Agnieszka Marczak1, Marta Denel-Bobrowska2, Aneta Rogalska2, Małgorzata Łukawska3, Irena Oszczapowicz3. 1. Department of Thermobiology, Institute of Biophysics, Faculty of Biology and Environmental Protection, Lodz University, Pomorska 141/143, 90-236 Lodz, Poland. Electronic address: aszwar@biol.uni.lodz.pl. 2. Department of Thermobiology, Institute of Biophysics, Faculty of Biology and Environmental Protection, Lodz University, Pomorska 141/143, 90-236 Lodz, Poland. 3. Department of Modified Antibiotics, Institute of Biotechnology and Antibiotics, 5 Staroscinska St., 02-516 Warsaw, Poland.
Abstract
BACKGROUND/AIM: In this study we investigated the effect of DOX and five of its derivatives containing a formamidine group (NCHNRR) at the 3' position with pyrrolidine (DOX-F PYR), piperidine (DOX-F PIP), morpholine (DOX-F MOR), N-methylpiperazine (DOX-F PAZ) and hexamethyleneimine (DOX-F HEX) ring on SKOV-3 ovarian cancer cells. We have focused on the anti-proliferative activity and the value of apoptosis induced by tested analogues. MATERIALS AND METHODS: The following methods were used: spectrophotometric assay with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); fluorimetric assays - double staining with Hoechst 33258 and propidium iodide (PI), measurement of caspase-3 activity; flow cytometry methods - phosphatidylserine (PS) externalization using Annexin V-FITC and PI fluorochromes, and TUNEL assay. RESULTS: All of the investigated derivatives were considerably more cytotoxic to the SKOV-3 cell line than DOX. The predominant type of cell death induced by the anthracycline analogues was apoptosis. Necrotic cells represented only a small percentage (<5%) of all cells. The number of apoptotic cells was dependent on the compound and the incubation time. Moreover, a significant increase in caspase-3 activity, DNA fragmentation, and morphological changes in ovarian cells were observed predominantly in new DOX analogues. CONCLUSIONS: All new formamidine derivatives of DOX were effective against ovarian cancer cells. They induced mainly the apoptotic pathway of cell death mediated by caspase-3. The most promising results were obtained for DOX-F MOR and DOX-F PAZ. The least potent was DOX-F HEX.
BACKGROUND/AIM: In this study we investigated the effect of DOX and five of its derivatives containing a formamidine group (NCHNRR) at the 3' position with pyrrolidine (DOX-F PYR), piperidine (DOX-F PIP), morpholine (DOX-F MOR), N-methylpiperazine (DOX-F PAZ) and hexamethyleneimine (DOX-F HEX) ring on SKOV-3 ovarian cancer cells. We have focused on the anti-proliferative activity and the value of apoptosis induced by tested analogues. MATERIALS AND METHODS: The following methods were used: spectrophotometric assay with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); fluorimetric assays - double staining with Hoechst 33258 and propidium iodide (PI), measurement of caspase-3 activity; flow cytometry methods - phosphatidylserine (PS) externalization using Annexin V-FITC and PI fluorochromes, and TUNEL assay. RESULTS: All of the investigated derivatives were considerably more cytotoxic to the SKOV-3 cell line than DOX. The predominant type of cell death induced by the anthracycline analogues was apoptosis. Necrotic cells represented only a small percentage (<5%) of all cells. The number of apoptotic cells was dependent on the compound and the incubation time. Moreover, a significant increase in caspase-3 activity, DNA fragmentation, and morphological changes in ovarian cells were observed predominantly in new DOX analogues. CONCLUSIONS: All new formamidine derivatives of DOX were effective against ovarian cancer cells. They induced mainly the apoptotic pathway of cell death mediated by caspase-3. The most promising results were obtained for DOX-F MOR and DOX-F PAZ. The least potent was DOX-F HEX.