NaAsO₂ Induces The Toxic Injuries Through The Hippo-YAP Signaling Pathway In PC12 Cells

Objective:To investigate the toxic injuries caused by NaAsO₂,the roles played by the Hippo-YAP signaling pathway in arsenic-induced neural injuries and the underlying mechanisms in PC12 cells.Methods:PC12 cells were randomly divided into four groups:the control group,the NaAsO₂ group（incubated with 10,20,30,40,50 and 60μM NaAsO₂ for 24 and 48h）,XMU-MP-1（inhibitor of Hippo-YAP pathway）group,XMU-MP-1+NaAsO₂ group（pretreatment with 3μM XMU-MP-1 for 4 h before incubation with 40 NaAsO₂ for 24 h）.CCK-8 viability assay,flow cytometry,optical or fluorescence microscope and western blotting assay were used to observe the effects of NaAsO₂ on the viability,the cell cycle,the apoptosis rates,the cell morphology,the apoptosis related proteins（P53,BAX,BCL-2）,the synaptic related proteins（SYN,PSD95）,the nucleus translocation of YAP proteins,and the Hippo-YAP signaling pathway-related proteins（MST1,p-MST1,LATS1,p-LATS1,YAP,p-YAP）.Results:1.Compared with control group,the cell viability were inhibited significantly in a dose-dependent manner.（p<0.05）.60μM NaAsO₂ significantly increased the S phase ratio（p<0.01）,and decreased the G0/G1 and G2/M phase ratio（p<0.05）.2.The morphology of PC12 cells was changed from long spindle to round shape and neurites became shorter than those in the control group.The nuclear translocation of YAP protein and the protein expressions of SYN and PSD95 were both suppressed markedly by 40μM NaAsO₂（p<0.01）.3.40μM NaAsO₂ significantly increased both the apoptosis rates and the expression of the P53 and BAX,and downregulated the expression of BCL-2compared to those of the control group（p<0.05）.4.30μM NaAsO₂significantly increased the protein expressions of MST1 and LATS1（p<0.05）.Furthermore,40μM NaAsO₂ significantly increased not only the protein expressions of MST1 and LATS1（p<0.05）,but also those of p-MST1,p-LATS1 and p-YAP（p<0.05）.However,the protein expressions of YAP was decreased markedly by 50 and 60μM NaAsO₂（p<0.01）.5.Blockage of the Hippo-YAP signaling pathways with XMU-MP-1 could reverse the decrease of cell viability,the S phase arrest,the changes of the morphology,and the apoptosis caused by NaAsO₂in PC12 cells.Conclusions:1.NaAsO₂ can inhibit the cell growth,disturb the cell cycle,damage the cell morphology,inhitbit the nuclear translocation of YAP protein,activate the Hippo signaling pathway and promote the cell apoptosis in PC12 cells.2.Blocking the Hippo signaling pathway may partly protect PC12 cells against the arsenic-induced injuries.