Investigations of Combined Strategies to reverse P-glycoprotein- and BCRP-mediated Multidrug Resistance in Human Ovarian Cancer Cells and Xenograft Tumors

Purpose : The ATP-binding cassette (ABC) transporters P-glycoprotein (Pgp) and BCRP are implicated in the multidrug resistance (MDR) of many tumors. Pgp-mediated MDR can be functionally inhibited using small molecule inhibitors or transcriptionally downregulated by MDR1 antisense oligodeoxynucleotides (ODNs). Interestingly, simultaneous inhibition of several ABC transporters including Pgp and BCRP by cyclosporin A has been shown to circumvent MDR in clinical trials. In this thesis, the novel multi-targeted tetrahydroisoquinolin-ethyl-phenyl-amine-based MDR inhibitors XR9577, WK-X-34, WK-X-50 and WK-X-84 were thoroughly in vitro and in vivo characterized for interaction with Pgp, BCRP and MRP-transporters and compared to treatments with MDR1 antisense ODNs. Methods : The novel MDR inhibitors and cyclosporin A were examined for cellular toxicity in several cell lines. Inhibition of BCRP-mediated mitoxantrone efflux was studied in BCRP-overexpressing MCF7/mx cells. Inhibition of Pgp function was assessed in 99m Tc-Sestamibi and daunorubicin transport assays. Reversal of Pgp- and BCRP-mediated resistance towards daunorubicin and mitoxantrone, respectively, were investigated in A2780/Adr and MCF7/mx cells. Potential MRP-interactions were evaluated in 5-CFDA efflux assays using selectively transfected MRP-1, -2 and -3 cell lines. Daunorubicin transport and Pgp surface expression in resistant A2780/Adr cells treated with MDR1 antisense ODNs were determined using flow cytometry, fluorescence microscopy and protein staining techniques. The in vivo performance and toxicity of WK-X-34 was evaluated by 99m Tc-Sestamibi imaging experiments using multidrug resistant human ovarian cancer (A2780/Adr) xenograft models. To study antisense treatments in vivo , A2780/Adr xenograft models were dosed intratumorally with MDR1 antisense ODNs for three days followed by either WK-X-34 or vehicle treatment. 99m Tc-Sestamibi distribution and accumulation were analyzed by imaging of the animals and gamma-counting of the isolated tissues. ...