Down-regulation of DNA topoisomerase IIα in human colorectal carcinoma cells resistant to a protoberberine alkaloid, berberrubine
Down-regulation of DNA topoisomerase IIα in human colorectal carcinoma cells resistant to a protoberberine alkaloid, berberrubine
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DOI：
10.1124/mol.61.4.879
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发表时间：
2002-04-01
影响因子：
3.6
通讯作者：
Chung, IK
中科院分区：
文献类型：
--
作者：
Kang, MR;Chung, IK
小檗红碱是一种在动物模型中表现出抗肿瘤活性的原小檗碱生物碱，在体外已被鉴定为DNA拓扑异构酶II的特异性毒物。为了更好地理解细胞对小檗红碱的反应机制，选择对小檗红碱具有抗性的人结肠直肠癌细胞（AMC 5）。所得细胞系（AMC 5/B1）在不存在MDR 1过表达的情况下对小檗红碱具有5.3倍的抗性。AMC 5/B1细胞系对拓扑异构酶II靶向药物具有交叉耐药，但对其他抗肿瘤药物无交叉耐药。对各种药物的交叉耐药模式使我们检查拓扑异构酶II的细胞内容物。AMC 5/B1细胞的拓扑异构酶II活性是亲本细胞的2.8倍。AMC 5/B1细胞系拓扑异构酶II α蛋白水平降低了5倍，拓扑异构酶II α mRNA水平降低了2.5倍。拓扑异构酶II α mRNA的降解动力学的比较表明，两种细胞系之间的mRNA稳定性没有差异。此外，拓扑异构酶II α启动子在AMC 5/B1细胞中的活性约为AMC 5亲本细胞中的25%时，进行瞬时转染实验与启动子-荧光素酶报告基因。这些结果表明AMC 5/B1细胞中拓扑异构酶II α的下调发生在转录水平。拓扑异构酶II α启动子区的核苷酸测序显示在AMC 5/B1细胞中没有突变。总之，AMC 5细胞对小檗红碱的耐药性与催化活性拓扑异构酶II α水平降低相关，表明拓扑异构酶II α是小檗红碱体内的细胞靶点。
Berberrubine, a protoberberine alkaloid that exhibits antitumor activity in animal models, has been identified as a specific poison of DNA topoisomerase II in vitro. To better understand the mechanisms of cellular response to berberrubine, human colorectal carcinoma cells (AMC5) were selected for resistance to berberrubine. The resulting cell line (AMC5/B1) was 5.3-fold resistant to berberrubine in the absence of MDR1 overexpression. The AMC5/B1 line was cross-resistant to topoisomerase II-targeted drugs but showed no cross-resistance to other antitumor drugs. The patterns of cross-resistance to various drugs led us to examine the cellular contents of topoisomerase II. Topoisomerase II activity was similar to2.8-fold lower in AMC5/B1 cells compared with parental cells. The AMC5/B1 line contained similar to5-fold decrease in topoisomerase IIalpha protein level and similar to2.5-fold decrease in topoisomerase IIalpha mRNA level. A comparison of the degradation kinetics of topoisomerase IIalpha mRNA demonstrated that there was no difference in mRNA stability between the two cell lines. Furthermore, the activity of topoisomerase IIalpha promoter in AMC5/B1 cells was about 25% of that in AMC5 parental cells when transient transfection experiments were performed with the promoter-luciferase reporter gene. These results indicate that down-regulation of topoisomerase IIalpha in AMC5/B1 cells occurs at the transcriptional level. Nucleotide sequencing of the topoisomerase IIalpha promoter regions revealed no mutations in AMC5/B1 cells. In summary, resistance to berberrubine in AMC5 cells is associated with decreased level of catalytically active topoisomerase IIalpha, suggesting that topoisomerase IIalpha is the cellular target of berberrubine in vivo.