Confocal scanning microspectrofluorometry reveals specific anthracyline accumulation in cytoplasmic organelles of multidrug-resistant cancer cells.
We used confocal microspectrofluorometry to investigate intracellular distribution of pirarubicin or THP-DOX in parental K562, CEM, and LR73 tumor cells and their corresponding multidrug-resistant (MDR) strains. Each spectrum of a recorded image was considered as a combination of cell autofluorescence and fluorescence of the drug. In the cytoplasm of parental K562, CEM, and LR73 cells, THP-DOX fluorescence emission profile was similar to that of free drug in aqueous buffer. The (I550nm/I600nm) ratio was 0. 50 +/- 0.1. However, in the cytoplasm of resistant cells the 550-nm band profile was modified. The I550nm/I600nm ratio was 0.85 +/- 0.2 in MDR K562 cells, which is significantly different from the ratio in sensitive cells (p<0.01). This appeared first to correspond to accumulation and self-oligomerization of THP-DOX in cytoplasmic organelles of MDR cells. Transfection of LR73 cells with the mdr1 gene conferred this characteristic on the resistant LR73R cells. Bodipy-ceramide, a trans-Golgi probe, was co-localized with the typical fluorescence emission peak at 550 nm observed in the cytoplasm of MDR cells. This organelle has been shown to be more acidic in MDR cells. Moreover, this specific pattern was similar to that observed when anthracycline is complexed with sphingomyelin. The typical fluorescence emission peak at 550 nm decreased in MDR cells incubated simultaneously in the presence of the drug and quinine, verapamil, or S9788. Growth inhibitory effect and nuclear accumulation of THP-DOX data obtained on LR73R and LR73D cell lines showed that only during reversion of resistance by verapamil and S9788 was an increase of nuclear THP-DOX accumulation observed. Our data suggest that characteristics of molecular environment, such as higher pH gradient or lipid structures, would be potential mechanisms of multidrug-resistance via the sequestration of anthracyclines.
Belhoussine
R
R
Université de Reims Champagne-Ardenne, UFR de Pharmacie, IFR53, Laboratoire de Spectroscopie Biomoléculaire, Reims, France.
Morjani
H
H
Millot
J M
JM
Sharonov
S
S
Manfait
M
M
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000032:analysis*; D016466:CHO Cells; D002467:Cell Nucleus / Q000737:chemistry / Q000187:drug effects; D006224:Cricetinae; D003593:Cytoplasm / Q000737:chemistry* / Q000187:drug effects; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000032:analysis; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D006056:Golgi Apparatus / Q000737:chemistry; D006801:Humans; D015591:Microspectrophotometry; D010880:Piperidines / Q000494:pharmacology; D011803:Quinine / Q000494:pharmacology; D014227:Triazines / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology
D000903:Antibiotics, Antineoplastic; D010880:Piperidines; D014227:Triazines; C077022:S 9788; D004317:Doxorubicin; D011803:Quinine; D014700:Verapamil; C027260:pirarubicin