The Effect Of Cyclooxygenase-2 Expression In Bladder Transitional Cell Carcinoma On The Phenotypes And Function Of Tumor Infiltrating Dendritic Cells In Vitro

Bladder tumor is the most common tumors in urinary system of domestic people. More than 90% of the bladder tumors belong to transitional cell carcinoma(TCC),which is characterised as high incidence rate and high recurrence rate in clinic.Many scientists have witnessed that the immunological function is lower in the patient with tumor and the function of tumor infiltrating dendritic cells(TiDC) is defective,the activity of the tumor infiltrating lymphocytic cell is lower than healthy people. It was widly accepted that the decrease immunological function in the local of bladder tumor could lead to bladder cancer immune escape.This may result in the remaining tumor cells surviving and keeping growth.It may be the main reason of the high recurrence rate in bladder cancer. Then the"Immunologic black hole theory "was brought up.Dendritic cells(DCs) constitute the major population of antigen-prsenting cells(APCs) ,which serve as professional antigen-presenting cells ,and could be considered the key cells in tumor antigen processing, are pivotal in the host immune response to tumor antigens. Dendritic cells(DCs) orchestrate the development of the immune response by integrating and relaying various signals through interactions with T lymphocytes,B lymphocytes and natural killer(NK) cells,which are potent initiators and modulators of the immune response and potent stimulators of T cell activation,are capable of eliciting antitumor immune responses.However,apparent local immunosuppression presents in bladder cancer ,the impairment of DC function may present a possible reason for this immunosuppression,so it is important to explore the mechanism of the impairment of TiDC function in BTCC. Cyclooxygenase (COX) is the rate-limiting enzyme for the production of prostaglandins. Two isoenzymes of COX have been identified: a constitutive form(COX-1) and an inducible isoenzymes (COX-2). COX-1 contitutively present in almost normal cells and tissues,which is involved in normal tissue homeostasis;and COX-2 is up-regulated in response to a variety of stimuli,including cytokines and growth factors. Accumulating evidences indicate that cyclooxygenase (COX)-2 is inducibly overexpessed in a variety of malignancies,which plays an important role in tumorigenesis .The overexpression of tumor COX-2 is associated with apoptosis resistance,decreased host immunity,increased angiogenesis,and enhanced invasion and metastasis. But the relationship between COX-2 expression and TiDC function inhibition in human bladder transitional cell carcinoma(BTCC) remains unknown. The aim of this study was to evaluate the relationship between COX-2 expression in human BTCC and tumor immune escape. Part ⅠExpression of Cyclooxygenase-2(COX-2) in Bladder Transitional Cell Carcinoma and the relationship between that and the Biological Behavior of Bladder Cancer Objective: To study the expression of cyclooxygenase-2(COX-2) in BTCC and the relationship between that and the biological behavior of bladder cancer and its clinical significance. Methods: Reverse transcriptase polymerase chain reaction(RT-PCR) was applied to detect the mRNA expression of COX-2 in 10 frozen specimens from BTCC , and 10 frozen specimens of normal bladder tissues obtained from brain death donors. Immunohistochemistry method (Powervision method ) was used to detect the expression of COX-2 in 72 cases BTCC ,and 10 specimens of normal bladder tissues.Furthermore ,the relationship between COX-2 expression level and cancer pathologic parameters was analyzed. Results: Determining by RT-PCR method,all of 10 frozen specimens from BTCC showed expressing COX-2mRNA,while only 1 of 10 frozen specimens of normal bladder tissues expressed COX-2mRNA.The results showed significant difference.Using Immunohistochemistry method ,no marked expression of COX-2 was observed in the 10 specimens of normal bladder tissues, while COX-2 protein expression was postive in 72 cases of BTCC,The intensity of COX-2 expression was associated with tumor grade,stage and recurrence. Conclusions : COX-2 overexpressed in BTCC and the abnormal expression of COX-2was closely correlated with the grade , stage and recurrence of tumors.This result indicated that COX-2 may play an important role in the development and progression of BTCC . Part ⅡThe effect of COX-2 expressed in BTCC on the phenotypes and function of TiDC in vitro Objective:To evaluate the effect of COX-2 expressed in BTCC on the phenotypes and function of TiDC in bladder tumor microenvironment and its role in bladder tumor immune escape. Furthermore,to investigate whether BTCC-issued COX-2 can be used as a novel target of bladder cancer chemo-therapy and chemo-prophylaxis. Method: 1. The time-effect relationship and concentration-effect relationship between Celebrex and the expression of COX-2mRNA and prostaglandin E2(PGE2) in bladder cancer T24 cell lines were studied by reverse transcriptase polymerase chain reaction(RT-PCR)and enzyme-linked immuno-absordent assay(ELISA). 2. Monocytes isolated from human peripheralblood by Ficoll-Hypaque solution were cultured in RPMI-1640 with 10%FCS,200ng/ml rhGM-CSF ,50ng/ml rhIL-4 , either T24 tumor cell supernatant(TSN) or TSN from T24 cells treated with specific COX-2 inhibitor ,celebrex, and rhTNF-α20ng/ml for 9 days,and a large number of DCs were generated.In vitro,DC morphology,phenotype,alloreactivity and interleukin(IL)-12 secretion were evaluated.In the same time ,after 6 day culture,DCs were pulsed with T24 cell-lysate,and then their ability to generate antitumor immune responses was assessed by determining the potent effect of cytotoxic T lymphocytes (CTL) response elicited by T24 cell-lysate-pulsed DC in vitro.The production of interferon-γ(INF-γ) by CTL was also assessed by ELISA assay. Results: 1. COX-2 overexpressed in T24 cell line and the basal levels of PGE2 synthesis in T24 cell lines was 2058.3 ±138.0pg/2×105cells.ml /24h. The expression of PGE2 was inhibited by Celebrex in a certain degree of time-dependent and dose-dependent manner,while the expression of COX-2mRNA was not inhibited by Celebrex. However,the production of PGE2 was dramatically decreased to 782.0±69.1 pg/2×105cells.ml /24hwhen T24 cell was treated with 60μmol/L Celebrex for 24h. 2. The cultured cell had typical dendritic cell morphologic feature under optical microscope and scanning electric microscope.There was no significant changes in the expression of CD1a and CD83 in DC(P>0.05).However,the expression of MHC class-Ⅱ( HLA-DR) and costimulating factor(CD80) on the surface of DC generated with TSN were down regulated,and the stimulating proliferation ability of allogeneic T lymphocytes and IL-12 production of these DCs were dramatically decreased(P<0.01). The production of INF-γand specific lysis of T24 cells by effector cells which was stimulated by T24 cell lysate-pulsed DCs generated with TSN has a significant decrement(P<0.01).The similar results of TSN-cultured DCs could be replicated by exogenous PGE2-cultured DCs. TSN from celebrex treated T24 cell could partially blocked the alteration in DC phenotype,alloreactivity and interleukin(IL)-12 secrtion;moreover, the production of INF-γand specific lysis of T24 cells by effector cells stimulated by T24 cell lysate-pulsed DCs generated with Celebrex treated-TSN is up-regulation.However,we found no effect on TSN-cultured DCs phenotypes and function when TSN-cultured DCs directly cocultured with Celebrex. Conclusions : 1. There are no effect of T24 tumor cell supernatant(TSN) on the morphology and maturation of DCs,but the expression of MHC class-Ⅱ( HLA-DR) and costimulating factor(CD80) on the surface of DCs generated with TSN are down regulated, the ability of antigen presenting and T cell activation of these DCs are also decreased.These changes seriously cripple the specific antitumor immune responses induced by DC;in the same time, DC generated with TSN inhibits the release of IL-12,switching the Th1/Th2 balance toward Th2-type responses,decreasing the level of IFN-γ,then the Th1 mediated antitumor immune response is significantly reduced. 2. Our study demonstrates that it is a paracrine PGE2 pathway to inhibit the phenotypes and function of TiDC in bladder tumor microenvironment, specific COX-2 inhibitor,celebrex,partially blocks the alteration in TiDC by decreasing the production of aracrine PGE2. Collectively,this study indicates:(1)The overexpression of COX-2 in BTCC is closely correlated with the grade , stage and recurrence of tumors .(2) COX-2 expressed by bladdertumor cell causes the abnormal phenotypes and inhibits the capability of presenting antigen and inducing the anti-tumor immunoresponse through PGE2 pathway in TiDC.These changes may be one of the important mechanismes of immune escape in bladder cancer.(3) Celebrex,specific COX-2 inhibitor,improves the function of TiDC through decreasing the production of PGE2 in the microenvironment.(4) BTCC-derived COX-2 may be an important target for chemo-prevention as well as pharmacological therapy in bladder cancer.