The Role And Mechanism Of PKC? In The Inhibition Of Autophagy In SK-N-SH Human Neuroblastoma Cells By TOCP

Objective: To investigate the regulatory role and mechanism of action of PKC? in TOCP-induced autophagy.Methods:1.The IC50 of TOCP in SK-N-SH cells after incubation for 48 h,as determined by MTT assay.2.After SK-N-SH cells was exposed to 0.75 mmol/L TOCP and then lysed,we examined the effect of TOCP exposure on proteasomal activity in SK-N-SH cells by a fluorescence spectrophotometer.Total cell lysates were analyzed respectively by immunoblotting using anti-PKC?,anti-p-PKC? antibody,anti-LC3 antibody,anti-OPTN antibody,anti-NBR1 antibody,anti-P62 antibody,anti-MAP2 antibody,anti-p-Tau antibody and anti-UB antibody.GAPDH was used as a loading control.Representative transmission electron micros copy images depicting the ultrastructure of the cells.3.SK-N-SH cells were exposed to PMA(100 nM)or SP(2 nM)for 30 min,followed by 0.75 mM TOCP for 48 h and then lysed,we examined the effect on proteasomal activity in SK-N-SH cells by a fluorescence spectrophotometer.Total cell lysates were analyzed respectively by immunoblotting using anti-PKC?,anti-p-PKC? antibody,anti-LC3 antibody,anti-OPTN antibody,anti-NBR1 antibody,anti-P62 antibody,anti-MAP2 antibody,anti-p-Tau antibody and anti-UB antibody.GAPDH was used as a loading control.Results:1.The LC50 of TOCP in SK-N-SH cells after incubation for 48 h incubation was approximately 0.86 mM,as determined by MTT assay.SK-N-SH cell was significantly inhibited.2.SK-N-SH cells at 80% confluence were exposed to PKC? activator(100 nM)or PKC? inhibitor(2 nM)for 0.5 h and then treated with TOCP(0.75 mM)for 48 h.we found that the TOCP,PMA,and TOCP+PMA treatment groups had increased p-PKC?/PKC?.The levels of the autophagy proteins P62 and LC3II/LC3 I were significantly downregulated in the SP and SP+TOCP groups and significantly up-regulated in the PMA and PMA+TOCP groups.The results of WB were further confirmed by immunofluorescence method to determine the protein expression levels of PKC? protein and autophagy-related proteins(LC3 and P62).3.the expression levels of the autophagy receptor proteins OPTN,NF-H and MAP2 were significantly downregulated and that of p-Tau was upregulated in the TOCP,PMA,and PMA+TOCP groups.we found that the TOCP,PMA,and PMA+TOCP treatment groups showed lower proteasomal activity than the control.As further confirmation of UPS impairment,TOCP treatment led to a dramatic accumulation of HMW ubiquitin-conjugated proteins in cells,as revealed by WB.PKC? activation can further promote the accumulation of HMW ubiquitin-conjugated proteins.Conclusions:1.TOCP induces autophagy in human neuroblastoma SK-N-SH cells.2.TOCP activates PKC? in human neuroblastoma SK-N-SH cells.3.PKC? activators could positively regulate TOCP-induced autophagy in the neuroblastoma SK-N-SH cells.TOCP degraded OPTN via PKC? activation and accumulated NBR1 proteins cumulatively.4.PKC? activators could downregulated the skeleton-associated proteins NF-H and MAP2 and up-regulated the p-tau protein.5.TOCP inhibits proteasomal activity and induces aggregation of high molecular weight(HMW)ubiquitinated proteins in human neuroblastoma SK-N-SH cells...