Growth Inhibition Of Imatinib-resistant K562Cells In Vivo And In Vivo By4-chlorobenzoyl Berbamine And Its Mechanisms

Objective:To explore the effects and mechanisms of4-chlorobenzoyl berbamine (BBD9) on Imatinib-resistant cell line K562(K562R) in vitro and in vivo.Methods:After treatment of imatinib(IM), BBD9and berbamine (BBM) on K562R and K562cells for72hours,cell viability was determined by MTT assay and IC50values were calculated. The expression of p210BCR-ABL was determined by Western Blot after treatment of BBD91μg/ml and BBM8μg/ml on K562R cells for48hours and BCR-ABL mRNA was determined by semi-quantative RT-PCR after treatment of BBD91μg/ml on K562R cells for various hours.IKK-α,cytoplasmic and nuclear NF-κBp65were determined by Western Blot after K562R cells were treated with BBD91μg/ml and BBM8μg/ml for48hours. Flow cytometry (FCM) was used to determine cell viability, apoptosis and necrosis, Western Blot was used to determine the expressions of PARP, Caspase-3,Caspase-9and LC3-Ⅱ after K562R cells were exposed to various concentrations of BBD9for48hours.Mouse xenograft model was used to determine in vivo anti-leukemia activity of BBD9. Tumor weight, tumor regression rate and mouse body weight were measured at the end of the experiment.(d35)Results:(1) IM resistance dectection:72h IC50value of IM on K562R and K562cells was0.933±0.015μg/ml and0.036±0.011μg/ml, respectively. K562R cells were about25times more resistant than K562cells.(2)In vitro cytotoxicity of BBM on K562R and K562cells:72h IC50value was7.81±0.711μg/ml and5.14±0.281μg/ml, respectively.(3) In vitro cytotoxicity of BBD9on K562R and K562cells:72h IC50value was1.02±0.161μg/ml and1.16±0.131μg/ml, respectively.(4) Effects of BBD9and BBM on expressions of BCR-ABL fusion protein p210BCR-ABL and mRNA:BBD9showed more potent effect in decreasing p210BCR-ABL, and24hours after treatment of BBD9at1μg/ml, BCR-ABL mRNA begun to decrease.(5) Effects of BBD9and BBM on expressions of IKKaand NF-KBp65:BBD9showed more potent effect in reducing IKKa and nuclear NF-KBp65but neither BBD9nor BBM reduced cytoplasmic NF-KBp65.(6) Effects of BBD9on cell death-related pathways:Both apoptosis and necrosis increased in accordance with the concentration of BBD9and cleaved Caspase-3, Csapase-9, PARP, and increased expression of LC3-Ⅱ were observed in a BBD9dose-dependent manner.(7) In vivo growth inhibition of BBD9on K562R cells:BBD9had better results than IM in terms of reducing tumor weight, promoting tumor regression and increasing body weight in nude mouse model, further more, no gross abnormalities of mouse organs were observed.Conclusions:(1) K562R cells were highly resistant to IM than K562cells, which was about25fold of the latter in terms of72h IC50value.(2) BBD9potently inhibited growth of K562R cells in vitro and in vivo and triggered apoptosis, necrosis and autophagy pathways and downregulated expressions of p210BCR-ABL, IKKaand inhibited NF-KBp65cytoplasm-to-nucleus translocation.(3) BBD9was of no obvious toxity to the mouse, which had the potential for further development.