Inhibition of canonical WNT/β-catenin signaling is involved in leflunomide (LEF)-mediated cytotoxic effects on renal carcinoma cells
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Yicheng Chen1, Qiaoli Huang2, Hua Zhou2, Yueping Wang3, Xian Hu4, Tao Li2
1Department of Urology, Sir Run-Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310016, China
2Department of Biology, College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua, Zhejiang 321004, China
3Department of Urology, Wuyi First People's Hospital, Wuyi, Zhejiang 321200, China
4Department of Plastic Surgery, Sir Run-Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310016, China
Tao Li, email: email@example.com
Keywords: leflunomide, renal cell carcinoma, cytotoxicity, WNT/β-catenin signaling
Received: February 05, 2016 Accepted: June 13, 2016 Published: July 6, 2016
Leflunomide (LEF), an inhibitor of dihydroorotate dehydrogenase (DHODH) in pyrimidine biosynthetic pathway, is an immunomodulatory agent approved for the treatment of rheumatoid arthritis. In this study, we show that LEF significantly reduced cell proliferation of renal carcinoma cells in a concentration-dependent manner. LEF at 50 μM induced S-phase arrest and autophagy. Higher doses of LEF (>50 μM) effectively induced cell apoptosis. Modulating the concentration of LEF resulted in distinct effects on the expression of regulatory proteins associated with cell cycle, apoptosis, and autophagy. In particular, high concentrations of LEF inhibited canonical WNT signaling by promoting nucleo-cytoplasmic shuttling and proteasome-dependent degradation of β-catenin. Mechanistic studies showed that the repression of AKT activation partly accounted for LEF-mediated WNT inhibition. Gene expression microarray revealed that LEF treatment greatly inhibited the expression of FZD10 gene, a receptor mediating WNT/β-catenin activation. In vivo xenograft study in NOD/SCID mice further validated the inhibitory effects of LEF on tumor growth and Wnt/β-catenin signaling. However, LEF treatment also triggered cell autophagy and elevated the expression of WNT3a, which ameliorated its cytotoxic effects. The combination of LEF with a WNT inhibitor IWP-2 or autophagy inhibitor HCQ could yield an enhanced anti-tumor outcome. Taken together, these results identify the potential utility and pharmacological feature of LEF in the chemotherapy of renal cell carcinoma (RCC).
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