br It seems that the presence
It seems that the presence of a chlorine Belinostat (PXD101) at the para position of C4-phenyl ring is desirable for BCRP inhibitory activity as D3, bearing 4-chlorophenyl, showed superior inhibition and C3 and C4 containing 4-chlorophenyl and 2,4-dichlorophenyl demonstrated moderate in-hibitory activity on BCRP.
3.4. Measurement of doxorubicin resistance reversal in MES-SA/DX5 cells
Measurement of doxorubicin resistance reversal proved that D3, D5,
D6, D2, C4 and C3 significantly decreased the IC50 of doxorubicin in
Fig. 6. Mitoxantrone efflux inhibition measured in BCRP-overexpressing HEK293 cells. (A) Control HEK293 cells were used for determination of the maximal drug-fluorescence accumulation equivalent of 100% inhibition. An assay was performed with mitoxantrone only, as a negative control, to determine the background fluorescence accumulation in BCRP-expressing cells and also in the presence of Ko143 used as a reference inhibitor with an inhibition percentage of 74% ( ± 14) at 1 μM. (B) BCRP-expressing HEK293 cells were seeded into 96-well microplates and, after overnight incubation, were consecutively treated first with 20 μM test compound, followed by 5 μM mitoxantrone. Compounds displaying good inhibition levels were further tested at lower concentrations. Data are mean ± SD of three independent experiments (*P < .05, **P < .01).
Fig. 7. Measurement of doxorubicin resistance re-versal in MES-SA/DX5 cells. Cells were seeded into 96-well microplates and incubated for 24 h, then treated with two or three different concentrations of compounds followed by doxorubicin addition. Plates were further incubated for 48 h, MTT assay was carried out, IC50 of doxorubicin was calculated in the absence or presence of synthesized compounds and reversal fold was calculated as doxorubicin's IC50/ doxorubicin's IC50 in presence of tested compound. Data are mean ± S.E.M. of 3–5 independent ex-periments. The difference between the IC50 values of doxorubicin in the absence and presence of the test compounds was significant (P ˂ 0.05).
MES-SA-DX5 cells at 5 μM. Results are shown in Fig. 7. These findings confirmed the flow cytometry results to a large extent and admitted the importance of chlorine and nitro substitutions on phenyl ring for P-gp modulatory activity.
3.5. Hexahydroquinoline derivatives as CS agents
Cytotoxicity of synthesized compounds in comparison with ver-apamil (used as a positive control) was evaluated against BHK-21 and MRP1-transfected BHK-21 cells at 20 μM (Fig. 8). CS in MRP1-BHK-21 cells was observed with the D compounds, the best one being D4. Comparing cell viability of BHK-21 and MRP1-BHK-21 cells shows that CS induced by compound D4 is comparable with that of verapamil at 20 μM. Compounds D5 and C5 were toxic for BHK-21 cells (cell viabi-lity ˂ 70%). Other compounds did not show any cytotoxic effect against control or resistant BHK-21 cells. The cytotoxicity of D3, D4, D5 and D6 against MRP1-overexpressing and wild type BHK-21 cells was in-vestigated at various concentrations and CS effects of the compounds were demonstrated in Fig. 9. The Selectivity Ratio (SR), defined as the ratio between IC50 in control and in MRP1-overexpressing cells, were of 1.6, 3.8, 2.7 and 3.2 for compounds D3, D4, D5 and D6, respectively. A SR value > 1 indicates that the compound elicits CS. Conversely, a SR
value < 1 indicates a resistance of the MDR line towards the com-pound, and it is likely for the compound to be effluxed by the ABC transporter responsible for the MDR phenotype (Pluchino et al., 2012). A SR ≥ 2 is generally accepted as significant to define a CS-promoting agent, characterizing D4, D5 and D6 as CS agents. D4 showed a lower SR than verapamil because it became cytotoxic for control BHK-21 at concentrations above 20 μM. Compound D6 is also very interesting since it was efficient at concentrations lower than 10 μM.