Inhibition of viral protein r on human colorectal cancer cells and the possible mechanisms / 中华消化外科杂志

ABSTRACT

Objective To investigate the inhibitive effects of viral protein r (Vpr) of human immunodeficiency virus 1 ( HIV-1 ) on human colorectal cancer cell line HCT-8,and to find the possible mechanisms.Methods The HCT-8 cells were divided into the control group,adv group and adv-Vpr group.HCT-8 cells were not treated in the control group; HCT-8 cells were treated with Adv or Adv-Vpr at different multiplicity of infection (MOI) in the Adv group or Adv-Vpr group,respectively.Cell proliferation was detected by MTT assay.Cell cycle,apoptosis and mitochondrial membrane potential were detected by flow cytometry.The expression of apoptosisrelated proteins was detected by Western blot.All data were analyzed by using the q test,t test and one-way or two-way analysis of variance.Results The proliferation of HCT-8 cells was significantly inhibited by Vpr.The MTT value of HCT- 8 cells in the Adv-Vpr group was 1.03 ± 0.04,which was significantly lower than 2.46 ± 0.15 in the Adv group and 2.51 ± 0.14 in the control group at 72 hours after Adv-Vpr transfection ( MOI =200) ( F =144.6,P ＜ 0.05).The ratio of HCT-8 cells in the G2/M phase was 37.31％ ± 5.90％ in the Adv-Vpr group,which was significantly higher than 18.30％ ± 6.04％ in the Adv group and 16.66％ ± 3.51％ in the control group ( F =10.08,P ＜ 0.05 ).The ratio of HCT-8 cells with decreased mitochondrial membrane potential was 32.07％ ±5.64％ in the Adv-Vpr group,which was significantly higher than 3.32％ ±0.79％ in the Adv group and 2.76％ ±1.43 ％ in the control group at 48 hours after Adv-Vpr transfection ( MOI =200) ( F =64.45,P ＜ 0.05).The apoptosis rate of HCT-8 cells was 37.62％ ±6.48％ in the Adv-Vpr group,which was significantly higher than 3.44％ ± 1.11％ in the Adv group and 2.93％ ± 1.07％ in the control group at 72 hours after Adv-Vpr transfection ( MOI =200) ( F =122.4,P ＜ 0.05 ).The results of Western blot showed that Vpr induced cleavage and activation of Caspase-9 and Caspase-3 and phosphorylation of Chk1-S345,while the expression levels of Fas,Fas-L,ERK1,ERK2 remained the same at 48 hours after Adv-Vpr treatment ( MOI =200).Conclusions Vpr inhibits the proliferation of the HCT-8 cells in vitro through G2/M phase arrest and apoptosis.Vpr plays its role by activating DNA damaging pathway and initiating mitochondria apoptotic pathway.Vpr is a potential therapeutic agent for colorectal cancer.