title: New insights in the cellular processing of platinum antitumor compounds, using fluorophore-labeled platinum complexes and digital fluorescence microscopy.
abstract: The cellular distribution and processing pathways of two platinum compounds, modeling the antitumor drug cisplatin (cDDP) in human osteosarcoma (U2-OS) cells is reported. A [Pt(en)Cl] entity has been covalently linked to a carboxyfluorescein diacetate (CFDA) moiety and to a dinitrophenyl (DNP) moiety. The two different constructs were administered to living cell cultures that were analyzed using digital fluorescence microscopy. The non-fluorescent CFDA construct becomes fluorescent after cellular uptake and subsequent acetate hydrolysis by esterases, and is therefore suitable to monitor platinum in living cells; the DNP construct can be visualized by immunocytochemistry and consequently serves as a control. Both complexes were readily internalized by the cells, and localized throughout the whole cell. After 2-3 h the complex accumulated in the nucleus, but 6-8 h after incubation a punctuate staining of a cytoplasmic region was observed, that persisted and became more pronounced after 24 h. The overall fluorescence in the cell decreased over time, implying a secretion of the platinum complex. Surprisingly, the accumulation remained visible after 72 h. Co-localization experiments with a Golgi apparatus-selective stain indicate the involvement of Golgi vesicles in intracellular processing of cisplatin-derived complexes. Immunocytochemical studies, using the DNP derivative, resulted in very similar images as obtained with the CFDA construct. CFDA-boc (a non-platinum-containing fluorescein derivative) was used as control: a faint staining throughout the whole cell was observed. Cisplatin-resistant U2-OS/Pt cells showed staining patterns very similar to the U2-OS cells using both platinum constructs. This study illustrates that only a very small portion of the platinum complex eventually remains bound to DNA, as after 24 h no significant fluorescence could be observed in the nucleus. Cisplatin-derived complexes with fluorescent tags afford a new insight into the cellular processing of these complexes and therefore may contribute to further unraveling of the mechanism of platinum antitumor complexes.
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Centre, The Netherlands.
authors.forename: C
authors.initials: C
authors.lastname: Molenaar
authors.forename: J M
authors.initials: JM
authors.lastname: Teuben
authors.forename: R J
authors.initials: RJ
authors.lastname: Heetebrij
authors.forename: H J
authors.initials: HJ
authors.lastname: Tanke
authors.forename: J
authors.initials: J
authors.lastname: Reedijk
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry* / Q000378:metabolism* / Q000493:pharmacokinetics; D001859:Bone Neoplasms / Q000378:metabolism; D002945:Cisplatin / Q000737:chemistry* / Q000378:metabolism* / Q000493:pharmacokinetics; D004140:Dinitrophenols / Q000737:chemistry / Q000276:immunology; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D009841:Oligonucleotides / Q000378:metabolism; D012516:Osteosarcoma / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D004140:Dinitrophenols; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D002945:Cisplatin