Identification and Characterization of Transporters in Human Gliomas

Abstract

Functional overexpression of the ATP binding cassette (ABC) transporters at the cell surface is thought to be responsible for clinical multidrug resistance (MDR) in tumours of the brain. Inhibition of ABC transporters by existing inhibitors has proven to be inconclusive. This research program hypothesized an alternative location for the ABC transporters in glioblastoma cells and also proposed to develop stationary phases for the identification of ABC transporters inhibitors. Expression profile investigation of P-glycoprotein (PGP), multidrug resistant protein 1 (MRP1), multidrug resistant protein 2 (MRP2) and the breast cancer resistant protein (BCRP) in glioblastoma multiforme cell lines and clinical patient specimens suggested varying levels of expression. Localisation studies by confocal microscopy confirmed cell surface expression and also indicated that BCRP was localised at the nucleus of the T98 and LN229 cells. Immunoblots of LN229 nuclear extracts indicated ~ 2 fold higher expression of BCRP as compared to cytoplasmic extracts. Immunohistochemistry studies with clinical samples confirmed the nuclear and perinuclear location of BCRP. IC50 value for Mitoxantrone (MTX); a BCRP substrate was calculated as 0.29 ± 0.020 μM for the LN229 cell line, and pre-treatment with the cell impermeant fumitremorgin C 3 (FTC, 5 μM) slightly reduced the IC50 value to 0.16 ± 0.087 μM. This refractoriness to FTC is in contrast with the literature showing a ~ 6-fold reduction in IC50 value of MTX upon pre-treatment with FTC in human breast cancer MCF-7 cell line with ectopic expression of BCRP. The results supported the notion that the nuclear presence of endogenously expressed BCRP actively extrudes MTX, and that because FTC is not able to inhibit the nuclear BCRP, significant reduction in the IC50 was not observed. The results suggest that the treatment of clinical MDR should be expanded to include inhibition of ABC transporters functioning at the nuclear membrane. Cellular membrane affinity chromatography columns were developed for the study of the MRP1, MRP2 and BCRP using Spodoptera frugiperda (Sf9) cells that had been stably transfected with human Mrp1, Mrp2 or Bcrp cDNA. The resulting columns and a control column were characterized using frontal affinity chromatography using [3H]-etoposide as the marker ligand and etoposide, benzbromarone and MK571 as the displacers on the CMAC(Sf9MRP1) column, etoposide and furosemide on the CMAC(Sf9MRP2) column and etoposide and fumitremorgin C on the CMAC(Sf9BCRP) column. The binding affinities obtained from the chromatographic studies were consistent with the data obtained using non-chromatographic techniques and the results indicate that the immobilized MRP1, MRP2 and BCRP transporters retained their ability to selectively bind known ligands. The results indicated that the CMAC(Sf9MRP1), CMAC(Sf9MRP2) and CMAC(Sf9BCRP) columns can be used for the study of binding to the MRP1, 4 MRP2 and BCRP transporters and that membranes from the Sf9 cell line can be used to prepare CMAC columns. This study expands our knowledge of the ABC transporters and makes a case for the finding that nuclear efflux proteins play a pivotal role in the overall MDR phenotype in CNS tumours. Also the CMAC columns developed and characterised provide a tool to study the binding of potential therapeutic candidates to ABC proteins