Combination of dl922-947 Oncolytic Adenovirus and G-Quadruplex Binders Uncovers Improved Antitumor Activity in Breast Cancer

Abstract

G-quadruplexes (G4s) are nucleic secondary structures characterized by G-tetrads. G4 motif stabilization induces DNA damage and cancer cell death; therefore, G4-targeting small molecules are the focus of clinical investigation. DNA destabilization induced by G4 ligands might potentiate the anticancer activity of agents targeting DNA or inhibiting its repair such as oncolytic viruses. This study represents the first approach combining G4 ligands, BRACO-19 (B19), pyridostatin (PDS), and the adenovirus dl922-947 in breast cancer cells. We demonstrated that G4 binders and dl922-947 induce cytotoxicity in breast cancer cells (MDA-MB-231 and MCF-7) and at higher doses in other neoplastic cell lines of thyroid (BHT-101 cells) and prostate (PC3 cells). G4 binders induce G4 motifs distributed in the S and G2/M phases in MCF-7 cells. G4 binder/dl922-947 combination increases cell cytotoxicity and the accumulation in subG0/G1. Indeed, G4 binders favor viral entry and replication with no effect on coxsackie and adenovirus receptor. Notably, dl922-947 induces G4 motifs and its combination with PDS potentiates this effect in MCF-7 cells. The agents alone or in combination similarly enhanced cell senescence. Additionally, PDS/dl922-947 combination inactivates STING signaling in MDA-MB-231 cells. Our results suggest that G4 binder/virotherapy combination may represent a novel therapeutic anticancer approach.

Keywords: G-quadruplex; STING; adenovirus; breast cancer; oncolytic virus; senescence.

Figure 1

G4 motif content and distribution in cell cycle phases. (A) MDA-MB231 and MCF-7 cells were treated with G4 ligands at the IC₅₀ values. The dot plot profiles in the upper fluorescein isothiocyanate (FITC) gated panel indicate the amount of positivity for G4 motif. The x axis reports PI positivity to specifically stain cell DNA. In the dot plot reported, the percent of positive cells for each experimental conditions is indicated in the gated region (R) for a single representative experiment, whereas the histograms (B) represent the mean ± SD of at least three independent experiments. The statistical analysis was performed by GraphPad Prism 7 by two-way ANOVA using Dunnet’s multiple comparisons test. In the panel (C), G4 motif distribution is reported by evaluation of PI positive cells (PI-W versus PI-H dot plot) in each phase of the cell cycle as the percent of G4 motif positivity. The statistical significance was calculated by GraphPad Prism 7 with two-way ANOVA using Tukey’s multiple comparisons test. The calculated p values are: *** p < 0.0001, ** p < 0.005, * p < 0.05.

Figure 2

Cytotoxic effects of G4 ligands/dl922-947 combination and regulation of the cell cycle progression. Cytotoxic effect of G4 ligands with dl922-947 determined by SRB assays in MDA-MB-231 and MCF-7 cells after six days of treatment (A). Figures report cell death vs. untreated cells as mean of three independent experiments generated with IC₂₅ and IC₅₀ values of PDS and B19 (in µM) and IC₅₀ value of dl922-947 (in p.f.u.). The black bars represent the treatment with G4 binders alone, whereas the gray bars report the combination of dl922-947 (at the IC₅₀) with G4 ligands at the IC₂₅ and/or IC₅₀. Statistical significance was calculated by GraphPad Prism 7 with two-way ANOVA using Tukey’s multiple comparison test. * calculated versus untreated cells; $ calculated vs. PDS alone (IC₂₅ and/or IC₅₀); & calculated versus dl922-947 alone; (*^& p < 0.05, ** p < 0.005, *** p < 0.0005, ****^,$ p < 0.0001). MDA-MB-231 (B) and MCF-7 (C) cells were treated with dl922-947, G4 ligands or their combination at the IC₅₀ values. After 6 days of treatment, cells were stained with PI. A representative flow cytometry profile of the cell cycle is shown for both cell lines, and the percent of cells in each phase of the cell cycle is indicated for the single experiment, whereas the bars in the histograms represent the mean ± SD of the least three independent experiments. The statistical significance was calculated by GraphPad Prism 7 with two-way ANOVA using Tukey’s multiple comparisons test (& p < 0.0001, ** p < 0.005, * p < 0.05).

Figure 2

Cytotoxic effects of G4 ligands/dl922-947 combination and regulation of the cell cycle progression. Cytotoxic effect of G4 ligands with dl922-947 determined by SRB assays in MDA-MB-231 and MCF-7 cells after six days of treatment (A). Figures report cell death vs. untreated cells as mean of three independent experiments generated with IC₂₅ and IC₅₀ values of PDS and B19 (in µM) and IC₅₀ value of dl922-947 (in p.f.u.). The black bars represent the treatment with G4 binders alone, whereas the gray bars report the combination of dl922-947 (at the IC₅₀) with G4 ligands at the IC₂₅ and/or IC₅₀. Statistical significance was calculated by GraphPad Prism 7 with two-way ANOVA using Tukey’s multiple comparison test. * calculated versus untreated cells; $ calculated vs. PDS alone (IC₂₅ and/or IC₅₀); & calculated versus dl922-947 alone; (*^& p < 0.05, ** p < 0.005, *** p < 0.0005, ****^,$ p < 0.0001). MDA-MB-231 (B) and MCF-7 (C) cells were treated with dl922-947, G4 ligands or their combination at the IC₅₀ values. After 6 days of treatment, cells were stained with PI. A representative flow cytometry profile of the cell cycle is shown for both cell lines, and the percent of cells in each phase of the cell cycle is indicated for the single experiment, whereas the bars in the histograms represent the mean ± SD of the least three independent experiments. The statistical significance was calculated by GraphPad Prism 7 with two-way ANOVA using Tukey’s multiple comparisons test (& p < 0.0001, ** p < 0.005, * p < 0.05).

Figure 2

Cytotoxic effects of G4 ligands/dl922-947 combination and regulation of the cell cycle progression. Cytotoxic effect of G4 ligands with dl922-947 determined by SRB assays in MDA-MB-231 and MCF-7 cells after six days of treatment (A). Figures report cell death vs. untreated cells as mean of three independent experiments generated with IC₂₅ and IC₅₀ values of PDS and B19 (in µM) and IC₅₀ value of dl922-947 (in p.f.u.). The black bars represent the treatment with G4 binders alone, whereas the gray bars report the combination of dl922-947 (at the IC₅₀) with G4 ligands at the IC₂₅ and/or IC₅₀. Statistical significance was calculated by GraphPad Prism 7 with two-way ANOVA using Tukey’s multiple comparison test. * calculated versus untreated cells; $ calculated vs. PDS alone (IC₂₅ and/or IC₅₀); & calculated versus dl922-947 alone; (*^& p < 0.05, ** p < 0.005, *** p < 0.0005, ****^,$ p < 0.0001). MDA-MB-231 (B) and MCF-7 (C) cells were treated with dl922-947, G4 ligands or their combination at the IC₅₀ values. After 6 days of treatment, cells were stained with PI. A representative flow cytometry profile of the cell cycle is shown for both cell lines, and the percent of cells in each phase of the cell cycle is indicated for the single experiment, whereas the bars in the histograms represent the mean ± SD of the least three independent experiments. The statistical significance was calculated by GraphPad Prism 7 with two-way ANOVA using Tukey’s multiple comparisons test (& p < 0.0001, ** p < 0.005, * p < 0.05).

Figure 3

Adenovirus AdGFP entry, dl922-947 replication, and CAR modulation by G4 binders in breast cancer cells. MDA-MB-231 (A) and MCF-7 (B) cells were infected with the non-replicating AdGFP and treated with G4 binders at different times. AdGFP and G4 binders were added concomitantly (24 h after seeding the cells black bars A and B); or AdGFP was added 24 h after G4 binders (AdGFP was added 48 h after seeding cells, gray bars A and B). GFP emission was evaluated by flow cytometry and flow cytometry profile of a representative experiment is reported. The statistical significance was calculated by GraphPad Prism 7 with two-way ANOVA using Tukey’s multiple comparisons test (** p < 0.0001, * p < 0.05). The statistical significance is indicated in the histograms that represent the mean of at least three independent experiments. To evaluate viral replication, MDA-MB-231 (C) and MCF-7 cells (D) were treated with dl922-947 (at the IC₅₀ value) or AdGFP (30 p.f.u. x cell, used as control at 48 hpi, not shown) in combination with G4 ligands (at their IC₅₀ values). After 24 and 48 h of incubation, supernatants and adherent cells were collected separately to evaluate extracellular release and intracellular viral particles, respectively. Viral DNA was extracted and used to quantify viral titer by Real-Time qPCR. The data represent the mean of three independent experiments (C). Statistical significance was calculated comparing the increased intracellular amplification with respect to the correspondent extracellular fraction (* p < 0.05, ** p < 0.005, *** p < 0.0001). To evaluate CAR modulation by G4 binders, MDA-MB-231 (E) and MCF-7 cells (F) were treated with G4 binders (at the IC₅₀ values) for 48 h. The images represent the expression of CAR evaluated by Western blot. As control, α-tubulin was used. Original blots are enclosed as supplemental Figure S5 for CAR. The histogram reports the mean of two independent experiments. The statistical significance was calculated by parametric paired two tailed T test, by GraphPad Prism 7 (* p < 0.05).

Figure 3

Adenovirus AdGFP entry, dl922-947 replication, and CAR modulation by G4 binders in breast cancer cells. MDA-MB-231 (A) and MCF-7 (B) cells were infected with the non-replicating AdGFP and treated with G4 binders at different times. AdGFP and G4 binders were added concomitantly (24 h after seeding the cells black bars A and B); or AdGFP was added 24 h after G4 binders (AdGFP was added 48 h after seeding cells, gray bars A and B). GFP emission was evaluated by flow cytometry and flow cytometry profile of a representative experiment is reported. The statistical significance was calculated by GraphPad Prism 7 with two-way ANOVA using Tukey’s multiple comparisons test (** p < 0.0001, * p < 0.05). The statistical significance is indicated in the histograms that represent the mean of at least three independent experiments. To evaluate viral replication, MDA-MB-231 (C) and MCF-7 cells (D) were treated with dl922-947 (at the IC₅₀ value) or AdGFP (30 p.f.u. x cell, used as control at 48 hpi, not shown) in combination with G4 ligands (at their IC₅₀ values). After 24 and 48 h of incubation, supernatants and adherent cells were collected separately to evaluate extracellular release and intracellular viral particles, respectively. Viral DNA was extracted and used to quantify viral titer by Real-Time qPCR. The data represent the mean of three independent experiments (C). Statistical significance was calculated comparing the increased intracellular amplification with respect to the correspondent extracellular fraction (* p < 0.05, ** p < 0.005, *** p < 0.0001). To evaluate CAR modulation by G4 binders, MDA-MB-231 (E) and MCF-7 cells (F) were treated with G4 binders (at the IC₅₀ values) for 48 h. The images represent the expression of CAR evaluated by Western blot. As control, α-tubulin was used. Original blots are enclosed as supplemental Figure S5 for CAR. The histogram reports the mean of two independent experiments. The statistical significance was calculated by parametric paired two tailed T test, by GraphPad Prism 7 (* p < 0.05).

Figure 4

Induction of G4 motif content and distribution in cell cycle phases. G4 motif induction was evaluated by flow cytometry. MCF-7 and MDA-MB-231 cells were treated with dl922-947, G4 binders, or their combination at the IC₅₀ values for 6 days. The histograms represent the mean ± SD of at least three independent experiments (A). Gating strategies were adopted to select in each phase of the cell cycle the percent of G4 structure positivity in MCF-7 cells (B). The statistical significance was calculated by GraphPad Prism 7 with two-way ANOVA using Sidak’s multiple comparisons test (* p < 0.001, & p < 0.0001; £ p < 0.05; * = statistical significance calculated vs. ctr, & = statistical significance calculated vs. PDS, £ = statistical significance calculated vs. dl922-947).

Figure 5

Induction of cell senescence. MDA-MB-231 and MCF-7 cells were treated for 72 h with dl922-947, G4 binders, or their combination at the IC₅₀ values. Senescent cells are indicated in the images by the arrows as blue SA-b-Gal positive stained cells. Doxorubicin was used as positive control. The images are representative of three independent experiments. The statistical significance indicated in the histograms was calculated by GraphPad Prism 7 with two-way ANOVA with Tukey’s multiple comparisons test (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05).

Figure 6

Expression of STING in breast cancer cells. The image represents the expression of STING evaluated by Western blot in MDA-MB-231 cells (A) and MCF-7 cells (B) treated for 24 h with G4 binders, dl922-947, and their combination at the IC₅₀. As control, α-tubulin was used. The histogram reports the mean of two or three independent experiments, respectively, for MDA-MB-231 and MCF-7 cells. Original blots are enclosed as supplemental Figure S6 for STING. The statistical significance was calculated by parametric paired two tailed t test, by GraphPad Prism 7 (* p < 0.05).