Role Of Monocyte Chemotactic Protein-1 In Bacillus Calmette-Guerin Induced Immunol Responses To Human Bladder Cancer

Purpose: To study the effect of MCP-1 (monocyte chemotactic protein-1) on the TIL (tumor infiltrating lymphocytes) proliferation and examine whether treatment of human bladder carcinoma cell line with MCP-1 enhance cytotoxicity by TIL.To explore the expression of Toll-like receptors(TLR2 ,TLR4) ,NFkB (nuclear factor kappa B) and MCP-1 in BCG-induced immunol response to bladder cancer,furthermore discuss the procedure and mechanism production of MCP-1.Material and methods: Human bladder transitional cell line EJ were maintained in RPMI 1640 with 15% heat-inactived fetal calf serum, 100u/ml penicilline and streptomycine and 200mmol/ml glutamine. The cell lines were maintained under sterile condition utilizing standard tissue culture incubators at 37℃,5%CO₂ moist atmosphere, passaged in common practice. Improved Rosenberg method was refered to separate TILs.The concentration was adjusted to 1× 10⁶,cell suspenions were incubated at 37 ℃ in a humidified atmosphere of 5%CO₂ in air. The number of viable TILwere determined by counting sample in a hemacytometer, presented with quantity/ml. MCP-1 concentration : O.lng/ml, lng/ml,10ng/ml,stirring TIL proliferation respectively. The cytoxicity of TILs supplemented with different concentration MCP-1 were assayed by MTT method which was describle by Sargent[21. Human bladder transitional cell lines EJ used as the target cells. EJ lines were passaged using 0.25% tripsin(Sigma) and 0.02% EDTA.,EJ cells were made into single cell suspension replate at 5X103 cells/well into flate bottom 96 well tissue culture plates incubator for 24h, growing with TILs which stirred different concentration MCP-1 for 15 days.(MCP-l concentration: (0ng/ml,0.1ng/ml,1.0ng/ml,10ng/ml). Effect cells/target cells:2:1,10:1,50:1 and then plused 0.1%MTT 15ul, the plate reincubated for 4h.Removed MTT and plused DMSO(Sigma) 150ul by mixing on a microshake for 10 minutes.The absorbance(A) at 570nm in each well was determined using a microplate auto-reader(Nanjing,China). repeated 3 times and measured its mean. The Ratio of EJ cells suppression (%) =(1 -effect/control) X100%.The bladder cancer samples of 30 patients from West China Hospital ,Sichuan University were collected and preserved in RNAlater(Ambison)at 4°C,10 urologic tumor-free patients were selected as controls,and 10 patients with superfical bladder cancer(grade Ti;stages2-3)who had previously undergone transurethral resection(TUR) received intravesical BCG (bacillus Calmette-Guerin) treatmentThe remaining 10 patients with primary cancer,including 1 case cystectomy,9 cases TUR, did not receive BCGThe patient was cystectomized because of primary multifocal tumor. The total RNA of each sample wasextracted.RT-PCR procedure was conducted using primer of MCP-l,TLR-2,TLR-4, NFKB(Premega),and products were analyzed with agarose gels electrophoresis,then the expression of MCP-1,TLR-2,TLR-4, NFkB was assessed by the analyzing system of gel imaging. Immunohistochemical staining was also performed by SP method for these cases with the NFKB(P65)-monocolonal antibody to detect the expression of NFKB(P65),compared the staining intensity and staining density, between BCG-treatmented bladder cancer and BCG-untreatmented bladder cancer samples.Result: The proliferation of TIL was significantly strengthed and the cytoxicity was increased by 16.4% in the condition of MCP-1 at concentration of lng/ml in 15days (p<0.01) .With RT-PCR ,MCP-l,TLR-2,TLR-4 and NFkB mRNA expression were detected in 9 of 10 cases of BCG-treatmented bladder cancer samples respectively (90%). The expression of MCP-1,TLR-2,TLR-4 and NFkB was increased in BCG-treatmented bladder cancer samples,compared with BCG-untreatmented bladder cancer samples and urologic tumor-free samples.Except TLR2 expression increased in BCG-untreatmented bladder cancer samples(40%),there was no difference between BCG-untreatmented bladder cancer samples and urologic tumor-free samples.In immunohistochemicaljthe staining intensity and staining density was increased in BCG-treatmented bladder cancer samples,compared with BCG-untreatmented bladder cancer samples,while urologic tumor-free samples were stained negatively.Conculusion: The proliferation and cytoxicity of TIL was increased byMCP-1 in a suitability concentration.The expression of MCP-1,TLR-2,TLR-4 and NFkB was increased in BCG-treatmented bladder cancer samples. Expression of MCP-1 may be depended on TLR2 andTLR4 activating NFkB.MCP-1 expression up-regulated to increase proliferation and cytoxicity of TIL was perhaps partially involved in antitumor mechanism of BCG...