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. Author manuscript; available in PMC: 2018 Nov 15.
Published in final edited form as: Mol Cancer Res. 2016 Sep 22;14(12):1229–1242. doi: 10.1158/1541-7786.MCR-16-0028

Increased expression of system xc− in glioblastoma confers an altered metabolism and chemoresistance

Monika D Polewski 1,2, Rosyli F Reveron-Thornton 3,4, Gregory A Cherryholmes 5,6, Georgi K Marinov 7, Kaniel Cassady 8,9, Karen S Aboody 10,11
PMCID: PMC6237285  NIHMSID: NIHMS817257  PMID: 27658422

Abstract

Glioblastoma multiforme (GBM) is the most aggressive malignant primary brain tumor in adults. Several studies have shown that glioma cells up-regulate the expression of xCT (SLC7A11), the catalytic subunit of system xc−, a transporter involved in cystine import, that modulates glutathione production and glioma growth. However, the role of system xc− in regulating the sensitivity of glioma cells to chemotherapy is currently debated. Inhibiting system xc− with sulfasalazine decreased glioma growth and survival via redox modulation, and use of the chemotherapeutic agent temozolomide together with sulfasalazine had a synergistic effect on cell killing. To better understand the functional consequences of system xc− in glioma, stable SLC7A11 knock-down and over-expressing U251 glioma cells were generated. Modulation of SLC7A11 did not alter cellar proliferation but over-expression did increase anchorage-independent cell growth. Knock-down of SLC7A11 increased basal ROS and decreased glutathione generation resulting in increased cell death under oxidative and genotoxic stress. Over-expression of SLC7A11 resulted in increased resistance to oxidative stress and decreased chemosensitivity to temozolomide. In addition, SLC7A11 over-expression was associated with altered cellular metabolism including increased mitochondrial biogenesis, oxidative phosphorylation and ATP generation. These results suggest that expression of SLC7A11 in the context of glioma contributes to tumorigenesis, tumor progression, and resistance to standard chemotherapy.

Implications:

SLC7A11, in addition to redox modulation, appears to be associated with increased cellular metabolism and is a mediator of temozolomide resistance in human glioma, thus making system xC− a potential therapeutic target in GBM.

Keywords: SLC7A11, glioma, oxidative stress, glutathione, chemoresistance, metabolism

Introduction

Glioblastoma multiforme (GBM), a grade IV astrocytoma, is the most common and aggressive primary brain tumor in adults; patient survival averages only 14 months after diagnosis (1). The standard-of-care for patients with newly diagnosed GBM includes aggressive safe tumor resection followed by radiotherapy with concomitant systemic chemotherapy using the alkylating agent temozolomide (TMZ) (2). Unfortunately, patients with high grade gliomas inevitably progress or relapse an average of only 6.9 months after treatment (3). Therapeutic options for recurrent GBM are limited and generally not effective. One of the main causes of treatment failure in GBM patients is resistance to post-operative radiation and chemotherapy. Many mechanisms contribute to the development of drug resistance, including DNA repair, drug uptake and efflux, apoptosis and glutathione-mediated cellular detoxification pathways (4). Thus, an improved understanding of the molecular mechanisms involved in glioma progression and survival, as well as mediators of TMZ resistance, could lead to development of more effective therapeutic strategies.

One of the mechanisms for chemo- and radiotherapy is to disproportionately increase intracellular reactive oxygen species (ROS) to induce cell cycle arrest, senescence and apoptosis (5). Accumulation of ROS can trigger apoptosis due to oxidative damage to DNA, macromolecules, lipids and mitochondria. However, up-regulation of antioxidant systems is observed in various tumors making them more resistant to chemotherapy (6). Approaches to maximally exploit ROS-mediated cell death by combining drugs that induce ROS generation with compounds that suppress cellular antioxidant capacity have been proposed years ago (7).

System xc−, a sodium-independent membrane transporter, couples the influx of extracellular cystine to the efflux of glutamate (8). Expression of system xc−, specifically the catalytic domain xCT, is up-regulated in gliomas, and several studies have shown over-expression confers a growth advantage, either through increased extracellular glutamate levels that promote neuronal cell death or through increased import of cystine that is converted to cysteine (8–11). Cysteine is the rate-limiting precursor for generating the major antioxidant glutathione (GSH). GSH can neutralize intracellular ROS or be conjugated by glutathione S-transferases to xenobiotic agents, which are then exported out of the cell (12, 13).

There is accumulating evidence that GSH and system xc− may mediate resistance of cancer to cytotoxin-based therapies (5). Radiation therapy-resistant glioma cells exhibited a 5-fold increase in the expression of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and glutathione reductase that maintain redox balance (14). High intracellular GSH levels in cancer cells have also been associated with drug resistance and detoxification of alkylating agents. System xc− has been shown to maintain intracellular GSH levels in ovarian cancer cells, resulting in cisplatin resistance (15). Inhibition of system xc− in pancreatic cancer cells led to growth arrest and over-expression led to gemcitabine resistance (16). Recent findings also support the hypothesis of a correlation between adaption to oxidative stress, low mitochondrial ROS, enhanced mitochondrial respiration and resistance to chemotherapy drugs (17).

In this study, we show that xCT (SLC7A11) was highly expressed in established glioma cell lines and that treating cells with sulfasalazine (SSZ), an inhibitor of system xc−, decreased glioma growth and increased ROS-mediated cell death. Inhibiting system xc− in U251 glioma cells with SSZ concomitant with TMZ treatment had a synergistic killing effect. However, stable knock-down of SLC7A11 in U251 glioma cells did not alter cell growth or viability under basal conditions, despite changes in redox balance. Over-expression of SLC7A11 resulted in anchorage-independence and resistance to oxidative stress, while SLC7A11 knock-down decreased anchorage-dependent cell growth and resistance to oxidative stress. Knocking-down expression of SLC7A11 in U251 glioma cells also increased their sensitivity to TMZ. In contrast, U251 cells in which SLC7A11 was over-expressed had decreased sensitivity to TMZ and reduced apoptosis. Over-expression of SLC7A11 in U251 glioma cells also resulted in an up-regulation of genes involved in cellular metabolism, increased mitochondrial biogenesis, oxidative phosphorylation, and ATP production while maintaining low cytoplasmic and mitochondrial ROS levels. These results suggest that high expression of SLC7A11/system xc− activity may confer resistance of glioma to TMZ treatment by increasing GSH production for redox balance and promoting cellular metabolism.

Materials and Methods

Cell Culture

Human glioma cell lines (U251 and U87) and normal primary human astrocytes (pNHA) were purchased from American Type Culture Collection (ATCC) and cultured as previously described (18). The primary high grade glioma line PBT017 was obtained and cultured as previously described (18). For genotoxic or oxidative stress studies, cells were treated 24 h after plating with either 300 μM TMZ for 72 h or 100 μM H2O2 for 6 h.

Orthotopic Transplantation and Histopathological Analysis

In vivo studies were carried out in an orthotopic U251, U87, and PBT017 human glioma mouse model. Tumors were established by stereotactic, intracranial injection of 2 × 105 cells into the frontal lobe of NOD-scid mice. At 4 weeks, mice were perfused transcardially with 4% paraformaldehyde in PBS. Brains were harvested and formalin-fixed paraffin-embedded sections were stained with hematoxylin-eosin to confirm the presence of tumors. To assess xCT protein expression in vivo, sections were incubated overnight with a goat anti-human polyclonal xCT antibody (LS-B4345, LifeSpan Biosciences, Inc.) followed by incubation with secondary antibodies conjugated to horseradish peroxidase and detected using a DAB Peroxidase Substrate Kit (Vector Laboratories, Inc.)

RNA Isolation, cDNA Synthesis, and Quantitative Real-time-PCR

Total RNA was extracted using Trizol reagent. Synthesis of cDNA was performed using the BioRad cDNA synthesis kit. SYBR green PCR master mix (Life Technologies) was used for quantitative real-time PCR (qRT-PCR) monitored with a C1000 Thermal Cycler (BioRad) as previously described.(19) Reaction conditions for qRT-PCR were as follows: 1 cycle of 3 min at 95ºC; 39 cycles of 10 s at 95ºC, 10 s at 55ºC, 30 s at 72 ºC; 1 cycle of 10 s at 95°C; and a melting curve of 5 s at 65°C - 95°C. A standard linear curve was generated using pooled sample DNA and the threshold exponential amplification cycle (CT) was calculated by system software. The following primer sequences were used: human SLC7A11: 5’-CTGAGGAGCTGCTGCTTTCAAA-3’ and 5’-AGGAGAGGGCAACAAAGATCGGAA-3’; and human GAPDH: 5’-ACCAAATCCGTTGACTCCGACCTT-3’ and 5’-TTCGACAGTCAGCCGCATCTTCTT-3’.

Western Blot Analysis

Total protein extraction and Western blot analysis were performed as previously described.(9) The following primary antibodies were used: polyclonal goat anti-xCT (GTX89082; GeneTex), and rabbit monoclonal anti-active caspase-3 (ab32042; Abcam). Immunoreactivity was detected with a polyclonal rabbit-anti goat horseradish peroxide (HRP)-conjugated secondary antibody, and a polyclonal rabbit-anti goat HRP-conjugated secondary antibody, respectively. Mouse β-actin (A1978; Sigma-Aldrich) was used as a loading control and detected with polyclonal goat- anti mouse HRP- conjugated secondary antibody (Cat. 1706516; BioRad).

Immunofluorescence Microscopy

Cells were fixed with 4% paraformaldehyde and stained with a rabbit polyclonal antibody to xCT (NB-300–318; Novus Biologics). Immune complexes were detected with an AlexaFlour-488 conjugated secondary antibody (Molecular Probes). Nuclei were counterstained with DAPI (Vectastain). Images were acquired with a LSM 510 Meta Inverted 2-photon confocal microscope.

Production of shSLC7A11 and SLC7A11 U251 Glioma Cell Lines

Lentivirus particles were produced by transfection of HEK 293T cells with either 15 μg of human TRC-pLKO.1-SLC7A11 shRNA (TRCN0000043123, TRCN0000043125, TRCN0000043126, TRCN0000288865, or TRCN0000380471), 15 μg of pLK01-non-targeting shRNA (Mission shRNA, Sigma-Aldrich) or 15 μg of a human SLC7A11-pLX304 plasmid (DNASU Plasmid Repository) using calcium phosphate coprecipitation. The culture medium was replaced with fresh 10% FBS in 1xDMEM after 8 h and supernatant was collected 48 h after transfection. After determination of viral titers, U251 cells were incubated with a viral vector containing the appropriate over-expressing RNA, shRNA, or control shRNA, using a multiplicity of infection of 0.5. Blasticidin (1.0 μg/mL) (Sigma-Aldrich) or puromycin (10 μg/mL) (Sigma-Aldrich) selection was used to obtain stable recombinant SLC7A11 over-expressing and shSLC7A11 knock-down U251 cells, respectively. Parental U251 cells served as controls for SLC7A11 over-expressing cells while cells transduced with an empty vector served as controls for the SLC7A11 knock-down cells.

ROS Production

Production of intracellular ROS under basal and treatment conditions was measured using the cell-permeant 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) (Invitrogen). To evaluate the direct production of mitochondrial ROS in cells, MitoTracker Red CM-H2XRos, which is dependent on the mitochondria membrane potential (ΔΨm), was used. At the indicated time-points, cells were incubated (6% CO2, 37ºC, 30 min) with either 5μM H2DCFDA or 500 nM MitoTracker Red. Media was aspirated; cells collected with Accutase and spun down at 1200 x g for 5 min. Cells were then resuspended in flow buffer (1% FBS in PBS) and analyzed using a BD Accuri C6 Flow Cytometer.

Glutamate Measurement

Glutamate was measured in media samples using the BioProfile 100 Plus (Nova Biomedical). SLC7A11 modified and control U251 cells were cultured in glutamate free DMEM for 24 h. After culture, 600 µL of media was removed from each sample dish and analyzed according to the manufacturer’s instructions.

Assessment of Mitochondria Function

Mitochondrial function was examined by staining with the mitochondrial membrane potential (ΔΨm)-sensitive fluorochrome MitoTracker Red CMXRos. Cells (200,000 cells/well) were plated (12-well plate), cultured overnight, and then incubated (60 minutes, 37ºC) with 500 nM of MitoTracker Red CMXRos. After two washes with PBS, cells were fixed with methanol:acetone (3:1) for 10 min. Cells were washed twice in PBS, mounted in Dako Fluorescent Mounting Medium and imaged on a LSM 510 Meta Inverted 2-photon confocal microscope.

Measurement of Apoptosis

Apoptosis ratios were analyzed using the Alexa Fluor 488 AnnexinV/Dead Cell Apoptosis kit (Invitrogen) according to the manufacturer’s instructions. Samples were analyzed on a BD Accuri C6 Cytometer, and Annexin V−/ PI− cells were used as unstained controls.

Quantification of Total Cellular ATP

To measure intracellular ATP, cells were lysed in buffer (200 mM Tris, 2 mM EDTA, 150 mM NaCl, 0.5% Triton X-100) and CellTiter-Glo Luminescence Viability Assay (Promega) was performed according to the manufacturer’s protocol. An ATP standard curve was generated by serial dilutions of a 1 mg ATP stock (Sigma Aldrich). Luminescence measured using a SpectraMax M3 (Molecular Devices).

Glutathione Measurement

At the indicated time-points, cells were lysed with 200 μl of MES buffer (0.4 M 2-(N-morpholino) ethanesulphonic acid, 0.1 M phosphate, 2 mM EDTA, pH 6.0) and sonicated. Protein concentrations were quantified using the BCA Protein Assay (Thermo Scientific). A Glutathione Assay Kit (Caymen Chemical) was used to quantify total GSH and glutathione disulfide (GSSG) according to manufacturer’s protocol. Absorbance was measured at 405 nm using a SpectraMax M3.

Cell Viability and Proliferation Assays

Cell counting kit-8 (CCK-8; Dojindo Molecular Technologies) was used to measure cell viability according to the manufacturer’s protocol. Absorbance was measured at 450 nm using a SpectraMax M3. A colorimetric immunoassay (Roche Diagnostics) was used according to the manufacturer’s protocol to quantify cell proliferation based on the measurement of BrdU incorporation during DNA synthesis. Absorbance was measured at 370 nm using a SpectraMax M3. Cell numbers at each time-point were determined by flow cytometry (Guava EasyCyte, Millipore) using Guava Viacount (Millipore).

Soft Agar Assay for Anchorage-Independent Cell Growth

Anchorage-independent growth was determined by seeding 2.5 × 104 cells per 12 well in 0.35% agar on top of a base layer containing 0.4% agar. Plates were incubated at 37 °C at 5% CO2 in a humidified incubator for 30 days and stained with 0.005% crystal violet for 1 h. Colonies >0.1 mm in diameter were counted under a microscopic field at ×10 magnifications.

Transmission Electron Microscopy

Cultured cells were pelleted and cryo-fixed in a Leica EM PACT2 high pressure freezer (~2,000 bars). In a Leica automated freeze substitution system AFS2, cryo-fixed specimens were freeze-substituted in anhydrous acetone containing 2% osmium tetroxide. The temperature progression was 8 h at −90°C, −90°C to −60°C at 5°C/h, −60°C for 16 h, −60°C to 0°C at 5°C/h. Cells were held at 0°C until time for further processing, when they are warmed to room temperature, rinsed in pure acetone, infiltrated and embedded in Epon812 at 60°C for 48 hours. Ultra-thin sections (~70 nm thick) were cut using a Leica Ultra cut UCT ultramicrotome with a diamond knife, picked up on 200 mesh nickel EM grids. For morphology, grids were stained with 2% uranyl acetate in 70% ethanol for 1 minute followed Reynold’s lead citrate staining for 1 minute. Electron microscopy was done on an FEI Tecnai 12 transmission electron microscope equipped with a CCD camera.

Oxygen Consumption and Extracellular Acidification Rate

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was determined using XF24 Extracellular Flux analyzer (Seahorse Bioscience, North Billerica, MA). Briefly, cells (20,000/well) were plated into XF24 polystyrene cell culture plates (Seahorse Bioscience) and incubated for 24 hours in a humidified 37°C incubator with 10% CO2 (DMEM medium with 10%FBS). The following day, cells were washed, fresh assay media (2mM L-Glutamine + 2mM Pyruvate + 25mM Glucose in XF Base Media; pH to ~7.35) was added and cells incubated in a 37°C/non-CO2 incubator for 60 minutes prior to the start of an assay. Sensor cartridges were calibrated and loaded to dispense three metabolic inhibitors sequentially at specific timepoints: oligomycin (inhibitor of ATP synthase, 8uM/port), followed by FCCP (a protonophore and uncoupler of mitochondrial oxidative phosphorylation, 18uM/port), followed by the addition of rotenone (mitochondrial complex I inhibitor, 25uM/port). Basal OCR and ECAR were measured, as well as changes in oxygen consumption caused by the addition of the metabolic inhibitors described above. Cells were treated with trypsin and then counted to determine the cell number in each well after the assay. OCR and ECAR were reported as normalized rates (pmoles/cell for OCR and mpH/cell for ECAR) or expressed as a percentage of the baseline oxygen consumption. Each datum was determined minimally in replicates of five. Several parameters were deducted from the changes in oxygen consumption including ATP turnover, proton leak, maximal respiratory capacity, and mitochondrial reserve capacity (= [maximum mitochondrial capacity] – [basal OCR]).

RNA-Sequence Data Generation and Analysis

Sequencing libraries were prepared with TruSeq RNA Sample Preparation Kit V2 (Illumina, San Diego) according to the manufacturer’s protocol with minor modifications. Briefly, 500 ng of total RNA from each sample was used for polyadenylated RNA enrichment with oligo dT magnetic beads, and the poly(A) RNA was fragmented with divalent cations under elevated temperature. First-strand cDNA synthesis produced single-stranded DNA copies from the fragmented RNA by reverse transcription. After second-strand cDNA synthesis, the double-stranded DNA underwent end repair, and the 3′ ends were adenylated. Finally, universal adapters were ligated to the cDNA fragments, and 10 cycles of PCR were performed to produce the final sequencing library. Library templates were prepared for sequencing using cBot cluster generation system (Illumina) with TruSeq SR Cluster V3 Kit. Sequencing run was performed in single read mode of 51 cycle of read1 and 7 cycles of index read using Illumina HiSeq 2500 platform with TruSeq SBS V3 Kits. Real-time analysis (RTA) software was used to process the image analysis and base calling. Sequencing runs generated approximately 40 million single reads for each sample. The refSeq annotation for the hg19 version of the human genome was used to create a transcriptome Bowtie (20) index (version 0.12.7), to which reads were aligned with the following settings: “-v 3 -a”. Gene expression levels were estimated using eXpress (21) (version 1.4.1), and the effective count values were used as input to DESeq (22) for evaluating differential expression.

Statistical Analysis

Experiments were performed in at least triplicate and repeated at least three independent times. Statistical significance was determined by difference of means between two groups and was calculated using Student’s t test. All reported p-values were two-sided; P<0.05 was considered significant.

Results

SLC7A11/xCT is Up-regulated in Glioma Cell Lines and Inhibition Induces ROS-Mediated Cell Death

The expression of SLC7A11 and xCT in human astrocytoma cell lines was assessed by qRT-PCR and Western blot analysis, respectively. Both the U87 and U251 established astrocytoma cell lines had greater SLC7A11 gene (Fig. 1A) and xCT protein expression (Fig. 1B) as compared to primary normal human astrocytes (pNHAs). Protein expression was also retained when U87 and U251 cells or a primary high grade glioma line, PBT017, were orthotopically transplanted into NOD-scid mice (Fig. 1C).

Fig. 1.

Fig. 1.

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SLC7A11/xCT is up-regulated in glioma cell lines in which inhibition of system xc− modulates cell survival and proliferation. (A) Real-time quantitative RT-PCR analysis of SLC7A11 expression in human U87 and U251 glioma cell lines compared to primary normal human astrocytes (pNHA). (B) Immunoblot analysis of xCT and β-actin (loading control) expression in U87 and U251 glioma cells compared to pNHAs. (C) Immunohistochemical staining for xCT in the orthotopic tumors; positive xCT staining indicated by arrows. Scale bar, 200 μm. (D) Proliferation of U251 cells treated with increasing doses of sulfasalazine (SSZ) for 72 h. (E) Intracellular ROS levels in U251 cells treated with or without (control) 500 μM SSZ for 72 h. Error bars indicate SD; *, P<0.05; **, P<0.01; ***, P<0.001. (F) Combination indices (CI) of U251 glioma cells treated with SSZ (50, 100, 200, 400, 800 or 1600 μM) and increasing doses of TMZ (12.5, 25, 50, 100, 200, 400, 800 μM) to determine drug interactions. CI<1, +1, and >1 indicate synergism, additive effect, and antagonism, respectively.

To determine if system xc− transport activity is necessary for the survival of glioma cells, we treated U251 glioma cells with increasing doses of the pharmacologic inhibitor SSZ. BrdU incorporation, which is a relative measure of cellular proliferation, showed a dose-dependent decrease with higher SSZ doses (1000 μM and 1500 μM) at multiple time points (Suppl. Fig 1A). After 72 h of SSZ treatment, there was a dose-dependent decrease in U251 cell numbers (Fig. 1D). A majority of the cells were lost at the higher doses for the cell count assay; therefore we chose to use 500 μM SSZ for subsequent assays. To determine whether the decrease in cell growth was due to apoptosis, Annexin V-PI staining was performed. Treatment of U251 cells with 500 μM SSZ resulted in a large increase in apoptotic cells (Suppl. Fig 1B). We also reasoned that inhibiting system xc− transport activity should result in decreased amounts of intracellular antioxidants (e.g., cysteine and GSH), and a subsequent increase in pro-oxidants. Intracellular ROS levels were measured by DCF staining to determine if there was a more pro-oxidant state that could contribute to the increased cell death observed after SSZ treatment. Indeed, treatment of the glioma cells with 500 μM SSZ resulted in significantly greater amounts of intracellular ROS compared to non-treated cells (Fig. 1E).

Synergistic Cytotoxicity in Glioma between the Chemotherapeutic Agent Temozolomide and an Inhibitor of System Xc−

We evaluated whether inhibition of system xc− could change the chemosensitivity or chemoresistance of U251 cells to TMZ, the standard chemotherapeutic agent used to treat newly diagnosed GBM patients. We first examined whether pharmacologically inhibiting system xc− with SSZ would increase the toxicity of TMZ. Parental U251 cells were treated for 3 days with increasing doses of SSZ (50, 100, 200, 400, 800, or 1600 μM) and increasing doses of TMZ (12.5, 25, 50, 100, 200, 400, or 800 μM). A combination index (CI)-isobologram equation was used to quantitatively determine drug interactions, where CI<1 indicates synergism, 1 indicates an additive effect, and >1 indicates antagonism (23). A synergistic effect was seen with all doses of TMZ and 50 μM of SSZ as well as other lower doses of both drugs (Fig. 1F), indicating that inhibition of system xc− may sensitize glioma cells to chemotherapy. SSZ doses at 800 μM or higher showed additive or antagonistic effects.

SLC7A11 Over-expression Confers Resistance to Oxidative Stress

To gain insight in the function of SLC7A11 in human glioma, we established stable U251 over-expressing and knock-down lines using lentiviral vector-mediated gene transfer and RNA silencing technology. The SLC7A11 over-expressing cells expressed 10-fold more SLC7A11 mRNA than did control cells (Fig. 2A), and this was accompanied by greater xCT protein expression, as shown by Western blot analysis (Suppl. Fig. 2A) and immunocytochemistry (Fig. 2B). Five different shRNA clones to target different regions of SLC7A11 were tested to obtain the most efficient knock-down compared to empty vector control. Because the shSLC7A11_ TRCN0000043126 construct suppressed gene and total protein expression the most in the transduced U251 cells (Suppl. Fig. 2B), which was confirmed by immunocytochemistry (data not shown), we chose it for further analysis. The SLC7A11-knock-down cell expressed significantly less SLC7A11 mRNA than did the cells transduced with an empty vector (Fig. 2A) Knock-down of xCT (SLC7A11) protein expression was confirmed by immunocytochemistry (Fig. 2B).

Fig. 2.

Fig. 2.

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Viability and proliferation are not affected in stable SLC7A11-knock-down and over-expressing U251 glioma cells. (A) Quantification of xCT (SLC7A11) mRNA in U251 glioma transduced with viral vector containing SLC7A11 over-expression construct or viral vector containing shSLC7A11 (TRCN0000043126). (B) Confocal microscopy images of SLC7A11 over-expression and knock-down U251 cell lines stained for xCT protein (green). Cells were counterstained with DAPI (blue) to visualize nuclei. Scale bars, 50 μm. (C) Amount of glutamate released into the media by SLC7A11 over-expressing and knock-down U251 cell lines relative to control. (D) Quantification of anchorage-independent cell growth in SLC7A11-modified glioma cells compared to controls. (E) Proliferation of SLC7A11-modified glioma cells at indicated time-points as measured by BrdU incorporation. (F) Viability of SLC7A11-modified glioma cells measured by CCK-8 assay at indicated time-points. Data shown are means from a representative experiment. Error bars indicate SD; *, P<0.05; **, P<0.01; ***, P<0.001.

To assess whether modification of SLC7A11 expression affects system xc− transport activity, we measured glutamate release into the media. SLC7A11 over-expressing cells released significantly more glutamate compared to control cells, while SLC7A11-knock-down cells released significantly less, indicating the cystine influx: glutamate efflux transporter was functional (Fig. 2C). We found that enhancement or suppression of SLC7A11 expression in established cell lines did not influence cell proliferation (Fig. 2E) or cell viability (Fig. 2F). However, a soft agar assay revealed that the SLC7A11 over-expressing cells had higher anchorage-independent cell growth while the SLC7A11 knock-down cells had lower anchorage-independent cell growth, indicating that the SLC7A11 over-expressing cells may be more tumorigenic (Fig. 2D).

Because system xc−, or more specifically its catalytic subunit xCT, plays an important role in regulating GSH levels, we evaluated intracellular GSH and ROS in the SLC7A11-modified U251 lines. Under basal conditions, the SLC7A11 knock-down cells had significantly lower intracellular GSH levels as compared to their respective controls (Fig. 3A), and this correlated with significantly higher intracellular ROS levels (Fig. 3B). Although the SLC7A11 over-expressing U251 cells did not have higher total intracellular GSH levels than the control, despite increased xCT expression, they did have significantly lower intracellular ROS levels under basal conditions (Fig. 3B). However, under oxidative stress conditions, mimicked by treatment with 100 μM H2O2, the SLC7A11 over-expressing cells had significantly increased intracellular GSH levels as compared to under basal conditions and to control cells treated with H2O2 (Fig. 3C). Consistent with increased GSH levels, H2O2-treated SLC7A11 over-expressing cells showed markedly less H2O2-induced intracellular ROS (Suppl. Fig. 3) and less H2O2-induced apoptosis compared to control cells (Fig. 3D).

Fig. 3.

Fig. 3.

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SLC7A11 over-expressing U251 glioma cells are more resistant to oxidative stress. (A) Relative intracellular glutathione (GSH) measured in SLC7A11-modified U251s under normal culture conditions. (B) Relative intracellular ROS measured in SLC7A11-modified U251s under normal culture conditions. (C) Relative intracellular GSH measured in SLC7A11 over-expressing U251 glioma after 6 h treatment with 100μM H2O2. (D) Flow cytometry analysis of Annexin V/propidium iodide staining of SLC7A11 over-expressing and control glioma after 6 h treatment with 100 μM H2O2. Data shown in (A), (B) and (C) are means from a representative experiment. Error bars indicate SD; *, P<0.05; **, P<0.01; ***, P<0.001.

SLC7A11 Over-expression Confers Resistance to Genotoxic Stress Induced by Temozolimide

Various types of cancer exhibit elevated GSH levels, which makes these cancers more resistant to chemotherapy (6). The combinatorial drug studies indicated that inhibition of system xc− with SSZ could increase cell killing with TMZ under low doses. We next examined whether genetic manipulation of SLC7A11 could modulate chemosensitivity to TMZ. The SLC7A11 over-expressing cells had greater viability than did control cells treated with increasing doses of TMZ, while the SLC7A11 knock-down cells were less viable than control cells (Fig. 4A). Consistent with this, the SLC7A11 over-expressing cells had a 6-fold higher IC50 value for TMZ (419 µM) as compared to control cells (64 µM). The increased resistance to TMZ upon SLC7A11 over-expression was further confirmed by higher cell proliferation rates compared to the control cells (Fig. 4 B). In addition, the SLC7A11 over-expressing cells exhibited much less TMZ-induced apoptosis when treated than did control cells, while the SLC7A11 knock-down cells showed a large increase in the percent of apoptotic cells (Suppl. Fig. 4A). To further test whether SLC7A11 confers resistance to TMZ by inhibiting apoptosis, we analyzed expression of cleaved caspase-3, a marker of apoptotic cells, by Western blot analysis. Expression of cleaved caspase-3 was markedly greater in all TMZ-treated cell lines, but was lower in the SLC7A11 over-expressing cells as compared to the SLC7A11 knock-down cells (Fig. 4C). To determine whether increased GSH levels contributed to the increased chemoresistance of the SLC7A11 over-expressing cells, we measured intracellular GSH levels cells exposed to 300 μm TMZ. Consistent with the effect of oxidative stress on increasing GSH levels (Fig. 3C) in SLC7A11 over-expressing cells, the genotoxic stress induced by TMZ resulted in a significant increase in intracellular GSH levels (Fig. 4D). TMZ treatment of the SLC7A11 knock-down cells did not alter GSH levels as compared to untreated cells (Suppl. Fig. 4B).

Fig. 4.

Fig. 4.

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SLC7A11 over-expression in U251 glioma conferred increased resistance to TMZ. (A) Viability of SLC7A11 over-expressing and knock-down U251 cells treated for 72 h with increasing doses of TMZ (IC50 values are indicated). (B) Proliferation of SLC7A11 over-expressing and knock-down U251 cells treated for 72 h with increasing doses of TMZ compared to respective controls. (C) Immunoblot analysis of cleaved caspase-3 and β-actin (loading control) in SLC7A11-modified U251 and respective control cells after treatment with 300 μM TMZ. (D) Relative intracellular GSH in SLC7A11 over-expressing U251 cells after treatment with 300 μM TMZ for 72 h. Data shown are means from a representative experiment. Error bars indicate SD; *, P<0.05; **, P<0.01; ***, P<0.001.

Differential Gene Expression in SLC7A11 Over-expressing and Knock-down Glioma Cells

RNA-sequence analysis was performed to assess gene expression changes upon SLC7A11 over-expression and knock-down in U251 glioma. Differential expression analysis revealed over 4,000 differential genes between SLC7A11 over-expression and respective control cells (Fig. 5A and Fig. 5B), and over 3,000 differential genes when SLC7A11 is knocked down in U251 cells (Fig. 5A and Suppl. Fig. 5A). Gene Ontology (GO) enrichment analysis shows the number of GO terms that are over-represented (or under-represented) in the SLC7A11 over-expressing glioma (Fig. 5C) and in the SLC7A11 knock-down glioma compared to respective controls (Suppl. Fig. 5B), with a complete list of enriched categories included in the Supplementary Files. Representative GO terms indicate that several genes involved in mitochondrial biogenesis and mitochondrial function are up-regulated in the SLC7A11 over-expressing glioma (Figure 5C), suggesting that SLC7A11 over-expressing U251 cells may have altered metabolic function.

Fig. 5.

Fig. 5.

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Gene expression changes upon SLC7A11 over-expression and knock-down in U251 cells. (A) Global similarity of gene expression profiles between SLC7A11 over-expression (OE), knock-down (KD) and matched control samples. Differential expression analysis was carried out on biological replicate samples for all pairs of conditions using DESeq (22); the heatmap shows the number of differential genes, which was used as a similarity metric between conditions. (B) Differentially expressed genes between SLC7A11 over-expressing and control U251 cells. (C) Representative Gene Ontology (GO) terms enriched in genes up-regulated upon SLC7A11 over-expression in U251 cells. GO enrichment analysis was carried out using FuncAssociate (37). Complete lists of enriched categories are provided in the Supplementary Data.

SLC7A11 Over-expressing Cells have Increased Mitochondrial Biogenesis

Morphological examination of mitochondria was performed utilizing a transmission electron microscope (TEM). The electron micrographs revealed relatively well-preserved mitochondria in the SLC7A11 modified and control cells (Fig. 6A). Magnification of the mitochondria (right panel of each set) shows typical double membranes, the intermembrane space, cristae, and a matrix. Quantification of the mitochondria in the cell lines reveals that the SLC7A11 over-expressing glioma has significantly higher number of mitochondria present compared to control cells (Fig. 6B), indicating that there is increased mitochondrial biogenesis. To further confirm increased mitochondria number and assess mitochondrial function, cells were stained with the mitochondrial membrane potential ΔΨm-sensitive fluorochrome, MitoTracker Red CMXRos. Fluorescent microscopic examination revealed a stronger MitoTracker Red CMXRos stain in the SLC7A11 over-expressing glioma (Fig. 6C). Analysis of the mean fluorescence intensity showed significantly higher MitoTracker Red CMXRos stain in the SLC7A11 over-expressing glioma compared to control cells (Fig. 6D). The increased accumulation and retention of the probe indicates higher mitochondrial membrane potential (ΔΨm) in the SLC7A11 over-expressing glioma.

Fig. 6.

Fig. 6.

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SLC7A11 over-expressing U251 glioma has increased mitochondrial biogenesis and increased mitochondrial membrane potential. (A) Transmission electron micrographs of SLC7A11-modified glioma cells and control cells. Representative mitochondria are indicated by a red arrow. Scale bars: 2 um (left micrograph) and 0.5 um (right micrograph) of each set. (B) Quantification of mitochondria per field of view (FOV) in SLC7A11 modified lines compared to their respective controls. (C) Fluorescent microscopic examination of the ΔΨm-dependent uptake and retention of MitoTracker Red CMXRos into mitochondria in SLC7A11 over-expressing glioma compared to parental control. Scale bars: 100 µm. (D) Quantification of the mean fluorescent intensity (MFI) per field of view (FOV) of MitoTracker Red CMXRos staining in the SLC7A11 over-expressing glioma compared to control cells. Data shown in (B) and (D) are means from a representative experiment. Error bars indicate SD; *, P<0.05; **, P<0.01.

SLC7A11 Over-expressing Cells have Increased Oxidative Phosphorylation

To assess cellular bioenergetics in the SLC7A11 over-expressing U251 cells, extracellular flux analysis was used to determine the oxygen consumption rate (OCR) which is a measurement of mitochondrial respiration. Basal respiration was significantly higher in the SLC7A11 over-expressing U251 glioma compared to control cells (Fig. 7A). Basal respiration is usually controlled by ATP turnover and only partly by substrate oxidation and proton leak (24). Therefore, we next examined ATP turnover by inhibiting ATP synthase using oligomycin. Treatment with oligomycin revealed that the SLC7A11 over-expressing cells had higher ATP-linked respiration (oligomycin-sensitive fraction) compared to control cells (Fig. 7B). The maximal respiratory capacity was measured in the presence of carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCC), an uncoupler that causes dissipation of the proton gradient by carrying protons across the inner mitochondrial membrane (17). FCCP causes rapid depolarization of mitochondria and acceleration of electron flux through the electron transport chain which resulted in significantly higher mitochondrial oxidative capacity in the SLC7A11 over-expressing glioma compared to parental U251 cells (Fig. 7C). The spare respiratory capacity is the ability of substrate supply and electron transport to respond to an increase in energy demand, and, therefore, maintenance of some spare respiratory capacity is a major factor defining cell survival (24). Although not statistically significant, the SLC7A11 over-expressing U251 glioma had increased reserve capacity (p=0.058) compared to the control cells (Fig. 7D). Together these data suggest that the SLC7A11 over-expressing glioma, which display higher basal OCR, ATP-linked respiration, mitochondrial oxidative capacity and spare respiratory capacity, have higher oxidative phosphorylation. Since mitochondria are one of the main producers of ROS and the primary producers of ATP, we performed analysis of ATP generation in the SLC7A11 over-expressing cells. Consistent with the OCR data, SLC7A11 over-expressing cells generate more ATP compared to control cells (Fig. 7E). Despite the increased ATP generation, the SLC7A11 over-expressing cells had lower mitochondrial ROS compared to the SLC7A11 knock-down cells or the control cells (Fig. 7F).

Fig. 7.

Fig. 7.

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Oxygen Consumption Rates (OCR) and respiration parameters in SLC7A11 over-expressing U251 glioma cells compared to parental U251 cells. (A) Baseline Respiratory Capacity (B) ATP-linked Respiration and (C) Maximal Respiratory Capacity and (D) Reserve Capacity calculated for the SLC7A11 over-expressing glioma compared to parental U251 cells. (E) Intracellular ATP normalized to cell number in SLC7A11 over-expressing glioma cells compared to control cells. (F) Mitochondrial ROS measured in SLC7A11-modified glioma cells by the MitoTracker Red CM-H2XRos dye which stains actively respiring cell, accumulates in the mitochondria and is dependent on the mitochondria membrane potential. Error bars indicate SD; *, P<0.05; **, P<0.01; ***, P<0.001.

Discussion

It has been suggested that system xc− may be a promising cancer target since it may sensitize tumors to conventional chemo/radiation-based therapies by lowering GSH levels (25). One of the causes of post-operative radiation and chemotherapy treatment failure in GBM patients is an increase in GSH levels that may decrease chemotherapy-associated oxidative stress and play a role in glutathione-mediated cellular detoxification pathways. In this work, we have expanded on the role system xc− plays in TMZ resistance in human glioma and have identified a novel mechanism that may contribute to glioma progression, via metabolic alteration. Inhibitors of system xc−, such as SSZ, have been put forward as possible effective therapeutic options. SSZ has been shown to deplete GSH levels by inhibiting the uptake of cystine, which lowers levels of intracellular cysteine, the rate-limiting precursor for GSH synthesis (8).

Supporting use of system xc− inhibitors, we show that SSZ treatment of U251 glioma resulted in ROS-mediated cell death. In addition, SSZ treatment in combination with TMZ treatment had a synergistic killing effect in U251 glioma cells. By inhibiting system xc− using SSZ, the efficacy of TMZ was increased at lower doses in vitro. Other pharmacological inhibitors of xCT, such as erastin, have been shown to sensitize glioma cells to TMZ (26). This suggests that use of specific system xc− inhibitors could have promise for avoiding or greatly reducing TMZ-associated toxicity in GBM patients. However, other factors contributing to the decreased cell growth and viability cannot be ruled out since SSZ has been noted to target several other pathways (27). SSZ has several immunomodulatory effects, including inhibition of NF-κB, and inhibition of GSH S-transferase, which is responsible for conjugating GSH to xenobiotics for detoxification (28). These off-target effects limit the conclusions drawn from SSZ inhibition studies of system xc− in glioma cells. Clinical trials studying the effects of SSZ for the treatment of malignant glioma in adults were terminated due to adverse events and toxicity (29).

We circumvented the problems associated with SSZ inhibition of system xc− by generating stable SLC7A11 knock-down and over-expressing U251 cell lines in order to better understand the role system xc− plays in glioma progression. In contrast to reports that inhibition of system xc− with SSZ led to decreased growth and cell cycle progression in glioma (8), we did not observe any changes in proliferation in the SLC7A11-modified lines. One possibility is that system xc− does not directly modulate cell growth and previous reports of transporter inhibition did not take into account the off-target effects associated with SSZ use. In addition, it is possible that the SLC7A11 knock-down cells are able to take up enough cystine to support proliferation, whereas a complete SLC7A11 knock-out would decrease glioma viability and growth. Although proliferation of the SLC7A11 knock-down cells was not affected, their cystine uptake was impaired as evidenced by the significant decrease in glutamate release and GSH generation, and significant increase in intracellular ROS. In terms of resistance to oxidative stress, over-expression of SLC7A11 conferred increased resistance to apoptosis, which we attributed to a significant increase in GSH generation. This suggests that the primary role of system xc− is in redox regulation rather than protein synthesis.

Because elevated GSH levels in cancer cells have been associated with drug resistance, we hypothesized that system xc− may play a role in modulating genotoxic stress induced by TMZ. We found that over-expression of SLC7A11 reduced the sensitivity of glioma to TMZ and decreased TMZ-induced apoptosis. Disruption of system xc− function by knocking down SLC7A11 expression to low levels increased the sensitivity to TMZ, resulting in pronounced apoptosis. The increased sensitivity may be partly due to the low levels of GSH because only the SLC7A11 over-expressing cells exhibited increased GSH production after treatment with TMZ. In addition, marked activation of caspase-3 in knock-down cells confirmed that suppression of SLC7A11 facilitated TMZ-induced activation of the apoptotic pathway.

It may be that the SLC7A11 knock-down cells exhibit the so-called “threshold concept for cancer therapy,” whereby an additional increase in ROS levels by ROS-generating agents, such as chemotherapy drugs, pushes the endogenous levels of ROS past a cellular tolerability threshold (30). TMZ has also been shown to generate ROS in human glioblastoma cell lines, including superoxides, cytosolic H2O2, and mitochondrial H2O2, which could be suppressed by pretreatment with antioxidants resveratrol, vitamin C, and iron (31). Concurrent treatment of glioblastoma cell lines with TMZ and valproic acid (VPA), an anticonvulsant and mood-stabilizing drug, showed an increase in ROS and GSH depletion resulting in higher apoptosis compared to TMZ or VPA alone (32). These data suggest that redox regulation and TMZ-triggered ROS bursts can contribute to the sensitivity of glioma cells to chemotherapy. Indeed, the SLC7A11 knock-down cells exhibited a significantly greater amount of endogenous ROS under basal conditions, and TMZ treatment augmented these levels because of impaired GSH production.

Recently, it has been shown that glioma chemoresistance to TMZ is linked to tighter mitochondrial coupling and low ROS production indicating that chemoresistance is related to a remodeling of the electron transport chain (17). Since mitochondrial respiration generates ROS, and system xc− has been shown to increase the antioxidant defense mechanisms to maintain redox balance, we hypothesized that SLC7A11 levels in glioma may have an impact on mitochondrial health and function. Over-expression of SLC7A11 in U251 glioma cells resulted in increased mitochondrial biogenesis and enhanced mitochondrial functions, indicated by increased mitochondrial membrane potential (ΔΨm). This was further confirmed by increased mRNA levels of several genes involved in mitochondrial biogenesis and function. While knock-down of SLC7A11 did not impair mitochondrial respiration, over-expression of SLC7A11 significantly increased the oxygen consumption rate (OCR) which is reflective of increased OXPHOS, and was accompanied by an increase in ATP generation. However, SLC7A11 over-expressing U251 glioma generated less cytosolic and mitochondrial H2O2 compared to SLC7A11 knock-down and control cells.

These results suggest that high expression of SLC7A11 may play a role in mitochondrial biogenesis and energetics. How SLC7A11 increases OXPHOS and the molecular signaling pathway involved have yet to be determined. The ability of cells to respond to stress is influenced by the energetic capacity of mitochondria, especially under conditions of increased energy demand. One possibility is that the SLC7A11 over-expressing cells rely more on oxidative phosphorylation due to an increased ATP demand. The generation of GSH relies on two ATP-dependent steps: synthesis of γ-glutamylcysteine and then subsequent addition of cysteine to the γ-glutamylcysteine. Thus, the SLC7A11 over-expressing cells may rely on higher respiratory rates for GSH production. Alternatively, it may be that the increased GSH generation in the SLC7A11 over-expressing cells prevents the accumulation of ROS-mediated defects in the mitochondria, thereby promoting enhanced mitochondrial respiration. Mitochondrial DNA has a high mutation rate and, as mtDNA mutations increase, the energy capacity of the cell declines until there is insufficient energy to sustain cellular function, indicating that the bioenergetic threshold of the cell has been reached (33). The molecular mechanisms involved and how the model of chemoresistance and mitochondria metabolism can be integrated warrants further examination. Whether other cancer cells that over-express SLC7A11 have increased mitochondrial respiration and whether OXPHOS is dependent on system xc− remains to be explored. However, our observations provide a potential new link between system xc−, redox balance, and OXPHOS to promote glioma progression and survival.

In conclusion, we have shown that over-expression of system xc− in glioma confers a survival advantage suggesting worse prognosis in GBM patients. Indeed, strong xCT/SLC7A11 expression in GBM patients correlated with an infiltrative phenotype on MRI and has been shown to be significantly associated with shorter progression-free and overall survival (34, 35). Microarray gene analysis of 60 human cancer cell lines used by the National Cancer Institute for drug screening (NCI-60) showed that SLC7A11 expression was negatively correlated with sensitivity of tumor cells to anti-cancer drugs (4). Additionally, cytotoxic drugs have been shown to activate SLC7A11 expression in various cancer cells (36). Our data shows that over-expression of SLC7A11 not only promoted resistance to oxidative stress, but also implicated system xc− in TMZ resistance and altered metabolism in GBM. In U251 cells, over-expression of SLC7A11 promoted increased GSH production under oxidative stress and genotoxic stress. The GSH neutralized intracellular ROS and increased the survival of the cells once stressed. Over-expression of SLC7A11 was also correlated with an increased mitochondrial metabolism, which may contribute to the increased chemoresistance in these cells to TMZ. Additional studies are warranted to explore the molecular mechanisms involved in the SLC7A11-mediated drug resistance and determine whether SLC7A11 may be a therapeutic target. Therefore, further investigation of manipulating the activity of this transporter either alone or in combination with other treatment modalities may lead to improved therapies and clinical outcomes of patients with GBM.

Supplementary Material

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Acknowledgements

We acknowledge the technical support of the City of Hope RNAi Core (Dr. Claudia M. Kowolik), the Light Microscopy Digital Imaging Core (Dr. Brian Armstrong and Tina Patel), Megan Gilchrist for staining the glioma orthotopic xenograft sections, and Dr. Keely L. Walker for critical reading and editing of the article.

Funding:

This work was supported by funding from the California Institute of Regenerative Medicine (TG2-01150), the Rosalinde and Arthur Gilbert Foundation, STOP Cancer, and the Cancer Center Support Grant (P30CA033572)

Footnotes

Conflict of Interest: K.S.A. is a shareholder, director, and officer of TheraBiologics Inc., a clinical-stage biopharmaceutical company focused on the development of stem cell-mediated cancer therapies.

Contributor Information

Monika D. Polewski, Department of Neurosciences, City of Hope National Medical Center and Beckman Research Institute, Duarte, California, 91010, USA Irell and Manella Graduate School of Biological Sciences, City of Hope National Medical Center and Beckman Research Institute, Duarte, California, 91010, USA.

Rosyli F. Reveron-Thornton, Department of Neurosciences, City of Hope National Medical Center and Beckman Research Institute, Duarte, California, 91010, USA Department of Biological Sciences, California State University, San Bernardino, California, 92407, USA.

Gregory A. Cherryholmes, Department of Cancer Immunotherapeutics and Tumor Immunology, City of Hope National Medical Center and Beckman Research Institute, Duarte, California, 91010, USA Irell and Manella Graduate School of Biological Sciences, City of Hope National Medical Center and Beckman Research Institute, Duarte, California, 91010, USA.

Georgi K. Marinov, Division of Biology, California Institute of Technology, Pasadena, California, 91125, USA

Kaniel Cassady, Departments of Diabetes Research and Hematology/Hematopoietic Cell Transplantation, City of Hope National Medical Center and Beckman Research Institute, Duarte, California, 91010, USA; Irell and Manella Graduate School of Biological Sciences, City of Hope National Medical Center and Beckman Research Institute, Duarte, California, 91010, USA.

Karen S. Aboody, Department of Neurosciences, City of Hope National Medical Center and Beckman Research Institute, Duarte, California, 91010, USA Division of Neurosurgery, City of Hope National Medical Center and Beckman Research Institute, Duarte, California, 91010, USA.

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Supplementary Materials

1
NIHMS817257-supplement-1.pdf (62.9KB, pdf)
2
NIHMS817257-supplement-2.pdf (357.7KB, pdf)
3
NIHMS817257-supplement-3.pdf (72.9KB, pdf)

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