Glutathione peroxidase 4 maintains a stemness phenotype, oxidative homeostasis and regulates biological processes in Panc‑1 cancer stem‑like cells

  • Authors:
    • Guiqin Peng
    • Zongwei Tang
    • Yongjia Xiang
    • Wanyi Chen
  • View Affiliations

  • Published online on: December 6, 2018     https://doi.org/10.3892/or.2018.6905
  • Pages: 1264-1274
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Abstract

Reactive oxygen species (ROS) have been widely accepted as critical molecules playing regulatory roles in various biological processes, including proliferation, differentiation and apoptotic/ferroptotic/necrotic cell death. Emerging evidence suggests that ROS may be involved in the induction of epithelial‑to‑mesenchymal transition (EMT), which has been reported to promote cancer stem‑like cell (CSC) generation. Recent data indicate that altered accumulation of ROS is associated with CSC generation, EMT and hypoxia exposure, but the underlying mechanisms are poorly understood. In the present study, we derived CSCs from Panc‑1 human pancreatic cancer cells and characterized them using serial replating assays and western blot analysis. Functional identification of viable cells was performed using the CCK‑8 assay and colony formation assays. The expression of various antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GPX), was measured by western blot analysis in Panc‑1 CSCs. The role of GPX4 in regulating biological processes of Panc‑1 CSCs was assessed by proliferation, sphere formation and invasion assays with or without oxidative stress. Manipulation of GPX4 expression by siRNA knockdown or an overexpression vector was performed to assess functions including proliferation, colony formation and invasion. EMT hallmark genes were detected after GPX4 alteration by RT‑qPCR and western blot analysis. Panc‑1 CSCs displayed more resistance to hypoxia exposure. Compared with the parental Panc‑1 cells, Panc‑1 CSCs expressed an obviously higher endogenous GPX4 level, indicating their role in maintaining homeostasis. During GPX4 knockdown, ROS accumulation was promoted following oxidative stress exposure to either H2O2 or erastin. Additionally, overexpression of GPX4 eliminated ROS induction by oxidative stress exposure and thus, exerted protective effects on physiological processes in the Panc‑1 CSCs. Knockdown of GPX4 arrested cell cycle progression at the G1/G0 phase; inhibited cell proliferation, colony formation, invasion and the stemness phenotype in the Panc‑1 CSCs; and decreased the EMT phenotype. Collectively, GPX4 plays a critical role in maintaining oxidative homeostasis and regulates several biological processes, including stemness and EMT, in Panc‑1 CSCs.

Introduction

Reactive oxygen species (ROS), which mainly encompass oxygen molecules containing one or more unpaired, unstable electrons, are important cellular signalling molecules involved in a variety of biological processes, including cell proliferation, differentiation and apoptosis (1). Both endogenous sources (such as mitochondria and peroxisomes) and exogenous sources (such as environmental agents, pharmaceuticals and irradiation) can induce ROS accumulation (2). ROS homeostasis is critical for maintaining normal physiological and biological processes. Accumulation of excess ROS, which is termed oxidative stress, can lead to irreversible injury to cells via damaged biomolecules, including DNA, resulting in genomic instability and genetic mutations that contribute to diseases such as tumourigenesis. In this case, the mechanisms for maintaining the low levels of intracellular ROS in mammalian cells are extremely important. The powerful scavenger antioxidant enzyme systems, including superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX), have been well studied. Once formed, ROS are rapidly converted by SODs to H2O2. Newly formed H2O2 is converted to H2O+O2 by GPX through coupling with reduced glutathione (GSH) and its subsequent conversion to oxidized glutathione (3). It has been reported that changes in GPX levels, especially in 5 of the 8 sub-members of the GPX family, are tightly associated with malignant phenotypes in several types of tumours (4). GPX1, GPX3 and GPX4 are reported to function as tumour suppressors in breast (5), colorectal (6), endometrial (7), pancreatic (8) and prostate (9) cancer. However, the regulatory roles that GPX family members play in tumours are still largely unknown.

Recently, the existence of cancer stem-like cells (CSCs) has been recognized (10). As a small proportion of cancer cells, CSCs exert potential roles in aggressive tumour phenotypes and malignant behaviours, such as chemoresistance, recurrence, relapse and metastasis. It is well established that, in stem cells, several signalling molecules are involved in and play critical roles in maintaining low levels of ROS, which may facilitate chemo/radiotherapy resistance and increased self-renewal capacity in stem cells (1115). In cancer cells, accumulated intracellular ROS may function as a tumour suppressor or enhancer depending on the cellular mechanisms activated (3,15). In comparison with normal stem cells or a heterogeneous population of cancer cells, the regulatory role of intracellular ROS in CSCs is still largely unknown. It has been reported that CSCs have lower intracellular ROS contents compared with a heterogeneous population of cancer cells (16), which may be due to upregulated expression of members of the free radical scavenging system, including the SODs or GPXs (17). Kim and colleagues showed that increased CD13, a CSC molecule, negatively regulates ROS, resulting in increased stemness in liver CSCs (18). This suggests an association between low levels of ROS with CSC stemness. However, the underlying mechanism for decreased ROS is not well understood in CSCs.

The EMT programme has been recognized as a major mechanism of tumour metastasis for 2 decades (19). Inactivation of E-cadherin, activation of vimentin, Slug and Snail are considered hallmarks of EMT programme activation (19). Surprisingly, it was observed that experimental activation of EMT, via either the overexpression of Twist1 or treatment with TGFβ, confers many of the characteristics of CSCs on non-CSC epithelial carcinoma cells (20,21). The association between EMT and CSCs indicates that EMT programme activation in non-CSC cells may enable their conversion into CSCs. In this case, both self-renewal capacity and an EMT phenotype are considered as CSC characteristics (22). However, little is known about whether inactivation of the EMT programme or activation of the MET programme affects biological processes in CSCs.

The established relationship between ROS and EMT (23,24), and the regulatory mechanisms between EMT and CSCs (25) suggests that maintenance of oxidative homeostasis regulates the EMT process and the stemness phenotype via balancing ROS in CSCs. Differential expression of endogenous oxidative stress scavengers may play a critical role in leading to the imbalance or rebalance of oxidative stress and the ultimate effect on biological processes.

In the present study, we demonstrated the regulatory role of GPX4 in maintaining oxidative homeostasis and thus affecting the EMT programme and stemness phenotype of Panc-1 CSCs. We describe a novel role of GPX4 in regulating biological processes in Panc-1 CSCs, which indicates a promising further in CSCs induced metastasis.

Materials and methods

Cell cultures

The human pancreatic cancer cell line, Panc-1, was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and frozen in our laboratory in liquid nitrogen and cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Paisley, UK), 100 U/ml of penicillin (Gibco; Thermo Fisher Scientific, Inc.), 100 µg/ml of streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO2 incubator at 37°C. For enriching CSCs from parental Panc-1 cells, a suspension culture supporting proliferation of undifferentiated cells was adopted (26). Briefly, Panc-1 cells were maintained in DMEM/Ham's Nutrient Mixture F-12 (F-12) (1:1) (Life Technologies; Thermo Fisher Scientific, Inc.) with the addition of epidermal growth factor (EGF, 20 ng/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), human fibroblast growth factor basic (hFGFb, 10 ng/ml; Sigma-Aldrich; Merck KGaA) and 2% B27 (Life Technologies; Thermo Fisher Scientific, Inc.) for 14–21 days. Medium was half-refreshed every 3 days until the spheres were observed.

Cell viability and apoptosis assays

Cell viability was determined by the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories Co., Ltd., Kumamoto, Japan) assay. Briefly, Panc-1 or Panc-1 cancer stem-like cells (Panc-1 CSCs) (5×104) were cultured in 96-well plates. A total of 10 µl of freshly prepared CCK-8 solution was added to the cell culture for a 2-h co-incubation. The absorbance was measured at 620 nm. The cell viability was expressed as OD (OD620).

The apoptosis of Panc-1 or Panc-1 CSCs was determined by FACS analysis of Annexin V-FITC/propidium iodide (PI; Roche, Basel, Switzerland)-double stained cells. Briefly, cells were grown in a 6-well plate in serum-free medium. After treatment, Panc-1 and Panc-1 CSCs were trypsinized, washed with phosphate-buffered saline (PBS), resuspended in 200 µl PBS with 10 µl RNAase (10 mg/ml) and incubated at 37°C for 30 min. At the end of incubation, 50 µl of Annexin V-FITC/PI labeling solution (BD Biosciences, San Jose, CA, USA) was added and cells were analyzed for apoptosis using a flow cytometry (BD FACSCanto II; BD Biosciences).

Hypoxia exposure

For hypoxia induction, cells were cultured under a hypoxic condition (1% O2, 5% CO2 and 94% N2) in a multigas incubator (MCO-19; Sanyo Scientific, Tokyo, Japan) for 6, 12 or 24 h. For normoxia incubation, cells were incubated under the condition of 20% O2, 5% CO2 and 75% N2.

Serial replating assay

For detecting self-renewal capacity of Panc-1 CSCs, CSCs were grown in 6-well ultra-low attachment plates (Corning Inc., Corning, NY, USA) at a density of 1,000 cells/ml in well-defined serum-free medium at 37°C in a humidified atmosphere of 95% air and 5% CO2. Fourteen days later, the spheres with a diameter of >40 µm were counted under an Olympus X71 (U-RFL-T) fluorescence microscope (Olympus Corp., Melville, NY, USA). Then, similarly, for secondary spheroids, the same number of CSCs from spheroids were reseeded for another 14 days. All the procedures were repeated 4 times.

siRNA transfection

Knockdown of the mRNA level of GPX4 was achieved by transient transfection of cells with siRNA duplexes (Thermo Fisher Scientific, Inc., Waltham, MA, USA), specific to the mRNA of GPX4. The relevant siRNA sequences were: GPX4 sense, 5′-UUCGAUAUGUUCAGCAAGAUU-3′ and antisense, 5′-UCUUGCUGAACAUAUCGAAUU-3′; negative control (NC) sense, 5′-GUUCAAUAUUAUCAAGCGGUU-3′ and antisense, 5′-CCGCUUGAUAAUAUUGAACUU-3′. According to the manufacturer's instructions, 5×105 cells were grown in 2 ml of serum-free medium. A siRNA/transfection reagent complex was formed at room temperature by combining siRNA oligomer (50 nM) with 5 µl (2 µg/ml) Lipofectamine™ 2000 transfection reagent (Thermo Fisher Scientific, Inc.) in 0.5 ml Opti-MEM medium (Thermo Fisher Scientific, Inc.) and this was applied to cells for 48 h until they were harvested. Cells transfected with NC siRNA were considered as the control cells. For erastin treatment, 48 h after transfection, the cells were cultured in media supplemented with 2 µM erastin or an equal amount of dimethyl sulfoxide (DMSO) (mock group) for 2, 4, 6 and 8 h, respectively. After 3 washes with PBS, the cells were harvested for further analysis.

For H2O2 treatment, 48 h after transfection, cells were cultured in media supplemented with 50, 100, 200 and 500 µM H2O2, respectively, for 24 h. After 3 washes with PBS, cells were harvested for further analysis.

Construction of the expression plasmid and transfection

The full-length complementary DNA (cDNA) of GPX4 (human GPX4 GenBank accession no. NC_000019) was obtained from RiboBio Co., Ltd. (Guangzhou, China) and ligated into the NheI-XhoI site of the pcDNA3.1 vector (Life Technologies; Thermo Fisher Scientific, Inc.). The NheI and XhoI restriction enzymes and T4 ligase were purchased from Takara Bio (Heidelberg, Germany). A total of 0.8 µg of the plasmid or empty vector was mixed with 4 µl Lipofectamine™ 2000 transfection reagent (Thermo Fisher Scientific, Inc.) in 0.5 ml Opti-MEM medium (Thermo Fisher Scientific, Inc.) and this was applied to cells for 4 h followed by medium-refresh. After 24 h, the culture medium was refreshed containing 500 µg/ml geneticin sulfate 418 (G418; Sigma-Aldrich; Merck KGaA) for antibiotic selection. Two weeks later, survival-transfected cells were collected and maintained in 250 µg/ml G418.

For the apoptosis assay, cells were pretreated with 20 µM caspase inhibitor Z-VAD-FMK (Promega Corporation, Madison, WI, USA), 1 µM Ferrostatin-1 (Ferr-1; Sigma-Aldrich; Merck KGaA), or 0.3 µM Necrostatin-1 (Nec-1; Sigma-Aldrich; Merck KGaA) for 12 h followed by co-incubation of H2O2 or erastin, respectively. Twenty-four hours later, the cells were washed 3 times with PBS and harvested for further analysis.

RT-qPCR

The mRNA expression of GPX4, E-cadherin, vimentin, Slug, Snail and the housekeeping gene β-actin was determined by RT-qPCR using primer oligomers as followed: GPX sense primer, 5′-CGATACGCTGAGTGTGGTTTGC-3′ and antisense primer, 5′-CATTTCCCAGGATGCCCTTG-3′; E-cadherin sense primer, 5′-CGAGAGCTACACGTTCACGG-3′ and antisense primer, 5′-GGGTGTCGAGGGAAAAATAGG-3′; vimentin sense primer, 5′-GACGCCATCAACACCGAGTT-3′ and antisense primer, 5′-CTTTGTCGTTGGTTAGCTGGT-3′; Slug sense primer, 5′-CGAACTGGACACACATACAGTG-3′ and antisense primer, 5′-CTGAGGATCTCTGGTTGTGGT-3′; Snail sense primer, 5′-TCGGAAGCCTAACTACAGCGA-3′ and antisense primer, 5′-AGATGAGCATTGGCAGCGAG-3′; β-actin sense primer, 5′-CATGTACGTTGCTATCCAGGC-3′ and antisense primer, 5′-CTCCTTAATGTCACGCACGAT-3′. Reverse transcription was carried out amplifying 1 µg total RNA using a reverse transcriptase kit (Guangzhou Ribobio Co., Ltd.) according to the manufacturer's instructions. PCR was then carried out using PowerUp SYBR™-Green Master Mix (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. Briefly, the PCR conditions were started by denaturation for 5 min at 95°C and followed by 35 cycles of 95°C for 10 sec and 60°C for 1 min. mRNA levels were normalized against β-actin mRNA and for relative quantification, 2−ΔΔCq method was used (27).

ROS measurement

Total cellular ROS levels were determined using the Image-iT LIVE Green Reactive Oxygen Species (ROS) Detection kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. A total of 1×105 cells were used for analysis. Cells were washed with ice-cold 1X PBS and incubated with 50 µl staining solution containing 25 µM 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) for 30 min in darkness. Cells were washed twice with ice-cold 1X PBS and ROS levels were measured at 495/529 nm (for carboxy-H2DCFDA) wavelengths on a Multiskan spectrum microplate reader (Thermo Fisher Scientific, Inc.).

Western blotting

Panc-1 or Panc-1 CSCs were washed twice with ice-cold 1X PBS, and then resuspended in lysis buffer containing 25 mM HEPES buffer (pH 7.6), 3 mM MgCl2, 40 mM KCl, 2 mM DTT, 5% glycerol and 0.5% NP-40, and 1X protease inhibitor, and were maintained in an ice bath for 30 min. After a 15-min centrifugation at 12,000 × g, at 4°C, the supernatant was collected as total lysate. The protein concentration was determined using the Bradford protein assay procedure (Sigma-Aldrich; Thermo Fisher Scientific, Inc.). Twenty micrograms of protein was subjected to 10% SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, Inc.), and incubated at room temperature overnight in PBS containing 5% dried milk, 0.05% Tween-20 and primary antibodies: Anti-superoxide dismutase 1 antibody (cat. no. ab13498; diluted at 1:2,000), anti-superoxide dismutase 2/MnSOD antibody (diluted at 1:2,000), anti-glutathione peroxidase 1 antibody (cat. no. ab22604; diluted at 1:1,000), anti-glutathione peroxidase 4 antibody (cat. no. ab125066; diluted at 1:2,000) (all from Abcam, Cambridge, MA, USA); anti-Nanog XP® rabbit monoclonal antibody (mAb) (cat. no. D73G4; diluted at 1:2,500), anti-c-Myc rabbit mAb (cat. no. D3N8F; diluted at 1:1,000), anti-Oct-4 antibody (cat. no. 2750; diluted at 1:1,000), anti-Sox2 antibody (cat. no. 3579; diluted at 1:2,000), anti-E-cadherin rabbit mAb (cat. no. 24E10; diluted at 1:2,000), anti-vimentin XP® rabbit mAb (cat. no. D21H3; diluted at 1:1,000), anti-Slug rabbit mAb (cat. no. C19G7; diluted at 1:1,000), anti-Snail rabbit mAb (cat. no. C15D3; diluted at 1:2,000), anti-β-actin mouse mAb (cat. no. 8H10D10; diluted at 1:5,000) (all from Cell Signaling Technology, Inc., Danvers, MA, USA). After 4 washes in PBS containing 0.05% Tween-20, the membrane was incubated with secondary antibody [anti-mouse IgG, HRP-linked antibody (cat. no. 7076; diluted at 1:5,000), anti-rabbit IgG, HRP-linked antibody (cat. no. 7074; diluted at 1:5,000) (both from Cell Signaling Technology, Inc,)] for 1 h, washed 4 times and the bound antibody was detected by chemiluminescence, and analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

All the data are expressed as means ± SD of 3 independent experiments. Unpaired Student's t-tests were used to compare the means of 2 groups. Differences between groups were analyzed by performing the ANOVA Kruskal-Wallis test, with Dunn's multiple group comparison test. P-values <0.05 were considered to indicate a statistically significant result.

Results

Characterization of CSCs derived from Panc-1 cells

After enriched Panc-1 CSCs were cultured for 7 and 21 days, it was obvious that the morphology of the pancreatic CSCs displayed sphere-like appearance (Fig. 1A). The ability of self-renew is the definition of a stem cell that is thought to be functionally mimicked by CSCs (2830) and serial-replating assay which is widely employed (31), was performed to assess self-renewal of derived cells. As shown in Fig. 1B, no obvious difference in sphere formation ability was found between the cells at the 1st to 4th passage. We then determined whether the spheroid cells have the capacity of high colony formation using Soft agar colony formation assay. Sphere-forming assay was performed to evaluate the stemness property of the Panc-1 cells. The results indicated that the stemness property of the Panc-1 cells was higher than that of their parental counterpart after a period of adaptation to 10% FBS containing medium, instead of serum-free medium (Fig. 1C). And as expected, the capacity of cell proliferation in the spheroid cells was significantly higher than that of their parental counterpart (Fig. 1D). Furthermore, 4 transcription factors which regulate the self-renewal capacity of CSCs (32) were detected by western blot analysis and the results showed that spheroid cells derived from Panc-1 cells displayed obviously higher expression levels of these 4 factors, compared to these levels in the parental counterpart (Fig. 1E).

Panc-1 CSCs display more resistant to hypoxic exposure and show induced expression levels of SOD1, SOD2, GPX1 and GPX4

Hypoxia has been reported to be a promoter of maintaining the stem-like phenotype of CSCs (33). To confirm whether CSCs derived from Panc-1 cells are resistant to hypoxic exposure, Panc-1 or Panc-1 CSCs were exposed to hypoxia for 6, 12 or 24 h and stained using Annexin V-FITC/PI double staining. As shown in Fig. 2A, in normoxia condition, no detectable difference between Panc-1 and Panc-1 CSCs in apoptotic cell death rate (Annexin V-FITC-positive/PI-negative and Annexin V-FITC-positive/PI-positive subpopulation) was noted. After being exposed to hypoxia for 12 and 24 h, the apoptotic cell death rate in the Panc-1 CSCs (4.3±0.5% for 12 h; 0.8±0.6% for 24 h) was obviously lower than that of the Panc-1 cells (9.8±0.9% for 12 h; 11.6±1.7% for 24 h). Then the effects of hypoxia on cell viability were assessed. It was observed that, in Panc-1 CSCs, hypoxia exposure failed to decrease cell viability after 24 h (Fig. 2B). By considering the roles of SODs and GPXs in maintaining low intracellular ROS level in mammalian cells (34), western blot analysis was performed to detect the SOD and GPX protein levels in the Panc-1 and Panc-1 CSCs after hypoxic exposure. Expectedly, SOD1, SOD2 and GPX1 levels were induced by hypoxic exposure (Fig. 2C). However, GPX4 presented a relative high expression level in the Panc-1 CSCs compared with that in the Panc-1 cells, and GPX4 expressing levels were not obviously affected by hypoxic exposure in the Panc-1 CSCs. To confirm whether GPX4 presents a high endogenous level beyond hypoxic exposure, SODs and GPXs were compared in the Panc-1 and Panc-1 CSCs. In Fig. 2D, it was observed that, in the Panc-1 CSCs, GPX4 presented a consistently high endogenous level, which was obviously higher than that noted in the Panc-1 cells and did not respond to hypoxic exposure. This demonstrated that GPX4 potentially play critical roles in regulating physiological processes in panc-1 CSCs, not only after hypoxic exposure.

Endogenous GPX4 regulates oxidative homeostasis and exerts a protective effect on cell viability

For identifying the transfecting efficiency of siRNA by Lipofectamine 2000, Alexa Fluor 488-labeled siRNA-NC was introduced into the Panc-1 CSCs and was detected immediately after 3 PBS washes. As shown in Fig. 3A (upper panels), siRNA-NC was successfully introduced into the Panc-1 CSCs. Then, the knockdown efficiency of the siRNA targeted to GPX4 mRNA (siRNA-GPX4) was measured by RT-qPCR and western blot analysis 2 days after transfection. The mRNA level of GPX4 was downregulated by >50%. In addition, a detectable downregulation in the protein level of GPX4 was also confirmed (Fig. 3A, lower panels).

By considering the inactivating ability of erastin to GPX enzymes, especially GPX4 (35), we examined the ROS accumulation of Panc-1 CSCs treated with targeted siRNA 3 days after transfection treated with 2 µM erastin (Fig. 3B). Cells transfected with siRNA-GPX4 presented significant high ROS levels at 2- or 4-h of erastin treatment, while the control cells presented no detectable ROS accumulation. We then examined the effects of GPX4 knockdown on the cell growth of Panc-1 CSCs, which were exposed to hypoxia for 12 h previously, using CCK-8 assay. There was no significant difference in cell viability between cells transfected with GPX4 and control siRNA up to 0 and 2 h after erastin treatment (Fig. 3C). However, at 4 h after erastin treatment, the viability of the siRNA-GPX4 transfected cells was significantly lower than that of the siRNA-NC transfected cells, suggesting that GPX4 exerts protective effect for cell viability against erastin treatment. We further clarify the ROS eliminating effect of GPX4 under oxidative stress condition induced by H2O2. As shown in Fig. 3D right panel, NAC pre-treatment scavenged ROS in cells. In Fig. 3D left panel, without NAC treatment (mock groups), GPX4 knockdown promoted the ROS accumulation at 50 and 100 µM (Fig. 3D).

Knockdown of GPX4 tightly regulates physiological processes and inhibits the stemness phenotype in the Panc-1 CSCs

Previously, we showed that the presence of GPX4 exerted protective effects to Panc-1 CSCs under oxidative stresses including H2O2 or erastin (Fig. 3). To further investigate whether GPX4 plays certain roles without oxidative stress in Panc-1 CSCs, its effects on cell proliferation was determined. By performing CCK-8 assay and cell counting, it was observed that, without disturbing cell viability, GPX4 knockdown decreased cell proliferation compared to that of the siRNA-NC (Fig. 4A). Flow cytometric analysis after PI staining further showed that GPX4 knockdown arrested the cell cycle at the G1/G0 phase, indicating cell cycle arrest at this phase (Fig. 4B). The results of functional analysis showed that knockdown of GPX4 significantly suppressed sphere formation ability (Fig. 4C), migration and invasion capacity (Fig. 4D and E) in Panc-1 CSCs, as compared with siRNA-NC cells. Collectively, our results revealed that knockdown of GPX4 suppressed the stemness phenotype of Panc-1 CSCs and inhibited in vitro cell functions.

Overexpression of GPX4 suppressed the stemness phenotype and exerts protective effects under oxidative stress

We further evaluated the effects of the overexpression of GPX4 on stemness characteristics and chemosensitivity in Panc-1 CSCs. After stable transfection of GPX4, its mRNA and protein levels were obviously overexpressed (Fig. 5A). Expectedly, after oxidative stress exposure, overexpression of GPX4 inhibited the accumulation of ROS after H2O2 or erastin treatment (Fig. 5B). By performing serial-replating assay with the existence of 100 µM H2O2, it was found that overexpression of GPX4 promoted sphere formation at passage 3 and 4, indicating that the self-renewal capacity of the Panc-1 CSCs was promoted by overexpression of GPX4 (Fig. 5C). Followed by detection of physiological processes under oxidative stress (100 µM H2O2), it was revealed that overexpression of GPX4 promoted cell proliferation (Fig. 5D), migration (Fig. 5E) and invasion (Fig. 5F). In order to ascertain whether overexpression of GPX4 inhibits oxidative stress-induced cell death in Panc-1 CSCs, the cells pretreated with apoptotic death inhibitor (Z-VAD-FMK), ferroptotic death inhibitor (Ferr-1) or necrotic death inhibitor (Nec-1) were treated with oxidative stress (100 µM H2O2 or 2 µM erastin) and stained using CFSE/PI followed by flow cytometric analysis. As shown in Fig. 5G, overexpression of GPX4 inhibited apoptotic cell death induced by H2O2, ferroptotic and necrotic cell death induced by erastin. All these data showed that overexpression of GPX4 exerts protective effects under oxidative stress.

GPX4 plays a critical role in the partial regulation of the EMT phenotype in Panc-1 CSCs

ROS level is reported to be directly and tightly related to EMT-programme activation (35). This promoted us to ascertain whether H2O2 or erastin-induced ROS accumulation regulates EMT-programme processes. Firstly, we tested the ROS eliminating effect of N-acetyl cysteine (NAC), a ROS scavenger, after H2O2 or erastin induction. As expected, NAC efficiently eliminated ROS after oxidative stress (Fig. 6A), thus, in further experiments, NAC was employed as a ROS scavenger. RT-qPCR results for detection of mRNA levels of EMT hallmark genes, including E-cadherin, vimentin, Slug and Snail showed that both H2O2 and erastin treatment upregulated E-cadherin and downregulated vimentin, Slug and Snail mRNA levels (Fig. 6B), while elimination of ROS by a scavenger inhibited these changes, indicating that the generated ROS are attributed to the regulation of EMT. Consistently, protein levels of E-cadherin, vimentin, Slug and Snail were consistent with the tendency of mRNA levels (Fig. 6C).

By considering that the difference between CSCs and non-CSCs is likely to be attributable largely to the cell biological programme termed EMT (37,38), the effects of modified GPX4 on EMT hallmark genes were further detected by RT-qPCR and western blot analysis. Both knockdown and overexpression of GPX4 upregulated E-cadherin, downregulated vimentin, Slug and Snail at the mRNA and protein levels, indicating its inhibitory effect on the EMT programme (Fig. 6D and E), which is consistent with the morphologic changes.

Discussion

ROS are recognized as critical cellular signalling molecules. The maintenance of ROS is critical for a wide array of biological processes, including cell viability, proliferation, apoptosis and angiogenesis (1). In tumours, emerging evidence suggests that accumulation of ROS is associated with the generation of CSCs and the activation of the EMT programme upon hypoxia exposure, and it is considered the most common characteristic associated with tumourigenesis and tumour progression (39). The antioxidant system is intensively studied in mammalian cells (40), however, the importance of a specific antioxidant enzyme has not been fully understood in Panc-1 CSCs. In the present study, we enriched CSCs from Panc-1 cells using serum-free medium. After comparison of several major antioxidant enzymes, including SOD1, SOD2, GPX1 and GPX4 after hypoxic exposure, we found that SOD1, SOD2 and GPX1 were upregulated in both Panc-1 and Panc-1 CSCs. Surprisingly, GPX4 displayed a much higher level of expression in Panc-1 CSCs compared with Panc-1 cells under normoxic conditions. This suggests the importance of GPX4 as a potential key regulator of oxidative homeostasis under normoxic conditions in Panc-1 CSCs. Knockdown and overexpression studies of GPX4 were employed to discern the importance of GPX4 in maintaining oxidative homeostasis in Panc-1 CSCs. According to our results, both overexpression and knockdown of GPX4 increased epithelial markers and decreased mesenchymal ones, and eliminated the characteristics of Panc-1 CSCs without oxidative stress, suggesting that GPX4 may be critical for maintaining oxidative homeostasis. In our hypothesis, both upregulation and downregulation of GPX4 inactivate the EMT programme in unknown mechanisms, which requires further investigation.

Both SODs and GPXs are critical scavenger antioxidative enzyme systems, which are responsible for eliminating accumulated intracellular ROS induced by hypoxia or oxidant exposure. Formed ROS are converted by SODs to H2O2 and subsequently converted by GPXs to H2O+O2 (3). In both Panc-1 and Panc-1 CSCs, hypoxia exposure stimulated the expression of SOD1, SOD2 and GPX1, which is consistent with a previous report (4). To avoid the involvement of SODs in ROS elimination, cells were treated with H2O2 to generate ROS in Panc-1 CSCs. As expected, without the involvement of SODs, GPX4 exerted a critical role in eliminating ROS in CSCs (41). After hypoxic exposure, both SODs and GPXs were upregulated in Panc-1 CSCs, which is consistent with a previous report (42). Notably, before hypoxia stimulation, endogenous GPX4 was relatively high in CSCs and there was no obvious upregulation after hypoxic exposure, suggesting a role for GPX4 under normal conditions. Erastin induces ROS accumulation and ferroptotic cell death (43). We showed that expression of GPX4 exerted antioxidant effects in response to erastin-induced oxidant injury, suggesting that GPX4 exerts protective effects mainly via eliminating ROS induced in a different manner.

EMT has been reported as an important regulator of cancer cells exhibiting stem cell-like properties (44), and EMT is closely linked to a stem cell phenotype in CSCs. In the present study, we demonstrated that changes in GPX4 expression led to decreases in the EMT phenotype and reduction in the stemness phenotype in Panc-1 CSCs. These results indicate that GPX4 potentially regulates the stemness phenotype via regulation of ROS accumulation and the EMT programme. There is still a limitation of this study that only a single cell line was used. For generalizing the results obtained, we intend to test the roles of GPX4 in another cellular system in vitro and in other cancer stem-like cells derived from other organs, and to confirm the roles of GPX4 in human pancreatic tumor tissues in further studies.

Collectively, we demonstrated that GPX4 is upregulated in Panc-1 CSCs compared with parental Panc-1 cells. The elevated levels of GPX4 are responsible for regulating oxidative homeostasis, maintaining the EMT programme and maintaining the CSC phenotype. These data suggest that GPX4 is a possible therapeutic target to prevent the development of resistance to oxidative stress.

Acknowledgements

We would like to thank Professor Hummin Zhao (Sichuan University) for the language editing.

Funding

The present study was funded by the Traditional Chinese Medicine Scientific Support program of Health and Family Planning Commision of Chongqing (no. ZY201703017).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

GP designed part of the experiments and performed the gene expressing and the cell culture relative experiments. ZT and YX performed the gene expression analysis and some cell culture relative experiments. YX wrote the draft of the manuscript. WC designed part of the experiments and supervised the whole procedure. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Bao B, Azmi AS, Li Y, Ahmad A, Ali S, Banerjee S, Kong D and Sarkar FH: Targeting CSCs in tumor microenvironment: The potential role of ROS-associated miRNAs in tumor aggressiveness. Curr Stem Cell Res Ther. 9:22–35. 2014. View Article : Google Scholar : PubMed/NCBI

2 

Klaunig JE, Kamendulis LM and Hocevar BA: Oxidative stress and oxidative damage in carcinogenesis. Toxicol Pathol. 38:96–109. 2010. View Article : Google Scholar : PubMed/NCBI

3 

Trachootham D, Alexandre J and Huang P: Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach? Nat Rev Drug Discov. 8:579–591. 2009. View Article : Google Scholar : PubMed/NCBI

4 

Jiao Y, Wang Y, Guo S and Wang G: Glutathione peroxidases as oncotargets. Oncotarget. 8:80093–80102. 2017. View Article : Google Scholar : PubMed/NCBI

5 

Cejas P, García-Cabezas MA, Casado E, Belda-Iniesta C, De Castro J, Fresno JA, Barriuso J, Espinosa E, Zamora P, Feliu J, et al: Phospholipid hydroperoxide glutathione peroxidase (PHGPx) expression is downregulated in poorly differentiated breast invasive ductal carcinoma. Free Radic Res. 41:681–687. 2007. View Article : Google Scholar : PubMed/NCBI

6 

Al-Taie OH, Uceyler N, Eubner U, Jakob F, Mörk H, Scheurlen M, Brigelius-Flohe R, Schöttker K, Abel J, Thalheimer A, et al: Expression profiling and genetic alterations of the selenoproteins GI-GPx and SePP in colorectal carcinogenesis. Nutr Cancer. 48:6–14. 2004. View Article : Google Scholar : PubMed/NCBI

7 

Falck E, Karlsson S, Carlsson J, Helenius G, Karlsson M and Klinga-Levan K: Loss of glutathione peroxidase 3 expression is correlated with epigenetic mechanisms in endometrial adenocarcinoma. Cancer Cell Int. 10:462010. View Article : Google Scholar : PubMed/NCBI

8 

Liu J, Du J, Zhang Y, Sun W, Smith BJ, Oberley LW and Cullen JJ: Suppression of the malignant phenotype in pancreatic cancer by overexpression of phospholipid hydroperoxide glutathione peroxidase. Hum Gene Ther. 17:105–116. 2006. View Article : Google Scholar : PubMed/NCBI

9 

Yu YP, Yu G, Tseng G, Cieply K, Nelson J, Defrances M, Zarnegar R, Michalopoulos G and Luo JH: Glutathione peroxidase 3, deleted or methylated in prostate cancer, suppresses prostate cancer growth and metastasis. Cancer Res. 67:8043–8050. 2007. View Article : Google Scholar : PubMed/NCBI

10 

Bonnet D and Dick JE: Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 3:730–737. 1997. View Article : Google Scholar : PubMed/NCBI

11 

Kensler TW, Wakabayashi N and Biswal S: Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol. 47:89–116. 2007. View Article : Google Scholar : PubMed/NCBI

12 

Ito K, Hirao A, Arai F, Takubo K, Matsuoka S, Miyamoto K, Ohmura M, Naka K, Hosokawa K, Ikeda Y, et al: Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nat Med. 12:446–451. 2006. View Article : Google Scholar : PubMed/NCBI

13 

Bell EL, Klimova TA, Eisenbart J, Moraes CT, Murphy MP, Budinger GR and Chandel NS: The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production. J Cell Biol. 177:1029–1036. 2007. View Article : Google Scholar : PubMed/NCBI

14 

Huang J, Yang J, Maity B, Mayuzumi D and Fisher RA: Regulator of G protein signaling 6 mediates doxorubicin-induced ATM and p53 activation by a reactive oxygen species-dependent mechanism. Cancer Res. 71:6310–6319. 2011. View Article : Google Scholar : PubMed/NCBI

15 

Juntilla MM, Patil VD, Calamito M, Joshi RP, Birnbaum MJ and Koretzky GA: AKT1 and AKT2 maintain hematopoietic stem cell function by regulating reactive oxygen species. Blood. 115:4030–4038. 2010. View Article : Google Scholar : PubMed/NCBI

16 

Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN, Qian D, Lam JS, Ailles LE, Wong M, et al: Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature. 458:780–783. 2009. View Article : Google Scholar : PubMed/NCBI

17 

Ye XQ, Li Q, Wang GH, Sun FF, Huang GJ, Bian XW, Yu SC and Qian GS: Mitochondrial and energy metabolism-related properties as novel indicators of lung cancer stem cells. Int J Cancer. 129:820–831. 2011. View Article : Google Scholar : PubMed/NCBI

18 

Kim HM, Haraguchi N, Ishii H, Ohkuma M, Okano M, Mimori K, Eguchi H, Yamamoto H, Nagano H, Sekimoto M, et al: Increased CD13 expression reduces reactive oxygen species, promoting survival of liver cancer stem cells via an epithelial-mesenchymal transition-like phenomenon. Ann Surg Oncol. 19 Suppl 3:S539–S548. 2012. View Article : Google Scholar : PubMed/NCBI

19 

Thompson EW, Newgreen DF and Tarin D: Carcinoma invasion and metastasis: A role for epithelial-mesenchymal transition? Cancer Res. 65:5991–5995. 2005. View Article : Google Scholar : PubMed/NCBI

20 

Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, et al: The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 133:704–715. 2008. View Article : Google Scholar : PubMed/NCBI

21 

Morel AP, Lièvre M, Thomas C, Hinkal G, Ansieau S and Puisieux A: Generation of breast cancer stem cells through epithelial-mesenchymal transition. PLoS One. 3:e28882008. View Article : Google Scholar : PubMed/NCBI

22 

Chi HC, Tsai CY, Tsai MM, Yeh CT and Lin KH: Roles of long noncoding RNAs in recurrence and metastasis of radiotherapy-resistant cancer. Int J Mol Sci. 18(pii): E19032017. View Article : Google Scholar : PubMed/NCBI

23 

Cannito S, Novo E, di Bonzo LV, Busletta C, Colombatto S and Parola M: Epithelial-mesenchymal transition: From molecular mechanisms, redox regulation to implications in human health and disease. Antioxid Redox Signal. 12:1383–1430. 2010. View Article : Google Scholar : PubMed/NCBI

24 

Giannoni E, Parri M and Chiarugi P: EMT and oxidative stress: A bidirectional interplay affecting tumor malignancy. Antioxid Redox Signal. 16:1248–1263. 2012. View Article : Google Scholar : PubMed/NCBI

25 

Bao B, Azmi AS, Ali S, Ahmad A, Li Y, Banerjee S, Kong D and Sarkar FH: The biological kinship of hypoxia with CSC and EMT and their relationship with deregulated expression of miRNAs and tumor aggressiveness. Biochim Biophys Acta. 1826:272–296. 2012.PubMed/NCBI

26 

Fu Z, Li G, Li Z, Wang Y, Zhao Y, Zheng S, Ye H, Luo Y, Zhao X, Wei L, et al: Endogenous miRNA sponge lincRNA-ROR promotes proliferation, invasion and stem cell-like phenytype of pancreatic cancer cells. Cell Death Discov. 3:170042017. View Article : Google Scholar : PubMed/NCBI

27 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI

28 

Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL and Wahl GM: Cancer stem cells-perspectives on current status and future directions: AACR workshop on cancer stem cells. Cancer Res. 66:9339–9344. 2006. View Article : Google Scholar : PubMed/NCBI

29 

Pradal R, Clarke MF and Morrison SJ: Applying the principle of stem-cell biology to cancer. Nat Rev Cancer. 3:895–902. 2003. View Article : Google Scholar : PubMed/NCBI

30 

Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, Pilotti S, Pierotti MA and Daidone MG: Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res. 65:5506–5511. 2005. View Article : Google Scholar : PubMed/NCBI

31 

Somervaille TC and Cleary ML: Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell. 10:257–268. 2006. View Article : Google Scholar : PubMed/NCBI

32 

Sharma N, Nanta R, Sharma J, Gunewardena S, Singh KP, Shankar S and Srivastava RK: PI3K/AKT/mTOR and sonic hedgehog pathways cooperate together to inhibit human pancreatic cancer stem cell characteristics and tumor growth. Oncotarget. 6:32039–32060. 2015. View Article : Google Scholar : PubMed/NCBI

33 

Yeung TM, Gandhi SC, Wilding JL, Muschel R and Bodmer WF: Cancer stem cells from colorectal cancer-derived cell lines. Proc Natl Acad Sci USA. 107:3722–3727. 2010. View Article : Google Scholar : PubMed/NCBI

34 

Galadari S, Rahman A, Pallichankandy S and Thayyullathil F: Reactive oxygen species and cancer paradox: To promote or to suppress? Free Radic Biol Med. 104:144–164. 2017. View Article : Google Scholar : PubMed/NCBI

35 

Yang WS, SriRamaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan VS, Cheah JH, Clemons PA, Shamji AF, Clish CB, et al: Regulation of ferroptotic cancer cell death by GPX4. Cell. 156:317–331. 2014. View Article : Google Scholar : PubMed/NCBI

36 

Shibue T and Weinberg RA: EMT, CSCs, and drug resistance: The mechanistic link and clinical implications. Nat Rev Clin Oncol. 14:611–629. 2017. View Article : Google Scholar : PubMed/NCBI

37 

Medema JP: Cancer stem cells: The challenges ahead. Nat Cell Biol. 15:338–344. 2013. View Article : Google Scholar : PubMed/NCBI

38 

Polyak K and Weinberg RA: Transitions between epithelial and mesenchymal states: Acquisition of malignant and stem cell traits. Nat Rev Cancer. 9:265–273. 2009. View Article : Google Scholar : PubMed/NCBI

39 

Ischenko I, Seeliger H, Kleespies A, Angele MK, Eichhorn ME, Jauch KW and Bruns CJ: Pancreatic cancer stem cells: New understanding of tumorigenesis, clinical implications. Langenbecks Arch Surg. 395:1–10. 2010. View Article : Google Scholar : PubMed/NCBI

40 

Hayashi R, Himori N, Taguchi K, Ishikawa Y, Uesugi K, Ito M, Duncan T, Tsujikawa M, Nakazawa T, Yamamoto M, et al: The role of the Nrf2-mediated defense system in corneal epithelial wound healing. Free Radic Biol Med. 61:333–342. 2013. View Article : Google Scholar : PubMed/NCBI

41 

Lu L, Oveson BC, Jo YJ, Lauer TW, Usui S, Komeima K, Xie B and Campochiaro PA: Increased expression of glutathione peroxidase 4 strongly protects retina from oxidative damage. Antioxid Redox Signal. 11:715–724. 2009. View Article : Google Scholar : PubMed/NCBI

42 

Perrella MA and Yet SF: Role of heme oxygenase-1 in cardiovascular function. Curr Pharm Des. 9:2479–2487. 2003. View Article : Google Scholar : PubMed/NCBI

43 

Maldonado EN, Sheldon KL, DeHart DN, Patnaik J, Manevich Y, Townsend DM, Bezrukov SM, Rostovtseva TK and Lemasters JJ: Voltage-dependent anion channels modulate mitochondrial metabolism in cancer cells: Regulation by free tubulin and erastin. J Biol Chem. 288:11920–11929. 2013. View Article : Google Scholar : PubMed/NCBI

44 

Scheel C and Weinberg RA: Phenotypic plasticity and epithelial-mesenchymal transitions in cancer and normal stem cells? Int J Cancer. 129:2310–2314. 2011. View Article : Google Scholar : PubMed/NCBI

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February-2019
Volume 41 Issue 2

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Spandidos Publications style
Peng G, Tang Z, Xiang Y and Chen W: Glutathione peroxidase 4 maintains a stemness phenotype, oxidative homeostasis and regulates biological processes in Panc‑1 cancer stem‑like cells. Oncol Rep 41: 1264-1274, 2019
APA
Peng, G., Tang, Z., Xiang, Y., & Chen, W. (2019). Glutathione peroxidase 4 maintains a stemness phenotype, oxidative homeostasis and regulates biological processes in Panc‑1 cancer stem‑like cells. Oncology Reports, 41, 1264-1274. https://doi.org/10.3892/or.2018.6905
MLA
Peng, G., Tang, Z., Xiang, Y., Chen, W."Glutathione peroxidase 4 maintains a stemness phenotype, oxidative homeostasis and regulates biological processes in Panc‑1 cancer stem‑like cells". Oncology Reports 41.2 (2019): 1264-1274.
Chicago
Peng, G., Tang, Z., Xiang, Y., Chen, W."Glutathione peroxidase 4 maintains a stemness phenotype, oxidative homeostasis and regulates biological processes in Panc‑1 cancer stem‑like cells". Oncology Reports 41, no. 2 (2019): 1264-1274. https://doi.org/10.3892/or.2018.6905