Tumor volumes were weekly measured.The mice were sacrificed at day 42 after treatment and tu-mor tissues were used to detect COX-2, VEGF and survivin, and microvessel density(MVD) by immunohistochemistry and RT-PCR.Results:1 Expression of FEZ1, Survivin in small cell lung carcinoma and squamous cell lung carcinomaThe immunohistochemical staining showed that the positive expression rate of FEZ1 were 72.22% and 39.19% in SCLC and SCC respectively and the positive expression rate of survivin were 75. 00% and 52.70% respectively in SCLC and SCC. The results of flow cytometry was consistent with the results of immunohistochemical staining, in the SCLC and SCC, the positive expression of FEZ1 and Survivin were significantly different. We further compared the apoptosis and proliferation status of different lung cancer, the apoptosis rate in normal tissue, SCC, SCLC were 4.61±2.16, 10.03±3.37, 11.47±4.30 respectively;the proliferation index were 40.09±9.68, 48.23±10.33, 52.08±8.60 respectively. The apoptosis ratio and proliferation index of normal lung tissue obviously lower than SCC and SCLC,the dif-ferences was significant (P<0.05). The apoptosis ratio and proliferation index went higher with the elevation of malignant degree of the lung cancer.2 Effect of different doses of celecoxib on tumor growth, survivin ex-pression and angiogenesis in lung cancer xenograft in nude mice All the mice developed tumors 4 to 13 days after cell inoculation. two mice died in the third celecoxib group during the experiment. After the treatment was finished, the tumor volume of each group was as follows: control group, 3124±657mm3;celecoxib group one, 2093.12±609 mm3;celecoxib group two, 1652±463 mm3; celecoxib group three, 1341±282 mm3. Different doses of celecoxib prevented the growth of tumors in nude mice. At the end of the experiment, celecoxib inhibited the tumor growth by 34.60%,49.40% and 59.71% in celecoxib group one, two, three, respec-tively (P < 0.05 compared with control group). Tumor treated with celecoxib grew slowly. Tumor in celecoxib group three grew more slowly than tumors in celecoxib group one.In order to explore the mechanisms of the inhibition on tumor growth of celecoxib we performed immunohistochemistry analysis of COX-2 protein and survivin protein in tumor samples. Mice treated with different doses of celecoxib had a statistically significant reduction in tumor levels of COX-2 and survivin protein in comparison with mice in control group (P < 0.05). COX-2 and survivin protein level in celecoxib group three was significantly lower than that in celecoxib group one(P < 0.05). the results of Western blot analysis was consistent with immunohistochemistry analysis. COX-2 and survivin protein expression was suppressed dose dependently.In comparison with control group, VEGF mRNA level was significantly reduced when mice were treated with different doses of celecoxib There was no difference in VEGF mRNA level among celecoxib-treated groups (P > 0.05). Different doses of celecoxib significantly inhibited xenograft tumor angiogenesis, but the inhibition was not dose-dependent.3 Effect of celecoxib combined with oxaliplatin on tumor growth, sur-vivin expression and angiogenesis in lung cancer xenograft in nude mice.After the treatment was finished, the tumor volume of each group was as follows: control group, 3124±657.41mm3;celecoxib group, 2093.12±608.93 mm~3;oxaliplatin group, 1736±336.46 mm3;combination use group, 1152±247.89 mm3. Both celecoxib and oxaliplatin prevented the growth of lung tumors in nude mice. At the end of the experiment, celecoxib and oxaliplatin inhibited the tumor growth by 34.60% and 46.70% respectively (P < 0.05 compared with control group). Administering both agents at the same time caused a 66.09% tumor inhibition (P < 0.05 compared with the other three group, respectively). Compared with tumor growth in control group, the tu-mors in all the treatment group grew slowly. Combination use of celecoxib and oxaliplatin promoted tumor inhibition. We could find from the tumor growth curve that celecoxib had a more obvious inhibition on tumor growth than oxaliplatin did at a early stage. At a late stage, oxaliplatin had a more significant effect.In order to explore the mechanisms of the inhibition on tumor growth of celecoxib and oxaliplatin, we performed Western blot analysis and immuno-histochemistry analysis of COX-2 protein and survivin protein in tumor sam-ples. COX-2 staining was found only in the cytoplasm. Mice treated with celecoxib had a statistically significant reduction in tumor levels of COX-2, survivin protein in comparison with mice in control group (P < 0.05). Survivin protein levels were significantly reduced in oxaliplatin group compared with that in control group (P < 0.05). In comparison with control group, COX-2 level in oxaliplatin group is significantly elevated (P < 0.05). Survivin protein level in combination group was significantly lower than that in control group(P < 0.05). the results of Western blot analysis was consistent with im-munohistochemistry analysis.In comparison with control group, VEGF mRNA level was significantly reduced when mice were treated with celecoxib alone, or in combination with oxaliplatin, and elevated when mice were treated with oxaliplatin alone (P < 0.05). There was no difference in VEGF mRNA level between celecoxib group and combination group (P > 0.05). Celecoxib alone, or in combination with oxaliplatin, significantly inhibited xenograft tumor angiogenesis, but not oxaliplatin alone. MVD values in celecoixb group and combination group were significantly lower than that of control group(P < 0.05).Conclusions:1 The positive staining rates of carcinoma for FEZ1 and Survivin are significantly different in SCLC and SCC, and is beneficial for clinical diagno-sis and treatment.2 Celecoxib can suppress tumour growth dose dependently partly by inhibiting suvivin expression. low dose of celecoxib can decrease xenograft lung cancer angiogenesis which play an important role in the metastatic po-tential of lung cancer 3 Celecoxib can suppress tumor growth, angiogenesis and expression of COX-2 and survivin,when being used alone, further, when being com-bined with oxaliplatin,it can enhance the effect of oxaliplatin against tumor.