Basic & Clinical Pharmacology & Toxicology, 2014, 114, 377–386

Doi: 10.1111/bcpt.12207

MiniReview

Selenium Cytotoxicity in Cancer Marita Wallenberg†, Sougat Misra† and Mikael Bj€ornstedt Division of Pathology F46, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden (Received 2 December 2013; Accepted 21 January 2013) Abstract: Selenium is an essential trace element with growth-modulating properties. Decades of research clearly demonstrate that selenium compounds inhibit the growth of malignant cells in diverse experimental model systems. However, the growth-modulating and cytotoxic mechanisms are diverse and far from clear. Lately, a remarkable tumour selective cytotoxicity of selenium compounds has been shown, indicating the potential of selenium in the treatment of cancer. Of particular interest are the redoxactive selenium compounds exhibiting cytotoxic potential to tumour cells. These selenium compounds elicit complex patterns of pharmacodynamics and pharmacokinetics, leading to cell death pathways that differ among compounds. Modern oncology often focuses on targeted ligand-based therapeutic strategies that are specific to their molecular targets. These drugs are initially efficient, but the tumour cells often rapidly develop resistance against these drugs. In contrast, certain redox-active selenium compounds induce complex cascades of pro-death signalling at pharmacological concentrations with superior tumour specificity. The target molecules are often the ones that are important for the survival of cancer cells and often implicated in drug resistance. Therefore, the chemotherapeutic applications of selenium offer great possibilities of multi-target attacks on tumour cells. This MiniReview focuses on the tumour-specific cytotoxic effects of selenium, with special emphasis on cascades of cellular events induced by the major groups of pharmacologically active selenium compounds. Furthermore, the great pharmacological potential of selenium in the treatment of resistant cancers is discussed.

History – the Discovery of Selenium Selenium is one of four elements discovered by Berzelius. The discovery is remarkable in many different ways. The soil in Sweden is very poor in selenium, but circumstances made the discovery possible. Selenium was first isolated from iron pyrite, a side product from the copper mine in Falun, Sweden. The pyrite was a raw material for the production of sulphur and sulphuric acid mainly for the war industry. The production facility was in Mariefred, south-west of Stockholm. As Berzelius was a part owner of the factory, he was very involved in the production process. In the reaction chambers, a red precipitate was accumulated. Berzelius first concluded that this precipitate was tellurium, but further investigations using the famous blow pipe, among other equipment, showed that the precipitate was a completely novel element with properties similar to both sulphur and tellurium, and Berzelius decided to name the new element after the Greek word for the moon, Selene, which in latinized form will be Selenium [1]. Chemical Properties Selenium has the atomic number 34 and atomic mass 78.96 and is present in group VI of the periodic table between Author for correspondence: Marita Wallenberg, Division of Pathology F46, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden (fax +46 8 58581020, e-mail [email protected]). † These authors have contributed equally to the manuscript.

sulphur and tellurium. Seventeen isotopes of the element have been recorded of which six are stable. The most abundant isotopes in nature are selenium with the mass numbers 80 (49.82%) and 78 (23.52%) [2]. Selenium with the mass number 77 has a low natural abundance (7.58%) but is stable with a nuclear spin of ½ why this isotope is useful for labelling and biophysical measurements [3]. Due to five possible oxidation states (
2, 0, +2, +4 and +6), selenium is exceptionally well represented in a variety of compounds with diverse chemical properties. Furthermore, selenium can be present in the place of sulphur in virtually all sulphur compounds, inorganic as well as organic. The latter is exemplified by the presence of the naturally occurring seleno-amino acids selenomethionine (SeMet) and selenocysteine (SeCys; fig. 1). These selenium analogues are biologically interesting because there are slight but important differences compared to the corresponding sulphur amino acids. These differences are explained by the fact that the atomic radius of selenium (1.17  A) is slightly larger and the electronegativity is slightly lower compared to sulphur. Another important difference is that selenols (corresponding to thiols) are relatively strong acids, and the pKa of the selenol is 5.24 compared to 8.25 of the thiol resulting in free selenolate at physiological pH compared to the less reactive thiol [4]. Selenium Metabolism The essential selenium molecule is present in the 21st aminoacid selenocysteine (SeCys), incorporated in 25 today known

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mammalian selenoprotein families. Selenium is important due to its localization within the active site of some selenoproteins with several functions including the maintenance of the redox balance. Detailed information concerning selenoproteins and their biosynthesis has been reviewed elsewhere [5]. Most selenium compounds, organic and inorganic, are readily absorbed from the diet and transported to the liver – the prime organ for selenium metabolism. The general metabolism of selenium compounds follows three major routes depending on the chemical properties, that is, redox-active selenium compounds, precursors of methylselenol and seleno-amino acids [6]. The most important naturally occurring redox-active selenium compounds are selenite and selenodiglutathione (GS-Se-SG; table 1 and fig. 1). Selenite is very reactive with thiols and will therefore be reduced already in the gastric mucosa. As a consequence, selenite ingested per orally will not appear in the blood. All available thiols will react with selenite, including free cysteine. An intermediate in the reaction with GSH is GS-Se-SG. The thioredoxin and glutaredoxin systems efficiently reduce selenite and GS-Se-SG to form the central selenium metabolite, selenide (HSe
) which under aerobic conditions and sufficient access to reducing power will redox cycle non-stoichiometrically to form large quantities of reactive oxygen species (ROS) (fig. 2) [7–9]. In the presence of S-adenosylmethionine, monomethylselenol will be formed non-enzymatically or enzymatically from selenide, and this methylated species is a superior substrate to the glutaredoxin and thioredoxin systems compared to selenide leading to efficient redox cycles [10]. Selenate (oxidation state +6) is the most oxidized selenium species in nature. This oxide is slowly

metabolized in the body to selenide by the thioredoxin system in the presence of glutathione (GSH) [11]. The predominant organic forms of selenium in plants include the SeCys and the methylated species, selenomethylselenocysteine (SeMSC) and SeMet. The two former require b-lyase to cleave the strong Se-C bond to form monomethylselenol and the latter c-lyase [12,13]. SeMet may also be metabolized by the trans-selenation pathway to form SeCys (table 1 and fig. 2). Selenide is very reactive and may redox cycle with oxygen, form elemental selenium, undergo methylation to monomethylselenol, dimethylselenide or trimethylselenonium or be incorporated into selenoproteins as SeCys. Monomethylselenol is a highly reactive species and believed to be the most efficient selenium compound to induce apoptosis in malignant cells. The methylation procedure is reversible, and there are methylation and dimethylation reactions in the cell catalysed by three classes of methyltransferases (MT-1, MT-2, MT-3), and demethylation is catalysed by demethylases [14] (fig. 2). The most predominant excretory urinary metabolites are Se-methyl-N-acetylgalactoseamine, Se-methyl-N-acetylglucosamine and trimethylselenonium [15,16]. Targeting Redox Regulation in Cancer Cells by Selenium Compounds at Therapeutic Doses Abnormal redox regulation in cancer cells is well documented at different stages of cancer. Both the enzymatic and nonenzymatic redox systems are modulated in cancer cells, providing certain growth and survival advantages necessary for the progression of the disease. Among the enzymatic systems,

Fig. 1. Molecular structure of the selenium compounds discussed in the review. All the structures were drawn using Symax Draw (version 4; Accelrys Inc, San Diego, CA, USA). Table 1. Sources of selenium compounds and their metabolic active form. Compound Selenite Selenodiglutathione Selenocystine Selenomethylselenocysteine Methylseleninic acid Selenomethionine

Source

Active form

Soils, water Organic intermediate metabolite Plants, meat Plants Synthetic compound Plants, meat

Reduced to selenide Reduced to selenide Reduced to selenide or cleaved by b-lyase to methylselenol Cleaved by b-lyase to methylselenol Reduced to methylselenol Transformed to SeCys via trans-selenation pathway or cleavage by c-lyase to methylselenol may occur

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Fig. 2. A schematic diagram describing cellular selenium metabolism in the mammalian system. The transport systems involved in selenium influx are not really well known. The organic forms share the transport systems of corresponding amino acid analouges and similar transport systems. Depending on the redox state of the extracellular environment, inorganic selenium compounds including selenite exhibit great variability in transport properties. One of the key transporters involved in the process is the xCT antiporter, regulating the extracellular redox milieu. Following uptake, different selenium compounds are metabolized into two central redox-active molecules – selenide (HSe
) and methylselenol (CH3Se
), either directly or via intermediate metabolism. Thiol-based redox systems including the thioredoxin systems and glutaredoxin systems play a key role in the metabolism of inorganic selenium compounds. The metabolism of organic selenium compounds includes trans-selenation pathway and several other enzymatic systems. The major excretory products are dimethylselenide, dimethyldiselenide, trimethylselenonium and selenosugars. Biosynthesis of selenoproteins is a key feature of all selenium compounds, irrespective of their chemical forms.

the expression of superoxide dismutase, catalase, glutathione peroxidase, peroxiredoxins and different selenoproteins with antioxidant functionalities is increased in many cancer types, while an opposite scenario prevails in other types of cancers [17]. Such opposing findings indicate complex and diverse functional regulation of antioxidant enzymes in different tumour types. In contrast, many tumour types harbour high levels of intracellular GSH. Increased GSH biosynthesis does not only combat the ROC-induced oxidative insults but also confers drug resistance. To this end, several cytostatic drugs have been designed to target intracellular GSH homeostasis (e.g. doxorubicin, placitaxel, buthionine sulphoximine) and antioxidant enzymes (auranofin, 2-methoxyestradiol). While the efficacy of single uses of these drugs resulted in variable outcome, drug resistance is still a major problem to be resolved for successful treatment. However, redox-based strategic chemotherapeutic application is a promising avenue, if the acquired drug resistance mechanisms can be successfully targeted. It is apparent that selective targeting of cellular antioxidant systems may profoundly affect the thiol- and nonthiol-mediated ROS scavenging survival axis of cancer cells with potential therapeutic applications. The question arises how redox-active selenium compounds are unique in this

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process? Redox cycling of intermediate selenium metabolites like hydrogen selenide (HSe
) and methyl selenol (CH3Se
) at higher concentration with GSH results in ROS generation, while GSH is scavenged in the process. Other thiol-containing and non-thiol antioxidants are also targets of these metabolites. Under such circumstances, not only the non-enzymatic redoxbased and redox-dependent defence systems are compromised, but also sustained exposure to these selenium intermediates becomes highly toxic to cancer cells. The oncogene, c-MYC, a regulator of GSH synthesizing enzymes in melanoma cells [18], is a target of a methylated selenium compound, methylseleninic acid (MSA) [19]. These observations suggest that apart from direct regulation of cellular thiol status, certain selenium compounds can indirectly inhibit the indispensable thiol biosynthetic pathway of certain types of cancers. Overall, redox-active selenium compounds comprise a promising class of chemotherapeutics in targeting thiol-based resistance paradox in cancer cells. The biological role of thiols in the form of cysteine is prominent not only for the cellular thiol homeostasis but also for numerous thiol-based redox-regulated processes within cells [20]. Thiol modification may cause structural changes of proteins, leading to activation or inactivation of their function. Many of these proteins are involved in metabolism, transcription and signalling pathways like the nuclear factor kappa b (NF-kb) and Jun N-terminal kinase (JNK)-signalling pathways [21]. Modification of critical thiol residues may lead to altered binding activity of transcription factors and their target gene expressions [22], calcium homeostasis [23], functionality of iron–sulphur cluster proteins [24], iron metabolism [25] and the synthesis of SeCys [26], among others. In 1941, Painter et al. showed that thiols were important for the reduction in selenite [27]. Since then, this finding has been proved both in in vitro and in vivo studies. In a classical paper, Seko et al. [28] found that reduction in selenite by GSH into selenide resulted in superoxide (O
2 ) formation and suggested the latter to be the cytotoxic cause of selenium-induced cell death. It has further been shown that addition of reduced extracellular GSH together with selenite increases the selenium uptake and cytotoxicity, leading to a decrease in intracellular GSH and an increase in the consumption of oxygen [29,30]. The toxicity of selenite and GS-Se-SG in cancer cells seems to be selective and dependent on a reduced extracellular environment. A recent study from our group shows that certain resistant tumour cells secrete high amounts of thiols (cysteine) into the media, and this efflux is regulated by the cystine/glutamate antiporter xCT. This efflux confers sensitivity to selenite [31]. A highly reduced extracellular environment of resistant tumour cells facilitates both the reduction in selenite to selenide and the subsequent selenium uptake. This antiporter has further been shown to be induced under oxidative stress, by increased oxygen supply, deprivation of cystine and by electrophilic compounds [32]. Many drug-resistant tumour cells overexpress xCT [33], which regulates the cysteine (reduced)/cystine (oxidized) homeostasis and GSH biosynthesis. This is beneficial for the tumour cells because increased GSH level facilitates detoxification of drugs. Another study

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suggests that the selectivity of selenite toxicity in prostate tumour cells compared to normal cells depends on the higher expression of manganese superoxide dismutase (MnSOD) in normal cells, rendering protection against selenite-induced superoxide formation [34]. Molecular Mechanisms of Selenium Cytotoxicity There is great variability in the mode of cell death induced by different selenium compounds. In general, cell death mechanisms in tumour cells are dependent on selenium species, exposure dose and time and the origin tissue of the tumour cells. Of significant importance is also the density of cells at the time of treatment in in vitro experiments. Usually, the organic selenium forms are considered to be less toxic than inorganic selenium compounds, due to the need of organic compounds to be metabolized in order to exert their cytotoxic potential. The cytotoxic mechanisms induced by selenium compounds in tumour cells at pharmacological concentrations have predominantly been ascribed to be caused by generation of ROS, including superoxide. However, this is not a general mechanism as the intermediate metabolites can directly modulate the functionalities of many signalling molecules. In many enzymes and transcription factors, cysteine residues located within the active and regulatory sites function as sensors for many redox regulatory processes (described in detail under section 4). Redox-active selenium compounds can directly or indirectly modulate the functionalities of these proteins upon direct interactions or by altering the cellular redox milieu. Therefore, the treatment concentration of selenium, subsequent metabolism and the interaction of these metabolites with protein and nonprotein thiols play important roles in the overall outcome. Based on the intermediate metabolites with known anticancer properties, selenium compounds can broadly be classified into two major groups. The first group consists of compounds that are primarily metabolized to selenide (HSe
), while the other group comprises compounds metabolized to methylselenol (CH3Se
). The group of redox-active selenium compounds that generate selenide under reduction includes selenite, GSSe-SG and to some extent CysSeSeCys (table 1). These are able to induce superoxide generation in the presence of thiols and oxygen, followed by formation of other ROS. Not all selenium compounds induce superoxide formation in vitro, because some of these also need to be metabolized before reduction into selenide or methylselenol implicated in superoxide formation [35]. In the presence of thiols, superoxide production targets the mitochondrial permeability transition pore, resulting in the release of cytochrome C and induction of apoptosis. Other important intracellular molecules in selenium toxicity are BCL-2 family proteins [36,37] and caspases which are activated by some selenium compounds [38]. Treatment with selenite and GS-Se-SG has been reported to induce single-strand breaks of DNA [39,40]. However, the group that generates methylselenol has instead been reported generally not to induce superoxide formation, but instead caspase-mediated apoptosis, and either DNA fragmentation [41] or no sign

of DNA damage [42]. This group consists of selenomethylselenocysteine (SeMSC), selenomethionine (SeMet), MSA and methylselenocyanate. The differences in the cytotoxicity between selenide- or methylselenol-generating compounds have been exemplified in several studies. In a prostate cancer cell line, selenite (5 lM) and MSA (3 and 5 lM) toxicity were compared thoroughly. Selenite treatment led to a superoxide-induced S-phase arrest, caspase-independent DNA fragmentation and apoptosis [43]. Selenite treatment further increased the expression of phosphorylated serine/threonine kinase (pAKT), JNK1 and p38 mitogen-activated protein kinase (p38 MAPK), which are kinases activated under cellular stress. In contrast, MSA caused G1-phase arrest, DNA fragmentation and a caspase-dependent poly (ADP-ribose) polymerase 1 (Parp-1) cleavage, leading to apoptosis. Compared to selenite, MSA instead decreased the phosphorylation of AKT and extracellular-signal-regulated kinase (ERK). Selenate, the highest oxidized form of selenium, is an inorganic redox-inactive selenium compound and is only toxic at very high doses, compared to selenite. Selenate is reduced slowly by thioredoxin in the presence of GSH [11]. New studies indicate that selenate may have other important functionalities as to inhibit adipogenesis via transforming growth factor b 1 (TGF-b 1) signalling, which selenite and MSA could not [44]. Selenium and Programmed Cell Death Impaired programmed cell death (PCD) is a key feature during the development and progression of cancer. PCD is a tightly regulated cell death process in contrast to necrosis which is caused by injury to the tissue or as an inflammatory response. There are multiple modalities of PCD that have been exemplified by the recent discoveries of several new pathways. Apart from previously known apoptosis (type I), autophagy (type II) and paraptosis (type III), the nomenclature now also includes mitotic catastrophy, anoikis, necroptosis, parthenos, pyroptosis and cornification, among others [45,46]. As with other chemotherapeutics, understanding the molecular mechanisms of selenium-induced programmed cell death pathways in cancer cells is equally important for their therapeutic usage. As several selenium compounds have been proved to target tumour cells selectively, and not their normal counterparts [40,47], it makes selenium even more interesting and promising to be used as an antitumour drug, compared to conventional cancer treatments used today. However, with ever increasing newly synthesized selenium compounds and different experimental model systems with diverse genetic expression conclusive determination of compound-specific cell death pathways is a challenging task. This complexity is also confounded by involvement of several intracellular molecular targets, apart from induction of ROS by several selenium compounds. The possible occurrence of genetic mutations or altered expression of important proteins having specific downstream interactions in cellular signalling pathways might lead to different cell death responses. In addition, as ROS is an important signalling molecule under normal conditions, with

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many intracellular molecular signalling targets, oxidative stress may activate several signalling pathways simultaneously. Lately, different selenium species have been described to induce programmed cell death by multiple mechanisms. In the context of PCD, selenite has been described to induce caspase-independent apoptosis via p53 and p38 in cervical cancer cells treated at 5 and 50 lM [48], superoxide formation and ER stress and subsequently p53-mediated apoptosis in prostate cancer cells. In promyelocytic leukaemia NB4 cells, selenite treatment (20 lM) has been reported to protect against autophagy via the PI3K/Akt signalling, leading to increased apoptosis [49]. Treatment with selenite (7 lM) to malignant glioma cells has been implicated in superoxide-induced mitophagic cell death [50]. Furthermore, GS-Se-SG at 8 lM concentration has been shown to selectively induce apoptosis in squamous carcinoma cells compared to normal mucosa cells by induction of Fas-ligand (Fas-L) expression, and activation of the stress pathway by increased expression of JNK and p38 [40]. This is also in line with recent findings in our laboratory, where GS-Se-SG (5 lM) was able to glutathionylate cell surface proteins and induce apoptosis more readily, compared to selenite (5 lM) [51]. CysSeSeCys treatment at various concentrations (5–40 lM) and time has been reported to induce a S-phase arrest and apoptosis via ERK and Akt in MCF-7 cells [52]. In renal cancer cells, treatment with SeMSC (10–200 lM) increased the TRAIL-mediated apoptosis in association with down-regulation of BCL-2 and caspase activation [36]. Another study has shown SeMet (50 lM) and selenite (2,5 lM) to induce ROS-dependent apoptosis, but only SeMet exerted phosphorylation of AKT and mammalian target of rapamycin (mTOR) in the lung cancer cell line A549 [53]. Suzuki et al. [38] compared the cytotoxicity of three selenium compounds at different concentrations, selenite (1–10 lM) SeMSC (10–1000 lM) and SeMet (10–1000 lM) using different tumour cell lines where they stated that all these selenium compounds induced apoptosis, but by different mechanisms. SeMet was found to be dependent on a functional p53 to induce apoptosis, while SeMSC instead induced a p53-independent apoptosis and by activation of caspases. There was also evidence of ER stress associated with all the compounds, but only selenite and SeMSC caused an activation of caspase 12, indicating the involvement of an ER stress-related pathway. Induction of ER stress by selenium compounds. ER stress is generally not considered to belong to the group of PCD modes, but is a regulated cellular response and condition that eventually may lead to PCD, by apoptosis which is tightly connected to the ER. The high ratio of oxidized and reduced glutathione (GSSG/GSH) in the range of 1:1–3:1 within the ER lumen is crucial for the protein folding and formation of disulphide bonds during protein synthesis. ER localized chaperones and oxidoreductases like binding immunoglobulin protein (BiP/GRP78), ERdj0 s and protein disulphide isomerase (PDI) regulate these processes. However, during dysfunctional protein biosynthetic pathways, misfolded proteins may be accumulated leading to ER stress, also

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recognized as unfolded protein response. Furthermore, there may be an up-regulation of ER stress markers as BiP, which is released by the ER transmembrane localized proteins serine/ threonine-protein kinase/endoribonuclease 1 (IRE1), eukaryotic translation initiation factor 2-a kinase 3 (PERK) or activating transcription factor 6 (ATF6) to orchestrate for survival, or the transcription factor C/EBP homology protein (CHOP/ Gadd153), which is up-regulated by ATF6. CHOP may further inhibit BCL-2, leading to initiation of the intrinsic apoptotic pathway. Another common marker of ER stress is caspase 12 which is activated by Ca2+ release from the ER. IRE1 may also activate the extrinsic apoptotic pathway by complex formation with TRAF2 and initiation of ASK1 and c-Jun (reviewed in [54]). Several recent papers have reported selenium to induce ER stress by targeting these above-mentioned markers, like for instance MSA [55] and a concentrationdependent ER stress induced by selenite in NB4 cells [56]. Selenite treatment led to either cell survival via AKT and ERK and removal of misfolded proteins or induced apoptosis by activation of GADD153/CHOP and ROS production. The involvement of selenium-induced ER stress is further confirmed by a recent study from our group, where especially seleno-DL-cystine induced ER stress, as determined by morphological studies using transmission electron microscopy, increased protein expression of BiP and CHOP, in addition to ubiquitinylation of proteins [51]. ER-resident selenoproteins, like selenoprotein K, M, N, S, T and 15 kDa selenoprotein, may also play important physiological roles [57]. For instance, selenoprotein K (SelK) was recently discovered to participate in degradation of misfolded proteins and maintenance of the ER homeostasis [58] and selenoprotein S to inherit peroxidase activity and to be involved in the endoplasmic-reticulum-associated protein degradation machinery [59]. p53 status and selenium cytotoxicity. Another aspect concerning selenium-induced cell death is the expression of oncogenes, because a functional expression of p53 seems to be important for the outcome of some selenium species during treatment. P53 is also a regulator of a p53-dependent apoptosis, which is initiated by transcriptional activation of plasma membrane proteins, or the mitochondrial proteins PERK, Bcl-2-associated X protein (Bax), phorbol-12-myristate13-acetate-induced protein 1 (NOXA) or p53 up-regulated modulator of apoptosis. Cytosol-located p53 may also interact with BCL-2 and Bax, while a p53-independent apoptosis is initiated by DNA damage and activation of caspase 2. P53 seems to be especially important to induce apoptosis by SeMet (50 lM) [38], but also to some extent in the case of selenite-induced activation of apoptosis, although selenite has been shown to increase the phosphorylation of p53 and that non-p53 expressing cells are able to diminish superoxide formation induced by selenite (5 lM) and following apoptosis [60]. However, p53 is not crucial for induction of cell death by SeMSC (50 lM) [38] and MSA (10 lM) [60]. A summary of the most pronounced cell death mechanisms of the above described selenium compounds has been summarized in fig. 3.

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Fig. 3. Summary of the cell death signalling pathways associated with the major selenium compounds discussed in the MiniReview.

Selenium Compounds as Chemotherapeutics General overview. The effects of selenium on cancer cells are highly concentration-dependent, because low to moderate levels may stimulate growth, whereas higher levels are cytotoxic (fig. 4). It has been found that neoplastic breast tissues contain higher amount of total selenium compared to its surrounding nonneoplastic tissues [61]. Similar results have been reported in colorectal and gastric cancer patients by the same group. In conjunction, several investigations clearly indicate increased expression of the selenoprotein thioredoxin reductase (TrxR), in the neoplastic tissues compared to their normal counterparts (reviewed in [6]). As neoplastic transformation is often associated with increased cell proliferation during the initiation and promotional phases of tumour development, trace concentrations of selenium as micronutrient may augment the transformation process by maintaining a sustained cell proliferation of malignant neoplasms. These observations together imply the importance of selenium in the established neoplastic tissues. From the above observations, it is tempting to deduce that cancer cells may be capable of effectively harness selenium from the plasma pool in which selenoprotein P functions as a major source of selenium. Such selectivity on the reliance of selenium in cancer biology originates the concept of manipulating its essentiality. This concept relies on the opposite spectrum in which redox-active selenium compounds at high dose induce ROS generation and subsequently oxidative stress, upon metabolism in cancer cells many of which harbour inherently high level of ROS [62]. This is reminiscent of a spillover situation in which the antioxidant defence mechanisms of cancer cells are overwhelmed by sustained ROS-mediated toxicity, leading to loss of structural and functional integrity and subsequent demise of cancer cells. In principle, such a

treatment strategy can apparently be highly effective, given that the confounding factors of successful chemotherapeutic application can be avoided to a greater extent. Selenium-specific cytotoxicity in cancer. Neither all selenium compounds are redox-active, nor do they exhibit equipotent toxicity to cancer cells. Also, there are discrete differences in their pharmacokinetic and pharmacodynamic properties depending on the route of administration, doses and duration of exposure. Distinct differences in the tissue-specific metabolism of these compounds can partially explain the variation in biological effects. Selenium compounds with growth-modulating properties are metabolized to many intermediate metabolites including HSe
or CH3Se
, as indicated previously. Both of these redox-active metabolites are reported to have distinct toxic effects as reflected by different pharmacodynamics. Therefore, it is of pivotal importance to select the right candidate molecule to achieve an optimum outcome in cancer treatment. Specificity to cancer cells is another important question pertaining to chemotherapeutic uses of selenium. A brief summary of the previous research findings has been discussed below, with major emphasis on the efficacy of different selenium compounds in inducing cancer cell death. In vitro cytotoxicity studies. Numerous in vitro studies with multiple cancer cell lines of distinct origin suggest variable responses of these cells to different selenium compounds. An important discretion shall be made with GS-Se-SG, an organic intermediate, which possesses high toxicity to most of cell lines. Results from our own laboratory suggest that HeLa cells are equally sensitive to selenite and GS-Se-SG, but quite resistant to seleno-DL-cystine. Similar findings have been

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Fig. 4. A schematic diagram (unscaled) representing the general dose–effect relationship of selenium. Severe deficiency of this micronutrient is detrimental for normal growth and development in mammals. Proper physiological functionalities of selenoproteomes require an optimum dose level. The chemopreventive effects of selenium are achieved at concentration just above the normal intake level. In contrast, the chemotherapeutic window resides at pharmacological concentrations. It is important to note that there is a narrow window between chemopreventive and chemotherapeutic dose level. The same paradigm also exists between chemotherapeutic dose and acute toxic dose level.

reported with HepG2 cells that exhibit variable sensitivity towards different selenium compounds [63]. Another study reported variable cytotoxic responses of different cell lines when treated with selenite, SeMSC and SeMet, with the latter being the least toxic [38]. One of the most remarkable, yet less recognized and perhaps less explored feature of cytotoxic effects of selenium is the specificity towards cancer cells. Several studies suggest that certain chemical forms of selenium exhibit higher toxicity to cancer cells compared to their normal counterparts [47,64–66]. Notably, we have previously shown that drug-resistant lung cancer cells are much more sensitive to selenite than their drug-non-resistant variants [67]. It is apparent that discrete differences in the toxicity profiles of different classes of selenium compounds to different cancer cell types warrant critical pharmacokinetics and pharmacodynamics evaluations prior to their chemotherapeutic uses. Animal model of cancer studies. The first in vivo study suggested anticancer effects of selenium in which dietary selenite (5 ppm) supplementation has resulted in 50% decrease in liver tumour development in rat when fed with an azo dye [68]. Subsequent studies by Ip and coworkers have

revealed that dietary selenite supplement results in significant abrogation of DMBA-induced mammary carcinogenesis [69]. Similar inhibitory effects of selenium have been reported by Medina and coworkers using primary alveolar nodules transplanted BALB/c mice [70]. Protective effects of selenium against the virus- and chemical-induced carcinogenesis have been reviewed by Milner [71]. From the above-mentioned studies and the references therein, the following conclusions can be made: (i) Not all selenium compounds exhibit equipotent anticancer properties. Most of the findings in animal models suggest that inorganic selenium (mainly selenite) is the predominant form of selenium with anticarcinogenic properties. (ii) There exists a clear dose dependency, with the higher dose being more effective against tumour development. (iii) The anticarcinogenic effects are mostly described during the initiation and progression stages of cancer, while limited information is available on the effects of selenium in the regression of established tumours. (iv) An efficient inhibition of tumourigenesis requires prolonged intake of selenium. Chemotherapeutic uses of selenium in human beings. It has been about 100 years since selenium was recognized as a

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chemotherapeutic agent in human beings. In early 1910, the efficacy chemotherapeutic uses of the colloidal selenium (as selenide salt) have been reported. One of the most striking results suggests that intravenous injection of colloidal suspension of erythro-selenium b resulted in marked improvement in carcinoma of the alimentary tract [72]. The author reports reduction in tumour-associated pain, tenderness, size of tumour and secondary deposits. It is also found that the patients experienced improvement of anaemia and cachexia, healing of the ulcerated surfaces, increased mobility and improved sleep. Although the author notes no perceivable indication on the efficacy of selenium in curing cancer, the treatment resulted in clear improvement in the quality of life for the patients. During the study period, some of the patients received more than 100 mg of selenium and were reported as ‘absolutely non-toxic’. From a pharmacodynamics perspective, this is perhaps one of the most interesting findings of the study. A subsequent study by Dr. A.T. Todd revealed remarkable results in many patients with cancer who received colloidal SSe and R.A.S (selenide of radium G and its disintegration product), as described elsewhere [73]. The study revealed that about 20% patients, suffering from certain forms of cancers, were restored to health. The findings of these studies hitherto elaborate superior outcome with limited side effects, suggesting selective actions of the selenium compounds in the target tumour tissue. Unfortunately, since then, there have been very limited efforts to test the efficacy of the described chemotherapeutic application of selenium. In 1966, Cavalieri et al. [74] reported that intravenous administration of radioactive selenite (as 75Se) resulted in preferential accumulation in the intrathorasic and intra-abdominal tumours. However, the study reports that 75Se could not differentiate between neoplastic and inflammatory lesions. In view of the established data on the role of inflammation in cancer progression and selenite being an anti-inflammatory agent, accumulation of selenium in the inflammatory lesions may be beneficial. However, such a hypothetical perspective should be carefully interpreted in the absence of any direct evidence. Furthermore, an anti-inflammatory effect may lead to an overinterpretation of the antitumour effect, because decreased inflammation may shrink the lesion. Apart from selenite, per oral administration of CysSeSeCys has been reported to be a promising chemotherapeutic agent against acute leukaemia and chronic myeloid leukaemia [75]. In all 4 patients receiving CysSeSeCy, rapid decrease in leucocyte counts and spleen size was reported. Such effects were obtained from patients unresponsive to other chemotherapeutics available then. Side effects like nausea and vomiting were reported without any appreciable systemic toxicity as apparent from post-mortem observations of the major organs. In this case, the patient received a total of 5.2 g of CysSeSeCy over a period of 50 days, suggesting remarkable tolerance against CysSeSeCy in human beings. The authors also mention that intravenous administration of CysSeSeCy appears to be better tolerated, indicating the possibility of an alternative delivery route.

From the above studies and other pre-clinical data in animal models and in vitro studies, it is conceivable that redox-active selenium compounds exhibit high potential for chemotherapeutic application. The chemotherapeutic window lies within the range far beyond the supplementation studies aimed at achieving prevention effects. It has been found that selenized yeast supplementation (3.2 mg/day) for about 1 year has resulted in no appreciable toxicity in biopsy-proven prostate cancer patients [76], suggesting extreme tolerance against selenized yeast. Our own experience from the ongoing clinical trial (NCT01959438) suggests a similar but striking tolerance against intravenous administration of sodium selenite with no major observed side effects in patients with cancer. Concluding Remarks We failed to find any convincing reports in the literature, the results of which convincingly support the antithesis of chemotherapeutic application of selenium. Retrospectively, one may wonder how much do we know to rule out any beneficial effects of selected selenium compounds as chemotherapeutics. As with others, we certainly believe that prevention is better than cure. However, with an ever increasing global incidence of cancer, the current focus can go far beyond the prevalent concept of using selenium as a nutritional supplement in chemoprevention. It is important to understand that chemoprevention and chemotherapeutic effects of selenium are not mutually exclusive, rather independent branches of science. This shall be distinguished accordingly. In the same note, it is of pivotal importance to gain better understanding of pharmacokinetics and pharmacodynamics of different redox-active selenium compounds in human beings. Several redox-based therapeutics have shown potentiating effects on the pharmacodynamics of other anticancer agents, but offer only modest chemotherapeutic benefit if used as single agent. Evaluating a similar paradigm with redox-active selenium compounds would probably be beneficial in designing targeted cancer therapy. Acknowledgements The authors would like to acknowledge Cancerfonden, Radiumhemmets forskningsfonder, Stichting af Jochnick Foundation, Stockholm County Council and Cancer- Och Allergifonden for funding. SM received a post-doctoral fellowship from Cancer- Och Allergifonden. References 1 Berzelius JJ. Unders€okning af en ny Mineral-kropp, funnen i de orenare sorterna af det i Falun tillverkade svaflet, in Afhandlingar i fysik, kemi och mineralogi. 1818, Stockholm. p. 42–144. 2 Bagnall KW. Selenium, tellurium and polonium. In: Bailar JC, Emeleus HJ, Nyholm R, Trotman-Dickenson AF (eds). Comprehensive Inorganic Chemistry. Pergamon Press, Oxford, 1973;935– 1008. 3 Stadtman T. Some selenium-dependent biochemical processes. Adv Enzymol 1979;48:1–28. 4 Huber RE, Criddle RS. Comparison of the chemical properties of selenocysteine and selenocystine with their sulfur analogs. Arch Biochem Biophys 1967;122:164–73.

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