Evaluation of cellular responses to photodynamic therapy with HPMA copolymer-Mce6 conjugates in human ovarian carcinoma cells

Photodynamic therapy (PDT), a new form of cancer treatment, involves the excitation of photosensitizers by a specific wavelength of light at the sight of neoplastic growth. We have previously shown combination chemotherapy and PDT with N-(2hydroxyprophyl)methacrylamide (HPMA) copolymer-bound anticancer drugs resulted in complete tumor ablation of human ovarian carcinomas heterotransplanted in nude mice. Additionally, HPMA copolymer-conjugated drugs administered by intravenous injection accumulated at significantly higher levels in tumor tissues compared to free drugs. In the following studies, cellular responses to PDT were examined utilizing the photosensitizing agent mesochlorin e6 monoethylenediamine (Mce6) or HPMA copolymer-Mce6 conjugates. First, the development of multridrug resistance, one of the major causes of failure for the treatment of human malignancies, was evaluated with Mce6 versus HPMA copolymer-bound Mce6. Prolonged exposure of human carcinoma A2780 cells to similar effective doses of free Mce6 or HPMA copolymer-Mce6 conjugates was investigated characterizing chronically treated cells by cytotoxicity analysis gene expression and anti-human P-glycoprotein IgG binding. Second, HPMA copolymer-Mce6 conjugates were synthesized to clucidate subcellular targets sensitive to PDT. Mce6 was bound to HPMA copolymers via a nondegradable dipeptide linker (P-GG-Mce6) or a lysosomally degradable spacer (P-GFLG-Mce6). Minimal nuclear localization signal (NLS) from simian virus large rumor antigen (PKKKRKC126K(FITC)C) was conjugated to HPMA copolymer-Mce6 conjugates (P-NLS(FITC)-GG-Mce6, P-NLS(FITC)-GFLG-Mc36) to direct delivery of HPMA copolymer-Mce6 conjugates to the nucleus. Low temperature and metabolic inhibitors were utilized to inhibit endocytin uptake of HPMA copolymer-Mce6 conjugates (P-GG_MCe6) resulting in plasma membrane bound P-GG-Mce6. Subcellular distribution analyzed by confocal microscopy and subcellular fractionation was correlated with cytotoxicity to A2780 cells. Third, mechanism of cell death were examined utilizing flow cytometry and ELISA. Inhibition studies incorporating actinomycin D, an inhibitor of RNA synthesis and cycloheximide, and inhibitor of protein synthesis, were used to determine the significance of gene and protein expression on cell death. Gene targets of A2780 cells treated with free Mce6 or HPMA copolymer-Mce6 conjugates were monitored by reverse transcription-polymerase chain reaction (RT-PCR). Collectively these studies validate the use of HPMA copolymer-Mce6 conjugates for the prolonged treatment of ovarian cancer and assist in the design of second-generation HPMA copolymer PDT delivery systems.