New Insights into Oxidative and Reductive Stress Responses and Their Relation to the Anticancer Activity of Selenium-Containing Compounds as Hydrogen Selenide Donors

Simple Summary Both oxidative and reductive stress can be destructive to cells. Unlike oxidative stress, reductive stress and the therapeutic opportunities underlying the mechanisms of reductive stress in cancer, as well as how cancer cells reply to reductive stress, have attracted little interest and are not properly characterized. Selenium compounds have been demonstrated to have chemotherapeutic effects against cancer, and their anticancer mechanism is thought to be related to the formation of their metabolites. One of them is hydrogen selenide (H2Se), a highly reactive molecule with reducing properties. Unfortunately, most chemotherapeutic selenium compounds were studied under aerobic conditions, although solid tumors exhibit hypoxic conditions. Here, we show the latest reports on the mechanism of cellular recognition and reaction to reductive and oxidative stress as well as the mechanisms through which different types of selenium compounds can release H2Se and thereby can selectively affect reductive stress under controlled conditions, which may be crucial for their anticancer activity. Abstract Redox balance is important for the homeostasis of normal cells, but also for the proliferation, progression, and survival of cancer cells. Both oxidative and reductive stress can be harmful to cells. In contrast to oxidative stress, reductive stress and the therapeutic opportunities underlying the mechanisms of reductive stress in cancer, as well as how cancer cells respond to reductive stress, have received little attention and are not as well characterized. Therefore, there is recent interest in understanding how selective induction of reductive stress may influence therapeutic treatment and disease progression in cancer. There is also the question of how cancer cells respond to reductive stress. Selenium compounds have been shown to have chemotherapeutic effects against cancer, and their anticancer mechanism is thought to be related to the formation of their metabolites, including hydrogen selenide (H2Se), which is a highly reactive and reducing molecule. Here, we highlight recent reports on the molecular mechanism of how cells recognize and respond to oxidative and reductive stress (1) and the mechanisms through which different types of selenium compounds can generate H2Se (2) and thus selectively affect reductive stress under controlled conditions, which may be important for their anticancer effects.


Introduction
Redox homeostasis, that is, the proper balance between pro-and antioxidants, is important for almost all natural processes [1]. 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 [2]. Under physiological

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 [23]. 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) Biology 2023, 12, 875 4 of 22 but also from exposure to radiation and toxins [10]. 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 [23]. 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.

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) [5,6]. The redox potential of redox pairs as well as the concentration of ROS vary depending on their localization in the subcellular compartments [14]. Thus, H 2 O 2 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) [5,6,24]. 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 [25][26][27][28]. NRF2 also induces the expression of detoxification enzymes and suppresses the induction of pro-inflammatory cytokine genes [29,30]. 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 [31,32]. 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 [33][34][35].
Stress responses are often controlled via ubiquitination, the specificity of which depends on many different E3 ligases conjugating activated ubiquitin to the substrate [36][37][38]. 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 [39].
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) [40], which is associated with the F-actin cytoskeleton and has been described as an important sensor of oxidative stress in the cell [25,41]. The stoichiometry of KEAP1 and NRF2 within the complex is 2:1, as demonstrated in an isothermal calorimetry study [42]. Then, NRF2 is ubiquitinated with the participation of E3 ligase cullin-3 (CUL3) and subsequently degraded via the proteasome ( Figure 1A). 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 [40,[43][44][45]. 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 AREs, which are enhancer sequences in the regulatory regions of NRF2 target genes, essential for the transcription and expression of antioxidant genes [41,46] ( Figure 1B).
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 [47]. 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 [48].
The importance of correct stress signaling can be illustrated by the deletion of VHL or KEAP1, which leads to embryonic or early postnatal death, respectively [49,50], and their mutations are a common cause of cancer, e.g., lung squamous cell carcinoma [47,51]. responsible for the multiple mechanisms of stress-sensing acting through KEAP1 [40,[43][44][45]. 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 AREs, which are enhancer sequences in the regulatory regions of NRF2 target genes, essential for the transcription and expression of antioxidant genes [41,46] ( Figure 1B).
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 [47]. 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 [48].
The importance of correct stress signaling can be illustrated by the deletion of VHL or KEAP1, which leads to embryonic or early postnatal death, respectively [49,50], and their mutations are a common cause of cancer, e.g., lung squamous cell carcinoma [47,51].  [41]. (A) Under normal conditions, NRF2 is bound to its cellular inhibitor, KEAP1, which is connected with the F-actin cytoskeleton and is inactive. NRF2 concentrations are mostly regulated by the proteasome [52,53]. (B) In the response to oxidative stress, NRF2 detaches from KEAP1, translocates to the nucleus and forms a heterodimer with MAF: the NRF2/MAF heterodimer binds to ARE and prompts the expression of different genes, including antioxidant. (Adapted from ref. [43]; reproduced with permission of Elsevier.)

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+  [41]. (A) Under normal conditions, NRF2 is bound to its cellular inhibitor, KEAP1, which is connected with the F-actin cytoskeleton and is inactive. NRF2 concentrations are mostly regulated by the proteasome [52,53]. (B) In the response to oxidative stress, NRF2 detaches from KEAP1, translocates to the nucleus and forms a heterodimer with MAF: the NRF2/MAF heterodimer binds to ARE and prompts the expression of different genes, including antioxidant. (Adapted from ref. [43]; reproduced with permission of Elsevier).

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 [3,10]. 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 [54], 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 [8].
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-6phosphate 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 [23,31,41]. 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) [55], triggers cardiomyopathy (through dysregulation of G6PD activity) [26], muscular dystrophy (due to cytoplasmic laminin aggregation and activation of the NRF2/KEAP1 pathway) [56], or diabetes (NADH/NAD + redox imbalance due to impairment of NAD + regeneration enzymes) and increases mortality [57]. 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 [10].
Despite these serious consequences, only recently Manford et al. have reported how reductive stress is sensed and alleviated [58]. 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 ( Figure 2). 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 [59]. 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 ( Figure 2). BEX proteins bind multivalently to the ubiquitination ligase via sites located at C186/Zn 2+ 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 [60]. 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. nanostructure of CdTe quantum dots coated with mesoporous silica (MSN), function ized with poly(2-vinylpyridine)-polyethylene glycol-folic acid (PPF), and loaded w ascorbic acid) controlled release of high levels of ascorbic acid can trigger apoptosi HepG2 cells (human hepatocellular carcinoma) through inducing reductive stress, w apparently limited damage to normal tissue. In vivo studies in mice bearing HepG2 tum confirmed that CTMP-AA exhibits a killing effect on tumor cells and inhibits tum growth. Figure 2. Scheme of reductive stress signaling. Reductive stress reverses oxidation state of thio Cys residues in FNIP1, leading to the recognition of FNIP1 by ligase complex CUL2/FEM1B Zn 2+ -dependent binding, ubiquitination, and degradation in proteasome (modified from [58]). creasing concentration of FNIP1 intensifies mitochondrial activity and starts the generation of R to neutralize reductive stress. The binding of reduced form FNIP1 to the ligase comp CUL2/FEM1B is controlled by BEX family of proteins (modified from [59]).

The Biological Significance of Selenium Compounds
Selenium is an essential trace element whose role is important in health and dise It is usually supplied in the daily diet in the form of inorganic (drinking water) or orga (plants, yeasts, and animal sources) compounds such as selenate (sodium selenate), s nite (sodium selenite), selenocysteine (Sec), selenomethionine (SeM), and less commo methylselenocysteine (MeSeCys) and CysSeSeCys [62]. Many cellular physiological p cesses depend on the level of selenium compound intake. At relatively low concentrati (about 55 µ g/day), Se is necessary for various physiological processes, while at high do (>400 µ g/day), it can have toxic effects and is also harmful to health when deficient (d below 20 µ g/day) [17]. via Zn 2+ -dependent binding, ubiquitination, and degradation in proteasome (modified from [58]). Decreasing concentration of FNIP1 intensifies mitochondrial activity and starts the generation of ROS to neutralize reductive stress. The binding of reduced form FNIP1 to the ligase complex CUL2/FEM1B is controlled by BEX family of proteins (modified from [59]).
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 ( Figure 2).
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 [61]. 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.

The Biological Significance of Selenium Compounds
Selenium is an essential trace element whose role is important in health and disease. It is usually supplied in the daily diet in the form of inorganic (drinking water) or organic (plants, yeasts, and animal sources) compounds such as selenate (sodium selenate), selenite (sodium selenite), selenocysteine (Sec), selenomethionine (SeM), and less commonly methylselenocysteine (MeSeCys) and CysSeSeCys [62]. Many cellular physiological processes depend on the level of selenium compound intake. At relatively low concentrations (about 55 µg/day), Se is necessary for various physiological processes, while at high doses (>400 µg/day), it can have toxic effects and is also harmful to health when deficient (dose below 20 µg/day) [17].
Dietary Se compounds are metabolized in vivo via different pathways to different selenium metabolites, but their pathways meet in a shared metabolite, recognized as hydrogen selenide (H 2 Se, mainly as HSe − at physiological pH), which is a reduced form of selenium ( Figure 3, section "Dietary selenium compounds as generators of H 2 Se") [20]. H 2 Se is a highly reducible molecule with very high chemical reactivity that cannot be readily detected in cells and animal models. It is efficiently taken up by human cells with an apparent K m in the nanomolar range through transport via ATPases, allowing it to be readily distributed in tissues [63].

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 [17,18,20], but their anti-cancer mechanism has not been fully elucidated. It is suggested that H2Se or methylselenol (CH3SeH), the common metabolites of several selenium compounds, including some chemically synthesized ones, may be the actual anticancer agents [18,19,[70][71][72][73]. Therefore, research on existing and new selenium-containing drugs with antitumor effects should focus on these molecules. Excessive amounts of selenide or methylselenol may be harmful to cells [17,20]. 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 [17,20]. However, in solid tumors, that is, under low-oxygen conditions, e.g., hypoxia, high H2Se concentrations were produced after administration of an external selenium source, without obvious increase in ROS being observed, and H2Se accumulation led to reductive stress in studied cells [74].
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 pro-oxidant.
The direct role of hydrogen selenide in cancer therapy with selenium compounds had not been investigated until 2016 [75,76] (see Section 4.1), probably due to the lack of well-characterized and controllable H2Se donors under physiological conditions, and the lack of detection methods for this highly reactive, reducible, and short-living molecule. The half-life for the oxidation of H2Se (HSe − at physiological pH) to elemental selenium in air-saturated water (without other reactants) and at pH 7 is about 30 s, and independent of its concentration [77]. H2Se has analogous physical and chemical properties to H2S (hydrogen sulfide) but also shows significant differences; therefore, it exerts different biological effects because selenium compounds are mostly more reactive than corresponding sulfur products [78]. Unlike H2Se, the half-life time of H2S in air-saturated water is a few Selenium compounds mediate physiological functions mainly through the incorporation of Se into selenoproteins, usually in the form of selenocysteine (Sec), the 21st amino acid. For the incorporation of selenocysteine into selenoproteins, selenium has to be converted to selenide, from which monoselenophosphate (H 2 SePO 3 − ) is formed to generate selenocysteyl-tRNA (Ser)Sec (SeC-tRNA) [64,65]. Finally, only selenium compounds that can be converted to selenide can serve as selenium sources for selenoproteins [62]. Most selenoproteins, such as GPxs and TrxRs, incorporate Sec into their active sites and exhibit antioxidant properties-they protect the cell from the harmful effects of ROS. On the other hand, SeM is randomly integrated into proteins. The antioxidant property of seleniumcontaining proteins (chemopreventive effect) has been used to prevent cancer, and this result occurs at low levels of dietary Se compounds [20,66]. Conversely, selenium shows an antitumor activity (chemotherapeutic effect) at relatively high levels [17]. Several Se compounds showed significant cytotoxicity (in the low micromolar range) against various malignant cells (lung, prostate, cervical, etc.) [2,17,18]. However, the effects of selenium application are highly dependent on the chemical form, cell type, and biomarker used to test efficacy [62].
The other physiological function of selenium compounds is exerted by selenium modification of tRNA, which has been found so far in bacteria ( Figure 3). The enzyme tRNA 2-selenouridine synthase (SelU) is responsible for a two-step process: (1) S2U-RNA (2-thiouridine RNA) geranylation (with ppGe) and (2) subsequent selenation of the resulting geS2U-RNA (with SePO 3 3− ) to Se2U-RNA (2-selenouridine RNA) [67,68]. Se2U or S2U in the wobble position of the anticodon fragment of some tRNA plays an important role in the precise reading of mRNA during translation through increasing the thermodynamic stability of RNA duplexes containing S2U-A or Se2U-A base pairs and restricting the formation of S2U-G or Se2U-G base pairs [69].
Excretion of excess selenium (detoxification) occurs through sequential methylation of H 2 Se to dimethylselenide ((CH 3 ) 2 SeH), which is excreted in the breath, and selenosugar and trimethylselenide ((CH 3 ) 3 Se + ), which are excreted in the urine.

Selenium Compounds as H 2 Se Donors That May Affect Redox Homeostasis in Healthy Organisms and Cancers
Several selenium compounds have been shown to have chemotherapeutic effects against cancer [17,18,20], but their anti-cancer mechanism has not been fully elucidated. It is suggested that H 2 Se or methylselenol (CH 3 SeH), the common metabolites of several selenium compounds, including some chemically synthesized ones, may be the actual anticancer agents [18,19,[70][71][72][73]. Therefore, research on existing and new selenium-containing drugs with antitumor effects should focus on these molecules. Excessive amounts of selenide or methylselenol may be harmful to cells [17,20]. 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 [17,20]. However, in solid tumors, that is, under low-oxygen conditions, e.g., hypoxia, high H 2 Se concentrations were produced after administration of an external selenium source, without obvious increase in ROS being observed, and H 2 Se accumulation led to reductive stress in studied cells [74].
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 pro-oxidant.
The direct role of hydrogen selenide in cancer therapy with selenium compounds had not been investigated until 2016 [75,76] (see Section 4.1), probably due to the lack of well-characterized and controllable H 2 Se donors under physiological conditions, and the lack of detection methods for this highly reactive, reducible, and short-living molecule. The half-life for the oxidation of H 2 Se (HSe − at physiological pH) to elemental selenium in airsaturated water (without other reactants) and at pH 7 is about 30 s, and independent of its concentration [77]. H 2 Se has analogous physical and chemical properties to H 2 S (hydrogen sulfide) but also shows significant differences; therefore, it exerts different biological effects because selenium compounds are mostly more reactive than corresponding sulfur products [78]. Unlike H 2 Se, the half-life time of H 2 S in air-saturated water is a few minutes. Sulfur and selenium belong to the same group in the periodic table of elements and have a similar atom radius (88 and 103 pm), similar covalent radius (102 and 116 pm), and similar electronegativity (2.58 and 2.55). They also show some differences, since a selenium atom is a better-reducing agent and oxidizes more easily, while a sulfur atom tends to form much stronger covalent linkages. The pK a1 values of H 2 S and H 2 Se vary significantly (6.88 and 3.89), while the pK a2 values of HX − do not differ considerably (14.15 and 15.1, respectively). H 2 S, the gaseous, membrane-permeable molecule, is the central compound of cellular sulfur chemistry and is one of three inorganic gaseous messengers in mammalian cells (the others are NO and CO). The formation of reactive species (RSS) from H 2 S is analogous to ROS so that the transfer of single electrons starting from H 2 S leads to the RSS thiyl radical (HS • ), hydrogen persulfide (H 2 S 2 ), supersulfide radical ( • S 2 − ), and finally to elemental sulfur [14]. Due to the higher reactivity of selenium compounds compared to the corresponding sulfur derivatives, it can be argued that all biologically relevant selenium compounds are "Reactive Selenium Species" RSeS) [78]. Therefore, H 2 Se as RSeS should generate oxidative stress similarly to ROS and RSS. On the other hand, H 2 Se, a molecule with strong reductive properties, is theoretically capable of inducing reductive stress through lowering cellular ROS below their physiological levels and disrupting their signaling functions. As a result, reductive stress can promote the production of ROS as redox pairs can reduce O 2 to form • O 2 − leading to DNA strand breaks and apoptosis in cancer cells [3,9].
However, under hypoxic conditions, H 2 Se accumulation can lead to reductive stress [74]. Thus, the toxicity of selenium-containing compounds likely combines the dependence on several mechanisms, such as affecting the redox state and eventually DNA damage/repair, gene expression, cell signaling pathways, cell cycle arrest, GSH levels, protein oxidation (in the presence of O 2 ), the angiogenesis process, metastasis, or others [17,70]. Additionally, the chemical form in which they are supplied to the organism (e.g., organic or inorganic) is also important and can have an influence on the effect exerted.

Dietary Selenium Compounds as Generators of H 2 Se
Selenomethionine (SeM), selenocysteine (SeC), selenite (SeO 3 2− ), and selenate (SeO 4 2− ) account for the majority of organic and inorganic Se in the diet. They are absorbed without regulation, metabolized in a different way in vivo, and they generate different selenium metabolites, but their common intermediate is H 2 Se (Figure 3) [62]. Organic selenium compounds released from selenoproteins (from plant and animal sources) such as SeM and SeC are absorbed via transcellular pathways. SeM can enter the trans-selenation pathway (analogous to the transsulfuration pathway) and be converted to SeC, which is subsequently reduced to H 2 Se (HSe − ) by selenocysteine β-lyase. Alternatively, SeM can be converted into methylselenol (CH 3 SeH) by a γ-lyase and then to H 2 Se by methylselenol demethylase.
Sec, SeM, and MeSeCys are seleno-amino acids that have been studied for their anticancer properties. Sec inhibits the growth of cancer cells (lung, breast, bladder, liver, and other) through inducing apoptosis and cell cycle arrest in phase S, increasing DNA fragmentation, and activating the caspase-3 via the mechanism related to the overproduction of ROS [18,79,80]. Inhibition of cell proliferation through apoptosis induction caused by SeMet has been observed in various cancer cell lines, such as colorectal, prostate, breast, lung, and melanoma cells [18,81]. However, MeSeCys is generally more toxic than SeM as shown in lung cancer (A549) and human neuroblastoma cells (SH-SY5Y), probably due to its quick metabolism to CH 3 SeH, a potentially antitumorigenic compound [71,72,82,83]. Therefore, the antitumor effect of these dietary organic selenium compounds is thought to be due to the formation of CH 3 SeH and its further oxidation rather than the involvement of H 2 Se, although this pathway cannot be excluded due to a lack of evidence.
On the other hand, SeO 3 2− and SeO 4 2− , the primary environmental inorganic Se sources, which are mainly supplied with drinking water, are metabolized differently. After absorption, inorganic selenate is reduced to selenite by ATP sulfurylase (Figure 3). SeO 3 2− reacts spontaneously with GSH to form selenodiglutathione (GSSeSG) [78], and subsequently, GSSeSG is decomposed to selenide in the presence of NADPH by the enzymatic cellular system in several steps. The enzymes involved in these redox reactions are GR, glutaredoxin (Grx), or the Trx/TrxR system (selenium proteins themselves) [62,84]. Next, in the presence of oxygen, H 2 Se is easily oxidized by O 2 to Se 0 . However, excess GSH can protect H 2 Se from oxidation through trapping O 2 and forming GSSG, so the role of GSH is to protect functional protein groups from oxidation. Therefore, depletion of intracellular GSH by H 2 Se can lead to oxidation and loss/decrease in the function of different proteins necessary for maintaining cell homeostasis. SeO 3 2− constitutes an inorganic form of selenium that has cytotoxic effects on several human cancer cell lines, including glioma, prostate, and lung [85][86][87][88]. For example, comparative proteomics and mass spectrometry studies demonstrated that sodium selenite induced significant growth inhibition and apoptosis in the human prostate cancer cell line PC-3 [89]. Na 2 SeO 3 increased intracellular ROS and decreased mitochondrial membrane potential, leading to activation of caspase-8, cleavage of PARP, and finally apoptosis of cells. Selenite-induced apoptosis was mediated mainly by the mitochondrial pathway but also involved ER stress and HIF-1α mediated pathways. Sodium selenite also inhibited the migration of PC-3 cells in vitro.
In 2016, a specific fluorescent probe (NIR-H 2 Se; ex/em = 633/650-750 nm) was developed for real-time monitoring of H 2 Se in living cells and in vivo [75] (Figure 4A). Thus, it was possible to track H 2 Se in cells, and H 2 Se was shown to play a key role in cell death induced by selenite (IC 50 = 5 µM after 24 h under normoxia). NIR-H 2 Se (10 µM) was used to detect the H 2 Se content in HepG2 cells (human hepatocellular carcinoma) during Na 2 SeO 3 -induced apoptosis under normoxic conditions via the mechanism of oxidative stress. However, in the presence of reduced oxygen, increased H 2 Se content and decreased ROS levels were observed, suggesting that cell death in a hypoxic environment occurs via a non-oxidative stress mechanism [75].  Later, a mitochondria-targeted fluorescent H2Se nanosensor, Mito-N-D-MSN, formulated from mesoporous silica nanoparticles (MSNs), was developed [90]. Mito-N-D-MSN was prepared through loading NIR-H2Se into MSN and modifying it with triphenylphosphonium ions (TPP) as a mitochondria-targeting moiety. The results showed that the mitochondrial H2Se content gradually increased after Na2SeO3 administration (at concentrations 2-10 µ M and 12 h) to HepG2 cells, while O2 content remained unchanged in HepG2 cells under hypoxic conditions, suggesting that the anticancer mechanism of selenite involves non-oxidative stress in the tumor microenvironment.
Next, H2Se release generated by Na2SeO3 under hypoxia (detected via NIR-H2Se probe) was shown to induce reductive stress in HepG2 cells and activate cell autophagy through regulating the redox of HMGB1 protein [73]. H2Se could disrupt the disulfide bond in HMGB1 and promote its secretion, which are the characteristic effects of reductive stress. The reduced HMGB1 protein was then secreted into the extracellular space, which subsequently activated autophagy through inhibiting the Akt/mTOR pathway in cells. Moreover, autophagy played a dual role; mild autophagy inhibited apoptosis, whereas excessive autophagy led to autophagy-associated cell death.
Sodium selenite, as an H2Se donor, was one of the selenium compounds tested in clinical trials where the combination of sodium selenite with commonly used anti-cancer therapies was applied [91][92][93][94][95]. The results obtained with the NIR-H 2 Se probe were confirmed with another fluorescent probe, Hcy-H 2 Se (ex/em = 470 nm/575 nm, Figure 4B) [76]. The compound (10 µM), which also showed high selectivity over H 2 S, was successfully applied to image H 2 Se generated from Na 2 SeO 3 in living cells HepG2 under both normoxic and hypoxic conditions and in solid tumors. The confocal fluorescence imaging results showed that H 2 Se gradually accumulated in a hypoxic environment, suggesting that the anticancer mechanism of Se in hypoxic solid tumors occurs via nonoxidative stress.

Chemically Synthesized Selenium Compounds as H2Se Suppliers
Later, a mitochondria-targeted fluorescent H 2 Se nanosensor, Mito-N-D-MSN, formulated from mesoporous silica nanoparticles (MSNs), was developed [90]. Mito-N-D-MSN was prepared through loading NIR-H 2 Se into MSN and modifying it with triphenylphosphonium ions (TPP) as a mitochondria-targeting moiety. The results showed that the mitochondrial H 2 Se content gradually increased after Na 2 SeO 3 administration (at concentrations 2-10 µM and 12 h) to HepG2 cells, while O 2 content remained unchanged in HepG2 cells under hypoxic conditions, suggesting that the anticancer mechanism of selenite involves non-oxidative stress in the tumor microenvironment.
Next, H 2 Se release generated by Na 2 SeO 3 under hypoxia (detected via NIR-H 2 Se probe) was shown to induce reductive stress in HepG2 cells and activate cell autophagy through regulating the redox of HMGB1 protein [73]. H 2 Se could disrupt the disulfide bond in HMGB1 and promote its secretion, which are the characteristic effects of reductive stress. The reduced HMGB1 protein was then secreted into the extracellular space, which subsequently activated autophagy through inhibiting the Akt/mTOR pathway in cells. Moreover, autophagy played a dual role; mild autophagy inhibited apoptosis, whereas excessive autophagy led to autophagy-associated cell death.
Sodium selenite, as an H 2 Se donor, was one of the selenium compounds tested in clinical trials where the combination of sodium selenite with commonly used anti-cancer therapies was applied [91][92][93][94][95].

Chemically Synthesized Selenium Compounds as H 2 Se Suppliers
Chemically synthesized organic selenium-containing compounds that exhibit antitumor activity belong to different chemical classes and are metabolized into different redox-active products. Their active antitumor metabolites, similar to the dietary selenium compounds, can be divided into two groups: generators of hydrogen selenide (H 2 Se) and methylselenol (CH 3 SeH) [73]. Both are redox active but exhibit a slightly different mechanism of action and toxicity, and their properties depend on the doses used [70]. Methylselenol precursors, such as diselenides, selenoethers, selenoesters, methylseleninic acid, 1,2-benzisoselenazole-3[2H]-one, and selenophene-based derivatives, as well as selenoamino acids and selol (contains dioxaselenolane rings and selenium at +4 oxidation stage), have been addressed in the other review [18]. Moreover, the research progress and different therapeutic applications of Se-containing compounds have been reviewed [96], and many various Se-containing compounds showed potent activities such as anticancer, antioxidant, antifibrinolytic, antiparasitic, antibacterial, antiviral, antifungal, and centralnervous-system-related effects.
The toxicity and complex role of H 2 Se in human cancers requires the development of compounds that are well-defined H 2 Se donors with controllable release properties. Inorganic selenide salts (e.g., Na 2 Se and NaHSe) are short-living H 2 Se donors that not only can be toxic [97,98] but also fail to mimic slow and well-regulated H 2 Se formation in vivo. Organic Se compounds as potential H 2 Se donors are generally considered safer (lower toxicity) than inorganic Se salts [20,81,[99][100][101][102][103][104]. For example, organic diselenides and phthalic selenoanhydride have shown promising biological activities, including radioprotection, free radical scavenging, and multidrug-resistance-reversing activity, but H 2 Se release was not directly observed in experiments with these compounds [81,[99][100][101][102][103][104].
There were not many well-characterized and controllable H 2 Se donors that would act under physiological conditions, and the study of H 2 Se biology encountered technical difficulties because there was no reliable assay for direct H 2 Se quantification. In this review, we focus on compounds whose mechanism of action is based on hydrogen selenide, and we show how this product is formed in cases where its presence is well documented.
Due to the chemical similarity of H 2 S and H 2 Se, many researchers have been inspired by H 2 S donors in the development of synthetic H 2 Se donors.
The first well-characterized synthetic donor for hydrolysis-based H 2 Se release was prepared through modifying the widely used H 2 S donor GYY4137 [105]. In 2019, based on the P==S motif, the Pluth group developed the analogous compound TDN1042, which contained the P==Se motif [106]. This small synthetic molecule releases H 2 Se directly and under controlled conditions ( Figure 5A). Using 31 P and 77 Se NMR-controlled experiments, the authors demonstrated the pH dependence of H 2 Se release (an increase in the rate at more acidic pH), and finally, they confirmed H 2 Se release using an electrophilic trapping reagent (dinitrofluorobenzene). Using HPLC, the authors observed the formation of both expected products: di(2,4-dinitrophenyl) selenide and the corresponding diselenide in the trapping solution. Proposed hydrolysis mechanism of TDN1042 resulting in H2Se release at acidic pH (modified from ref. [106]); (B) hydrolysis of cyclic compound Cat-PSe to release H2Se at pH 7.4 (modified from [107]); (C) structure of 2AP-PSe used in the study [107].
Considering the study of Pluth et al. and inspired by other synthetic H2S donors (compounds activated by bio-thiols [108]), Kang and coworkers proposed Cys-activated H2Se donors, that is, selenocarbonyl-containing derivatives with C==Se motif [109]. Thus, the C==Se motifs in selenocyclopropenones and selenoamides can be selectively activated by the -SH moiety of Cys for controllable H2Se release at pH 7.4, and these reactions occur with different rates for those compounds ( Figure 6A). In the qualitative assay, H2Se release from selenocyclopropenone was observed via the formation of Se 0 from the oxidation of H2Se and using the H2Se-selective gas detector. In addition, an electrophilic iodoacetamide was used to trap H2Se in this Cys-activated system, and the expected products (diacetamide selenide and diselenide) were observed via HRMS (high-resolution mass spectrometry) analysis. For the quantitative assay in aqueous solution, Cy7-CI (11-chloro-1,1′din-propyl-3,3,3′,3′-tetramethyl-10,12-trimethyleneindatricarbocyanine iodide) was used for direct H2Se trapping. The selectivity of Cy7-CI was evaluated towards H2Se, R-SH, and R-Se, and the reaction rates were measured via HPLC and evaluated from the optical response ( Figure 6B); that is, the initial absorbance peak of Cy7-CI at 780 nm was shifted to 710 nm after the reaction with H2Se. Cys-triggered (1 mM) H2Se release from arylselenoamides (25 μM) in living cells was also confirmed via confocal fluorescence imaging in HeLa cells (human cervical cancer) using the previously reported fluorescent probe NIR-H2Se (Figure 4, [75]). In addition, mechanistic studies of Cys-triggered H2Se release from both selenocyclopropenone and arylselenoamide were performed. Figure 5. Donors for hydrolysis-based H 2 Se release. (A) Proposed hydrolysis mechanism of TDN1042 resulting in H 2 Se release at acidic pH (modified from ref. [106]); (B) hydrolysis of cyclic compound Cat-PSe to release H 2 Se at pH 7.4 (modified from [107]); (C) structure of 2AP-PSe used in the study [107].
Due to the relatively slow H 2 Se release at pH 7.4, the team decided to modify this compound, and in further study, they incorporated ortho-substituted phenols into the phosphorus center of TDN1042 to form cyclic-PSe compounds (e.g., 2AP-PSe, 2-aminophenol derivative; Cat-PSe) as H 2 Se donors ( Figure 5B,C), and H 2 Se release was monitored via 31 P and 77 Se NMR spectroscopy using alkylative trapping experiments as before [107]. The mechanism of H 2 Se release was shown on the example of the Cat-PSe compound reaction ( Figure 5B). In addition, a colorimetric method based on the reaction of H 2 Se with NBD-Cl (4-chloro-7-nitrobenzofurazan, λmax = 350 nm) was used to generate NBD-SeH (λmax = 551 nm), a compound that can be used for colorimetric detection of free H 2 Se. Moreover, TOF-SIMS (time of flight secondary ion mass spectroscopy) was used to demonstrate that 2AP-PSe is cell permeable at 10 µM concentration and in a dose-dependent manner from 0 to 25 µM. In this assay, total intracellular Se content as a function of H 2 Se donor concentration was measured.
It was also shown that 2AP-PSe can exert antioxidant activities through reducing ROS and H 2 O 2 content in living HeLa cells.
Considering the study of Pluth et al. and inspired by other synthetic H 2 S donors (compounds activated by bio-thiols [108]), Kang and coworkers proposed Cys-activated H 2 Se donors, that is, selenocarbonyl-containing derivatives with C==Se motif [109]. Thus, the C==Se motifs in selenocyclopropenones and selenoamides can be selectively activated by the -SH moiety of Cys for controllable H 2 Se release at pH 7.4, and these reactions occur with different rates for those compounds ( Figure 6A). In the qualitative assay, H 2 Se release from selenocyclopropenone was observed via the formation of Se 0 from the oxidation of H 2 Se and using the H 2 Se-selective gas detector. In addition, an electrophilic iodoacetamide was used to trap H 2 Se in this Cys-activated system, and the expected products (diacetamide selenide and diselenide) were observed via HRMS (high-resolution mass spectrometry) analysis. For the quantitative assay in aqueous solution, Cy7-CI (11-chloro-1,1 -din-propyl-3,3,3 ,3 -tetramethyl-10,12-trimethyleneindatricarbocyanine iodide) was used for direct H 2 Se trapping. The selectivity of Cy7-CI was evaluated towards H 2 Se, R-SH, and R-Se, and the reaction rates were measured via HPLC and evaluated from the optical response ( Figure 6B); that is, the initial absorbance peak of Cy7-CI at 780 nm was shifted to 710 nm after the reaction with H 2 Se. Cys-triggered (1 mM) H 2 Se release from arylselenoamides (25 µM) in living cells was also confirmed via confocal fluorescence imaging in HeLa cells (human cervical cancer) using the previously reported fluorescent probe NIR-H 2 Se (Figure 4, [75]). In addition, mechanistic studies of Cys-triggered H 2 Se release from both selenocyclopropenone and arylselenoamide were performed. Recently, selenium-derived nucleotides have been proposed as a new class of selenium-containing compounds that are H2Se donors with anticancer potential. These compounds have been shown to act through a novel mechanism of H2Se release in cells, and the involvement of the HINT1 protein in this process has been demonstrated [110]. HINT1 is a hydrolase involved in nucleoside/nucleotide metabolism. This cytoplasmic enzyme is widely expressed in all organisms and belongs to the histidine triad superfamily (HIT), whose members are characterized by the sequence motif H-X-H-X-H-X-X-X (H-a histidine residue, X-a hydrophobic amino acid) [111]. Previously, HINT1 was shown to be involved in the metabolism of phosphorothioate oligonucleotide drugs catalyzing the conversion of 5′-O-thiophosphate nucleosides (NMPS) to NMP and H2S in vitro and in cells [112,113]. By analogy, studies have shown that deoxyguanosine selenophosphate (dGMPSe) undergoes HINT1-assisted hydrolysis to the corresponding phosphate and H2Se in a similar manner both in vitro and intracellularly, according to the equation presented in Figure 7A. Recently, selenium-derived nucleotides have been proposed as a new class of seleniumcontaining compounds that are H 2 Se donors with anticancer potential. These compounds have been shown to act through a novel mechanism of H 2 Se release in cells, and the involvement of the HINT1 protein in this process has been demonstrated [110]. HINT1 is a hydrolase involved in nucleoside/nucleotide metabolism. This cytoplasmic enzyme is widely expressed in all organisms and belongs to the histidine triad superfamily (HIT), whose members are characterized by the sequence motif H-X-H-X-H-X-X-X (H-a histidine residue, X-a hydrophobic amino acid) [111]. Previously, HINT1 was shown to be involved in the metabolism of phosphorothioate oligonucleotide drugs catalyzing the conversion of 5 -O-thiophosphate nucleosides (NMPS) to NMP and H 2 S in vitro and in cells [112,113]. By analogy, studies have shown that deoxyguanosine selenophosphate (dGMPSe) undergoes HINT1-assisted hydrolysis to the corresponding phosphate and H 2 Se in a similar manner both in vitro and intracellularly, according to the equation presented in Figure 7A.
The release of H 2 Se in the living cells and the tube after administration of dGMPSe was observed using a fluorogenic SF7 probe (5 µM, ex/em = 485/528 nm, Figure 7B), which has been used previously to detect the release of H 2 S in cells [113,114]. Moreover, the toxicity of dGMPSe to HeLa cells was determined (IC 50 = 8 µM after 24 h), and the induction of tumor cell death has been shown to be related to H 2 Se release, which was observed after only 6 h of incubation in cells at dGMPSe concentration of 10 µM. In contrast to dGMPSe, dGMPS was not cytotoxic to HeLa cells even at millimolar concentrations. Because of the substrate specificity of HINT1, the reaction with the enzyme can be extended to other nucleoside 5 -O-selenophosphates {(d)NMPSe}. Consistent with the activity shown for various nucleoside phosphorothioates of the ribo-and deoxy-series (higher activity for purine ribonucleosides), the cellular toxic effect toward AMPSe and GMPSe may also be greater than for dGMPSe [115]. The release of H2Se in the living cells and the tube after administration of dGMPSe was observed using a fluorogenic SF7 probe (5 μM, ex/em = 485/528 nm, Figure 7B), which has been used previously to detect the release of H2S in cells [113,114]. Moreover, the toxicity of dGMPSe to HeLa cells was determined (IC50 = 8 µ M after 24 h), and the induction of tumor cell death has been shown to be related to H2Se release, which was observed after only 6 h of incubation in cells at dGMPSe concentration of 10 µ M. In contrast to dGMPSe, dGMPS was not cytotoxic to HeLa cells even at millimolar concentrations. Because of the substrate specificity of HINT1, the reaction with the enzyme can be extended to other nucleoside 5′-O-selenophosphates {(d)NMPSe}. Consistent with the activity shown for various nucleoside phosphorothioates of the ribo-and deoxy-series (higher activity for purine ribonucleosides), the cellular toxic effect toward AMPSe and GMPSe may also be greater than for dGMPSe [115].
Recently, Lukesh and coworkers reported that γ-keto selenides provide a useful platform for H2Se donation via an α-deprotonation/β-elimination pathway that is highly dependent on both pH and alpha-proton acidity [116]. This new class of slow-release donors functioned via an alternative mechanism compared to the first compound reported by the Pluth team [106], and the rate of H2Se release was enhanced under neutral to slightly basic conditions ( Figure 8B), in contrast to the increased hydrolysis at more acidic pH for TDN1042. To unequivocally confirm H2Se release from γ-keto selenides, an additional trapping experiment was performed. Using HRMS, the resulting product, volatilized H2Se, was captured with electrophilic iodoacetamide and easily detected in the obtained spectrum. Further evidence for the release of H2Se from γ-keto selenides was the formation of more stable selenodiglutathione (GSSeSG) upon its reaction in a redox buffer consisting of glutathione and glutathione disulfide in an ammonium bicarbonate buffer Recently, Lukesh and coworkers reported that γ-keto selenides provide a useful platform for H 2 Se donation via an α-deprotonation/β-elimination pathway that is highly dependent on both pH and alpha-proton acidity [116]. This new class of slow-release donors functioned via an alternative mechanism compared to the first compound reported by the Pluth team [106], and the rate of H 2 Se release was enhanced under neutral to slightly basic conditions ( Figure 8B), in contrast to the increased hydrolysis at more acidic pH for TDN1042. To unequivocally confirm H 2 Se release from γ-keto selenides, an additional trapping experiment was performed. Using HRMS, the resulting product, volatilized H 2 Se, was captured with electrophilic iodoacetamide and easily detected in the obtained spectrum. Further evidence for the release of H 2 Se from γ-keto selenides was the formation of more stable selenodiglutathione (GSSeSG) upon its reaction in a redox buffer consisting of glutathione and glutathione disulfide in an ammonium bicarbonate buffer (observed directly via mass spectrometry). In a cellular environment, the γ-keto selenides compounds were evaluated for their growth inhibitory activity against HeLa and HCT116 cells (human colon cancer). The investigated H 2 Se donors 1, 4, 5 ( Figure 8A) showed low micromolar activity against both studied cancer cell lines (IC 50 = 3.7-10.6 µM in HeLa; IC 50 = 4.1-10.1 µM in HCT116 and >50 µM in the case of control, compound 3 for both cell lines), which is consistent with the anticancer activity of the previously reported H 2 Se donors [74,110].
(observed directly via mass spectrometry). In a cellular environment, the γ-keto selenides compounds were evaluated for their growth inhibitory activity against HeLa and HCT116 cells (human colon cancer). The investigated H2Se donors 1, 4, 5 ( Figure 8A) showed low micromolar activity against both studied cancer cell lines (IC50 = 3.7-10.6 µ M in HeLa; IC50 = 4.1-10.1 µ M in HCT116 and >50 µ M in the case of control, compound 3 for both cell lines), which is consistent with the anticancer activity of the previously reported H2Se donors [74,110]. The selenium compounds discussed in this review are presented in Table 1.  The selenium compounds discussed in this review are presented in Table 1.

Conclusions
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 H 2 Se donors meet these requirements.
However, there are few detailed studies on the chemical biology of H 2 Se, and there are not many reports about the essential physiological functions of H 2 Se, 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 H 2 Se through continuously releasing the unstable biomolecule under physiologically relevant conditions.
In this review, we present only H 2 Se donors where the release of this product has been documented using various techniques such as fluorescence imaging, HRMS, 77 SeNMR, or others. Previously (before 2016), there were some compounds for which H 2 Se release was supposed but for which there was no technical evidence. Since then, several H 2 Se donors have been developed for which H 2 Se release has been well documented, although not all of them have been demonstrated to show anticancer activity in the cellular model. However, the possibility of using several new H 2 Se donors provides an opportunity to explain their influence on redox homeostasis of cancer cells and to answer the question of whether Se is an antioxidant or a pro-oxidant. So far, it has been shown that depending on aerobic conditions, selenium compounds can act as generators of oxidative stress (normoxia) or reductive stress (hypoxia). However, 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 Na 2 SeO 3 was studied so deeply (apoptosis under oxidative stress and autophagy under reductive stress). Many other mechanisms are possible, including different types cell death such as ferroptosis, necroptosis, etc.; influence on DNA repair or damage; and other various cell signaling pathways.
In conclusion, future studies are needed to show the utility of selenium compounds as H 2 Se donors in cancer therapy and in the selective induction of reductive stress in cells and in vivo, as few data are available to date.