4-氯苯甲酰小檗胺体内外抑制伊马替尼耐药K562细胞增殖的作用及机制

Inhibitory effect of 4-chlorobenzoyl berbamine on imatinib-resistant K562 cells in vitro and in vivo

目的研究4-氯苯甲酰小檗胺(BBD9)体内外抑制伊马替尼(IM)耐药k562细胞(K562/IR)的增值作用,并探讨其机制。方法 MTT法测定BBD9和小檗胺(BBM)IC50以比较药效。0.5μg/ml BBD9或8μg/ml BBM处理体外培养K562/IR细胞48 h Western blot检测p210Bcr-Abl,IKKα,胞浆和胞核NF-κBp65表达水平。不同浓度BBD9处理体外培养的K562/IR48 h后流式测定细胞存活,凋亡,坏死比例;Western blotting检测PARP、Caspase3、Caspase9和LC3II表达水平。裸鼠移植瘤模型组1(15 mg/kg BBD9)、组2(30 mg/kg BBD9)、组3(100 mg/kg IM)和对照组(不给药)中移植瘤的瘤质量、瘤消退率和裸鼠体质量检测。结果 BBD9和BBM IC50分别为0.73μg/ml和5.43μg/ml。0.5μg/ml BBD9或8μg/ml BBM处理48 h后p210Bcr-Abl,IKKα和胞核NF-κBp65表达均下降且以BBD9更明显(P<0.05),胞浆NF-κBp65基本一致。细胞凋亡,坏死均和BBD9呈计量依赖;活化的PARP,Caspase3,Caspase9和LC3 II也随着药物浓度增加而升高(P<0.05)。裸鼠移植瘤瘤质量、瘤消退率、和体质量等指标BBD9给药组优于IM给药组(P<0.05)。结论 BBD9对K562/IR体内外均能起效,抑制p210Bcr-Abl,IKKα表达和胞浆NF-κBp65向胞核转位,并激活凋亡,坏死,自噬等多条死亡通路。

Objective To observe the inhibitory effect of 4-chlorobenzoyl berbamine（BBD9） on imatinib-resistant cell line K562（K562/IR） in vitro and in vivo and explore the mechanisms.Methods The IC50 of BBD9 and berbamine（BBM） was determined by MTT assay.The expressions of p210Bcr-Abl,IKKα,cytoplasmic and nuclear NF-κBp65 were determined using Western blotting in K562/IR cells following a 48-h exposure to 0.5 μg/ml BBD9 or 8 μg/ml BBM.Flow cytometry was used to analyze the cell viability,apoptosis and necrosis;Western blotting was employed to determine the expressions of PARP,caspase-3,caspase-9 and LC3II in K562/IR cells exposed to different concentrations of BBD9 for 48 h.In nude mouse models bearing K562/IR cell xenograft,the tumor weight,tumor regression,and body weight changes of the mice were measured after treatments with 15 mg/kg and 30 mg/kg BBD9 and 100 mg/kg imatinib.Results The IC50 of BBD9 and BBM was 0.73 μg/ml and 5.43 μg/ml,respectively.In K562/IR cell cultures,the expressions of p210Bcr-Abl,IKKα and nuclear NF-κB p65 were all decreased following BBD9 and BBM treatments,but BBD9 produced more potent effect;cytoplasmic NF-κB p65 showed no obvious changes after the treatments.The cell apoptosis and necrosis increased with the concentrations of BBD9,which also dose-dependently increased the levels of cleaved caspase-3,csapase-9,PARP,and LC3II expression.In the tumor-bearing mouse model,BBD9 showed stronger effects than imatinib in reducing the tumor weight,promoting tumor regression,and increasing the body weight.Conclusion BBD9 can effectively inhibit the growth of K562/IR cells in vitro and in vivo by activating cell apoptosis,necrosis and autophage pathways,down-regulating expressions of p210Bcr-Abl and IKKα and suppressing the cytoplasm-to-nucleus translocation of NF-κBp65.