Multidrug Resistance

We have exploited the optical detection sensitivity and the high resolution of NSOM to detect the cellular localization and effect of ABC proteins associated with MDR [24-26]. Drug resistance can be associated with several cellular mechanisms ranging from reduced drug uptake to reduction of drug sensitivity due to genetic alterations. MDR is therefore a phenomenon that indicates a variety of strategies that cancer cells are able to develop in order to resist the cytotoxic effects of anticancer drugs. Decades of studies demonstrate that there are different ways in which tumor cells can develop resistance. MDR can result from (1) decreased influx of cytotoxic drugs [27], (2) overexpression of drug transporters that belong to the ABC family of proteins including the Pgp, MDR-associated protein (MRP1), and the breast cancer resistance protein 1 (BCRP1), and (3) changes in cellular physiology affecting the structure of the plasma membrane, the cytosolic pH, and the rate and extent of intracellular transport through membranes [28].

In particular, P-gp (a transmembrane glycoprotein of 170 kDa) was the first protein associated with MDR. This protein is strongly homologous to a family of ABC protein membrane transporters, which are capable of translocating drugs and other xenobiotic compounds out of the cell. P-gp is a broad-spectrum multidrug efflux pump that has 12 transmembrane regions and two ATP-binding sites [29]. The transmembrane regions bind hydrophobic drug substrates that are either neutral or positively charged, and are probably presented to the transporter directly from the lipid bilayer. Two ATP hydrolysis events, which do not occur simultaneously, are needed to transport one drug molecule [30]. Binding of substrate to the transmembrane regions stimulates the ATPase activity of P-gp, causing a conformational change that releases substrate to either the outer leaflet of the membrane (from which it can diffuse into the medium) or the extracellular space [31]. Hydrolysis at the second ATP site seems to be required to reset the transporter so that it can bind the substrate again, completing one catalytic cycle.

One of the salient features of P-gp is its broad substrate recognition pattern. Over the past decade the substrate list expanded from the original description of P-gp as conferring resistance to the vinca alkaloids and anthracyclines, to the current very large list of compounds, which includes structurally unrelated anticancer agents, antihuman immunodeficiency virus (HIV) agents, and fluorophores. A classification of the drug interaction with P-gp has been done on four categories: agonists, partial agonist, antagonists, and nonsubstrates [32]. An agonist would be both an ATPase activator and a transport substrate. Some typical examples are the classical substrates of P-gp, that is, the anthracyclines and the vinca alkaloids. A partial agonist would be a molecule that stimulates the P-gp ATPase activity but which does not show any significant transport substrate features. To this group belong verapamil and progesterone, both of which activate P-gp at the catalytic level, but inhibit at the transport level.

An antagonist would inhibit the action of P-gp, and inhibit both at the ATPase and the transport level. An example of a well-known antagonist is vanadate, which inhibits the ATPase activity of P-gp by binding to the catalytic site, thus acting as a noncompetitive inhibitor of transport. Another group of drugs, which inhibit the P-gp-associated ATPase activity are the cyclosporins. Nonsubstrates would simply be drugs that do not appear to interact with P-gp, neither at the ATPase level nor at the transport site. Methotrexate belongs to this group.

For a better understanding of the localization of the MDR proteins and their association with the MDR substrates, a technique that is capable of mapping the distribution of the MDR proteins and monitoring their effect on the localization of therapeutic regimes in cells is required. NSOM, being a highly sensitive and specific technique that offers nanoscale resolution, is the most appropriate technique to determine the localization of the MDR proteins and their substrates in cancer cells. Unlike confocal microscopy, which is suitable for imaging inside a cell, NSOM imaging is capable of simultaneously acquiring fluorescence and topography images thus allowing real space mapping with a subwavelength resolution of cellular components localized mainly on the surface of the cell. We investigated the distribution and the localization of MDR proteins (Pgp) and MDR substrates (doxorubicin and verapamil) in living and fixed malignant rat prostate tumor cells AT3B-1 and MLLB-2 cells developed from the AT-3 and MAT-LyLu cell lines, respectively [33]. Chinese hamster ovarian (CHO) cells that are non-MDR-expressing were used as a control.

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