Visualization and quantification of the reversal of MDR by PSC833 in vitro and in vivo

1. WT cells MDR cells 140 120 100 80 60 40 20 0 Doxorubicin accumulation in MDA-MB-435 cells 5 uM DOX 3ug/ml PSC833 WT MDR MDR Wt cells 100 mdr cells 90 80 70 Fluorescent intensity of Dox 60 50 40 30 * 20 10 0 5 µMMTX 3 µg/ml PSC833 + 5 µMMTX Mitoxantrone accumulation in MDA-MB-435 cells 5 uM MTX 3ug/ml PSC833 WT MDR N= 15 cells/bar Fluorescent intensity of MTX * 5 µM MTX 3 µg/ml PSC833 + 5 µMMTX Visualization and quantification of the reversal of MDR by PSC833 in vitro and in vivo Fei Shen and Leonard C. Erickson Department of Pharmacology & Toxicology and IU Simon cancer center Abstract Multidrug Resistance (MDR) is one of the major reasons for anti-cancer chemotherapy failure. The molecular mechanisms of MDR in cancer cells are involved in the over-expression of ATP-Binding Cassette (ABC) transporters on cell membranes. These transporters mediate the efflux of the structurally and functionally unrelated anti-neoplastic drugs from cells and thereby decrease intracellular drug accumulation. Using confocal microscopy we have conducted in vitro and in vivo studies on the roles of P-glycoprotein (Pgp), one of the ABC transporters, in the uptake and efflux of doxorubicin (DOX) and mitoxantrone (MX), and on the effects of Pgp modulators in MDR transduced human cancer MDA-MB-435 cells (MDR). IC50s of DOX and MX in MDA-MB-435 wild type (WT) cells are 0.60 ± 0.04 µM and 0.16 ± 0.008 µM, respectively. The MDR cells were about 9- and 8-fold more resistant to DOX and MX than the WT cells. Intracellular accumulation of DOX and MX in the MDR cells was only 19% and 33% of that in the WT cells. Lower DOX net uptake in the nuclei and stronger DOX efflux in the cytoplasm were the main reasons for the decreased DOX intracellular accumulation in the MDR cells. Compared to the WT cells, MDR cells had lower net uptake of MX in both nuclei and cytoplasm, which was the major cause of the reduced MX intracellular accumulation in the cells. In MDA-MB-435 tumor xenografts in living mice, the accumulation of DOX and MX in MDR tumors was 68% and 42% of that in WT tumors. Pgp inhibitor, PSC833 increased the accumulation of DOX and MX in MDR cells to 65 % and 85% of that in the WT cells in vitro and reversed the fluorescent intensity of DOX and MX in MDR tumors to 94 % of that in the WT tumors in vivo. Taken together, Pgp causes reduced intracellular accumulation by decreasing drug net uptake and/or increasing drug efflux and the Pgp inhibitor PSC833 reverses the effects of Pgp in cancer cells. In addition, Pgp processes individual anticancer compounds differently, which may be related to the anti-neoplastic mechanisms of these drugs. Florescent intensity of Mitoxantrone and Doxorubicin in MDA-MB-435 Xenograft Tumors Cytoxicity of mitoxantrone and doxorubicin and modulation of drug resistance by PSC833 in human MDA-MB-435 cancer cells *Statistically significant difference (p < 0.05) in fluorescent intensity of doxorubicin in MDA-MB-435wt tumors and MDA-MB-435mdr tumors. • Summary: • Human MDA-MB-435mdr cancer cells were 8- and 9-fold more resistant to mitoxantrone and doxorubicin than the wild type cells. • Accumulation of mitoxantrone and doxorubicin in MDA-MB-435mdr cells were 33% and 19% of that in the wild type cells. • Uptake of mitoxantrone in the cytoplasm and nuclei of MDA-MB-435mdr cells was less and slower than that in the same intracellular compartments of MDA-MB-435wt cells. • Accumulation of mitoxantrone and doxorubicin in MDA-MB-435mdr xenograft tumors were 42% and 68% of that in the wild type xenograft tumors. • Pgp inhibitor, PSC833 increased the accumulation and uptake of mitoxantrone and doxorubicin in MDA-MB-435mdr cells. *Statistically significant difference (P < 0.05) in drug resistance a Resistance factor: IC50 of mitoxantrone and doxorubicin in MDR cells divided by IC50 of same drug in the WT cells.