Studies On Mechanisms Of Cadmium's Inhibition Of XIAP Pathway Contributing To Apoptosis Via Inducing Mitochondrial ROS In Neuronal Cells

The present study,using cellular and molecular biology techniques and methods including cell culture,TUNEL and/or DAPI staining,ROS fluorescent labeling,MTS cell viability assay,immunocytochemical analysis,RNA interference,Western blotting,etc.,and employing PC12 cells and primary murine neurons as experimental objects,studied the molecular mechanisms of cadmium(Cd)'s inhibition of XIAP pathway contributing to apoptosis by inducing mitochondrial ROS in neurons cells.The detailed results were summarized as follows:1 Cd-induced ROS inhibits XIAP pathway leading to neuronal apoptosisPC12 cells and primary murine neurons were exposed to Cd(10 and 20 ?M)for 4 or 24 h following pretreatment with NAC(5 mM)or Embelin(20 ?M)for 1 h.The expression of cellular proteins was analyzed by Western blotting.XIAP expression was detected by immunofluorescence staining,ROS level was labeled by ROS fluorescence,and apoptotic cells were assayed by DAPI/TUNEL staining.The results showed that Cd induced ROS and leaded to apoptosis in neuronal cells in a concentration-dependent manner;XIAP pathway was inhibited,and cleavages of caspase-3 and PARP were increased;pretreatment with NAC significantly reduced Cd neurotoxicity,whereas pretreatment with Embelin potentiated the toxic effects of Cd.These results uncover that Cd-induced ROS inhibits XIAP pathway leading to neuronal apoptosis.2 XIAP exerts a pivotal role in Cd-induced ROS-dependent neuronal apoptosisPC12 cells,infected with lentiviral shRNA GFP,shRNA XIAP,EGFP and FLAG-XIAP,were pretreated with Nutlin-3a(10 ?M)for 1 h and then exposed to Cd(10 ?M)for 4 or 24 h.The expression of cellular proteins was analyzed by Western blotting,cell viability was detected by MTS assay,ROS level was labeled by ROS fluorescence,and apoptotic cells were assayed by DAPI staining.The results showed that Nutlin-3a resisted Cd-induced neuronal apoptosis by blocking combination of MDM2 to p53.Down-regulation of XIAP significantly reduced Cd-induced ROS and apoptosis in the cells.Overexpression of XIAP markedly resisted Cd-induced ROS and apoptosis as well.These results suggest that XIAP exerts a pivotal role in Cd-induced ROS-dependent neuronal apoptosis.3 Cd-induced mitochondrial ROS inhibits XIAP pathway contributing to neuronal apoptosisPC12 cells and primary murine neurons,or PC12 cells infected with shRNA XIAP or shRNA GFP were pretreated with Embelin(20 ?M)and/or Mito-TEMPO(10 ?M)for 1 h and then exposed for Cd(10 ?M)for 4 or 24 h.The expression of cellular proteins was analyzed by Western blotting,cell viability was detected by MTS assay,ROS level was labeled by ROS fluorescence,and apoptotic cells were assayed by DAPI staining.The results showed that Mito-TEMPO significantly ameliorated Cdand/or Embelin-induced inactivation of XIAP pathway and apoptosis in neuronal cells.Down-regulation of XIAP enhanced neuroprotective effect of Mito-TEMPO.These results imply that Cd inhibits XIAP pathway leading to neuronal apoptosis by inducing mitochondrial.