Selenite-Induced Toxicity in Cancer Cells Is Mediated by Metabolic

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Selenite-induced Toxicity in Cancer Cells is mediated by Metabolic Generation of Endogenous Selenium Nanoparticles Peng Bao, Zheng Chen, Renzhong Tai, Han-Ming Shen, Francis Luke Martin, and Yong-Guan Zhu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr501086e • Publication Date (Web): 08 Jan 2015 Downloaded from http://pubs.acs.org on January 15, 2015

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Journal of Proteome Research

Selenite-induced Toxicity in Cancer Cells is mediated by Metabolic Generation of Endogenous Selenium Nanoparticles Peng Bao1,5, Zheng Chen1, Ren-Zhong Tai2, Han-Ming Shen3, Francis L. Martin4, Yong-Guan Zhu1,5,* 1

State Key Lab of Urban and Regional Ecology, Research Center for

Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, P.R. China; 2Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, 201204, P.R. China; 3Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 117597, Singapore;

4

Centre for

Biophotonics, LEC, Lancaster University, Lancaster LA1 4YQ, UK; 5Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, P.R. China

Address correspondence to Professor Yong-Guan Zhu, Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, P.R. China. Tel: +86 592 6190997; Fax: +86 592 6190977; E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT Selenite has been a touted cancer chemopreventative agent but generates conflicting outcomes. Multiple mechanisms of selenite cytotoxicity in cancer cells are thought to be induced by metabolites of selenite. We observed that intracellular metabolism of selenite generates endogenous selenium nanoparticles (SeNPs) in cancer cells. Critical proteins that bind with high-affinity to elemental selenium during SeNPs self-assembly were identified through proteomics analysis; these include glycolytic enzymes, insoluble tubulin and heat shock proteins 90 (HSP90). Sequestration of glycolytic enzymes by SeNPs dramatically inhibits ATP generation, which leads to functional and structural disruption of mitochondria. Transcriptome sequencing showed

tremendous

down-regulation

of

mitochondrial

respiratory

NADH

dehydrogenase (complex I), cytochrome c oxidase (complex IV) and ATP synthase (complex V) in response to glycolysis-dependent mitochondrial dysfunction. Sequestration of insoluble tubulin led to microtubule depolymerization, altering microtubule dynamics. HSP90 sequestration led to degradation of its downstream effectors via autophagy, ultimately resulting in a cell-signaling switch to apoptosis. Additionally, the surface effects of SeNPs generated oxidative stress, thus contributing to selenite cytotoxicity. Herein, we reveal that the multiple mechanisms of selenite-induced cytotoxicity are caused by endogenous protein-assisted self-assembly of SeNPs, and suggest that endogenous SeNPs could be potentially primary cause of selenite-induced cytotoxicity.

Keywords: Chemoprevention; Self-assembly; Selenite; Selenium nanoparticles; Glycolysis; Mitochondrial dysfunction; Tubulin; Heat shock proteins

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INTRODUCTION

Sodium selenite, an inorganic selenium (Se) compound, possesses anti-cancer properties and has been used in chemoprevention studies.1-3 Selenite induces cell death in human cancer cells via several mechanisms, including generation of reactive oxygen species (ROS); this is mediated by intracellular redox cycling of selenide (a Se metabolite) with oxygen and cellular thiols.4,5 Selenite primarily mediates induction

of

apoptosis

via

mitochondrial-dependent

intrinsic

pathways.1,6,7

Metabolites of selenite in cancer cells, including selenodiglutathione (GSSeSG) and hydrogen selenide (HSe-), are believed to be cytotoxic and induce oxidative stress.4 In addition to ROS generation, one needs to consider that selenite in cancer cells could result in autophagy inhibition, impairment of protein synthesis, DNA strand breaks and microtubule depolymerization, leading to cell cycle arrest, inhibition of cell proliferation and apoptosis.1,7-10 Currently, little is known regarding the multiple mechanisms of selenite-induced cytotoxicity. Selenite is reduced in cells into selenide (H2Se) by a number of intermediate steps in the presence of glutathione, with simultaneous production of superoxide radical.11,12 Selenide is then transformed by methyltransferase into methylselenol (CH3SeH), and further methylated metabolites. Additionally, selenide is an intermediate for selenoproteins, Se-sugar and elemental Se.7 Reactions of selenide and oxygen that give rise to elemental Se are assumed to be a source of superoxide radicals.6 Microprobe X-ray absorption near-edge structure spectroscopy (µ-XANES) shows that highly-localized Se species in selenite-treated lung cancer A549 cells are mostly elemental Se.13 Elemental Se is a metabolite in selenite-treated trout or rat hepatocytes.14,15 The role of elemental Se in selenite-induced cytotoxicity remains obscure. 3



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This study was stimulated by observations that bacterial reduction of selenite/selenate to elemental Se can result in intracellular SeNPs precipitation and accumulation by binding to high-affinity proteins.16-18 Most SeNPs-bound proteins in bacteria are non-inducible; these are ribosomal proteins and selenite reduction-related oxidoreductases.18 We hypothesize that elemental Se gives rise in cancer cells to endogenous self-assembled SeNPs, which have toxicological activity. It is of interest to investigate if there are protein-assisted SeNPs self-assemblies in cancer cells, and if so, the consequences if high-affinity proteins are sequestered by SeNPs. This is of significance given environmental depletion of trace elements such as Se and the possible implications of this in the incidence of certain cancers, e.g., of the prostate. We report novel mechanisms underlying the cytotoxic effects of selenite in non-small lung cancer H157 cells, via intracellular protein-assisted SeNPs self-assemblies. Sequestration of high-affinity proteins by SeNPs results in multiple cytotoxic effects, including glycolysis inhibition, glycolysis-dependent mitochondrial dysfunction, microtubule depolymerization, and autophagy inhibition. Additionally, surface effects of SeNPs result in ROS generation and DNA damage. We find that selenite generates similar mechanisms in a number of other cancer cell lines. These studies set out to attain deep insights into the mechanisms underlying selenite-induced cytotoxicity towards stimulating interest in exploiting its positive effects in chemoprevention.

MATERIALS AND METHODS

Cell Lines and Culture Human non-small lung cancer H157 cells purchased from Institute of Basic Medical Sciences (Chinese Academy of Medical Sciences) was the main cell line used. Lung 4


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cancer A549 cells and human gastric carcinoma MGC-803 cells were gifts from Professor Zi-Jian Wang (State Key Lab of Urban and Regional Ecology, Chinese Academy of Sciences). Identification and Distribution of SeNPs in H157 Cells H157 cells treated with selenite (10 µM) for 24 h were fixed and dropped directly onto a copper wire mesh on the sample holder of a scanning transmission X-ray microscope (STXM) (BL08U STXM, Shanghai Synchrotron Radiation Facility, Chinese Academy of Sciences, Shanghai, P.R. China). Using dual-energy absorption for elemental mapping, two absorption-contrast images are scanned separately at energies E2=1394 eV and E1=1385 eV, where E2 is focused and E1 is below the absorption edge of elemental Se. H157 cells treated with selenite (10 µM) for 24 h were prepared for transmission electron microscopy (TEM, HITACHI H-7500). Ultrathin sections were cut perpendicular to the plane of the monolayer, double-stained with lead citrate and uranyl acetate. Characterization of SeNPs Extracts from H157, A549 or MGC-803 Cells Cell culture suspensions treated with selenite (10 µM) for 24 h were filtered, then centrifuged at 11,000 rpm/min to collect suspended SeNPs. Adherent cells were digested with pancreatic enzyme and ultrasonication for 60 s at 200 W after centrifugation. The ruptured cell mixture was centrifuged at 4,000 rpm/min to remove cell debris, and then the supernatant was centrifuged at 11,000 rpm/min to collect SeNPs. These two portions of SeNPs were collected for field emission scanning electron microscopy (FESEM, HITACHI SU8020). SeNPs were initially washed with PBS and dehydrated under room temperature.

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Proteome Analysis of SeNPs High-Affinity Proteins: Protein Identification, Quantification, Database Construction, and Bioinformatic Data Analysis SeNPs were extracted from H157 cells treated with selenite (10 µM) for 24 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate SeNPs binding proteins according to standard protocols.19 Samples were subsequently loaded onto an Agilent C18 trap, followed by nano-LC-ESI-MS/MS analysis. MS/MS data were searched against a human protein database (2011-12-14, 33256 entries) downloaded from the NCBI database using the SEQUEST (v.28) program (Thermo, USA). Western Blot Assay To analyze SeNPs binding proteins, SeNPs collected from H157, A549 or MGC-803 cells were resolved by 4 × SDS-PAGE samples loading buffer and electrophoresis, then transferred to nitrocellulose membranes. The membranes were probed with anti-HSP90α (1:1000), anti-β-tubulin (1:500), anti-Pyruvate kinase M2 (1:100), anti-LDH-A (1:200), anti-α-enolase (1:2000), anti-Aldolase (1:400), anti-Annexin A2 (1:500), anti-α-Actinin-1 (1:400), anti-α-Actinin-4 (1:2000), anti-Ribosomal protein S40 (1:1000) or anti-Peroxiredoxin (1:1000) overnight [see Electronic Supporting Information (ESI), Table S1]. Bound primary antibodies were detected using appropriate secondary antibodies followed by detection with a super signal enhanced chemiluminescence kit. RNA Extraction, Sequencing and Assembly of Nuclear Genome A 100 ml of H157 cell culture was pelleted and mRNA extracted using TruSeq RNA Sample Prep Kit, following two 24-h treatments with or without selenite (10 µM). Aliquots of mRNA were pooled for conversion into cDNA and sequenced to obtain transcriptomic data. The quality of cDNA used for sequencing was assessed by 6


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PicoGreen assay (Quant-iT PicoGreen dsDNA Assay Kit, Invitrogen, P7589) and an Agilent Bioanalyzer (Agilent 2100 Bioanalyzer, Agilent, 2100; Agilent High Sensitivity DNA Kit, Agilent, 5067-4626); all quality-control metrics were satisfied. Assembly was performed and followed by measuring and comparing their quality. Annotated transcripts were available from the Ensembl database. Indirect Immunofluorescence Microscopy H157 cells were cultured until adhered to slides for 24 h. After culture with or without selenite for another 24 h, slides were fixed in 4% paraformaldehyde and permeabilized using 0.1% Triton X-100. The slides were then blocked with 2% BSA, and incubated with β-tubulin antibody overnight at 4°C. Cells were incubated with FITC-conjugated secondary antibody for 60 min at room temperature, after washing three times with PBS. After a second round of PBS washing, the cells were stained with DAPI for 5 min; the slides were again washed three times and mounted in anti-fading medium. Images were visualized and photographed using a Nikon E-2100 microscope. Microtubule Polymerization Assay Approximately 2 × 106 cells were collected and twice washed with PBS. Then the cells were re-suspended in hypotonic buffer at 37°C for 5 min. After centrifugation (14,000 g, 10 min at 25°C), supernatants containing soluble tubulin were collected and pellets containing insoluble tubulin were re-suspended in RIPA buffer and disrupted by ultrasonication on ice. Lysates were centrifuged (12,000 g, 10 min at 4°C), and supernatants containing insoluble tubulin were collected.10

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RESULTS

Formation of SeNPs in H157 Cells We initially searched for the presence of SeNPs in H157 cells using STXM and TEM. Figure 1 shows absorption-contrast images at two photon energies. Figure 1A was obtained at E1=1385 eV and shows various intense absorption areas compared with Figure 1B, which was obtained at E2=1394 eV. Absorbance ranges were from 193.2 to 408.9 (Figure 1A), and from 261.7 to 558.1 (Figure 1B). We converted absorption contrasts of the two images into optical densities and utilized subtraction analysis to calculate elemental Se distribution after the two images were registered. Then, intracellular elemental Se distributions were acquired and shown in color pixels overlapped on the original image (within blue ellipsoid areas, Figure 1C). Distributions of endogenous SeNPs in H157 cells are shown in selenite-exposed H157 cells compared with control H157 cells (Figure 1D-G). Endogenous SeNPs were present both in the cytoplasm and organelles, although they were probably compressed during ultrathin section cutting (Figure 1F, G). The combined results shown by STXM and TEM demonstrate the presence of elemental Se in H157 cells as endogenous self-assembled SeNPs. Intracellular SeNPs Self-Assembly is Not Cell-Type Specific for H157 cells Cancer cell lines of different origins were examined to determine whether this finding is typical. SeNPs extracted from selenite (10 µM)-treated (for 24 h) H157, A549 or MGC-803 cells were characterized by field emission-scanning electron microscopy (FE-SEM) (see ESI, Figure S1A-F). Characteristics of SeNPs extracted from selenite-exposed H157, A549 and HGC-803 cell lines are shown in ESI Figure S1 A-F and summarized in Table S2. An EDX spectrum of electron-dense SeNPs shows that the ratios of Se were 29.2%, 51.9% and 29.9% for H157, A549 and MGC-803 8


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cells, respectively (see ESI, Figure S2). This result implies that SeNPs are self-assembled through facilitation with a biomacromolecule, e.g., proteins. Dose-dependent effects of sodium selenite on viability of H157, A549 and MGC-803 cells show that the former two generate more SeNPs and are much more sensitive than MGC-803 cells (see ESI, Figure S1G). Identification of SeNPs High-Affinity Proteins in H157 Cells We analyzed and identified SeNPs high-affinity proteins using TEM and SDS-PAGE, respectively (Figure 2A, B). Nano-LC-LTQ results showed a qualitative characterization of high-affinity proteins associated with self-assembled SeNPs in H157 cells. In three parallel SeNPs samples and based on their abundance, proteins were identified (Figure 2C). The protein types with the highest number of peptides matched are glycolytic enzymes with 1358 peptides and ≈22.1% abundance in triplicate samples. Eleven different enzymes and relevant isomers in the glycolytic process were identified: phosphoglycerate kinase, transketolase, triosephosphate, L-lactate

dehydrogenase,

glucose-6-phosphate

fructose-bisphosphate

1-dehydrogenase,

aldolase,

glucose-6-phosphate

enolase,

isomerase

and

glyceraldehyde-3-phosphate dehydrogenase, respectively, along with two key enzymes, 6-phosphofructokinase and pyruvate kinase (see ESI, Table S3). The abundance of glycolytic enzymes binding to SeNPs implies strong inhibition of glycolysis. Actin, tubulin, HSP and annexin A2 were other proteins showing significant binding to SeNPs; the number of peptides for each were 893 (14.5%), 712 (11.56%), 470 (7.63%) and 231 (4.01%), respectively (Figure 2C). Elongation factors together with ribosomal proteins (138 peptides, 2.24%) binding with SeNPs might result in the inhibition of translation. ROS-associated antioxidants (122 peptides, 1.98%) bound to SeNPs might lead to reduced cell resistance to ROS. It is also noted 9



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that c-myc promoter-binding protein-1 isoform (119 peptides, 1.93%) was found in SeNPs high-affinity proteins (Figure 2C); this implies possible negative effects of selenite as a chemopreventative agent as a result of possible protooncogene overexpression and cancer cell proliferation. Thirteen types of high-affinity proteins were chosen and analyzed by Western blot (see ESI, Figure S3). These results indicate that intracellular SeNPs self-assembly and the critical proteins sequestered might interfere with cancer cell metabolism and proliferation. Genome-Wide Expression Analysis in H157 Cells following Exposure to Selenite To evaluate the transcription response of SeNPs high-affinity proteins to selenite exposure, cDNA was obtained using RNA extracted from 24 h selenite-treated vs. untreated cell cultures, respectively. Samples were amplified prior to paired-end deep sequencing. Of 51,796,643 and 53,498,910 pairs of reads generated, 80.4% and 75.3% had at least one end mapped to the reference genome (see ESI Tables S4-S6). Selenite exposure significantly (P

Selenite-Induced Toxicity in Cancer Cells Is Mediated by Metabolic

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