New insights in the cellular processing of platinum antitumor compounds, using fluorophore-labeled platinum complexes and digital fluorescence microscopy

J Biol Inorg Chem. 2000 Oct;5(5):655-65. doi: 10.1007/s007750000153.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / chemistry*
  • Antineoplastic Agents / metabolism*
  • Antineoplastic Agents / pharmacokinetics
  • Bone Neoplasms / metabolism
  • Cisplatin / chemistry*
  • Cisplatin / metabolism*
  • Cisplatin / pharmacokinetics
  • Dinitrophenols / chemistry
  • Dinitrophenols / immunology
  • Fluoresceins / metabolism
  • Fluorescent Dyes / metabolism*
  • Golgi Apparatus / metabolism
  • Humans
  • Microscopy, Fluorescence
  • Molecular Structure
  • Oligonucleotides / metabolism
  • Osteosarcoma / metabolism
  • Tumor Cells, Cultured

Substances

  • Antineoplastic Agents
  • Dinitrophenols
  • Fluoresceins
  • Fluorescent Dyes
  • Oligonucleotides
  • Cisplatin