A number of organelles contained within mammalian cells have been implicated in the selective sequestration of chemical entities including drug molecules. Specifically, weakly basic molecules have been shown to selectively associate with either the mitochondrial compartment or lysosomes; however, the structural basis for this differentiation has not been understood. To investigate this, we have identified a series of seven weakly basic compounds, all with pK a near neutrality, which have different sequestration sites within the multidrug-resistant HL-60 human leukemic cell line. Three of the compounds were selectively sequestered into the mitochondria of the cells, whereas the remainder were predominantly localized within lysosomes. Using specific chemical inhibitors to disrupt either mitochondrial or lysosomal accumulation capacity, we demonstrated that accumulation of these compounds into respective organelles are not competitive processes. Comparison of the permeability characteristics of these compounds as a function of pH revealed striking differences that correlate with the intracellular sequestration site. Only those compounds with significantly reduced permeability in the ionized state relative to the un-ionized state had the capacity to accumulate within lysosomes. Alternatively, those compounds with relatively pH-insensitive permeability selectively accumulated into mitochondria. Using novel quantitative assays for assaying drug accumulation into subcellular organelles, we demonstrated a correlation between these permeability characteristics and the lysosomal versus mitochondrial accumulation capacity of these compounds. Together, these results suggest that the selective accumulations of weakly basic compounds in either lysosomes and mitochondria occur via exclusive pathways governed by a unique permeability parameter.

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...

The acquisition of multi-drug resistance (MDR) in cancer cells subjected to anticancer agents remains a formidable obstacle to successful therapeutic outcomes in cancer patients. As the name implies, the resistance phenotype (MDR) is not typically limited to the drug initially used to eradicate cancer but is often transferred to structurally unrelated chemotherapeutic agents. The mechanisms underlying the development of MDR have been extensively studied and are considered multifactorial. Interestingly, recent observations have shown that altered intracellular distribution of drugs may play an important role in the establishment of the MDR phenotype. Such intracellular redistribution events may reduce the opportunity for a drug molecule to permeate into a drug target-containing compartment and thus limit its therapeutic effect. This review summarizes cases in which intracellular redistribution of drugs has been associated with the emergence of MDR in cancer cells. The review also provides a general overview regarding intracellular compartmentalization mechanisms of drugs in cells, which will include some of the known factors/conditions that influence the accumulation of drugs into specific cellular compartments. Finally, potential strategies for overcoming this resistance phenotype are discussed.

Many weakly basic drugs incubated with cells have been shown to specifically accumulate in lysosomes. The mechanistic basis and substrate specificity for this sequestration have not been rigorously evaluated; however, conditions are favorable for a pH-partitioning type accumulation. In some circumstances, this compartmentalization can be very extensive, which can impact the therapeutic efficacy of a drug. Despite the pharmaceutical importance, direct quantitative assessments of drug accumulation in lysosomes have not been previously described. We report here a novel magnetic capture technique that allows for quick and efficient isolation of lysosomes from cultured HL-60 cells that have been preincubated with model compounds. The amount of compound associated with the isolated fraction is determined by HPLC. Extensive biochemical and morphological characterizations of isolated lysosomes, together with HPLC data, allowed for estimates to be made regarding the concentration of model compounds in lysosomes. The corresponding theoretically determined concentration values, based on pH-partitioning theory, were also calculated for comparison purposes. Interestingly, experimentally determined values were approximately 3-15 times higher than theoretically predicted values. This finding suggests that mechanisms, in addition to pH-partitioning, may play a significant role in the accumulation of drugs in lysosomes.

A number of multidrug-resistant (MDR) cancer cells have been shown to have acquired an increased capacity to sequester weakly basic anticancer drugs in their lysosomes relative to drug-sensitive counterparts. In this report we have comparatively evaluated the concentrations of the anticancer agent daunorubicin (DNR) in intracellular compartments of drug-sensitive and MDR HL-60 cell lines, both of which do not express common efflux transporters such as P-glycoprotein at the plasma membrane. Our results suggest that lysosomal sequestration plays a significant role in the emergence of MDR since it effectively limits the drug's ability to interact with target molecules located in the nucleus. Using a series of weakly basic structural isomers with variable basicity, we illustrate that the magnitude of the pKa value correlates with the degree of lysosomal sequestration. Accordingly, a series of structurally modified forms of DNR with reduced basicity were synthesized, and their intracellular distribution was evaluated. Consistent with model compounds, derivatives of DNR with lowered pKa values showed visibly reduced lysosomal sequestration in two separate MDR cell lines. Collectively, this work highlights the importance of understanding the intracellular localization of drugs and proposes a rational strategy to manipulate it.