The Study Of The Anticancer Effect Of Polyphyllin I In Bladder Cancer And Its Underlying Mechanism

Background:Bladder cancer is the 10th most common cancer worldwide.Its incidence varies globally,with a high prevalence in well-developed regions such as Europe and North America.In 2018,there are an estimated 549,000 new cases and 200,000 deaths worldwide.Meanwhile,bladder cancer occurs more frequently in men than in women and among the aged populations,especially those between 50 and 70 years old.In addition to surgical treatment,chemotherapy also plays a crucial role in reducing disease recurrence and improving prognosis in patients with bladder cancer.However,it is so frustrating that a considerable part of bladder cancer patients failed to benefit from these drugs due to chemo-resistance and severe side effects.Polyphyllin I(PPI),an extract from the rhizome of Paris polyphylla,was discovered to have a potent anticancer activity among a broad spectrum of malignant diseases.However,to date,no studies have studied the antitumor effect of PPI in bladder cancer.Purpose:The present study aimed to evaluate the anticancer effect of PPI in bladder cancer,and explore the underlying molecular mechanism of this phenomenon.Methods:For in vitro experiments,we used bladder cancer cell lines to investigate the cytotoxic activity of PPI.Using the CCK8 assay,we determined the IC50 of PPI in different bladder cancer cell lines and screened out the most susceptible cell lines.Next,we used those susceptible cell lines,to conduct the following experiments,including the colony formation assay,the cell apoptosis assay,the cell cycle assay,and the detection of mitochondrial membrane potential.To investigate the underlying mechanism of the cytotoxicity of PPI,we firstly performed RNA-seq analysis to screen the signaling pathways involved in the antitumor effect of PPI against bladder cancer cells.Then from RNA-seq analysis,we found out the activated signaling pathway and further validated the activated signaling using western blotting and immunofluorescence assay.We also silenced the key gene in the activated signaling pathway using the corresponding small interfering RNA to test the alternation of the cytotoxicity of PPI in bladder cancer cells.For in vivo experiments,we used the orthotopic bladder tumor mice model to determine the therapeutic potential of PPI.We randomly divided the tumor-bearing mice into two groups and treated with PPI or saline intraperitoneally.We measured the weight of mice every two days and also detected the size of orthotopic bladder tumors using the in vivo imaging system every week.By the end of the in vivo experiment,the expression level of the key protein in the activated signaling pathway was detected in the harvested orthotopic bladder tumor samples by immunohistochemistry.Results:A relatively low concentration of PPI can exert potent cytotoxicity effect in bladder cancer cell lines,among which,T24 and UMUC3 are the most susceptible cell types.PPI exerts its inhibitive effect on colony formation and promotes the cell apoptosis,the decrease of mitochondrial membrane potential and the cell cycle arrest in a dose-and time-dependent manner in bladder cancer cells.Besides,the expression level of Cleaved-Caspase3 and p21 protein was elevated after PPI treatment in bladder cancer cells.By whole transcriptome sequencing,a total of 6254 different expression genes(DEGs)were spotted in T24 pairs and 1304 DEGs were identified in UMUC3 pairs,with 338 overlapping DEGs between T24 and UMUC3 cells.KEGG database-dependent pathway enrichment analysis suggested that these overlapping DEGs were enriched in FOXO3 signaling pathways.Furthermore,our western blotting analysis showed that the FOXO3 transcription factor,a key member of the FOXO family,and its downstream target proteins,BIM and NOXA,was markedly upregulated after treated with PPI in bladder cancer cells.We also found the PPI-induced nuclear translocation of the FOXO3 transcription factor by immunofluorescence assay.Since the FOXO3 signaling plays an important role in PPI-induced cytotoxicity in bladder cancer cells,we silenced the expression of FOXO3 protein in T24 cells and treated them with PPI again.The result indicates that the silence of FOXO3 can partially inverse the cytotoxicity of PPI in bladder cancer cells.The in vivo experiments indicate that PPI treatment can markedly inhibit the growth of bladder cancer tumor and doesn't significantly alter the body weight of PPI-treated mice compared to control group mice.Moreover,the FOXO3 expression in bladder cancer xenografts was dramatically increased in the PPI-treated group compare to control group.Conclusion:Our findings suggested that PPI exerted a potent cytotoxic effect against bladder cancer cells in vitro by inducing apoptosis and cell cycle arrest,and also had a powerful antitumor activity in vivo.Moreover,we unveiled that the cytotoxic effect and antitumor activity of PPI against bladder cancer were partly mediated via the activation of FOXO3/BIM or NOXA signaling axes.Taken together,PPI may serve as a promising chemotherapy agent for bladder cancer treatment.