NCX-4040, a Unique Nitric Oxide Donor, Induces Reversal of Drug-Resistance in Both ABCB1- and ABCG2-Expressing Multidrug Human Cancer Cells

Abstract

The emergence of multidrug resistance (MDR) in the clinic is a significant problem for a successful treatment of human cancers. Overexpression of various ABC transporters (P-gp, BCRP and MRP's), which remove anticancer drugs in an ATP-dependent manner, is linked to the emergence of MDR. Attempts to modulate MDR have not been very successful in the clinic. Furthermore, no single agent has been found to significantly inhibit their functions to overcome clinical drug resistance. We have previously shown that nitric oxide (^●NO) inhibits ATPase functions of ABC transporters, causing reversal of resistance to clinically active anticancer drugs. In this study, we have used cytotoxicity and molecular docking studies to show that NCX4040, a nitric oxide donor related to aspirin, inhibited the functions of ATPase which resulted in significant reversal of resistance to both adriamycin and topotecan in P-gp- and BCRP-expressing human cancer cell lines, respectively. We also used several other cytotoxic nitric oxide donors, e.g., molsidomine and S-nitroso glutathione; however, both P-gp- and BCRP-expressing cells were found to be highly resistant to these NO-donors. Molecular docking studies showed that NCX4040 binds to the nucleotide binding domains of the ATPase and interferes with further binding of ATP, resulting in decreased activities of these transporters. Our results are extremely promising and suggest that nitric oxide and other reactive species delivered to drug resistant tumor cells by well-designed nitric oxide donors could be useful in sensitizing anticancer drugs in multidrug resistant tumors expressing various ABC transporters.

Keywords: NCX4040; P-gp protein; adriamycin; breast cancer resistance protein; nitric oxide; nitric oxide-donors; topotecan.

Figure 1

Structures of NCX4040 and NO-donors.

Figure 2

Cytotoxicity of MS in (A) WT OVCAR-8 and NIH/ADR-RES Cells; and (B) WT MCF-7 and MXR cells. Cytotoxicity of SIN-1 in (C) WT OVCAR-8 and NIH-RES/ADR Cells; and (D) WT MCF-7 and MXR cells. **, *** p values < 0.005, < 0.001 compared to concentration-matched samples.

Figure 3

Cytotoxicity of GSNO in (A) WT OVCAR-8 and NIH-RES/ADR Cells; and (B) WT MCF-7 and MXR cells. **, *** p values < 0.005, <0.001 compared to concentration-matched samples.

Figure 4

Cytotoxicity of GSNO in (A) MXR cells and (B) WT MCF-7 cells in the presence of 500 nm Ko143. Cytotoxicity of GSNO in (C) NCI/ADR-RES cells in the presence of Verapamil (10 µM). Ko143 or Verapamil was preincubated with cells for 2h in the complete medium before adding various concentrations of GSNO as detailed in the methods section. *** p values < 0.001 compared with concentration-matched samples.

Figure 5

Cytotoxicity of NCX4040 in (A) WT OVCAR-8 and NCI-RES/ADR cells, (C) WT MCF-7 and MXR cells. Effects of verapamil (10 µM, (B)) and Ko143 (0.5 µM, (D)) on cytotoxicity of NCX4040 in NCI/ADR-RES and in MXR cells, respectively. ** and *** p values, <0.005 and <0.001, respectively compared to concentration-matched samples.

Figure 6

Reversal of adriamycin (A) and topotecan (C) resistance by NCX4040 (2 µM) in NCI/ADR-RES and MCF-7/MXR cells, respectively and effects of NCX 4040 (5 × 10⁻⁸M) in corresponding sensitive WTOVCAR-8 (B) and WT MCF-7 (D) cells. ** and *** p values < 0.005 and <0.001, respectively compared to concentration-matched samples.

Figure 7

Reversal of adriamycin (A) and topotecan (B) resistance by NCX4040 (2 µM, and 5 µM) in NCI/ADR-RES and MCF-7/MXR cells, respectively. *, ** and *** p values 0.05, 0.005 and 0.001, respectively compared to concentration-matched samples. ##, ### p values <0.005 and <0.001 compared to drug alone and $$ and $$$ p values <0.005 and <0.001 compared to concentration-matched drug in the presence 2.0 µM NCX4040.

Figure 8

Effect of NCX4040 (5 µM) on accumulations of ADR in OVCAR WT (A) and NCI/ADR-RES cells (B) in the presence or absence of 10 µM verapamil. Effects of NCX4040 (5 µM) on accumulations of Hoechst 33342 in MCF-7 WT(C) and MCF-7/MXR cells (D) in the presence or absence of Ko143 (500 mM). The relative cellular fluorescence values in the presence of Ko143 × 10. *, ** and *** p values <0.05, <0.005 and <0.001, respectively compared to drug (dye) alone.

Figure 9

Effects of NO/NCX4040 (5 µM) on P-gp and BCRP proteins in NCI/ADR-RES and MCF-7/MXR cells following 4, 24 and 48 h treatment. Lanes 1 and 5 are untreated controls, 2 and 6 at 4 h; 3 and 7 at 24 h; and 4 and 8 at 48 h. A representative Western Blot is shown. The uncropped Western blots have been shown in Figure S1.

Figure 10

NCX4040 docked on (A) P-gp (pdb ID: 6C0V.pdb) (B) BCRP (pdb ID: 6hbu.pdb). NCX4040 is shown in solid spheres and the proteins are represented by ribbon diagrams. Residues of the protein that are in contact or making H-bonds are shown in right panels.