Chemotherapeutic potential of diazeniumdiolate-based aspirin prodrugs in breast cancer

Abstract

Diazeniumdiolate-based aspirin prodrugs have previously been shown to retain the anti-inflammatory properties of aspirin while protecting against the common side effect of stomach ulceration. Initial analysis of two new prodrugs of aspirin that also release either nitroxyl (HNO) or nitric oxide (NO) demonstrated increased cytotoxicity toward human lung carcinoma cells compared to either aspirin or the parent nitrogen oxide donor. In addition, cytotoxicity was significantly lower in endothelial cells, suggesting cancer-specific sensitivity. To assess the chemotherapeutic potential of these new prodrugs in treatment of breast cancer, we studied their effect both in cultured cells and in a nude mouse model. Both prodrugs reduced growth of breast adenocarcinoma cells more effectively than the parent compounds while not being appreciably cytotoxic in a related nontumorigenic cell line (MCF-10A). The HNO donor also was more cytotoxic than the related NO donor. The basis for the observed specificity was investigated in terms of impact on metabolism, DNA damage and repair, apoptosis, angiogenesis and metastasis. The results suggest a significant pharmacological potential for treatment of breast cancer.

Keywords: Anti-inflammatory; Anticancer; Aspirin; DEA/NO; Diazeniumdiolate; IPA/NO; NONOate; NSAID; Nitric oxide; Nitroxyl.

Figure 1

Effect of NONO-aspirin prodrugs (10–200 μM), aspirin (50–200 μM) or parent diazeniumdiolates (200 μM) on the viability of A) MDA-MB-231, B) MDA-MB-468, C) MCF-7 or D) MCF-10A cells. Cells were treated for 48 h at 37 °C, and cell survival was determined using the MTT assay. Figures are representative of three trials with data plotted as mean percentage versus untreated cells ± SD of the four replicates per plate). Vehicle (DMSO or 10 mM NaOH) was added as a control to otherwise untreated cells.

Figure 1

Effect of NONO-aspirin prodrugs (10–200 μM), aspirin (50–200 μM) or parent diazeniumdiolates (200 μM) on the viability of A) MDA-MB-231, B) MDA-MB-468, C) MCF-7 or D) MCF-10A cells. Cells were treated for 48 h at 37 °C, and cell survival was determined using the MTT assay. Figures are representative of three trials with data plotted as mean percentage versus untreated cells ± SD of the four replicates per plate). Vehicle (DMSO or 10 mM NaOH) was added as a control to otherwise untreated cells.

Figure 2

Effect of NONO-aspirin prodrugs or aspirin in nude mice implanted with 7.5 × 10⁵ MDA-MB-231 cells stably transfected with GFP. The cells were allowed to grow for 14 d, and the animals (40) were randomly divided into four groups (control, aspirin, IPA/NO-aspirin or DEA/NO-aspirin). Treated groups were injected daily with equimolar doses of DEA/NO-aspirin or IPA/NO-aspirin (16 mg/kg) or aspirin (9 mg/kg) or with DMSO for the next five weeks. Tumor size was then measured using in vivo fluorescent imaging for quantification of the GFP tag: A) qualitative image of individual animals, B) quantitative analysis of fluorescence intensity at the primary tumor site (n = 5), C) qualitative image of individual brains, D) quantitative analysis of fluorescence intensity due to metastasis to the brain (n = 7). *p < 0.001 vs. control.

Figure 3

Effect of NONO-prodrugs on inhibition of GAPDH in A) MDA-MB-231 or B) MCF-10A cells. The cells were treated with varied concentrations (0, 25, 50, 75, 100 μM) of IPA/NO-aspirin (blue) or DEA/NO-aspirin (green) for 1 h at 37 °C. GAPDH activity was assessed by measuring fluorescence intensity at 570 nm. Data are plotted as mean ± SD (n = 3).

Figure 4

Effect of NONO-aspirin prodrugs, aspirin or parent diazeniumdiolates (100 μM) on oxidation of DCF in MDA-MB-231 cells. DCF loaded cells were treated with IPA/NO-aspirin (blue), DEA/NO-aspirin (green), IPA/NO (magenta), DEA/NO (brown), aspirin (red) or DMSO (0.1%; black) at 37 °C, and the fluorescence at 535 nm was measured in a time-dependent manner. Data are plotted as mean ± SD (n = 4).

Figure 5

Effect of the NONO-aspirin prodrugs on DNA damage in MDA-MB-231 cells as determined using the Comet assay after 8 h at 37 °C: A) 50 μM IPA/NO-aspirin, B) 75 μM DEA/NO-aspirin, C) 0.1% DMSO or D) 100 μM H₂O₂. Tail length is quantified in E (mean ± SEM, n ≥ 150).

Figure 5

Effect of the NONO-aspirin prodrugs on DNA damage in MDA-MB-231 cells as determined using the Comet assay after 8 h at 37 °C: A) 50 μM IPA/NO-aspirin, B) 75 μM DEA/NO-aspirin, C) 0.1% DMSO or D) 100 μM H₂O₂. Tail length is quantified in E (mean ± SEM, n ≥ 150).

Figure 6

Effect of NONO-aspirin prodrugs on caspase-3 activity of A) MDA-MB-231, B) MDA-MB-468 or C) MCF-7 cells. The cells were treated with various concentrations (0, 25, 50, 100 μM) of IPA/NO-aspirin (blue) or DEA/NO-aspirin (green) for 24 h at 37 °C. Caspase-3 activity was determined by recording the fluorescence (λ_ex 485 nm, λ_em 535 nm) of the product formed by cleavage of the substrate N-Ac-DEVD-N′-MC-R11. Data are plotted as mean ± SD (n = 3).

Figure 6

Effect of NONO-aspirin prodrugs on caspase-3 activity of A) MDA-MB-231, B) MDA-MB-468 or C) MCF-7 cells. The cells were treated with various concentrations (0, 25, 50, 100 μM) of IPA/NO-aspirin (blue) or DEA/NO-aspirin (green) for 24 h at 37 °C. Caspase-3 activity was determined by recording the fluorescence (λ_ex 485 nm, λ_em 535 nm) of the product formed by cleavage of the substrate N-Ac-DEVD-N′-MC-R11. Data are plotted as mean ± SD (n = 3).

Figure 7

Effect of NONO-aspirin prodrugs on PARP cleavage in MDA-MB-231 cells. The cells were treated with 50 μM IPA/NO-aspirin (blue) or 75 μM DEA/NO-aspirin (green), respectively at 37 °C, and protein was collected at various time points (1–44 h). Lanes 1–6 (20 μg of protein): IPA/NO-aspirin at 1, 3, 6, 10, 20, 44 h; Lanes 6–12: DEA/NO-aspirin at 1, 6, 10, 20, 30, 44 h; Lane 15: control. Cleaved PARP levels at different time points were quantified by Western blot with respect to control, which was treated as 100%. Protein levels are normalized to the loading control HPRT.

Figure 8

Effect of NONO-aspirin prodrugs on p53 phosphorylation in MDA-MB-231 cells. The cells were treated with 50 μM IPA/NO-aspirin (blue) or 75 μM DEA/NO-aspirin (green), respectively at 37 °C, and protein was collected at various time points (1–44 h). P-p53 was quantified by Western blot. Lanes 1–6 (20 μg of protein): IPA/NO-aspirin at 1, 3, 6, 10, 20, 44 h; Lanes 6–12: DEA/NO-aspirin at 1, 6, 10, 20, 30, 44 h; Lane 15: control. p-P53 protein at different time points was quantified by Western blot with respect to control, which was treated as 100%. Protein levels are normalized to the loading control HPRT.

Figure 9

Effect of NONO-aspirin prodrugs on inhibition of angiogenesis in HUVECs. Cells in Matrigel were treated with A) DMSO, B) 1 μM or C) 10 μM IPA/NO-aspirin, D) 1 μM or E) 10 μM DEA/NO-aspirin at 37 °C, and the extent of tube formation was measured after 12 h using a microscope.

Figure 10

Effect of NONO-aspirin prodrugs on E-cadherin expression in MDA-MB-231 cells. The cells were treated with vehicle (black) or 100 μM IPA/NO-aspirin (blue) or DEA/NO-aspirin (green) for 3, 6 or 12 h at 37 °C. Relative A) mRNA or B) protein expression was measured using RT-PCR and Western blot techniques, respectively. E-cadherin mRNA and protein levels were quantified with respect to control (HPRT), which was treated as 1 for mRNA and 100% for protein. In A, data are plotted as mean ± SD (n = 3).

Figure 10

Effect of NONO-aspirin prodrugs on E-cadherin expression in MDA-MB-231 cells. The cells were treated with vehicle (black) or 100 μM IPA/NO-aspirin (blue) or DEA/NO-aspirin (green) for 3, 6 or 12 h at 37 °C. Relative A) mRNA or B) protein expression was measured using RT-PCR and Western blot techniques, respectively. E-cadherin mRNA and protein levels were quantified with respect to control (HPRT), which was treated as 1 for mRNA and 100% for protein. In A, data are plotted as mean ± SD (n = 3).

Scheme 1

Structures of IPA/NO-aspirin and DEA/NO-aspirin