COX-2 Inhibition Demonstrates The Reversal Effect Of Malignant Biology Behaviors In Bladder Cancer

Bladder cancer is common type of malignant tumor. In China, it causes first place of the incidence and mortality in the urological and genital tumor. Tumor recurrence is easily occurred in bladder cancer with the relapse rate post operation of 60-80%, and the grade of malignancy and the potence of infiltrate is enhanced with the number of relapase increasing. At present, the aggregate efficiency of chemical therapy for bladder cancer is about 15-20%. Thus, new and more effective treatment modalities are needed. It was reported that COX-2 expression was closely related to carcingensis of clon cancer, pancreatic cancer, prostate cancer, ostoesarcoma, small cell lung cancer and COX-2 inhibitor could restrain tumr proliferation. Up to now, the concrete mechanism of COX-2 in carcingensis is not clear. So, elucidating the effects of COX-2 on baladder cancer will further our understanding of the etiology of bladder cancer and lay the therotics and experimential basis for cancer prevement and treatment.[Objective]The aims of current study were to evaluate the reversal effect of malignant biology behaviors by cyclooxygenase-2 (COX-2) inhibition andto elucidate the role of COX-2 in carcinogenesis of human bladde cancer.[Materials and methods!1. Detection of COX-2 expression in clinical specimenCOX-2 expression and microvascular density were examined in 96 cases of bladder transitional cell carcinoma specimens and 10 cases of normal bladder tissues by immunohistochemical staining, then the relationship between COX-2 expression and biological behaviors of bladder cancer was analyzed.2. Transfection of COX-2 anti-sense eukaryotic expression vectors into COX-2 highly expressed human bladder cancer cell line 5637(1) Using lipofectamine 2000 reagent, the COX-2 highly expressed human bladder cancer cell line 5637 was transfected anti-sense eukaryotic expression vectors pcDNA3.1/hCOX2 (-) and control plasmid pcDNA3.1. Single stable clones were screened by limited dilition after G418 screening for 2 months;(2) Semi-quantitative RT-PCR and Western blot were used to testify the protein and mRNA level in the transfected 5637-AS cells.3. Biological characteristics of the transfected cells(1) MTT method was used to draw the growth curves of the transfected cells. Cell cycle distribution was studied by flow cytometry;(2) ELISA was used to detect the amount of PGE2 in supernatant;(3) PKA activity was examined by PKA detection kit;(4) Soft agar assay was used to examine the tumorigenesis of the transfected cells in vitro. Transwell chamber assay was used to determine the invasiveness of the transfectants;(5) Tumor implantations experiment was carried out to determine the tumorigenesis of the transfected cells in vivo;(6) Western blot was conducted to examine the expression ofendothelium-specific angiogenic factors VEGFn Ang-1 andAng-2;(7) HUVECs proliferation and migration experiment were conducted to study influence of transfected cells on the proliferation and migration ability of endothelial cells;(8) An immunohistochemistry SP method was used to detect microvessel density of tumor grafts.4. The reversal effect of COX-2 inhibitor on bladder cancer(1) MTT method was used to elucidate the effect of COX-2 inhibitor on proliferation of bladder cancer in vitro, and FCM was used to analysis cell cycle distrubtion and apoptosis. Transmission electron microscope was used to observe the ultrastructure of apoptotic cells;(2) Tumor implantations experiment was carried out to determine the influence of COX-2 inhibitor on tumorigenesis of the bladder cancer cells in vivo.[Results]1. COX-2 expression in bladder cancer specimensThe positive rate of COX-2 expression in tumor tissues was 46.9%, whileCOX-2 expression was negative in 10 normal bladder tissues. COX-2expression was significantly correlated to the tumor grades, stages andrecurrence. MVD was higher in COX-2 positive expression patients thanthat in COX-2 negative expression patients.2. Cell transfection and identificationpcDNA3.1/hCOX2(-) or pcDNA3.1 was stably transfected into bladder cancer 5637 cells using lipofectamine 2000 reagent. The transfected cells were screened by G418 for 2 months. Semi-quantitative RT-PCR and Western blot suggested that in pcDNA3.1/hCOX2(-) transfected 5637-AS cells COX-2 expression was decreased, while in pcDNA3.1 transfected 5637-P cells COX-2 expression remain the same level as untransfected cell.3. Biological characteristics of the transfected cells3.1 Growth and proliferation characteristics of the transfected cellsMTT assay suggested that the 5637-AS cell which transfected with pcDNA3.1/h COX2(-) proliferated more slowly than the 5637 cells with prolonged double time, while the growth qnd proliferation characteristics of 5637-P that transfected with pcDNA3.1 changed slightly when compared with 5637 cells.3.2 Cell cycle distribution of the transfected cellsIn flow cytometry, 5637-AS cells were found to arrested inG₁ phase with increased ratio of G₀/G₁; while the cell cycle distrubtion of 5637-P changed slightly when compared with 5637 cells.3.3 PGE2 release in the culture supernatant of the transfected cellsIn ELISA, the PGE2 level of 5637-AS cells was significatly decreased when compared with 5637 cells and 5637-P.3.4 Detection of protein kinase A activity5637-AS cells had a similar total PKA and a significant lower active PKA activity than the 5637 cells in per μg cell extract.3.5 Soft agar assaySoft agar assay suggested that 5637-AS cells formed fewer colonies in the soft agar than 5637 cells. There was no significant difference in the colony numbers between 5637-P and 5637 cells.3.6 Transwell chamber assayTranswell chamber assay suggested that the cell number of the 5637-AS cells that migrated through the filter was less than the 5637 and 5637-P cells, while the cell number of 5637-P cells was similar to 5637 cells.3.7 Tumor implantation testOne-way AVONA showed that the volume and weight of tumor transgrafts in 5637-AS was significantly lower than in 5637 and 5637-P.Similar pattern was shown in MVD of transgrafts. There was no significant difference in HE coloration of transgraft organs.3.8 Detction of endothelium-specific angiogenic factorsWestern blot indicated that VEGF and angiopoietin-1(Ang-1) expression was much lower in 5637-AS cells than in 5637 and 5637-P cells, while the expression of angiopoietin-2(Ang-2) remianed the same level among three cell lines.3.9 HUVECs proliferation and migration experimentConditioned culture medium from 5637-AS cells were shown to inhibit the proliferation and migration ability of HUVECs compared to those from parental 5637 and empty-vector transfected 5637-P cells. 4. The reverse effect of COX-2 inhibitor on bladder cancer4.1 The influence of COX-2 inhibitor on proliferation in vitroMTT assay showed that COX-2 inhibitor could significantly reduced the proliferation and growth in bladder cancer in vitro. Among NSAIDs, the inhibition effect of celecoxib was the highest. And the inhibition effect of celecoxib in 5637-AS was lower than in 5637 and 5637-P. The inhibition effect of aspirin, indomethacin, sulindac and nimesulide was similar among three cell lines.4.2 The influence of COX-2 inhibitor on cell cycle distributionIn flow cytometry, celecoxib and indomethacin increased the ratio of G₀/G₁ while decreased the ratio of S and G₂/M phase in 5637 cells, and the effect caused by celecoxib was larger than indomethacin. In 5637-AS cells, above effects were not observed.4.3 The influence of COX-2 inhibitor on apoptosisCOX-2 inhibitor celecoxib and indomethacin both induced apoptosis in bladder cancer cells. Transmission electron microscope obtained results showed that in the COX-2 inhibitor treated cells nuclear fragmentation, chromosomal condensation, cell shrinkage were visible. Subsequent ruffling...