Experimental Study Of Inhibitory Effect On Implanted Lewis Lung Carcinoma Of C57BL/6 Mice By Combination Of Cyclooxygenase-2 Inhibitor Celecoxib And Gemcitabine

ObjectiveTo study the inhibitory effect of selective cyclooxygenase-2 inhibitor celccoxibcombined with gemcitabine on subcutaneouslv implanted Lewis lung carcinoma(LLC) ofC57BL/6 Mice and to explore the possible mechanisms.Materials and MethodsForty C57BL/6 mice weighting 20±2g, 4～5 weeks old were used and all were male.The LLC cell line was cultivated in a humidified atmosphere of 5% CO₂ at 37 C in DMEMmedium, supplemented with 15% calf serum and antibiotics. Tumor models wereestablished by hypodermical injection of 1.2×10⁶ tumor cells into the right armpit of themice, and tumor growth was calculated by measuring the long and short diameters. Theexpression of COX-2 protein of LLC cell line and the tumor samples was detected byWestern blot.The mice were divided randomly into four groups and each group consists of 10 mice.Celecoxib group and combined group were given with the diet supplemented withcelecoxib 1mg/g from the next day of the LLC cells injected throughout the wholeexperiment. Gemcitabine group and combined group were given with gemcitabine100mg/kg/0.2ml intraperitoneal injection weekly when the tumors reached 0.2cm in longdiameter(about the 7th day ), and equal volume of saline was given to control groupmeantime. Tumors growth was delineated the tumor growth curve. Four weeks afterimplantation, all mice were sacrificed. The weight of tumors and mice was measured andthe inhibition rates obtained; Immunohistochemistry method was used to examine the expression of VEGF, MMP-2 and to detect MVD of tumor samples. The apoptosis oftumor cells was measured by TUNEL staining and the apoptosis index(AI) was calculated.Analysis: Results were expressed as mean values±standard deviation((?)±s). T test andone-way ANOVA test were used to evaluate differences among the groups, where a valueless than 0.05 (P＜0.05) denote statistical significance. The software package SPSS13.0was used in the statistical analysis.ResultsCyclooxygenase-2 protein was detected both in LLC cell line and tumor samples.Both COX-2 inhibitor and gemcitabine could lead to a significant reduction of tumorgrowth compared with the control group (P＜0.01). And it is more effective when they wereused in combination(Q＞1.15). Compared with the control group, the implanted tumorswere significantly reduced in weight of mice treated with gemcitabine(P＜0.01),celecoxib(P＜0.01) and combined treatment(P＜0.01), and with corresponding inhibitionrates of 54.18%, 49.52% and 88.59%.Expression of VEGF, MMP-2 and MVD in combined group tumors were lower thanthat of control group(P＜0.01, P＜0.01, P＜0.01), gemcitabine group(P＜0.01, P＜0.05,P＜0.01) and celecoxib group(P＜0.05, P＜0.01, P＜0.01), respectively. Expression ofMMP-2 in gemcitabine group was lower than that of control group(P＜0.01) and celecoxibgroup(P＜0.05). The TUNEL staining of tumor showed that the AI (%) of gemcitabinegroup, celecoxib group and combined group were 8.90±1.84, 7.29±1.28 and 17.28±1.74,respectively, which were significantly higher than that of control group2.85±0.56((P＜0.01)). The AI of combined group was also significantly higher than that ofgemcitabine group(P＜0.01) or celecoxib group(P＜0.01).ConclusionBoth selective COX-2 inhibition celecoxib and gemcitabine can significantly inhibitthe growth of implanted Lewis lung carcinoma.Combination use of celecoxib and gemcitabine has significantly synergistic antitumoreffect on implanted Lewis lung carcinoma compared with gemcitabine or celecoxib alone. The synergistic antitumor effect may be achieved by angiogenesis inhibition and apoptosisinduction of tumor. The above results suggest combination of COX-2 inhibitors andgemcitabine represent a potential new therapeutic strategy against lung cancer.