1. 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. 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 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 Pre-treatmentMDA-MB -435 cellIC 50 of MTX (µM)RF a IC 50 of DOX (µM)RF a None WT0.2 ± 0.011.00.60 ± 0.041.0 MDR1.6 ± 0.138.0*5.29 ± 0.858.8* PSC833 (3mg/ml) WT0.2 ± 0.041.00.57 ± 0.041.0 MDR 0.2 ± 0.001.03.71 ± 0.476.5* 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 drug resistance a Resistance factor: IC 50 of mitoxantrone and doxorubicin in MDR cells divided by IC 50 of same drug in the WT cells. 5 uM MTX 3ug/ml PSC833 WT MDR 5 µM MTX3 µg/ml PSC833 + 5 µM MTX * 0 20 40 60 80 100 120 140 WT cells MDR cells N= 15 cells/bar Fluorescent intensity of MTX Mitoxantrone accumulation in MDA-MB-435 cells Pretreatment MDA-MB- 435 tumors Fluorescent Intensity of MTX (Mean ± SE) % of WT tumors Fluorescent Intensity of DOX (Mean ± SE) % of WT tumors None WT 91.1 ± 27.3 10075 ± 4.7100 MDR38.0 ± 5.442*51 ± 6.068* PSC833 WT81.2 ± 12.6 100 80 ± 4.6100 MDR75.8 ± 5.3 94 76 ± 6.394 Florescent intensity of Mitoxantrone and Doxorubicin in MDA-MB-435 Xenograft Tumors *Statistically significant difference (p < 0.05) in fluorescent intensity of doxorubicin in MDA-MB-435wt tumors and MDA-MB-435mdr tumors. Summary: 1.Human MDA-MB-435mdr cancer cells were 8- and 9-fold more resistant to mitoxantrone and doxorubicin than the wild type cells. 2.Accumulation of mitoxantrone and doxorubicin in MDA-MB-435mdr cells were 33% and 19% of that in the wild type cells. 3.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. 4.Accumulation of mitoxantrone and doxorubicin in MDA-MB-435mdr xenograft tumors were 42% and 68% of that in the wild type xenograft tumors. 5.Pgp inhibitor, PSC833 increased the accumulation and uptake of mitoxantrone and doxorubicin in MDA-MB-435mdr cells. MDR WT Fluorescent intensity of Dox 5 uM DOX * 3ug/ml PSC833 0 10 20 30 40 50 60 70 80 90 100 Wt cells mdr cells 3 µg/ml PSC833 + 5 µM MTX 5 µM MTX Doxorubicin accumulation in MDA-MB-435 cells MDR