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Optimizing chemotherapy by measuring reversal of P-glycoprotein activity in plasma membrane vesicles
Sabine Köhler1, Wilfred D Stein
1Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. wdstein@vms.huji.ac.il
Abstract:
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 microM for verapamil. An even greater difference between these two drugs, nearly 1,000-fold, could be shown also in intact cells by calcein AM uptake experiments.
Insights
Multidrug resistance (MDR) in cancer hinders chemotherapy. A new assay measures how plasma binding affects chemotherapy blockers, revealing XR9576 is significantly more effective than verapamil.
Area of Science:
- Pharmacology
- Cancer Biology
- Biochemistry
Background:
- Multidrug resistance (MDR) in cancer cells is a significant challenge in chemotherapy.
- P-glycoprotein is a key protein responsible for pumping drugs out of cancer cells, causing MDR.
- Existing chemotherapy blockers can be ineffective due to variable plasma binding in patients.
Purpose of the Study:
- To develop and validate a 96-well kit for assaying inter-person differences in plasma binding of chemotherapy blockers.
- To compare the effectiveness of the blocker XR9576 against the classical blocker verapamil in the presence of human plasma.
Main Methods:
- Utilized membrane vesicles from a human lymphoblastoid cell line (CEM Col1000).
- Measured rhodamine uptake into vesicles with varying concentrations of verapamil and XR9576 in human plasma.
- Conducted calcein AM uptake experiments in intact cells to further compare drug efficacy.
Main Results:
- The developed assay effectively measures inter-person differences in blocker plasma binding.
- XR9576 demonstrated significantly higher efficacy compared to verapamil, with K(m) values ranging from 2.66 to 45 nM versus 0.7 to 5.5 µM, respectively.
- In intact cells, XR9576 showed nearly 1,000-fold greater effectiveness than verapamil.
Conclusions:
- The new 96-well kit provides a reliable method for assessing chemotherapy blocker efficacy considering individual plasma binding variations.
- XR9576 represents a more potent modulator of P-glycoprotein compared to verapamil, offering potential advantages in overcoming MDR.
- This research contributes to personalized medicine approaches in cancer chemotherapy by enabling the selection of more effective drug modulators.