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2-Methoxyestradiol Inhibits the Anaphase-Promoting Complex and Protein Translation in Human Breast Cancer Cells

¹ Lineberger Comprehensive Cancer Center, ² Department of Biochemistry and Biophysics, and ³ Carolina Cardiovascular Biology Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; ⁴ Departments of Medicine, ⁵ Surgery, and ⁶ Biochemistry and Molecular Biology, Indiana University School of Medicine; and ⁷ Walther Cancer Institute, Indianapolis, Indiana

Requests for reprints: Nancy Klauber-DeMore, MD 3010 Old Clinic Building, CB #7213, Chapel Hill, NC 27599. Phone: 919-966-8007; Fax: 919-966-8806; E-mail: nancy_demore{at}med.unc.edu.

2-Methoxyestradiol (2ME2), an estradiol metabolite with antiproliferative and antiangiogenic activities, is in phase I/II clinical trials for breast cancer. 2ME2 inhibits microtubule polymerization and causes cells to arrest in G₂-M. The purpose of this study was to further elucidate the molecular mechanism of 2ME2. MDA-MB-435 breast cancer cells were treated with 2ME2 (2 µmol/L) or vehicle alone. RNA was extracted and genomic profiling was done using 22k Agilent microarrays. Expression Analysis Systematic Explorer was used to determine enrichment of Gene Ontology categories. Protein isolates were subjected to Western blot analysis. Protein synthesis was measured with a [³⁵S]methionine pulse assay. An MDA-MB-435 cell line with two ß-tubulin mutations (2ME2R) was used to determine whether novel mechanisms were tubulin-dependent. Gene Ontology categories enriched include genes that regulate the mitotic spindle assembly checkpoint, apoptosis, and the cytosolic ribosome. The target of the mitotic spindle assembly checkpoint is the anaphase-promoting complex (APC). APC inhibition was confirmed by measuring protein levels of its targets securin and cyclin B1, which were increased in 2ME2-treated cells. Because gene expression in the cytosolic ribosome category was decreased, we evaluated whether 2ME2 decreases protein translation. This was confirmed with a pulse assay, which showed decreased isotope incorporation in 2ME2-treated cells, which was maintained in the tubulin-resistant 2ME2R cells. APC inhibition was not maintained in 2ME2R cells. 2ME2 induces tubulin-dependent cell cycle arrest through regulation of genes involved in the mitotic spindle assembly checkpoint, which results in inhibition of the APC and tubulin-independent inhibition of protein translation. [Cancer Res 2007;67(2):702–8]

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