title: Doxorubicin attached to HPMA copolymer via amide bond modifies the glycosylation pattern of EL4 cells.
abstract: To avoid the side effects of the anti-cancer drug doxorubicin (Dox), we conjugated this drug to a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone. Dox was conjugated via an amide bond (Dox-HPMA(AM), PK1) or a hydrazone pH-sensitive bond (Dox-HPMA(HYD)). In contrast to Dox and Dox-HPMA(HYD), Dox-HPMA(AM) accumulates within the cell's intracellular membranes, including those of the Golgi complex and endoplasmic reticulum, both involved in protein glycosylation. Flow cytometry was used to determine lectin binding and cell death, immunoblot to characterize the presence of CD7, CD43, CD44, and CD45, and high-performance anion exchange chromatography with pulsed amperometric detector analysis for characterization of plasma membrane saccharide composition. Incubation of EL4 cells with Dox-HPMA(AM) conjugate, in contrast to Dox or Dox-HPMA(HYD), increased the amounts of membrane surface-associated glycoproteins, as well as saccharide moieties recognized by peanut agglutinin, Erythrina cristagalli, or galectin-1 lectins. Only Dox-HPMA(AM) increased expression of the highly glycosylated membrane glycoprotein CD43, while expression of others (CD7, CD44, and CD45) was unaffected. The binding sites for galectin-1 are present on CD43 molecule. Furthermore, we present that EL4 treated with Dox-HPMA(AM) possesses increased sensitivity to galectin-1-induced apoptosis. In this study, we demonstrate that Dox-HPMA(AM) treatment changes glycosylation of the EL4 T cell lymphoma surface and sensitizes the cells to galectin-1-induced apoptosis.
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: Lubomir
authors.initials: L
authors.lastname: Kovar
authors.forename: Tomas
authors.initials: T
authors.lastname: Etrych
authors.forename: Martina
authors.initials: M
authors.lastname: Kabesova
authors.forename: Vladimir
authors.initials: V
authors.lastname: Subr
authors.forename: David
authors.initials: D
authors.lastname: Vetvicka
authors.forename: Ondrej
authors.initials: O
authors.lastname: Hovorka
authors.forename: Jiri
authors.initials: J
authors.lastname: Strohalm
authors.forename: Jan
authors.initials: J
authors.lastname: Sklenar
authors.forename: Petr
authors.initials: P
authors.lastname: Chytil
authors.forename: Karel
authors.initials: K
authors.lastname: Ulbrich
authors.forename: Blanka
authors.initials: B
authors.lastname: Rihova
mesh_terms: D000577:Amides / Q000737:chemistry; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis; D015153:Blotting, Western; D045744:Cell Line, Tumor / Q000187:drug effects; D049109:Cell Proliferation; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000494:pharmacology; D004337:Drug Carriers; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D037483:Galectin 1 / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D051917:Leukosialin / Q000378:metabolism; D016399:Lymphoma, T-Cell / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D011109:Polymethacrylic Acids / Q000494:pharmacology*
chemical_list: D000577:Amides; D000903:Antibiotics, Antineoplastic; D004337:Drug Carriers; D037483:Galectin 1; D051917:Leukosialin; D011109:Polymethacrylic Acids; C055176:doxorubicin-N-(2-hydroxypropyl)methacrylamide copolymer conjugate; D004317:Doxorubicin