Redox homeostasis, that is, the proper balance between pro- and antioxidants, is important for almost all natural processes.
. The functioning of mammalian cells depends on several oxidation-reduction reactions to generate energy (e.g., synthesis of ATP) and to produce important cellular factors (such as nucleic acids) from nutrients to assist their biological functions. These redox reactions are critical for the homeostasis of normal cells as well as for the proliferation, development, and survival of tumor cells.
Under physiological conditions, when cells are exposed to reductive or oxidative insults, cellular redox buffers have sufficient capacity to maintain cellular oxidants and reductants at physiological levels. The balance between molecular redox pairs, such as GSSG/GSH, NAD(P)+
/NAD(P)H, and FAD/FADH2
(glutathione disulfide/reduced glutathione, nicotinamide adenine dinucleotide(phosphate)/reduced nicotinamide adenine dinucleotide(phosphate), and flavin adenine dinucleotide/reduced flavin adenine dinucleotide) is essential for the regulation of various signaling processes responsible for maintaining intracellular homeostasis, and, collectively, these ratios design the cellular redox state.
. These redox pairs function as cofactors or substrates for the enzymatic or non-enzymatic neutralization of electrons and reactive oxygen species (ROS), including maintenance of a relatively reducing environment in cells and participation in cellular energetics. For instance, NADPH is an important electron source for the biosynthesis of fatty acids and nucleic acids, NADH delivers electrons for oxidative phosphorylation (oxPHOS) in mitochondria, and NAD+
serves as an electron scavenger to support glycolysis.
When the equilibrium between cellular oxidation and reduction potential is shifted in favor of the oxidizing fraction, a phenomenon called oxidative stress is observed. It occurs when the cell’s ability to defend itself against antioxidants is overwhelmed by the massive production of pro-oxidants such as ROS/RNS/RSS (reactive oxygen species/reactive nitrogen species/reactive sulfur species). Redox imbalance towards pro-oxidative conditions is involved in cancer development as oxidative stress causes genomic instabilities, favoring cancer metastasis and progression
On the other hand, if the cellular redox balance is shifted towards reduction, reductive stress is observed, defined as excessive accumulation or production of reducing agents, such as GSH, NADH, NADPH, as well as the free thiol groups in proteins present in cysteine residues. These cysteines in reduced form, which are present in excess in proteins, can lead to the activation of the “Unfolded Protein Response” (UPR), which impairs the activity of endogenous oxidoreductases. In addition, reductive stress lowers cellular ROS levels below their physiological levels, disrupting their signaling functions. From another perspective, reductive stress may also promote the production of ROS, as redox couples can reduce O2 to ●O2 − in an oxygen environment.
Imbalance of redox status may result from contact with infectious factors or certain diseases. Oxidative stress plays a role in the development of many human diseases, including cancer, as well as aging. Different levels of redox balance affect the regulation of cellular processes in tumors in different ways. Cancer cells generally exhibit a more oxidized environment, meaning that their redox balance is shifted toward higher levels of ROS, which plays a critical role in tumor development, e.g., initiation, progression, migration, invasion, and metastasis. Moderate concentration of ROS promotes the proliferation and metastasis of cancer cells, favoring tumor progression. This is because higher levels of ROS may be the result of more intense oxidative phosphorylation in mitochondria, which means higher ATP production, since cancer cells require a lot of energy for growth. However, oxidative stress, when enormously high, is toxic through oxidative damage to intracellular biomacromolecules in cancer cells. Depending on the stage of cancer development, cells are able to adapt to high ROS levels via altering their metabolism through various mechanisms. These include the activation of antioxidant transcription factors, the elevation of NADPH via the pentose phosphate pathway (PPP), and reductive glutamine and folate metabolism, all of which allow cancer cells to survive. Several antioxidant enzymes and molecules are overexpressed under oxidative stress conditions in cancer and often this metabolic reprogramming leads to a state of ‘pseudohypoxia’.
On the other hand, reductive stress impairs cellular signaling and function and has been associated with cancer, diabetes, and cardiomyopathy. Reductive stress can lead to disruption of mitochondrial homeostasis, decrease metabolism, influence resistance to anti-cancer therapies, alter the formation of disulfide bonds in proteins leading to activation of UPR/ER stress, and finally be harmful to cells. Redox biology, therefore, seeks to understand the mechanisms of regulation and maintenance of homeostasis, as well as the processes that are perturbed in various disease progress where oxidative or reductive stress is a problem.
Chemotherapeutic agents are designed to kill cancer cells because, as antioxidants, they usually act by shifting the redox balance toward reductive stress, which paradoxically can force cells to produce an excess of ROS and, consequently generate oxidative stress. This mechanism resembles an uncontrolled amplification of cellular antioxidant signaling leading to reductive stress . However, under hypoxic conditions, which are a feature of the microenvironment of solid tumors, only a small amount of O2 is present, limiting the production of ROS.. Therefore, it is of great interest to understand how selective induction of reductive stress may influence therapeutic treatment and disease progression in cancer, particularly under conditions of limited O2 levels. Consequently, it is important to recognize that the effects of chemotherapeutic agents may differ under different oxygen conditions. Over the years, many selenium-containing compounds have been investigated as anticancer chemotherapeutic agents, but the specific mechanism of their anticancer activity has not been fully elucidated. It has been suggested that their metabolites, such as methylselenol (CH3SeH) and/or hydrogen selenide (H2Se), may be responsible for their anticancer effects. What is important, cancer cells have been found to be significantly more sensitive than normal cells to the antiproliferative effects of many selenium-containing compounds. Hydrogen selenide is a highly reactive and reducing molecule and thus can induce a reductive environment in cells. Therefore, selenium compounds that are H2Se donors may selectively induce reductive stress and be useful in anticancer and redox homeostasis research.
Unlike oxidative stress, reductive stress is a phenomenon that is not sufficiently described. Little attention has been paid to reductive stress and the therapeutic possibilities underlying the mechanisms of reductive stress in cancer, as well as how cancer cells respond to reductive stress, and there are many conflicting hypotheses in the literature. This concept was first described by Gores et al. in 1989. The authors performed experiments in which they induced hypoxia using chemicals and blocked mitochondrial respiration and ATP production in rat hepatocytes. They concluded that inhibition of respiration leads to “reductive stress”, which can contribute to lethal cellular damage due to low oxygen levels and the formation of toxic oxygen species. These findings called for further studies on this new concept and the mechanism of reductive stress.
On the other hand, the term oxidative stress was first used in 1970 by Paniker et al. during studies on GSH/GSSG pairs in H2O2-stimulated normal and GR-deficient human erythrocytes. Since then, the number of reports of oxidative stress in the literature has increased significantly, from 14 in 1980 and 242 in 1990 to 12,356 in 2010 and 29,069 in 2022 (based on PubMed).
2. Redox Network in Mammalian Cells
Optimal levels of ROS are critical for intracellular signal transduction for proper cellular functions. Redox signaling may play a crucial role in immune response, stem cell biology, cancer, and aging.
Imbalance of redox status is observed in the development of many human diseases such as obesity, diabetes, neurodegenerative diseases, and cancer. This imbalance may result from exposure to infectious factors (e.g., viruses and bacteria) but also from exposure to radiation and toxins.
In tumors, redox imbalance and the subsequent disruption of redox signaling are associated with the proliferation and development of cancer cells and their resistance to radio- and chemotherapy.
Organisms have preserved stress response pathways that recognize and mitigate a wide range of adverse conditions to protecting cell populations from harm.
Due to their quick activation, stress responses must be turned off shortly after cellular homeostasis is reinstated; otherwise, the cell faces far-reaching consequences, including death. The molecular responses to oxidative and reductive stress are shown below, demonstrating the ability of the cellular machinery to respond rapidly.
2.1. Detection and Response to Oxidative Stress
Oxidative stress manifests as increased production of ROS/RNS/RSS generated by enzymes such as nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidase) and nitric oxide synthases (NOSs) or the system of electron transport chain in mitochondria (mtETC).
The redox potential of redox pairs as well as the concentration of ROS vary depending on their localization in the subcellular compartments.
concentration was found to be about 80 pM in the cytosol, 20 nM in the mitochondria, 700 nM in the endoplasmic reticulum, and 1–5 μM in the extracellular space. To counteract ROS/RNS/RSS accumulation, various antioxidant systems are active (oxidative stress response), including non-enzymatic ones such as GSH, ascorbate, and α-tocopherol (vitamins); enzymatic ones such as superoxide dismutases (SODs), catalase, glutathione reductase (GR), glutathione peroxidases (GPxs), thioredoxins (Trxs), and peroxiredoxins (Prxs); and others, such as nuclear factor erythroid 2-related factor (NRF2).
NRF2 is the major transcription factor of antioxidant defense that regulates many genes encoding antioxidant response through inducing the expression of proteins that scavenge oxidizing molecules and convert oxidized proteins to their functional reduced state.
NRF2 also induces the expression of detoxification enzymes and suppresses the induction of pro-inflammatory cytokine genes.
Stem cells that are unable to shut down the oxidative stress response are unable to establish the physiological ROS required for signal transduction and differentiation.
On the other hand, NRF2 function has been shown to be overactivated in many cancers and such aberrant NRF2 activation in cancer cells strongly correlates with negative clinical prognosis.
Stress responses are often controlled via ubiquitination, the specificity of which depends on many different E3 ligases conjugating activated ubiquitin to the substrate.
This posttranslational modification enables mechanistically diverse, quantitative, and reversible regulation of various cellular processes, including signal transduction, migration, cell division, and differentiation. On the other hand, abnormal ubiquitination leads to a broad spectrum of developmental diseases, cancer, and neurodegeneration.
In oxidative stress, stabilization of NRF2 transcription factor activity is critical. Under normal conditions, in the absence of activation signals and when high levels of this transcription factor are not required, NRF2 interacts with KEAP1 (Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1),
which is associated with the F-actin cytoskeleton and has been described as an important sensor of oxidative stress in the cell.
The stoichiometry of KEAP1 and NRF2 within the complex is 2:1, as demonstrated in an isothermal calorimetry study.
Then, NRF2 is ubiquitinated with the participation of E3 ligase cullin-3 (CUL3) and subsequently degraded via the proteasome. When cells are exposed to oxidative stress, the formation of the NRF2/KEAP1/CUL3 complex is inhibited by ROS-dependent oxidation of Cys residues in KEAP1. Several reports have demonstrated that Cys151/273/288/226/613/622/624 may be responsible for the multiple mechanisms of stress-sensing acting through KEAP1.
Consequently, the sequestration and ubiquitination of NRF2 are stopped, and NRF2 translocates and accumulates in the nucleus, where it heterodimerizes with MAF (musculoaponeurotic fibrosarcoma oncogene homolog) protein. The heterodimers recognize the ARE
s, which are enhancer sequences in the regulatory regions of NRF2 target genes, essential for the transcription and expression of antioxidant genes.
Similarly, the E3 CUL2/VHL complex constrains HIF-1α (hypoxia-inducible factor 1) until hypoxic stress occurs, and this released transcription factor then initiates angiogenesis.
HIF-1α is a key transcriptional regulator of cellular metabolism under hypoxic conditions, which also occur in solid tumors. This transcription factor is involved in regulating the expression of many genes responsible for the differences in metabolism compared with the corresponding normal tissue, and there is evidence that (pseudo)hypoxia increases cellular GSH levels via both HIF-1α-dependent and -independent mechanisms.
The importance of correct stress signaling can be illustrated by the deletion of VHL
, which leads to embryonic or early postnatal death, respectively,
and their mutations are a common cause of cancer, e.g., lung squamous cell carcinoma.
2.2. Detection and Response to Reductive Stress
Reductive stress manifests as ROS depletion below their physiological level, due to prolonged antioxidant signaling or mitochondrial inactivity that reverses oxidation. Reductive stress manifests as excessive levels of reduced NAD+ (NADH), reduced NADP+ (NADPH), and GSH. It is as damaging as oxidative stress and has been associated with many pathological processes.
For example, elevated GSH levels lead to increased resistance to chemotherapeutic agents in numerous cancers, as some cancer cells can develop drug resistance through redox resetting,
which can promote cancer migration and metastasis. Similar to ROS, the distribution of NAD(H), NADP(H), and GSH/GSSG is highly compartmentalized in the cell. Unexpectedly, the production of ROS can also increase because of reductive stress, i.e., via activation of NOXs, one-electron transfers to oxygen or through ETC complexes I and IV.
Reductive stress may be the result of, among other things, excessive activation of NRF2 as a result of an oxidative stress response that is not shut down quickly enough, which may occur in cancer. This can lead to overexpression of, e.g., G6PD (glucose-6-phosphate dehydrogenase), the NADPH-producing enzyme. Therefore, similar to rapid activation, stress responses have to be turned off shortly after cellular homeostasis has been reestablished. Thus, cells which do not shoot down the oxidative stress response are incapable of collecting the physiological ROS levels necessary for signaling and are not able to differentiate because of the consequential reductive stress.
An extended lack of ROS, named reductive stress, also blocks glucose homeostasis and insulin signaling (manifesting as increased expression of an antioxidant enzyme such as GPX1),
triggers cardiomyopathy (through dysregulation of G6PD activity),
muscular dystrophy (due to cytoplasmic laminin aggregation and activation of the NRF2/KEAP1 pathway),
or diabetes (NADH/NAD+
redox imbalance due to impairment of NAD+
regeneration enzymes) and increases mortality.
Some proteins, which are redox-sensitive and are associated with cell growth and survival, such as PTEN (phosphatase and tensin homolog; tumor suppressor), NF-κB (nuclear transcription factor kappa B that is involved in inflammatory and immune responses and in the regulation of expression of many other genes related to cell survival, proliferation, and differentiation), Ref1 (redox factor-1), p53 (transcription factor of genes associated with cell cycle, DNA repair, apoptosis, etc.; tumor suppressor protein), and PPATδ (peroxisome proliferator-activated receptor δ; modulates glucose and lipid metabolism), have also been shown to be involved in the reductive stress response in cancer.
Despite these serious consequences, only recently Manford et al. have reported how reductive stress is sensed and alleviated.
. They found that cells detect and respond to reductive stress through ubiquitinating and degrading the mitochondrial gatekeeper FNIP1 (Folliculin-interacting protein 1). The complex E3 ligase CUL2/FEM1B (Cullin-2 ligase/Fem-1 homolog B) can bind its target (FNIP1) in a redox state-dependent manner. Reductive stress, as caused by prolonged antioxidant signaling or mitochondrial inactivity, reverses the oxidation of Cys residues in FNIP1. This means that the Cys residues are in reduced form, which allows the ligase CUL2/FEM1B to recognize and bind FNIP1 through direct interaction with FEM1B. Based on structural data from X-ray crystallography, these interactions were found to occur via zinc ions at the interface between FEM1B and the cysteine residue C186 in FNIP1, which is critical for the recognition of the FEM1B substrate.
As a result, CUL2/FEM1B-dependent FNIP1 ubiquitination and proteasomal degradation of FNIP1 happen, followed by restoration of mitochondrial production to generate ROS and maintain redox homeostasis. It is speculated that regulation of this process can occur with the involvement of BEX proteins (brain-expressed and X-linked proteins), which are pseudosubstrate inhibitors of E3 ligase, and participate in the reductive regulation of the stress response. BEX proteins bind multivalently to the ubiquitination ligase via sites located at C186/Zn2+
and R126 of FEM1B. Recently, the small synthetic ligand EN106 was shown to target a cysteine residue in FEM1B that is essential for substrate recognition.
Through covalent interactions with C186, EN106 is able to bind FEM1B E3 ligase and disrupt the recognition of its key reductive stress substrate, FNIP1. It may be of future interest to determine whether EN106 can be used therapeutically to inhibit the formation of the CUL2/FEM1B/FNIP1 complex and disrupt reductive stress signaling through stabilizing FNIP1 in certain cancers.
In summary, the key to the reductive stress response is the ability of the E3 ligase CUL2/FEMB1 to distinguish the reduced from the oxidized form of FNIP1, so the E3 ligase is able to discriminate targets based on redox state with zinc ions at the interface (it selectively recruits the reduced Cys residues of FNIP), and this interaction is controlled by BEX family pseudosubstrate inhibitors. Finally, the degradation of FNIP1 leads to the activation of mitochondria to recalibrate ROS.
Thus, the reductive stress response relies on ubiquitin-dependent regulation that turns mitochondrial activity on and off according to redox demand and involves metabolic control in the coordination of stress and developmental signaling.
Recent evidence suggests that reductive stress may be an avenue for therapeutic intervention in cancer.
Under hypoxic conditions, CTMP-AA nanocarrier (a core–shell nanostructure of CdTe quantum dots coated with mesoporous silica (MSN), functionalized with poly(2-vinylpyridine)-polyethylene glycol-folic acid (PPF), and loaded with ascorbic acid) controlled release of high levels of ascorbic acid can trigger apoptosis of HepG2 cells (human hepatocellular carcinoma) through inducing reductive stress, with apparently limited damage to normal tissue. In vivo studies in mice bearing HepG2 tumor confirmed that CTMP-AA exhibits a killing effect on tumor cells and inhibits tumor growth.
3. Selenium compounds as H2Se donors that may affect redox homeostasis in healthy organisms and cancers
Several selenium compounds have been shown to have chemotherapeutic effects against cancer,
but their anti-cancer mechanism has not been fully elucidated. It is suggested that H2
Se or methylselenol (CH3
SeH), may be the actual anticancer agents.
Se and CH3
SeH are the common metabolites of several selenium compounds, including dietary (inorganic: sodium selenate and sodium selenite, as well as organic: selenocysteine, selenomethionine and less commonly methylselenocysteine, and CysSeSeCys 
) and some chemically synthesized ones. Therefore, research on existing and new selenium-containing drugs with antitumor effects should focus on these molecules. In oxygen environment, these two forms readily oxidize and can lead to the formation of superoxide and other reactive oxygen species with additional toxic effects. The most of the chemotherapeutic selenium compounds have been studied under aerobic conditions, and the generation of oxidative stress has been assumed the main mechanism of their anticancer activity under these conditions.
However, in solid tumors, that is, under low-oxygen conditions e.g., hypoxia, high H2
Se concentrations were produced after administration of an external selenium source, without obvious increase in ROS being observed, and H2
Se accumulation led to reductive stress in studied cells.
Thus, the anticancer effects of various selenium compounds are related to their influence on the redox homeostasis of cancer cells, but the question arises whether Se is an antioxidant or a prooxidant.
Se is essential for cell survival at relatively low concentrations (about 55 µg/day) but is toxic at high doses (> 400µg/day), so the complex role of H2Se in human cancers, require the development of well-defined H2Se donors with controllable release properties. Inorganic selenide salts (e.g., Na2Se and NaHSe) are short-living H2Se donors, that not only can be toxic  but also fail to mimic slow and well-regulated H2Se formation in vivo. Organic Se compounds as potential H2Se donors are generally considered safer (lower toxicity) than inorganic Se salts.
There is not known many well-characterized and controllable H2Se donors that would act under physiological conditions, and the study of H2Se biology encountered technical difficulties because there was no reliable assay for direct H2Se quantification. Here, the research focus on compounds whose mechanism of action is based on hydrogen selenide, and formation of this product is well documented. These compounds are collected in Table 1.
Table 1. Selenium compounds as H2Se donors and studies of their biological effects.
||Detection method of H2Se release
||Mechanism of H2Se release
(under clinical trials) 
Fluorescence imaging: NIR-H2Se
Fluorescence imaging: Hcy-H2Se
Fluorescence imaging: Mito-N-D-MSN (nanoprobes mitochondria-targeted)
Grx (GSH), Trx, TrxR (NADPH)
HepG2 cells (cytotoxicity IC50=5 μM after 24h, reductive stress; H2Se release), mice
HepG2 cells (H2Se release)
HepG2 cells (H2Se release)
31P and 77Se NMR and electrophilic trapping reagent
2AP-PSe, Cat-PSe (P=Se motif)
31P and 77Se NMR and electrophilic trapping reagent; colorimetric detection with NBD-CI
HeLa cells (antioxidant activity)
selenocyclopropenones and arylselenoamides (C=Se motif)
H2Se-selective gas detector; electrophilic trapping reagent and HRMS analysis; Cy7-CI trapping; fluorescence imaging (NIR-H2Se)
Cys at pH 7.4
(Cys-mediated H2Se release)
dGMPSe (2'-deoxyguanosine selenophosphate)
Fluorescence imaging: SF7
HeLa cells (cytotoxicity IC50=8 µM after 24h; H2Se release)
Trapping reagent and HRMS (high-resolution mass spectrometry)
neutral to slightly basic conditions
HeLa and HCT116 cells (cytotoxicity IC50= 3.7–10.6 µM )
Many pathological conditions, including cancer, may be due to an imbalance of oxidative and reductive byproducts. To protect their cell populations from damage, organisms have conserved stress response pathways that recognize and mitigate a variety of adverse conditions. Because of their rapid activation, stress responses should be terminated soon after cellular homeostasis is restored. Otherwise, the cell is threatened with dire consequences, even death.
Reductive stress is not as well-characterized of a phenomenon as oxidative stress. It has only recently gained more attention, particularly because of its potential importance and therapeutic intervention in cancer. Understanding the regulation of responses to reductive stress both in normal cells and in the tumor microenvironment can provide new therapeutic approaches for cancer treatment and anticancer drug resistance. Therefore, compounds that can selectively and controllably induce reductive stress in cells are being sought. Selenium-containing compounds which are H2Se donors meet these requirements.
However, there are few detailed studies on the chemical biology of H2Se, and there are not many reports about the essential physiological functions of H2Se, the cellular objects, and the therapeutic perspectives. The lack of clarity on these key questions was largely due to the lack of small molecule donors that could effectively enhance the bioavailability of H2Se through continuously releasing the unstable biomolecule under physiologically relevant conditions. Several H2Se donors have been developed for which H2Se release has been well documented, although not all of them have been demonstrated to show anticancer activity in the cellular model. It is too early to compare their efficacy in cancer therapy with other selenium-containing compounds as well the mechanisms responsible for their toxicity in cells as only Na2SeO3 was studied so deeply (apoptosis under oxidative stress and autophagy under reductive stress).
In conclusion, future studies are needed to show the utility of selenium compounds as H2Se donors in cancer therapy and in the selective induction of reductive stress in cells and in vivo, as few data are available to date.