Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns

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 LN229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN229)) and (CMAC(LN229)), respectively. Pgp, MRP1and BCRP transporters co-immobilized on both columns was characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs 3.7μM), verapamil (0.6 vs 0.7μM) and prazosin (0.099 vs 0.033μM) 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 8 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(LN229) column and decreased it (−5%) on the NMAC(LN229), 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.

The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

“…Mitochondria were solubilized using the previously described protocol for the synthesis of CMAC and NMAC columns [24,26]. The solubilization buffer was Tris buffer [10 mM, pH 7.4], supplemented with 2% (w/v) CHAPS, 10% glycerol, 500 mM NaCl, 5 mM 2-mercaptoethanol, 100 μM benzamidine, 1:100 dilution of protease inhibitor cocktail, 50 μg/ml TPCK, 100 μM PMSF and 100 μM ATP with 10 ml utilized in the synthesis of the MMAC columns and 3 ml of solubilization in the preparation of the MMAC-OT columns.…”

“…Western blot analysis was performed according to previously reported method [26], which involved equal protein loading of 20 μg/well, separation using SDS-PAGE, blocking in 5% non-fat milk and incubated with the primary antibody, followed by incubation with a secondary antibody conjugated with horseradish peroxidase. The detection of immune-reactive bands was performed by using the ECL Plus Western Blotting Detection System (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK).…”

“…In addition to CMAC column, more recently, the development and characterization of immobilized nuclear membrane fragments from the LN-229 cell line has been carried out to study multiple ATP binding cassette (ABC) transporters, breast cancer resistance protein (BCRP), P-glycoprotein (Pgp) and multidrug resistance protein 1 (MRP1) [26]. Similar to the mitochondria, nuclear membranes are also comprised of an inner and outer membranes.…”

“…Similar to the mitochondria, nuclear membranes are also comprised of an inner and outer membranes. The resulting nuclear membrane affinity chromatography column (NMAC), was characterized and the presence of the three transporters was demonstrated [26,27]. …”

Cellular membrane affinity chromatography (CMAC) in drug discovery from complex natural matrices

“…Since the successful immobilization of nAChRs on the IAM stationary phase, multiple transmembrane proteins have been used to prepare CMACs with IAM.PC (immobilized artificial membrane phosphatidylcholine). The examples of transmembrane proteins immobilized on IAM.PC particles include: ligand-gated ion channels ( x  y nAChR x = 3,4,7; y = 2,3,4; GABA A , NMDA, combination of nAChR, NMDA) [11,13,[15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30], G-protein coupled receptors (opioid, -adrenergic, P2Y1, Histamine 1-4, CB 1 , CB 2 ) [31][32][33][34][35] and drug transporters (ABC: Pgp, MRP1, MRP2, BCRP; SLC: hOCT and hOCT SNPs, hOAT) [36][37][38][39]. IAM.PC mimics the cell membrane environment and keeps the experimental conditions close to the physiological state.…”