The Mechanisms Of Andrographolide Enhancing TRAIL-induced Apoptosis In Bladder Cancer Cells

Background : Bladder cancer is the most common malignant carcinoma of human urinary system,of which bladder transitional epithelial cancer has the highest incidence.Current treatments have limited efficacy in advanced bladder cancer patients.Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)is an effective chemotherapeutic agent that specifically impairs cancer cells while sparing normal cells;however,bladder cancer cells can develop resistance to TRAIL.Recent studies have shown that Andrographolide(Andro)enhances TRAIL-induced tumor cell apoptosis in multiple cancer cell lines.However,the combination therapy of TRAIL with Andro has not been implemented in bladder cancer treatment,and its molecular mechanisms remains unclear.Objectives : To provide evidence of Andrographolide,a diterpenoid lactone derived from Andrographis paniculata,as an effective sensitizer to overcome TRAIL resistance in bladder cancer cells.To further investigate the molecular mechanisms of Andro's sensitization effect on TRAIL in bladder cancer cells.Methods:For bioinformatics analysis,we analyzed mRNA expression levels of targeted genes between tumor tissues and adjacent normal bladder tissues based on data from the Oncomine and the TCGA(The Cancer Genome Atlas)database.We conducted GSEA(Gene Set Enrichment Analysis)to assess the association between the predefined gene sets and the particular phenotypes.For in vitro experiments,cell viabilities were assessed by MTS assays;protein expression levels were detected through Western blot assays;cell apoptosis rates were assessed by flow cytometry using an Annexin V-FITC/PI apoptosis kit.We used cell counting assays,cell colony formation assays to determine cell proliferation rates;we used cell Wound Healing assays to assess cell migration ability;we used real-time quantitative PCR(qPCR)methods to detect the mRNA expression levels of the targeted genes.Results:Our results showed that the combination treatment of Andro and TRAIL retarded growth,attenuated proliferation,decreased colony formation,inhibited migration and promoted caspases-mediated apoptosis in T24 cells.The combination treatment of TRAIL and Andro can further activate caspase8 and caspase9.Additionally,the sensitization of TRAIL in bladder cancer cells by Andro is achieved through up-regulation of death receptors(DR4 and DR5)in a P53-dependent manner.Crucially,Andro is also capable of inactivating NF-?B signaling pathway via transcriptional down-regulation NF-?B p65/RelA,which is further contributed to enhancement of TRAIL-mediated cytotoxicity.Besides,the combination therapy further reduced the mRNA expression levels of anti-apoptotic genes,such as Bcl-2and XIAP.Conclusion:These results indicated that non-toxic doses of Andrographolide sensitized bladder cancer cells to TRAIL-mediated apoptosis,suggesting it as an effective therapeutic agent for TRAIL resistant human bladder cancers.