Nitric oxide-donor/PARP-inhibitor combination: A new approach for sensitization to ionizing radiation

Abstract

Recently, clinical development of PARP inhibitors (PARPi) expanded from using them as a single agent to combining them with DNA-damaging therapy to derive additional therapeutic benefit from stimulated DNA damage. Furthermore, inhibiting PARP in cancers with BRCA1/2 mutations has been shown to be an effective synthetic lethality approach either as a single agent or in combination with the different DNA damaging agents: chemotherapy or ionizing radiation (IR). However, inherited BRCA1/2 mutations account only for 5-10% of breast cancers, 10-15% of ovarian cancers, and lesser for the other cancers. Hence, for most of the cancer patients with BRCA1/2-proficient tumors, sensitization to DNA-damaging agents with PARPi is significantly less effective. We recently demonstrated that moderate, non-toxic concentrations of NO-donors inhibited BRCA1 expression, with subsequent inhibition of error-free HRR and increase of error-prone non-homologous end joining (NHEJ). We also demonstrated that the effect of NO-dependent block of BRCA1 expression can only be achieved in the presence of oxidative stress, a condition that characterizes the tumor microenvironment and is also a potential effect of IR. Hence, NO-donors in combination with PARPi, with effects limited by tumor microenvironment and irradiated area, suggest a precise tumor-targeted approach for radio-sensitization of BRCA1/2-proficient tumors. The combination with NO-donors allows PARPi to be successfully applied to a wider variety of tumors. The present work demonstrates a new drug combination (NO-donors and PARP-inhibitors) which demonstrated a high potency in sensitization of wide variety of tumors to ionizing radiation treatment.

Keywords: Homologous recombination repair; Ionizing radiation; Nitric oxide donors; PARP inhibitors; Synthetic lethality.

Graphical abstract

Fig. 1

Dose-dependent downregulation of BRCA1 protein by NO-donors: DETA and SNAP. (A) A-549 and H-1299 cells were incubated with different concentrations of DETA and SNAP. After 12 h of incubation, cells were lysed and total cell lysates were probed for antibodies against BRCA1 and β-Tubulin (as a loading control). (B) Analysis of WB results of three independent experiments. Results were expressed as fold changes of control. Experimental data are presented as the mean ± SD. The P-value was calculated with the Student t-test and shown as: *– p < 0.01.

Fig. 2

Combined treatment of NO-donors (SNAP, DETA) and PARP inhibitor ABT-888 with IR. (A) Clonogenic analysis for A-549 and H-1299 cells treated with NO-donors (SNAP and DETA) with or without IR (4Gy). (B) Clonogenic analysis for A-549 and H-1299 cells treated with the combination of ABT-888 and IR (4Gy). Experimental data are presented as the mean ± SD for quadruplicate samples. The P-value was calculated with the Student t-test and shown as: **– p < 0.05 and ***– p < 0.001.

Fig. 3

Combining treatment of NO-donors (SNAP or DETA) with PARP-inhibitor ABT-888 induces sensitization to IR. A-549 and H-1299 cells were pretreated 4 h before IR with 200 μM of SNAP, 100 μM of DETA, 10 μM of ABT-888 or the combination of NO donor and ABT-888. Controls were pretreated with vehicle (DMSO). (A) Experimental data are presented as the mean ± SD for quadruplicate samples. (B) Experimental data was normalized to Vehicle control. Sensitization by ABT-888/SNAP and ABT-888/DETA combinations was compared with sensitization activity of ABT-888 for the different radiation doses. The P-value was calculated with the Student t-test and shown as: **– p < 0.05 and ***– p < 0.001.

Fig. 4

Effects of pre-treatment with ABT-888, DETA and their combination on IR-induced γ-H2AX foci formation and DNA double-strand breaks (DSBs). A-549 and H-1299 cells were treated with 100 μM of DETA, 10 μM of ABT-888 or combination of drugs followed 4 h later by a single IR exposure. (A) γ-H2AX foci formation was analyzed using the immunofluorescence assay for A-549 and H-1299 cells at non-irradiated controls, 4 h, and 24 h after a single IR exposure. (B) Level of DNA DSBs was measured by Neutral Comet Assay for A-549 and H-1299 cells at non-irradiated controls and 24 h after a single IR exposure. (C) Experimental data of three independent Neutral Comet Assays are represented as the mean ± SD. At least 50–60 comets were scored for each sample and analyzed using the ImageJ software with the OpenComet plugin. The P-value was calculated with the Student t-test and shown as: **– p < 0.05 and ***– p < 0.001.

Fig. 5

Combination of DETA with ABT-888 enhances IR-stimulated apoptotic cell death. (A) Flow cytometric analysis of apoptosis in A-549 cell lines at different time-points after 4Gy IR using Annexin V-fluorescein isothiocyanate (FITC)/Propidium iodide (PI) assay. Quadrants: Q3 (normal cells), Q4 (early apoptotic cells), Q2 (late apoptotic/necrotic cells). Numbers represent a percentage of cells in the relevant quadrants. (B) Summary of flow cytometry analysis for A-549 and H-1299 cell lines. Columns represent the means ± SD values for apoptotic cells (Q2+Q4) obtained from three individual experiments. The P-value was calculated with the Student t-test and shown as: **– p < 0.05 and ***– p < 0.001.

Fig. 6

Sensitization to IR by NO-donor/PARP-inhibitor combination is BRCA1-dependent. A-549 and H-1299 cells were transfected with corresponding siRNA and 24 h after transfection cells were trypsinized and one part of the cells were subjected to clonogenic assay (as described in Fig. 3), the second part was subjected for Annexin V-fluorescein isothiocyanate (FITC)/PI assay (as described in Fig. 5), and the last part was lysed and total cell lysates were probed for antibodies against BRCA1, RBL2, and β-Tubulin (as a loading control). Transfection with AllStars siRNA was used as a negative control; (A) Block of RBL2 expression significantly reduced effect of sensitization to IR by DETA/ABT-888 combination. Inserted Western blots and graphs demonstrates RBL2 protein expression downregulation by siRNA transfection. Graphs represent WB results of three independent experiments. Results were expressed as fold changes of control. Experimental data are presented as the mean ± SD; (B) Block of BRCA1 expression significantly stimulated effect of sensitization to IR by ABT-888 pretreatment. Inserted Western blots and graphs demonstrate effect of BRCA1 protein downregulation by siRNA transfection. Graphs represent WB results of three independent experiments. Results were expressed as fold changes of control. Experimental data are presented as the mean ± SD (C) Summary of flow cytometric analysis of apoptosis for A-549 and H-1299 cell lines: non-irradiated controls and 72 h after 4Gy of IR (as shown in Fig. 5). Columns represent the means ± SD values for apoptotic cells obtained from three individual experiments. The P-value was calculated with the Student t-test and shown as: **– p < 0.05 and ***– p < 0.001.

Fig. 7

A proposed stimulation of synthetic lethality by the combination of NO-donor and PARP-inhibitor.