Abstract
Background: Multidrug resistance (MDR) is frequently associated with expression of anti-apoptotic proteins of the Bcl-2 family and/or ATP-binding cassette (ABC) transporter proteins. We previously showed that the peripheral benzodiazepine receptor (pBR) ligand, PK11195, promotes mitochondrial apoptosis and blocks P-glycoprotein (Pgp)-mediated drug efflux, establishing PK11195 as a promising MDR reversal agent. We have now assessed its effect on other ABC transporters, namely multidrug resistance protein (MRP) and breast cancer resistance protein (BCRP), and have explored the mechanism by which PK11195 blocks drug efflux, focusing on Pgp as a paradigm.
Methods: Flow cytometry was used to measure efflux of the cytotoxic drug, mitoxantrone (MIT), that was inhibited by cyclosporine A (CSA; to assess Pgp function), MK-571 (to assess MRP function), or Ko143 or GF120918 (to assess BCRP function). MIT-induced toxicity was determined after 24 hours by flow cytometry with DiOC6(3) plus propidium iodide staining, or by 3H-thymidine incorporation. Hematopoietic cell lines were virally transduced with human pBR to assess the effect of pBR expression on PK11195 action. Specific 3H-PK11195 binding was determined by competition with 1000-fold excess unlabeled PK11195. Microsome and plasma membranes were prepared using sucrose gradients. ATPase activity was measured as the liberation of inorganic phosphate detected by absorbance at 800nM. Conformation-specific and conformation-insensitive anti-Pgp monoclonal antibodies were used to analyze PK11195 effects on Pgp by flow cytometry.
Results: Using a panel of human acute myeloid leukemia and multiple myeloma cell lines with endogenous or ectopic expression of one or more ABC transporter proteins, and using primary leukemia cell samples, we found that PK11195 broadly inhibits ABC transporter function, affecting not only Pgp but also MRP and BCRP. PK11195 blocked efflux often more effectively than CSA in Pgp+, MRP+ and BCRP+ cells, blocked efflux as well as the MRP modulator, MK-571, in MRP+ cells, and inhibited as well or better than the BCRP modulator, GF120918, in BCRP+ cells. Compared to parental cell lines, sublines over-expressing Pgp, MRP, or BCRP showed relative MIT-resistance in cytotoxicity assays. PK11195 co-treatments significantly increased MIT cytotoxicity in such cell lines expressing relatively high levels of one or more clinically relevant drug efflux protein even when anti-apoptotic proteins were also expressed. Ectopic expression models confirmed that pBR can directly mediate chemosensitizing by PK11195, presumably via mitochondrial activities, but that pBR expression is unnecessary for PK11195-mediated efflux inhibition. PK11195 bound plasma membrane sites in Pgp+ cells, stimulated Pgp-associated ATPase activity, and caused conformational changes of Pgp, suggesting that PK11195 modulates Pgp-mediated efflux by direct transporter interaction(s). PK11195 and CSA bound non-competitively in Pgp+ cells, indicating that PK11195 interacts with Pgp at sites that are distinct from CSA-binding sites.
Discussion: PK11195 promotes chemotherapy-induced apoptosis by a pBR-dependent mitochondrial mechanism, and broadly blocks drug efflux by a pBR-independent, ABC transporter-dependent mechanism. Since PK11195 concentrations that are effective in vitro can be safely achieved in vivo, PK11195 may be a useful clinical chemosensitizer in patients with MDR+ malignancies.
The Peripheral Benzodiazepine Receptor Ligand 1-(2-Chlorophenyl-methylpropyl)-3-isoquinoline-carboxamide Is a Novel Antagonist of Human Constitutive Androstane Receptor