HPMA copolymer-bound doxorubicin induces apoptosis in ovarian carcinoma cells by the disruption of mitochondrial function.
N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-bound doxorubicin has showed greater potency than free doxorubicin in the treatment of ovarian cancer in vivo and in vitro. The promising activity of the conjugate demonstrated in clinical trials has generated considerable interest in understanding the mechanism of action of this macromolecular therapeutic. In this study, the involvement of the mitochondrial pathway in HPMA copolymer-bound doxorubicin-induced apoptosis in the human ovarian cancer cell line A2780 was investigated. Through a series of in vitro assays, including confocal microscopy, flow cytometry, and spectrofluorimetry, a significant decrease in mitochondrial membrane potential in A2780 cells treated with HPMA copolymer-bound doxorubicin was found. The most dramatic changes in mitochondrial membrane potential were observed between 2 and 12 h of continuous drug exposure. The potential of the mitochondrial membrane remained collapsed when drug treatment continued up to 24 h. For the first time, it was shown that HPMA copolymer-bound doxorubicin induces apoptosis in ovarian cancer cells by simultaneous activation of both caspase-dependent and caspase-independent pathways of DNA damage. This was determined by monitoring the translocation of the mitochondrial proteins cytochrome c and apoptosis-inducing factor to cytosol. The altered balance between anti-apoptotic and pro-apoptotic members of the Bcl-2 family of proteins was responsible for the mitochondrial function distraction. HPMA copolymer-bound doxorubicin induced a time-dependent decrease in the expression of the anti-apoptotic Bcl-2 and Bcl-xL proteins, which control cell survival. At the same time, the expression level of pro-apoptotic members (Bax, Bad) of the Bcl-2 family was increased under the chosen experimental conditions. Altogether, these results indicate that HPMA copolymer-bound doxorubicin induced apoptosis in ovarian cancer cells through the mitochondrial pathway.
Malugin
Alexander
A
Department of Pharmaceutics and Pharmaceutical Chemistry and Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112, USA.
Kopecková
Pavla
P
Kopecek
Jindrich
J
D000178:Acrylamides / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology*; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects*; D051336:Mitochondrial Membranes / Q000187:drug effects; D010051:Ovarian Neoplasms / Q000188:drug therapy; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
D000178:Acrylamides; D019253:Proto-Oncogene Proteins c-bcl-2; C482378:N-(2-hydroxypropyl)methacrylamide co-polymer-doxorubicin conjugate; D004317:Doxorubicin