Identification of proteins and cellular pathways targeted by 2-nitroimidazole hypoxic cytotoxins

Abstract

Tumour hypoxia negatively impacts therapy outcomes and continues to be a major unsolved clinical problem. Nitroimidazoles are hypoxia selective compounds that become entrapped in hypoxic cells by forming drug-protein adducts. They are widely used as hypoxia diagnostics and have also shown promise as hypoxia-directed therapeutics. However, little is known about the protein targets of nitroimidazoles and the resulting effects of their modification on cancer cells. Here, we report the synthesis and applications of azidoazomycin arabinofuranoside (N₃-AZA), a novel click-chemistry compatible 2-nitroimidazole, designed to facilitate (a) the LC-MS/MS-based proteomic analysis of 2-nitroimidazole targeted proteins in FaDu head and neck cancer cells, and (b) rapid and efficient labelling of hypoxic cells and tissues. Bioinformatic analysis revealed that many of the 62 target proteins we identified participate in key canonical pathways including glycolysis and HIF1A signaling that play critical roles in the cellular response to hypoxia. Critical cellular proteins such as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the detoxification enzyme glutathione S-transferase P (GSTP1) appeared as top hits, and N₃-AZA adduct formation significantly reduced their enzymatic activities only under hypoxia. Therefore, GAPDH, GSTP1 and other proteins reported here may represent candidate targets to further enhance the potential for nitroimidazole-based cancer therapeutics.

Keywords: Click chemistry; Head and neck tumour; Hypoxia; Nitroimidazole; Proteomics.

Fig. 1

N₃-AZA synthesis, cytotoxicity and click chemistry principle to isolate 2-NI target proteins. (A) Synthesis of N₃-AZA. Reagents and conditions: (a) Ts-AZA, NaN₃, DMSO, 50 °C, overnight, 69%; (b) IAZA, NaN₃, DMF, 100 °C, 2 h, 93%. N₃-AZA shows preferential cytotoxicity in hypoxic FaDu (B), A549 (C), A172 (D) and PC3 cells (E), with statistically significant differences between their normoxic and hypoxic IC₅₀ values. Data represents mean ± S.E.M. from at least three independent experiments. (F) Experimental design for isolation and visualization of N₃-AZA bound proteins. (G) Click chemistry was performed on cell extracts collected from N₃-AZA (or DMSO) treated normoxic and hypoxic FaDu cells using a biotin alkyne. Western blotting showed that the signal for Streptavidin-HRP is only present in drug treated hypoxic samples. Drug bound proteins could successfully be isolated using streptavidin-mutein beads, with no significant background binding. Representative immunoblots are displayed from three independent experiments.

Fig. 2

Mass spectrometric analysis of N₃-AZA target proteins. (A) Venn diagram showing the distribution of proteins identified by mass spectroscopic analysis based on the different treatment conditions. (B) Comparison of PSM values for proteins identified in eluates from N₃-AZA treated normoxic and hypoxic cells. Data represent cumulative averages for each protein from three independent experiments. (C) Enrichment analysis demonstrated that the likelihood N₃-AZA labelling is generally dependent on the abundance of target proteins. (D and E) Upstream regulatory analysis by IPA identified 2 clusters of 8 proteins, each under the regulation of a common upstream regulator HSF1 (D) or HIF1A (E). 5 of HSF1 downstream targets are implicated in protein folding while 7 of HIF1A downstream targets are involved in carbohydrate metabolism.

Fig. 3

Effects of N₃-AZA on GAPDH and GSTP1 protein levels, GAPDH localization and their enzymatic activity. (A) Lysates prepared from N₃-AZA (or 0.02% DMSO) treated normoxic and hypoxic FaDu cells were processed for western blotting. N₃-AZA treatment did not alter GAPDH and GSTP1 protein levels regardless of O₂ conditions. Representative immunoblots and quantitation [mean ± S.E.M.] from three independent experiments are displayed. (B) Cells treated with N₃-AZA (or 0.02% DMSO) under normoxia and hypoxia were processed for immunocytochemistry to monitor GAPDH localization; no change in cellular localization of GAPDH was observed in response to N₃-AZA treatment. The micrographs are representative of at least three independent experiments; scale bar = 20 μm. (C and D) FaDu cells treated with N₃-AZA (or 0.02% DMSO) under normoxia and hypoxia were processed for GAPDH activity assay (C) or GST activity assay (D); the enzymatic activities of GAPDH and GST were significantly reduced only in N₃-AZA treated hypoxic cells. Data represent mean ± S.E.M. from three independent experiments.

Fig. 4

N₃-AZA click chemistry as a hypoxia marker. (A–D) FaDu cells, treated with different concentration of N₃-AZA (or 0.02% DMSO vehicle control) were incubated under normoxia or hypoxia (0.1% O₂ or <0.1% O₂), and click chemistry was performed on paraformaldehyde fixed cells using a fluorescently tagged alkyne. N₃-AZA click staining was present only in drug treated hypoxic cells. Intensity of N₃-AZA click staining increased with drug concentration and decreased with O₂ levels. (E) N₃-AZA click staining is concentrated in nucleoli. Micrographs displayed are representative of at least three independent experiments; scale bar = 20 μm.

Fig. 5

Pimonidazole immunostaining is comparable to that of N₃-AZA. (A) Representative micrographs from three independent experiments showing that N₃-AZA click staining and pimonidazole immunostaining overlaps in hypoxic FaDu cells co-treated with both compounds. (B) The micrographs were processed with IMARIS software to quantify channel intensities from N₃-AZA click staining and pimonidazole immunostaining. The ratios of signal (hypoxia):background (normoxia) intensities for cells co-treated with N₃-AZA and pimonidazole [or vehicle control (0.02% DMSO) i.e. columns labelled 0 μM, see Fig. S7 for micrographs of cells not treated with N₃-AZA or pimonidazole] are shown (mean ± S.E.M.). N₃-AZA click staining generated a higher signal to noise ratio compared to pimonidazole immunostaining. (C–F) In vivo comparison of N₃-AZA click staining with pimonidazole immunostaining. Both are concentrated in the same regions of a mouse subcutaneous tumour section (C–E) and of a primary mouse head and neck tumour section (F). Representative micrographs are displayed from at least three independent experiments; scale bar represents 20 μm (A), 1 mm (C–E) and 200 μm (F).