| Objective:Nifurtimox is an effective drug for the treatment of Chagas disease caused by trypanosomal infection.In recent years,it has received widespread attention for its anti-cancer effect in the treatment of neuroblastoma(NB).This study reports the effects of nifurimox treatment on the proliferation and apoptosis of human neuroblastoma cell lines and attempts to elucidate its underlying mechanism.Methods:SH-SY5 Y cells were treated with nifurtimox,and cell proliferation was detected by CCK-8.The optimal concentration of nifurtimox to inhibit SY5 Y cells was selected;and CCK-8 was measured to determine the inhibition of SY5 Y growth by nifurtimox at the selected concentration at different times;apoptosis and apoptosis-related protein expression were detected by using flow cytometry and western blotting.DCFDA fluorescence assay were used for the measurement of intracellular reactive oxygen species(ROS);Western Blotting were performed to detect the expression of MYCN;SH-SY5 Y cells were inoculated subcutaneously in NCr nude mice,and were administered with Nifurtimox or normal saline in the treatment group and the control group,respectively.Tumor growth was observed and immunohistochemical differences between the treatment group and the control group were detected by immunohistochemistry.Results:After 0,5,10,15,20,25μg/ ml of nifurtimox treatment for 48 h,the cell activity(percentage in the control group)was 96.23±4.54、89.35±7.73、50.28±6.22、33.80±5.24、13.95±3.72、5.13±2.07,with significant differences(P < 0.05).The apoptosis rates of nifurtimox treated with 0,10 and 20 μg/ml for 48 h increased significantly(P <0.05),and the apoptosis rates(%)of the three groups were 3.58±0.8、46.8±1.6、64.1±2.1.Secondly,western blotting results revealed changes in apoptosis-related protein expression in cells treated with nifurtimox for 48 h at three concentrations(0,10,20μg/ml).The ratio of cleaved Cas3/Cas3 expression to GAPDH was 0.3±0.02,0.37±0.03 and 0.62 ± 0.04,respectively,with significant differences(P < 0.05).The expression levels of Bax/GAPDA were 0.15±0.01,0.71±0.03 and 0.85±0.03,respectively(P <0.05).The expression levels of Bcl2/GAPDA were 0.6±0.01,0.58±0.02 and 0.55±0.01,respectively,with statistically significant differences(P < 0.05).After 0 and 10 h/ml of nifurtimox treatment,ROS production multiples(Fold Change)were 1.0±0.04 and1.48±0.22,respectively,with statistically significant differences between groups(P <0.05).Western blotting showed a decrease in MYCN proteins after 10 μg/ ml nifurtimox treatment.After in vivo culture,the tumor was removed,and immunohistochemical HE staining showed that the tumor cells of nude mice with NB underwent nifurtimox intragastric administration,and the tumor cell hypertrophy disappeared after nifurtimox treatment.Conclusion:Nifurtimox can inhibit the proliferation of SH-SY5 Y cells and promote the apoptosis of SH-SY5 Y cells.Western Blotting showed that the expression of pro-apoptotic proteins Caspase3 and Bax increased and the expression of pro-apoptotic proteins Bcl2 decreased after treatment with nifurtimox.In addition,cells treated with nifurtimox had an increase in intracellular reactive oxygen species,which also promoted apoptosis.Western Blotting showed that nifurtimox inhibited the expression of MYCN proteins.Nifurtimox also inhibited tumor progression by inhibiting proliferation and inducing apoptosis in nude mice.Immunohistochemistry showed that the tumor cells in the treated group showed different degrees of apoptosis compared with the control group.In summary,nifurtimox can inhibit the proliferation of SH-SY5 Y cells by regulating the expression of apoptotic protein,promoting the generation of intracellular reactive oxygen species,and inducing the apoptosis of SH-SY5 Y cells in vivo and in vitro to inhibit its proliferation.It can be a potential drug for treating NB.In addition,nifurtimox inhibits the expression of MYCN,which is strongly associated with NB high risk,providing a new approach for the treatment of NB at high risk. |
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