Antisense Inhibition Of COX-2 And NSAIDs Reduce Malignant Phenotype Of Bladder Cancer Cells

Bladder cancer is the most common tumor in urinary system of domestic people which has the clinical characteristic of high incidence rate, high recurrence rate and being difficult to treat. Most superficial cases are treated conservatively by transurethral removal of the tumor, followed by adjuvant intravesical therapy. Although recent advances in the treatment of bladder cancer have been developed, but new strategy in the management of it is needed.Cyclooxygenase (COX), a key enzyme in the conversion of arachidonic acid to prostaglandins and other eicosanoids, exists as two isoforms COX-1 and COX-2. COX-1 is constitutively expressed whereas COX-2 is a highly inducible gene that is activated by cytokines, growth factors, phorbolesters, oncogens, and chemical carcinogens, and has a putative role in carcinogenesis and inflammation. Increased COX-2 expression has been demonstrated in several cancers, including esophageal, gastric, colorectal, pancreatic, lung, breast, prostatic and bladder tumours.Several epidemiological studies have shown that the regular intake of non-steroidal anti-inflammatory drugs (NSAIDs) decreases the risk of colon cancer. This results may be related to the inhibition of cyclooxygenases. According to these findings, the COX-2-selective NSAIDs shows a chemopreventive effect both in animal tumour models and in patients. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, is the only non-steroidal anti-inflammatory drug so far which has been approved by the FDA for adjuvant treatment of patients with familial adenomatous polyposis. The molecular mechanism responsible for the anticarcinogenic effects of celecoxib is still not fully understood.In this study we focused on the reducing of malignant phenotype in bladder cancer cells with antisense COX-2 RNA and NSAIDs in vitro, in vivo. This has established a groundwork for the further exploiture and application of NSAIDs as an alternative or adjunct to conventional chemotherapy regimens in an effort to prevent or delay progressionin the patients with bladder cancer. The main methods and results are as follows:1. Construction of the antisense COX-2 RNA and to be transfected into the bladder cancer cell linesObjective: To construct eukaryotic antisense RNA expression vector of COX-2 and to transfect the bladder cancer cell lines T24.Method: Firstly, by way of PCR, human COX-2 cDNA gene was amplified with plasmid pCB7/hCOX-2 template, and was cloned into plasmid pMD18-T at the restriction endonuclease sites of EcoR I and Xba I. Secondly, pMD18-T/COX-2 and pcDNA3.1 were digested with EcoR I and Xba I, then the COX-2 cDNA was lignated with pcDNAS.l at EcoR 1 and Xba I restriction sites by T4 lignase, and the eukaryotic expression plasmid pcDNA3.1/COX-2 as for tumor suppression gene COX-2 formed .Using lipofectamine-mediated DNA transfection technique, an antisense COX-2 RNA gene expression plasmid (pcDNA3.1/COX-2 as) was transfected into cultured human bladder cancer cell lines T24. After selected by G418, positive clones were selected to culture continually and identified by fluorocytometry assay and indirect immunofluorescence staining.Results: Human COX-2 cDNA was inserted reversely to eukaryotic expression vector pcDNA3.1 at the restriction endonuclease sites of EcoR I and Xba I. The recombinant was named as pcDNA3.1/COX-2 as. It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the constructed eukaryotic antisense RNA expression vector pcDNA3/COX-2 as was correct . Then pcDNA3.1/COX-2 as was transfected into cultured human bladder cancer cell lines T24. It was identified that the expression of COX-2 was significantly suppressed in T24 cells transfected by pcDNA3/COX-2 as compared with the untransfected T24 cells.Conclusions: To construct eukaryotic antisense RNA expression vector of COX-2 successfully and be named as pcDNA3.1/COX-2 as. The antisense COX-2 RNA gene was successfully introduced into T24 cells, which could remarkably inhibit the COX-2 activity. So we got the T24-AS to use it in our further study.2. Anticarcinogenic effects of antisense COX-2 RNA and NSAIDs on Bladder Cancer cell in vitroObjective: To investigate the extent to which the anticarcinogenic effect of NSAIDs(Indomethacin and Celecoxib) is dependent on COX-2 expression.Method: To detect the apoptosis and proliferation of human bladder cancer cells T24 and T24-AS treated with or without NSAIDs(IN, Celecoxib) by MTT, flow cytometry assay and To detect expression of apoptosis gene (Bax, Bcl-2, Bcl-xl, Bak, survivin) in T24 and T24-AS cells by RT-PCR. To detect expression of COX-2 by Western blot analysis, fluorocytometry assay and indirect immunofluorescence staining in T24 and T24-AS cells.Results: The proliferation level of T24-AS to compared with T24 decreased and treatment of these cells with IN and Celecoxib can make it decrease more. The expression level of Bcl-2, Bax, Bak, Bcl-xl in T24-AS was the same as in T24, but we found the expression level of survivin increased. The expression level of COX-2 decreased in T24-AS compared with T24 or T24-P and treatment with IN and Celecoxib can make it decrease more. Treatment of these cells with IN and Celecoxib dose-dependly reduced cell survival which was accompanied by an induction of a G0/G1 phase block and apoptosis. Induction of apoptosis in T24-AS cells was more marked than in T24 cells .Conclusions: These data indicate that apoptosis inducing effects of IN and Celecoxib partly depend on COX-2 expression of the cells, whereas induction of a cell cycle block and antiproliferation occurred COX-2 independently.3. Growth inhibition of NSAIDs on human bladder cancer cell xenografts in nude miceObjective: To establish the nude mice xenografts models of T24 and T24-AS and study the growth inhibition effects of NSAIDs.Method: Each of 42 intact athymic nude mice (5 to 7 weeks old) was injected into 1.5 X 106 bladder cancer cells. The mice were divided equally into seven groups. Four groups of them received oral indomethacin or celecoxib, 3mg/kg and lOmg/kg respectively per mouse per day, and the other group received the same amount of normal saline. After 30 days, we killed the nude mice and record the weight and volume of the xenografts of every one. Histological hematoxylin-osin staining and immunocytochemical SABC technique were employed to detect the histopathological changes.To detect expression of COX-2 in human bladder cancer cell xenografts by RT-PCR and western blot.Results: On the day 7 after transplantation, xenografts was found in nude mice injected bladder cancer cell T24 or T24-P cells. Two days later, in the rest of the mices xenografts was found too. The expression level of COX-2 decreased in T24-AS bladder cancer cellxenografts xenografts decreased compared with T24 or T24-P and decreased in T24 and T24-P after administration of NSAIDs too. The weight and volume of the xenografts of nude mice injected with T24 and T24-P was bigger than with T24-AS. In the groups of T24 and T24-AS after treatment with IN and Celecoxib the weight and volume of the xenografts decreased more and showed dose-dependly.Conclusions: COX-2 could stimulate the growth of T24 xenografts and IN or Celecoxib could inhibit the tumor growth of both T24 and T24-P.In conclusion, we successfully constructed eukaryotic antisense RNA expression vector of COX-2 and got the stable transduction T24 cells using it. We assessed the effect of NSAIDs as a function of COX-2 expression without the interference of other genetic factors. We have obtained additional evidence that the apoptosis-inducing effects of celecoxib and indomethacin only partly depend on COX-2 expression of the cells, whereas induction of a cell cycle block and antiproliferation occurred COX-2 independently.