Down-regulation of DNA topoisomerase IIalpha in human colorectal carcinoma cells resistant to a protoberberine alkaloid, berberrubine.

摘要

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 approximately 2.8-fold lower in AMC5/B1 cells compared with parental cells. The AMC5/B1 line contained approximately 5-fold decrease in topoisomerase IIalpha protein level and approximately 2.5-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.