关键词: 肾母细胞瘤, 青蒿琥酯, AMPK/mTOR/ULK1信号通路, 增殖, 凋亡, 自噬

Abstract: Objective To investigate the effects of artesunate on the proliferation, apoptosis and autophagy of nephroblastoma cells and its molecular mechanism. Methods Human nephroblastoma cell line SK⁃NEP⁃1 was cultured in vitro and treated with artesunate at concentrations of 0, 12.5, 25.0, 50.0, 100.0 and 150.0 μmol/L, respectively; the cell viability of each group was determined by CCK⁃8 method and the optimal concentration of artesunate was screened. SK⁃NEP⁃1 cells were randomly divided into control group, artesunate (100.0 μmol/L) group, AMPK inhibitor Dorsomorphin (10.0 μmol/L) group, and artesunate (100.0 μmol/L)+Dorsomorphin (10.0 μmol/L) group. After treated with artesunate and Dorsomorphin alone or in combination, the cell viability of each group was detected by CCK⁃8 method. The colony formation condition of each group was detected by plate clone formation assay. The apoptosis condition of cells in each group was detected by flow cytometry. The autophagy of cells in each group was detected by MDC fluorescence staining. The expression of apoptosis⁃related proteins ［B⁃cell lymphoma⁃2 (Bcl⁃2), cleaved poly ADP ribose polymerase (PARP), Bcl⁃2⁃related X protein (Bax)］, autophagy⁃related proteins ［light chain 3 (LC3) Ⅱ, LC3Ⅰ, Beclin⁃1］ and AMPK/mTOR/ULK1 signaling pathway⁃related proteins in each group were detected by Western blot. Results Artesunate at different concentrations could inhibit the activity of SK⁃NEP⁃1 cells (all P<0.05), and the inhibitory effect was enhanced with the increase of concentration. After treating SK⁃NEP⁃1 cells with 100.0 μmol/L artesunate and 10.0 μmol/L Dorsomorphin, respectively,  compared to the control group, the artesunate group had higher apoptosis rate, larger relative amount of autophagic vacuoles, increased protein expression levels of Bax and cleaved PARP, LC3Ⅱ/LC3Ⅰ, Beclin⁃1, p⁃AMPK/AMPK, and p⁃ULK1/ULK1 (all P<0.05), whereas its cell viability, colony formation rate, protein expression levels of Bcl⁃2 and p⁃mTOR/mTOR decreased (all P<0.05); the apoptosis rate, relative amount of autophagic vacuoles, protein expression levels of Bax and cleaved PARP, LC3Ⅱ/LC3Ⅰ, Beclin⁃1, p⁃AMPK/AMPK, p⁃ULK1/ULK1 in Dorsomorphin group were decreased (all P<0.05), whereas its cell viability, colony formation rate, protein expression levels of Bcl⁃2 and p⁃mTOR/mTOR were increased (all P<0.05). The combined intervention of artemisinin and Dorsomorphin reversed the anti⁃tumor effect of artesunate in SK⁃NEP⁃1 cells. Conclusions Artesunate may enhance autophagy of nephroblastoma cells by activating AMPK/mTOR/ULK1 signaling pathway, thereby inhibiting its proliferation and promoting apoptosis.

Key words: Nephroblastoma, Artesunate, AMPK/mTOR/ULK1 signaling pathway, Proliferation, Apoptosis, Autophagy