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Autophagy inhibition enhances leflunomide-induced cytotoxicity in human bladder cancer cells

Abstract Background Dihydroorotate dehydrogenase (DHODH) is one of the essential enzymes in the de novo biosynthesis of pyrimidine and might be a potential therapeutic target for cancer suppress. The anti-proliferative and apoptosis-inducing effects of leflunomide, a potent DHODH blocker, have been demonstrated in multiple human cancers. This study aims to investigate the cytostatic effects of leflunomide on bladder cancer and the involved mechanism. Methods Two human bladder cancer cell lines, T24 and 5637, were used in this study. After incubation with varied doses of leflunomide, the cell viability, apoptosis and cell cycle assay were determined with MTS, cell colony assay and flow cytometry. Western blot was used to evaluate the expression changes of cleaved-PARP, proteins involved in Akt/mTOR/P70S6K signaling pathway and cell autophagy pathway. AVO stain assay was performed to detect the autophagosome. Moreover, the cytostatic effects of leflunomide were further investigated after the modulation of cell autophagy with autophagy agonist rapamycin and inhibitor chloroquine. Results Our data demonstrated that leflunomide markedly inhibited the growth of both bladder cancer cells via inducing cell apoptosis and cell cycle arrest in S phase in a time- and dose-dependent manner. After leflunomide treatment, the phosphorylation levels of Akt, mTOR and p70S6K proteins in both cells were significantly down-regulated. Furthermore, AVO stain assay revealed the decline of autophagosome under the incubation of leflunomide. Modulation of autophagy with rapamycin and chloroquine observably attenuated and enhanced the cytostatic effects of leflunomide, respectively. Conclusions Leflunomide significantly reduced the cell viability of bladder cancer cells via Akt/mTOR/P70S6K signaling pathway. In addition, cell autophagy was demonstrated to be involved, combination leflunomide with autophagy modifier exerted enhanced antitumor effects in bladder cancer, which offered novel ideas for bladder cancer treatment. Legal entity responsible for the study The author. Funding Has not received any funding. Disclosure The author has declared no conflicts of interest.