Abstract
BackgroundP-glycoprotein is responsible for the ATP-dependent export of certain structurally unrelated compounds including many chemotherapeutic drugs. Amplification of P-glycoprotein activity can result in multi-drug resistance and is a common cause of chemotherapy treatment failure. Therefore, there is an ongoing search for inhibitors of P-glycoprotein. Observations that cyclosporin A, and certain other substances, inhibit both the proteasome and P-glycoprotein led us to investigate whether anthracyclines, well known substrates of P-gp, also inhibit the function of the proteasome.MethodsProteasome function was measured in cell lysates from ECV304 cells incubated with different doses of verapamil, doxorubicin, daunorubicin, idarubicin, epirubicin, topotecan, mitomycin C, and gemcitabine using a fluorogenic peptide assay. Proteasome function in living cells was monitored using ECV304 cells stably transfected with the gene for an ubiquitin/green fluorescent protein fusion protein. The ability of the proteasome inhibitor MG-132 to affect P-glycoprotein function was monitored by fluorescence due to accumulation of daunorubicin in P-glycoprotein overexpressing KB 8-5 cells.ResultsVerapamil, daunorubicin, doxorubicin, idarubicin, and epirubicin inhibited 26S chymotrypsin-like function in ECV304 extracts in a dose-dependent fashion. With the exception of daunorubicin, 20S proteasome function was also suppressed. The proteasome inhibitor MG-132 caused a dose-dependent accumulation of daunorubicin in KB 8-5 cells that overexpress P-glycoprotein, suggesting that it blocked P-glycoprotein function.ConclusionOur data indicate that anthracyclines inhibit the 26S proteasome as well as P-glycoprotein. Use of inhibitors of either pathway in cancer therapy should take this into consideration and perhaps use it to advantage, for example during chemosensitization by proteasome inhibitors.
Highlights
P-glycoprotein is responsible for the ATP-dependent export of certain structurally unrelated compounds including many chemotherapeutic drugs
Our data indicate that anthracyclines inhibit the 26S proteasome as well as Pglycoprotein
Verapamil is an inhibitor of 20S and 26S proteasome function In order to test the hypothesis that the P-gp inhibitor verapamil inhibits proteasome function, proteasome extracts of ECV304 and PC-3 cells were incubated with different concentrations of the drug (0, 50, 100 and 200 μM) and immediately tested for their chymotrypsin-like activity against the fluorogenic substrate SucLLVY-7-amido-4methylcoumarin
Summary
P-glycoprotein is responsible for the ATP-dependent export of certain structurally unrelated compounds including many chemotherapeutic drugs. Multi-drug-resistance (MDR) is a common reason for chemotherapy treatment failure in breast cancer, leukemia, and non-Hodgkin lymphoma patients. MDR can often be attributed to over-expression of the mdr gene that codes for an ATP-dependent, transmembrane P-glycoprotein (P-gp) efflux pump pathway, which rapidly exports man structurally un-related drugs from the cell, including anthracyclines [1,2]. Insights into substrate cross specificity of P-gp could offer a basis for the development of more selective P-gp inhibitors. They could indicate reasons for the toxicity of these inhibitors, and why they affect cellular functions other than those related to P-gp
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