Curcumin modulates efflux mediated by yeast ABC multidrug transporters and is synergistic with antifungals

Abstract

Curcumin (CUR), a natural product of turmeric, from rhizomes of Curcuma longa, is a known agent of reversal of drug resistance phenotypes in cancer cells overexpressing ATP-binding cassette (ABC) transporters, viz., ABCB1, ABCG2, and ABCC1. In the present study, we evaluated whether CUR could also modulate multidrug transporters of yeasts that belong either to the ABC family or to the major facilitator superfamily (MFS). The effect of CUR on multidrug transporter proteins was demonstrated by examining rhodamine 6G (R6G) efflux in Saccharomyces cerevisiae cells overexpressing the Candida albicans ABC transporters Cdr1p and Cdr2p (CaCdr1p and CaCdr2p, respectively) and the MFS transporters CaMdr1p and S. cerevisiae Pdr5p. CUR decreased the extracellular concentration of R6G in ABC transporter-expressing cells but had no effect on methotrexate efflux mediated through the MFS transporter CaMdr1p. CUR competitively inhibited R6G efflux and the photolabeling of CaCdr1p by [(125)I]iodoarylazidoprazosin, a drug analogue of the substrate prazosin (50% inhibitory concentration, 14.2 microM). Notably, the mutant variants of CaCdr1p that displayed abrogated efflux of R6G also showed reduced modulation by CUR. Drug susceptibility testing of ABC protein-expressing cells by spot assays and checkerboard tests revealed that CUR was selectively synergistic with drug substrates such as R6G, ketoconazole, itraconazole, and miconazole but not with fluconazole, voriconazole, anisomycin, cycloheximide, or FK520. Taken together, our results provide the first evidence that CUR modulates only ABC multidrug transporters and could be exploited in combination with certain conventional antifungal drugs to reverse multidrug resistance in Candida cells.

FIG. 1.

Effects of CUR on efflux of substrates in yeast cells. (A) Extracellular R6G concentrations in S. cerevisiae control cells (AD1-8u⁻) (diamonds) and in cells overexpressing CaCdr1p (AD-CDR1) (squares), incubated either with R6G (10 μM) alone (filled symbols) or with R6G plus CUR (100 μM) (open symbols). Filled triangles represent AD-CaMDR1 cells. Energy-dependent R6G efflux was initiated by adding 2% glucose (arrow) and was quantified by measuring the absorbance of the supernatant at 527 nm. Values are means and standard deviations (error bars) for three independent experiments. (B) [³H]MTX accumulation in S. cerevisiae control cells (AD1-8u⁻) and in cells overexpressing CaMdr1p (AD-CaMDR1). Cells were incubated either with [³H]MTX (25 μM; specific activity, 8.60 Ci/mmol) alone (shaded bars) or with [³H]MTX plus CUR (100 μM) (open bars). The solid black bar represents AD-CDR1 cells. The accumulated [³H]MTX was measured, 40 min after the initiation of efflux, using a liquid scintillation counter (Beckman). Values are means ± standard deviations (error bars) for three independent experiments. (C) [³H]FLC accumulation in S. cerevisiae control cells and in cells overexpressing CaCdr1p. Cells were incubated with either [³H]FLC (100 nM; specific activity, 19 Ci/mmol) alone (filled bars) or [³H]FLC plus CUR (100 μM) (open bars). The accumulated [³H]FLC was measured 40 min after the addition of glucose (2%). Values are means ± standard deviations (error bars) for three independent experiments. (D) Extracellular R6G concentrations in C. albicans strain CAI4. Cells were incubated either with R6G (10 μM) alone (filled triangles) or with R6G plus CUR (100 μM) (open triangles). Energy-dependent R6G efflux was initiated by adding 2% glucose (arrow) and was quantified by measuring the absorbance of the supernatant at 527 nm. Values are means and standard deviations (error bars) for three independent experiments. (E) Extracellular R6G concentrations in S. cerevisiae control cells (AD1-8u⁻) (diamonds) and in cells overexpressing CaCdr2p (AD-CDR2) (squares) incubated either with R6G (10 μM) alone (filled symbols) or with R6G plus CUR (100 μM) (open symbols). (F) Extracellular R6G concentrations in S. cerevisiae control cells (AD1-8u⁻) (diamonds) and in cells overexpressing ScPdr5p (AD-PDR5) (squares), incubated either with R6G (10 μM) alone (filled symbols) or with R6G plus CUR (100 μM) (open symbols). Energy-dependent R6G efflux was initiated by the addition of 2% glucose (arrows) and was quantified by measuring the absorbance of the supernatant at 527 nm. Values are means and standard deviations (error bars) for three independent experiments.

FIG. 2.

Effect of CUR on the viability of S. cerevisiae cells as determined by an MTT assay. Shown is the percentage of survival among control cells (AD1-8u⁻) (open circles) and among cells overexpressing ABC or MFS transporters: AD-CDR1 (open inverted triangles), AD-CDR2 (open triangles), AD-PDR5 (open diamonds), and AD-CaMDR1 (open squares) cells. The experiments were conducted in triplicate, and the values are means ± standard deviations for three independent experiments.

FIG. 3.

Effects of pure curcuminoids on R6G transport in S. cerevisiae cells overexpressing CaCdr1p. (A) Structures of curcumin I, curcumin II, and curcumin III and competition assays with R6G. CaCdr1p-overexpressing S. cerevisiae cells were incubated either with 10 μM R6G alone or with 10 μM R6G plus curcumin I, II, or III (10 to 100 μM). R6G efflux was monitored 40 min after the addition of glucose (2%). Extracellular R6G was quantified by measuring the absorbance at 527 nm. The data are plotted using GraphPad Prism. Values are means and standard deviations (error bars) for three independent experiments. (B) Structures of the various substrates used.

FIG. 4.

Biochemical analysis of CaCdr1p in the presence of CUR. (A) Lineweaver-Burk plot of CaCdr1p-mediated R6G efflux in the presence of CUR 5 min after the addition of 2% glucose. Filled diamonds, open squares, and filled triangles represent 0, 50, and 100 μM CUR, respectively. The rate of each reaction was calculated as nanomoles of R6G released per minute per 5 × 10⁶ cells. (B) Effect of CUR or R6G on the photoaffinity labeling of CaCdr1p with [¹²⁵I]IAAP. The autoradiogram represents the amounts of [¹²⁵I]IAAP incorporated into CaCdr1p in the presence of the indicated concentrations of CUR or R6G. The graph represents the amounts of [¹²⁵I]IAAP incorporated into CaCdr1p in the presence of the indicated concentrations of CUR. (C) Effect of CUR on the ATPase activity of CaCdr1p. PMs from cells overexpressing CaCdr1p were incubated with or without 100 μM CUR and varying concentrations of ATP (0.5 mM to 7 mM) in the ATPase buffer. The assay was performed essentially as described in Materials and Methods. The data are plotted using GraphPad Prism. (D) Effect of CUR (100 μM) on the expression of CaCdr1p. Western blot analyses were performed with an anti-GFP monoclonal antibody. Equal loading of protein was assessed by using a Coomassie-stained gel.

FIG. 5.

Synergistic effects of CUR on drug resistance. Control (AD1-8u⁻) and CaCdr1p-expressing (AD-CDR1) S. cerevisiae cells were grown overnight on YEPD plates and then resuspended in normal saline to an optical density at 600 nm of 0.1. The following stock solutions of drugs were used: R6G at 1 mg/ml in dimethyl sulfoxide, FLC at 1 mg/ml in water, VORI at 5 mg/ml in water, CYH at 0.1 mg/ml in water, MCZ at 1 mg/ml in methanol, KTC at 1 mg/ml in methanol, ANISO at 1 mg/ml in dimethyl sulfoxide, FK520 at 1 mg/ml in ethanol, MTX at 1 mg/ml in 10 mM Tris-Cl, and CUR at 11 mg/ml in dimethyl sulfoxide. Five microliters of a fivefold serial dilution of each strain was spotted onto YEPD plates as described previously (27) either in the absence (control) (i) or in the presence of antifungals, alone or in combination with CUR (ii through iv). (ii) R6G (0.209 μM), ITC (0.141 μM), KTC (0.037 μM), or MCZ (0.167 μM), alone or in combination with CUR (75.6 μM). (iii) FLC (6.52 μM), ANISO (2.97 μM), CYH (0.28 μM), FK520 (12.6 μM), or VORI (5.72 μM), alone or in combination with CUR (75.6 μM). (iv) MTX (11 μM) or CUR (75.6 μM), alone or in combination.

FIG. 6.

R6G transport in S. cerevisiae cells overexpressing CaCdr1p or its mutant variants. (A and C) Extracellular R6G concentrations (expressed as percentages of those with wild-type CaCdr1p [AD-CDR1 cells]) in S. cerevisiae cells overexpressing CaCdr1p or mutant variants of CaCdr1p, incubated with 10 μM R6G alone. Energy-dependent R6G efflux was initiated by adding 2% glucose and was quantified by measuring the absorbance of the supernatant at 527 nm. (B and D) Percentages of inhibition of R6G efflux by CUR (100 μM), calculated by taking the level of R6G efflux with each mutant CaCdr1p variant in the absence of CUR as 100%. Values are means ± standard deviations (error bars) for three independent experiments.