Vasohibin-1 Recombinant Adenovirus On Proliferation And Apoptosis Of Human Ovarian Cancer SKOV-3 Cells In

Background/Objective:Because of the clinical manifestation of ovarian cancerwas not typical at early stage and lack effective screening, most ovarian cancer wasdiagnosed at advanced stage. Despite debulking surgery and adjuvant combinationchemotherapy with platinum and taxane have significantly improved the prognosisof patients, but there were70%patients with recurring into2years, while the5-year survival rate of recurrence patients with clinical stage III and IV was only23%. For this reason, effective and novel strategies for ovarian cancer treatment isvery necessary. This study is to investigate the effects of the endogenous angiogenesis inhibitor vasohibin-1on proliferation and apoptosis of the human ovarian cancer SKOV-3cells, in order to provide new ideas for the gene therapy of ovarian cancer.Methods:SKOV-3cells were cultured in RPMI1640media supplemented with10%fetal bovine serum (FBS),100mg/ml streptomycin and100units/ml penicillin at37℃in a humidified atmosphere of5%CO2. Cells growth were in monolayers, and chose cells in logarithmic growth phase.(1) SKOV-3cells were transfected by vasohibin-1recombinant adenovirus (Ad-vasohibin-1) at different multiplicity of infection (MOI), MOI=25,50,100,200, then observed the green fluorescence protein (GFP) and cells’states under fluorescence microscope. Calculated the infection rate and ensured the best MOI.(2) MOI=50,75,100,SKOV-3cells was transfected by Ad-vasohibin-l,we found GFP became bright with the MOI increasing and time prolong.(3) SKOV-3cells were transfected by vasohibin-1recombinant adenovirus at a MOI of75.The expression of vasohibin-1protein in SKOV-3cells was confirmed by western blot.(4) Cell Count Kit-8(CCK-8) was used to examine the effects of Ad-vasohibin-1at different MOI (25,50,100,200) and different time points (24h, 48h and72h) on proliferation of SKOV-3cells.(5) The apoptosis effect was determined by Hoechst33258Staining Kit.Results:(1)The green fluorescence could be observed under fluorescence microscope after incubating24h, and the green fluorescence became most brightly after incubating48h. The cell death was happened at MOI of100.The GFP was disappeared at ten days.(2) The GFP became brightly with the MOI increasing and time prolong.(3) Compared with controls (negative control and blank control), the vasohibin-1protein was significantly increased after vasohibin-1recombinant adenovirus transfecting SKOV-3cells for48h(P<0.05).(4) There was no statistical difference on inhibition between Ad-vasohibin-1group and negative control group at a MOI of25after Ad-vasohibin-1transfecting SKOV-3cells for24h (P=0.065); but the other groups had statistical significance and the inhibition of Ad-vasohibin-1on the proliferation of SKOV-3cells increased with the growth of MOI and with the time prolonging(P<0.05).(5) Ad group cell showed typical characteristics of apoptosis:the nucleus shrink, chromatin condensed chromatin, nuclear fragmentation, the brightness increased, which showed dense granular or blocky; while the cells in NC group and BC group stained evenly blue, only a few cells appeared the phenomenon.Conclusion:This study suggests that vasohibin-1could over-expression in SKOV-3cells and over-express vasohibin-1could inhibit the proliferation and induce apoptosis of SKOV-3cells, indicating a potential target for therapeutic intervention in ovarian cancer.