Optimizing chemotherapy by measuring reversal of P-glycoprotein activity in plasma membrane vesicles

The appearance of multidrug resistance (MDR) of cancer cells is a major obstacle to successful chemotherapy. Several proteins have been identified that pump chemotherapeutic drugs out of cells, thus bringing about MDR. One representative pump is the P-glycoprotein, whose function can be inhibited by blockers (also known as reversers, modulators or chemosensitizers). In clinical application, many of these blockers are often not effective because they become bound to the plasma of the patients. The extent of plasma binding of the blocker varies in different persons and we have developed a 96-well kit to assay such inter-person differences. The assay uses membrane vesicles isolated from a human lymphoblastoid cell line (CEM Col1000). Uptake of rhodamine into the vesicles was measured with different concentrations of the blockers verapamil and XR9576 in presence of human plasma. The reverser XR9576 is nearly 30 times more effective than the classical blocker verapamil, the relevant K_m values ranging from 2.66 to 45 nM for XR 9576 and 0.7 to 5.5 μM for verapamil. An even greater difference between these two drugs, nearly 1000-fold, could be shown also in intact cells by calcein AM uptake experiments.