The positive inotropic agent DPI-201106 selectively reverses ABCB1-mediated multidrug resistance in cancer cell lines

Abstract

The overexpression of ABCB1 in cancer cells is a major factor contributing to the development of multidrug resistance (MDR) and treatment failure in cancer patients. Therefore, re-sensitization of MDR cancer cells to anticancer drugs remains an important aspect in chemotherapy. The progress in developing clinically applicable synthetic inhibitors of ABCB1 has been slow, mostly due to complications associated with intrinsic toxicities and unforeseen drug-drug interactions. Here, we explored the drug-repositioning approach for cancer therapy by targeting ABCB1-mediated MDR in human cancer cells. We found that DPI-201106, a positive inotropic agent, selectively inhibits the drug efflux function of ABCB1, and in doing so, re-sensitizes ABCB1-overexpressing MDR cancer cells to conventional anticancer drugs. Furthermore, the ATPase activity of ABCB1 and docking analysis of DPI-201106 in the drug-binding pocket of ABCB1 were determined to confirm the interaction between DPI-201106 and ABCB1 protein. In summary, we revealed an additional action and a potential clinical application of DPI-201106 to reverse ABCB1-mediated MDR in human cancer cells, which may be beneficial for cancer patients who have developed multidrug resistance and no longer respond to conventional chemotherapy, and should be further investigated.

Keywords: ABCB1; DPI-201106; Modulator; Multidrug resistance; Sodium channel.

Fig. 1.. DPI-201106 selectively inhibits ABCB1-mediated drug efflux.

The accumulation of fluorescent calcein in HEK293 cells (A and B, left panels), HEK293 cells transfected with human ABCB1 (A, right panel) or human ABCC1 (B, right panel), human KB-3–1 epidermal cancer cells (D, left panel) and ABCB1-overexpressing KB-V-1 cancer cells (D, right panel), as well as in human OVCAR-8 ovarian cancer cells (E, left panel) and ABCB1-overexpressing NCI-ADR/RES cancer cells (E, right panel), or fluorescent PhA in HEK293 cells (C, left panel) and HEK293 cells transfected with human ABCG2 (C, right panel), was measured in the absence (solid lines) or presence of 500 nM DPI-201106 (shaded, solid lines) or 20 μM verapamil, a reference inhibitor for ABCB1 (A, D and E, dotted lines), 25 μM MK-571, a reference inhibitor for ABCC1 (B, dotted lines) or 3 μM Ko143, a reference inhibitor for ABCG2 (C, dotted lines), and analyzed immediately by flow cytometry as described previously [52] Representative histograms of three independent experiments are shown. (F) The concentration-dependent inhibition of ABCB1-mediated calcein-AM efflux by DPI-201106 (empty circles) or verapamil (filled circles) was determined in ABCB1-transfected MDR19-HEK293 cells. Values are presented as mean ± SD calculated from at least three independent experiments.

Fig. 2.. DPI-201106 reverses ABCB1-mediated paclitaxel resistance in ABCB1-overexpressing MDR cell lines.

Drug-sensitive human KB-3–1 epidermal cancer cells (A, left panel) and ABCB1-overexpressing variant KB-V-1 cancer cells (A, right panel), drug-sensitive human OVCAR-8 ovarian cancer cells (B, left panel) and ABCB1-overexpressing variant NCI-ADR/RES cancer cells (B, right panel), as well as parental HEK293 cells (C, left panel) and HEK293 cells transfected with human ABCB1 (C, right panel) were treated with paclitaxel in the presence of DMSO (empty circles), or DPI-201106 at 50 nM (filled circles), 100 nM (empty squares), 200 nM (filled squares), 500 nM (empty triangles) or 5 μM of verapamil (filled triangles) as a positive control. Points, mean values from at least three independent experiments; bars; SEM.

Fig. 3.. Effect of DPI-201106 on ABCB1 protein expression in human KB-V-1 epidermal cancer cells.

Immunoblot detection and quantification of human ABCB1 in (A) drug-sensitive human KB-3–1 epidermal cancer cells and ABCB1-overexpressing variant KB-V-1 cancer cells or (B) drug-sensitive human OVCAR-8 ovarian cancer cells and ABCB1-overexpressing variant NCI-ADR-RES cancer cells, treated with DMSO (vehicle control) or increasing concentrations (0.05 – 0.5 μM) of DPI-201106 for 72 h as described previously [52]. α-Tubulin was used as an internal loading control. Values are presented as mean ± SD calculated from three independent experiments.

Fig. 4.. DPI-201106 enhances drug-induced apoptosis in ABCB1-overexpressing cancer cells.

(A) Drug-sensitive human epidermal KB-3–1 cancer cells (top panels) and ABCB1-overexpressing MDR variant KB-V-1 cancer cells (lower panels) were treated with either DMSO (control), 500 nM DPI-201106 (+ DPI-201106), 500 nM colchicine (+ colchicine) or a combination of 500 nM colchicine and 500 nM of DPI-201106 (+ colchicine + DPI-201106) for 48 h. Cells were isolated and analyzed by flow cytometry as described previously [22]. Representative dot plots and the mean values of three independent experiments are shown. (B) Quantification of colchicine-induced apoptosis in human epidermal KB cell lines. Values are presented as mean ± SD calculated from three independent experiments. ^**p < 0.01, versus the same treatment in the absence of DPI-201106.

Fig. 5.. DPI-201106 inhibits Vi-sensitive ATPase activity of ABCB1.

(A) The effect of DPI-201106 on ABCB1 ATP hydrolysis was determined as described previously [1]. Points, mean values from at least three independent experiments; bars; SD. (B) Binding modes of DPI-201106 with homology modeled ABCB1 protein structure was predicted by Acclerys Discovery Studio 4.0 software as described in Materials and methods. DPI-201106 is shown as a molecular model with the atoms colored as carbon- gray, hydrogen-light gray, nitrogen-blue and oxygen-red. The same color scheme is used for interacting amino acid residues. Dotted lines indicate proposed interactions.

Fig. 6.. A graphic illustration of DPI-201106 attenuating the drug efflux function of ABCB1 in cancer cells.

Anticancer drugs (white circles) are actively transported out of cancer cells by ABCB1, resulting in MDR phenotype (upper panel). In contrast, the presence of DPI-201106 (white rectangle) blocks ABCB1-mediated drug efflux and restores drug accumulation to a sufficient level that causes apoptosis in cancer cells overexpressing ABCB1.