Computational Studies of Au(I) and Au(III) Anticancer MetalLodrugs: A Survey.

Owing to the growing hardware capabilities and the enhancing efficacy of computational methodologies, computational chemistry approaches have constantly become more important in the development of novel anticancer metallodrugs. Besides traditional Pt-based drugs, inorganic and organometallic complexes of other transition metals are showing increasing potential in the treatment of cancer. Among them, Au(I)- and Au(III)-based compounds are promising candidates due to the strong affinity of Au(I) cations to cysteine and selenocysteine side chains of the protein residues and to Au(III) complexes being more labile and prone to the reduction to either Au(I) or Au(0) in the physiological milieu. A correct prediction of metal complexes' properties and of their bonding interactions with potential ligands requires QM computations, usually at the ab initio or DFT level. However, MM, MD, and docking approaches can also give useful information on their binding site on large biomolecular targets, such as proteins or DNA, provided a careful parametrization of the metal force field is employed. In this review, we provide an overview of the recent computational studies of Au(I) and Au(III) antitumor compounds and of their interactions with biomolecular targets, such as sulfur- and selenium-containing enzymes, like glutathione reductases, glutathione peroxidase, glutathione-S-transferase, cysteine protease, thioredoxin reductase and poly (ADP-ribose) polymerase 1.

Repurposing of auranofin: Thioredoxin reductase remains a primary target of the drug.

Auranofin is a gold (I)-containing compound used for the treatment of rheumatic arthritis. Auranofin has anticancer activity in animal models and is approved for clinical trials for lung and ovarian carcinomas. Both the cytosolic and mitochondrial forms of the selenoprotein thioredoxin reductase (TrxR) are well documented targets of auranofin. Auranofin was recently reported to also inhibit proteasome activity at the level of the proteasome-associated deubiquitinases (DUBs) UCHL5 and USP14. We here set out to re-examine the molecular mechanism underlying auranofin cytotoxicity towards cultured cancer cells. The effects of auranofin on the proteasome were examined in cells and in vitro, effects on DUB activity were assessed using different substrates. The cellular response to auranofin was compared to that of the 20S proteasome inhibitor bortezomib and the 19S DUB inhibitor b-AP15 using proteomics. Auranofin was found to inhibit mitochondrial activity and to an induce oxidative stress response at IC50 doses. At 2-3-fold higher doses, auranofin inhibits proteasome processing in cells. At such supra-pharmacological concentrations USP14 activity was inhibited. Analysis of protein expression profiles in drug-exposed tumor cells showed that auranofin induces a response distinct from that of the 20S proteasome inhibitor bortezomib and the DUB inhibitor b-AP15, both of which induced similar responses. Our results support the notion that the primary mechanism of action of auranofin is TrxR inhibition and suggest that proteasome DUB inhibition is an off-target effect. Whether proteasome inhibition will contribute to the antineoplastic effect of auranofin in treated patients is unclear but remains a possibility.

Small Molecules to Target the Selenoprotein Thioredoxin Reductase.

The selenoprotein thioredoxin reductase (TrxR) enzymes are the only identified proteins that maintain thioredoxin (Trx) proteins in a reduced state under physiological conditions, and consequently play a pivotal role in the regulation of various cellular redox signaling pathways involved in cell differentiation, growth, and death. The elevated expression of TrxR enzymes has been observed in different types of cancer cells, and this overexpression is of pathological significance in maintaining tumor phenotypes, such as uncontrolled proliferation and resistance to apoptosis. Herein, we discuss recent advances in the study of TrxR, including classic assays of TrxR, the emerging chemical tools of TrxR, and small molecules that target TrxR as potential anticancer agents.

Selenoprotein S protects against adipocyte death through mediation of the IRE1α-sXBP1 pathway.

As the most conserved branch of the unfolded protein response (UPR), the inositol-requiring enzyme 1a (IRE1a)/X-box binding protein 1 (XBP1) pathway plays crucial roles in cell survival and cell death by upregulating UPR-associated genes involved in protein entry into the endoplasmic reticulum (ER) and ER-associated degradation (ERAD). Selenoprotein S (SelS) is localized to the ER membrane and involved in ERAD. Although SelS plays an important role in restoring ER stress, the SelS-dependent protective mechanisms against cell death remain unclear. Here, using an inducible SelS knockdown (KD) 3T3-L1 cell model, we showed that SelS KD resulted adipocyte death, which was associated with imbalance of the Bcl-2 family members. Furthermore, SelS KD decreased spliced XBP1 (sXBP1), increased IRE1α and p-JNK, suggesting a role of SelS in the modulation of the IRE1α-sXBP1 pathway. Moreover, adipocyte death induced by SelS suppression can be inhibited by overexpression of sXBP1. Thus, it is proposed that SelS promotes cell survival through the IRE1α-XBP1 signaling pathway.

Selenoprotein-U (SelU) knockdown triggers autophagy through PI3K-Akt-mTOR pathway inhibition in rooster Sertoli cells.

Selenium (Se) is a major component of male reproduction which exerts its effects via selenoproteins. Selenoprotein U (SelU), a newly identified protein, is expressed highly in eukaryotes and possesses a conserved motif similar to that existing in other thiol-dependent redox regulating selenoproteins; however its function is unknown. To investigate the role of SelU in testis autophagic and/or apoptosis cell death mechanisms, we established a Sertoli cell (SC) model isolated from 45 day old layer roosters. Small interfering RNA (siRNA) technology was used to develop SelU-knockdown (SelU-KD) and normal (N) SC models. Consequent to transfection, electron microscopy, qPCR, and western blot were performed. The results show that the mRNA and proteins of autophagy and anti-apoptosis genes increased while that of anti-autophagic mammalian target of rapamycin (mTOR) and pro-apoptosis genes decreased significantly in SelU-KD in contrast to N cells. Simultaneously, in contrast to N cells the expression of phosphoinositide-3-kinase (PI3K) and protein kinase B (PKB/Akt) both at the mRNA and protein levels decreased significantly in SelU-KD cells. In-addition, SelU depletion altered the expression of regulatory factors and increased the mRNA of TSC (tuberous sclerosis complex) genes as compared to N cells. Extensive autophagosome formation and lysosome degradation with an intact cytoskeleton were observed in SelU-KD cells. Our data indicate that SelU deprivation elicits autophagy and reduces the expression of important growth factors in SCs by disrupting the PI3K-Akt-mTOR signaling pathway. However SelU attenuation did not induce apoptosis in rooster SCs. Taken together, we conclude that SelU is essential for the survival and normal functioning of SCs.

Selenoprotein S silencing triggers mouse hepatoma cells apoptosis and necrosis involving in intracellular calcium imbalance and ROS-mPTP-ATP.

Selenoprotein S (SelenoS) is one of the cellular endoplasmic reticulum (ER) and membrane located selenoproteins, and it has the main functions of anti-oxidation, anti-apoptosis and anti-ER stress. To investigate the effect of SelenoS silencing on mouse hepatoma cell death and the intracellular biological function of SelenoS, we knocked down SelenoS in Hepa1-6 cells, and detected ER stress, intracellular calcium homeostasis, mitochondrial dynamics, apoptosis and necrosis. To further explore whether reactive oxygen species (ROS) has an effect on apoptosis and necrosis under SelenoS silencing, we used NAC (2.5 mM) to pretreat cells, and detected ΔΨm, ATP, and apoptosis and necrosis rates. SelenoS silencing broke the intracellular calcium homeostasis, induced mitochondrial dynamic disorder, ROS accumulation, loss of ΔΨm and ATP, and triggered apoptosis and necrosis in mouse hepatoma cells. The clearance of ROS alleviated the loss of ΔΨm and ATP caused by silencing of SelenoS, reduced cell necrosis and increased apoptosis. However, SelenoS silencing did not cause ER stress in Hepa1-6 cells. These results indicate that SelenoS silencing triggers mouse hepatoma cells apoptosis and necrosis through affecting intracellular calcium homeostasis and ROS-mPTP-ATP participates in cell death transformation from apoptosis to necrosis to rise damage.

The Antagonistic Effect of Selenium on Cadmium-Induced Damage and mRNA Levels of Selenoprotein Genes and Inflammatory Factors in Chicken Kidney Tissue.

Selenium (Se) is a necessary trace mineral in the diet of humans and animals. Cadmium (Cd) is a toxic heavy metal that can damage animal organs, especially the kidneys. Antagonistic interactions between Se and Cd have been reported in previous studies. However, little is known about the effects of Se against Cd toxicity and on the mRNA levels of 25 selenoprotein genes and inflammatory factors in chicken kidneys. In the current study, we fed chickens with a Se-treated, Cd-treated, or Se/Cd treated diet for 90 days. We then analyzed the mRNA expression of inflammatory factors (including prostaglandin E synthase (PTGES), nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2)) and 25 selenoprotein genes (Gpx1, Gpx2, Gpx3, Gpx4, Txnrd1, Txnrd2, Txnrd3, Dio1, Dio2, Dio3, SPS2, Sepp1, SelPb, Sep15, Selh, Seli, Selm, Selo, Sels, Sepx1, Selu, Selk, Selw, Seln, Selt). The results demonstrated that Cd exposure increased the Cd content in the chicken kidneys, renal tubular epithelial cells underwent denaturation and necrosis, and the tubules became narrow or disappeared. However, Se supplementation reduced the Cd content in chicken kidneys and induced normal development of renal tubular epithelial cells. In addition, we also observed that Se alleviated the Cd-induced increase in the mRNA levels of inflammatory factors and ameliorated the Cd-induced downtrend in the mRNA levels of 25 selenoprotein genes in chicken kidneys.

Selenoprotein T is a novel OST subunit that regulates UPR signaling and hormone secretion.

Selenoprotein T (SelT) is a recently characterized thioredoxin-like protein whose expression is very high during development, but is confined to endocrine tissues in adulthood where its function is unknown. We report here that SelT is required for adaptation to the stressful conditions of high hormone level production in endocrine cells. Using immunofluorescence and TEM immunogold approaches, we find that SelT is expressed at the endoplasmic reticulum membrane in all hormone-producing pituitary cell types. SelT knockdown in corticotrope cells promotes unfolded protein response (UPR) and ER stress and lowers endoplasmic reticulum-associated protein degradation (ERAD) and hormone production. Using a screen in yeast for SelT-membrane protein interactions, we sort keratinocyte-associated protein 2 (KCP2), a subunit of the protein complex oligosaccharyltransferase (OST). In fact, SelT interacts not only with KCP2 but also with other subunits of the A-type OST complex which are depleted after SelT knockdown leading to POMC N-glycosylation defects. This study identifies SelT as a novel subunit of the A-type OST complex, indispensable for its integrity and for ER homeostasis, and exerting a pivotal adaptive function that allows endocrine cells to properly achieve the maturation and secretion of hormones.

Gene expression of selenoproteins can be regulated by selenoprotein K silencing in chicken myoblasts.

The aim of the present study was to clarify the effect of Selenoprotein K (Selk) silencing on the mRNA expression of 25 selenoproteins in chicken myoblasts. The specific small interfering RNA (siRNA) for Selk gene was designed and transfected into chicken myoblasts. Post-transfection mRNA expression of 25 selenoproteins was determined at various time periods i.e., 24, 48 and 72 h. Moreover, based on the results of expression of 25 selenoproteins, correlation analysis and principal component analysis (PCA) were used for further analysis. The results showed that the designed siRNA effectively inhibited Selk expression (decreased by 20, 29 and 43 % on 24, 48 and 72 h, respectively) and the mRNA expression levels of the 23 selenoproteins were influenced by silencing Selk differently (P < 0.05). Time-dependent pattern of mRNA expression after siRNA treatment in three groups were found similar: one group including Gpx1, Gpx2, Gpx3, Gpx4, Txnrd1, Txnrd2, Txnrd3, Sepw1, Selh, Sepp1, Selo and Sepx1, another group including Sepn1, Sels, Selt, Selm and Sep15 and other group including Dio2 and Dio3. The results of correlation analysis showed that Gpx1, Gpx2, Gpx3, Gpx4, Dio1, Dio3, Sepn1, Sels, Sepw1, Selt, Selh, Sep15, Seli and Selu had a positive correlation with Selk, while Dio2 and Sepp1 had a negative correlation with Selk. PCA data also indicated that Txnrd1, Txnrd2, Dio2, Selpb, Sepp1and Selo may play special roles in response to Selk silencing. In summary, these results indicated that different selenoproteins possess and exhibits distinct responses to silencing of Selk in chicken myoblasts.

Endogenous and exogenous pathways maintain the reductive capacity of the phagosome.

Although endosomes, lysosomes, and phagosomes require a reductive environment for the optimal activity of disulfide reductases and other thiol-dependent enzymes, how these reductive environments are established and maintained remain unknown. Our goal in this study was to begin to elucidate the redox control systems responsible for maintaining redox-sensitive enzymatic activities in the phagolysosome of murine macrophages. Through the use of specific inhibitors and genetic knockdown of known redox enzymes, we identified redox pathways that influence phagosomal disulfide reduction. In particular, known inhibitors of the NADPH-dependent selenoprotein, thioredoxin reductase, were shown to inhibit phagosomal disulfide reduction and phagosomal proteolysis. This was supported by the observation that conditional deletion of the selenocysteine tRNA in macrophages decreased phagosomal disulfide reduction capacity. In addition, pharmacologic inhibition of the pentose phosphate pathway decreased rates of disulfide reduction and proteolysis in the phagosome, implicating NADPH as a source of phagosomal reductive energy. Finally, by analyzing the effect of extracellular redox couples, such as cysteine:cystine on thiol-dependent phagosomal processes, we demonstrated that the extracellular space can additionally supply the phagosome with reductive energy. Collectively, these data demonstrate that defined cytosolic reductive pathways act in concert with the uptake of cysteine from the extracellular space to support thiol-dependent chemistries in the phagosome.

Selenoprotein H suppresses cellular senescence through genome maintenance and redox regulation.

Oxidative stress and persistent DNA damage response contribute to cellular senescence, a degeneration process critically involving ataxia telangiectasia-mutated (ATM) and p53. Selenoprotein H (SelH), a nuclear selenoprotein, is proposed to carry redox and transactivation domains. To determine the role of SelH in genome maintenance, shRNA knockdown was employed in human normal and immortalized cell lines. SelH shRNA MRC-5 diploid fibroblasts under ambient O2 displayed a distinct profile of senescence including ß-galactosidase expression, autofluorescence, growth inhibition, and ATM pathway activation. Such senescence phenotypes were alleviated in the presence of ATM kinase inhibitors, by p53 shRNA knockdown, or by maintaining the cells under 3% O2. During the course of 5-day recovery, the induction of phospho-ATM on Ser-1981 and γH2AX by H2O2 treatment (20 µm) subsided in scrambled shRNA but exacerbated in SelH shRNA MRC-5 cells. Results from clonogenic assays demonstrated hypersensitivity of SelH shRNA HeLa cells to paraquat and H2O2, but not to hydroxyurea, neocarzinostatin, or camptothecin. While SelH mRNA expression was induced by H2O2 treatment, SelH-GFP did not mobilize to sites of oxidative DNA damage. The glutathione level was lower in SelH shRNA than scrambled shRNA HeLa cells, and the H2O2-induced cell death was rescued in the presence of N-acetylcysteine, a glutathione precursor. Altogether, SelH protects against cellular senescence to oxidative stress through a genome maintenance pathway involving ATM and p53.

The PACAP-regulated gene selenoprotein T is abundantly expressed in mouse and human ß-cells and its targeted inactivation impairs glucose tolerance.

Selenoproteins are involved in the regulation of redox status, which affects several cellular processes, including cell survival and homeostasis. Considerable interest has arisen recently concerning the role of selenoproteins in the regulation of glucose metabolism. Here, we found that selenoprotein T (SelT), a new thioredoxin-like protein of the endoplasmic reticulum, is present at high levels in human and mouse pancreas as revealed by immunofluorescence and quantitative PCR. Confocal immunohistochemistry studies revealed that SelT is mostly confined to insulin- and somatostatin-producing cells in mouse and human islets. To elucidate the role of SelT in ß-cells, we generated, using a Cre-Lox strategy, a conditional pancreatic ß-cell SelT-knockout C57BL/6J mice (SelT-insKO) in which SelT gene disruption is under the control of the rat insulin promoter Cre gene. Glucose administration revealed that male SelT-insKO mice display impaired glucose tolerance. Although insulin sensitivity was not modified in the mutant mice, the ratio of glucose to insulin was significantly higher in the SelT-insKO mice compared with wild-type littermates, pointing to a deficit in insulin production/secretion in mutant mice. In addition, morphometric analysis showed that islets from SelT-insKO mice were smaller and that their number was significantly increased compared with islets from their wild-type littermates. Finally, we found that SelT is up-regulated by pituitary adenylate cyclase-activating polypeptide (PACAP) in ß-pancreatic cells and that SelT could act by facilitating a feed-forward mechanism to potentiate insulin secretion induced by the neuropeptide. Our findings are the first to show that the PACAP-regulated SelT is localized in pancreatic ß- and δ-cells and is involved in the control of glucose homeostasis.

A diterpenoid derivate compound targets selenocysteine of thioredoxin reductases and induces Bax/Bak-independent apoptosis.

We have previously shown that the natural diterpenoid derivative S3 induced Bim upregulation and apoptosis in a Bax/Bak-independent manner. However, the exact molecular target(s) of S3 and the mechanism controlling Bim upregulation are still not clear. Here, we identify that S3 targets the selenoproteins TrxR1 and TrxR2 at the selenocysteine residue of the reactive center of the enzymes and inhibits their antioxidant activities. Consequently, cellular ROS is elevated, leading to the activation of FOXO3a, which contributes to Bim upregulation in Bax/Bak-deficient cells. Moreover, S3 retards tumor growth in subcutaneous xenograft tumors by inhibiting TrxR activity in vivo. Our studies delineate the signaling pathway controlling Bim upregulation, which results in Bax/Bak-independent apoptosis and provide evidence that the compounds can act as anticancer agents based on mammalian TrxRs inhibition.

Overexpression of human selenoprotein H in neuronal cells enhances mitochondrial biogenesis and function through activation of protein kinase A, protein kinase B, and cyclic adenosine monophosphate response element-binding protein pathway.

Mitochondrial biogenesis is activated by nuclear encoded transcription co-activator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which is regulated by several upstream factors including protein kinase A and Akt/protein kinase B. We have previously shown that selenoprotein H enhances the levels of nuclear regulators for mitochondrial biogenesis, increases mitochondrial mass and improves mitochondrial respiratory rate, under physiological condition. Furthermore, overexpression of selenoprotein H protects neuronal HT22 cells from ultraviolet B irradiation-induced cell damage by lowering reactive oxygen species production, and inhibiting activation of caspase-3 and -9, as well as p53. The objective of this study is to identify the cell signaling pathways by which selenoprotein H initiates mitochondrial biogenesis. We first confirmed our previous observation that selenoprotein H transfected HT22 cells increased the protein levels of nuclear-encoded mitochondrial biogenesis factors, peroxisome proliferator-activated receptor γ coactivator-1α, nuclear respiratory factor 1 and mitochondrial transcription factor A. We then observed that total and phosphorylation of protein kinase A, Akt/protein kinase B and cyclic adenosine monophosphate response element-binding protein (CREB) were significantly increased in selenoprotein H transfected cells compared to vector transfected HT22 cells. To verify whether the observed stimulating effects on mitochondrial biogenesis pathways are caused by selenoprotein H and mediated through CREB, we knocked down selenoprotein H mRNA level using siRNA and inhibited CREB with napthol AS-E phosphate in selenoprotein H transfected cells and repeated the measurements of the aforementioned biomarkers. Our results revealed that silencing of selenoprotein H not only decreased the protein levels of PGC-1α, nuclear respiratory factor 1 and mitochondrial transcription factor A, but also decreased the total and phosphorylation levels of protein kinase A, protein kinase B, and CREB. Similarly, CREB inhibition reduced CREB activation and PGC-1α protein levels in selenoprotein H transfected cells. Moreover, selenoprotein H transfection increased the activity of mitochondrial complexes and prevented the ultraviolet B induced fall of mitochondrial membrane potential. We conclude that the effects of selenoprotein H on mitochondrial biogenesis and mitochondrial function are probably mediated through protein kinase A-CREB-PGC-1α and Akt/protein kinase B-CREB-PGC-1α pathways.

Pyrroloquinoline quinone modulates the kinetic parameters of the mammalian selenoprotein thioredoxin reductase 1 and is an inhibitor of glutathione reductase.

Pyrroloquinoline quinone (PQQ) is a redox active cofactor for bacterial quinoproteins. Dietary PQQ also has prominent physiological effects in mammals although no mammalian quinoprotein has yet been conclusively identified. Here we found that PQQ has substantial effects on the redox active mammalian selenoprotein thioredoxin reductase 1 (TrxR1). PQQ efficiently inhibited the activity of TrxR1 with its main native substrate thioredoxin and acted as a low efficiency substrate in a Sec-dependent TrxR1-catalyzed reduction. Interestingly, PQQ also stimulated redox cycling of TrxR1 with another quinone substrate, juglone, as much as 13-fold (k(cat)/K(m) increased from 105 min(-1) µM(-1) to 1331 min(-1) µM(-1) for juglone in the presence of 50 µM PQQ, mainly through a lowered apparent K(m) for juglone). Glutathione reductase was also inhibited by PQQ but in contrast to the effects of PQQ on TrxR1, its quinone reduction was not further stimulated. These results reveal that glutathione reductase and the mammalian selenoprotein TrxR1 are direct PQQ protein targets, although not being genuine quinoproteins. These findings may help explain several of the effects of PQQ seen in mammals.

A new strategy for assessing selenoprotein function: siRNA knockdown/knock-in targeting the 3'-UTR.

Selenocysteine insertion into protein in mammalian cells requires RNA elements in the 3'-untranslated regions (3'-UTRs) of selenoprotein genes. The occurrence of these conserved sequences should make selenoproteins particularly amenable for knockdown/knock-in strategies to examine selenoprotein functions. Herein, we utilized the 3'-UTR of various selenoproteins to knock down their expression using siRNAs and then knock in expression using constructs containing mutations within the target region. Thioredoxin reductase 1 (TR1) knockdown in a mouse kidney cell line resulted in the cells growing about 10% more slowly, being more sensitive to UV radiation, and having increased apoptosis in response to UV than control cells. The knockdown cells transfected with a construct encoding the wild-type TR1 gene and having mutations in the sequences targeted by siRNA restored TR1 expression and catalytic activity, rendered the knockdown cells less sensitive to UV, and protected the cells against apoptosis. We also applied this technique to other selenoproteins, selenophosphate synthetase 2 and glutathione peroxidase 1, and found that mRNA and protein levels were restored following transfection of knockdown cells with the corresponding knock-in constructs. In addition to important new insights into the functions of key mammalian selenoproteins, the data suggest that the RNAi-based knock-in technology could distinguish phenotypes due to off-targeting and provide a new method for examining many of the subtleties of selenoprotein function not available using RNAi technology alone.