Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 Contents lists available at ScienceDirect 2 3 4 5 6 journal homepage: www.elsevier.com/locate/freeradbiomed 7 8 9 Original Contribution 10 11 12 13 14 15 16 Yulei Wang 1,a,b, Wenfeng Fang c,1, Ying Huang a,d, Fen Hu a, Qi Ying e, Wancai Yang f,e,n, 17 18 Q1 Bin Xiong a,b,nn 19 a Department of Oncology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, 430071, China 20 b Hubei Key Laboratory of Tumor Biological Behaviors, Hubei Cancer Clinical Study Center, Wuhan, Hubei, 430071, China 21 c State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, 510060, China d 22 Department of Oncology, the Fifth Hospital, Wuhan, Hubei, 430051, China e Department of Pathology, University of Illinois at Chicago, IL 60612, USA 23 f Department of Pathology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan, 453003, China 24 25 26 art ic l e i nf o a b s t r a c t 27 28 Article history: Selenium is an essential trace element and has been extensively studied for preventive effects on 29 Received 25 August 2014 cancers. Recent emerging evidence has also shown that selenium at supranutritional dosage has a 30 Received in revised form preferential cytotoxicity in cancer cells and chemotherapeutic drug-resistant cells, but the underlying 31 14 November 2014 mechanisms remain largely unknown. This study was to investigate the roles of two distinct 32 Accepted 20 November 2014 representatives of selenium-containing proteins, selenium-binding protein 1 (SBP1) and glutathione 33 peroxidase 1 (GPX1), in selenite-mediated cancer-specific cytotoxicity. We found that there was a 34 Keywords: Q3 significantly inverse correlation between SBP1 and GPX1 protein level in human breast cancers and 35 Selenium-binding protein 1 adjacent matched nontumor tissues (Pearson r ¼–0.4347, P ¼0.0338). Ectopic expression of GPX1 Glutathione peroxidase 1 36 enhanced selenite cytotoxicity through down-regulation of SBP1, and SBP1 was likely to be a crucial Selenium-containing protein 37 determinant for selenite-mediated cytotoxicity. Reduction of SBP1 in cancer cells and epirubicinSelenoprotein resistant cells on selenite exposure resulted in a dramatic increase in the generation of hydrogen 38 Selenite peroxide and superoxide anion, which in turn caused oxidative stress and triggered apoptosis. 39 Cytotoxicity Furthermore, knockdown SBP1 by small interfering RNA increased selenite sensitivity by elevating Breast cancer 40 extracellular glutathione (GSH), which spontaneously reacted with selenite and led to the rapid Reactive oxygen species 41 depletion of selenium (IV) in growth medium and the high-affinity uptake of selenite. In conclusion, Hydrogen peroxide 42 Superoxide anion these findings would improve our understanding of the roles of selenium-containing proteins in 43 Glutathione selenite-mediated cytotoxicity, and revealed a potent mechanism of the selective cytotoxicity of selenite 44 in cancer cells and drug-resistant cells, in which SBP1 was likely to play an important role in modulating 45 the extracellular microenvironment by regulating the levels of extracellular GSH. 46 & 2014 Elsevier Inc. All rights reserved. 47 48 49 Introduction 50 51 Selenium is an essential trace element that is involved in a 52 variety of physiological functions of the human body [1]. Over the Abbreviations: Se, sodium selenite; SBP1, selenium-binding protein 1; GPX1, 53 glutathione peroxidase 1; GSH, glutathione; NAC, N-acetyl-L-cysteine; selenium past decades, selenium supplementation at nutritional dosage (nM 54 (IV), the selenium in þ 4 oxidation state; ROS, reactive oxygen species; PBS, range) has been extensively studied for its chemo-preventive 55 0 0 phosphate-buffered saline; DCFH-DA, 2 ,7 -dichlorofluorescein diacetate; DHE, effects against various cancers [2–7], implicating that selenium56 dihydroethidium; DAN, 2,3-diaminonaphthalene; DTNB, 5,50 -dithiobis-(2-nitrobinding proteins (selenoproteins) are likely to play crucial roles in 57 benzoic acid). n selenium-mediated cancer prevention [3–9]. On the other hand, Corresponding author at: Department of Pathology, Xinxiang Medical Univer58 sity, Xinxiang, china. selenium at supranutritional dosage (μM range) is toxic to mam59 nn Corresponding author at: Department of Oncology, Zhongnan Hospital of mals, but efficiently induces apoptosis in numerous cancer cell 60 Wuhan University, Wuhan, Hubei, 430071, China. lines through multiple mechanisms [10–15]. More interestingly, 61 E-mail addresses: [email protected] (W. Yang), selenium has been shown to be far more toxic to malignant [email protected] (B. Xiong). 62 1 cells and, particularly, chemotherapeutic drug-resistant cells than These authors contributed equally to this work. 63 64 http://dx.doi.org/10.1016/j.freeradbiomed.2014.11.015 65 0891-5849/& 2014 Elsevier Inc. All rights reserved. 66

Free Radical Biology and Medicine

Reduction of selenium-binding protein 1 sensitizes cancer cells to selenite via elevating extracellular glutathione: A novel mechanism of cancer-specific cytotoxicity of selenite

Please cite this article as: Wang, Y; et al. Reduction of selenium-binding protein 1 sensitizes cancer cells to selenite via elevating extracellular glutathione: A novel mechanism of.... Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.11.015i

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Y. Wang et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

normal cells [16–20]. The question whether selenium-containing proteins are responsible for the selective cytotoxicity of selenium remains largely unclear. Selenium-binding protein 1 (SBP1, SELENBP1, or hSP56) and glutathione peroxidase 1 (GPX1 or GPx-1) are two distinct representatives of selenium-containing proteins. SBP1 belongs to the class of selenium-binding proteins (SBPs) that covalently binds selenium by a mechanism that has not yet been fully defined [21,22], whereas GPX1 is a class of selenium-containing proteins in which selenium is an essential component in the form of the 21st amino acid, selenocysteine (Sec), that is inserted cotranslationally into these proteins in response to an in-frame UGA codon, which typically serves as a termination codon but is recognized as a site encoding Sec in a process that requires a Sec insertion sequence (SECIS) element in the 30 -untranslated region of selenoprotein messenger RNA (mRNA), a specific Sec tRNA (tRNASec) and a variety of selenoprotein-specific translation factors [23–25]. We and others have recently reported that GPX1 negatively regulates SBP1 at transcriptional and translational levels [26], whereas SBP1 forms a physical interaction with GPX1 and inhibits GPX1 enzyme activity without any significant effects on either GPX1 mRNA or protein levels [26,27]. These findings represent a unique example of molecular cross talk between members of distinct families of selenium-containing proteins [28]. The human SBP1 gene, located on chromosome 1 at q21–22 [29], is the homologue of the mouse SP56 gene that was originally reported as a 56-kDa mouse protein binding 75selenium stably [21,22]. The human SBP1 protein contains 472 amino acids, resides both in the cytoplasm and in the nucleus [29,30], and is ubiquitously expressed in various tissue types, including heart, lung, liver, kidney, and tissues of the digestive tract. Recently, the cysteine residue (Cys57) has been identified to be required for SBP1 to bind the selenium [31]. The physiological function of SBP1 is still largely unknown, although it has been reported to be a potential participant in intra-Golgi transport [32] and in ubiquitination-mediated protein degradation pathways [33]. We and others have recently reported that the levels of SBP1 are significantly down-regulated or even lost in various tumors as compared to the corresponding normal tissues, including those of the lung [30], esophagus [34], stomach [35,36], liver [27], colon [37,38], prostate [39], ovary [40], and breast [41]. Reduced expression of SBP1 is also associated with poor clinical prognosis among patients with lung adenocarcinoma [30], gastric cancer [36], hepatocellular carcinoma [27], colon cancer [37,38], and breast cancer [41]. Furthermore, our in vitro and in vivo studies have also shown that increasing SBP1 expression in colorectal cancer cells attenuates cancer cell migration and tumor growth in nude mice [42]. Consistent with these findings, knockdown of SBP1 by small interfering RNA (siRNA) in the nontransformed human liver cells results in a substantial increase in cell proliferation index and migration capacity [27], suggesting that SBP1 acts as a tumor suppressor [43], which is further supported by the recent study that SBP1 negatively regulates HIF-1α expression and suppresses the malignant phenotype of prostate cancer cells [44]. GPX1 is the first and best-characterized member of the GPXs family that detoxifies both hydrogen and lipid peroxides using reducing equivalents from glutathione (comprehensively reviewed in Ref. [25]). This ubiquitously expressed antioxidant enzyme has been linked to affecting breast cancer risk by the initial study that leucine (Leu)-containing allele due to the presence of the single nucleotide polymorphism (SNP) at codon 198 is more frequently associated with breast cancer than the proline (Pro)-containing allele [45]. In contrast to the proline-containing variant, which is distributed almost equally between the cytoplasm and the mitochondria, leucine-containing GPX1 is predominantly located in the cytoplasm [46], and is less responsive to increasing nutritional

supplementation of selenium than a proline-containing variant [45]. These findings provide feasible molecular evidence for the role of GPX1 Pro198Leu polymorphisms in determining breast cancer risk. Additionally, GPX1 is one of the selenoproteins most readily affected by selenium levels; therefore, this antioxidant enzyme is presumably believed to play an essential role in the cancer preventive effects of selenium supplementation [25]. However, the role of GPX1 in the cytotoxic effects of selenium at supranutritional dosage is still largely undefined. In this study, we determined the in vivo expression patterns of SBP1 and GPX1 in breast cancers and adjacent noncancerous tissues, and then investigated the roles of SBP1 and GPX1 in selenite-induced cytotoxicity in vitro. We found that there was a significantly inverse correlation between SBP1 and GPX1 protein level in human breast tissues (Pearson r ¼ –0.4347, P ¼ 0.0338). SBP1, but not GPX1, was found to be a critical mediator in selenitemediated cytotoxicity. Furthermore, the selective cytotoxic effects of selenite on malignant and chemotherapeutic drug-resistant cells might be explained by expression levels of SBP1, loss of which resulted in the high-affinity uptake of selenite through elevating the levels of extracellular GSH, thus increasing intracellular reactive oxygen species (ROS) generation and further enhancing the consequent cell death induced by selenite.

Materials and methods Cell culture and reagents Human breast cancer cell line MCF-7 and human colon cancer cell line HCT116 were obtained from ATCC (American Type Culture Collection, USA). Stable MCF-7-vector and MCF-7-GPX1 cells were previously generated by the introduction of empty vector or construct expressing human GPX1 into MCF-7 cells, respectively [26]. HCT116-SBP1 and HCT116-vector cells derived from HCT116 stably transfected with pIRES2-SBP1 or vector, respectively, were also described previously [26]. The epirubicin-resistant MCF-7/Epi cells were established by chronic exposure of the parental MCF-7 to stepwise increases in epirubicin concentration up to 12 μM [47]. HCT116 and their derivative cells were cultured in McCoy's 5A medium (GIBCO, China), and parental and derivative MCF-7 cells in modified Eagle's medium (GIBCO, China). The medium was supplemented with 10% (v/v) fetal bovine serum and antibiotics (1  105 U/mL penicillin and 1  105 μg/mL streptomycin). All cell lines were maintained in humidified incubator at 37 1C with 5% CO2. Sodium selenite (Sigma-Aldrich, USA) dissolved in phosphate-buffered saline (PBS) at 1 mol/L stock solution was stored at –20 1C until used. NAC (N-acetyl-L-cysteine) and GSH (reduced glutathione) were purchased from Beyotime Institute of Biotechnology (China). DTNB (5,50 -dithiobis-(2-nitrobenzoic acid)) was obtained from Sigma-Aldrich (China). Breast cancer tissue specimens Clinical samples were used under the agreement of the patients treated by surgery in Zhongnan Hospital of Wuhan University (Wuhan, China) during October 2009 to June 2011. After collection, samples were stored at –80 1C until used. Twelve pairs of tumor tissues and adjacent normal tissues were collected from 12 female patients with average age of 41.2 ranging from 29 to 68 years, who did not receive any preoperative chemotherapy or radiotherapy before surgery, and were then diagnosed as breast cancer. Ethical approval was obtained from the ethics committee of Zhongnan Hospital of Wuhan University. The detailed clinical and pathological characteristics of the 12 pairs of tumor tissues were described in Supplementary Table 1.

Please cite this article as: Wang, Y; et al. Reduction of selenium-binding protein 1 sensitizes cancer cells to selenite via elevating extracellular glutathione: A novel mechanism of.... Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.11.015i

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Plasmids and transfection

Measurement of intracellular reactive oxygen species (ROS)

The construct encoding human HA-tagged SBP1 was generated previously [42]. The pLNCX-GPX1 expression plasmid was a gift from Dr. Alan M. Diamond at University of Illinois at Chicago [45]. The short hairpin RNA (shRNA) targeting human SBP1 (shSBP1) was purchased from Genecopoeia (Guangzhou, China). The small interfering RNA (siRNAs) against human SBP1 (1, 50 -AAG GAG GGC TGA AGT TGA A-30 ; and 2, 50 -AGG ACG AGC TGC ATC ACT C-30 ) and GPX1 (50 -CGC AAC GAT GTT GCC TGG A-30 ), and scrambled control siRNA [38] were obtained from RiboBio (Guangzhou, China). Transfection of plasmids, shRNAs, or siRNAs into cells was performed with Lipofectamine 2000 (Invitrogen, China) according to the manufacture's instruction. Stable shSBP1 or vector-transfected MCF-7 cells were established under the selection of 1.5 μg/mL puromycin (Invitrogen, China).

DCFH-DA (20 ,70 -dichlorofluorescein diacetate, Sigma-Aldrich, USA) and DHE (dihydroethidium, Sigma-Aldrich, USA) were used to detect the intracellular ROS levels. DCFH-DA was more sensitive to hydrogen peroxide production induced by sodium selenite, whereas DHE was used to detect the levels of superoxide anion. Cells in 24-well plates were treated with selenite for the indicated time, and then washed twice with PBS and incubated with 10 μM DCFH-DA or DHE in dark at 37 1C for 30 min. Cells were then washed thoroughly with serum-free medium to remove the remnant probes. For DCFH-DA staining, cells were harvested, washed twice with PBS, and resuspended in PBS. The fluorescence intensity of DCF, the oxidized form of DCFH-DA by hydrogen peroxide, was immediately monitored by flow cytometry (FACScan, BD Biosciences, USA) with excitation and emission wavelengths at 480 and 535 nm, respectively. For DHE staining, cell nuclei were then stained by incubation of cells in Hoechst 33258 (10 μg/mL) for 20 min. Following a thorough wash with PBS, cells were observed under a Nikon fluorescence microscope and the images were captured under the same parameters (e.g., exposure time and gain value) to detect Hoechst 33258 (λEx ¼340 nm) and DHE (λEx ¼485 nm) staining. The average DHE fluorescence intensity per cell was quantified using NIH ImageJ analysis software. Cell number was determined by counting the number of Hoechststained nuclei. An average of 100 cells per field and at least five random fields were selected for each experimental group and analyzed in a blind manner.

Immunoblotting assay Cells were harvested and suspended in RIPA lysis buffer (Beyotime Institute of Biotechnology, China) containing a protease inhibitor cocktail (Sigma-Aldrich, USA). After incubation on ice for 20 min, cell lysates were centrifuged at 10,000g for 15 min at 4 1C. The protein concentration of the supernatant was quantified using a BCA protein assay kit (Beyotime Institute of Biotechnology, China). Equal aliquots of cell lysates were separated by 12% or 10% SDS-PAGE gels and transferred to Immun-Blot PVDF membranes (Bio-Rad Laboratories, USA). Blots were probed with primary antibodies specific for SBP1, GPX1 (Medical and Biological Laboratories, Japan), JNK1, phosphorylated-JNK1 (p-JNK1), β-catenin, cyclin D1 (Santa Cruz Biotechnology, USA), c-Myc, and the loading control β-actin (Sigma-Aldrich, USA). All secondary antibodies against rabbit or mouse IgG were purchased from Santa Cruz Biotechnology. Signal was detected using BeyoECL Plus (Beyotime Institute of Biotechnology, China).

Cell viability assay The number of 1  104 cells per well was seeded in a 96-well plate. After 24 h of incubation, the medium was replaced with fresh culture medium containing the indicated final concentrations of sodium selenite. At least three independent experiments were performed. Following 48 h exposure, the relative number of viable cells was determined by MTS assay (3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) according to the manufacturer's protocol (CellTiter 96 Non-Radioactive Cell Proliferation Assay Kit, Promega, China), as described previously [10].

Nuclear staining with Hoechst dye This method that was used to distinguish apoptotic cells from viable ones has been described previously [48]. Apoptotic cells were characterized by the presence of highly condensed or fragmented nuclei. Briefly, cells in 24-well plates were treated with 10 μM selenite for 48 h, and then washed twice with PBS and fixed with 3.7% (v/v) paraformaldehyde in PBS for 20 min at room temperature and permeated with 0.1% Triton X-100 for 15 min at room temperature. The fixed cells were then stained with Hoechst 33258 (10 μg/mL; Sigma-Aldrich, USA) for 20 min, washed twice with PBS, and then analyzed at 340 nm excitation using a Nikon fluorescence microscope (Japan).

Quantification of extracellular GSH levels When reaching about 80% of confluence, cells were incubated with fresh medium for the indicated time, and the medium was collected and centrifuged at 10,000g for 10 min at 4 1C. The concentrations of total glutathione (T-GSH), reduced glutathione (GSH), and oxidized glutathione disulfide (GSSG) in the supernatant were measured by an enzymatic method according to the manufacture's instruction of the commercial assay kit (Beyotime Institute of Biotechnology, China). Briefly, T-GSH was determined using DTNB-GSSG reductase recycling. GSSG was measured by monitoring the generation rate of 5-thio-2-nitrobenzoic acid (TNB) from the reaction of reduced GSH with DTNB. The rate of TNB formation was measured at 412 nm over 25 min. Pure GSH was used to generate standard curves. The concentration of extracellular reduced GSH was obtained by subtracting GSSG from T-GSH. Assays were conducted in triplicate by using three independently generated supernatants. Determination of selenium (IV) and total selenium concentrations The levels of total selenium or selenium (IV) in samples (culture medium or cell lysates) were determined by using 2,3diaminonaphthalene (DAN, Sigma-Aldrich, USA), which was able to form Se-DAN fluorometric complexes by specifically interacting with selenium in the þ 4 oxidation state (designated as selenium (IV)). Following the indicated treatment for the indicated time, cells were harvested and then lysed by brief sonication. The concentrations of total selenium in the lysates were measured according to the procedure described previously [49,50]. The values of total selenium concentrations in cells were expressed as nanogram (selenium)/100 mg total proteins, whose concentrations were quantified using a BCA protein assay kit (Beyotime Institute of Biotechnology, China). For determination of selenium (IV) levels in growth medium, culture medium was collected after the treatment of cells with selenite for indicated time points, and 50 μL of collected medium was incubated with 2 mL mL of

Please cite this article as: Wang, Y; et al. Reduction of selenium-binding protein 1 sensitizes cancer cells to selenite via elevating extracellular glutathione: A novel mechanism of.... Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.11.015i

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P¼ 0.0338) (Fig. 1C), consistent with the findings in breast cancer cell lines [26].

2.5 mM mM EDTA and 500 μL of 6.3 mM DAN (dissolved in 0.1 M HCL) at 55 1C for 30 min. Cyclohexane (2 mL) was added to extract the Se-DAN complexes and the fluorescence was recorded using a Tecan GENios microplate reader (λEx ¼ 360 nm; λEm ¼525 nm). The linear standard curves of sodium selenite in corresponding fresh culture medium (0, 2, 4, 6, 8, 10, and 12 μM) were determined in parallel with every set of measurements. These experiments were performed at least in triplicate.

Reduction of SBP1 was required for the role of GPX1 in selenite-mediated cytotoxicity Although selenium has been shown to efficiently exert its cytotoxic effects in malignant cells [10–15], the roles of GPX1 and SBP1 in selenium-induced cytotoxicity remain unknown. To clarify this, we chose human breast cancer MCF-7 cells, in which endogenous mRNA and protein levels of GPX1 were undetectable [26,45], and employed two previously generated MCF-7 transfectants stably expressing vector or GPX1 (designated as MCF-7vector and MCF-7-GPX1, respectively) [26]. The redox-active selenium, sodium selenite (Se), was used throughout this study, and selenite concentrations used in this study were easily achievable with no or minor reversible side effects in humans (reviewed in Ref. [8]). As shown in Figs. 2A and B, ectopic expression of GPX1 markedly suppressed SBP1 protein [26] and resulted in a significant decrease in cell viability following selenite treatment, compared with MCF-7-vector group (Fig. 2B). Our previous studies have shown that selenite inhibits cell growth and induces apoptosis in colon cancer cells through activating JNK1 and suppressing β-catenin signaling [10]. Therefore, we detected the alterations of these related proteins in MCF-7-vector and MCF-7-GPX1 cells in response to selenite treatment. We found that forced expression of GPX1 increased the cytotoxic effects of selenite, as evidenced by the observations that 7.5 and 10 mM selenite caused the activation of JNK1 and suppression of β-catenin, c-Myc, and cyclin D1 only in MCF-7-GPX1, but not in MCF-7-vector, cells (Fig. 2C). We then determined whether GPX1 was an independent player or dependent on the down-regulation of SBP1 to enhance seleniteinduced cytotoxicity. To this end, we chose human colon cancer HCT116 cells for the following experiments because they had undetectable SBP1 protein levels as a result of hypermethylation in SBP1 promoter region [42]. Surprisingly, neither overexpression (Fig. 2D) nor knockdown (Fig. 2F) of GPX1 affected selenite cytotoxicity in HCT116 cells (Figs. 2E and G), thus leading us to hypothesize that down-regulation of SBP1 was likely to be

Statistical analysis The significance of the differences between groups was determined with ANOVA test or Student t test, and Po 0.05 was considered significant. The results were expressed as the mean 7SD from at least three independent experiments.

Results SBP1 protein levels were reduced in breast cancers and were inversely correlated with GPX1 We have previously reported that SBP1 is suppressed at transcriptional and translational levels by ectopic expression of GPX1 in human breast cancer GPX1-null MCF-7 cells in vitro [26] and there is an inverse correlation of SBP1 and GPX1 in prostate cancers [39]. Herein, we determined the expression patterns of SBP1 and GPX1 in human breast cancers and their matched adjacent normal tissues using immunoblotting assay, and found that SBP1 protein levels were significantly down-regulated in most of breast cancers relative to the matched normal tissues (Figs. 1A and B), consistent with prior reports that SBP1 was frequently reduced in various types of cancers [27,30,34–41]. However, in comparison with their adjacent normal tissues, GPX1 levels were markedly up-regulated in 10 of the 12 paired breast cancer tissues (Figs. 1A and C). Interestingly, SBP1 levels were significantly and negatively correlated with GPX1 analyzed in each individual case by Pearson correlation test analysis (Pearson r¼ –0.4347,

#1 N

T

#2 N T

N

#3 T

#4 N

T

#5 #6 N T N T

#7 N

T

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#11 N T

#12 N T SBP1 GPX1

p=0.0002

1.5 1.0 0.5 0.0

Normal

Tumor

10.0

p=0.0311

Relative SBP1 levels

2.0

Relative GPX1 levels

β-Actin

Relative SBP1 levels

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8.0 6.0 4.0 2.0 0.0

Normal

Tumor

2.0

Pearson r = -0.4347, p=0.0338 Normal Tumor

1.5 1.0 0.5 0.0 0.0

2.0

4.0

6.0

8.0

10.0

Relative GPX1 levels Fig. 1. SBP1 protein levels were reduced in breast cancers and were inversely correlated with GPX1. (A) Twelve pairs of breast cancers and adjacent matched normal tissues were extracted and subjected to immunoblotting assay for the protein levels of SBP1 and GPX1. β-Actin was used as a loading control. (B, C) Relative intensity of SBP1 (B) and GPX1 (C) in (A) was determined using Quantity One software, and normalized to β-actin, respectively. The relative intensity of SBP1 or GPX1 in normal tissues was set to 1.0. Paired t test was performed and P values were indicated in each top panel. (D) The correlation between the levels of SBP1 and GPX1 in breast tissues including normal and tumor was analyzed by Pearson correlation test with Pearson r ¼–0.4347, P¼ 0.0338.

Please cite this article as: Wang, Y; et al. Reduction of selenium-binding protein 1 sensitizes cancer cells to selenite via elevating extracellular glutathione: A novel mechanism of.... Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.11.015i

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SBP1 β-Actin

MCF-7-vector

MCF-7-GPX1

5

Vector GPX1

120

HCT116 Vector GPX1 SBP1 GPX1 β-Actin

Cell Viability (%)

GPX1

140 120 100 80 60 40 20 0

100 80 60 40 20 0

0.0 2.5 5.0 7.5 10.0 12.5 15.0

0

Se (μM)

5.0 7.5 10. 0

Se (μM)

HCT116 siCon siGPX1

p-JNK1 JNK1 β-Catenin c-Myc Cyclin D1 SBP1

120

GPX1 β -Actin

siCon siGPX1

100 80 60 40 20 0 0 5.0 7.5 10.0 Se (μM)

GPX1 β-Actin

120 100 80 60 40 20 0

*

HA-SBP1

β-Actin

5.0 7.5 10 Se (μM)

vector

GPX1

0

5.0 7.5 10

MCF-7-vector (vector)

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HA-SBP1 SBP1

Cell Viability (%)

MCF-7-vector

Cell Viability (%)

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Y. Wang et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

MCF-7-GPX1

Fig. 2. Reduction of SBP1 was required for the role of GPX1 in selenite-mediated cytotoxicity. (A) Stable MCF-7-vector and MCF-7-GPX1 cells were lysed and subjected to immunoblotting assay for GPX1 and SBP1 levels. β-Actin was used as a loading control. (B, C) Following 48 h treatment of stable MCF-7-vector and MCF-7-GPX1 cells with indicated concentrations of selenite, cell viability assay was performed at least three times (B); cells were extracted and subjected to immunoblotting assay for assessing the levels of the indicated proteins (C). β-Actin was used as a loading control. (D, E) After 24 h of transfection of HCT116 with vector or pLNCX-GPX1, cells were harvested to identify the efficacy of transfection using immunoblotting assay (D); cell viability was determined following the treatment of indicated concentrations of selenite for another 48 h (E). These experiments were performed at least in triplicate. (F, G) Twenty-four hours post transfection with 10 nM scramble siRNA (siCon) or GPX1 siRNA (siGPX1), the efficiency of GPX1 knockdown in HCT116 cells was assessed by immunoblotting assay (F); cell viability assay was performed following the exposure of selenite for another 48 h (G). The values were expressed as mean 7 SD from at least three independent experiments. (H) Stable MCF-7-vector and MCF-7-GPX1 cells were transfected with empty vector or construct expressing HA-SBP1. Twenty-four hours after transfection, cells were extracted for immunoblotting assay (top panel); cells were then treated with 10 mM selenite for another 48 h, and cell viability assay was performed (bottom panel). The values were expressed as mean7 SD from three independent experiments. n indicates Po 0.05.

required for the effects of GPX1 overexpression on seleniteinduced cytotoxicity. To verify this hypothesis, we restored the expression of SBP1 in MCF-7-GPX1 cells by ectopically expressing HA-SBP1 (Fig. 2H, top panel), and treated these tranfectants with 10 mM selenite for 48 h. Cell viability assay showed that the restoration of SBP1 completely abolished the effects of GPX1 on the selenite-induced cytotoxicity (Fig. 2H, bottom panel), demonstrating that inhibition of SBP1 is essential for the effects of ectopic expression of GPX1 on selenite-induced cytotoxicity. SBP1 was a crucial determinant for selenite-induced cytotoxicity To further confirm the role of SBP1 in selenite-induced cytotoxicity, we employed SBP1-null HCT116 cells stably expressing vector or SBP1 (designated as HCT116-vector and HCT116-SBP1, respectively; Fig. 3A), and exposed these stable transfectants to various concentrations of selenite for 48 h. Increased expression of SBP1 significantly attenuated selenite-induced cytotoxic effects, as illustrated by these observations from cell viability assay that HCT116-SBP1 cells were much more resistant to selenite than HCT116-vector cells (Fig. 3B), as well as the results from immunoblotting assay showing that overexpression of SBP1 abolished the effects of selenite on the activation of JNK1 and the suppression of

β-catenin, c-Myc, and cyclin D1 (Fig. 3C). In contrast, we knocked down the levels of SBP1 in GPX1-null MCF-7 by using specific short hairpin RNAs (shRNAs) against SBP1 (Fig. 3D), and found that reduced expression of SBP1 dramatically decreased cell viability (Fig. 3E) and increased the apoptosis (Fig. 3F) in MCF-7 cells following selenite treatment, suggesting that SBP1 is a crucial player in selenite-mediated cytotoxicity and functions in a GPX1independent manner. Selenite has been previously shown to have a preferential cytotoxicity in chemotherapeutic drug-resistant cells [19,20]. To verify this notion, we used epirubicin, a widely used agent for breast cancer therapy, to establish epirubicin-resistant MCF-7 cells (designated as MCF-7/Epi), and then treated these cells and their parental MCF-7 cells with various concentrations of selenite for 48 h. As expected, MCF-7/Epi cells were much more sensitive to selenite than the parental MCF-7 (Fig. 3G). To determine the involvement of SBP1 in the increased sensitivity of MCF-7/Epi to selenite, the protein levels of SBP1 and GPX1 in MCF-7/Epi were evaluated by immunoblotting assay. As shown in Fig. 3H, GPX1 expression was still undetectable in MCF-7/Epi cells, while SBP1 was significantly down-regulated in MCF-7/Epi as compared to parental MCF-7 cells. This result suggests that the down-regulation of SBP1 may contribute to the elevated sensitivity of MCF-7/Epi to selenite. To

Please cite this article as: Wang, Y; et al. Reduction of selenium-binding protein 1 sensitizes cancer cells to selenite via elevating extracellular glutathione: A novel mechanism of.... Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.11.015i

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Fig. 3. SBP1 was a crucial determinant for selenite-induced cytotoxicity. (A) Stable HCT116-vector and HCT116-SBP1 cells were lysed and subjected to immunoblotting assay for SBP1 levels. β-Actin was used as an internal control. (B, C) Following 48 h of treatment with indicated concentrations of selenite, cell viability was assessed and the values are presented as mean7 SD from three independent experiments (B); cell extracts were subjected to immunoblotting assay for assessing the levels of the indicated proteins (C). β-Actin was used as a loading control. (D–F) MCF-7 cells were stably transfected with control shRNA (shCon) or two sets of SBP1 shRNA (shSBP1-1#, shSBP1-2#), respectively. Transfection efficacy of SBP1 knockdown was assessed by immunoblotting assay (D). Stable transfectants were treated with indicated concentrations of selenite for 48 h, and cell viability assay was performed (E). The values are expressed as mean 7 SD from at least three independent experiments. After the 48-h incubation in the absence or presence of 10 mM selenite, cells were fixed and stained with Hoechst 33258 as described under Materials and methods. Arrows in figures indicate the apoptotic cells characterized by the presence of highly condensed or fragmented nuclei (F). (G) Parental MCF-7 and epirubicin-resistant MCF-7/Epi cells were treated with indicated concentrations of selenite for 48 h, and cell viability was determined. The values are presented as mean 7 SD from three independent experiments. (H) HCT116, MCF-7, and MCF-7/Epi cells were lysed and subjected to immunoblotting assay for SBP1 and GPX1 levels. β-Actin served as a loading control. (I, J) MCF-7/Epi cells were transiently transfected with empty vector or construct expressing HA-SBP1, respectively. Twenty-four hours post transfection, SBP1 levels were determined using immunoblotting assay. The last lane is parental MCF-7 without transfection (I). β-Actin was used as a loading control. Transfectants were treated with PBS or 10 mM selenite for 48 h, and cell viability assay was performed (J). The values are presented as mean 7 SD from three independent experiments. * indicates Po 0.05.

confirm this, MCF-7/Epi cells were transiently transfected with constructs encoding vector or HA-SBP1 (Fig. 3I), and were then treated with 10 μM selenite for 48 h. Cell viability assay showed that overexpression of SBP1 almost completely reversed the sensitive property of MCF-7/Epi in response to selenite (Fig. 3J). Collectively, these findings suggest that loss of SBP1 may account for the selective cytotoxicity of selenite in cancer cells and chemotherapeutic drugresistant cells.

Reduction of SBP1 increased selenite-induced ROS generation Selenite has been reported to induce apoptosis in cancer cells via generating high levels of ROS [12–17], including hydrogen peroxide and superoxide anion. Indeed, exposure of selenite to MCF-7/Epi cells for 2 h resulted in a significant increase in levels of hydrogen peroxide (H2O2) (Fig. 4A) and superoxide anion (O.– 2) (Fig. 4B). However, cotreatment of MCF-7/Epi with 5 mM NAC, a

Please cite this article as: Wang, Y; et al. Reduction of selenium-binding protein 1 sensitizes cancer cells to selenite via elevating extracellular glutathione: A novel mechanism of.... Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.11.015i

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Fig. 4. Reduction of SBP1 increased selenite-induced ROS generation. (A, B) In the presence or absence of pretreatment of 5 mM NAC for 1 h, MCF-7/Epi cells were exposed to PBS or 7.5 mM selenite alone or in combination with 5 mM NAC for another 2 h, and then stained with DCFH-DA (A) or DHE (B) probes for determining the levels of intracellular hydrogen peroxide and superoxide anion, respectively. The DCF fluorescence intensity of 104 cells acquired on every FACS assay was analyzed by using WinMDI 2.9 software (A). (C) In the presence or absence of pretreatment of 5 mM NAC for 1 h, MCF7/Epi cells were incubated with 7.5 mM selenite alone or in combination with 5 mM NAC. Cell viability was measured after 48 h of selenite treatment. Values are presented as mean 7 SD from three independent experiments. The symbol (*) indicates P o0.05. (D–F) Twenty-four hours after transient transfection of MCF-7 with 10 nM scramble or SBP1 siRNA (siCon or siSBP1, respectively), whole-cell lysates were immunoblotted with antibodies against SBP1 and β-actin, serving as a loading control (D); followed by treatment with vehicle (PBS) or 10 μM selenite for 16 h, intracellular hydrogen peroxide was measured as described under Materials and methods and values are expressed as mean7 SD from at least three independent experiments (E); following selenite exposure for 16 h, the levels of intracellular superoxide anion were determined by DHE staining. Cell nuclei were stained using Hoechst 33258, and images were captured using a Nikon fluorescence microscope (40  magnification; F, left panel). The DHE fluorescence intensity was quantified by using ImageJ software (F, right panel). The DHE intensity of MCF-7-siCON cells treated with vehicle (PBS) was set to 1.0. The symbol (*) indicates Po 0.05 in comparison with MCF-7-siCon group treated with selenite. The symbol (#) indicates P o 0.05 relative to MCF-7-siCon group without treatment.

well-known antioxidant, dramatically abolished the formation of ROS induced by selenite (Figs. 4A and B), and consequently blocked the cytotoxic effects of selenite (Fig. 4C), again demonstrating that production of excessive ROS is crucial for selenitemediated cytotoxicity. Next, we examined whether SBP1 attenuated cytotoxicity of selenite through inhibiting the formation of ROS. We used two sets of specific siRNAs against human SBP1 to knock down the expression of SBP1 in MCF-7 cells (Fig. 4D). As shown in Figs. 4E and F, treatment of selenite for 16 h only slightly elevated the levels of hydrogen peroxide (about 1.1-fold induction relative to that in cells treated with PBS) and superoxide anion (about 1.5-fold induction) in MCF-7 cells with control siRNA transfection (MCF-7-siCon), whereas knockdown of SBP1 (MCF7-siSBP1) resulted in a dramatic increase in the levels of seleniteinduced hydrogen peroxide (about 1.8-fold induction) and superoxide anion (about 3.3-fold induction). In addition, those cells with higher levels of superoxide anion were found to develop vacuolization (Fig. 4F), suggesting excessive levels of superoxide anion might be the driving force of morphological changes, which might be an early event in selenite-induced cytotoxicity. Taken together,

our findings show that down-regulation of SBP1 significantly enhances selenite-induced ROS production. 3.5. Reduction of SBP1 accelerated metabolism and uptake of extracellular selenite The notion that selenium uptake is a prerequisite for its cytotoxic effects has been well documented by Olm et al. [18]. To determine whether SBP1 affects selenium uptake, we first monitored the depletion rate of extracellular selenium (IV) by using DAN, which specifically interacted with selenium in the þ4 oxidation state (namely selenium (IV)), including selenite [49,50]. Interestingly, a time-course study revealed that the concentration of selenium (IV) in growth medium that was used to incubate selenite-sensitive MCF-7/Epi cells decreased rapidly in a liner manner during the first 4 h, while seleniteresistant MCF-7 cells almost did not absorb selenium during 24 h (Fig. 5A). This finding might explain the above observation that MCF7/Epi showed increased levels of ROS at as early as 2 h after the addition of 7.5 μM selenite (Figs. 4A and B), whereas ROS levels remained unchanged in the parental MCF-7 cells treated with 10 μM

Please cite this article as: Wang, Y; et al. Reduction of selenium-binding protein 1 sensitizes cancer cells to selenite via elevating extracellular glutathione: A novel mechanism of.... Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.11.015i

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Fig. 5. Reduction of SBP1 accelerated metabolism and uptake of extracellular selenite. (A) Selenium (IV) contents were determined in growth medium collected at the indicated time after the addition of 10 and 7.5 μM selenite into parental MCF7 and epirubicin-resistant MCF-7/Epi cells, respectively. Values were presented as mean 7SD from at least three independent experiments. (B, C) Stable scramble or SBP1 shRNA-transfected MCF-7 cells were exposed to 10 μM selenite for the indicated time, and the growth medium was collected for the determination of selenium (IV) contents (B); the total intracellular selenium absorbed by cells was analyzed at 24 h (C).

selenite for as long as 16 h (data not shown). More importantly, knockdown of SBP1 was found to markedly accelerate the depletion and subsequent uptake of selenium in growth medium (Figs. 5B and C), thereby increasing selenite-induced ROS formation and cell death. Reduction of SBP1 enhanced selenite-mediated cytotoxic effects through elevating extracellular GSH levels Selenite has been shown to enter into cells directly through low-affinity multiple anion transport channels, but its intermediate, hydrogen selenide produced by spontaneous reaction of selenite with GSH through Painter reaction [51], is able to cross the cell membrane efficiently and is more toxic than selenite [52,53]. The presence of extracellular GSH, therefore, would enhance cytotoxicity of selenite by accelerating selenium uptake [18,54]. To verify whether extracellular GSH was involved in the effects of SBP1 on selenium uptake, the levels of the extracellular reduced form of GSH were measured. Selenite-sensitive MCF-7/Epi cells secreted  2 and  15 μM of reduced GSH into growth medium after 4 h and 16 h incubation, respectively, whereas the GSH levels secreted by selenite-resistant MCF-7 were nearly undetectable even at 16 h (Fig. 6A). The significant differences in the levels of extracellular GSH between MCF-7/Epi and parental MCF-7 cells possibly explain the findings in Fig. 5A that selenium (IV) concentrations in growth medium decreased rapidly following treatment of MCF-7/Epi with 7.5 μM selenite, while almost all of selenite still stayed in the growth medium after 24 h of exposure of MCF-7 cells to 10 μM selenite. Further experiments showed that knockdown of SBP1 led to a remarkable increase in the levels of extracellular GSH (Fig. 6B), suggesting that reduction of SBP1 may

enhance selenite cytotoxicity through up-regulating the levels of extracellular GSH, which accelerates the depletion and the subsequent uptake of selenium in growth medium, eventually leading to a dramatic increase in selenite-induced formation of ROS and cell death (a proposed model summarized in Fig. 6E). To validate this hypothesis, we artificially modulated the extracellular levels of GSH by using GSH or DTNB (a cell-impermeable oxidant scavenging thiols including GSH). As expected, the addition of DTNB dramatically abolished the effects of SBP1 knockdown on selenium (IV) depletion in growth medium (Fig. 6C) and selenite cytotoxicity (Fig. 6D). Conversely, the presence of 75 μM GSH increased the depletion of selenium (IV) in growth medium, and enhanced selenite cytotoxicity in both MCF-7-shCon and MCF-7shSBP1 cells (Figs. 6C and D), as reported previously [18,54]. More interestingly, SBP1 knockdown failed to further enhance selenite cytotoxicity (Fig. 6D) when uptake rates of selenite were adjusted to the same extent by the addition of GSH (Fig. 6C), suggesting that SBP1 alters selenite cytotoxicity predominantly through modulating the extracellular GSH and the subsequent uptake of selenite, rather than through physically binding the intracellular free selenium. Our findings together suggest that SBP1 knockdown enhances selenite cytotoxicity by elevating extracellular levels of GSH.

Discussion Selenium is an essential trace element with potent preventive effects on various cancers [2–7]. However, selenium at supranutritional doses (μM range) has emerged as a promising anticancer drug with a high degree of selective cytotoxicity in tumor cells and chemotherapeutic drug-resistant cells [16–20]. As for the roles of selenium-containing proteins in the selective cytotoxicity of selenium, the effects of thioredoxin reductase 1 (TrxR1) on selenium cytotoxicity, one selenoprotein that is frequently up-regulated in cancer cells and functions as one of the major redox regulators in mammalian cells (reviewed In Ref. [8]), have been extensively investigated previously [7,8,20,51,55,56]. Overexpression of this enzyme has been shown to protect cells from selenite-induced cytotoxicity [55]; in contrast, reduction of TrxR1 enhances selenite toxicity in cancer cells by elevating extracellular GSH through a mechanism that is independent of thioredoxin [56]. These findings provide a novel strategy for further enhancing cancer-specific cytotoxicity of selenium by combining the selenite treatment and TrxR1 inhibition, although the preferential cytotoxicity of selenium is hard to be explained by the alterations of TrxR1 in malignant cells and chemotherapeutic drug-resistant cells. In the present study, we showed that reduction of selenium-binding protein 1, not GPX1, accounted for the selective cytotoxicity of selenite in breast cancers, which sensitized cancer cells and drugresistant cells to selenite via elevating extracellular glutathione (a proposed model summarized in Fig. 6E). Our previous studies have shown that GPX1 inhibits SBP1 expression at transcriptional and translational levels in vitro, and SBP1 forms a physical interaction with GXP1 and inhibits GPX1 enzyme activity [26]. In this study, we found that there also was an inverse correlation between SBP1 and GPX1 expression in breast tissues including tumor and adjacent normal tissues (Pearson r ¼ –0.4347, P¼ 0.0338), consistent with our previous findings in prostate cancers [39], suggesting that the regulation of SBP1 by GPX1 may also exist generally in vivo. SBP1 has been reported to contain several potential hypoxia response elements, a consensus sequence recognized by transcription factor HIF-1α, in its proximal and distal promoter [57]. As an antioxidant enzyme, GPX1 can modulate the levels of ROS and, thus, has a profound impact on ROS-mediated transcriptional regulation involving HIF-1α [25].

Please cite this article as: Wang, Y; et al. Reduction of selenium-binding protein 1 sensitizes cancer cells to selenite via elevating extracellular glutathione: A novel mechanism of.... Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.11.015i

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Fig. 6. Reduction of SBP1 enhanced selenite-mediated cytotoxic effects through elevating extracellular GSH levels. (A) When reaching about 80% of confluence, MCF-7 and MCF-7/Epi cells were incubated in fresh medium for the indicated time, and growth medium was collected for the analysis of extracellular reduced GSH levels. Values are represented as the mean7 SD from three independent experiments. (B) Stable scramble or SBP1 shRNA-transfected MCF-7 cells were incubated in fresh medium for 16 h, and reduced GSH levels excreted by cells were determined. Values are expressed as the mean 7 SD from four independent experiments. (C, D) In the presence or absence of 75 μM GSH or 350 μM DTNB, cells were treated with 10 μM selenite for the indicated time, and the selenium (IV) contents in growth medium were determined (C); cell viability was measured at 48 h (D). (E) A proposed model for the roles of selenium-containing proteins (SBP1 and GPX1) in selenite-mediated cytotoxicity.

However, whether HIF-1α mediates the transcriptional inhibition of SBP1 by GPX1 is not clear and needs further investigation, and the results from which will help us better understand the physiological relevance of the molecular cross talk between members of the distinct classes of selenium-containing proteins [28]. Despite multiple mechanisms that contribute to the selenitemediated cell cycle arrest and apoptosis in numerous cancer cell lines [10–17], the leading ones have been shown to be redox effects [8], resulting in the formation of ROS, including hydrogen peroxide and superoxide anion [12–17]. Indeed, the presence of high doses of NAC (a well-known antioxidant at 5 mM) significantly abolished selenite-induced ROS production and cytotoxicity (Figs. 4A–C). The protective effects of NAC, however, were further found to function in a strict concentration-dependent manner because the cotreatment of low doses of NAC (100 μM) dramatically enhanced selenite-mediated cytotoxicity (Supplementary Fig. 1B). The potential mechanism for this observation will be discussed later. GPX1 detoxifies hydrogen peroxide to nontoxic water [25]; therefore up-regulation of this enzyme protects cancer cells from oxidative stress- and anticancer agent-induced cell death [58,59]. However, herein we found that neither overexpression nor reduction of GPX1 had any significant effects on seleniteinduced cytotoxicity in SBP1-null HCT116 cells (Figs. 2D–G). In line with our findings, a previous study has shown that seleniteinduced apoptosis in prostate cancer cells is only inhibited by overexpression of manganese superoxide dismutase, an antioxidant enzyme that converts superoxide anion (O.– 2 ) to hydrogen peroxide (H2O2), but not by other antioxidant enzymes including GPX1, catalase, and copper–zinc superoxide dismutase [12]. This

finding, together with other studies [13–16], strongly suggests that production of superoxide anion in mitochondria is much more crucial for selenium-mediated cytotoxicity than hydrogen peroxide. However, overexpression of GPX1 in MCF-7 pronouncedly enhanced the cytotoxic effects of selenite (Figs. 2B and C). These observed effects of GPX1 on selenite cytotoxicity were further found to be the direct consequence of down-regulation of SBP1 (Figs. 2A and H), which was further shown to be an independent player in selenite-mediated cytotoxicity, at least independently of GPX1 (Fig. 3). Interestingly, we showed that reduction of SBP1 played a critical role in the increased sensitivity of MCF-7/Epi to selenite (Figs. 3I and J). Even though the exact mechanisms by which the stepwise selection of epirubicin resulted in the inhibition of SBP1 were still unclear, the down-regulation of SBP1 occurred in a GPX1independent manner because GPX1 remained undetectable in MCF-7/Epi cells (Fig. 3H), suggesting that other mechanisms might be responsible for the down-regulation of SBP1, such as the methylation in SBP1 promoter [34,42]. Given the established roles of SBP1 in selenite-mediated cytotoxicity, this finding led us to the conclusion that frequent reduction or even loss of SBP1 in cancer tissues [27,30,34–41] and in chemotherapeutic drug-resistant cells was likely to account for the cancer-specific cytotoxicity of selenite. To date, cancer-specific cytotoxicity has been presumably linked to the ability of cancer cells to uptake selenium in a high affinity due to the presence of the relatively high levels of the extracellular reduced form of GSH [18,52–54]. Here, we found that SBP1 knockdown enhanced selenium uptake and the resulting cytotoxicity of selenite by elevating extracellular levels of GSH (Fig. 5 and Fig. 6). As reported previously [18,54], the addition of GSH at either low or high

Please cite this article as: Wang, Y; et al. Reduction of selenium-binding protein 1 sensitizes cancer cells to selenite via elevating extracellular glutathione: A novel mechanism of.... Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.11.015i

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doses did facilitate selenium uptake (Fig. 6C and Supplementary Fig. 1A). Similarly, cotreatment of NAC also resulted in the highaffinity uptake of selenium (Supplementary Fig. 1A). Consistent with roles of GSH or NAC in assisting selenium uptake, low doses of GSH (75 μM) or NAC (100 μM) dramatically increased selenite-induced cytotoxicity (Supplementary Fig. 1B). However, these dramatic effects were significantly attenuated by the presence of high doses of GSH (2 mM) or NAC (5 mM) (Supplementary Fig. 1B), suggesting that excessive doses of antioxidant GSH and NAC can efficiently eliminate the excessive production of ROS (Figs. 4A and B) after the highaffinity uptake of selenite. Moreover, our findings showed effects of SBP1 on modulating extracellular GSH levels, supporting the role for SBP1 as a regulator of extracellular redox environment. Furthermore, knockdown of SBP1 in GPX1-null MCF-7 cells caused a significant increase in the steady-state levels of intracellular superoxide anion (Fig. 4F), which was consistent with low levels of intracellular GSH in MCF-7-shSBP1 cells (data not shown). It is, therefore, reasonable to assume that SBP1 knockdown may have roles in assisting the efflux of GSH, leading to an increase in the steady-state levels of intracellular ROS. However, this intriguing assumption reveals a need for the continued investigation of the molecular mechanisms responsible for the roles of SBP1 in modulating both intra- and extracellular redox states. Taken together, our findings have revealed a potent mechanism of the selective cytotoxicity of selenite in cancer cells and in drugresistant cells, in which SBP1 is likely to play an important role in modulating the extracellular microenvironment by regulating the levels of extracellular reduced form of GSH. Our findings provide additional evidence that SBP1 may serve as a promising biomarker for predicting the efficacy of supranutritional dosages of selenium in cancer therapy, and also suggest that those individuals with lower levels or even loss of SBP1 in tumor tissues would probably receive more benefit from future clinical trials that use selenium as a therapeutic drug in cancer therapy.

Conflict of interests The authors declared no competing interests.

Acknowledgments We thank Weihuang Liu at Wuhan University School of Medicine for helping us analyze the results of intracellular hydrogen peroxide levels obtained by flow cytometry. This work was supported in part by grants from National Natural Science Foundation of China (Grants 91229115 and 81272251 to W.Y. and 81201917 to W.F.) and Specialized Research Fund for the Doctoral Program of Higher Education (20120171120116 to W.F.), as well as Doctoral Candidate Research Fund of Wuhan University (20103030101000208 to Y.W.) and State Scholarship Fund of China (201206270012 to Y.W.)

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