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.

Inhibitors of vacuolar H+-ATPase impair the preferential accumulation of daunomycin in lysosomes and reverse the resistance to anthracyclines in drug-resistant renal epithelial cells

“…Although several authors have reported that anthracyclines can be detected in other acidic compartments, such as the trans-Golgi network or recycling endosomes [7,9,11], all of the fluorescent drugs tested have always been found solely in the more acidic lysosomal compartment of the various MDR cell lines analysed [7][8][9][10][11]. It has been suggested that the intracellular trapping of drugs in the expanded acidic organelle compartments of drug-resistant cells [10,26], plus the active drug export mediated by membranous ATP-dependent P-gps or MRPs, contributes to the reduced accumulation of drugs in MDR cells. Altan et al [11] have reported that lysosomes and recycling endosomes are not acidified in drug-sensitive MCF-7 breast tumour cells, and that the cytosol of drug-sensitive MCF-7 cells is more acidic than the cytosol of their drug-resistant MCF-7\ADR counterparts [11].…”

“…lysosomes and recycling endosomes) from drug-resistant MCF-7\ADR breast cancer cells, but not in those from their drug-sensitive MCF-7 counterparts exhibiting a vesicular acidification defect [11]. These authors and others have also shown that disruption of the pH gradient between the cytoplasm and the acidic organelles by a variety of lysosomotropic agents leads to the release of chemotherapeutic drugs accumulated in the acidic organelles from drug-resistant cells [10,11]. This intravesicular relocation of anthracyclines can obviously be expected to impair their cytotoxic action, which involves nuclear effects, including binding of DNA and interference with topoisomerase II [13].…”

“…As a consequence, the intracellular concentration and distribution of drugs may differ considerably in drug-sensitive and drug-resistant cells. Chemotherapeutic drugs such as the anthracyclines are distributed mainly in the cytoplasm and nucleus of drug-sensitive cells, but can accumulate preferentially in the acidic organelles of a variety of drug-resistant cancer cells [6][7][8][9][10][11]. Schindler et al [12] have proposed a ' protonation, sequestration and secretion (PSS) ' model that could account for the relative sensitivity of tumour cells to anthracyclines.…”

“…Briefly, cells were incubated sequentially with or without 20 nM concanamycin A (CCM A) for 24 h and with 50 µM daunomycin for 2 h at 37 mC. After rinsing, the cells were resuspended in a lysis buffer containing 0.3 % Nonidet P40 and incubated for 5 min at 4 mC as described in [10]. Pelleted nuclei resuspended in 400 µl of PBS were fixed using IntraPrep TM fixation and permeabilization reagents (Immunotech, Marseilles, France).…”

Fluorescence lifetime‐resolved pH imaging of living cells

“…In some mammalian cells, the morphology and transportation activities of the cellular acidic vesicles are strongly dependent on the pH gradients across the vesicular membranes (5–7). Several studies on multidrug‐resistant cells also implicate the correlation between intracellular pH gradients and cellular activity in expelling chemotherapeutic drugs to the endocytic vesicles (8–10). Moreover, neuronal disorders such as seizure and cytotoxic edema are reported to connect to pathologic changes of the pH in glial cells (11, 12).…”