PMC

Representative examples of dose–response uptake and efflux analysis in transporter-expressing cells and their parental counterparts via the carbocyanine dyes JC-1 (T3168) and DiOC₂(3) (D14730). (A) Comparison of JC-1 efflux in ABCB1, ABCC1, and ABCG2 transporter-expressing cells (dashed line) as compared with parental cellular fluorescence retention (solid line). (B) Normalized percentage response of each transporter-expressing cell line using parental fluorescence to establish the activity range. Each cell line effectively pumped out all of the T3168 substrate. (C) Comparison of D14730 ABCB1, ABCC1, and ABCG2 transporter-expressing cell line efflux (dashed line) as compared with parental cellular fluorescence retention (solid line). (D) The normalized percentage response readily demonstrates the ABCB1/ABCC1 (solid circles/solid triangles) efflux specificity of this cyanine substrate.

Illustration of curve validation cutoff criteria for efflux inhibition. The curve fit was first validated by excluding curves in which the standard error of the log IC₅₀ was greater than 15% of the value. A positive Hill slope was also required. A baseline fluorescence change also needed to be maintained where bottom to top needed to be at least 50 MCF units. For substrates with lower fluorescence (top between 50 and 500 MCF units), a fold change in fluorescence of 2.5 from top to bottom was required. For substrates with high fluorescence (top >500 MCF units), the fold change was reduced to a 2-fold change to offset possible exclusion of curves with high baselines that still maintained workable fluorescence ranges.

The fluorescence retention of each substrate at 100 nM is plotted for ABCB1, ABCC1, and ABCG2 transporter-expressing cells (black bars, overlay) and their parental counterparts (gray bars). (A) High ABCB1 expression in the CCRF-ADR 5000 cell line facilitated efficient efflux of most membrane-permeable substrates, indicated by the low-level fluorescence in comparison with the parental CCRF-CEM cells. (B) Greater substrate efflux specificity was observed for ABCC1-overexpressing SupT1-Vin cells as compared with ABCB1. (C) ABCG2 overexpression in Ig-MXP3 cells demonstrated the most specific substrate–pump interactions. Many of the responses were at overall lower levels compared with the other two pumps.

The 31 substrates (100 nM) chosen for further study were plotted as percentage response of transporter-expressing cells (black bars) as compared with the parental MCF cellular retention values (gray bars) to determine substrates with both high efflux responses and reasonable fluorescence ranges. (A) High efflux responses were observed for the majority of the substrates in ABCB1 with the exceptions of C2925 and D20350. (B) ABCC1 closely mirrors the efflux activity of ABCB1 with some selectivity observed, such as substrate C2925, which appears not to be an ABCB1 substrate. (C) The efflux profile of ABCG2 is significantly more substrate specific than either of the other two pumps. Fewer than a dozen substrates have responses above 75%. Substrate D20350 appears to be ABCG2 selective at approximately 65% response.

Representative dose–response curves of the efflux inhibition of BODIPY FL histamine (B22461). The concentration of each inhibitor ranged from 7.6 nM to 50 μM. All IC₅₀ values are given as the average of multiple runs (n ≥ 2; subsequent curves not shown). (A) B22461 efflux in ABCB1-overexpressing CCRF-ADR 5000 cells was inhibited by both mometasone (closed squares) and nicardipine (closed circles), with IC₅₀ values of 9.1 ± 8.4 and 3.2 ± 1.0 μM, respectively. Weak nonquantitative inhibition was also noted for pimozide (closed diamonds) and verapamil (closed upward-pointing triangles). (B) The ABCC1 (SupT1-Vin) efflux inhibition profile of B22461 closely mirrored that of ABCB1; however, pimozide and verapamil inhibition levels were within the measured concentration range, affording IC₅₀ values of 11.1 ± 6.4 and 12.4 ± 4.9 μM, respectively. The IC₅₀ value for mometasone was 3.7 ± 1.0 μM, and that for nicardipine was 1.2 ± 0.9 μM. (C) Inhibition of B22461 efflux in ABCG2 (Ig-MXP3) was seen with mometasone, nicardipine, and pimozide, with IC₅₀ values of 4.4 ± 3.4, 4.3 ± 2.3, and 14.2 ± 4.3 μM, respectively. Over multiple runs, a weak inhibition was also noticed with niclosamide (open downward-pointing triangles), albeit with lower than average Hill slopes and relatively high baseline fluorescence.

Heat map of inhibition responses for seven inhibitors against fluorescent substrates in each of the ABC transporter-overexpressing cell lines. Values are represented as IC₅₀ values without error values for the sake of clarity. ABCB1 (CCRF-ADR 5000) demonstrated the highest number of effluxed fluorescent substrates of the three tested transporter pumps. Mometasone and nicardipine inhibition was observed for all of the shown substrates, which included some of the lowest IC₅₀ values. For example, sub-micromolar inhibition of the cyanine DiOC₆(3) (D273) was observed with both mometasone (0.3 ± 0.1 μM) and nicardipine (0.8 ± 0.9 μM). Pimozide and verapamil were also shown to be inhibitors of a majority of the substrates, albeit with higher IC₅₀ values. No activity was seen with niclosamide in ABCB1, and only BODIPY FL EDA (D2390) was inhibited by lasalocid (IC₅₀ = 9.0 ± 6.2 μM). Similar inhibitory activity was seen in ABCC1 (SupT1-Vin) as compared with ABCB1 with the BODIPY, cyanine, and single fluorescein substrates. Of the rhodamine/rosamine substrates shown, only CellTracker Orange CMTMR (C2927), rhodamine B, hexyl ester, perchlorate (R6, R648MP), and tetramethylrosamine chloride (T639) were shown to have inhibitable efflux activity. As in ABCB1, mometasone, nicardipine, pimozide, and verapamil were the common inhibitors. Again, no activity was seen with niclosamide in ABCC1, and only D2390 was inhibited by lasalocid (IC₅₀ = 13.5 ± 2.1 μM). Much less activity was seen in ABCG2 (Ig-MXP3), and except for the three-pump cross-substrate JC-1 (T3168), the inhibition profiles seemed to be more selective. T3168 efflux was inhibited by lasalocid, mometasone,nicardipine, and pimozide in a range of IC₅₀ values. Interestingly, T3168 in ABCG2 was the only substrate efflux inhibited by niclosamide (IC₅₀ = 0.8 ± 0.6 μM). The remaining active substrates were all members of the BODIPY fluorophore subset and included the nicardipine-inhibited three-pump cross-substrates BODIPY FL histamine (B22461), BODIPY FL prazosin (B7433), and BODIPY FL forskolin (B7469). Once again, D2390 was inhibited by lasalocid (IC₅₀ = 4.2 ± 2.4 μM).