IL-8 And IL-8R Neutralizing Antibodies Inhibit The Proliferation And Migration Of Androgen Independent Prostate Cancer Cells

BackgroundProstate cancer(Prostate cancer,PCa)is one of the most common causes of cancer-ralated death in men.The vast majority of these patients present with locally advanced or metastatic disease,rendering the cancer inoperable.Radiotherapy,alone or in combination with hormone ablation therapy,is a safe and effective treatment for patients with organ-confined PCa.However,neither therapy is effective in improving the prognosis of advanced PCa which is uniformly fatal.As many as 30% of patients treated with radiotherapy,chemotherapy and/or hormone ablation therapy develop a recurrence of their cancer.Bone metastasis,an incurable complication of PCa,was detected in >90% of patients who died from PCa.Especially in recent years,with the use of hormone therapy in patients with PCa,more and more develop into androgen independent prostate cancer.Even tumors without metastasis in time,it is still a great challenge to the clinical treatment,so it is also called the patients with refractory prostate cancer.IL-8 is a proinflammatory cytokine,which is highly expressed in prostate cancer and prostate cancer,and it has a close relationship with the occurrence and development of tumor.IL-8 can be secreted by prostate cancer tumor cells and infiltrating inflammatory cells,which promote cancer cell proliferation and migration by binding with IL-8 receptor on the surface of tumor cells.Studies have showed that the tumor promoting effect of IL-8 could be stopped by blocking the signal of IL-8 producing in the above cells.But it is very difficult to completely block the IL-8 secretion signal in vivo.In this study,we used androgen independent prostate cancer cell line DU145 model to investigate the effects of IL-8 and IL-8R on cancer cell apoptosis,proliferation and migration.It will provide a new target and theoretical basis for the clinical application of IL-8 antibody or IL-8R antibody to treat hormone independent PCa.Objective Our study aims to make clear that DU145 cells could express IL-8,by flow cytometry,MTT method,Transwell test and immunofluorescence experiments we may demonstrate that IL-8 or IL-8R antibodies can promote DU145 cell apoptosis and inhibit the proliferation and migration.This will provide a new target and theoretical basis for the clinical application of IL-8 antibody or IL-8R antibody to treat hormone independent PCa.Methods1.Detect the level of IL-8 in culture supernatant: DU145 cells were cultured 0h,12 h,24h and 48 h within supernatant 500 ul,divided into 0h group(name as the control group),12 h group,24 h group and 48 h group of four groups,the level of IL-8 in different culture time was detected by IL-8 ELISA test kit,with culture medium was the blank control group.2.Detect the apoptosis of DU145 cells: the apoptosis of DU145 cells dealt with IL-8 antibody,IL-8R antibody and control 1640 was detected by flow cytometry.3.Detect the proliferation activity of DU145 cells: Adding IL-8 and IL-8R antibody after DU145 cell clone formation decreased,respectively(1.31 + 0.10)and(1.07 + 0.14),the two group OD values were significantly lower than the control group(3.02 + 0.08),and there were significant differences.4.Detect the migration ability of DU145 cells: the migration ability of DU145 cells dealt with IL-8 antibody,IL-8R antibody and control 1640 was detected by Transwell test.5.Detect the expression of HMGB1 protein in DU145 cells: the expression of HMGB1 protein in DU145 cells dealt with IL-8 antibody,IL-8R antibody and control 1640 was detected by immunofluorescence confocal microscopy.Results1.The results for ELISA: DU145 cells were cultured for 0h,12 h,24h and 48 h,and IL-8 concentration in supernatant was(0.03±0.02)ng/ml,(1.21±0.05)ng/ml,(1.21±0.05)ng/ml,(3.10±0.05)ng/ml,(3.91±0.06)ng/ml,respectively.With the increase of culture time,the concentration of IL-8 was increased,and there was a statistical difference between each time group(P<0.001).2.The results for flow cytometry: compared with the control,the apoptosis rate of DU145 cells dealt with IL-8/IL-8R antibody was(9.92±1.02)% and(10.34±1.07)%,respectively,and there were significant differences(T=21.2,P<0.001;T=12.5,P<0.001).3.The results for MTT: the OD value of DU145 cell dealt with IL-8/IL-8R antibody was significantly lower than that in the control group,and there is significant difference(T=9.3,P<0.001;T=5.4,P<0.001)..4.The results for Transwell: the clone formation of DU145 cell dealt with IL-8/IL-8R antibody was(49.67 + 3.54)and(49.83 + 3.44),respectively,which was both lower than that in untreated group,and there were significant differences(T=19.3,P<0.001;T=9.3,P<0.001).5.The results for immune fluorescence detection: without the antibody group,the cells were spindle-like and had rich cytoplasm,HMGB1 showed red large granular.With IL-8/IL-8R antibody treated,the cells showed a round shape and cytoplasm was reduced,HMGB1 expression signal was weak and only present particle or dust shape.Conclusions1.1640 cultured androgen independent prostate cancer DU145 cells can secrete IL-8,and with the time prolonged,IL-8 secretion increased;2.IL-8 antibody or IL-8R antibody can promote the apoptosis of DU145 cells in vitro,and inhibit the proliferation and migration of the tumor cells.