Impact of the APE1 Redox Function Inhibitor E3330 in Non-small Cell Lung Cancer Cells Exposed to Cisplatin: Increased Cytotoxicity and Impairment of Cell Migration and Invasion

Abstract

Elevated expression levels of the apurinic/apyrimidinic endonuclease 1 (APE1) have been correlated with the more aggressive phenotypes and poor prognosis of non-small cell lung cancer (NSCLC). This study aimed to assess the impact of the inhibition of the redox function of APE1 with E3330 either alone or in combination with cisplatin in NSCLC cells. For this purpose, complementary endpoints focusing on cell viability, apoptosis, cell cycle distribution, and migration/invasion were studied. Cisplatin decreased the viability of H1975 cells in a time- and concentration-dependent manner, with IC₅₀ values of 9.6 µM for crystal violet assay and 15.9 µM for 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. E3330 was clearly cytotoxic for concentrations above 30 µM. The co-incubation of E3330 and cisplatin significantly decreased cell viability compared to cisplatin alone. Regarding cell cycle distribution, cisplatin led to an increase in sub-G1, whereas the co-treatment with E3330 did not change this profile, which was then confirmed in terms of % apoptotic cells. In addition, the combination of E3330 and cisplatin at low concentrations decreased collective and chemotactic migration, and also chemoinvasion, by reducing these capabilities up to 20%. Overall, these results point to E3330 as a promising compound to boost cisplatin therapy that warrants further investigation in NSCLC.

Keywords: E3330; apoptosis; apurinic/apyrimidinic endonuclease 1; cisplatin; cytotoxicity; invasion; migration; non-small cell lung cancer.

Figure 1

Chemical structure of cisplatin (A) and (2E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)methylene] undecanoic acid (E3330) (B).

Figure 2

Cytotoxic effects of cisplatin (1–50 µM) in H1975 cells. The viability of cells treated with cisplatin for 48 h and 72 h was assessed by crystal violet (CV) staining assay (A) and for 72 h by MTS reduction assay (B). Values represent mean ± SD (n = 3–4) and are expressed as percentages of the vehicle-treated control cells.

Figure 3

Evaluation of E3330 (5–50 µM) cytotoxicity in H1975 cells. The cell viability of E3330-exposed cells (72 h) was evaluated by CV staining (A) and MTS reduction (B) assays. Values represent mean ± SD (n = 3) and are expressed as percentages of the vehicle-treated control cells.

Figure 4

Impact of E3330 on the viability of H1975 cells treated with cisplatin. Cells were pre-incubated with E3330 (30 µM) for 3 h and then simultaneously exposed to E3330 and cisplatin (5–20 µM) for 72 h. The effects in terms of cell viability were evaluated using the CV staining assay (A) and MTS reduction assay (B). Values represent mean ± SD (n = 3–5) and are expressed as percentages relative to vehicle-treated control cells. * p < 0.05 and ** p < 0.01 relative to respective cisplatin-treated cells (Student’s t-test).

Figure 5

Cell cycle progression and apoptosis of H1975 cells treated with E3330 and/or cisplatin. Cells were pre-incubated with E3330 (30 µM) for 3 h and then cisplatin (20 µM) was added for co-incubation for further 72 h. After this exposure period, cell DNA content analysis with PI staining was performed by flow cytometry. (A) Representative flow cytometry histograms. (B) Sub-G1, G0/G1, S, and G2/M populations’ summary results. The percentage of apoptotic cells was determined by PI and Annexin V staining after the same incubation profile as in the cell DNA content analysis. (C) Representative flow cytometry dot-plots. (D) Percentage of viable cells, cells undergoing early and late apoptosis, and necrotic cells summary results. (E) Summary results demonstrate the percentage of apoptotic cells (Annexin V positive cells). Values represent mean ± SD (n = 3), * p < 0.05 (one-way ANOVA with Tukey’s test).

Figure 6

Viability of H1975 cells exposed to low concentrations of cisplatin or E3330 in culture medium with 2% FBS. (A) Effect of cisplatin (0.1–5 μM; 24 h) on cell viability, in the presence of 2% FBS, evaluated by the CV assay. Effect of E3330 (10–30 μM; 24 h) on cell viability in the presence of 2% FBS evaluated by both CV (B) and MTS assays (C). Values for cell viability represent mean ± SD (n = 4–7) and are expressed as percentages relative to vehicle-treated control cells.

Figure 7

E3330 effect on collective and chemotactic migration of H1975 cells exposed to cisplatin. Collective cell migration was evaluated by the wound-healing assay (A) and chemotaxis was measured using a transwell assay (C). Representative microscopy images of the wound-healing assay (40×, B) and the chemotaxis assay (migrating cells stained with crystal violet—100×, D). Scale bars = 200 μm. Values for the wound-healing assay represent mean ± SD (n = 3) and are expressed as percentage of wound closure, calculated relative to the initial width; * p < 0.05 and ** p < 0.01 (Student’s t-test). Values for the chemotaxis assay represent mean ± SD (n = 3) and are expressed as percentages relative to vehicle-treated control cells; * p < 0.05 (Student’s t-test).

Figure 8

Effect of E3330 on the invasiveness of cisplatin-treated H1975 cells. (A) Transwell chemoinvasion was assessed after a pre-incubation period of 3 h with E3330 and a subsequent period of 16 h with both compounds. Values represent mean ± SD (n = 5) and are expressed as percentages relative to vehicle-treated control cells; * p < 0.05 and ** p < 0.01 (Student’s t-test). (B) Representative microscopy images of invading cells stained with crystal violet (100×). Scale bars = 100 μm.