title: Differences in the intracellular distribution of acid-sensitive doxorubicin-protein conjugates in comparison to free and liposomal formulated doxorubicin as shown by confocal microscopy.
abstract: OBJECTIVE
  To investigate differences in the cellular uptake and intracellular distribution of protein-bound doxorubicin in comparison to free doxorubicin and a liposomal formulation (CAELYX) METHODS: LXFL 529 lung carcinoma cells were incubated with an acid-sensitive transferrin and albumin conjugate of doxorubicin, a stable albumin doxorubicin conjugate, and free and liposomal doxorubicin for up to 24 h. The uptake of doxorubicin was detected with confocal laser scanning microscopy (CLSM). To investigate the intracellular localization of the anticancer drug, lysosomes, Golgi apparatus, and mitochondria were also stained by various organelle-specific fluorescent markers. In vitro efficacy of the doxorubicin derivatives was examined with the BrdU incorporation assay.
  
  
  RESULTS
  The acid-sensitive albumin and transferrin doxorubicin conjugates showed enhanced cytotoxicity in comparison to liposomal doxorubicin, whereas the stable albumin-doxorubicin conjugate showed only marginal activity. Of all compounds tested, doxorubicin showed the highest cytotoxicity. CLSM studies with specific markers for lysosomes, mitochondria, and the Golgi apparatus demonstrated that protein-bound doxorubicin or liberated doxorubicin was accumulated in the mitochondria and Golgi compartments, but not in the lysosomes after 24 h. Free doxorubicin showed a time-dependent intracellular shift from the nucleus to the mitochondria and Golgi apparatus. Fluorescence resulting from incubation with CAELYX was primarily detected in the nucleus.
  
  
  CONCLUSIONS
  Our results indicate that other organelles in addition to the cell nucleus are important sites of accumulation and interaction for protein-bound doxorubicin or intracellularly released doxorubicin as well as for free doxorubicin.
authors.affiliation: Tumor Biology Center, Department of Medical Oncology, Clinical Research, Freiburg, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Beyer
authors.forename: B
authors.initials: B
authors.lastname: Rothern-Rutishauser
authors.forename: C
authors.initials: C
authors.lastname: Unger
authors.forename: H
authors.initials: H
authors.lastname: Wunderli-Allenspach
authors.forename: F
authors.initials: F
authors.lastname: Kratz
mesh_terms: D000970:Antineoplastic Agents / Q000493:pharmacokinetics*; D002467:Cell Nucleus / Q000378:metabolism*; D004317:Doxorubicin / Q000493:pharmacokinetics*; D004337:Drug Carriers; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism*; D008081:Liposomes; D018613:Microscopy, Confocal / Q000379:methods; D008928:Mitochondria / Q000378:metabolism*; D012709:Serum Albumin / Q000493:pharmacokinetics; D014168:Transferrin / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D004337:Drug Carriers; D008081:Liposomes; D012709:Serum Albumin; D014168:Transferrin; D004317:Doxorubicin