Drug sequestration in cytoplasmic organelles does not contribute to the diminished sensitivity of anthracyclines in multidrug resistant K562 cells

Abstract

Cells that acquire multidrug resistance (MDR) are characterized by a decreased accumulation of a variety of drugs. In addition, sequestration of drugs in intracellular vesicles has often been associated with MDR. However, the nature and role of intracellular vesicles in MDR are unclear. We addressed the relationship between MDR and vesicular anthracycline accumulation in the erythroleukemia cell line K562 and a drug-resistant counterpart K562/ADR that overexpresses P-glycoprotein. We used four anthracyclines (all of which are P-glycoprotein substrates): daunorubicin and idarubicin, which have good affinity for DNA and as weak bases can accumulate inside acidic compartments; hydroxyrubicin, which binds to DNA but is uncharged at physiological or acidic pH and thus cannot accumulate in acidic compartments; and WP900, an enantiomer of daunorubicin, which is a weak DNA binder but has the same pK a and lipophilicity as daunorubicin. The intrinsic fluorescence of anthracyclines allowed us to use macroand micro-spectrofluorescence, flow cytometry, and confocal microscopy to characterize their nuclear or intravesicular accumulation in living cells. We found that vesicular accumulation of daunorubicin, WP900 and idarubicin, containing a basic 3 0 -amine was predominantly restricted to lysosomes in both cell lines, that pH regulation of acidic compartments was not defective in human K562 cells, and that vesicular drug accumulation was much more pronounced in the parental tumor cell line than in the multidrug-resistant cells. These results indicate that vesicular anthracycline sequestration does not contribute to the diminished sensitivity to anthracyclines in multidrugresistant K562 cells.Keywords: multidrug resistance; daunorubicin; anthracycline; intravesicular accumulation.Multidrug resistance (MDR) is a form of resistance to anticancer agents derived from natural products such as the anthracyclines, vinca alkaloids, and epipodophyllotoxins. MDR is often characterized by a decrease in the intracellular accumulation of the drug relative to that in wild-type cells caused by an increase in the ATP-dependent efflux of the cytotoxic agent through the cellular plasma membrane. Two plasma membrane drug transporter proteins, P-glycoprotein (P-gp) and the MDR-associated protein MRP 1 , have been cloned and shown by transfection to induce the MDR phenotype in tumor cells [1 -5]. Both proteins create a concentration gradient of the anthracycline daunorubicin (DNR) across the cellular plasma membrane such that the intracellular drug concentration is lower than the extracellular concentration [6 -9].Changes in drug accumulation alone cannot fully account for anthracycline resistance in many multidrug-resistant cells and other mechanisms may contribute to the MDR phenotype. Drug sequestration in cytoplasmic organelles has been described for several resistant cells in vitro and in vivo [10][11][12][13][14]. Vesicular drug sequestration has been associated with the over expression of P-gp [10-13,15] or the MRP or lung-resistance-rela...

Identification of Key Structural Characteristics of Schisandra chinensis Lignans Involved in P-Glycoprotein Inhibition

“…Only a few reports have been made on the comparison of the P-gp inhibitory activity of enantiomers. For example, enantiomers of verapamil, omeprazole, and propranolol displayed equal affinities to P-gp, suggesting that their recognition of P-gp is nonchiral. − On the other hand, stereoselective P-gp inhibition by mefloquine was observed in rat cells but not in human cells. The (+)-stereoisomer of mefloquine was up to 8-fold more effective than its antipode in increasing cellular accumulation of [ 3 H]vinblastine in GPNT (rat brain capillary endothelial) cells, while in Caco-2 (human colon carcinoma) cells, both enantiomers were equally effective .…”

Acute Doxorubicin Insult in the Mouse Ovary Is Cell- and Follicle-Type Dependent

“…It is unclear whether these structures play a role in the apparent sensitivity of granulosa cells to DXR-induced DNA damage or are in fact a defense mechanism. One frequently observed feature of multidrug resistant cancer cells is compartmentalization of DXR and other chemotherapy agents into acidic intracellular compartments [31], [35], [61], [63], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82], [83], [84], [85], [86], [87], [88], [89], [90], [91], [92], [93], [94], [95], [96], [97]; this compartmentalization is directly linked to limiting nuclear accumulation of DXR and therefore decreasing chemotherapy toxicity. Exceptions exist, however, such as the uterine drug-sensitive MES-SA cell line which accumulates DXR in lysosomes where the drug-resistant MES-SA/Dx5 cell line does not [76].…”

Efflux Kinetics and Intracellular Distribution of Daunorubicin Are Not Affected by Major Vault Protein/Lung Resistance-Related Protein (Vault) Expression

“…The sequestration of anthracyclines in cytoplasmic structures/vesicles has been described in several drug-resistant cell lines and has been associated with the expression of P-gp, MRP1, and/or MVP (32)(33)(34)(35)(36)(37)(38)(39)(40). However, much is still unclear about the sequestration process and its role in MDR.…”