Vesicular Anthracycline Accumulation in Doxorubicin-Selected U-937 Cells: Participation of Lysosomes

Abstract

The U-A10 cell line, a doxorubicin-selected variant of human U-937 myeloid leukemia cells, exhibits a redistribution of anthracyclines into a expanded vesicular compartment. The acidic nature of this compartment was confirmed by vital staining with a pH sensitive dye, LysoSensor yellow/blue DND-160. Identification of the vesicular compartment was performed by immunofluorescence analysis. Staining for the LAMP-1 and LAMP-2 antigens showed that the vesicles are enlarged lysosomes that are eccentrically placed near the nucleus of U-A10 cells. By contrast, the expression of the multidrug resistance-associated protein and the P-glycoprotein was observed predominately on the plasma membrane of the drug-resistant cells. The accumulation of daunorubicin into cellular compartments was quantified using radiolabeled drug. Exposing cells to 3[H]-daunorubicin and then isolating intact nuclei showed that nuclei from U-A10 cells accumulated twofold to threefold less anthracycline than nuclei from U-937 cells. However, when nuclei were isolated first and then exposed to 3[H]-daunorubicin, little difference in net nuclear drug accumulation was detected. Cytoplasts prepared from U-A10 and U-937 cells were exposed to 3[H]-daunorubicin to measure cytoplasmic drug accumulation. At external daunorubicin concentrations of 100 ng/mL or higher, cytoplasts from U-A10 cells accumulated significantly more daunorubicin than cytoplasts from U-937 cells. Moreover, studies with the lysosomotropic agent chloroquine showed that U-A10 cells accumulated twofold more chloroquine and showed twofold enhanced sensitivity to this agent as compared with parental U-937 cells. Fluorescence microscopy showed that chloroquine affects vesicular anthracycline sequestration in U-A10 cells with an associated increase in daunorubicin nuclear fluorescence. Although chloroquine did not alter anthracycline cytotoxicity in parental cells, it restored daunorubicin and doxorubicin sensitivity to U-A10 cells. Taken together, these studies demonstrate that U-A10 cells exhibit a redistribution of the lysosomal compartment. The trapping of drug into an expanded acidic vesicular compartment results in decreased nuclear drug accumulation and decreased cytotoxicity. Lysosomotropic agents, such as chloroquine, warrant further study as modulators of this acquired drug-resistance phenotype.

Evaluating the Roles of Autophagy and Lysosomal Trafficking Defects in Intracellular Distribution-Based Drug-Drug Interactions Involving Lysosomes

“…9,10 In addition, some cancer cells have been shown to acquire an enhanced capacity to sequester anticancer drugs in their lysosomes, and this is considered to be a mechanism for multidrug resistance. [11][12][13] Drug accumulation in lysosomes also has potential toxicological ramifications. The extensive lysosomal accumulation of a number of cationic amphiphilic drugs has been associated with the development of drug-induced phospholipidosis; the long-term consequences of this, if any, are controversial and not completely understood.…”

Cepharanthine potently enhances the sensitivity of anticancer agents in K562 cells

“…Cellular pH in tumor-derived cells was measured in several studies. The vesicles in which drugs accumulated have been identified as lysosomes, (3,4) endosomes, (5) and vesicles of the Golgi compartment, (6,7) all of which are acidic organelles. Many anticancer agents, such as doxorubicin (ADM), daunorubin, vincristine (VCR), and mitoxantrone, are weak bases with pK a values between 7.4 and 8.4.…”

“…(10) Agents that disrupt organelle acidification should increase drug sensitivity. Treatments of resistant cells with reagents that are known to increase the pH of endomembrane compartments block the vesicular accumulation of chemotherapeutic drugs (4,5) and increase the cytotoxicity of anticancer agents.…”

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

“…Logically, any weakly basic compound will tend to accumulate in any membrane-bound compartment that has a lower internal pH than external pH. Previous studies have indicated that drugs accumulate in Golgiderived vesicles [13,31,32], endosomes [13] and lysosomes [13,33,34]: all of which are more acidic than the cytosol. The cytosol of most cells has a pH of 7.2-7.3, by contrast, the lumena of recycling endosomes and trans-Golgi network have a pH of 6.0-6.3, and the pH of lysosomes can be lower than 4.5.…”