Study On The Activity Of BRD4 Degradation Agent In Bladder Cancer

Bladder cancer is the second most prevalent malignant tumor of the urinary system and remains a significant challenge worldwide.Although current treatments such as surgery and adjuvant chemotherapy are effective for some patients,the overall treatment outcomes of bladder cancer remain unsatisfactory.Therefore,it is crucial to explore new and effective treatment strategies.The recurrence and metastasis of bladder cancer are frequently attributed to its intricate molecular regulatory mechanism.Identifying aberrant gene regulation and novel therapeutic targets can enhance our comprehension of the pathogenesis and biological traits of bladder cancer.The BET protein family plays a critical role as transcriptional regulators and can reshape chromatin structure.Specifically,BRD4,a key member of this family,has been shown to regulate the biological activity of tumor cells in a variety of cancers.BET inhibitors have been developed to block the binding of BET proteins to chromatin,thereby exhibiting potent anti-tumor activity.In various types of tumors,BET inhibitors have shown to be effective in inhibiting tumor growth.Therefore,understanding the mechanism of action of BRD4 in bladder cancer can enhance our comprehension of its involvement in tumor development.In this study,we will explore the expression and function of BRD4 in bladder cancer and evaluate the effect of BRD4 protein degradation by drugs on bladder cancer biology using in vitro and in vivo experiments.Our findings will provide a better understanding of the molecular regulatory mechanisms of bladder cancer,and further supplement and improve its treatment strategies.Methods:To investigate the differential expression of BRD4 m RNA between bladder cancer tissue and adjacent tissue,we utilized TCGA and GEO databases.Additionally,we used techniques such as Western blot and q PCR to study the degradation activity of compounds on BRD4 protein and the effect on the expression of downstream proteins of BRD4.We also used CCK-8 method to detect the inhibitory activity of compounds on cell proliferation,flow cytometry to study the effect of compounds on cell cycle and apoptosis,and TUNEL staining for apoptotic cells induced by compounds.In addition,we constructed a PDX mouse model to evaluate the anti-tumor biological activity of compounds against bladder cancer in vivo.Results:1.By analyzing TCGA and GEO databases,revealed a significant up-regulation of BRD4 gene expression in bladder cancer tissues compared with adjacent tissues(p<0.05).Furthermore,by analysis of clinical data with Kaplan-Meier survival analysis revealing that patients with high BRD4 expression had a lower overall survival rate than those with low BRD4 expression(p<0.05).2.We observed that QCA570 compound can induce BRD4 protein degradation in bladder cancer cells in a dose-dependent manner,with BRD4 degradation rate >80% in bladder cancer cell lines at 3 n M,and DC50 of approximately 1 n M.In the degradation kinetics assay,BRD4 degradation achieved its maximum in 5637,T24,UM-UC-3 cells within 1 hour,and in J82 and EJ-1 cell lines within 3 hours.In the ligand competition analysis,we verified that QCA570 induced BRD4 through the proteasome pathway,which conforms to the design principle of PROTACs.3.By analyzing TCGA and GEO databases,we discovered that EZH2 gene expression was significantly up-regulated in bladder cancer tissues compared with adjacent tissues(p<0.05),suggesting that EZH2 gene is significantly associated with bladder cancer risk.q PCR assay revealed that following exposure to QCA570 compound,EZH2 and c-MYC genes m RNA levels were significantly down-regulated in bladder cancer cells(p<0.01).Similarly,Western blot analysis demonstrated that EZH2 and c-MYC protein expression levels were down-regulated by QCA570compound(p<0.05).4.Using CCK-8 assay,we demonstrated that QCA570 effectively reduced the survival rate of five tested BC cell lines,with IC50 values ranging from 2 to 30 n M.Moreover,5637 cells and J82 cells were especially sensitive to QCA570 treatment,with IC50 values of 2.6 and 10.8 n M respectively,superior to JQ1 anti-proliferation effect.Using PI single-stained flow cytometry,we found that QCA570 induced cell cycle arrest in G2 / M phase in 5637 cells,further confirming the functional effect of QCA570.In addition,we observed that after treating the cells with QCA570 compound for 24 hours,the cell migration ability was significantly inhibited(p<0.05).5.Using Annexin V-FITC/PI double-stained flow cytometry,we demonstrated that QCA570 increased cell apoptosis in a dose-dependent manner.In J82 and 5637 cells,more than 50% of cells were induced to undergo apoptosis by QCA570,while JQ1 had negligible effect on cell apoptosis(p<0.01).Western blot analysis of Caspase-3expression and TUNEL assay for DNA fragmentation further confirmed that QCA570 induced more cell apoptosis than JQ1 in BC cells.6.In this study,we established patient-derived xenograft(PDX)mouse models to evaluate the anti-tumor efficacy of QCA570 in vivo.Our data demonstrated that QCA570 significantly suppressed tumor growth at a dose of 1 mg/kg.Furthermore,we performed immunohistochemistry(IHC)staining of Ki-67 protein in tumor tissues and found that QCA570 markedly reduced Ki-67 expression,indicating decreased cell proliferation.We also measured the protein levels of EZH2 and c-MYC by western blotting and observed that QCA570 downregulated both proteins in tumor tissues.Conclusion:BRD4 may be a potential therapeutic target for bladder cancer.Compound QCA570 can degrade BRD4 protein,induce downregulation of EZH2 and C-MYC genes,and significantly inhibit bladder tumor proliferation.It can serve as a potential therapeutic drug.