Molecular Mechanisms of Cytotoxicity of NCX4040, the Non-Steroidal Anti-Inflammatory NO-Donor, in Human Ovarian Cancer Cells

Abstract

NCX4040, the non-steroidal anti-inflammatory-NO donor, is cytotoxic to several human tumors, including ovarian tumor cells. We have found that NCX4040 is also cytotoxic against both OVCAR-8 and its adriamycin resistant (NCI/ADR-RES) tumor cell lines. Here, we have examined mechanism(s) for the cytotoxicity of NCX4040 in OVCAR-8 and NCI/ADR-RES cell lines. We found that NCX4040 induced significant apoptosis in both cell lines. Furthermore, NCX4040 treatment caused significant depletion of cellular glutathione, causing oxidative stress due to the formation of reactive oxygen/nitrogen species (ROS/RNS). Significantly more ROS/RNS were detected in OVCAR-8 cells than in NCI/ADR-RES cells which may have resulted from increased activities of SOD, glutathione peroxidase and transferases expressed in NCI/ADR-RES cells. NCX4040 treatment resulted in the formation of double-strand DNA breaks in both cells; however, more of these DNA breaks were detected in OVCAR-8 cells. RT-PCR studies indicated that NCX4040-induced DNA damage was not repaired as efficiently in NCI/ADR-RES cells as in OVCAR-8 cells which may lead to a differential cell death. Pretreatment of OVCAR-8 cells with N-acetylcysteine (NAC) significantly decreased cytotoxicity of NCX4040 in OVCAR-8 cells; however, NAC had no effects on NCX4040 cytotoxicity in NCI/ADR-RES cells. In contrast, FeTPPS, a peroxynitrite scavenger, completely blocked NCX4040-induced cell death in both cells, suggesting that NCX4040-induced cell death could be mediated by peroxynitrite formed from NCX4040 following cellular metabolism.

Keywords: DNA damage; NCX4040; nitric oxide; peroxynitrite; reactive oxygen species.

Figure 1

Structure of NCX4040 and its known/proposed mechanisms of cytotoxicity.

Figure 2

Cytotoxicity of NCX4040 in (A) and topotecan (TPT, (B)) in WT OVCAR-8 and NCI/ADR-RES cells at 48 h drug exposures, respectively. * p and ** p-values, <0.05 and <0.005, respectively, compared to concentration-matched treatment.

Figure 3

Percent GSH depletion in OVCAR-8 (A,B) and in NCI/ADR-RES cells (C,D) and concentration dependence of GSH depletion by NCX4040 for 4 h (E). Representative plots (A–D) are shown here. *** p-values, <0.001, compared to concentration-matched NCI/ADR-RES.

Figure 3

Percent GSH depletion in OVCAR-8 (A,B) and in NCI/ADR-RES cells (C,D) and concentration dependence of GSH depletion by NCX4040 for 4 h (E). Representative plots (A–D) are shown here. *** p-values, <0.001, compared to concentration-matched NCI/ADR-RES.

Figure 4

Formation of Mitosox positive cells in OVCAR-8 and NCI/ADR-RES cells during incubation with NCX4040 (25 µM) with time (E). Plots (A–D) were obtained at 2 h of drug treatment and representative plots are shown here. *** p-values <0.001 compared to time-matched control and NCI/ADR-RES.

Figure 4

Formation of Mitosox positive cells in OVCAR-8 and NCI/ADR-RES cells during incubation with NCX4040 (25 µM) with time (E). Plots (A–D) were obtained at 2 h of drug treatment and representative plots are shown here. *** p-values <0.001 compared to time-matched control and NCI/ADR-RES.

Figure 5

Detection of DNA-double strand breaks produced following treatment with different concentrations of NCX4040 in OVCAR-8 and NCI/ADR-RES cells for 60 min and with different concentrations (C). TPT (10 µM) was used as a control. Representative plots (A,B) are shown here. * and *** p-values, <0.05, and <0.001, respectively, compared to concentration-matched treatment.

Figure 6

Detection of CaspaTag positive cells (A,B) OVCAR-8 and (C,D) in NCI/ADR-RES cells and with different concentrations of NCX4040 for 24 h (E). Representative plots (A–D) are shown here. ** and *** p-values, <0.005, and <0.001, respectively, compared to concentration-matched treatment.

Figure 7

Detection of Annexin positive cells (A,B) OVCAR-8, and (C,D) in NCI/ADR-RES cells and with different concentrations of NCX4040 for 24 h (E). Representative plots (A–D) are shown here. *** p-values, <0.001, compared to concentration-matched treatment.

Figure 8

Effects of NAC (2 mM) and FeTPPS (5 µM) on the cytotoxicity of NCX4040 in OVCAR-8 (A) and NCI/ADR-RES (B) cells following 48 h exposure. NAC or FeTPPS were preincubated with cells for 20–30 min before adding NCX4040. *, ** and *** p-values, <0.05, <0.005 and <0.001, respectively, compared to concentration-matched drug alone treatment. ##, ### p-values, <0.005 and <0.001, respectively, compared to concentration-matched drug alone treatment. $, $$$ p-values, <0.05, and <0.001, respectively, compared to concentration-matched drug + NAC treatment.

Figure 9

RT-PCR analysis of apoptosis- and oxidative stress-related genes differentially expressed by NCX4040 (5 µM) in OVCAR-8 (A) and NCI/ADR-RES (B) cells and DNA damage and DNA repair genes in OVCAR-8 (C) and NCI/ADR-RES (D) following 4, 24 and 48 h NCX4040 treatment. RT-PCR was carried out as described in the methods section. *, ** and *** p-values, <0.05, <0.005 and <0.001, respectively, compared to actin controls.

Figure 10

Proposed formation of reactive free radical species, ^●NO, O₂^●−, and O=N-O-O⁻ following cellular metabolism of NCX4040 in tumor cells and role of peroxynitrite in tumor cell death.