Intracellular localization and trafficking of fluorescein-labeled cisplatin in human ovarian carcinoma cells.
OBJECTIVE
We sought to identify the subcellular compartments in which cisplatin [cis-diamminedichloroplatinum (DDP)] accumulates in human ovarian carcinoma cells and define its export pathways.


METHODS
Deconvoluting digital microscopy was used to identify the subcellular location of fluorescein-labeled DDP (F-DDP) in 2008 ovarian carcinoma cells stained with organelle-specific markers. Drugs that block vesicle movement were used to map the traffic pattern.


RESULTS
F-DDP accumulated in vesicles and were not detectable in the cytoplasm. F-DDP accumulated in the Golgi, in vesicles belonging to the secretory export pathway, and in lysosomes but not in early endosomes. F-DDP extensively colocalized with vesicles expressing the copper efflux protein, ATP7A, whose expression modulates the cellular pharmacology of DDP. Inhibition of vesicle trafficking with brefeldin A, wortmannin, or H89 increased the F-DDP content of vesicles associated with the pre-Golgi compartments and blocked the loading of F-DDP into vesicles of the secretory pathway. The importance of the secretory pathway was confirmed by showing that wortmannin and H89 increased whole cell accumulation of native DDP.


CONCLUSIONS
F-DDP is extensively sequestered into vesicular structures of the lysosomal, Golgi, and secretory compartments. Much of the distribution to other compartments occurs via vesicle trafficking. F-DDP detection in the vesicles of the secretory pathway is consistent with a major role for this pathway in the efflux of F-DDP and DDP from the cell.
Safaei
Roohangiz
R
Department of Medicine, and the Rebecca and John Moores Cancer Center, University of California-San Diego, San Diego, CA 92093, USA. rsafaei@ucsd.edu
Katano
Kuniyuki
K
Larson
Barrett J
BJ
Samimi
Goli
G
Holzer
Alison K
AK
Naerdemann
Wiltrud
W
Tomioka
Mika
M
Goodman
Murray
M
Howell
Stephen B
SB
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D001692:Biological Transport; D020126:Brefeldin A / Q000494:pharmacology; D027682:Cation Transport Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002945:Cisplatin / Q000378:metabolism / Q000494:pharmacology*; D003287:Contrast Media; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D000073840:Copper-Transporting ATPases; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D019793:Fluorescein; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions; D013449:Sulfonamides / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D000077191:Wortmannin
D000730:Androstadienes; D000970:Antineoplastic Agents; D027682:Cation Transport Proteins; D003287:Contrast Media; D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D013449:Sulfonamides; D020126:Brefeldin A; D003300:Copper; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D002945:Cisplatin; D019793:Fluorescein; D000077191:Wortmannin