Influence of 2-Nitroimidazoles in the Response of FaDu Cells to Ionizing Radiation and Hypoxia/Reoxygenation Stress

Abstract

Cellular adaptations to hypoxia promote resistance to ionizing radiation (IR). This presents a challenge for treatment of head and neck cancer (HNC) that relies heavily on radiotherapy. Standard radiosensitizers often fail to reach diffusion-restricted hypoxic cells, whereas nitroimidazoles (NIs) [such as iodoazomycin arabinofuranoside (IAZA) and fluoroazomycin arabinofuranoside (FAZA)] can preferentially accumulate in hypoxic tumours. Here, we explored if the hypoxia-selective uptake of IAZA and FAZA could be harnessed to make HNC cells (FaDu) susceptible to radiation therapy. Cellular response to treatment was assessed through clonogenic survival assays and by monitoring DNA damage (immunofluorescence staining of DNA damage markers, γ-H2AX and p-53BP1, and by alkaline comet assay). The effects of reoxygenation were studied using the following assays: estimation of nucleoside incorporation to assess DNA synthesis rates, immunofluorescent imaging of chromatin-associated replication protein A as a marker of replication stress, and quantification of reactive oxygen species (ROS). Both IAZA and FAZA sensitized hypoxic HNC cells to IR, albeit the former is a better radiosensitizer. Radiosensitization by these compounds was restricted only to hypoxic cells, with no visible effects under normoxia. IAZA and FAZA impaired cellular adaptation to reoxygenation; high levels of ROS, reduced DNA synthesis capacity, and signs of replication stress were observed in reoxygenated cells. Overall, our data highlight the therapeutic potentials of IAZA and FAZA for targeting hypoxic HNC cells and provide rationale for future preclinical studies.

Keywords: DNA damage; head and neck cancer; hypoxia; nitroimidazole; radiosensitizer.

Figure 1

IAZA and FAZA sensitize hypoxic FaDu cells to IR. (A,B) Chemical structures for IAZA and FAZA. (C) Successful induction of hypoxia was confirmed by probing for HIF-1α. (D) IAZA and FAZA significantly reduced clonogenicity in hypoxic irradiated cells. (E,F) Sensitization by IAZA and FAZA appear to be synergistic at higher radiation dosage. The calculated sensitizer enhancement ratios (SER) for IAZA and FAZA are indicated. Data show mean from at least three independent experiments; error bars represent standard error of the mean (S.E.M.).

Figure 2

Co-treatment of IAZA or FAZA with 5 Gy IR increases DNA damage only under hypoxia. (A) Representative micrographs showing immunofluorescent staining of γ-H2AX in FaDu cells treated with vehicle control (DMSO) or drugs (IAZA or FAZA; 100 µM), with or without IR (5 Gy), under normoxia and hypoxia (<0.1% O₂); scale bar = 20 µm. (B) Representative histograms showing quantification of γ-H2AX staining intensity; dotted line shows the threshold for background levels of γ-H2AX staining. (C) Treatment with IAZA and FAZA increases the percentage of γ-H2AX-positive population in hypoxic irradiated samples. (D) Alkaline comet assay shows significant increase in DNA damage in hypoxic drug-treated irradiated cells. Data show mean ± S.E.M. (C,D). The no-radiation arms of these data were previously reported in ref. [15] and are used here for comparison purposes.

Figure 3

IAZA-/FAZA-treated hypoxic cells display high levels of γ-H2AX after reoxygenation, but not a corresponding increase in DNA damage. (A) Representative micrographs showing immunofluorescent staining of γ-H2AX and phopho-53BP1 in FaDu cells treated with vehicle control (DMSO) or drugs (IAZA or FAZA; 100 µM), with or without IR (5 Gy), under normoxia and hypoxia (<0.1% O₂), followed by a 24 h reoxygenation and recovery period; scale bar = 20 µm. (B) Drug-treated reoxygenated cells showed increased percentage of γ-H2AX-positive population, but no increase in comet tail moment (C). (D) No effects were seen on total PP2A protein levels in hypoxia/reoxygenation group when cells were treated with IAZA or FAZA. (E) Quantification of PP2A immunoblots. Data show mean ± S.E.M from three independent replicates.

Figure 4

IAZA-/FAZA-treated hypoxic cells display signs of replication stress upon reoxygenation. (A) Representative micrographs showing click-EdU incorporation rates in FaDu cells treated with vehicle control (DMSO) or drugs (IAZA or FAZA; 100 µM) under normoxia and hypoxia (<0.1% O₂), followed by a 24 h reoxygenation and recovery period. (B) Intensity of EdU click-stained micrographs was quantified and plotted as intensity versus %population histograms. Hypoxic exposure by itself increased cell population with low EdU staining; incubation with IAZA and FAZA under hypoxia further increased “low EdU stained” cell fractions. (C) Drug-treated reoxygenated cells showed higher levels of detergent extraction resistant chromatin-bound RPA. (D) Quantification of nuclear RPA staining. Data show mean ± S.E.M. from three independent replicates; scale bar = 20 µm.

Figure 5

Reoxygenation in the presence of NAC reduced γ-H2AX and RPA-positive populations in drug-treated hypoxic cells. (A) Representative micrographs showing a reduction in γ-H2AX and RPA staining in response to NAC incubation during reoxygenation; scale bar = 20 µm. (B) Representative histograms showing quantification of γ-H2AX staining intensity; dotted line shows the threshold for background levels of γ-H2AX staining. (C) Percentage of γ-H2AX-positive population in hypoxia/reoxygenated cells treated with IAZA or FAZA (100 μM) was decreased in the NAC-treated group; data show mean ± S.E.M. from two independent replicates. Quantification of γ-H2AX (D) and RPA (E) staining intensity are shown; a minimum of 125 cells were analyzed for each condition.