Reconsidering the Role of Cyclooxygenase Inhibition in the Chemotherapeutic Value of NO-Releasing Aspirins for Lung Cancer

Abstract

Nitric oxide-releasing aspirins (NO-aspirins) are aspirin derivatives that are safer than the parent drug in the gastrointestinal context and have shown superior cytotoxic effects in several cancer models. Despite the rationale for their design, the influence of nitric oxide (NO^•) on the effects of NO-aspirins has been queried. Moreover, different isomers exhibit varying antitumor activity, apparently related to their ability to release NO^•. Here, we investigated the effects and mode of action of NO-aspirins in non-small-cell lung cancer (NSCLC) cells, comparing two isomers, NCX4016 and NCX4040 (-meta and -para isomers, respectively). NCX4040 was more potent in decreasing NSCLC cell viability and migration and exhibited significant synergistic effects in combination with erlotinib (an epidermal growth factor receptor inhibitor) in erlotinib-resistant cells. We also studied the relationship among the effects of NO-aspirins, NO^• release, and PGE₂ levels. NCX4040 released more NO^• and significantly decreased PGE₂ synthesis relative to NCX4016; however, NO^• scavenger treatment reversed the antiproliferative effects of NCX4016, but not those of NCX4040. By contrast, misoprostol (a PGE₂ receptor agonist) significantly reversed the antiproliferative effect of NCX4040, but not those of NCX4016. Furthermore, misoprostol reversed the antimigratory effects of NCX4040. Overall, these results indicate that PGE₂ inhibition is important in the mode of action of NO-aspirins.

Keywords: NCX4016; NCX4040; cyclooxygenase; erlotinib; nitric oxide; non-small-cell lung cancer; prostaglandin.

Figure 1

Chemical structure of the NO-aspirins used in this study. The structure of aspirin is highlighted in red, and the nitro (nitric oxide-releasing) group is shown in blue. The linker group is shown in black.

Figure 2

Effect of NO-aspirins on non-small-cell lung cancer cell migration. Assays were conducted by evaluating the migration of H1299 cells through an 8 μM-pore cell culture insert. Cells were stained with DAPI after 6 h of migration. (A) Representative images of migration of H1299 cells exposed to aspirin (ASA; 1 and 2 mM), NCX4016 (100 and 200 μM), and NCX4040 (25 and 50 μM). (B) Quantitation of migration of H1299 cells exposed to aspirin (ASA; from 0.3 to 2 mM), NCX4016 (from 25 to 200 μM), and NCX4040 (from 6.3 to 50 μM). The bar graph summarizes the results from four independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001, and **** p < 0.0001, compared with the control group, calculated by one-way ANOVA and Dunnett’s post-test.

Figure 3

The relationship between nitric oxide (NO^•) release by NO-aspirins and their effects on non-small-cell lung cancer (NSCLC) cells. Intracellular NO^• release by NO-aspirins was measured using a DAF-2 fluorescent probe. H1299 cells were pre-loaded with DAF-2 prior to incubation in culture plates. After 24 h, cells were exposed to the different drugs. Cell viability was measured by MTT reduction. Cell migration was evaluated using an 8 µM-pore cell culture insert and fluorescence microscopy. (A) Representative graph showing the relative fluorescence induced by NO^• release from NO-aspirins. (B) Slope of fluorescence over time, induced by NO^• release from NO-aspirins. SNAP was used as a positive control for NO^• release. * p < 0.05 and **** p < 0.00001, compared with the negative control, calculated by one-way ANOVA and Dunnett’s post-test. (C) Cell proliferation after exposure of H1299 cells to NCX4040 and NCX4040 + C-PTIO for 96 h. (D) Cell proliferation after exposure of H1299 cells to NCX4016 and NCX4016 + C-PTIO for 96 h. ** p < 0.01 and **** p < 0.00001, between the indicated groups, calculated by one-way ANOVA and Tukey post-test. (E) Representative images of H1299 cell migration following exposure to NO-aspirins alone or in the presence of C-PTIO. (F) Quantitation of migration of H1299 cells exposed to NO-aspirins alone and in the presence of C-PTIO. Bar graphs summarize the results of at least three independent experiments.

Figure 4

Relationship between the inhibition of prostaglandin E₂ (PGE₂) synthesis by NO-aspirins and their effects on non-small-cell lung cancer (NSCLC) cells. PGE₂ levels were measured in H1299 cells exposed to NO-aspirins for 24 h. To evaluate cell viability, cells were exposed to NO-aspirins or NO-aspirins + misoprostol (MSP) for 96 h, then cell viability was measured by MTT reduction. Cell migration was evaluated after 6 h of exposure using an 8 μM-pore cell culture insert and fluorescence microscopy. (A) PGE₂ levels in the supernatant of H1299 cells. * p < 0.05; ** p < 0.01, and *** p < 0.0001, compared with the control group, calculated by one-way ANOVA and Dunnett’s post-test. (B) Cell viability of H1299 exposed to NCX4016 or NCX4016 + MSP for 96 h. (C) Cell viability of H1299 cells exposed to NCX4040 or NCX4040 + MSP for 96 h. (D) Representative images of H1299 cell migration following exposure to NO-aspirins alone or in the presence of MSP. (E) Quantitation of the migration of H1299 cells exposed to NO-aspirins alone and in the presence of MSP. Bar graphs summarize the results of at least three independent experiments. For panels (B), (C), and (E), * p < 0.05; ** p < 0.01, and *** p < 0.0001, between the indicated groups, calculated by one-way ANOVA and Tukey post-test.