title: Mechanisms of cytotoxicity in human ovarian carcinoma cells exposed to free Mce6 or HPMA copolymer-Mce6 conjugates.
abstract: It is essential to understand cellular responses on photodynamic therapy (PDT) to design delivery systems that maximize cytotoxic effects coupled with minimal induction of side effects or protective mechanisms (or both). Here, we investigated mechanisms of toxicity in human ovarian carcinoma A2780 cells treated with structurally diverse N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (P)-mesochlorin e6 monoethylenediamine (Mce6) conjugates that possessed differential subcellular accumulation or covalent attachments of photosensitizers (or both). Apoptosis and necrosis were observed after photoactivation, with increased apoptotic responses observed in cells exposed to conjugates possessing Mce6 linkage via a lysosomally degradable tetrapeptide spacer (HPMA copolymer-Mce6 conjugates containing Mce6 bound via glycylphenylalanylleucylglycine [GFLG] linker [P-GFLG-Mce6], HPMA copolymer-Mce6 conjugates containing Mce6 bound via a GFLG spacer and containing nuclear localization sequence, PKKKRKV132K(FITC)C [NLS(fluorescein-5-isothiocyanate [FITC])] bound via a thioether linkage [P-NLS(FITC)-GFLG-Mce6]). Furthermore, the induction of necrosis was more pronounced in cells exposed to conjugates containing both a nuclear localization sequence (NLS) and Mce6 bound by a degradable linker (P-NLS(FITC)-GFLG-Mce6). Caspase-independent mechanisms of cell death were identified in cells treated with nuclear-targeted conjugates possessing Mce6 attached using a nondegradable tether (HPMA copolymer-Mce6 conjugates containing Mce6 bound via a GG spacer and containing NLS(FITC) bound via a thioether linkage [P-NLS(FITC)-GG-Mce6]), whereas low levels of apoptosis and necrosis were detected in cells exposed to photoactivated nontargeted HPMA copolymer-Mce6 conjugates containing Mce6 coupled through a nondegradable spacer (HPMA copolymer-Mce6 conjugates containing Mce6 bound via GG linker [P-GG-Mce6]). Variations in gene expression were observed in cells on PDT. Specifically, HSP70 expression was solely detected in cells treated with P-GFLG-Mce6, whereas the loss of detection of several genes were observed in cells treated with P-NLS(FITC)-GFLG-Mce6. Variations in cellular responses on PDT using different HPMA copolymer-Mce6 conjugates will prove useful in the design of optimal HPMA copolymer PDT delivery systems.
authors.affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry/CCCD, University of Utah, Salt Lake City, UT, USA.
authors.forename: Monica
authors.initials: M
authors.lastname: Tijerina
authors.forename: Pavla
authors.initials: P
authors.lastname: Kopecková
authors.forename: Jindŕich
authors.initials: J
authors.lastname: Kopecek
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008652:Mesoporphyrins; D008689:Methacrylates / Q000737:chemistry / Q000494:pharmacology* / Q000633:toxicity; D015394:Molecular Structure; D009336:Necrosis; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology*; D010778:Photochemotherapy; D017319:Photosensitizing Agents; D011166:Porphyrins / Q000737:chemistry / Q000494:pharmacology* / Q000633:toxicity; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D061945:Caspase Inhibitors; D008652:Mesoporphyrins; D008689:Methacrylates; D017319:Photosensitizing Agents; D011166:Porphyrins; D012333:RNA, Messenger; C088297:meso-chlorin e(6) monoethylene diamine; D020169:Caspases; C032802:hydroxypropyl methacrylate