Significance of nitroimidazole compounds and hypoxia-inducible factor-1 for imaging tumor hypoxia

Abstract

A tumor-specific microenvironment is characterized by hypoxia, in which oxygen tension is considerably lower than in normal tissues. The hypoxic status of various solid tumors has been attributed as an indicator of adverse prognosis due to tumor progression toward a more malignant phenotype with increased metastatic potential and resistance to treatment. Various exogenous and endogenous markers for hypoxia are currently available and studied in relation to each other, tumor architecture, and tumor microenvironment. Over the last few decades, various methods have been suggested to assess the level of oxygenation in solid tumors. Among them, nitroimidazole compounds have provided promising information on tumor hypoxia. To quantify the extent of hypoxia requires that nitroimidazole binding be primarily dependent on oxygen concentration as well as nitroreductase levels in the tumor cells. Furthermore, recent progress in molecular biology has highlighted a transcription factor, hypoxia-inducible factor (HIF)-1, whose activity is induced by hypoxia. HIF-1 plays a central role in malignant progression by inducing the expression of various genes, whose functions are strongly associated with malignant alteration of the entire tumor. The cellular changes induced by HIF-1 are extremely important therapeutic targets of cancer therapy, particularly in the therapy against refractory cancers. In this review, we will discuss the significance of pimonidazole and HIF-1 as exogenous and endogenous hypoxia markers, respectively, as well as their evaluation and imaging of tumor hypoxia.

Figure 1

Regulation of hypoxia‐inducible factor (HIF)‐1. In the presence of oxygen (normoxia), prolyl hydroxylase (PHD) hydroxylates proline residues on HIF‐1α, allowing HIF‐1α to interact with a ubiquitin–protein ligase complex (VHL, CLU2, and Elongin‐B and Elongin‐C) through VHL. Ubiquitination of HIF‐1α makes it a target for degradation by the 26S proteasome. Growth signals through receptor tyrosine kinases (Rec‐Tyr) and Ras activate the PI3K–Akt pathway and the Ras–Raf–MAP kinase pathway, respectively, increasing the translation of HIF‐1α. When oxygen supply is not enough to activate PHD or when HIF‐1α expression exceeds the capacity of ubiquitin–proteasome degradation, HIF‐1α binds to ubiquitously expressing HIF‐1β to form a heterodimer. The heterodimer then translocates to the nucleus and binds to hypoxia‐responsive elements in the promoter and enhancer region of target genes, inducing the expression of various HIF1‐responsive genes. HRE, hypoxia‐responsive element; Ub, ubiquitin.

Figure 2

Tumor microenvironment. Tumor hypoxia arises in regions with impaired oxygen delivery. The regions proximal to blood vessels are Ki‐67‐, FDG‐, and glucose transporter (Glut)‐1‐positive but radiosensitive, whereas the diffusion‐limited regions are Ki‐67‐, FDG‐, and Glut‐1‐negative and radioresistant. In hypoxic regions, HIF‐1‐active regions (red/pink) are located closer to the blood vessels than pimonidazole (Pimo)‐positive regions (light green). Pimo‐positive regions are located next to necrotic regions (dark blue) and barely express HIF‐1α; they possess little HIF‐1 activity. CA9, carbonic anhydrase 9; FMISO, fluoromisonidazole.

Figure 3

Representative hypoxic cell radiosensitizers and bioreductive prodrugs containing a nitroheterocyclic group.

Figure 4

Oxygen‐dependent bioreductive metabolism of nitroimidazoles in cells and proposed mechanism for selective covalent binding reaction of hydroxylamine intermediates with cellular nucleophiles under hypoxic conditions.

Figure 5

Imaging of hypoxia‐inducible factor (HIF)‐1 active cells. (a) Protein transduction domain (PTD)–oxygen‐dependent degradation (ODD) fusion protein consisting of three domains: PTD, ODD, and a functional domain. PTD enables the fusion protein to diffuse and enter the cell. ODD is derived from ODD_548–603 of the HIF‐1α protein and endows the fusion protein with the same oxygen‐dependent degradation regulation as the HIF‐1α protein. Thus, PTD–ODD is degraded quickly in normoxia (aerobic conditions) but is stabilized and functional in hypoxia. (b) The PTD–ODD–enhanced GFP (EGFP) fusion protein (probe) was labeled with a near‐infrared fluorescent dye and injected into a tumor‐bearing mouse. Fluorescence was detected in the whole body shortly after i.v. injection of the labeled probe. By 6 h after probe injection, the fluorescence was predominantly detected in the tumor, suggesting that the PTD–ODD probe could be a potential probe for imaging HIF‐1 activity.