Molecular Mechanisms of Apoptosis of Leukemia Cell Induced by Reovirus

LI Chen, MO Jing, SHU Li-ping, WU Xi-jun, XU Jian-wei, YANG Yan, HE Zhi-xu

Abstract

To investigate the molecular mechanism of apoptosis of HL60 cells induced by oncolytic virus Reovirus type 3 (Reo3).  Methods  HL60 cells were infected with Reo3 at different multiplicity of infection (MOI) with the uninfected HL60 cells as control group. After 48 h of infection, the activity of HL60 cells infected with virus at different MOI was detected by CCK8 method to investigate the influence of MOI to cell activity. Simultaneously, the apoptotic rate of HL60 cells was detected by flow cytometry, and the activation level of double-stranded RNA-dependent protein kinase (PKR) and the expression of apoptotic-related protein in HL60 cells were detected by Western blot. Before infection with Reo3 for 48 h, HL60 cells were treated with 2-aminopurine (2-AP), a specific inhibitor of PKR, for 24 h. Afterward, the apoptotic level and expression of apoptotic related proteins were detected.  Results  Activity of HL60 cells was obviously inhibited after infected with Reo3 with a MOI of 1 for 48 h. The cell survival rate was (24.333±3.396)% and the apoptotic rate was (29.96±2.06)%. Both rates were all higher than those in the control group (P < 0.05). Western blot results showed that the expression levels of PKR, p-PKR, Bax, Caspase3 and cleaved Caspase3 in HL60 cells infected with Reo3 were higher than those in the control group (P < 0.05), while the expression level of Bcl-2 was lower (P < 0.05). Compared with the group without inhibitor, the apoptotic rate of HL60 cells pretreated with 2-AP decreased (P < 0.05), the phosphorylation level of PKR and the expression level of apoptotic-related protein also decreased (P < 0.05).  Conclusion  Oncolytic virus Reo3 could activate PKR in HL60 cells and thus induce apoptosis of HL60 cells.

 

Keywords: Reovirus, HL60 cells, Apoptosis, Apoptosis-related proteins, PKR, 2-aminopurine

 

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References


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