β-Elemene induced ferroptosis via TFEB-mediated GPX4 degradation in EGFR wide-type non-small cell lung cancer

Abstract

Introduction: β-Elemene (β-ELE), derived from Curcuma wenyujin, has anticancer effect on non-small cell lung cancer (NSCLC). However, the potential target and detail mechanism were still not clear. TFEB is the master regulator of lysosome biogenesis. Ferroptosis, a promising strategy for cancer therapy could be triggered via suppression on glutathione peroxidase 4 (GPX4). Weather TFEB-mediated lysosome degradation contributes to GPX4 decline and how β-ELE modulates on this process are not clear.

Objectives: To observe the action of β-ELE on TFEB, and the role of TFEB-mediated GPX4 degradation in β-ELE induced ferroptosis.

Methods: Surface plasmon resonance (SPR) and molecular docking were applied to observe the binding affinity of β-ELE on TFEB. Activation of TFEB and lysosome were observed by immunofluorescence, western blot, flow cytometry and qPCR. Ferroptosis induced by β-ELE was observed via lipid ROS, a labile iron pool (LIP) assay and western blot. A549^{TFEB KO} cells were established via CRISPR/Cas9. The regulation of TFEB on GPX4 and ferroptosis was observed in β-ELE treated A549^WT and A549^{TFEB KO} cells, which was further studied in orthotopic NOD/SCID mouse model.

Results: β-ELE can bind to TFEB, notably activate TFEB, lysosome and transcriptional increase on downstream gene GLA, MCOLN1, SLC26A11 involved in lysosome activity in EGFR wild-type NSCLC cells. β-ELE increased GPX4 ubiquitination and lysosomal localization, with the increase on lysosome degradation of GPX4. Furthermore, β-ELE induced ferroptosis, which could be promoted by TFEB overexpression or compromised by TFEB knockout. Genetic knockout or inactivation of TFEB compromised β-ELE induced lysosome degradation of GPX4, which was further demonstrated in orthotopic NSCLC NOD/SCID mice model.

Conclusion: This study firstly demonstrated that TFEB promoted GPX4 lysosome degradation contributes to β-ELE induced ferroptosis in EGFR wild-type NSCLC, which gives a clue that TFEB mediated GPX4 degradation would be a novel strategy for ferroptosis induction and NSCLC therapy.

Keywords: Ferroptosis; Lysosome; Non-small cell lung cancer; TFEB; β-Elemene.

Graphical abstract

Fig. 1

β-ELE binds to TFEB and induces TFEB activation. (A) Different concentrations of β-ELE (50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM) flowed over the surface of a CM5 chip immobilized with TFEB protein. Interaction profile is recorded in real-time in a sensorgram. (B) The binding curves from (A). (C) Molecular docking simulation of β-ELE to HLH-ZIP region of TFEB by the Autodock Vina program. It showed the interaction of β-ELE (shown in orange color and stick model) with active site residues of TFEB (grey). The amino acids in position 308, 309, 312, 313 forms an H-bond with β-ELE (Hydrogen bonds were represented as grey dotted lines and their distances were labeled in angstrom). (D) The sensorgram of vehicle with HLH-ZIP region of TFEB. (E) β-ELE treated on A549, NCI-H460, and SPC-A-1 cells for 6, 12, 24, and 48 h, the protein expressions of TFEB, p-TFEB (Ser122), p-TFEB (Ser211) and 14-3-3 were analyzed by western blot. (F) β-ELE treated on A549, NCI-H460, and SPC-A-1 cells for 12, 24, and 48 h, the protein expression of TFEB in nuclei and cytosol were detected by western blot. α-tubulin served as a cytoplasmic marker protein, Lamin B1 served as a nuclear marker protein. (G) β-ELE treated on A549, NCI-H460, and SPC-A-1 cells for 24 h, the localization of TFEB (red fluorescence) in nuclei (blue fluorescence) was observed by immunofluorescence. Bar = 10 μm. Data are presented as means ± SEM from three independent experiments.

Fig. 2

β-ELE promotes lysosomal biogenesis. (A, D, G) β-ELE treated on A549 (A), NCI-H460 (D), and SPC-A-1 (G) cells for 24 h. The fluorescent signals were detected by confocal microscopy after staining with LysoTracker Red (50 nM) for 30 min. Bar = 10 μm. (B, E, H) β-ELE treated on A549 (B), NCI-H460 (E), and SPC-A-1(H) cells for 24 h. Cells were then loaded with LysoTracker Red (50 nM) for 30 min. Fluorescence intensity of 10,000 cells per sample was measured by flow cytometry. (C, F, I) β-ELE treated on A549 (C), NCI-H460 (F), and SPC-A-1 (I) cells for 24 h, the amount of mRNA encoding MCOLN1, SLC26A11, and GLA were determined by real-time PCR. mRNA was normalized with ACTB. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3

β-ELE promoted GPX4 degradation in NSCLC cells. (A) Quantification of GPX4 mRNA levels by qPCR in A549 cells treated with β-ELE (120 μg/mL) for different time points (24, 48 h). mRNA was normalized with ACTB, and “ns” means no statistical difference. (B) A549 cells were treated with β-ELE (120 μg/mL) after with or without the pretreatment of proteasome inhibitor MG132 (1 μM) or lysosomal degradation inhibitor BafA1 (160 nM)、CQ (20 μM) for 1 h. The changes of SLC7A11 and GPX4 protein levels were detected by western blot. (C) A549 cells were transfected with GPX4-GFP (10 μg/10 cm dish), then treated with β-ELE (120 μg/mL) for 6, 12, and 24 h. Cells were harvested and lysed with RIPA buffer, cell lysates were then subjected to immunoprecipitation (IP) via GFP-nanobeads and analyzed by western blotting with antibodies as indicated (GPX4, ubiquitin). (D) β-ELE (120 μg/mL) treated on A549 cells for 24 h, the co-localization of LAMP1 and GPX4 was detected by immunofluorescence. A green fluorescent signal representing GPX4 was distributed in both the cytoplasm and nucleus, and the red fluorescent signal representing LAMP1 was mainly distributed in the cytoplasm. Bar = 10 μm. (E) A549 cells were treated with β-ELE (120 μg/mL) for 24 h. Lysosomes and lysosome-free cell lysates were collected, the protein level of GPX4 was measured by western blot. LAMP1 and GAPDH were used as loading controls of lysosomal and non-lysosomal fractions, respectively. (F) Co-localization coefficients in (D) were calculated by Olympus Fluoview FV31S-DT Software. Each point represents the mean ± SEM, n = 4, *P < 0.05.

Fig. 4

TFEB promotes GPX4 degradation in β-ELE treated NSCLC. (A) A549 and A549^{TFEB KO} cells were transfected with or without TFEB (10 μg/10 cm dish) plasmid for 24 h, then treated with β-ELE (120 μg/mL) for 24 h. The protein level of TFEB and GPX4 were detected by western blot. (B) A549 cells transfected with or without TFEB^{S122A, S211A} (10 μg/10 cm dish), TFEB^{S122D, S211D} (10 μg/10 cm dish), then treated with β-ELE (120 μg/mL) for 24 h. S122A and S211A are dephosphorylation-mimicking mutations of TFEB, while S122D and S211D are phosphorylation-mimicking mutations of TFEB. The protein level of TFEB and GPX4 were detected by western blot. (C, D) A549 cells were co-transfected with GPX4-GFP (10 μg/10 cm dish) and TFEB (10 μg/10 cm dish) (C), A549 and A549^{TFEB KO} cells were transfected with GPX4-GFP (10 μg/10 cm dish) (D), then treated with β-ELE for 24 h. Cells were lysed with RIPA buffer, cell lysates were then subjected to IP via GFP-nanobeads and analyzed by western blot with antibodies as indicated (TFEB, GPX4). (E) A549 and A549^{TFEB KO} cells were treated with β-ELE (120 μg/mL) for 24 h, the co-localization of GPX4 and LAMP1 was detected by immunofluorescence. Bar = 10 μm. (F) A549 cells and A549^{TFEB KO} cells were treated with or without β-ELE (120 μg/mL) for 24 h. Lysosomes and lysosome-free cell lysates were collected and the protein level of GPX4 was measured by western blot. LAMP1 and GAPDH were used as loading controls of lysosomal and non-lysosomal fractions respectively. (G) Co-localization coefficients from (E) were calculated by Olympus Fluoview FV31S-DT Software. Each point represents the mean ± SEM. n = 3, ***P < 0.001.

Fig. 5

β-ELE induces ferroptosis in A549 cells. (A) A549 cells were treated with β-ELE (120 μg/mL) for 24 h, and stained with BODIPY™ 581/591 C11 (10 μM) for 30 min. Upon oxidation, its excitation maximum shifts from 581 to 500 nm and the emission maximum shifts from 591 to 510 nm. The level of lipid peroxidation was detected by flow cytometry. (B) The level of lipid peroxidation from (A) was calculated by FlowJo. (C) A549 cells were treated with β-ELE for 24 h, CA-AM was added to cells at the final concentration of 0.5 µM, followed by adding iron chelator deferiprone (DFP, 100 μM) for 1 h or left untreated. The mean fluorescence was detected fluorescence microplate reader (Exc = 488 nm, Em = 525 nm). (D) The amount of LIP was reflected via difference on mean fluorescence of each sample with or without deferiprone from (C). (E) A549 cells were treated with β-ELE for 6, 12, 24 and 48 h, the protein level of SLC7A11, GPX4, Ferritin and FTH were detected by western blot. GAPDH was used as a loading control. (F) A549 cells were treated with β-ELE after 1 h in the presence or absence of ferroptosis inhibitors Ferrostatin-1 (2 µM) or Liproxstatin-1 (50 nM). Cell viability was detected by MTT assay. Each point represents the mean ± SEM. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6

TFEB promotes β-ELE induced ferroptosis in NSCLC. (A) β-ELE (120 µg/mL) was applied to A549 and A549^{TFEB KO} cells for 24 h, the proliferation inhibition of the cells was detected by MTT assay. (B) A549 and A549^{TFEB KO} cells were transfected with Sh-GPX4 (constructed in psi-LVRU6MP vector with mCherry, Gene ID = 2879, HSH118768-LVRU6MP, GeneCopoeia) for 8 h, then removed medium and treated with β-ELE (120 µg/mL) for 24 h. Cells were stained with BODIPY™ 581/591 C11 (10 μM) for 30 min. After washed cells with three times of PBS, cells were resuspended in PBS at the density of 0.5 × 10⁶ cells/mL, the level of lipid peroxidation was detected by flow cytometry. (C) A549 cells were transfected with vector (pcDNA3.1) or TFEB, then treated β-ELE (120 µg/mL) for 24 h, the inhibition on proliferation was detected by MTT assay. (D) A549 cells were transfected with TFEB or GPX4, or co-transfected with GPX4 and TFEB, then treated with β-ELE (120 µg/mL) for 24 h, and the process of detecting lipid peroxidation is the same as that in (B). (E) A549 cells were transfected with vector (pcDNA3.1) or TFEB, then treated with β-ELE (120 μg/mL) for 24 h, the protein level of TFEB, SLC7A11 and GPX4 were detected by western blot. GAPDH was used as a loading control. (F) The transfection and drug treatment were same as in (E). The fluorescent signals were detected by flow cytometry after staining with LysoTracker Red (50 nM) for 30 min. Each point represents the mean ± SEM.n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7

Knock out of TFEB attenuates anticancer effect of β-ELE in orthotopic NSCLC NOD/SCID mouse model. Male NOD/SCID mice aged 4–5 weeks old were used in the study. A549-luci or A549^{TFEB KO}-luci cells (1 × 10⁶ in 100 μL 0.9% normal saline) were injected into the right lung of each mouse. After five days later, the mice were divided into 4 groups (6 mice pre group) and administered with β-ELE (120 mg/kg) by tail vein injection. On day 21 after administration, mice were sacrificed and tumor tissue samples were frozen in -80 °C or fixed in 4% PFA for IF or IHC respectively. The experiment was performed as described in materials and methods. (A) Representative images of bioluminescence at indicated time points (Day1, Day21) after β-ELE administration. (B) Bioluminescence intensity of mice. The signals are expressed in radiance (Ph/s). n = 6. (C) Co-localization of GPX4 and LAMP1 in tumor was detected by immunofluorescence. Bar = 10 μm. (D) Co-localization coefficients from (C) were calculated by Olympus Fluoview FV31S-DT Software. n = 4. (E) Immunohistochemical staining analysis. PCNA positive cells in the sections are stained brown. The nuclei are stained blue. Representative figures from each group are shown. (F) Each tumor tissue section was randomly selected for the calculation of the mean IOD value of the positive region through Image Pro Plus software, which indicated the amount of PCNA expression. Bar = 50 μm. Each point represents the mean ± SEM. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary figure 1

Figure S1. β-ELE statistically decreased the P-TFEB and increase the amount of nuclear TFEB, and had no effect on mRNA level of TFEB in NSCLC cells. (A) Semi-quantitative analysis of the expression of TFEB, p-TFEB (Ser122), p-TFEB (Ser211) and 14-3-3 in A549, NCI-H460, SPC-A-1 cells treated without or with β-ELE for different times (0, 6, 12, 24, or 48 h) in figure 1E. GAPDH was used as an internal standard for protein loading. (B) Semi-quantitative analysis of the expression of TFEB in nucleus and cytosol in A549, NCI-H460, and SPC-A-1 cells treated with or without β-ELE for 12, 24, and 48 h in figure 1F. Lamin B1 and α-tubulin were used as loading controls of the nuclear and cytoplasmic fraction respectively. (C) A549, NCI-H460, and SPC-A-1 cells were treated with β-ELE for 24 and 48h, the amount of mRNA encoding TFEB were determined by qPCR. The result is expressed as a fold change of the control. The values are presented as the means ± SEM. n= 3. *P<0.05, **P<0.01, ***P<0.001.

Supplementary figure 2

Figure S2. Lysosome inhibitors statistically decreased the expression of GPX4 in β-ELE treated A549 cells, and β-ELE statistically increased the expression of lysosomal GPX4 and the ubiquitination of GPX4. (A) Semi-quantitative analysis of the expression of SLC7A11 and GPX4 in A549 cells or cells treated with β-ELE in the presence or absence of MG132, BafA1 or CQ in figure 3B. GAPDH was used as an internal standard for protein loading. (B) Semi-quantitative analysis of the expression of ubiquitin in GPX4 transfected A549 cells without or with β-ELE for different times (0, 6, 12, 24h) in figure 3C. (C) Semi-quantitative analysis of the expression of GPX4 in lysosome and lysosome free fraction in β-ELE treated or untreated A549 cells in figure 3E. LAMP1 and GAPDH were used as loading controls of lysosome and lysosome free fractions respectively. The result is expressed as a fold change of the control. The values are presented as the means ± SEM. n= 3. *P<0.05, **P<0.01, ***P<0.001.

Supplementary figure 3

Figure S3. β-ELE statistically increased β-ELE induced decline, ubiquitination and lysosomal expression of GPX4.(A) Validation of TFEB knockout cell lines. CRISPR/Cas9-derived TFEB knockout A549 cells and WT A549 cells were lysed, and protein lysates were immunoblotted for TFEB. Immunoblotting of GAPDH served as a loading control. (B) Semi-quantitative analysis of the expression of TFEB and GPX4 in β-ELE untreated or treated A549 cells and A549^{TFEB KO} cells transfected with or without TFEB in figure 4A. GAPDH were used as loading control. (C) Semi-quantitative analysis of the expression of TFEB and GPX4 in β-ELE untreated or treated A549 cells and cells transfected with TFEB^{S122A, S211A} or TFEB^{S122D, S211D} in figure 4B. GAPDH was used as loading control. (D) Semi-quantitative analysis of the expression of ubiquitin in GPX4 transfected or GPX4 and TFEB co-transfected A549 cells in the absence or presence of β-ELE in figure 4C. (E) Semi-quantitative analysis of ubiquitin in GPX4 overexpressed A549 and A549^{TFEB KO} cells in the absence or presence of β-ELE in figure 4D. (F) Semi-quantitative analysis of the expression of GPX4 in lysosome and lysosome free fractions extracted from A549 cells and A549^{TFEB KO} cells treated with or without β-ELE in figure 4F. LAMP1 and GAPDH were used as loading controls of lysosome and lysosome free fractions respectively. The result is expressed as a fold change of the control. The values are presented as the means ± SEM. n= 3, *P<0.05, **P<0.01, ***P<0.001.

Supplementary figure 4

Figure S4 β-ELE statistically decreased ferroptosis related proteins. Relative density of protein bands of SLC7A11, GPX4, Ferritin, FTH in figure 5E were quantified, and normalized to GAPDH. The result is expressed as a fold change of the control. The values are presented as the means ± SEM. n= 3, *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary figure 5

Figure S5. β-ELE induces ferroptosis in NCI-H460 cells. (A) NCI-H460 cells were treated with β-ELE for 24 h, and stained with BODIPY™ 581/591 C11 (10 μM) for 30 min. The level of lipid peroxidation was detected by flow cytometry. (B) The level of lipid peroxidation from (A) was calculated by FlowJo. (C) NCI-H460 cells were treated with β-ELE for 24 h, CA-AM was added to cells at the final concentration of 0.5 µM, followed by adding iron chelator deferiprone (100 μM) for 1 hour or left untreated. The mean fluorescence was detected fluorescence microplate reader (Exc=488 nm, Em=525 nm). (D) The amount of LIP was reflected via difference on mean fluorescence of each sample with or without deferiprone from (C). (E) NCI-H460 cells were treated with β-ELE for 6, 12, 24 and 48 h, the protein levels of SLC7A11, GPX4, Ferritin and FTH were detected by western blot. (F) NCI-H460 cells were treated with β-ELE in the presence or absence of ferroptosis inhibitors Ferrostatin-1 or Liproxstatin-1 for 1 h. Cell viability was detected by MTT assay. (G) Semi-quantitative analysis of the expression of SLC7A11, GPX4, Ferritin, FTH in figure S5E. The values are presented as the means ± SEM. n= 5, *P<0.05, **P<0.01, ***P<0.001.

Supplementary figure 6

Figure S6. β-ELE induces ferroptosis in SPC-A-1 cells. SPC-A-1 cells were treated with β-ELE for 24 h, and stained with BODIPY™ 581/591 C11 (10 μM) for 30 min. The level of lipid peroxidation was detected by flow cytometry. (B) The level of lipid peroxidation from (A) was calculated by FlowJo. (C) SPC-A-1 cells were treated with β-ELE for 24 h, CA-AM was added to cells at the final concentration of 0.5 µM, followed by adding iron chelator deferiprone (100 μM) for 1 hour or left untreated. The mean fluorescence was detected fluorescence microplate reader (Exc=488 nm, Em=525 nm). (D) The amount of LIP was reflected via difference on mean fluorescence of each sample with or without deferiprone from (C). (E) SPC-A-1 cells were treated with β-ELE for 6, 12, 24 and 48 h, the protein level of SLC7A11, GPX4, Ferritin and FTH were detected by western blot. (F) SPC-A-1 cells were treated with β-ELE in the presence or absence of ferroptosis inhibitors Ferrostatin-1 or Liproxstatin-1 for 1 h. Cell viability was detected by MTT assay. (G) Semi-quantitative analysis of the expression of SLC7A11, GPX4, Ferritin, FTH in figure S5E. The values are presented as the means ± SEM.n= 3, *P<0.05, **P<0.01, ***P<0.001.

Supplementary figure 7

Figure S7. TFEB inactivation and lysosome inhibition compromise β-ELE induced cytotoxicity, and TFEB statistically increased β-ELE induced decline on GPX4 in NSCLC cells. (A) A549 cells were transfected with pcDNA, pcDNA-TFEB^S122D, pcDNA-TFEB^S211D, pcDNA-TFEB^{S211D, S122D}, then treated with β-ELE (120 µg/mL) for 24 h, cell viability was detected by MTT assay. (B) Semi-quantitative analysis of TFEB, SLC7A11 and GPX4 in β-ELE untreated or treated A549 cells and cells transfected with TFEB in figure 6E. (C) A549 cells were treated with β-ELE with or without proteasome inhibitor MG132, lysosomal inhibitors CQ, BafA1 for 24 h, the changes of cell viability were detected by MTT assay. n = 6, *P<0.05,**P<0.01, ***P<0.001.

Supplementary figure 8

Figure S8. β-ELE has no obvious effect on autophagy related proteins, and mainly induces K63-polyubiquitination in A549 cells. (A) A549 cells treated β-ELE or 6, 12, 24, and 48 h, the protein expressions of cleaved-PARP, Bcl-2, LC3, Atg7, Atg5 and P62 were analyzed by western blot. (B)A549 cells were co-transfected with GPX4-GFP and HA-Ub-WT, or GPX4-GFP and HA-K48-Ub, or GPX4-GFP and HA-K63-Ub. Then, β-ELE (120 μg/mL) applied on cells for 24 h. Cells were lysed with RIPA buffer, cell lysates were then subjected to immunoprecipitation via GFP-nanobeads and analyzed by western blot with antibodies as indicated.