Intracellular drug sequestration events associated with the emergence of multidrug resistance: a mechanistic review.

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.

A new approach for enhancing differential selectivity of drugs to cancer cells.

The degree to which anticancer agents selectively target cancer cells is a key determinant in successful therapeutic outcomes. Inhibitors of the Hsp90 molecular chaperone represent an important new class of anticancer agents. We propose here a novel mechanism by which physiochemical properties of Hsp90 inhibitors can be optimized to increase selectivity towards cancer cells. The basis for this approach relies on differential intracellular pH gradients that have been shown to exist between normal and transformed cells. Five Hsp90 inhibitors containing basic or neutral properties were evaluated in antiproliferation assays using cells with variable lysosomal pH. Inhibitors with basic functionalities had reduced activity in cells with normal (low) lysosomal pH but showed significantly greater activity in cells with abnormally elevated lysosomal pH (similar to what has been recorded in many types of cancer cells). Conversely, such selectivity enhancement was not observed for neutral inhibitors. The mechanistic basis for the observed selectivity was demonstrated quantitatively by determining the concentration of inhibitors within relevant intracellular compartments. Collectively, these findings suggest that Hsp90 inhibitors with optimal basicity and physicochemical properties have enhanced selectivity toward cancer cells than their neutral counterparts. It is anticipated that these findings may be applicable to other classes of anticancer agents for improvement of differential selectivity.

Niemann-Pick C1 protein facilitates the efflux of the anticancer drug daunorubicin from cells according to a novel vesicle-mediated pathway.

Niemann-Pick C1 (NPC1) is a late endosomal/lysosomal membrane protein originally reported on for its role in cholesterol trafficking in mammalian cells. NPC1 has been shown recently to share significant structural homology with a family of prokaryotic permeases and was proposed to play a role in intracellular drug transport; however, the mechanism for this has not been fully understood. We provide evidence here that is consistent with NPC1's involvement in a vesicle-mediated clearance of the anticancer agent daunorubicin from cells. In experiments with human fibroblasts, we demonstrate that lysosomal efflux of daunorubicin, as well as dextran molecules, are significantly reduced in cells with mutated and dysfunctional NPC1 compared with wild-type fibroblasts. Furthermore, we show that NPC1 is implicated in a lysosomal drug sequestration phenotype exhibited by the multidrug-resistant (MDR) human leukemic HL-60 cancer cell line. Evaluations of cholesterol trafficking, NPC1 mRNA levels, and protein expression are all consistent with a loss of NPC1 activity that is associated with the emergence of the MDR phenotype in this cell line. Collectively, this work proposes a novel role for NPC1 in a vesicle-mediated pathway responsible for the clearance of drugs from cells and provides an explanation for a drug sequestration phenotype exhibited by the MDR HL-60 cell line.

A novel assay reveals that weakly basic model compounds concentrate in lysosomes to an extent greater than pH-partitioning theory would predict.

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 chemical strategy to manipulate the intracellular localization of drugs in resistant cancer cells.

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.

A cell fractionation approach for the quantitative analysis of subcellular drug disposition.

PURPOSE: The purpose of this work was to develop and validate a method that can be used to quantify drugs associated with intracellular compartments. METHODS: The human leukemic cell line U-937 was used to evaluate the distribution of model compounds with known and different subcellular distribution profiles. Lysotracker Red is a lysosomal vital stain and doxorubicin is an anticancer agent with a strong propensity for nuclear accumulation in U-937 cells. After incubation with compounds, cells were separated into fractions containing nuclei, cytosol, and cytoplasmic organelles (lysosomes, mitochondria, Golgi apparatus). Compounds contained within isolated fractions were subsequently extracted and analyzed by high-performance liquid chromatography. Diffusion of compounds from isolated organelles was also investigated. RESULTS: Using this approach we have shown that the model compounds Lysotracker Red and doxorubicin preferentially accumulated within lysosomes and nuclei, respectively. We have reproducibly determined concentrations of these compounds in each of the cellular fractions. We have also shown that diffusion of these compounds from isolated cellular compartments was minimal during the time required to complete the experimental procedure. CONCLUSIONS: The analytical approach described in this manuscript yielded reproducible quantitative data regarding the intracellular distribution of model compounds in U-937 cells. With the aid of a relatively sensitive analytical assay, this technique should be useful for most drugs that have a specific concentrative mechanism for organelle accumulation similar to Dox and LTR.

Weak base permeability characteristics influence the intracellular sequestration site in the multidrug-resistant human leukemic cell line HL-60.

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.

Separate roles for the Golgi apparatus and lysosomes in the sequestration of drugs in the multidrug-resistant human leukemic cell line HL-60.

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.