Nitric oxide reverses drug resistance by inhibiting ATPase activity of p-glycoprotein in human multi-drug resistant cancer cells

Abstract

Background: Development of resistance to chemotherapy drugs is a significant problem in treating human malignancies in the clinic. Overexpression of drug efflux proteins, including P-170 glycoprotein (P-gp), an ATP-dependent efflux protein, is one of the main mechanisms responsible for multi-drug resistance (MDR). Because our previous studies have shown that nitric oxide (^˙NO) or its related species inhibit the ATPase activities of topoisomerase II, we hypothesized that ^˙NO should also inhibit the ATPase activity of P-gp and increase drug accumulation in MDR cells, causing a reversal of drug resistance.

Results: Cytotoxicity and cellular accumulation studies showed that ^˙NO significantly inhibited the ATPase activity of P-gp in isolated membranes and in NCI/ADR-RES tumor cells, causing an increase in drug accumulation and reversals of adriamycin and taxol resistance in the MDR cells. While ^˙NO had no effects on topoisomerase II-induced, adriamycin-dependent DNA cleavage complex formation, it significantly inhibited adriamycin-induced DNA double-strand breaks. Electron spin resonance studies showed an increase in adriamycin-dependent hydroxyl radical formation in the presence of an NO-donor.

Conclusions: The reversal of drug resistance is due to inhibition of the ATPase activity by ^˙NO, resulting in enhancement of the drug accumulation in the MDR cells. Furthermore, DNA damage was not responsible for this reversal of adriamycin resistance. However, formation of adriamycin-dependent toxic free radical species and subsequent cellular damage may be responsible for the increased cytotoxicity of adriamycin by ^˙NO in NCI/ADR-RES cells.

General significance: Appropriately designed NO donors would be ideal for the treatment of P-gp-overexpressing tumors in the clinic.

Keywords: Adriamycin; Free radical; Multi-drug resistance; Nitric Oxide; P-gp protein; Taxol.

Figure 1:

Western blot analysis (A) for P-gp proteins in OVCAR-8 (lane-1) and NCI/ADR-RES (lane-2) tumor cells and confocal microscopy studies for P-gp proteins in OVCAR-8 and NCI/ADR-RES tumor cells (B).

Figure 2:

The relative accumulation of ADR in OVCAR-8 and NCI/ADR-RES tumor cells (panel A) and effects of PPNO and PSC 388 on ADR accumulation in OVCAR-8 (panel B) and in NCI/ADR-RES tumor cells (panel C). Values represent three separate experiments. *** p values ≤ 0.0001compared with control (no treatment). ^### p values ≤ 0.0001 from ADR alone.

Figure 3:

Cytotoxicity of ADR (■-■) in OVCAR-8 tumor cells (panel A) in the presence of PPNO (●-●,50 µM) and DETNO (▲-▲, 50 µM) and in NCI/ADR-RES tumor cells following 72 h of drug treatment (Panel B). Cells were counted as described in the methods section. Panel C shows the effects of PPNO and DETNO on the reversal of Taxol resistance under similar conditions. Values represent three separate experiments carried out in triplicate. ***, ** and ^* p values ≤ 0.001, 0.005, and ≤ 0.05, respectively, compared with concentration-matched samples. ###, ##, and # values ≤ 0.001, 0.005 and ≤ 0.05, respectively, from concentration-matched samples.

Figure 4:

Inhibition of the ATPase activity of P-gp in isolated MDR1 membranes by PPNO. The inhibition studies were carried out according to the manufacturer’s instructions. Inhibitors (Na Vanadate and cyclosporine A), and various concentrations of PPNO were incubated with verapamil-activate membranes for 30 min prior to the addition of ATP. ** p values ≤ 0.005 compared to 0.02, 0.2 and 2.0 µM PPNO, respectively.

Figure 5:

Panel A: Formation of cleavage complexes in NCI/ADR-RES cancer cells by ADR alone (□) and in the presence of PPNO (100 µM; ■). Cells were preincubated with PPNO for 1–2 h and then incubated with ADR (5.0 and 25.0 µM) for 1 h in complete medium (2 mL). * p values ≤ 0.05 compared with control. Panel B: Flow cytometric analysis for ADR-induced double-strand breaks using the γ-H2AX phosphorylation assay following treatment with ADR (5 and 25 µM) alone (□) and in the presence of PPNO (100 µM; ■). Cells were preincubated with PPNO for 1h before adding ADR and then incubated for 1 h in complete medium (2 mL). The SDS-KCl precipitation assay and the γ-H2AX analysis were carried out as described in the methods section. **, p value ≤ 0.005 compared to adriamycin alone and *, p value ≤ 0.05 compared to control.

Figure 6:

Spin trapping of hydroxyl radicals formed from adriamycin (2.5 µM) by ESR in the presence of DMPO (100 mM) and NADPH (1 mM) in NCI/ADR-RES tumor cells in the presence (C) or in the absence (B) of PPNO (100 µM) and (A) no adriamycin. The cells were incubated with PPNO in PBS-G buffer at 37°C for 90 min, collected by centrifugation, washed once with PBS-G buffer, and suspended in PBS-G buffer containing 25 µM DETPAC and adding DMPO and ADR (5 µM). The reactions were initiated by adding NADPH (1 mM) as described in the methods section and the resulting spectra were recorded. The DMPO-OH adduct had the following hyperfine coupling constants: a^N = a^H = 14.9 G. Spectra (D) was obtained in the presence of DMSO (0.5 M) and 10 µM ADR showing the presence of both DMPO-OH (o) and DMPO-CH₃ (c) adducts with a^N = 16.3 G and a^H = 23.6 G. The receiver gain for A and B was 3 × 10⁴, and 1 × 10⁴ for C and D. Insert: Quantification of DMPO-OH adducts formed in the presence (C) and absence (B) of PPNO using low field line of the DMPO-OH adduct spectrum.

Figure 7:

Effects of DETNO (50 µM) on ADR- and Taxol-induced cell death of NCI/ADR-RES tumor cells. NCI/ADR-RES cells were treated with 25 µM ADR or 0.5 µM taxol with and without DETNO for 72 h in the complete media. Cells were collected and analyzed by flow cytometry as described in the methods section. Panel A, in absence of DETNO; Panel B, in the presence of DETNO.