We previously reported that the folate homeostasis modulates the Breast Cancer Resistance Protein (BCRP) and the Multidrug Resistance Protein 1 (MRP1) expression differently in cell lines from various origins. Of Note, folate deprivation provoked a loss of BCRP and MRP1 function in 2008/MRP1 ovarian and MCF-7/MR breast cancer cells, whereas in Caco-2 colon cancer cells an increased BCRP expression was observed. In order to understand the relevance of these results, we determined the subcellular localization of BCRP in Caco-2 and MCF-7/MR cells, and the expression of BCRP and MRPs in other HF and LF cells: KB, OVCAR-3, IGROV-1, ZR75-1/R/MTX, SCC-11B, SCC-22B and WiDr grown either in standard RPMI medium (2.3 μM supraphysiological folic acid; HF) or adapted to more physiological concentrations (1 to 5 nM folic acid or leucovorin; LF). BCRP and MRPs mRNA levels were determined by Real time PCR.

BCRP was highly expressed in WiDr cells and almost absent in OVCAR-3, IGROV-1 and ZR75-1/R/MTX cells; LF conditions induced 2.4 and 1.6-fold increase in BCRP expression in KB and WiDr LF cells, respectively, and 2.8-fold decrease in SCC-11B LF cells. Similar expression of MRP1 was observed in all cell lines; KB, ZR75-1/R/MTX and SCC-11B LF cells showed a small increase in the expression of this transporter compared with the HF cells. MRP3 was highly expressed in WiDr cells and almost undetectable in ZR75-1/R/MTX; MRP3 expression was 17.2 and 10.8-fold increased in KB and SCC-11B LF cells, respectively, but 15.1-fold decreased in IGROV-1 LF cells. The highest levels of MRP4 were detected in two ovarian cancer cell lines IGROV-1 and OVCAR-3; a 1.9 and 5.1-fold increase in MRP4 expression was found in KB and OVCAR-3 LF cells, respectively, and a 1.7-fold decrease in SCC-11B LF cells. High levels of MRP5 were found in SCC-22B, followed by ZR75-1/R/MTX; a slight increase in MRP5 expression was observed in KB and OVCAR-3 LF cells.

Regarding BCRP localization in Caco-2 cells, immunofluorescent staining observed with a confocal laser scan microscope revealed that in these cells (both HF and LF), BCRP was mainly located in the cytoplasm. In contrast, in MCF-7/MR cells, where folate deprivation induced loss of BCRP expression, BCRP was mainly located in the plasma membrane. These results suggest that, in Caco-2 cells, BCRP is not extruding its substrates (e.g. mitoxantrone and folates) out of the cell, but rather to an intracellular compartment where folates can be kept as a storage.

Collectively, we report here that a mechanism of adaptation to LF conditions involving upregulation of BCRP and/or MRPs is not restricted to Caco-2 cells, but can also be observed in cancer cells from different origins. Whether that overexpression is also associated with an intracellular relocalization of the transporters in those cells needs to be addressed.

Supported by FCT (SFRH/BD/16883/2004).

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA