In Vitro Experimental Studies And A Pilot Clinical Study Of The Anti-tumor Effect Of Selective COX-2 Inhibitor Celecoxib On Bladder Cancer

Bladder tumor is the most frequent urinary malignancy in Chinese people, of which most is non-muscle invasive tumor. Bladder cancer is characterized of high recurrence, owing probably to the local immunodeficiency in mucous membrane of urinary bladder, which makes residual tumor cells be able to escape from immunological surveillance, surviving and forming recurrence. For this reason, intravesical chemotherapy or immunotherapy is used postoperatively for the prevention of tumor recurrence and progress. Bacillus Calmette-Guerin (BCG) is currently the most effective intravesical immunotherapeutic agent, which takes the anti-tumor effect by the mechanism of effective activation of local immune response in mucous membrane of urinary bladder. However, treatments like intravesical BCG therapy are still not able to completely control recurrence and progress of bladder cancer. Prophylaxis of recurrence and progress of bladder cancer is a tough problem unsettled.In recent years, Cyclooxygenase 2 (COX-2) has gradually become a research focus of bladder cancer. Studies suggested that COX-2 was almost not expressed in normal human urothelial cells, but it was significantly over expressed in human bladder cancer cells. COX-2 affects cell differentiation, apoptosis, vascularization of bladder cancer, and is related with tumor grading and staging, tumor progress and prognosis. Therefore COX-2 has been supposed to be a new therapeutic target for the treatment of bladder cancer.Issues concerning COX-2 and bladder cancer have been a focal point of the recent researches in our laboratory. In this study, we investigated the anti-tumor effect and potential mechanisms involved by studying the in vitro impact of selective COX-2 inhibitor celecoxib on immunological functions of BCG-infected human cord blood derived dendritic cells (CBDCs) andin vitro proliferation and apoptosis of human bladder tumor cell line T24, and tried to understand potential adjunctive therapeutic effect of COX-2 by a pilot clinical study on intravesical BCG therapy for non-muscle invasive bladder cancer.Part I. Impact of Selective COX-2 Inhibitor Celecoxib on In Vitro Immunological Function of BCG-infected Human CBDCs1. Firstly, mononuclear cells were isolated from human cord blood and induced to differentiate to dendrtic cells (DCs) in vitro. Then DCs were stimulated mature using BCG. Results of identification showed that the cultured cells were morphology- and phenotype-typical DCs.2. DCs were co-cultured with complete medium (as blank), BCG, BCG plus PGE2, and BCG plus celecoxib. Th1-type cytokine IL-12 and Th2-type cytokine IL-10 were measured in culture supernatant of each group by enzyme-linked immunosorbent assay (ELISA). Results demonstrated that after BCG infection, both IL-12 and IL-10 production of DC increased significantly compared with blank group, and no dominant immunological drift of the Th response occured. Celecoxib stimulated a dose-dependent increased IL-12 and decreased IL-10 production, meanwhile PGE2 was on the contrary. The results indicated that Celecoxib induced Th response drift to Th1, meanwhile PGE2 induced a drift to Th2 type response.2. Allogenic mixed lymphocyte reactivity (MLR) was determined by MTT assay of DCs in each group. Results showed that celecoxib improved but PGE2 inhibited MLR of BCG-infected DCs.3. Cytotoxicity of CTLs activted by DCs of each group was measured by LDH release assay. Results suggested that celecoxib enhanced but PGE2 suppressed cytotoxicity of the BCG-infected DC-activated CTLs on human bladder cancer cell line T24.Part II: Impact of Celecoxib on Proliferation and Apoptosis of Human Bladder Cancer Line T24 in Vitro1. Adherent T24 cells was respectively co-cultured with different concentrations of celecoxib, PGE2 and PI-3K inhibitor LY294002, and their proliferation activity was determined using MTT assay. Adherent T24 cells were also co-cultured with CM, DMSO (0.1%), PGE2, celecoxib and LY294002 respectively, then apoptosis rate was detected using flow cytometry. Results showed celecoxib significantly and dose-dependently inhibited the proliferation and incresed apoptosis of adherent T24 cells; meanwhile PGE2 and LY294002 had no dominant impact on T24 cell proliferation and apoptosis. 2. Suspension cultured T24 cells were co-cultured respectively with CM, DMSO (0.1%), PGE2, celecoxib and LY294002, apoptosis (anoikis, detachment-induced apoptosis) of T24 cells was then measured using flow cytometry. And after co-cultured with CM, PGE2 and celecoxib, activation of PI-3K/Akt pathway were determined by Western blot. Results suggested that celecoxib and LY294002 significantly increased anoikis of T24 cells, meanwhile PGE2 had no obvious effect on T24 cell anoikis. Western blot showed celecoxib significantly inhibited but PGE2 increased Akt phosphorylation, indicating reduced and enhanced activation of PI-3K/Akt pathway by celecoxib and PGE2 respectively, which might be a mechanism of enhancement and inhibition of anoikis respectively.Part III. A Pilot Clinical Study of Impact of Oral Celecoxib Capsule on Efficacy of Intravesical BCG for Non-muscle Invasive Bladder CancerA total of 42 patients with non-muscle invasive bladder cancer enrolled this study atfter transurethral resection of bladder cancer, and were randomized into BCG alone group or combination treatment group and treated with intravesical therapy alone or intravesical BCG therapy plus oral celecoxib capsule treatment respectively. Urine cytokine levels of IL-12,IL-10 were determined by ELISA before and after treatment. And patients were followed by tumor recurrence, tumor progress, and treament related adverse reactions. Factors affecting treatment response to celecoxib were analyzed. Results demonstrated that:1. No significant difference of urine IL-12 and IL-10 was found between the two group prior treatment, but post treatment, IL-12 level and its increase quantity was significantly higher, and IL-10 level its increase quantity was significantly lower in combination group than in BCG alone group. These results demonstrated an induction of Th1-polarized immunological drift of BCG-activated local immune response in bladder wall and an enhancement of local anti-bladder tumor immune function in bladder by celecoxib.2. It is too early to discuss the effect of celecoxib on tumor recurrence and progress of intravesical BCG treated patients, since the follow-up is not enough. Long-term follow-up is needed for final conclusions。3. No obvious difference was found in treatment related adverse reactions between the two groups.4. Patients of older age (≥65 years), or of male sex, or with low stage and grade tumor, or with single and small size tumor, had better response to celecoxib treatment. Mechanisms involved are to be investigated.Summary and Conclusions:1. Celecoxib can enhance BCG-activated Th1-type and suppress Th2-type immune response, induce Th1-polarized immunological drift and thus increase BCG-activated anti-bladder tumor effect, by mechanism of downregulating PGE2 expression through COX-2 inhibition, and inhibiting PGE2-induced Th2-type cytokine IL-10 secretion and its antagonism on Th1-type cytokine IL-12 secretion.2. Celecoxib has double anti-tumor effects including enhancement of BCG-activated DC mediated immune response, and direct inhibition of proliferation and induction of apoptosis of bladder tumor cells. Both are related with COX-2.3. Induction of bladder tumor anoikis by celecoxib is of important clinical significance, which indicates potential prophylatic effect on tumor recurrence and metastasis postoperatively. Celecoxib may induce anoikis of bladder cancer via its inhibition of PI-3K/Akt pathway activation.4. Results of the pilot clinical study confirmed conclusions of in vitro experiments, which showed that celecoxib could induce Th1-polarized immunological drift and increase BCG-activated anti-bladder tumor effect, indicating that celecoxib might increase efficacy of intravesical BCG therapy for the treatment of bladder cancer. Analysis showed that age, gender, stage and grade, tumor number and size are factors affecting responsivity to celecoxib treatment, but mechanisms involved are to be investigated.