Beclin1Inhibition Promotes TOCP-induced Apoptosis In Human Neuroblastoma SH-SY5Y Cells

Objective:To explore the relationship between autophagy and apoptosis in tri-ortho-cresylphosphate (TOCP)-induced neurotoxicity in human neuroblastoma SH-SY5Y cells,and to illustrate mechanism of neurotoxicity caused by TOCP.Methods:1. pGenesil-1-Beclin1-shRNA eukaryotic expression vector was constructed byusing RNA interference, then transfecting the recombinant plasmid into SH-SY5Ycells, screening stable cell lines expressing Beclin1-shRNA by G418(sh-Beclin1cellline).The mRNA and protein expression of Beclin1were detected by RT-PCR andWestern blot.2. The sh-Beclin1cells and control SH-SY5Y cells were treated with differentconcentrations of TOCP (0,0.5mM) for48h. Monodansylcadaverine (MDC) stainingmeasured the number of autophagic vacuoles, and the protein expression of LC3-IIwas detected by western blotting.3. After sh-Beclin1cells and control SH-SY5Y cells treated with differentconcentrations of TOCP (0,0.25,0.5,1.0mM) for48h, Hoechst33258was used toobserve nuclear morphology of cells, flow cytometry analysised the cell cycle of twocell lines and western blot detected the expression of apoptosis-related genes Bax,Bcl-2and Caspase-3.Results:1. The result of DNA sequencing confirmed the correct construction of theeukaryotic expression vector pGenesil-1-Beclin1-shRNA. Fluorescence microscopy,RT-PCR and western blot demonstrated that the SH-SY5Y cell lines with inhibitedBeclin1expression were established successfully.2. The results of MDC staining and western blot showed that the number ofautophagic vesicles and LC3-II protein expression reduce significantly aftersh-Beclin1cells were treated with TOCP (0.5mmol/L) for48h compared with thecontrol SH-SY5Y cells.3. The sh-Beclin1cells and control SH-SY5Y cells were stained by Hoechst33258after treatment of TOCP (0,1.0mM) for48h. Compared with theSH-SY5Y cells，the blue fluorescent of nuclei enhanced, chromatin aggregation andnucleus fragmentation were observed by inversed fluorescent microscope insh-Beclin1cells, the number of nuclear condensations and DNA fragments increasedobviously.4. The sh-Beclin1cells and control SH-SY5Y cells were treated with variousconcentrations of TOCP (0,0.25,0.50,1.0mM) for48h, and sub-G1, G1, S and G2/Mphases were determined by flow cytometry. at48h. Results showed that cell cyclearrest in sub-G1phase, a marker of apoptotic cell death, was more pronounced bytreatment with0.25,0.50and1.0mM TOCP, and the number of cells in sub-G1phaseincreased in a dose-dependent manner in sh-Beclin1cells treated with TOCP(P<0.05),and sub-G1phase was not found in SH-SY5Y cells.5. The sh-Beclin1cells and control SH-SY5Y cells were treated with variousconcentrations of TOCP (0.25,0.50,1.0mM) for48h. The result of Western blotshowed that the ratio of bcl-2/bax and the level of Cleaved-caspase-3expression donot change significantly in SH-SY5Y cells compared with control; in sh-Beclin1cellsthe ratio of bcl-2/bax decreases obviously in a dose-dependent manner（P<0.05）andthe protein expression of cleaved-caspase3did not have any difference comparedwith control.Conclusions:1. Beclin1played a crucial role in TOCP-induced autophagy, and inhibition ofBeclin1could reduce TOCP-induced autophagy in SH-SY5Y cells.2. Beclin1inhibition promoted TOCP-induced mitochondria-mediatedcaspase3-independent apoptosis in human neuroblastoma SH-SY5Y cells.