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. 2019 Dec 29;21(1):238.
doi: 10.3390/ijms21010238.

The Selective Class IIa Histone Deacetylase Inhibitor TMP195 Resensitizes ABCB1- And ABCG2-Overexpressing Multidrug-Resistant Cancer Cells to Cytotoxic Anticancer Drugs

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The Selective Class IIa Histone Deacetylase Inhibitor TMP195 Resensitizes ABCB1- And ABCG2-Overexpressing Multidrug-Resistant Cancer Cells to Cytotoxic Anticancer Drugs

Chung-Pu Wu et al. Int J Mol Sci. .
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Abstract

Multidrug resistance caused by the overexpression of the ATP-binding cassette (ABC) proteins in cancer cells remains one of the most difficult challenges faced by drug developers and clinical scientists. The emergence of multidrug-resistant cancers has driven efforts from researchers to develop innovative strategies to improve therapeutic outcomes. Based on the drug repurposing approach, we discovered an additional action of TMP195, a potent and selective inhibitor of class IIa histone deacetylase. We reveal that in vitro TMP195 treatment significantly enhances drug-induced apoptosis and sensitizes multidrug-resistant cancer cells overexpressing ABCB1 or ABCG2 to anticancer drugs. We demonstrate that TMP195 inhibits the drug transport function, but not the protein expression of ABCB1 and ABCG2. The interaction between TMP195 with these transporters was supported by the TMP195-stimulated ATPase activity of ABCB1 and ABCG2, and by in silico docking analysis of TMP195 binding to the substrate-binding pocket of these transporters. Furthermore, we did not find clear evidence of TMP195 resistance conferred by ABCB1 or ABCG2, suggesting that these transporters are unlikely to play a significant role in the development of resistance to TMP195 in cancer patients.

Keywords: P-glycoprotein; TMP195; breast cancer resistance protein; chemoresistance; histone deacetylase inhibitor; modulators.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
TMP195 re-sensitizes ABCB1-overexpressing cells to paclitaxel and ABCG2-overexpressing cells to mitoxantrone. The chemosensitization effect of TMP195 was examined by treating cells with increasing concentrations of paclitaxel (a known substrate drug of ABCB1), or mitoxantrone (a known substrate drug of ABCG2) or etoposide (a known substrate drug of ABCC1) in the presence of DMSO (open circles) or TMP195 at 1 μM (open squares), 2 μM (filled squares), 3 μM (open triangles) or 5 μM (filled triangles). (A) Drug-sensitive parental KB-3-1 (left panel) and the ABCB1-overexpressing multidrug resistance (MDR) variant KB-V-1 (right panel) human epidermal cancer cells, as well as (B) HEK293 cells (left panel) and HEK293 cells transfected with human ABCB1 (MDR1, right panel) were used to determine the effect of TMP195 on ABCB1-mediated paclitaxel resistance. (C) Drug-sensitive parental S1 (left panel) and the ABCG2-overexpressing MDR variant S1-M1-80 (right panel) human colon cancer cells, as well as (D) HEK293 cells (left panel) and HEK293 cells transfected with human ABCG2 (R482, right panel) were used to determine the effect of TMP195 on ABCG2-mediated mitoxantrone resistance. (E) Drug-sensitive parental COR-L23/P (left panel) and the ABCC1-overexpressing MDR variant COR-L23/R (right panel) human lung cancer cells, as well as (F) HEK293 cells (left panel) and HEK293 cells transfected with human ABCC1 (MRP1, right panel) were used to determine the effect of TMP195 on ABCC1-mediated etoposide resistance. Points, mean values from at least three independent experiments; error bars, SEM.
Figure 1
Figure 1
TMP195 re-sensitizes ABCB1-overexpressing cells to paclitaxel and ABCG2-overexpressing cells to mitoxantrone. The chemosensitization effect of TMP195 was examined by treating cells with increasing concentrations of paclitaxel (a known substrate drug of ABCB1), or mitoxantrone (a known substrate drug of ABCG2) or etoposide (a known substrate drug of ABCC1) in the presence of DMSO (open circles) or TMP195 at 1 μM (open squares), 2 μM (filled squares), 3 μM (open triangles) or 5 μM (filled triangles). (A) Drug-sensitive parental KB-3-1 (left panel) and the ABCB1-overexpressing multidrug resistance (MDR) variant KB-V-1 (right panel) human epidermal cancer cells, as well as (B) HEK293 cells (left panel) and HEK293 cells transfected with human ABCB1 (MDR1, right panel) were used to determine the effect of TMP195 on ABCB1-mediated paclitaxel resistance. (C) Drug-sensitive parental S1 (left panel) and the ABCG2-overexpressing MDR variant S1-M1-80 (right panel) human colon cancer cells, as well as (D) HEK293 cells (left panel) and HEK293 cells transfected with human ABCG2 (R482, right panel) were used to determine the effect of TMP195 on ABCG2-mediated mitoxantrone resistance. (E) Drug-sensitive parental COR-L23/P (left panel) and the ABCC1-overexpressing MDR variant COR-L23/R (right panel) human lung cancer cells, as well as (F) HEK293 cells (left panel) and HEK293 cells transfected with human ABCC1 (MRP1, right panel) were used to determine the effect of TMP195 on ABCC1-mediated etoposide resistance. Points, mean values from at least three independent experiments; error bars, SEM.
Figure 2
Figure 2
TMP195 enhances drug-induced apoptosis in ABCB1-overexpressing cancer cells and ABCG2-overexpressing cancer cells. Dot plots (upper panel) and quantification (lower panel) of (A) drug-sensitive KB-3-1 cells and the MDR variant KB-V-1 cells treated with either DMSO (control), 10 μM of TMP195 (+TMP195), 500 nM of colchicine (+colchicine), or a combination of 500 nM of colchicine and 10 μM of TMP195 (+colchicine +TMP195), and (B) drug-sensitive S1 and the MDR variant S1-M1-80 cells treated with either DMSO (control), 10 μM of TMP195 (+TMP195), 5 μM of topotecan (+topotecan) or a combination of 5 μM of topotecan and 10 μM of TMP195 (+topotecan +TMP195). Cells were treated with respective regimens, isolated, and analyzed by flow cytometry as described previously [30]. Representative dot plots and quantifications of apoptotic cell populations are presented as mean ± SD calculated from at least three independent experiments. ** p < 0.05; ** p < 0.01; *** p < 0.001, versus the same treatment in the absence of TMP195.
Figure 3
Figure 3
TMP195 modulates the drug efflux function, but not the protein expression, of ABCB1 and ABCG2 in human multidrug-resistant cancer cells. The effect of TMP195 on ABCB1-mediated transport of calcein-AM, a known substrate of ABCB1, was determined in (A) ABCB1-overexpressing KB-V-1 human epidermal cancer cells and (B) human HEK293 cells transfected with human ABCB1 (MDR19-HEK293), whereas the effect of TMP195 on ABCG2-mediated transport of pheophorbide A (PhA), a known substrate of ABCG2, was determined in (C) ABCG2-overexpressing S1-M1-80 human colon cancer cells and (D) HEK293 cells transfected with human ABCG2 (R482-HEK293). Respective drug-sensitive parental cell lines were used as controls (A–D, right panels). Intracellular fluorescence of calcein (A and B) or PhA (C and D) was measured in cells treated with DMSO (A–D, solid lines), 20 μM of TMP195 (A–D, filled solid lines), 20 μM of verapamil as a positive control for ABCB1 inhibition (A and B, dotted lines), or 1 μM of Ko143 as a positive control for ABCG2 inhibition (C and D, dotted lines) as indicated and analyzed by flow cytometry. Representative histograms of at least three independent experiments are shown. The effect of TMP195 on the protein expression of ABCB1 or ABCG2 was determined by treating (E) KB-V-1 cancer cells or (F) S1-M1-80 cancer cells with increasing concentrations of TMP195 (0–5 μM) as indicated for 72 h before processing for immunoblotting. α-Tubulin was used as an internal loading control. Immunoblot detection (upper panels) and quantification values (lower panels) are presented as mean ± SD calculated from at least three independent experiments.
Figure 3
Figure 3
TMP195 modulates the drug efflux function, but not the protein expression, of ABCB1 and ABCG2 in human multidrug-resistant cancer cells. The effect of TMP195 on ABCB1-mediated transport of calcein-AM, a known substrate of ABCB1, was determined in (A) ABCB1-overexpressing KB-V-1 human epidermal cancer cells and (B) human HEK293 cells transfected with human ABCB1 (MDR19-HEK293), whereas the effect of TMP195 on ABCG2-mediated transport of pheophorbide A (PhA), a known substrate of ABCG2, was determined in (C) ABCG2-overexpressing S1-M1-80 human colon cancer cells and (D) HEK293 cells transfected with human ABCG2 (R482-HEK293). Respective drug-sensitive parental cell lines were used as controls (A–D, right panels). Intracellular fluorescence of calcein (A and B) or PhA (C and D) was measured in cells treated with DMSO (A–D, solid lines), 20 μM of TMP195 (A–D, filled solid lines), 20 μM of verapamil as a positive control for ABCB1 inhibition (A and B, dotted lines), or 1 μM of Ko143 as a positive control for ABCG2 inhibition (C and D, dotted lines) as indicated and analyzed by flow cytometry. Representative histograms of at least three independent experiments are shown. The effect of TMP195 on the protein expression of ABCB1 or ABCG2 was determined by treating (E) KB-V-1 cancer cells or (F) S1-M1-80 cancer cells with increasing concentrations of TMP195 (0–5 μM) as indicated for 72 h before processing for immunoblotting. α-Tubulin was used as an internal loading control. Immunoblot detection (upper panels) and quantification values (lower panels) are presented as mean ± SD calculated from at least three independent experiments.
Figure 4
Figure 4
TMP195 stimulates ATPase activity of ABCB1 and ABCG2. The effect of TMP195 on vanadate-sensitive ATPase activity of (A) ABCB1 and (B) ABCG2 was determined by the endpoint Pi assay as described in the Section 4. Data are presented as a mean ± SD from at least three independent experiments as a percentage of basal activity taken as 100%.
Figure 5
Figure 5
Docking of TMP195 in the drug-binding pockets of ABCB1 and ABCG2. Binding modes of TMP195 with (A) the inward-open structure of human ABCB1 (PDBID:6QEX) and (B) structure of ABCG2 (PDB: 5NJ3) obtained after exhaustive docking using AutoDock Vina software as described in Section 4. TMP195 is presented as a molecular model with atoms colored as carbon–green, nitrogen–blue, oxygen–red, florine–white. The docking scores of the first nine poses (tighter binding) are shown on the left. Cartoon representation shows all nine binding poses in the side- and bottom-view of each transporter. Binding-cavity is shown in dark gray from the bottom-view, and TM helix numbers are specified. TMP195 is presented in green sticks. The lowest energy poses for TMP195 in the transmembrane region of ABCB1 and ABCG2 are presented in dark gray lines to illustrate the residues that are within 4 Å of the ligand. Figures were prepared using the Pymol molecular graphics system, Version 1.7 Shrödinger, LLC.
Figure 6
Figure 6
Drug-sensitive parental and multidrug-resistant cells overexpressing ABCB1 or ABCG2 are equally sensitive to TMP195. The cytotoxicity of TMP195 was determined in (A) drug-sensitive human epidermal cancer cell line KB-3-1 (open circles) and KB-V-1, the ABCB1-overexpressing multidrug-resistant variant (filled circles), (B) drug-sensitive human ovarian cancer cell line OVCAR-8 (open circles) and NCI-ADR-RES, the ABCB1-overexpressing multidrug-resistant variant (filled circles), (C) drug-sensitive human colon cancer cell line S1 (open circles) and S1-M1-80, the ABCG2-overexpressing multidrug-resistant variant (filled circles), (D) drug-sensitive human lung cancer cell line H460 (open circles) and H460-MX20, the ABCG2-overexpressing multidrug-resistant variant (filled circles), as well as (E) parental pcDNA-HEK293 cells (open circles) and HEK293 cells transfected with human ABCB1 (MDR19-HEK293, filled circles) or human ABCG2 (R482-HEK293, open squares). Points, mean values from at least three independent experiments; error bars; SD.
Figure 7
Figure 7
A simplified schematic diagram of TMP195 reversing drug resistance in cancer cells overexpressing ABCB1 or ABCG2 by attenuating the function of ABCB1 and ABCG2. The ATP-binding cassette proteins ABCB1 (brown) and ABCG2 (green) reduce intracellular drug concentration by actively transporting ABCB1 substrate drug (red circles) and ABCG2 substrate drug (blue circles) out of the cancer cell, which leads to multidrug resistant phenotype. By binding to the drug-binding pocket(s) of ABCB1 and ABCG2, TMP195 (triangle) attenuates the drug efflux function of both ABCB1 and ABCG2, thus restoring the chemosensitivity of cancer cells to these chemotherapeutic drugs.

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