| Objective BCR-ABL fusion protein with high a tyrosine kinaseactivity plays a key role in the pathology of chronic myeloid leukemia(CML). Imatinib, a specific inhibitor of tyrosine kinase, is now being usedin the common treatment of CML with an obvious effect, but the resistancehas become a problem nowadays. In the previous study of our group, arecombinant adenovirus Ad5F35-SD has been successfully constructedwhich can specifically bind phospho-BCR-ABL Y177（Y177-p）site. Thedeath effector domain (DED) is the critical factor for activation ofCaspases8-induced apoptosis signal. The binding of Y177-p with SH2domain can inhibit proliferation signals and the binding of DED domaincan activate Caspase8thus induce apoptosis. These tumor suppressingfunctions have been demonstrated in imatinib-sensitive CML cells andanimal models. In this study， we further investigated the effects ofAd5F35-SD on imatinib-resistant CML cells K562/G01which will be anew target in the treatment of CML Methods1. To assess the infection efficiency, optimal infection timeand MOI of Ad5F35-SD and Ad5F35-SmD. With MTT assay,colonyformation assay and flowcytometric analysis, we explore the effect ofAd5F35-SD on K562/G01cells proliferation.2To detected its effects on the apoptosis of Ad5F35-SD on K562/G01cells by Wright’s stain,flowcytometric analysis and expression level ofcaspases8and PARP by western-blot. We used caspases8inhibitorZ-IETD-FMK treating cells infected with Ad5F35-SD, aiming toinvestigating the apoptosis pathway involved.Results1The highest infection efficiency of K562/G01cells withAd5F35-SD was about60%when the infection time was48h and with theMOI105cfu/ml. The high infection efficiency was the basis of the followingtests.2Compared to the control, the inhibitory rate on cells proliferation was48.12%±2.07%（p<0.05）, the cloning formation efficiency41.2%（p<0.05）,and the percentage of cells at G2phase was28.94±0.24%（p<0.05）for K562/G01cells infected AD5F35-SD, respectively. All theresults indicated the obvious inhibition of cell growth, whereas in theAd5F35-SmD group, we didn’t find any statistic difference.3. In Ad5F35-SD infected cells, irregular cells, empty bubbles, nucleargather and fragments were observed. Higher apoptosis of13.44±2.30％（p<0.05）were detected by flow cytometric analysis. Western blot showed the fragments of caspases-8and PARP (89Kb and43Kbrespectively), indicating a cell apoptosis. No obvious difference wasobserved in Ad5F35-SmD group. After treatment of caspases8inhibitorZ-IETD-FMK in Ad5F35-SD group, cell apoptosis decreased to1.08±0.76％（p﹥0.05）, but caspases8、PARP didn’t presented fragmentswhich indicated that the apoptosis wasn’t obvious.Conclusions In summary, our results indicated that recombinantadenovirus Ad5F35-SD has inhibited K562/G01cells proliferation andinduced apoptosis by caspases8pathway. These results provides a noveltarget for treatment of CML and imatinib-resistant patient especially. |
PDF Full Text Request