雷公藤红素提高人白血病细胞化疗敏感性的实验研究

Effect of celastrol on enhancing chemosensitivity of human leukemia cells

目的探讨雷公藤红素提高人白血病多药耐药细胞株(K562/A02)化疗敏感性的作用及其机制。方法白血病细胞株K562和多药耐药细胞株K562/A02,培养至对数生长期分成6组,分别为K562/A02阴性组、K562/A02+62.5μmol/L雷公藤红素组、K562/A02+125μmol/L雷公藤红素组以及K562阴性组、K562+62.5μmol/L雷公藤红素组、K562+125μmol/L雷公藤红素组;设置K562+硼替佐米4nmol/L为阳性对照组。采用Annexin V-FITC/PI双染法检测细胞凋亡率;采用反转录PCR法检测各组细胞中核转录因子-κB(nuclear factor-kappa B,NF-κB)、survivin、死亡受体5(death receptor 5,DR5)mRNA表达水平;应用流式细胞术检测DR5蛋白阳性表达率。结果K562阴性组与K562+62.5μmol/L雷公藤红素组细胞凋亡率[(5.110±0.803)%、(6.283±0.889)%]以及K562/A02阴性组与K562/A02+62.5μmol/L雷公藤红素组细胞凋亡率[(7.203±0.275)%、(8.753±0.951)%]比较差异均无统计学意义(P>0.05);K562/A02阴性组NF-κB mRNA(0.863±0.073)、survivin mRNA(0.989±0.018)、DR5 mRNA(0.114±0.037)表达水平高于K562阴性组(0.262±0.074、0.267±0.056、0.050±0.011)(P<0.05);K562+62.5μmol/L雷公藤红素组、K562+125μmol/L雷公藤红素组NF-κB mRNA(0.210±0.053、0.128±0.076)、survivin mRNA(0.199±0.064、0.228±0.030)表达水平均低于K562阴性组,DR5mRNA表达水平(0.312±0.075、0.464±0.073)表达水平高于K562阴性组(P<0.05);K562/A02+62.5μmol/L雷公藤红素组、K562/A02+125μmol/L雷公藤红素组NF-κB mRNA(0.492±0.040、0.359±0.069)、survivin mRNA(0.516±0.042、0.348±0.047)表达水平均低于K562/A02阴性组,DR5mRNA表达水平(0.470±0.034、0.519±0.048)均高于K562/A02阴性组(P<0.05);K562+62.5μmol/L雷公藤红素组、K562+125μmol/L雷公藤红素组DR5蛋白阳性表达率[(5.438±0.517)%、(6.250±0.897)%]高于K562阴性组[(0.093±0.015)%](P<0.05),K562/A02+62.5μmol/L雷公藤红素组、K562/A02+125μmol/L雷公藤红素组DR5蛋白阳性表达率[(19.900±0.526)%、(20.703±1.045)%]高于K562/A02阴性组[(6.980±0.925)%](P<0.05)。结论雷公藤红素提高K562/A02耐药细胞株化疗敏感性的作用可能与下调NF-κB、survivin表达,上调DR5表达有关。

Objective To investigate the role and mechanism of celastrol in enhancing the chemosensitivity of human leukemia multi-drug resistance cell line K562/A02.Methods Human leukemia cell line K562 and human leukemia multidrug resistance cell line K562/A02 in logarithmic growth phase were divided into 6 groups as K562/A02 negative group,K562/A02+62.5μmol/L celastrol group,K562/A02+125μmol/L celastrol group,K562 negative group,K562+62.5μmol/Lcelastrol group and K562+125μmol/L celastrol group.K562+4 nmol/L bortezomib was as positive control group.The apoptosis rate was detected by Annexin V-FITC/PI double staining method.The levels of nuclear factorkappa B(NF-κB),survivin and death receptor 5(DR5)mRNA were detected by reverse transcription-PCR in all groups.The positive expression rate of DR5 protein was detected by flow cytometry.Results There were no significant differences in the apoptosis rates of K562 cells between K562 negative group and K562+62.5μmol/L celastrol group((5.110±0.803)% vs(6.283±0.889)%),and of K562/A02 cells between K562/A02 negative group and K562/A02+62.5μmol/L celastrol group((7.203±0.275)% vs(8.753±0.951)%)(P>0.05).The mRNA levels of NF-κB(0.863±0.073),survivin(0.989±0.018)and DR5(0.114±0.037)in K562/A02 negative group were significantly higher than those in K562 negative group(0.262±0.074,0.267±0.056,0.050±0.011)(P<0.05).The mRNA levels of NF-κB(0.210±0.053,0.128±0.076)and survivin(0.199±0.064,0.228±0.030)in K562+62.5μmol/L celastrol group and K562+125μmol/L celastrol group were significantly lower than those in K562 negative group(P<0.05).The mRNA levels of NF-κB(0.492±0.040,0.359±0.069)and survivin(0.516±0.042,0.348±0.047)in K562/A02+62.5μmol/L celastrol group and K562/A02+125μmol/L celastrol group were significantly lower than those in K562/A02 negative group(P<0.05).The mRNA levels of DR5 were significantly higher in K562+62.5μmol/L celastrol group(0.312±0.075),K562+125μmol/L celastrol group(0.464±0.073),K562/A02+62.5μmol/L celastrol group(0.470±0.034)and K562/A02+125μmol/L celastrol group(0.519±0.048)than those in K562 negative group and K562/A02 negative group(P<0.05).The positive rates of DR5 protein was significantly higher in K562+62.5μmol/L celastrol group((5.438±0.517)%)and K562+125μmol/L celastrol group((6.250±0.897)%)than that in K562 negative group((0.093±0.015)%)(P<0.05),and in K562/A02+62.5μmol/L celastrol group((19.900±0.526)%)and K562/A02+125μmol/L celastrol group((20.703±1.045)%)than that in K562/A02 negative group((6.980±0.925)%)(P<0.05).Conclusion Celastrol could enhance the chemosensitivity of human leukemia multi-drug resistance cell line K562/A02,which may be correlated with the down-regulation of NF-κB and survivin expression as well as up-regulation of DR5 expression.