GSTZ1 knockout promotes sorafenib resistance in HCC

To further evaluate whether GSTZ1 is related to sorafenib resistance in HCC, we found that overexpression of GSTZ1 through an adenovirus system^19,20 (Fig. ​(Fig.2A)2A) increased the sensitivity of HCC cells to sorafenib and inhibited cell proliferation via morphological observation (Fig. ​(Fig.2B).2B). Conversely, knockout of GSTZ1 in HepG2 and SNU449 cells via the CRISPR/Cas9 system^19,20 (Fig. ​(Fig.2C)2C) decreased sorafenib sensitivity and weakened its growth inhibition effect (Fig. ​(Fig.2D).2D). Next, we analyzed the cell viability of GSTZ1 overexpression (OE) and knockout (KO) cells treated with sorafenib over a series of time points or at different concentrations for 24h by cell growth curve. As expected, GSTZ1 overexpression significantly enhanced the sensitivity of HCC cell lines to sorafenib (Fig. 2E, F). As expected, the IC₅₀ value of GSTZ1-OE cells was decreased compared to that of the control groups, whereas the IC₅₀ value of GSTZ1-KO groups was increased (Fig. 2G, H). Taken all together, our results showed that GSTZ1 deficiency enhanced sorafenib resistance in HCC.

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Fig. 2

GSTZ1 knockout promotes sorafenib resistance in HCC cells.

A Overexpression of GSTZ1 was confirmed by immunoblot assay. B Morphological phase-contrast images (top) and quantification (bottom) of GSTZ1-OE cells after treatment with or without sorafenib (10μM) for 24h. C Knockout of GSTZ1 was confirmed by immunoblot assay. D Morphological phase-contrast images (top) and quantification (bottom) of GSTZ1-KO cells after treatment with or without sorafenib (10μM) for 24h. Scale bar=10μm. E, F Cell growth curve. GSTZ1-OE (E) and GSTZ1-KO (F) cells were treated with or without sorafenib (10μM). G, H The IC₅₀ of GSTZ1-OE (G) and GSTZ1-KO (H) cells were determined using the CCK-8 assay. For western blotting, 50μg protein was loaded per well. DMSO: dimethyl sulfoxide, Sora: sorafenib. Values represent the mean±SD (n=3). *p<0.05, **p<0.01, Student’s t-test (two groups).

GSTZ1 overexpression enhances sorafenib-induced ferroptosis in HCC

Recent studies indicate that ferroptosis plays a key role in the chemoresistance of human cancers^22–24. We confirmed that sorafenib-induced cell death in HCC cell lines was blocked by ferrostatin-1 (Fer-1, an inhibitor of ferroptosis), deferoxamine (DFO, an iron chelator), and N-acetyl-L-cysteine (NAC, an antioxidant), but not by bafilomycin A1 (Baf-A1, an inhibitor of autophagy), ZVAD-FMK, and necrosulfonamide (NSA, an inhibitor of necroptosis). This suggested that ferroptosis, rather than apoptosis, is essential for sorafenib-induced cell death in HCC (Supplementary Fig. 1A, B), consistent with findings of the previous studies^11,25,26. To determine whether GSTZ1 played a role in ferroptosis to reduce sorafenib resistance in HCC, transmission electron microscopy (TEM) was used to observe morphological changes in sorafenib-induced HCC cells with or without GSTZ1 depletion. GSTZ1-OE cells treated with sorafenib displayed smaller mitochondria, diminished or vanished mitochondria crista, and condensed mitochondrial membrane densities compared to parental cells, whereas GSTZ1-KO alleviated the abnormalities of mitochondrial morphology and cell death induced by sorafenib (Fig. ​(Fig.3A).3A). To further verify this observation, we measured ROS, iron, and lipid peroxidation levels, which are the primary cause of ferroptosis²⁷, after interference with GSTZ1 expression. Results showed that GSTZ1 overexpression significantly increased ROS, iron, and MDA levels in sorafenib-induced HCC cell lines (Fig. 3B–D and Supplementary Fig. 1C, D), whereas GSTZ1 knockout decreased their levels. In addition, we assessed mRNA and protein expression levels of ferroptosis-associated genes. Results showed that GSTZ1-OE decreased the expression levels of ferroptosis-related genes in sorafenib or erastin (an inducer of ferroptosis)-treated hepatoma cells, including GPX4, FTL, and SLC7A11. In contrast, GSTZ1-KO increased the levels of these above genes (Fig. 3E–G and Supplementary Fig. 1E, F). Interestingly, ferroptosis-associated genes were also enhanced in HepG2-SR and SNU449-SR cells (Fig. 4A, B), which had relatively lower levels of GSTZ1 than the parental cells. Meanwhile, GSTZ1 overexpression in drug-resistant cells reduced the levels of ferroptosis-related genes (Fig. ​(Fig.4C),4C), increased the accumulation of iron and MDA levels (Fig. 4D, E), decreased the intracellular GSH level (Fig. ​(Fig.4F),4F), and enhanced the inhibition of sorafenib to resistant cells (Fig. ​(Fig.4G).4G). The GSTZ1 Ser14→Ala mutation has the greatest detrimental effect on GSTZ1 activity²⁸. We then generated GSTZ-KO and SR cells expressing S14A GSTZ1 (Supplementary Fig. 2A, B) and found that upon sorafenib stimulation, iron and MDA levels were reduced in the cells expressing S14A GSTZ1 compared to wild-type (WT)-transduced cells (Supplementary Fig. 2C–F). This suggests that GSTZ1 S14A failed to sensitize hepatoma cells to sorafenib-induced ferroptosis (Supplementary Fig. 2G, H). To further identify the role of ferroptosis in sorafenib resistance caused by GSTZ1 deficiency, we examined the curative effects of sorafenib by interfering with the ferroptosis pathway. We observed that ferrostatin-1 inhibited sorafenib-induced GSTZ1-OE cell death, whereas erastin promoted GSTZ1-KO cell death (Fig. ​(Fig.4H).4H). Together, these results suggested that GSTZ1 increased the sensitivity of hepatoma cells to sorafenib by inducing ferroptosis.

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Fig. 3

GSTZ1 overexpression enhances sorafenib-induced ferroptosis in HCC.

A Representative TEM images of the mitochondrial morphology in GSTZ1-OE SK-Hep1 and GSTZ1-KO HepG2 cells treated with 10μM sorafenib for 24h. Red arrows indicate mitochondria. Scale bar=1μm. B Representative images (top) and quantification (bottom) of ROS level in GSTZ1-OE and GSTZ1-KO cells treated with sorafenib for 24h. Scale bar =20μm. C, D The intracellular iron (C) and MDA (D) levels in GSTZ1-OE and GSTZ1-KO cells treated with sorafenib for 24h. E–G mRNA and protein levels of target genes associated with ferroptosis in GSTZ1-OE and GSTZ1-KO cells treated with sorafenib or erastin, determined via qRT-PCR (E, F) and western blotting (G), respectively. For western blotting, 50μg protein was loaded per well. Values represent the mean±SD (n=3). The qRT-PCR data determined from three independent experiments. ns: no significant difference, *p<0.05, **p<0.01, ***p<0.001, Student’s t-test (two groups).

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Fig. 4

GSTZ1 overexpression sensitizes hepatoma cells to sorafenib-induced ferroptosis.

A, B mRNA (A) and protein (B) levels of ferroptosis-related genes in sorafenib-sensitive and -resistant cells were assayed using qRT-PCR and western blotting, respectively. C Western blotting for assessment of protein levels of ferroptosis-related genes in sorafenib-resistant HCCs with adenoviruses expressing GFP (AdGFP) or GSTZ1 (AdGSTZ1). D–F Iron (D), MDA (E) and GSH (F) levels in GSTZ1-OE sorafenib-resistant cells. G Cell viability was determined using CCK-8 assay. H Morphology (left) and quantification (right) of indicated HCC cells treated with sorafenib (10μM for 24h) alone or in combination with Fer-1 (1μM for 24h) or erastin (10μM for 24h). Scale bar=10μm. For western blotting, 50μg protein was loaded per well. Fer-1: ferrostatin-1, SR sorafenib resistant. Values represent the mean±SD (n=3). The qRT-PCR data determined from three independent experiments. *p<0.05, **p<0.01, ***p<0.001, Student’s t-test (two groups).

GSTZ1 sensitizes hepatoma cells to sorafenib-induced ferroptosis through the NRF2 signaling pathway

Previous studies have shown that GSTZ1 deficiency leads to the accumulation of endogenous metabolite succinylacetone, contributing to NRF2 activation via alkylating Kelch-like ECH-associated protein 1 (KEAP1) cysteine residues^19,20. Activation of NRF2 pathway plays a critical role in protecting HCC cells against sorafenib-induced ferroptosis²⁶. To verify whether GSTZ1 regulated sorafenib-induced ferroptosis through the NRF2 signaling pathway, we blocked the NRF2 pathway using brusatol (an inhibitor of NRF2) or Flag-tagged KEAP1²⁹ (a cytosolic inhibitor of NRF2) in GSTZ1-KO cells. We then observed the characteristic indicators related to ferroptosis, including iron, MDA, ROS, and 4-HNE levels. The results demonstrated that NRF2 inhibition significantly increased the accumulation of these indicators in GSTZ1-KO cells (Fig. 5A–D right, Fig. 5E, F bottom, and Supplementary Fig. 3A–C bottom), whereas NRF2 activation using tertiary butylhydroquinone (tBHQ, an activator of NRF2) and Myc-tagged NRF2 yielded opposite results in GSTZ1-OE cells (Fig. 5A–D left, Fig. 5E–F top and Supplementary Fig. 3A–C top). Moreover, the morphological images also indicated that NRF2 inhibition increased the efficiency of sorafenib for growth inhibition in GSTZ1-depleted HCCs, whereas NRF2 activation decreased that in GSTZ1-OE cells (Fig. ​(Fig.6A).6A). Importantly, western blotting revealed that the expression of ferroptosis-related proteins changed accordingly in GSTZ1-OE and -KO cells when treated with tBHQ or brusatol (Fig. ​(Fig.6B6B and Supplementary Fig. 3D). The above data suggested that GSTZ1 depletion alleviated sorafenib-induced ferroptosis via activation of the NRF2 pathway.

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Fig. 5

GSTZ1 knockout cells are insensitive to sorafenib-induced ferroptosis through the activation of NRF2.

A–D Levels of iron (A, B), and MDA (C, D) were assayed. GSTZ1-OE cells were treated with sorafenib alone or in combination with tBHQ (100μM for 3h) (left). GSTZ1-KO cells were treated with sorafenib alone or in combination with Bru (40nM for 24h) (left). Expressing Flag-KEAP1 plasmid was transfected into GSTZ1-OE cells with sorafenib treatment (right). Expressing Myc-NRF2 plasmid was transfected into GSTZ1-KO cells with sorafenib treatment (right). E Representative images (top) and quantification (bottom) of ROS level in GSTZ1-OE cells treated with sorafenib alone or in combination with tBHQ (top) and GSTZ1-KO cells treated with sorafenib alone or in combination with Bru (bottom). Scale bar=20μm. F 4-HNE-induced protein modification was examined. For western blotting, 50μg protein was loaded per well. tBHQ: tertiary butylhydroquinone, Bru brusatol, 4-HNE 4-hydroxy-2-nonenal. Values represent the mean±SD (n=3). *p<0.05, **p<0.01, ***p<0.001, Student’s t-test (two groups).

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Fig. 6

RSL3 enhances the sensitivity of GSTZ1-KO and sorafenib-resistant cells to sorafenib.

A, B Morphological changes (A) and protein level (B) of ferroptosis-related genes. GSTZ1-OE SK-Hep1 cells were treated with sorafenib alone or in combination with tBHQ (top). GSTZ1-KO HepG2 cells were treated with sorafenib alone or in combination with Bru (bottom). Scale bar=10μm. C–E Iron (C), MDA (D), and GSH (E) levels in GSTZ1-KO and sorafenib-resistant cells treated with sorafenib alone or in combination with RSL3 (500nM for 24h). F Cell growth curve of GSTZ1-KO and sorafenib-resistant cells treated with sorafenib alone or in combination with RSL3. RSL3: Ras-selective lethal small molecule 3, tBHQ tertiary butylhydroquinone, Bru brusatol, DMSO dimethyl sulfoxide, Sora sorafenib, MDA malondialdehyde. Values represent the mean±SD (n=3). *p<0.05, **p<0.01, ***p<0.001, Student’s t-test (two groups).

As GPX4 is involved in ferroptosis and a transcriptional target gene of NRF2^30,31, we utilized RSL3 to further examine whether ferroptosis is involved in the sensitivity of HCC cells to sorafenib. Interestingly, simultaneous treatment with sorafenib and RSL3 increased the accumulation of iron and MDA levels, decreased the intracellular GSH level, enhanced sorafenib-induced ferroptosis, and dramatically inhibited cell growth compared to sorafenib or RSL3 alone in GSTZ1-KO and SR cells (Fig. 6C–F). Collectively, these findings indicated that the inhibition of NRF2 could markedly sensitize GSTZ1-deficient hepatoma cells to sorafenib treatment. Furthermore, targeting GPX4 also improved the response of hepatoma cells to sorafenib.

Reagents and antibodies

Erastin (HY-15763), Ferrostatin-1 (HY-100579), Deferoxamine (HY-B0988), and Necrosulfonamide (HY-100573) were purchased from MedChemExpress (MCE; Shanghai, China). Sorafenib (S7397), ZVAD-FMK (S7023), Bafilomycin A1 (S1413), and RSL3 (S8155) were obtained from Selleckchem (Houston, TX, USA). N-acetyl-L-cysteine (NAC, S0077) was from Beyotime (Shanghai, China). Brusatol (Bru, MB7292) was obtained from Meilunbio (Dalian, China). Tertiary butylhydroquinone (tBHQ, 112941) was obtained from Sigma (Shanghai, China). Antibodies raised against GPX4 (ab125066), NRF2 (ab62352), 4-HNE (ab46545), NQO1 (ab34173), and β-actin (ab6276) were obtained from Abcam (Cambridge, MA, USA), anti-SLC7A11 (NB300-318) was from Novusbio (Centennial, CO, USA), anti-FTL (10727-1-AP) was from Proteintech (Shanghai, China), and anti-GSTZ1 (GTX106109) was from GeneTex (San Antonio, CA, USA).

Generation of SR cell lines

To establish SR cells, HepG2 and SNU449 cells were cultured by exposing cells with sorafenib at 5% of IC₅₀ concentration and the concentration was gradually increased at 10% of IC₅₀ until the maximum tolerated doses (10μM) was reached. SR cells (HepG2-SR and SNU449-SR) were cultured continuously at 1μM concentration of sorafenib to maintain the acquired resistance.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from HCC cell lines using TRIzol reagent (Invitrogen, Rockville, MD, USA) according to the manufacturer’s instructions. The purified RNA sample was reverse-transcribed into cDNA using the PrimeScript™ RT Reagent Kit (RR047A, TaKaRa, Tokyo, Japan) with gDNA Eraser. Complementary DNA from cell samples was amplified with the specific primers (Supplementary Table. 1). Briefly, Real-time qPCR was performed to quantity mRNA levels, using the SYBR Green qPCR Master Mix (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. The CT values were normalized to that of β-actin, and the relative mRNA expression levels of genes were calculated using 2^–ΔΔCt method. Each sample was analyzed in triplicate.

Western blot analysis

Protein samples from cells and animal tissues were lysed in Cell Lysis Buffer (Beyotime, Jiangsu, China) containing 1mM of phenylmethanesulfonyl fluoride (PMSF, Beyotime). The concentration of the protein homogenates was determined using the BCA assay Kit (Dingguo, Beijing, China). Equal volumes of protein samples were separated by SDS-poly acrylamide gel electrophoresis and electro-transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocked with 5% non-fat milk dissolved in TBST (10mM Tris, 150mM NaCl, and 0.1% Tween-20; pH 7.6), for 2h at room temperature, the membranes were incubated with the primary antibodies overnight at 4°C. Thereafter, membranes were incubated with the secondary antibodies coupled to horseradish peroxidase (HRP) for 2h at room temperature. Protein bands were visualized with enhanced Chemiluminescence substrate Kits (ECL, New Cell & Molecular Biotech Co, Ltd, China).

Transmission electron microscope assay

Cells were collected and fixed with 2.5% glutaraldehyde. Subsequently, cells were post-fixed in 2% tetroxide and dehydrated through a series of gradient ethanol. Samples were embedded in epoxy resin, cut into thin slices, and placed onto nickel grid. Images were acquired using Hitachi-7500 transmission electron microscope (Hitachi, Tokyo, Japan).

Intracellular ROS measurements

Cells were planted on coverslips in a 12-well plate, and then treated with the varying concentrations of test compound or drug. After 24h, cells were incubated at a final concentration of 5µM CellROX® Orange reagent (Life Technologies, Carlsbad, USA) for 30min at 37°C, after which they were washed, dyed with DAPI, mounted with Anti-fade Mounting Medium, and immediately analyzed for fluorescence intensity under Leica Confocal Microscope (TCS SP8, Germany) with a ×40 objective lens.

Measurement of total iron contents in hepatoma cells and liver tissues

The iron concentration was assessed using the Iron Assay Kit (MAK025; Sigma) according to the manufacturer’s protocol. Briefly, tissues (10mg) or cells (2×10⁶) were rapidly homogenized in 5 volumes of iron assay buffer. Tissues or cell homogenates were centrifuged at 15,000×g for 10min at 4°C and removed insoluble material. To measure total iron, add 50μL samples to 96-well plate, increase the volume to 100μL per well with iron assay buffer, and then add 5μL iron reducer to each well to reduce Fe³⁺ to Fe²⁺. And then samples were mixed using a horizontal shaker and incubated at 25°C for 30min. Subsequently, 100μL Iron Probe was added and incubated for 1h at 25°C. During each incubation, the plate was kept from light. Thereafter, the absorbance was detected at 593nm using a microplate reader.

Detection of malondialdehyde (MDA)

Analysis of lipid peroxidation was assessed by quantification of MDA concentration in cell lysates using Lipid Peroxidation MDA Assay Kit (S0131, Beyotime) in accordance with the manufacturer’s instructions.

Reduced GSH assay measurement

Reduced GSH levels were determined using the GSH and GSSG Assay Kit (Beyotime, S0053) following the manufacturer’s protocol.

Reporter plasmids and mutagenesis

Human Nrf2 expression plasmid was constructed by subcloning PCR amplification and inserted it into pcDNA-5Myc vector (a gift from prof. Wei Lv, Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences). Human Gstz1 and Keap1 expression plasmids were constructed by subcloning PCR amplification and inserted into pSEB-3Flag vector (a gift from Dr T-C He, University of Chicago, USA). The Ser to Ala mutant of Gstz1 (S14A) was constructed by site-directed mutagenesis, with WT Gstz1-3Flag plasmid as a template.

Cell growth curve and cell viability assay

For cell growth curve analysis, cells were seeded at 1×10⁴ cells/well in 96-well microtiter plates with three replicate per group and cultured overnight in a humidified incubator at 37°C and 5% CO₂. The plate was scanned and phase-contrast images were acquired after over a series of time points post treatment, and then quantified time-lapse curves were plotted using IncuCyte ZOOM software (Essen BioScience, Ann Arbor, MI, USA).

For cell viability assay, cells were seeded at 1000 cells per well in 96-well plates with fresh medium and analyzed by using the Cell Counting Kit-8 (CCK-8) (CK04, Dojindo, Japan) according to the manufacturer’s instructions. The microplates were incubated at 37°C for an additional 2h. Absorbance (Abs) was read at 450nm using a microplate reader (Thermo Fisher, USA). The blank group only contained medium, and the cells without any treatment were used as the control group. Herein, cell viability=(Abs of the experimental group-Abs of the blank group)/(Abs of the control group-Abs of the blank group) × 100%.

Cell growth rate and cell death

Cell growth was analyzed at 24, 48, 72 or 96h post culture. The cell growth rate was calculated by dividing the final number of cells by the number of cells seeded at day 0 and expressed as a percentage. Cell death was analyzed by propidium iodide (Invitrogen, USA) staining followed by flow cytometry.

Half-maximal inhibitory concentration assay (IC₅₀)

The cells were planted in 96-well plates at 1.0×10⁴ cells per well. The corresponding concentrations of sorafenib were given to cells for 24h. After 24h, CCK-8 (Dojindo, Japan) was used to measure sorafenib sensitivity at 450nm using a microplate reader (Thermo Fisher, USA) after incubating for 2h at 37°C.

Animal experiments

Heterozygous 129-Gstz1^tm1Jmfc/Cnbc mice (EM: 04481) were obtained from the European Mouse Mutant Archive and were crossed to breed wild-type (WT) and Gstz1^−/− mice. All mice were maintained under individual ventilation cages conditions in the laboratory animal center of Chongqing Medical University. For subsequent studies, mice were divided into five groups as follows: WT+DMSO (control), WT+Sora, Gstz1^−/−+DMSO, Gstz1^−/−+Sora, and Gstz1^−/−+Sora+RSL3. Each group included three male and three female mice. At 2 weeks of age, all mice were administered an intraperitoneal injection of diethylnitrosamine (DEN; Sigma, St. Louis, MO, USA) at a dose of 75mg/kg. At the third week, the mice were intraperitoneally administered carbon tetrachloride (CCl₄; Macklin, Shanghai, China) at 2ml/kg twice a week for 12 weeks. In the WT+Sora and Gstz1^−/−+Sora group, the mice at 22 weeks were administered intraperitoneally sorafenib (30mg/kg) every 2 days for 4 weeks until euthanasia. In the Gstz1^−/−+Sora+RSL3 group, in addition to sorafenib administration as described above, the mice were injected intraperitoneally with RSL3 (10mg/kg) every 2 days for 4 weeks at the same weeks. Body weight of each mice was measured every week and retro-orbital blood was collected before sacrifice. All mice were euthanized at 26 weeks of age. The liver weight and number of liver tumors were measured. Protein and mRNAs levels of hepatic tumors were detected by western blotting and qRT-PCR analysis, respectively. The intrahepatic Iron and MDA levels were measured with Iron Assay Kit and MDA Assay Kit, respectively. Samples of liver tumor were collected for further study or fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned for hematoxylin-eosin staining (H&E) and immunohistochemistry. All animal procedures were approved by the Research Ethics Committee of Chongqing Medical University (reference number: 2017010).

We wish to thank Dr. T.-C. He (University of Chicago, USA) for providing the plasmids pAdEasy system, and Prof. Ding Xue (Tsinghua University) for supplying the CRISPR/Cas9 system.