The inhibition of ABC transporters in cancer multidrug resistance by heterocyclic cyclohexanone analogues of curcumin

Abstract

Twenty-four heterocyclic cyclohexanone analogues of curcumin were investigated for inhibitory effects against the major ATP-binding cassette (ABC) transporters involved in cancer multidrug resistance (MDR). The main purpose was to identify superior inhibitors compared to the chemically and metabolically unstable parent compound, curcumin. As some of these analogues have been reported to show potent anticancer activity, the identification of ‘dual-role’ chemosensitisers with both MDR-reversal and anticancer activity was an exciting possibility. Flow cytometry screening demonstrated that the cyclohexanone analogues inhibited the four ABC transporters (P-gp, BCRP, MRP1 and MRP5) investigated in this study. At 20 1M, two analogues (C10 and RL92) completely inhibited P-gp; four (A12, A13, B11 and RL92) completely inhibited BCRP; and two (A13 and RL92) inhibited both MRP1 and MRP5. These analogues were more potent than, or equally potent to, curcumin, and two, A13 and RL92, were able to inhibit multiple transporters. Cell proliferation assays confirmed that the P-gp and BCRP inhibitors reversed ABC transporter-mediated drug resistance in transporter-overexpressing cell lines, although issues with cytotoxicity prevented resistance reversal effects to be thoroughly examined for MRP1 and MRP5 inhibitors. Evidence from these assays indicates that the analogues were not substrates of the transporters that they inhibited. From both the flow cytometry and resistance reversal assays, it was evident that the analogues were most promising as BCRP inhibitors, and thus, further studies focused on BCRP. Using cell-free membrane vesicles, A12, A13, B11, RL92 were found to potently (sub-1M IC50) and directly inhibit BCRP in a non-substrate-specific manner, and without requiring bioactivation. These analogues did not appear to alter cellular BCRP protein expression although A13 and RL92 rapidly decreased cell-surface expression of the transporter, but only at high concentrations (20 1M). From these results, it is evident that the cyclohexanone analogues can be considered as alternatives to curcumin as ABC transporter inhibitors. These analogues are particularly promising as inhibitors of BCRP, due to their potent, non-cell-line or substrate-specific inhibition of this transporter. The demonstration of potent and broad ABC transporter inhibition by some analogues such as A13 was particularly encouraging as this compound was reported to have anticancer activity superior to curcumin in a number of different cancer cell lines. Thus A13 should be further investigated as a dual-role chemosensitiser. Useful structure-activity inhibitory data was also gained from this study which, when considered along with cytotoxicity-screening data, enables the proposal of a new cyclohexanone compound, F14, with hopefully superior ABC transporter inhibition and cytotoxic activity than A13.