Anthracyclines, proteasome activity and multi-drug-resistance

Abstract

Background: P-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.

Methods: Proteasome 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.

Results: Verapamil, 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.

Conclusion: Our 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.

Figure 1

Verapamil is an inhibitor of 26S proteasome function. Incubation of crude extracts of ECV304 cells containing proteasomes with different doses of verapamil (50, 60, 80, 100, 200 μM) inhibited proteolysis of the chymotrypsin-like substrate SucLLVY-AMC in a dose-dependent manner, indicating inhibition of 26S proteasome function.

Figure 2

Anthracyclines are inhibitors of proteasome function. Incubation of ECV304 cells stably transfected with an Ub-GFP fusion protein with daunorubicin (100 μM, 16 h), caused accumulation of GFP throughout the cytoplasm (lower picture), indicating proteasome inhibition in living cells while untreated controls cells showed only little accumulation of GFP (A/B). Daunorubicin accumulated in the perinuclear region (C).

Figure 3

MG-132 treatment of KB 8-5 causes intracellular accumulation of anthracyclines. Incubation of KB 8-5 cells, which overexpress P-gp, with increasing doses of MG-132 (0, 6.25, 12.5, 25, 50 μM) caused a dose-dependent accumulation of daunorubicin, as measured by fluorescence, indicating inhibition of P-gp function by this proteasome inhibitor.

Figure 4

Accumulation of doxorubicin in the presence or absence of MG-132 (25 μM) in the cytoplasm and the nuclear fraction of ECV304 cells.