Hypoxia is one of the hallmarks of the tumor microenvironment and can be used in the design of targeted therapies. Cellular adaptation to hypoxic stress is regulated by hypoxia-inducible factor 1 (HIF-1). Hypoxia is responsible for the modification of cellular metabolism that can result in the development of more aggressive tumor phenotypes. Reduced oxygen concentration in hypoxic tumor cells leads to an increase in oxidoreductase activity that, in turn, leads to the activation of hypoxia-activated prodrugs (HAPs). The same conditions can convert a non-fluorescent compound into a fluorescent one (fluorescent turn off–on probes), and such probes can be designed to specifically image hypoxic cancer cells. This review focuses on the current knowledge about the expression and activity of oxidoreductases, which are relevant in the activation of HAPs and fluorescent imaging probes. The current clinical status of HAPs, their limitations, and ways to improve their efficacy are briefly discussed. The fluorescence probes triggered by reduction with specific oxidoreductase are briefly presented, with particular emphasis placed on those for which the correlation between the signal and enzyme expression determined with biochemical methods is achievable.

The effect of polypyridyl Ru(II) complexes on the ability of cancer cells to migrate and invade, two features important in the formation of metastases, is evaluated. In vitro studies are carried out on breast cancer cell lines, MDA-MB-231 and MCF-7, as well as melanoma cell lines A2058 and A375. Three Ru(II) complexes comprising two 4,7-diphenyl-1,10-phenanthroline (dip) ligands and as a third ligand 2,2'-bipyridine (bpy), or its derivative with either 4-[3-(2-nitro-1H-imidazol-1-yl)propyl] (bpy-NitroIm), or 5-(4-{4'-methyl-[2,2'-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-carbaldehyde semicarbazone (bpy-SC) moiety attached are examined. The low sub-toxic doses of the studied compounds greatly affected the cancer cells by inhibiting cell detachment, migration, invasion, transmigration, and re-adhesion, as well as increasing cell elasticity. The molecular studies revealed that the Ru(II) polypyridyl complexes impact the activity of the selected integrins and upregulate the expression of focal adhesion components such as vinculin and paxillin, leading to an increased number of focal adhesion contacts.

Primary tumor targeting is the dominant approach in drug development, while metastasis is the leading cause of cancer death. Therefore, in addition to the cytotoxic activity of a series of Ru(II) polypyridyl complexes of the type [Ru(dip)₂L]²⁺ (dip: 4,7-diphenyl-1,10-phenanthroline while L = dip; bpy: 2,2′-bipyridine; bpy-SC: bipyridine derivative bearing a semicarbazone 2-formylopyridine moiety; dpq, dpq(CH₃)₂, dpb: quinoxaline derivatives) their ability to inhibit cell detachment was investigated. In vitro studies performed on lung cancer A549 cells showed that they accumulate in cells very well and exhibit moderate cytotoxicity with IC₅₀ ranging from 4 to 13 µM. Three of the studied compounds that have dip, bpy-SC, or dpb ligands after treatment of the cells with a non-toxic dose (<¹/₂IC₅₀) enhanced their adhesion properties demonstrated by lower detachment in the trypsin resistance assay. The same complexes inhibited both MMP-2 and MMP-9 enzyme activities with IC₅₀ ranging from 2 to 12 µM; however, the MMP-9 inhibition was stronger. More detailed studies for [Ru(dip)₂(bpy-SC)]²⁺, which induced the greatest increase in cell adhesion, revealed that it is predominately accumulated in the cytoskeletal fraction of A549 cells. Moreover, cells treated with this compound showed the localization of MMP-9 to a greater extent also in the cytoskeleton. Taken together, our results indicate the possibility of a reduction of metastatic cells escaping from the primary lesion to the surrounding tissue by prevention of their detachment and by influencing the activity of MMP-2 and MMP-9.

The purpose of this study was to investigate the correlation between the spectroscopic and photophysical properties of Ru(ii) polypyridyl complexes and their photodynamic activity in vitro.

Hypoxia, defined as a reduction in oxygen availability resulting from an imbalance between oxygen supply and demand, represents an attractive tumor vulnerability that can be exploited to activate diagnostic and therapeutic agents. Despite the expected selectivity of hypoxiaactivated prodrugs (HAPs), clinical studies have demonstrated relatively low efficacy. This limitation is attributed to multiple factors, as hypoxiainduced cellular responses are highly complex and strongly patientdependent, highlighting the need for reliable hypoxia markers to enable appropriate patient stratification. In this study, we investigated the relationships between the expression levels of selected oxidoreductases involved in activation of HAPs, the readout of recently developed fluorescent hypoxia sensor, and the cytotoxicity of HAP - evofosfamide under rigorously controlled oxygen conditions (21% O₂ vs 1% O₂) across a panel of six human cancer cell lines (A375, A2058, MCF7, PANC1, A549, and HT29). Additionally, we assessed HIF1α expression and compared results obtained using three laboratory models of hypoxia: a hypoxic chamber and chemical induction with CoCl₂ or deferoxamine. Our results indicate that, although the expression of oxidoreductases shows some correlation with both the fluorescence signal from the hypoxia sensor and the cytotoxicity of evofosfamide, no correlation is observed between the hypoxia sensor readout and the prodrug’s activity. This implies that a single hypoxia marker may be insufficient for robust patient stratification and supports the need for a multiparametric framework that captures diverse aspects of the hypoxic tumor phenotype and activation biology of the prodrug.