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  • br Fig Flow cytometric detection of rhodamine accumulation

    2019-09-21


    Fig. 3. Flow cytometric detection of rhodamine 123 accumulation in MES-SA/DX5 cells. Cells were sus-pended in growth medium and then treated with verapamil (positive control) or synthesized com-pounds (P-gp inhibitors). After incubation for 20 min, 5 μM rhodamine 123 was added for 20 min at 37 °C. Cells were centrifuged, washed and re-suspended in ice-cold PBS. The fluorescence caused by the presence of rhodamine 123 in α-CEHC was measured by a flow cytometer. Effects of synthesized compounds bearing 3-(pyridin-2-yl)ethyl carbox-ylate (C) and 3-(pyridin-2-yl)propyl carboxylate (D) are shown. Data are mean ± S.E.M. of 3–5 in-dependent experiments. Gmean of fluorescence in-tensity in the presence of the test compound was significantly different from control untreated cells (P
    Fig. 4. Flow cytometric detection of rhodamine 123 accumulation in MES-SA cells. Cells were suspended in growth medium and then treated with verapamil (positive control) or synthesized compounds (P-gp inhibitors). After incubation for 20 min, 5 μM rho-damine 123 was added for 20 min at 37 °C. Cells were centrifuged, washed with ice-cold PBS and suspended in PBS. The fluorescence caused by the presence of rhodamine 123 in cells was measured by a flow cytometer. Effects of synthesized compounds bearing pyridin-2-ylethyl carboxylate (C) and pyr-idin-2-ylpropyl carboxylate (D) are shown. Error bars indicate S.E.M. of 3–5 independent experiments. Gmean of fluorescence intensity of untreated control cells was 1798 ± 6.4 (S.E.M).
    Fig. 5. Intracellular calcein fluorescence accumulation measured in MRP1-overexpressing Flp-In HEK293. (A) Control Flp-In HEK293 cells were used for determination of the maximal drug-fluorescence accumulation equivalent of 100% inhibition. An assay was performed with calcein-AM only, as a negative control, to determine the background fluorescence accumulation in MRP1-expressing cells. Verapamil was used as a reference modulator and showed inhibition percentages of 41% ( ± 5) and 82% ( ± 7) at 20 and 35 μM, respectively. (B) MRP1-expressing HEK293-Flp-In™ cells were seeded into 96-well microplates and, after overnight incubation, were consecutively treated first with 20 μM compounds as indicated, followed by 0.2 μM calcein-AM addition. Compounds displaying good inhibition levels were further assayed at lower concentrations. Assays were performed in triplicate. Data are mean ± SD of three independent experiments (*P < .05, **P < .01).
    classified into two groups according to their inhibition power at 10 μM; D5 (68 ± 12%) and D6 (46 ± 7%) bearing 2,3-dichlorophenyl and 3-chlorophenyl, might be classified as effective inhibitors, while C5 (37 ± 14%), D1 (36 ± 2%), C4 (25 ± 1%), C1 (21 ± 3%), and D3 (9 ± 0.4%) containing 2,3-dichlorophenyl, 3-nitrophenyl, 2,4-di-chlorophenyl, 3-nitrophenyl and 4-chlorophenyl moieties, respectively, could be ranked as moderately active inhibitors. Therefore, it might be stated that the lipophilic chlorine atom is substantial for the activity and increasing the number of chlorine atoms on phenyl ring improves the modulatory activity on MRP1 transporter in both C and D com-pounds due to increased lipophilicity, as the inhibition percentage for compounds C4 (bearing 2,4-dichlorophenyl) and C5 (bearing 2,3-di-chlorophenyl) was higher than that of C3 (bearing 4-chlorophenyl). The same result was observed for compound D5 (bearing 2,3-di-chlorophenyl) in comparison with compounds D3 (bearing 4-chlor-ophenyl) and D6 (bearing 3-chlorophenyl).
    3.3.3. Flow cytometric detection of mitoxantrone accumulation in BCRP-overexpressing HEK293
    We measured the accumulation of the fluorescent probe in BCRP-overexpressing HEK293 cells by flow cytometry using mitoxantrone, a fluorescent substrate of BCRP. Results are shown in Fig. 6. Control