title: Investigating the mechanism of enhanced cytotoxicity of HPMA copolymer-Dox-AGM in breast cancer cells.
abstract: Recently we have described an HPMA copolymer conjugate carrying both the aromatase inhibitor aminoglutethimide (AGM) and doxorubicin (Dox) as combination therapy. This showed markedly enhanced in vitro cytotoxicity compared to the HPMA copolymer-Dox (FCE28068), a conjugate that demonstrated activity in chemotherapy refractory breast cancer patients during early clinical trials. To better understand the superior activity of HPMA copolymer-Dox-AGM, here experiments were undertaken using MCF-7 and MCF-7ca (aromatase-transfected) breast cancer cell lines to: further probe the synergistic cytotoxic effects of AGM and Dox in free and conjugated form; to compare the endocytic properties of HPMA copolymer-Dox-AGM and HPMA copolymer-Dox (binding, rate and mechanism of cellular uptake); the rate of drug liberation by lysosomal thiol-dependant proteases (i.e. conjugate activation), and also, using immunocytochemistry, to compare their molecular mechanism of action. It was clearly shown that attachment of both drugs to the same polymer backbone was a requirement for enhanced cytotoxicity. FACS studies indicated both conjugates have a similar pattern of cell binding and endocytic uptake (at least partially via a cholesterol-dependent pathway), however, the pattern of enzyme-mediated drug liberation was distinctly different. Dox release from PK1 was linear with time, whereas the release of both Dox and AGM from HPMA copolymer-Dox-AGM was not, and the initial rate of AGM release was much faster than that seen for the anthracycline. Immunocytochemistry showed that both conjugates decreased the expression of ki67. However, this effect was more marked for HPMA copolymer-Dox-AGM and, moreover, only this conjugate decreased the expression of the anti-apoptotic protein bcl-2. In conclusion, the superior in vitro activity of HPMA copolymer-Dox-AGM cannot be attributed to differences in endocytic uptake, and it seems likely that the synergistic effect of Dox and AGM is due to the kinetics of intracellular drug liberation which leads to enhanced activity.
authors.affiliation: Centre for Polymer Therapeutics, Welsh School of Pharmacy, Redwood Building, Cardiff University, King Edward VII Ave., Cardiff CF10 3XF, UK.
authors.forename: Francesca
authors.initials: F
authors.lastname: Greco
authors.forename: María J
authors.initials: MJ
authors.lastname: Vicent
authors.forename: Siobhan
authors.initials: S
authors.lastname: Gee
authors.forename: Arwyn T
authors.initials: AT
authors.lastname: Jones
authors.forename: Julia
authors.initials: J
authors.lastname: Gee
authors.forename: Robert I
authors.initials: RI
authors.lastname: Nicholson
authors.forename: Ruth
authors.initials: R
authors.lastname: Duncan
mesh_terms: D000616:Aminoglutethimide / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D002746:Chlorpromazine / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D019394:Ki-67 Antigen / Q000235:genetics; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology; D008689:Methacrylates / Q000493:pharmacokinetics / Q000494:pharmacology*; D018613:Microscopy, Confocal; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D013778:Tetrazolium Salts; D013844:Thiazoles; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; C518961:HPMA copolymer-Dox-AGM; D019394:Ki-67 Antigen; D008689:Methacrylates; D019253:Proto-Oncogene Proteins c-bcl-2; D013778:Tetrazolium Salts; D013844:Thiazoles; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D000616:Aminoglutethimide; D003571:Cytochalasin B; D004317:Doxorubicin; C022616:thiazolyl blue; D002746:Chlorpromazine