Cariprazine, A Dopamine D₂/D₃ Receptor Partial Agonist, Modulates ABCG2-Mediated Multidrug Resistance in Cancer

Abstract

Multidrug resistance (MDR) is a continuing clinical problem that limits the efficacy of chemotherapy in cancer. The over expression of the ATP-binding cassette (ABC) family G2 (ABCG2) transporter is one of the main mechanisms that mediates MDR in cancer. Molecular modeling data indicated that cariprazine, a dopamine D₂/D₃ receptor partial agonist, had a significant binding affinity for ABCG2 transporter with a Glide XP score of -6.515. Therefore, in this in vitro study, we determined the effect of cariprazine on MDR resulting from the overexpression of ABCG2 transporters. Alone, cariprazine, at concentrations up to 20 μM, did not significantly decrease cell viability. Cariprazine, at concentrations ranging from 1 to 10 μM, did not significantly alter the cytotoxicity of mitoxantrone (MX) in the parental non-small cell cancer cell line, H460 and colon cancer cell S1. However, cariprazine (1⁻20 μM) significantly enhanced the efficacy of ABCG2 substrate antineoplastic drug MX in the ABCG2-overexpressing MDR cell line, H460-MX20 and S1M1-80, by reducing the resistance fold from 28 to 1 and from 93 to 1.33, respectively. Cariprazine, in a concentration-dependent (1⁻20 μM), significantly increased the intracellular accumulation of Rhodamine 123 in S1M1-80. Interestingly, 10 or 20 μM of cariprazine significantly decreased the expression levels of the ABCG2 protein in the colon and lung cancer cell lines, suggesting that cariprazine inhibits both the function and expression of ABCG2 transporters at nontoxic concentrations. Overall, our results suggest that cariprazine, via several distinct mechanisms, can resensitize resistant cancer cells to mitoxantrone.

Keywords: ABCG2; Cariprazine; Colon cancer; Dopamine D3-preferring D2/D3 receptor partial; Lung cancer; Multidrug resistance.

Figure 1

(A) Chemical structure of cariprazine. (B) 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay results are shown for H460 and H460-MX20 cells incubated with cariprazine and (C) S1 and S1M1-80 cells incubated with cariprazine. The data points represent the mean ± SD of at least three independent experiments repeated in triplicate.

Figure 2

The effect of cariprazine on the efficacy of MX in the ABCG2 overexpressing cell line H460-MX20. (A) MTT cytotoxicity is expressed as the percentage change in cell survival. (B) The IC₅₀ values for MX alone (control) or in combination with different concentrations of cariprazine or with 5 μM of nilotinib. (C) Microscopic images illustrating the synergistic effects of cariprazine on the efficacy of MX at different concentrations. Scale bar: 20×. The data points represent the means ± SD of at least three independent experiments, each with triplicate determinations. * p < 0.05; ** p < 0.01 vs. control cells incubated with MX alone.

Figure 3

The effect of cariprazine on the efficacy of MX in the ABCG2 overexpressing cell line S1M1-80. (A) MTT cytotoxicity is expressed as the percentage change in cell survival. (B). The IC₅₀ values for MX alone (control) or in combination with different concentrations of cariprazine or with 5 μM of nilotinib in S1M1-80 cells. (C) Microscopic images illustrating the synergistic effects of cariprazine on the efficacy of MX at different concentrations. Scale bar: 20×. The data points represent the means ± SD of at least three independent experiments, each with triplicate determinations. *** p < 0.001 vs. control cells incubated with MX alone.

Figure 4

The combination index values for the combination of 1, 10 or 20 μM of cariprazine, respectively, with 0.1, 0.3, 1, 3, 10, 30, or 100 μM of MX in (A): H460-MX20 cancer cells and (B) S1M1-80 cancer cells. The Fa values range from 0 to 1. CI < 1, synergism; CI = 1, additive effect and CI > 1, antagonism. The data represent the mean ± SD of three independent experiments in triplicate.

Figure 5

The effects of cariprazine on the expression level of the ABCG2 transporter protein. (A) H460-MX20 cells were incubated with 10 or 20 μM cariprazine, respectively. (B) The columns represent the mean of the western blot quantification values. The error bars represent the SEM. At least two additional experiments showed similar results to the representative figure results. ** p < 0.01.

Figure 6

The effect of cariprazine, and nilotinib on the intracellular accumulation of rhodamine 123 in ABCG2 overexpressing S1M1-80 cells. (A) The accumulation of rhodamine 123 was increased in a dose-dependent manner in S1M1-80 cells incubated with cariprazine. Red filled, control; black, 1 μM; orange, 10 μM; green, 20 μM; blue, nilotinib, 5 μM. (B) Rhodamine 123 levels were expressed as the units of mean fluorescent intensity. The data shown as means ± SD of triplicate determinations. ** p < 0.05; *** p < 0.001 vs. the control.

Figure 7

Model for the binding of cariprazine with ABCG2 transporter. (A) XP-Glide predicted binding mode of cariprazine in drug binding cavity of human ABCG2 transporter (PDB ID: 6ETI). Important amino acids are depicted as sticks with the atoms colored as carbon = green, hydrogen = white, nitrogen = blue, oxygen = red. The ligand cariprazine is shown with the same color scheme as above except for the carbon atoms, represented in orange and the chlorine atoms represented in dark green. The red dotted lines represent hydrogen bond. (B) A schematic diagram of the protein–ligand interaction is shown for cariprazine. Blue circles depict polar amino acids and green circles depict hydrophobic amino acids. The purple arrows depict side chain donor/acceptor interactions.