Abstract 3546: Role of the “1C” Aldo-Keto Reductases in Resistance to Doxorubicin in MCF-7 breast cancer cells

Abstract Metabolism of anthracyclines by members of the aldo-keto reductase (AKR) family of enzymes presents a potential mechanism of drug resistance by converting anthracyclines such as daunorubicin, doxorubicin, or epirubicin to less cytotoxic 13′-OH metabolites. The exact mechanism and relative role of AKRs in anthracycline resistance remains unclear. Changes in anthracycline sensitivity may, in part, be the result of altered expression of AKRs in anthracycline-resistant cancers. Anthracycline-resistant cell lines were created in our laboratory by selection of MCF-7 cells for survival in increasing doses of doxorubicin (MCF-7DOX-2) or epirubicin (MCF-7EPI). “Co-cultured control” MCF-7CC cell lines were also created by an identical selection procedure in the absence of drug. Drug resistance relative to MCF-7CC cells was established at a specific threshold dose, after which the magnitude of resistance increased with increasing selection dose. Microarray and confirmatory Q-PCR studies revealed that AKR1C2 and AKR1C3 transcripts were overexpressed in MCF-7DOX2 and MCF-7EPI lines relative to MCF-7CC cells at or above the threshold dose. At the highest selection dose, the AKR1C2/3 inhibitor 5β-cholanic acid (5βC) almost completely restored sensitivity to doxorubicin in MCF-7DOX2 cells, but had little effect on MCF-7EPI and MCF-7CC cells. HPLC analysis showed altered intracellular levels of doxorubicin in MCF-7DOX2 and MCF-7EPI relative to MCF-7CC. Upon addition of 5βC, intracellular levels of doxorubicin increased in MCF-7CC and MCF-7DOX2 but not in MCF-7EPI cells (p &amp;lt; 0.05), likely due to the strong overexpression of the Abcb1 drug exporter in the latter cell line. Surprisingly, cellular levels of doxorubicinol (the 13′-OH metabolite of doxorubicin) were undetectable by HPLC in all cell lines. Laser scanning confocal microscopy further revealed that, in contrast to its nuclear location in MCF-7CC cells, doxorubicin was found primarily outside the nucleus when added to MCF-7DOX-2 and MCF-7EPI cells. Doxorubicinol, on the other hand, remained extra-nuclear in all three cell lines (in the absence or presence of 5βC). Doxorubicin localization to the nucleus was restored in MCF-7DOX-2 cells (but not MCF-7EPI cells) upon addition of 5βC. Doxorubicin stained isolated nuclei from MCF-7CC cells much more strongly than doxorubicinol; consistent with this finding, doxorubicin showed a higher DNA binding affinity than doxorubicinol in a fluorescent intercalator displacement assay. Taken together, our findings suggest that selection of breast tumour cells for anthracycline resistance results in overexpression of specific AKR isoforms. These AKRs convert the parent compound entering the cytoplasm to a less toxic 13-OH metabolite, with a lower affinity for DNA. The metabolite may then diffuse from cells or be further metabolized, preventing its detection by HPLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3546.</jats:p

Role of aldo-keto reductases and other doxorubicin pharmacokinetic genes in doxorubicin resistance, DNA binding, and subcellular localization

Abstract Background Since proteins involved in chemotherapy drug pharmacokinetics and pharmacodynamics have a strong impact on the uptake, metabolism, and efflux of such drugs, they likely play critical roles in resistance to chemotherapy drugs in cancer patients. Methods To investigate this hypothesis, we conducted a whole genome microarray study to identify difference in the expression of genes between isogenic doxorubicin-sensitive and doxorubicin-resistant MCF-7 breast tumour cells. We then assessed the degree of over-representation of doxorubicin pharmacokinetic and pharmacodynamic genes in the dataset of doxorubicin resistance genes. Results Of 27,958 Entrez genes on the array, 7.4 per cent or 2,063 genes were differentially expressed by ≥ 2-fold between wildtype and doxorubicin-resistant cells. The false discovery rate was set at 0.01 and the minimum p value for significance for any gene within the “hit list” was 0.01. Seventeen and 43 per cent of doxorubicin pharmacokinetic genes were over-represented in the hit list, depending upon whether the gene name was identical or within the same gene family, respectively. The most over-represented genes were within the 1C and 1B families of aldo-keto reductases (AKRs), which convert doxorubicin to doxorubicinol. Other genes convert doxorubicin to other metabolites or affect the influx, efflux, or cytotoxicity of the drug. In further support of the role of AKRs in doxorubicin resistance, we observed that, in comparison to doxorubicin, doxorubincol exhibited dramatically reduced cytotoxicity, reduced DNA-binding activity, and strong localization to extra nuclear lysosomes. Pharmacologic inhibition of the above AKRs in doxorubicin-resistant cells increased cellular doxorubicin levels, restored doxorubicin cytotoxicity and re-established doxorubicin localization to the nucleus. The properties of doxorubicinol were unaffected. Conclusions These findings demonstrate the utility of using curated pharmacokinetic and pharmacodynamic knowledge bases to identify highly relevant genes associated with doxorubicin resistance. The induction of one or more of these genes was found to be correlated with changes in the drug’s properties, while inhibiting one specific class of these genes (the AKRs) increased cellular doxorubicin content and restored drug DNA binding, cytotoxicity, and subcellular localization.</p