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
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(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,
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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.