Novel Methylselenoesters Induce Programed Cell Death via Entosis in Pancreatic Cancer Cells.

Redox active selenium (Se) compounds have gained substantial attention in the last decade as potential cancer therapeutic agents. Several Se compounds have shown high selectivity and sensitivity against malignant cells. The cytotoxic effects are exerted by their biologically active metabolites, with methylselenol (CH3SeH) being one of the key executors. In search of novel CH3SeH precursors, we previously synthesized a series of methylselenoesters that were active (GI50 < 10 µM at 72 h) against a panel of cancer cell lines. Herein, we refined the mechanism of action of the two lead compounds with the additional synthesis of new analogs (ethyl, pentyl, and benzyl derivatives). A novel mechanism for the programmed cell death mechanism for Se-compounds was identified. Both methylseleninic acid and the novel CH3SeH precursors induced entosis by cell detachment through downregulation of cell division control protein 42 homolog (CDC42) and its downstream effector ß1-integrin (CD29). To our knowledge, this is the first time that Se compounds have been reported to induce this type of cell death and is of importance in the characterization of the anticancerogenic properties of these compounds.

Heterogeneous Rupturing Dendrimers.

Utilizing macromolecular scaffolds as templates for the production of small molecules that are distinctively different from the original monomer feedstock has many potential applications. Herein, as a proof-of-concept, a family of dendrimers displaying internally queued disulfide bridges were synthesized and exploited as flawless macromolecular templates that selectively rupture into a set of monomeric mercaptans. Disassembly was accomplished in a reducing environment, using DTT as an external stimulus, and the thiol constituents were successfully isolated. Their composition was dictated by three dendritic regions, i.e., (i) the symmetrical trithiol of the core (C3), (ii) the interior-asymmetric trithiols (CD2), and (iii) the periphery-asymmetric monothiols (DB2), in which B functionality is of an orthogonal nature. Taking into account the steady state between disulfides and thiols in all living cells, the collapse of the dendrimers to a multitude of smaller thiols was intracellularly assessed as a means to disrupt the balance of reactive oxygen species (ROS) often elevated in cancer cells. Indeed, the fragmentation induced a significant increase of ROS in human lung carcinoma A549 cells. These findings can potentially alter the perception of dendrimers being limited to carriers to being prodrugs for intracellular delivery of ROS with the potential to fight cancer.

Novel Methylselenoesters as Antiproliferative Agents.

Selenium (Se) compounds are potential therapeutic agents in cancer. Importantly, the biological effects of Se compounds are exerted by their metabolites, with methylselenol (CH3SeH) being one of the key executors. In this study, we developed a new series of methylselenoesters with different scaffolds aiming to modulate the release of CH3SeH. The fifteen compounds follow Lipinski's Rule of Five and with exception of compounds 1 and 14, present better drug-likeness values than the positive control methylseleninic acid. The compounds were evaluated to determine their radical scavenging activity. Compound 11 reduced both DPPH and ABTS radicals. The cytotoxicity of the compounds was evaluated in a panel of five cancer cell lines (prostate, colon and lung carcinoma, mammary adenocarcinoma and chronic myelogenous leukemia) and two non-malignant (lung and mammary epithelial) cell lines. Ten compounds had GI50 values below 10 µM at 72 h in four cancer cell lines. Compounds 5 and 15 were chosen for further characterization of their mechanism of action in the mammary adenocarcinoma cell line due to their similarity with methylseleninic acid. Both compounds induced G2/M arrest whereas cell death was partially executed by caspases. The reduction and metabolism were also investigated, and both compounds were shown to be substrates for redox active enzyme thioredoxin reductase.

Selenium compounds as therapeutic agents in cancer.

BACKGROUND: With cancer cells encompassing consistently higher production of reactive oxygen species (ROS) and with an induced antioxidant defense to counteract the increased basal ROS production, tumors have a limited reserve capacity resulting in an increased vulnerability of some cancer cells to ROS. Based on this, oxidative stress has been recognized as a tumor-specific target for the rational design of new anticancer agents. Among redox modulating compounds, selenium compounds have gained substantial attention due to their promising chemotherapeutic potential. SCOPE OF REVIEW: This review aims in summarizing and providing the recent developments of our understanding of the molecular mechanisms that underlie the potential anticancer effects of selenium compounds. MAJOR CONCLUSIONS: It is well established that selenium at higher doses readily can turn into a prooxidant and thereby exert its potential anticancer properties. However, the biological activity of selenium compounds and the mechanism behind these effects are highly dependent on its speciation and the specific metabolic pathways of cells and tissues. Conversely, the chemical properties and the main molecular mechanisms of the most relevant inorganic and organic selenium compounds as well as selenium-based nanoparticles must be taken into account and are discussed herein. GENERAL SIGNIFICANCE: Elucidating and deepening our mechanistic knowledge of selenium compounds will help in designing and optimizing compounds with more specific antitumor properties for possible future application of selenium compounds in the treatment of cancer. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.

Metal- and Semimetal-Containing Inhibitors of Thioredoxin Reductase as Anticancer Agents.

The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide disulfide oxidoreductases playing a central role in cellular redox homeostasis and signaling pathways. Recently, these selenoproteins have emerged as promising therapeutic targets for anticancer drug development, often being overexpressed in tumor cells and contributing to drug resistance. Herein, we summarize the current knowledge on metal- and semimetal-containing molecules capable of hampering mammalian TrxRs, with an emphasis on compounds reported in the last decade.

Selenium induces a multi-targeted cell death process in addition to ROS formation.

Selenium compounds inhibit neoplastic growth. Redox active selenium compounds are evolving as promising chemotherapeutic agents through tumour selectivity and multi-target response, which are of great benefit in preventing development of drug resistance. Generation of reactive oxygen species is implicated in selenium-mediated cytotoxic effects on cancer cells. Recent findings indicate that activation of diverse intracellular signalling leading to cell death depends on the chemical form of selenium applied and/or cell line investigated. In the present study, we aimed at deciphering different modes of cell death in a single cell line (HeLa) upon treatment with three redox active selenium compounds (selenite, selenodiglutathione and seleno-DL-cystine). Both selenite and selenodiglutathione exhibited equipotent toxicity (IC50 5 µM) in these cells with striking differences in toxicity mechanisms. Morphological and molecular alterations provided evidence of necroptosis-like cell death in selenite treatment, whereas selenodiglutathione induced apoptosis-like cell death. We demonstrate that selenodiglutathione efficiently glutathionylated free protein thiols, which might explain the early differences in cytotoxic effects induced by selenite and selenodiglutathione. In contrast, seleno-DL-cystine treatment at an IC50 concentration of 100 µM induced morphologically two distinct different types of cell death, one with apoptosis-like phenotype, while the other was reminiscent of paraptosis-like cell death, characterized by induction of unfolded protein response, ER-stress and occurrence of large cytoplasmic vacuoles. Collectively, the current results underline the diverse cytotoxic effects and variable potential of redox active selenium compounds on the survival of HeLa cells and thereby substantiate the potential of chemical species-specific usage of selenium in the treatment of cancers.

Effects of redox modulation by inhibition of thioredoxin reductase on radiosensitivity and gene expression.

The thioredoxin system is a promising target when aiming to overcome the problem of clinical radiation resistance. Altered cellular redox status and redox sensitive thiols contributing to induction of resistance strongly connect the ubiquitous redox enzyme thioredoxin reductase (TrxR) to the cellular response to ionizing radiation. To further investigate possible strategies in combating clinical radiation resistance, human radio-resistant lung cancer cells were subjected to a combination of single fractions of γ-radiation at clinically relevant doses and non-toxic levels of a well-characterized thioredoxin reductase inhibitor, the phosphine gold(I) compound [Au(SCN)(PEt(3))]. The combination of the TrxR-inhibitor and ionizing radiation reduced the surviving fractions and impaired the ability of the U1810 cells to repopulate by approximately 50%. In addition, inhibition of thioredoxin reductase caused changes in the cell cycle distribution, suggesting a disturbance of the mitotic process. Global gene expression analysis also revealed clustered genetic expression changes connected to several major cellular pathways such as cell cycle, cellular response to stress and DNA damage. Specific TrxR-inhibition as a factor behind the achieved results was confirmed by correlation of gene expression patterns between gold and siRNA treatment. These results clearly demonstrate TrxR as an important factor conferring resistance to irradiation and the use of [Au(SCN)(PEt(3))] as a promising radiosensitizing agent.

Extracellular thiol-assisted selenium uptake dependent on the x(c)- cystine transporter explains the cancer-specific cytotoxicity of selenite.

The selenium salt selenite (SeO(3)(2-)) is cytotoxic in low to moderate concentrations, with a remarkable specificity for cancer cells resistant to conventional chemotherapy. Our data show that selenium uptake and accumulation, rather than intracellular events, are crucial to the specific selenite cytotoxicity observed in resistant cancer cells. We show that selenium uptake depends on extracellular reduction, and that the extracellular environment is a key factor specific to selenite cytotoxicity. The extracellular reduction is mediated by cysteine, and the efficacy is determined by the uptake of cystine by the x(c)(-) antiporter and secretion of cysteine by multidrug resistance proteins, both of which are frequently overexpressed by resistant cancer cells. This mechanism provides molecular evidence for the existence of an inverse relationship between resistance to conventional chemotherapy and sensitivity to selenite cytotoxicity, and highlights the great therapeutic potential in treating multidrug-resistant cancer.

Selenium compounds are substrates for glutaredoxins: a novel pathway for selenium metabolism and a potential mechanism for selenium-mediated cytotoxicity.

The Grx (glutaredoxin) proteins are oxidoreductases with a central function in maintaining the redox balance within the cell. In the present study, we have explored the reactions between selenium compounds and the glutaredoxin system. Selenite, GS-Se-SG (selenodiglutathione) and selenocystine were all shown to be substrates of human Grx1, implying a novel role for the glutaredoxins in selenium metabolism. During the past few years, selenium has further evolved as a potential therapeutic agent in cancer treatment, and a leading mechanism of cytotoxicity is the generation of ROS (reactive oxygen species). Both selenite and GS-Se-SG were reduced by Grx1 and Grx2 in a non-stoichiometric manner due to redox cycling with oxygen, which in turn generated ROS. The role of Grx in selenium toxicity was therefore explored. Cells were treated with the selenium compounds in combination with transient overexpression of, or small interfering RNA against, Grx1. The results demonstrated an increased viability of the cells during silencing of Grx1, indicating that Grx1 is contributing to selenium toxicity. This is in contrast with TrxR (thioredoxin reductase), which previously was shown to protect cells from selenium cytotoxicity, verifying a diverse role between Grx and TrxR in selenium-mediated cytotoxicity. Furthermore, selenium treatment led to a marked increase in protein glutathionylation and cysteinylation that potentially can influence the activity and function of several proteins within the cell.

Chelation of lysosomal iron protects against ionizing radiation.

Ionizing radiation causes DNA damage and consequent apoptosis, mainly due to the production of hydroxyl radicals (HO•) that follows radiolytic splitting of water. However, superoxide (O2•-) and H2O2 also form and induce oxidative stress with resulting LMP (lysosomal membrane permeabilization) arising from iron-catalysed oxidative events. The latter will contribute significantly to radiation-induced cell death and its degree largely depends on the quantities of lysosomal redox-active iron present as a consequence of autophagy and endocytosis of iron-rich compounds. Therefore radiation sensitivity might be depressed by lysosome-targeted iron chelators. In the present study, we have shown that cells in culture are significantly protected from ionizing radiation damage if initially exposed to the lipophilic iron chelator SIH (salicylaldehyde isonicotinoyl hydrazone), and that this effect is based on SIH-dependent lysosomal stabilization against oxidative stress. According to its dose-response-modifying effect, SIH is a most powerful radioprotector and a promising candidate for clinical application, mainly to reduce the radiation sensitivity of normal tissue. We propose, as an example, that inhalation of SIH before each irradiation session by patients undergoing treatment for lung malignancies would protect normally aerated lung tissue against life-threatening pulmonary fibrosis, whereas the sensitivity of malignant lung tumours, which usually are non-aerated, will not be affected by inhaled SIH.

Expression profiles of thioredoxin family proteins in human lung cancer tissue: correlation with proliferation and differentiation.

AIMS: Lung cancer is one of the most common causes of cancer lethality worldwide. Despite recent progress, long-term survival remains poor. The aim of this study was to explore the expression pattern of the thioredoxin superfamily of proteins as potential new diagnostic and/or predictive markers. METHODS AND RESULTS: The expression of thioredoxin 1 (Trx1), thioredoxin reductase 1 (TrxR1), the isoforms TrxR1-v.2,3,5, glutaredoxin 1 (Grx1) and glutaredoxin 2 (Grx2) was examined by immunohistochemistry on paraffin-embedded sections from 42 cases of non-small cell lung cancer patients. Additional cases of lung cancer from tissue microarray were examined and the immunoreactivity was compared. All proteins except TrxR1 showed a significant correlation with the degree of differentiation in adenocarcinoma. Trx1 and TrxR1-v.2,3,5 also showed a significant correlation with differentiation in squamous carcinoma. Furthermore, Grx1 and Grx2 showed a clear inverse correlation with proliferation. The proliferation rate was further analysed in vitro in stably transfected Grx2 overproducing cells, showing that the proliferative effect of Grx2 is strictly dependent on subcellular localization. CONCLUSIONS: The thioredoxin family of proteins is important for growth and differentiation of lung cancer cells. The correlation with differentiation and proliferation of these enzymes makes them promising predictive/diagnostic markers.

Organic selenium compounds as potential chemotherapeutic agents for improved cancer treatment.

Selenium(Se)-containing compounds have attracted a growing interest as anticancer agents over recent decades, with mounting reports demonstrating their high efficacy and selectivity against cancer cells. Typically, Se compounds exert their cytotoxic effects by acting as pro-oxidants that alter cellular redox homeostasis. However, the precise intracellular targets, signalling pathways affected and mechanisms of cell death engaged following treatment vary with the chemical properties of the selenocompound and its metabolites, as well as the cancer model that is used. Naturally occurring organic Se compounds, besides encompassing a significant antitumor activity with an apparent ability to prevent metastasis, also seem to have fewer side effects and less systemic effects as reported for many inorganic Se compounds. On this basis, many novel organoselenium compounds have also been synthesized and examined as potential chemotherapeutic agents. This review aims to summarize the most well studied natural and synthetic organoselenium compounds and provide the most recent developments in our understanding of the molecular mechanisms that underlie their potential anticancer effects.

Selenite and methylseleninic acid epigenetically affects distinct gene sets in myeloid leukemia: A genome wide epigenetic analysis.

Selenium compounds have emerged as promising chemotherapeutic agents with proposed epigenetic effects, however the mechanisms and downstream effects are yet to be studied. Here we assessed the effects of the inorganic selenium compound selenite and the organic form methylseleninic acid (MSA) in a leukemic cell line K562, on active (histone H3 lysine 9 acetylation, H3K9ac and histone H3 lysine 4 tri-methylation, H3K4me3) and repressive (histone H3 lysine 9 tri-methylation, H3K9me3) histone marks by Chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq). Both selenite and MSA had major effects on histone marks but the effects of MSA were more pronounced. Gene ontology analysis revealed that selenite affected genes involved in response to oxygen and hypoxia, whereas MSA affected distinct gene sets associated with cell adhesion and glucocorticoid receptors, also apparent by global gene expression analysis using RNA sequencing. The correlation to adhesion was functionally confirmed by a significantly weakened ability of MSA treated cells to attach to fibronectin and linked to decreased expression of integrin beta 1. A striking loss of cellular adhesion was also confirmed in primary patient AML cells. Recent strategies to enhance the cytotoxicity of chemotherapeutic drugs by disrupting the interaction between leukemic and stromal cells in the bone marrow are of increasing interest; and organic selenium compounds like MSA might be promising candidates. In conclusion, these results provide new insight on the mechanism of action of selenium compounds, and will be of value for the understanding, usage, and development of new selenium compounds as anticancer agents.

Methylseleninic Acid Sensitizes Ovarian Cancer Cells to T-Cell Mediated Killing by Decreasing PDL1 and VEGF Levels.

Redox active selenium (Se) compounds at sub toxic doses act as pro-oxidants with cytotoxic effects on tumor cells and are promising future chemotherapeutic agents. However, little is known about how Se compounds affect immune cells in the tumor microenvironment. We demonstrate that the inorganic Se compound selenite and the organic methylseleninic acid (MSA) do not, despite their pro-oxidant function, influence the viability of immune cells, at doses that gives cytotoxic effects in ovarian cancer cell lines. Treatment of the ovarian cancer cell line A2780 with selenite and MSA increases NK cell mediated lysis, and enhances the cytolytic activity of T cells. Increased T cell function was observed after incubation of T cells in preconditioned media from tumor cells treated with MSA, an effect that was coupled to decreased levels of PDL1, HIF-1α, and VEGF. In conclusion, redox active selenium compounds do not kill or inactivate immune cells at doses required for anti-cancer treatment, and we demonstrate that MSA enhances T cell-mediated tumor cell killing via PDL1 and VEGF inhibition.

Bacterial thioredoxin and thioredoxin reductase as mediators for epigallocatechin 3-gallate-induced antimicrobial action.

Epigallocatechin 3-gallate (EGCG) is the most abundant catechin in green tea and may combat bacteria with few side-effects. Its selectivity for different bacterial infections remains unclear, and hence the identification of the underlying mechanism is of practical importance. Both the thioredoxin (Trx) system and the glutathione/glutaredoxin (Grx) system support bacterial growth. Some pathogenic bacteria are naturally deficient in the Grx system. We analyzed the effect of green tea extract (GTE) and EGCG on wild-type and null mutants of Escherichia coli with either Trx or Grx system deficiency and found that GTE and EGCG selected the Trx system as a target and killed the mutant that is exclusively dependent on Trx/Trx reductase (TrxR). EGCG inhibited the activity of both Trx1 and TrxR of E. coli in a dose-dependent and time-dependent manner. The IC50 values of EGCG for the reduced forms of E. coli Trx1/TrxR were ~ 3-4-fold lower than those for their non-reduced forms. The IC50 value of EGCG for the E. coli Trx1 system was 56-fold lower than that for the mammalian Trx1 system. The inhibition by EGCG of both Trx1 and TrxR of E. coli was irreversible. EGCG-induced inactivation of E. coli Trx1 was a second-order process, and that of E. coli TrxR was an affinity-labeling process. The covalent binding sites for EGCG in E. coli Trx1 were Trp(28) , Trp(31) and Cys(32) , and in E. coli TrxR were Cys(135) and Cys(138) . Moreover, the sensitivity of Staphylococcus aureus to EGCG was similar to that of an E. coli mutant with Grx system deficiency. EGCG-induced inactivation of Trx/TrxR in S. aureus coincided with suppressed growth of this virulent pathogen. Our findings suggest a role for EGCG-dependent Trx/TrxR inactivation in potentiating antibacterial activity of EGCG.

A ribonucleotide reductase inhibitor with deoxyribonucleoside-reversible cytotoxicity.

Ribonucleotide Reductase (RNR) is the sole enzyme that catalyzes the reduction of ribonucleotides into deoxyribonucleotides. Even though RNR is a recognized target for antiproliferative molecules, and the main target of the approved drug hydroxyurea, few new leads targeted to this enzyme have been developed. We have evaluated a recently identified set of RNR inhibitors with respect to inhibition of the human enzyme and cellular toxicity. One compound, NSC73735, is particularly interesting; it is specific for leukemia cells and is the first identified compound that hinders oligomerization of the mammalian large RNR subunit. Similar to hydroxyurea, it caused a disruption of the cell cycle distribution of cultured HL-60 cells. In contrast to hydroxyurea, the disruption was reversible, indicating higher specificity. NSC73735 thus defines a potential lead candidate for RNR-targeted anticancer drugs, as well as a chemical probe with better selectivity for RNR inhibition than hydroxyurea.

Glutaredoxin mediated redox effects of coenzyme Q10 treatment in type 1 and type 2 diabetes patients.

The possible beneficial effects of coenzyme Q10 (CoQ10) supplementation on disease progression and oxidant status in diabetes remains debated. In the present study, patients with type 1 and type 2 diabetes were treated with oral CoQ10, 100 mg twice daily for 12 weeks. We assessed total antioxidant capacity, intra- and extracellular levels of the redox regulating protein glutaredoxin 1 (Grx1), CoQ10, oxidized LDL-cholesterol, lipid profile and HbA1c. We have previously shown that extracellular Grx1 is increased in patients with type 2 diabetes compared to healthy subjects. In the present study, CoQ10 treatment significantly decreased serum Grx1 activity as well as total antioxidant capacity independent of type of diabetes, indicating an improvement to a less oxidized extracellular environment. The effect on serum Grx1 activity was more prominent in patients not on statin treatment. Conversely, intracellular Grx1 activity as well as mRNA levels increased independent of statin treatment. There was a significant improvement in oxidized LDL-cholesterol and lipid profile, with a tendency to improved metabolic control (HbA1c). Additionally, we describe for the first time that CoQ10 is a direct substrate for glutathione, and that Grx1 catalyzes this reaction, thus presenting a novel mechanism for CoQ10 reduction which could explain our findings of an increased intracellular Grx1. In conclusion, 12 weeks CoQ10 treatment significantly improved the extracellular redox balance and lipid profile, indicating that prolonged treatment may have beneficial effects also on clinical outcome in diabetes.

Protective effects of the thioredoxin and glutaredoxin systems in dopamine-induced cell death.

Although the etiology of sporadic Parkinson disease (PD) is unknown, it is well established that oxidative stress plays an important role in the pathogenic mechanism. The thioredoxin (Trx) and glutaredoxin (Grx) systems are two central systems upholding the sulfhydryl homeostasis by reducing disulfides and mixed disulfides within the cell and thereby protecting against oxidative stress. By examining the expression of redox proteins in human postmortem PD brains, we found the levels of Trx1 and thioredoxin reductase 1 (TrxR1) to be significantly decreased. The human neuroblastoma cell line SH-SY5Y and the nematode Caenorhabditis elegans were used as model systems to explore the potential protective effects of the redox proteins against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. 6-OHDA is highly prone to oxidation, resulting in the formation of the quinone of 6-OHDA, a highly reactive species and powerful neurotoxin. Treatment of human cells with 6-OHDA resulted in an increased expression of Trx1, TrxR1, Grx1, and Grx2, and small interfering RNA for these genes significantly increased the cytotoxic effects exerted by the 6-OHDA neurotoxin. Evaluation of the dopaminergic neurons in C. elegans revealed that nematodes lacking trxr-1 were significantly more sensitive to 6-OHDA, with significantly increased neuronal degradation. Importantly, both the Trx and the Grx systems were also found to directly mediate reduction of the 6-OHDA-quinone in vitro and thus render its cytotoxic effects. In conclusion, our results suggest that the two redox systems are important for neuronal survival in dopamine-induced cell death.

Alteration of thioredoxin and glutaredoxin in the progression of Alzheimer's disease.

Oxidative stress has an important role in the pathological process of most neurodegenerative disorders, including Alzheimer's disease (AD). The glutaredoxin (Grx) and thioredoxin (Trx) systems are central in maintaining a reduced environment in the cell and thus render protection against oxidative stress. Here, we show that Trx1 and Grx1 were released to the cerebrospinal fluid in 120 cases examined, and that the levels of these proteins increased significantly in the early stages of AD in comparison to mild cognitive impairment (MCI). Trx1 and Grx1 levels correlated with the established AD biomarkers tau and phospho-tau (p-tau). Moreover, by determining the levels of Trx1 and Grx1, discrimination between MCI converters and patients with stable MCI were possible. By applying the protein levels of Trx1 together with conventional diagnostic markers (Mini-Mental State Examination, tau, and p-tau) to a stepwise regression model, MCI stable, MCI converter, mild AD, and moderate AD was correctly diagnosed in 32 out of 33 cases. In order to further evaluate the involvement of these systems in AD, the immunoreactivity of Trx1, Trx2, Grx1, and Grx2 were investigated and the expression pattern was shown to be altered in hippocampus tissue sections from AD patients compared to controls. In conclusion, we introduce members of the thioredoxin super family of proteins as promising early biomarkers in the diagnosis of AD, suggesting their potential involvement in the pathogenesis of the disease.

Methylselenol formed by spontaneous methylation of selenide is a superior selenium substrate to the thioredoxin and glutaredoxin systems.

Naturally occurring selenium compounds like selenite and selenodiglutathione are metabolized to selenide in plants and animals. This highly reactive form of selenium can undergo methylation and form monomethylated and multimethylated species. These redox active selenium metabolites are of particular biological and pharmacological interest since they are potent inducers of apoptosis in cancer cells. The mammalian thioredoxin and glutaredoxin systems efficiently reduce selenite and selenodiglutathione to selenide. The reactions are non-stoichiometric aerobically due to redox cycling of selenide with oxygen and thiols. Using LDI-MS, we identified that the addition of S-adenosylmethionine (SAM) to the reactions formed methylselenol. This metabolite was a superior substrate to both the thioredoxin and glutaredoxin systems increasing the velocities of the nonstoichiometric redox cycles three-fold. In vitro cell experiments demonstrated that the presence of SAM increased the cytotoxicity of selenite and selenodiglutathione, which could neither be explained by altered selenium uptake nor impaired extra-cellular redox environment, previously shown to be highly important to selenite uptake and cytotoxicity. Our data suggest that selenide and SAM react spontaneously forming methylselenol, a highly nucleophilic and cytotoxic agent, with important physiological and pharmacological implications for the highly interesting anticancer effects of selenium.