Point of
VIEW
The Great Multidrug-Resistance Paradox Vivien Y. Chen and Gus R. Rosania*
Department of Pharmaceutical Sciences, University of Michigan College of Pharmacy, 428 Church Street, Ann Arbor, Michigan 48109
H
eat-shock proteins (Hsp) are a family of proteins that facilitate cells’ survival when they encounter stressful conditions, ranging from heat to toxic chemicals. One of these proteins, Hsp90, is a molecular chaperone that facilitates the folding of aggregated or otherwise misfolded proteins in the cytosol. Geldanamycin (GM; Figure 1) is a small-molecule natural product that binds to and inhibits Hsp90 in mammalian cells. GM is selectively toxic to certain cancer cells. This suggests that Hsp could serve as a molecular target for candidate anticancer drugs. To elucidate the molecular mechanisms rendering certain cancer cells more (or less) sensitive to Hsp90 inhibition, medicinal chemists are synthesizing and studying even more selective GM derivatives. These compounds are interesting because they could be good chemotherapeutic agents. They are also noteworthy because their chemical structure and mechanism of action may be keys to understanding the reason some cell lines respond differently to some small molecules than to others. The article by Duvvuri et al. (1) on page 309 of this issue of ACS Chemical Biology highlights a relationship between the subcellular transport properties of a family of GM derivatives and their cell-growth inhibitory activity against multidrug-resistant (MDR) cancer cells. These particular MDR cells lack significant expression of drug efflux pumps. They possess highly acidic lysosomes but were derived from a cell line with neutral lysosomes. GM derivatives with weakly basic functional groups are protowww.acschemicalbiology.org
nated and ionized at low pH. The neutral form of the molecule is membranepermeant, but the charged ionic state of the molecule is membrane-impermeant and thus becomes trapped in acidic organelles, such as lysosomes (Figure 2). By identifying MDR clones with acidic lysosomes derived from a drug-sensitive parent cell line with neutral lysosomes, Duvvuri et al. (1) were able to address how pH-dependent ion trapping affects differential sensitivity to GM by relating physicochemical properties of various GM derivatives to their lysosomal sequestration and to their growth-inhibitory activity. A cancer cell can become resistant to anticancer drugs in many ways, and one is to overexpress drug transporters at the plasma membrane to remove drugs from the cell (2). But, do transporters confer drug resistance by preventing drug molecules from accumulating inside the cell? As observed by Duvvuri et al. (1), comparing the total intracellular drug mass in MDR cells often reveals a decrease in intracellular drug content that is not sufficient to explain the resistant phenotype. In other studies looking at the cellular pharmacokinetics of drug-resistant cancer cells, intracellular drug concentrations have been measured and found to be much greater than extracellular concentrations (3, 4). This is the great multidrug-resistance paradox. So, if the amount of drug present in the cell cannot account for how drug efflux pumps affect drug resistance, then what does? As shown by Duvvuri et al. (1), drug-resistant cells that sequester drug in intracellular compartments
A B S T R A C T Much of the attention devoted to the elucidation of multidrug-resistance mechanisms in tumor cells has focused on transmembrane drug transporters and their ability to pump drug molecules from the cytosol to the extracellular medium. However, intracellular drug concentrations often remain high in drug-resistant cells and therefore do not explain how drug pumping at the plasma membrane confers multidrug resistance. Recent work indicates how drug sequestration in cytoplasmic organelles can account for these paradoxical results and how cellular pharmacokinetics may be exploited to target the activity of small molecules to specific cell types.
*To whom correspondence should be addressed. E-mail:
[email protected].
Published online June 16, 2006 10.1021/cb600215q CCC: $33.50 © 2006 by American Chemical Society
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O H3CO
O NH OH
H3C H3CO H3 C
O
CH3
H3CO CH3 OCONH2
Figure 1. Structure of geldanamycin (GM). GM inhibits mammalian Hsp90 and is selectively toxic to certain cancer cells.
are killed by the drug if the sequestration mechanism is perturbed by making lysosomes more alkaline. Intracellular drug sequestration is therefore a plausible explanation for the great multidrug-resistance paradox and could account for the selective cytotoxic activity of certain GM derivatives against specific cancer cell lines (Figure 2). Beyond the observed structure–localization–activity relationship, the pH-dependent partitioning mechanism may be used prospectively to optimize the selectivity of anticancer agents against cancer cells. In the case of GM, the experimental results argue that localization of small molecules can be targeted to the intracellular site of action (the cytosol) by optimizing the pH-dependent octanol/water partition property of the molecules (1). Lipophilicity differences between different ionic states of a drug are good surrogates for the relative rates of transport of those ionic states across cell membranes, with hydrophilic ions being less membrane-permeant than more hydrophobic ions. For GM, a strong case can be made for why neutral molecules are preferable to weakly basic molecules when the goal is to avoid drug sequestration in lysosomes (1). This is also a good case for why molecules with a high octanol/water partition coefficient in their charged state may be less prone to sequestration and resistance than molecules that are more lipophilic when protonated and charged in an acidic microenvironment. 272
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For understanding subcellular transport as a mechanism conferring differential sensitivity to anticancer agents, the role of transmembrane diffusive pathways in relation to active transport mechanisms governing microscopic drug distribution across membranes should be considered. Many studies have focused exclusively on the interaction between molecules and drug efflux pumps to look at how chemical structure links to drug resistance (5). However, in the case of hydrophobic molecules, drug– transporter interactions are less relevant, because hydrophobic molecules can shortcircuit active transport mechanisms by diffusing directly across phospholipid bilayers. Nevertheless, when multidrug resistance cannot be explained by differences in the intracellular concentration of drug, then differences in the molecular interaction between the small molecule and its target, or differences in the biochemical pathways leading to cell death, are invoked. However, when the focus is on the role of intracellular diffusive transport mechanisms in the differential activity of small molecules, attention shifts from how specific functional groups on a molecule affect interaction with specific proteins to the effect of local pH gradients on drug biodistribution. In tumor cells, pH gradients may be present between lysosomes and the cytosol, as well as between the cytosol and the extracellular tumor microenvironment (6, 7). At the lysosomal membrane, ATPcoupled proton transporters maintain the acidity of the lysosomal lumen. Tumors are often anaerobic, so glycolysis contributes directly to the acidification of the extracellular microenviroment (8). If pH is a major determinant of intracellular, microscopic drug distribution between lysosomes and cytosol, then pH could also affect the partitioning of drugs between cytosol and the extracellular microenvironment. For weakly basic, hydrophobic molecules, an acidic extracellular medium would also facilitate drug efflux from tumor cells. Hence, a CHEN AND ROSANIA
molecule’s site of action can be as much of a determinant of drug activity as its mechanism of action. As for the broader relevance of ion trapping and other passive transport mecha-
Figure 2. Mechanism and illustration of drug sequestration in the cytosol. a) The iontrapping mechanism. A molecule contains a functional group that becomes ionized and charged at low pH. The neutral form of the molecule (M) is membrane-permeant, whereas the protonated charged form (MHⴙ) is impermeant. At cytosolic pH, the molecule exists mostly in the M form, which is driven into the lysosomes by its concentration gradient across the lysosomal membrane. In the low-pH environment of the lysosome, the equilibrium distribution of the molecule is shifted to the MHⴙ form, which is unable to diffuse down its concentration gradient to the outside of the lysosome because it is membrane-impermeant. Thus, the trapped ion ends up accumulating at high concentrations in the lysosomes. b) The sequestration of doxorubicin in cytosolic compartments in K562 human erythroleukemic cells was directly imaged using confocal microscopy. Cells are pulsed with doxorubicin (whose intrinsic fluorescence allows direct observation) and then incubated in drugfree media to allow for efflux. Sequestration in cytoplasmic vesicles can be seen by comparing the images taken after 0 and 8 h following the doxorubicin pulse. Asterisks indicate cell nuclei. Cytoplasmic vesicles where doxorubicin is sequestered can be seen as bright dots in the nuclear periphery. www.acschemicalbiology.org
Point of
VIEW nisms, physicochemical properties influencing subcellular drug transport are attracting attention as a way to rationally optimize the activity and specificity of small, bioactive molecules in living cells. In the context of subcellular transport theory, cellular pharmacokineticists are developing computational models for calculating the intracellular distribution properties of small molecules as a function of chemical structure and physicochemical features (9–11). To the extent that many bioactive small molecules are hydrophobic and therefore distribute intracellularly through passive diffusion, the ion-trapping mechanism should be an important determinant of small-molecule activity and specificity for not only GM derivatives but also a wide variety of anticancer agents. To conclude, the great multidrugresistance paradox offers a potentially useful mechanism for targeting small molecules to specific subcellular locations. In the past few years, statistical methods for determining the significance of quantitative structure localization relationships between small molecules and subcellular distribution have been developed (12–14). In addition, it is now possible to measure intracellular drug concentrations in different subcellular compartments after organellar isolation and biochemical analysis (15, 16). With fluorescent molecules, high-throughput, microscopy-based screening instruments allow direct visualization of the relationship between chemical structure and subcellular distribution, both in fixed-endpoint and kinetic experiments, across large collections of compounds. As a result, a truly original and unique conceptual and experimental framework is emerging from these studies, with the site of action being increasingly recognized as a determinant of the activity and specificity of small molecules in living cells. www.acschemicalbiology.org
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16. Chen, V. Y., Posada, M. M., Blazer, L. L., Zhao, T., and Rosania, G. R. (2006) The role of the VPS4a-exosome pathway in the intrinsic egress route of a DNAbinding anticancer drug, Pharmacol. Res., in press.
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