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Ferroptosis and hypoxia-inducible factor 1α (HIF-1α) have critical roles in human tumors. The aim of the present study was to investigate the associations between ferroptosis, HIF-1α and cell growth in non-small cell lung cancer (NSCLC) cells. The lung cancer cell lines SW900 and A549 were evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the expression of HIF-1α. Cell Counting Kit-8, flow cytometry and Transwell migration assays were used to measure cell viability, apoptosis and invasion, respectively. The production of reactive oxygen species (ROS) and levels of malondialdehyde (MDA), glutathione (GSH) and ferrous ion (Fe2+) were determined using detection kits. The expression levels of glutathione peroxidase 4 (GPX4) and Yes-associated protein 1 (YAP1) were detected using RT-qPCR and western blotting. The results showed that the expression of HIF-1α was significantly upregulated in NSCLC cells compared with normal human bronchial epithelial cells. Small interfering RNA specific to HIF-1α (si-HIF-1α) significantly decreased the proliferation and invasion of NSCLC cells and increased their apoptosis. si-HIF-1α also increased the levels of ROS, MDA and Fe2+ but decreased GSH and GPX4 levels in A549 cells. Additionally, si-HIF-1α increased phosphorylated (p-)YAP1 levels, suppressed GPX4 and YAP1 expression, and attenuated the YAP1 overexpression-induced changes in YAP1, p-YAP1 and GPX4 levels and cell viability. The ferroptosis antagonist ferrostatin-1 partially attenuated the effects of si-HIF-1α on the NSCLC cells, while the ferroptosis agonist erastin further inhibited NSCLC growth by blocking HIF-1α expression. In conclusion, the silencing of HIF-1α induces ferroptosis by suppressing Hippo-YAP pathway activation in NSCLC cells. The present study provides novel insights into the malignant progression of NSCLC and suggests that HIF-1α is an effective target for the treatment of NSCLC.
Non-small cell lung cancer (NSCLC) is the most common type of cancer, accounting for ~85% of all cases of lung cancer, and is a major cause of cancer-associated mortality worldwide (
Ferroptosis is an iron-dependent form of oxidative cell death triggered by the accumulation of lethal levels of lipid-based reactive oxygen species (ROS) and lipid peroxidation (
Hypoxia plays a critical role in tumourigenesis, metastasis and chemotherapy resistance in solid tumours (
The Hippo-Yes-associated protein (YAP) pathway influences a wide variety of tumour types, including NSCLC (
In the present study, the proliferation, invasion, ferroptosis and oxidative stress levels of NSCLC cells were investigated after the silencing HIF-1α with or without treatment with the ferroptosis antagonist ferrostatin-1 (Fer-1) or ferroptosis agonist erastin. Expression levels of the Hippo-YAP pathway-associated protein YAP1 and ferroptosis marker protein glutathione peroxidase 4 (GPX4) were also detected. HIF-1α and ferroptosis were investigated as a therapeutic target and strategy, respectively, for the treatment of NSCLC.
SW900 (cat. no. HTB-59) and A549 (cat. no. CRM-CCL-185) human NSCLC and BEAS-2B (cat. no. CRL-9609) human bronchial epithelial cell lines acquired from the American Type Culture Collection were used in the study. The SW900 and A549 cells were maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 4.5 g/l glucose, 4 mmol/l L-glutamine, 10% foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The BEAS-2B cells were cultured in RPMI-1640 containing 10% FBS and 1% penicillin-streptomycin. All cells were cultured in triplicate in 12-well plates (1×105/cm2) at 37°C with 5% CO2.
Small interfering RNA (siRNA) specific to HIF-1α (si-HIF-1α, 5′-CGAUGGAAGCACUAGACAAAG-3′), siRNA negative control (si-NC, 5′-CACUGAUUUCAAAUGGUGCUAUU-3′) and a sequence for the overexpression (oe) of YAP1 (oe-YAP1; Shanghai GeneChem Co., Ltd.) were cloned into lentiviral vectors (Lenti-Mix: pMDLg/pRRE, pVSV-G, pRSV-Rev; Wuhan GeneCreate Biological Engineering Co., Ltd.). Lentivirus packaging plasmids (50 ng/µl) expressing si-HIF-1α, si-NC, oe-YAP1 and negative control for YAP1 overexpression (oe-NC) were constructed and transfected into SW900 and A549 cells using HighGene transfection reagent (ABclonal Biotech Co., Ltd.) at 37°C for 72 h. The mRNA expression and protein levels of HIF-1α and YAP1 were confirmed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively.
To induce ferroptosis, NSCLC cells were treated with 5 µg/ml erastin (Beijing Solarbio Science & Technology Co., Ltd.) for 24 h. Treatment with Fer-1 (10 µmol/l; ABclonal Biotech Co., Ltd.) for 24 h was used to inhibit ferroptosis (
The proliferation of SW900 and A549 cells was detected using a CCK-8 assay (Beyotime Institute of Biotechnology). Cells (2×10 cells/well) were seeded into 96-well plates (100 µl/well) and cultured for 24, 48, 72 and 96 h. The cells were then incubated with 10 µl CCK-8 solution for 2 h. A microplate reader (Wuxi Hiwell-Diatek Instruments Co., Ltd.) was used to determine the optical density at 450 nm (OD450).
NSCLC cell apoptosis was detected using an Annexin V-FITC Apoptosis Detection Kit (Beyotime Institute of Biotechnology). In brief, 2×105 cells were resuspended in 500 µl binding buffer and incubated with 5 µl Annexin V-FITC for 30 min at 4°C in the dark, followed by incubation with 5 µl propidium iodide for 5 min at 25°C. The apoptosis rate was then detected using flow cytometry with a CytoFLEX S instrument (Beckman Coulter, Inc.) and Cell Quest software (version 5.2.1; BD Biosciences).
Cell invasion was evaluated using Matrigel-coated Transwell chambers (Corning, Inc.). NSCLC cells (1×106/ml) were seeded into the upper chambers filled with serum-free RPMI-1640. The lower chambers were filled with complete RPMI-1640, and the cells cultured for 48 h at 37°C with 5% CO2. Cells that adhered to the surface of the lower chamber were fixed and stained with 1% crystal violet (Beyotime Institute of Biotechnology) at room temperature for 20 min. Cells were counted in digital photographs captured using a CKX53 microscope (Olympus Corporation).
RNA was isolated from NSCLC and BEAS-2B cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). First-strand cDNA was synthesised using a Fastingking gDNA Dispelling RT SuperMix kit (Tiangen Biotech Co., Ltd.) according to the manufacturer's protocol. SYBR Green PCR Master Mix (Xiamen Life Internet Technology Co., Ltd.) and an MX3000P Fast RT-PCR instrument (Agilent Technologies, Inc.) were used for qPCR analysis. The thermocycling conditions were as follows: 95°C for 3 min; followed by 40 cycles of 95°C for 12 sec and 62°C for 40 sec. The expression levels of HIF-1α and YAP1 were determined using the 2−∆∆Cq method (
Proteins were isolated from NSCLC and BEAS-2B cells using a radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology). The isolated protein samples were quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology) and then protein samples (25 µg) were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Beyotime Institute of Biotechnology). Immunoblotting analysis was performed using polyvinylidene fluoride membranes (Beyotime Institute of Biotechnology). The membranes were blocked with 5% non-fat milk for 6 min at room temperature. For the immunoblotting analysis, the membranes were incubated with specific primary antibodies, namely anti-HIF-1α (1:500; cat. no. ab51608; Abcam), anti-GPX4 (1:5,000; cat. no. ab125066; Abcam), anti-YAP1 (1:5,000; cat. no. ab52771; Abcam), anti-phosphorylated (p)-YAP1 (1:25,000; cat. no. ab76252; Abcam) and anti-GAPDH (1:2,500; cat. no. ab9485; Abcam) antibodies, at 4°C overnight. Horseradish peroxidase-tagged goat anti-rabbit IgG H&L (1:10,000; cat. no. A0516; Beyotime Institute of Biotechnology) was used as the secondary antibody and incubated with the membranes at room temperature for 1 h. An enhanced chemiluminescence system (Pierce; Thermo Fisher Scientific, Inc.) and automatic digital gel image analysis system (Tanon 3500; Tanon Science & Technology Co., Ltd.) were used to analyse the target proteins.
The levels of malondialdehyde (MDA), glutathione (GSH) and ROS in A549 cells were determined using corresponding commercial enzyme-linked immunosorbent assay kits (cat. no. BC0025 for MDA, Beijing Solarbio Science & Technology Co., Ltd.; cat. no. E-BC-K030-M for GSH, Elabscience Biotechnology, Inc.; and cat. no. CA1410 for ROS, Beijing Solarbio Science & Technology Co., Ltd.). A microplate reader was used to measure the OD450 value.
The concentration of Fe2+ in A549 cells was determined using an iron colorimetric assay kit (cat. no. K390-100; BioVision, Inc.). A microplate reader was used to determine the OD450 value.
All experiments were performed in triplicate. Statistical analyses were conducted using GraphPad Prism 7.0 software (GraphPad Software, Inc.). Experimental data are expressed as the mean ± standard deviation. Differences between two groups were analysed using an unpaired t-test. Differences among three or more experimental groups were analysed using one-way analysis of variance, followed by Tukey's test. P<0.05 was considered to indicate a statistically significant difference.
The expression levels of HIF-1α in SW900 and A549 NSCLC cell lines and BEAS-2B normal bronchial epithelial cells were detected using RT-qPCR and western blotting. The mRNA and protein expression levels of HIF-1α in the NSCLC cells were significantly higher than those in the normal cells (P<0.01). In addition, the mRNA and protein expression levels of HIF-1α in A549 cells were higher than those in SW900 cells (P<0.05;
The effects of HIF-1α silencing on the malignant progression of NSCLC were investigated. The CCK-8 assay showed that HIF-1α silencing significantly suppressed the proliferation of SW900 and A549 cells compared with the proliferation in the respective si-NC group (P<0.05;
Lipid peroxidation, GSH depletion and iron accumulation are reported to be critical events in ferroptosis (
The underlying mechanism by which si-HIF-1α induced ferroptosis in NSCLC cells was then investigated. The Hippo-YAP pathway regulates ferroptosis in cancer (
To confirm that si-HIF-1α-induced ferroptosis plays an important role in NSCLC treatment, the proliferation and invasion of A549 cells, and the oxidative stress and iron accumulation in these were cells investigated after treatment with Fer-1 or erastin. As shown in
In recent years, ferroptosis has emerged as a strategy for cancer treatment. The present study evaluated the ability of HIF-1α silencing to induce ferroptosis in NSCLC cells and investigated whether the underlying mechanism involves the inhibition of Hippo-YAP pathway activation.
Hypoxia is a common condition in the tumour environment due to the high proliferation rate of tumour cells (
As HIF-1α contributes to the inhibition of ferroptosis in hepatocellular carcinoma, it has been reported as a potential biomarker of poor post-operative outcomes in this disease (
The Hippo signalling pathway has tumour suppressing effects, and YAP acts as an oncogene in most tumour cells (
The present study had some limitations. First, the pathological mechanisms affecting the growth of cancer cells are complex, and ferroptosis is only one such mechanism; the other mechanisms that may be involved were not evaluated. Second, HIF-1α affects NSCLC cell ferroptosis and may be involved in multiple pathways in addition to the Hippo-YAP pathway. Finally, additional experimental data obtained from techniques such as confocal microscopy or fluorescent probe analyses are required to confirm the results.
In conclusion, HIF-1α expression is upregulated in NSCLC cells. The silencing of HIF-1α inhibited the proliferation and invasion of NSCLC cells and induced their ferroptosis; it also suppressed activation of the Hippo-YAP pathway. Moreover, erastin further enhanced the effect of si-HIF-1α whereas Fer-1 counteracted the effect of si-HIF-1α. These results suggest that HIF-1α silencing promotes ferroptosis in NSCLC cells via the inhibition of Hippo-YAP pathway activation. Therefore, HIF-1α is a potential target for NSCLC therapy.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
SZ, JM and YC contributed to the conception and the design of the study. SZ, JM and JZ were responsible for the acquisition, analysis and interpretation of the data. SZ, JZ and YC confirm the authenticity of all the raw data. SZ and JM contributed to manuscript drafting and critical revisions of intellectual content. SZ obtained the funding. YC approved the final manuscript for publication. All authors have read and approved the final version of the manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
non-small cell lung cancer
reactive oxygen species
vascular endothelial growth factor
Yes-associated protein 1
glutathione peroxidase 4
reverse transcription-quantitative polymerase chain reaction
Cell Counting Kit-8
optical density
malondialdehyde
glutathione
Expression of HIF-1α in NSCLC cells. (A) Expression of HIF-1α mRNA in SW900 and A549 NSCLC cell lines and normal BEAS-2B cells determined using reverse transcription-quantitative polymerase chain reaction. (B) Results of the western blot analysis of HIF-1α protein. In (A) and (B), **P<0.01 and ***P<0.001 vs. BEAS-2B; and #P<0.05 and ##P<0.01 vs. SW900. (C) mRNA and (D) protein expression of HIF-1α in NSCLC cells transfected with si-HIF-1α. In (C) and (D), **P<0.01 and ***P<0.001 vs. si-NC. HIF-1α, hypoxia-inducible factor 1α; NSCLC, non-small cell lung cancer; si-HIF-1α, small interfering RNA to HIF-1α; si-NC, small interfering RNA negative control.
Effect of si-HIF-1α on the proliferation, invasion and ferroptosis of NSCLC cells. Results of (A) Cell Counting Kit-8, (B) flow cytometry and (C) Transwell invasion assays of NSCLC cells transfected with si-HIF-1α. (D) Levels of ROS, MDA, Fe2+ and GSH in A549 cells transfected with si-HIF-1α. Scale bar, 50 µm. (E) Protein expression of GPX4 in A549 cells transfected with si-HIF-1α. *P<0.05, **P<0.01 and ***P<0.001 vs. si-NC. si-HIF-1α, small interfering RNA to hypoxia-inducible factor 1α; NSCLC, non-small cell lung cancer; ROS, reactive oxygen species; MDA, malondialdehyde; Fe2+, ferrous iron; GSH, glutathione; GPX4, glutathione peroxidase 4; si-NC, small interfering RNA negative control; OD, optical density.
si-HIF-1α inhibits Hippo-YAP pathway activation in non-small cell lung cancer cells. (A) Relative mRNA expression of YAP1 in A549 cells transfected with oe-NC and oe-YAP1. ***P<0.001 vs. oe-NC. (B) Relative expression of YAP1 mRNA in cells transfected with si-HIF-1α and/or oe-YAP1. (C) Cell Counting Kit-8 assay results showing the viability of A549 cells. (D) Fold-change in the p-YAP1/YAP1 ratio and GPX4 protein expression assessed using western blot analysis. *P<0.05 and ***P<0.001 vs. si-NC; ^^P<0.01 and ^^^P<0.001 vs. oe-YAP1. si-HIF-1α, small interfering RNA to hypoxia-inducible factor 1 α; YAP, Yes-associated protein; p-, phosphorylated; oe-YAP1, overexpression of YAP1; oe-NC, overexpression negative control; si-NC, small interfering RNA negative control; GPX4, glutathione peroxidase 4; OD, optical density.
si-HIF-1α promotes ferroptosis by inhibiting Hippo-YAP pathway activation in non-small cell lung cancer cells. (A) Cell Counting Kit-8 assay results. (B) Quantified Transwell invasion assay results and (C) representative images of invaded A549 cells. Scale bar, 50 µm. (D) ROS, MDA, Fe2+ and GSH levels in A549 cells. (E) Relative mRNA expression of HIF-1α and YAP1 in A549 cells. (F) Protein levels of HIF-1α, p-YAP1/YAP1 and GPX4 in A549 cells. **P<0.01 and ***P<0.001 vs. si-NC. ^P<0.05, ^^P<0.01 and ^^^P<0.001 vs. si-HIF-1α. HIF-1α, hypoxia-inducible factor 1 α; si-HIF-1α, small interfering RNA to HIF-1α; YAP, Yes-associated protein; p-, phosphorylated; ROS, reactive oxygen species; MDA, malondialdehyde; Fe2+, ferrous iron; GSH, glutathione; GPX4, glutathione peroxidase 4; PBS, phosphate-buffered saline; Fer-1, ferrostatin-1; si-NC, small interfering RNA negative control; OD, optical density.