br Intracellular GSH depletion in MRP transfected BHK
3.5.2. Intracellular GSH depletion in MRP1-transfected BHK-21 cell lines We measured the intracellular GSH content of both BHK-21 and MRP1-transfected BHK-21 in order to establish if group D compounds act as GSH depletion inducers in MRP1-overexpressing Imatinib (STI571) as we previously described (Trompier et al., 2004; Barattin et al., 2010; Genoux-Bastide et al., 2011; Lorendeau et al., 2014; Dury et al., 2017; Pérès et al., 2017). Intracellular GSH content was measured after 3 h incubation time after which the cellular GSH depletion is maximal, the GSH efflux mediated by MRP1 being rapid and massive after the ad-
dition of the CS agent.
Group D compounds, which selectively killed overexpressing MRP1-cells, indeed induced GSH depletion, as shown Fig. 10, confirming a CS mechanism triggered by GSH efflux, on the contrary to compounds C that did not show significant difference on cell survival between control BHK-21
Fig. 8. Cell viability of control BHK-21 (dark gray) and MRP1-transfected BHK-21 (light gray) cells treated with 20 μM of verapamil or synthesized compounds. Error bars indicate SD of triplicate experiments.
Fig. 9. Cytotoxicity and collateral sensitivity evaluation of D3, D4, D5 and D6 at various concentrations. Cell viability of control BHK-21 (●) and MRP1-transfected BHK-21 (■) cells in the presence of verapamil (A) or synthesized compounds D3 (B), D4 (C), D5 (D) and D6 (E). Cells were seeded into 96-well microplates at a density of 2 × 103 cells/well and incubated overnight, then treated with test compounds at the concentration range of 0.5–100 μM. After 72 h of incubation, MTT assay was performed. Verapamil was used as the positive control. Error bars indicate SD of triplicate experiments.
Fig. 10. Effect of verapamil and synthesized com-pounds on the total intracellular glutathione content. Control BHK-21 (dark gray) and MRP1-transfected BHK-21 (light gray) cells were incubated with ver-apamil or synthesized compounds (D3, D4, D5 and D6) at 1 μM and 20 μM for 3 h. Total intracellular glutathione contents were determined relative to control BHK-21 and MRP1-transfected BHK-21cells in the absence of any compounds. Error bars show SD of triplicate experiments.
and MRP1-transfected BHK-21 cells. Therefore, the length of the linker between pyridine ring and carboxylate moiety seems to be crucial for triggering GSH efflux mediated by MRP1. Fig. 10 shows that a dose-related depletion of intracellular glutathione content in MRP1-BHK-21 was ob-served after exposure to compounds D3, D4, D5 and D6 after 3 h incubation suggesting that a GSH efflux occurred. The percentage of glutathione de-pletion exposed to 20 μM of D3, D4 and D6 was of 36.5%, 66.0% and 65.1%, respectively. The GSH efflux ability of the compounds D4 and D6 were comparable to that of verapamil (60.5%) and significantly higher than that of D3. The GSH efflux induced by these compounds are correlated with their selective cytotoxicity towards MRP1-BHK-21 cells, with compound D4 and D6 exhibiting a higher CS activity than compound D3.
Since no CS was observed for D1 and D2 derivatives, it seems that nitro substitution on the phenyl moiety is not favorable for CS activity, whereas, compounds with mono halogen substituted group on phenyl
ring (compounds D3, D4 and D6) triggered collateral sensitivity. Moreover, mono halogen substituted group at position 3 of phenyl ring (3-flourophenyl of compound D4 and 3-chlorophenyl of compound D6) significantly enhances CS compared to mono halogen substituted group at position 4 of phenyl ring (4-chlorophenyl of compound D3). It is noted that 2,3-dichloro substituted group of compound D5, like for compound C5, induced cytotoxic effect for both parental and MRP1-overexpressing cancer cells. Moreover, mono halogen substituted group at position 3 of phenyl ring increases CS activity for MRP1-overexpressing cells.
When the chemical structure of group D compounds is compared to other CS agents such as verapamil (Trompier et al., 2004) and dimers of flavonoids (Dury et al., 2017), the two hydrophobic parts of the D compounds are separated by the presence of linker which might be necessary for the flexibility of the molecule. Besides, the presence of halogen group resulting in the increasing of hydrophobicity of the CS