Studies on effect of recombinant adenovirus PUMA on sensitivity of human glioblastoma cells to temozolomide through enhanced apoptosis pathways in vitro

Objective To investigate the effect of PUMA on the sensitivity of human glioblastoma U87MG cells to temozolomide and its related mechanisms.Methods U87MG cells under culture were divided into the normal control group,mock group and experiment group.After 24 hours of culture,the mock and experiment group were transfected with mock vector(Ad-△BH3)and the recombinant adenovirus carrying PUMA(Ad-PUMA)respectively at a multiplication of infection(MOI)of 50.The proliferation activity of cells and IC50 were detected by MTT assay,the apoptosis effect and the change of cell cycle determined by Hoechst stain and flow cytometry(FCM)technology respectively.The expression of related proteins was revealed by the method of Western blot.Results Exotic PUMA gene was expressed in U87MG cells transfected with Ad-PUMA,which could inhibit the proliferation of U87MG cells.The inhibition rate of proliferation 24,48,72 h after transfection was 17.3%,35.6%,43.3% and apoptosis rate was 20.3%,31.4%,45.4% respectively.Results:The TMZ IC50 values of PBS group,Ad-PUMA and Ad-△BH3 group cells were(15±1.9),(2.3±0.14)and(14.4±1.6)μmol/L respectively,with the sensitivity to the TMZ of Ad-PUMA group cells increased by 7.0 folds.PUMA overexpressing U87MG cells showed the DNA synthesis was inhibited and arrested in G2 phrase.The results of Western blot showed that after 72 h of transfection the PUMA and Bax protein showed increased expression(P0.01).There was no significant difference in the expression of p53 among these groups(P0.05).Conclusions PUMA gene transfection greatly enhances the sensitivity of U87MG cells to TMZ-induced apoptosis and can effectively inhibit the proliferation and promote the apoptosis of U87MG cells.The mechanism of apoptosis mainly relates to induce cell cycle G2 arrest and apoptosis in vitro.