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Li Cheng,1,* Hao Wang,2,* Zicheng Wang,3 Houbao Huang,1 Dong Zhuo,1 Jian Lin4 1Department of Urology, The First Affiliated Hospital, Yijishan Hospital of Wannan Medical College, Wuhu, Anhui Province, People’s Republic of China; 2Department of Geriatrics, Peking University First Hospital, Beijing, People’s Republic of China; 3Department of Urology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui Province, People’s Republic of China; 4Department of Urology, Peking University First Hospital, Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Li Cheng Email chenglibjmu@163.comIntroduction: Bladder cancer is a lethal human malignancy. Currently, treatment for bladder cancer is limited. The anti-tumor effects of leflunomide have attracted much more concern in multiple human cancers.Materials and Methods: This study evaluated the anti-tumor effects of leflunomide on cell viability, colony formation, apoptosis, and cell cycle in two human bladder carcinoma cell lines, 5637 and T24. Meanwhile, the underlying mechanism including PI3K/Akt signaling pathway and autophagy modulation was also identified.Results: Leflunomide markedly inhibited the growth of both bladder cancer cell lines and induced apoptosis and cell cycle arrest in S phase. The phosphorylation levels of Akt and P70S6K in both cell lines were significantly down-regulated with leflunomide treatment. Furthermore, the deceased formation of autophagosomes and the accumulation of LC3II and P62 suggested the blockade of autophagy by leflunomide. Modulation of autophagy with rapamycin and chloroquine markedly attenuated and enhanced the cytostatic effects of leflunomide, respectively.Conclusion: Leflunomide significantly reduced the cell viability of bladder cancer cells via inducing apoptosis and cell cycle arrest and suppressing the PI3K/Akt signaling pathway. In addition, the blockade of autophagy was observed, and autophagy inhibition enhanced leflunomide-mediating anti-tumor effects. Our data presented here offer novel ideas for comprehensive therapeutic regimes on bladder cancer.Keywords: leflunomide, autophagy, PI3K/Akt pathway, anti-tumor, bladder cancer

Background Let-7 is one of the earliest discovered microRNAs(miRNAs) and has been reported to be down-regulated in multiple malignant tumors. The effects and molecular mechanisms of let-7i in bladder cancer are still unclear. This study was to investigate the effects and potential mechanisms of let-7i on bladder cancer cells. Methods Total RNA was extracted from bladder cancer cell lines. The expression levels of let-7i and HMGA1 were examined by quantitative real-time PCR. Cell viability was detected using the CCK-8 and colony formation assays, while transwell and wound healing assays were used to evaluate migration ability. Luciferase reporter assay and western blot were used to confirm the target gene of let-7i. Results Compared with the SV-40 immortalized human uroepithelial cell line (SV-HUC-1), bladder cancer cell lines T24 and 5637 had low levels of let-7i expression, but high levels of high mobility group protein A1 (HMGA1) expression. Transfection of cell lines T24 and 5637 with let-7i mimic suppressed cell proliferation and migration. Luciferase reporter assay confirmed HMGA1 may be one of the target genes of let-7i-5p. Protein and mRNA expression of HMGA1 was significantly downregulated in let-7i mimic transfected cell lines T24 and 5637. Conclusions Up-regulation of let-7i suppressed proliferation and migration of the human bladder cancer cell lines T24 and 5637 by targeting HMGA1. These findings suggest that let-7i might be considered as a novel therapeutic target for bladder cancer.

Bufalin has been demonstrated to possess a wide range of pharmacological effects. Among these is its antitumour effect, which has been confirmed in multiple organs or tissues and provoked many concerns. However, its cytostatic effect and underlying mechanism in bladder cancer has not thoroughly been elucidated. This study aimed to investigate the hypothesis that Bufalin induces cell apoptosis and inhibits cell growth in bladder cancer through the inactivation of Na+/K+-ATPase (NKA). In the current study, it was demonstrated that Bufalin remarkably inhibited cell viability and induced cell apoptosis in bladder cancer cell line T24. Subsequently, we found that the expression of NKA was significantly supressed in Bufalin-treated cells and the NKA-α3 isoform was most sensitive to Bufalin among all α subunits of NKA. By transfection with NKA-α3 overexpressing plasmids, the expression of the NKA-α3 subunit was upregulated and NKA-α3 overexpression was found to markedly attenuated Bufalin-induced cell apoptosis in T24 cells, suggesting NKA-α3 played a critical role in Bufalin-induced cell apoptosis. Taken together, the present study confirmed that Bufalin promotes tumour apoptosis and inhibits tumour growth in bladder cancer in vitro, and this antitumour effect may be ascribed to the inactivation of NKA.