Doxorubicin-gallium-transferrin conjugate overcomes multidrug resistance: evidence for drug accumulation in the nucleus of drug resistant MCF-7/ADR cells.

ABSTRACT

Development of multidrug resistance (MDR) in cancer cells decreases net doxorubicin (ADR) uptake as a result of increased efflux, increased intracellular sequestration, and decreased membrane permeability. In this study, we investigated whether conjugation of ADR to transferrin (Tf) could overcome MDR in breast cancer cells. The multidrug resistant MCF-7/ADR breast cancer cell line was over 1000-fold more resistant to ADR, than its parental MCF-7 cell line, as determined by 3[H]-thymidine assay. The MCF-7/ADR cell line also expressed both MDR1 and MRP genes, as detected by RT-PCR. The ADR was coupled using a glutaraldehyde technique to human transferrin saturated with either ferric chloride (Fe-Tf) or gallium nitrate (Ga-Tf). These conjugates were tested for cytotoxicity on both MCF-7 and MCF-7/ADR cells after 6 days of incubation. The doxorubicin-gallium-transferrin conjugate (ADR-Ga-Tf) exhibited approximately the same inhibitory effect as ADR on MCF-7 cells with IC50s of 2.34 x 10(-3) microM and 1.42 x 10(-3) microM, respectively. However in MCF-7/ADR cells ADR-Ga-Tf reversed resistance to free ADR and decreased 100-fold the IC50 from 8.98 microM with free ADR to 9.52 x 10(-2) microM. ADR-Fe-Tf was 10-fold more inhibitory to MCF-7/ADR cells than free ADR. Compared to Ga-Tf, ADR-Ga-Tf was 500- and 3000-fold more inhibitory to MCF-7 and MCF-7/ADR cells, respectively. These results demonstrated that ADR-Ga-Tf reverses resistance to free ADR and Ga-Tf in MCF-7/ADR cells. The distribution of ADR in both cell lines was examined by fluorescence microscopy. It was noted that ADR mainly accumulated in the cytoplasm around the nucleus in MCF-7/ADR cells, but in both the cytoplasm and nucleus of MCF-7 cells. However the conjugate of ADR-Ga-Tf allowed ADR to accumulate in the cytoplasm and nucleus of both the MCF-7/ADR and MCF-7 cells. Further investigation of MDR1 and MRP genes expression by RT-PCR demonstrated that Ga-Tf decreased expression of the MRP more than the MDR1 gene. Therefore the reversal of resistance to ADR by the ADR-Ga-Tf conjugate is mediated by the transferrin receptor transmembrane transport mechanism, redistribution of ADR into the nucleus of ADR resistant MCF-7/ADR cells and inhibition of MRP gene expression.