Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using
K-L Habicht1,2, N S Singh1, M A Khadeer1
1Biomedical Research Center, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224, USA.
Abstract:
Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7microM), verapamil (0.6 vs. 0.7microM) and prazosin (0.099 vs. 0.033microM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (-5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences.
Insights
This study developed novel affinity chromatography columns to analyze drug efflux transporters in glioblastoma cells. Researchers found distinct differences in transporter behavior between nuclear and cellular membranes, offering new insights into multi-drug resistance.
Area of Science:
- Biochemistry
- Pharmacology
- Cell Biology
Background:
- Glioblastoma multiforme exhibits aggressive behavior and poor prognosis, largely due to multi-drug resistance.
- ATP-Binding Cassette (ABC) efflux transporters, including Pgp, MRP1, and BCRP, are key contributors to this resistance.
- Understanding the differential expression and function of these transporters in various cellular compartments is crucial.
Purpose of the Study:
- To develop and characterize nuclear and cellular membrane affinity chromatography columns (NMAC and CMAC) using LN-229 glioblastoma cells.
- To compare the binding affinities of known transporter substrates on both column types.
- To investigate potential differences in transporter behavior, particularly BCRP, between nuclear and cellular membranes.
Main Methods:
- Immobilization of nuclear and cellular membrane fragments from LN-229 cells onto an IAM stationary phase.
- Characterization of co-immobilized Pgp, MRP1, and BCRP transporters using established marker ligands.
- Selective transporter analysis via competitive binding assays and saturation of non-targeted transporter sites.
Main Results:
- No significant differences in binding affinities for estrone-3-sulfate, verapamil, and prazosin were observed between NMAC(LN-229) and CMAC(LN-229) columns.
- Selective screening revealed differential allosteric interactions of AZT and rhodamine with BCRP on nuclear versus cellular membranes.
- AZT showed a positive allosteric interaction with BCRP on the CMAC column (+15% retention) but a negative one on the NMAC column (-5% retention).
Conclusions:
- Significant differences exist in the behavior of BCRP expressed in nuclear versus cellular membranes of glioblastoma cells.
- The developed NMAC and CMAC columns are effective tools for probing these subtle differences in transporter function.
- Findings provide a foundation for understanding and potentially overcoming multi-drug resistance in glioblastoma.
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