title: 2-methoxyestradiol inhibits the anaphase-promoting complex and protein translation in human breast cancer cells.
abstract: 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 micromol/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 beta-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.
authors.affiliation: Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Bhati
authors.forename: Yesim
authors.initials: Y
authors.lastname: Gökmen-Polar
authors.forename: George W
authors.initials: GW
authors.lastname: Sledge
authors.forename: Cheng
authors.initials: C
authors.lastname: Fan
authors.forename: Harikrishna
authors.initials: H
authors.lastname: Nakshatri
authors.forename: David
authors.initials: D
authors.lastname: Ketelsen
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Dial
authors.forename: Cam
authors.initials: C
authors.lastname: Patterson
authors.forename: Nancy
authors.initials: N
authors.lastname: Klauber-DeMore
mesh_terms: D000077584:2-Methoxyestradiol; D064173:Anaphase-Promoting Complex-Cyclosome; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D048708:Cell Growth Processes / Q000187:drug effects; D045744:Cell Line, Tumor; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D014176:Protein Biosynthesis / Q000187:drug effects*; D014404:Tubulin / Q000235:genetics / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology; D043743:Ubiquitin-Protein Ligase Complexes / Q000037:antagonists & inhibitors*
chemical_list: D014404:Tubulin; D050257:Tubulin Modulators; D004958:Estradiol; D000077584:2-Methoxyestradiol; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome