Separate Roles for the Golgi Apparatus and Lysosomes in the Sequestration of Drugs in the Multidrug-resistant Human Leukemic Cell Line HL-60

Abstract

The sequestration of drugs away from cellular target sites into cytoplasmic organelles of multidrug-resistant (MDR) cancer cells has been recently shown to be a cause for ineffective drug therapy. This process is poorly understood despite the fact that it has been observed in a large number of MDR cancer cell lines. Analysis of drug sequestration in these cells has traditionally been done using fluorescent anthracycline antibiotics (i.e. daunorubicin, doxorubicin). This narrow selection of substrates has resulted in a limited understanding of sequestration mechanisms and the intracellular compartments that are involved. To better characterize this phenotype, we chose to examine the sequestration of molecules having different acid/base properties in the MDR HL-60 human leukemic cell line. Here we show that weakly basic drug daunorubicin is sequestered into lysosomes according to a pH partitioning type mechanism, whereas sulforhodamime 101, a zwitterionic molecule, is sequestered into the Golgi apparatus through a drug transporter-mediated process. Quantitative intracellular pH measurements reveal that the lysosome-tocytosol pH gradient is expanded in the MDR line. Moreover, the MDR cells overexpress the multidrug resistance-related protein (MRP1), which is localized to the Golgi apparatus. These results demonstrate, for the first time, that two distinct mechanisms for intracellular compartmentalization are operational in a single MDR cell line.The resistance of tumor cells to anticancer agents remains a major cause of treatment failure in patients with cancer. MDR 1 is a term used to describe a resistance phenotype in which cells become simultaneously resistant to different drugs with no obvious structural similarities or mechanisms of action (1). The emergence of MDR is multifactorial. Decreased drug accumulation and/or increased efflux, increased detoxification, increased DNA repair, and altered cell cycle regulation have all been implicated (2). Interestingly, many MDR cell lines have demonstrated the capacity to compartmentalize drugs away from intracellular target sites (3). In drug-sensitive cell lines, chemotherapeutic agents are localized to a significant extent within the cell nucleus. In contrast, the MDR cells compartmentalize drug within distinct cytoplasmic organelles. This sequestration serves to protect the MDR cell from the cytotoxic effects of drugs since cellular targets are often associated with the nucleus.There are at least two mechanistic explanations that can be used to rationalize the exaggerated drug sequestration capacity of subcellular compartments contained within MDR cancer cells (3). The first involves members of the ATP binding cassette superfamily of transporter proteins such as P-glycoprotein (P-gp) and the multidrug resistance-related protein 1 (MRP1). The overexpression of these transporter proteins in MDR cell lines is well known, and they are traditionally thought to participate in drug efflux at the plasma membrane (4). Recent evidence suggests that these transporters may als...

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Weak Base Permeability Characteristics Influence the Intracellular Sequestration Site in the Multidrug-resistant Human Leukemic Cell Line HL-60

“…Our laboratory investigated the intracellular drug sequestration pathways associated with the emergence of this drug-resistant phenotype. We have recently shown that selective accumulation of anticancer drugs in cellular organelles of the MDR human leukemic cell line HL-60 can be associated with decreased drug effectiveness (3). Moreover, we also have shown that different organelles are able to sequester drugs with different structural attributes according to independent mechanisms.…”

“…We along with others have shown that certain weakly basic compounds (i.e. daunorubicin, doxorubicin) with pK a values near neutrality are selectively sequestered into lysosomes of MDR cell lines (3,4). Alternatively, other weakly basic compounds, also with pK a near neutrality, specifically accumulate within the mitochondria of cells (i.e.…”

Depletion of major vault protein increases doxorubicin sensitivity and nuclear accumulation and disrupts its sequestration in lysosomes

“…Because anthracyclines, such as doxorubicin, are sequestered in lysosomes (20,24,26,30,31) and this is in turn thought to lead to drug resistance, we hypothesized that doxorubicin is relocalized to lysosomes in the UMUC-3 bladder cancer cell line. To test this, we stained cells with a pH-dependent lysosomal marker before analysis.…”