川楝素通过下调c-kit表达抑制GIST-T1细胞增殖并促进其凋亡
摘要
Objective To investigate the effects of toosendanin (TSN) on the proliferation and apoptosis of gastrointestinal stromal tumor (GIST) cells, and to explore the underlying mechanisms. Methods GIST-T1 cells were treated with TSN at different concentrations, and then CCK-8 assay was used to detect the effects of TSN on the proliferation of GIST-T1 cells. Subsequently, the experimental concentrations were further divided into 0, 15, 30 nmol/L TSN groups, with 50 nmol/L imatinib (IM) as the positive control. Flow cytometry and Hoechst 33342/PI fluorescence double staining were performed to detect cell apoptosis and cell cycle. Finally, the expression of c-KIT, PI3K, AKT and mTOR at mRNA and protein levels were determined by qRT-PCR and Western blotting. Results TSN inhibited the proliferation of GIST-T1 cells (P < 0.01), and its IC50 was 18.75 nmol/L at 48 h and 17.93 nmol/L at 72 h. As compared with the control group, the 15 and 30 nmol/L TSN groups as well as IM group induced cell apoptosis (P < 0.01), though the apoptotic rate showed no significance among different drug groups; and 30 nmol/L TSN induced S-phase arrest (P < 0.01). In addition, qRT-PCR results suggested that 15 nmol/L TSN down-regulated the mRNA expression of c-KIT, PI3K, AKT and mTOR (P < 0.05, P < 0.01), and Western blotting confirmed the inhibitory effect of 15 nmol/L TSN on the expression of these proteins and their phosphorylated proteins (P < 0.05, P < 0.01). Conclusion TSN inhibits the proliferation of GIST-T1 cells and promotes apoptosis by down-regulating c-KIT expression, which may function through PI3K/AKT/mTOR pathway.