title: The same drug but a different mechanism of action: comparison of free doxorubicin with two different N-(2-hydroxypropyl)methacrylamide copolymer-bound doxorubicin conjugates in EL-4 cancer cell line.
abstract: Doxorubicin is one of the most potent anti-tumor drugs with a broad spectrum of use. To reduce its toxic effect and improve its pharmacokinetics, we conjugated it to an HPMA copolymer carrier that enhances its passive accumulation within solid tumors via the EPR effect and decreases its cytotoxicity to normal, noncancer cells. In this study, we compared the antiproliferative, pro-survival, and death signals triggered in EL-4 cancer cells exposed to free doxorubicin and doxorubicin conjugated to a HPMA copolymer carrier via either enzymatically (PK1) or hydrolytically (HYD) degradable bonds. We have previously shown that the intracellular distribution of free doxorubicin, HYD, and PK1 is markedly different. Here, we demonstrated that these three agents greatly differ also in the antiproliferative effect and cell death signals they trigger. JNK phosphorylation sharply increased in cells treated with HYD, while treatment with free doxorubicin moderately decreased and treatment with PK1 even strongly decreased it. On the other hand, treatment with free doxorubicin greatly increased p38 phosphorylation, while PK1 and HYD increased it slightly. PK1 also significantly increased ERK phosphorylation, while both the free doxorubicin and HYD conjugate slightly decreased it. Long-term inhibition of JNK significantly increased both proliferation and viability of EL-4 cells treated with free doxorubicin, showing that the JNK signaling pathway could be critical for mediating cell death in EL-4 cells exposed to free doxorubicin. Both activation of caspase 3 and decreased binding activity of the p50 subunit of NFkappaB were observed in cells treated with free doxorubicin and HYD, while no such effects were seen in cells incubated with PK1. Analysis of the expression of genes involved in apoptosis and regulation of the cell cycle demonstrated that free doxorubicin and HYD have very similar mechanisms of action, while PK1 has very different characteristics.
authors.affiliation: Department of Immunology and Gnotobiology, Institute of Microbiology ASCR, v.v.i., VídenskA 1083, 142 20 Prague 4, Czech Republic. lkovar@biomed.cas.cz
authors.forename: Lubomír
authors.initials: L
authors.lastname: Kovár
authors.forename: Jirí
authors.initials: J
authors.lastname: Strohalm
authors.forename: Petr
authors.initials: P
authors.lastname: Chytil
authors.forename: Tomás
authors.initials: T
authors.lastname: Mrkvan
authors.forename: Marek
authors.initials: M
authors.lastname: Kovár
authors.forename: Ondrej
authors.initials: O
authors.lastname: Hovorka
authors.forename: Karel
authors.initials: K
authors.lastname: Ulbrich
authors.forename: Blanka
authors.initials: B
authors.lastname: Ríhová
mesh_terms: D000178:Acrylamides / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival; D004317:Doxorubicin / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology*; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D020128:Inhibitory Concentration 50; D016399:Lymphoma, T-Cell / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation
chemical_list: D000178:Acrylamides; D016328:NF-kappa B; C482378:N-(2-hydroxypropyl)methacrylamide co-polymer-doxorubicin conjugate; D004317:Doxorubicin; D020929:Mitogen-Activated Protein Kinase Kinases; D053148:Caspase 3