Effects Of Celecoxib On β-catenin,VEGF And MVD Of Xenograft Tumor Of Colon Cancer In Nude Mice

Objective:To detect the expression ofβ-catenin, the key molecule of cellular Wnt signal pathway; and the vascular endothelial growth factor (VEGF),with which the blood vessel to develop, and the Microvessel density(MVD)by administer the cox-2 inhibitor-celecoxib to xenograft tumor of colon cancer in nude mice. To research the effect of cox-2 inhibitor-celecoxib on theβ-catenin,VEGF and MVD. To detect the effect and research some mechanism of celecoxib anti-tumor, providing prepare woke for the using of cox-2 inhibitor in clinic.Method:Buy 30 BALB/C 4-5 week-old gynec nude mice, each about 20-24 gram, raised in specified-pathogens free environment, provide them with suitable temperature,humidity,germfree food and water. Raise human colon cancer cell strain HT-29 in 37℃and 5%CO2 incubator in D/F12 nutrient medium, which content 10% newborn calf serum,100U/ml penicilin and 100U/ml streptomycin. Dispose the HT-29 cell which in logarithmic growth phase with nutrient medium to 1×107/ml cell suspension. After skin degerming, injection of 0.2ml the suspension by 1ml syringe in right shoulder subcutaneouly to establish a xenograft animal model. Measure the tumor by sliding caliper regularly, count the approximate volume. After 16 days,mice were randomly divided into two groups:control group, celecoxib group(40mg/kg/d, suspension intragastric administration).At the time of administration, make sure that grasp the mouse tightly to against injury. Nude mice were raised in specified- pathogens free environment for 40 days. Tumour volumes and weight were measured every week for counting the rate of tumor-inhibited. Mice were sacrificed 40 days after the treatment. Some of xenograft cancer tissues were keeping in 4% paraform for immunohistochemistry, and some were keeping in liquid nitrogen pre-emergence. Xenograft cancer tissues were used to measure the expression level of COX-2,β-catenin and the microvessel density (MVD) by immunohistochemistry, mRNA level of VEGF by RT-PCR method. Compare each index in the two groups by SAS 6.12 t text. Analyze the dependablity of the indexes in celecoxib group by linear correlation analysis.Results:1 All the 30 nude mice have the tumor 5-11days after cell inoculation. At the 16th day, the average of tumor diameter is 5mm. There is no liver metastasis in each mouse by dissect them.2 After the treatment was finished, the tumor volume of each group was as follows: control group, 1238±241mm3;celecoxib group, 852±153mm3;Celecoxib prevented the growth of tumors in nude mice obviously. At the end of the experiment, celecoxib inhibited the tumor growth by 32.54%. Compare with control group, tumor in celecoxib group is grow slower by t test(P<0.05).3 COX-2 staining was mainly found in the cytoplasm which scattered as brown stain. Compare with control group, cox-2 expression is lower in celecoxib group, by using spectrodensitometry to analyze the tumor immunohistochemis- try slide of COX-2 protein in samples (P<0.05).β-catenin staining was mainly found in the cytoplasm/membrane, Compare with control group,β-catenin in celecoxib group is lower(P<0.05).4 Treat the production of RT-PCR with electrophoretic analysis: we can get about 178bp amplication brand, when add VEGF-mRNA primer, get 231bp amplication brand, when add GAPDH primer. Analyze the gray scale of this two brands, the expression of VEGF-mRNA in celecoxib group is lower (P<0.05).5 Counting the microvessels after staining by immunoh- istochemistry, MVD in celecoxib group is less than control group (P < 0.05).6 Comparing the indexes in celecoxib group, we get the positive correlation of COX-2and VEGF-mRNA and MVD(r>0.9,P<0.05), the positive correlation ofβ-catenin and VEGF-mRNA(r=0.95,P<0.05).Conclusion 1,COX-2 inhibitor celecoxib inhibit the growth of xenograft tumor of colon cancer in nude mice。2,inhibit the growth of colon cancer cell by inhibiting the abnormal expression ofβ-catenin which reduce cell proliferation by Wnt signal pathway;3,decrease micro vessels by direct inhibitting the expression of VEGF in tumor, thus inhibit the growth of colon cancer cells.4,decrease micro vessels by indirect inhibitting the inducement of VEGF byβ-catenin, thus inhibit the growth of colon cancer cells.