Genetic Analysis of the Multidrug Resistance Phenotype in Tumor Cells

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Resistance to chemotherapy occurs in cancer cells because of intrinsic or acquired changes in expression of specific proteins. We have studied resistance to natural product chemotherapeutic agents such as doxorubicin, Vinca alkaloids, and taxol and more recently, histone deacetylase inhibitors and targeted kinase inhibitors. In most cases, cells become simultaneously resistant to multiple drugs because of reductions in intracellular drug concentrations. For the natural product drugs, this cross-resistance is frequently due to expression of an energy-dependent drug efflux system (ABC transporter) known as P-glycoprotein (P-gp), the product of the MDR1 or ABCB1 gene, or to other members of the ABC transporter family, including ABCG2 and ABCB5. In collaboration with the group of Suresh Ambudkar, we have examined the basis of directional transport of compounds out of cells by P-glycoprotein. These studies have revealed a set of amino acid residues in the transmembrane regions of P-glycoprotein which can be altered to change the direction of transport of certain rhodamine compounds from out of the cell to into the cell. This process is concentration- and ATP-dependent, and gives important insight into how directionality of transport is determined in P-glycoprotein. Further studies are underway in the Ambudkar laboratory to clarify the role of specific residues and the mechanism by which the direction of transport is reversed. In collaboration with Jean-Pierre Gillet (University of Nemours, Belgium), we have further explored the role of ABCB5 in cancer drug resistance, in the malignant phenotype in melanoma (where it is frequently mutated), and as a partner as a heterodimer with other ABC B-type transporters. In pigmented cells that express ABCB5 there are both full-length and partial transcripts generated from different promoters. The partial transcripts encode a half-transporter which appears to be able to dimerize with other half transporters (notably ABCB6 and ABCB9). The localization and potential function of these heterodimeric transporters is under study. Ferroptosis is a non-apoptotic form of cell death caused by lethal lipid peroxidation. Several small molecule ferroptosis inducers (FINs) have been reported, yet little information is available regarding their interaction with the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp, ABCB1) and ABCG2. We thus sought to characterize the interactions of FINS with P-gp and ABCG2 which may provide information regarding oral bioavailability and brain penetration and predict drug-drug interactions. P-gp overexpression conferred resistance to FIN56 and the erastin derivatives imidazole ketone erastin and piperazine erastin. P-gp-mediated resistance to imidazole ketone erastin and piperazine erastin was also reversed in UO-31 renal cancer cells by CRISPR-mediated knockout of ABCB1. The FINs ML-162, GPX inhibitor 26a, and PACMA31 at 10 micromolar were able to increase intracellular rhodamine 123 fluorescence over 10-fold in P-gp-expressing MDR-19 cells. GPX inhibitor 26a was able to increase intracellular purpurin-18 fluorescence over 4-fold in ABCG2-expressing R-5 cells. We conclude that expression of P-gp may reduce the efficacy of these FINs in cancers that express the transporter and may prevent access to sanctuary sites such as the brain. The ability of some FINs to inhibit P-gp and ABCG2 suggests potential drug-drug interactions.