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. 2025 Feb 21;15:6422. doi: 10.1038/s41598-025-91202-w

Endogenous IL-33 inhibits apoptosis in non-small cell lung cancer cells by regulating BCL2/BAX via the ERK1/2 pathway

Liping Liu 1,2, Haoge Luo 1,2, Yingdong Xie 1,2, Ying Wang 1,2, Shiying Ren 1,2, Haiyang Sun 1,2, Zhuoyuan Xin 1,✉, Dong Li 1,2,✉
PMCID: PMC11845513  PMID: 39984631

Abstract

Lung cancer remains a leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for 85% of cases. Although targeted therapies have improved treatment outcomes, drug resistance poses a significant challenge, underscoring the need for novel therapeutic strategies. Interleukin-33 (IL-33), a member of the IL-1 superfamily, functions both as a nuclear protein and a cytokine, binding to its receptor, ST2. While IL-33 is known to promote tumour cell migration and metastasis, its role in regulating apoptosis remains incompletely understood. In this study, we focused on endogenous IL-33, employing lentiviral transfection to overexpress both the full-length and mature forms of IL-33 in lung cancer cells. We examined its effects on apoptosis in vitro and investigated the underlying molecular mechanisms. Our findings reveal that endogenous IL-33 inhibits apoptosis in lung cancer cells by modulating the expression of BCL2 and BAX via the ERK1/2 pathway in an autocrine manner. These results uncover a novel mechanism of IL-33-mediated tumour survival and provide a foundation for the development of IL-33/ST2-targeted therapies in NSCLC.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-91202-w.

Keywords: IL-33, ST2, Non-small-cell lung cancer, Apoptosis

Subject terms: Lung cancer, Cytokines

Introduction

Lung cancer remains a leading cause of morbidity and mortality worldwide, with approximately two and a half million new cases diagnosed annually and nearly two million deaths attributed to it each year1. Lung cancer accounts for 12.4% of new cancer cases and 18.7% of all cancer-related deaths globally2. It is classified into two main subtypes, small cell lung cancer (SCLC), which accounts for 15%, and non-small cell lung cancer (NSCLC), which constitutes 85% 3. Traditional therapies, such as surgery and chemotherapy, remain effective treatment options; however, immunotherapy is increasingly garnering attention as a promising alternative4, 5. However, resistance to targeted therapies often develops in some NSCLC patients, limiting their efficiency6, 7. As a result, the prognosis for NSCLC patients remains unsatisfactory, necessitating new therapeutic approaches to optimise treatment8.

Interleukin-33 (IL-33), a member of the IL-1 superfamily, functions as both a nuclear transcription factor and a released cytokine9, 10. Under physiological conditions, IL-33 is stored in the cell nucleus in its full-length (flIL-33) form to maintain barrier function11. When cells are damaged or under stress, flIL-33 is released from the nucleus and cleaved into a more bioactive mature form by enzymes such as neutrophil elastase or cathepsin G12. In this processed form, mature (m) IL-33 acts as an alarmin cytokine by binding to its receptor, ST2 13. Mature IL-33 plays a critical role in various inflammatory diseases, including asthma, arthritis, and inflammatory bowel disease14,15– 17. The functions of flIL-33 are still largely unknown, but there are reports indicating that nuclear flIL-33 also exerts negative regulatory functions in immune cells as a transcription factor18, 19.

Studies have identified that the IL-33/ST2 axis promotes the growth and metastasis of colorectal, breast, gastric, and ovarian cancers20–26. Recent research has also highlighted its potential role in the progression and prognosis of NSCLC27. Previous studies have demonstrated that IL-33 significantly enhanced cell migration and invasiveness via the AKT pathway28. However, it remains unclear whether these effects are driven by exogenous or endogenous IL-33, particularly the functions of full-length IL-33 (flIL-33). The role of endogenous IL-33 in the apoptosis of lung cancer cells also remains largely unexplored.

In this study, we investigated the effect of endogenous IL-33 on apoptosis of lung cancer cells. Our results demonstrated that endogenous IL-33 significantly inhibited the apoptosis in A549 cells by regulating BCL2 and BAX expression through the ERK1/2 pathway in an ST2-dependent manner. These findings offer novel insights into the mechanisms underlying IL-33-mediated pro-tumour effects and may help identify new therapeutic targets for NSCLC.

Materials and methods

Cell culture

The Lung cancer cell line cells A549 and LLC1 were grown in Dulbecco’s Modified Eagle Medium (DMEM, BI, Israel) supplemented with 10% fetal bovine serum (FBS, BI, Israel) and 1% penicillin/streptomycin (Solarbio, China). The cells were maintained in an incubator (Thermo Fisher Scientific, MA, US) at 37 °C with 5% CO₂ in a humidified atmosphere. Cells were acquired from our laboratory and genotypic authentication was confirmed for both cell lines.

Lentivirus infection

Human Control lentivirus, flIL33 (1-270 amino acids) lentivirus, and cIL33 (112–270 amino acids) lentivirus were purchased from Sangon Biotech (Shanghai) Co., Ltd. Similarly, mouse control lentivirus, flIl33 (1-266 amino acids) lentivirus, and cIl33 (109–266 amino acids) lentivirus were purchased from Sangon Biotech (Shanghai) Co., Ltd. The plasmid vector was labelled with HA. Lentiviruses were transfected into A549 and LLC1 cells using polybrene (Yeasen, China). Following 72 h of transfection, puromycin (Sigma-Aldrich, MI, US) was added to select the transfected cells. After one week of selection, monoclonal cell populations with stable expression were established.

Small interfering RNA (siRNA) transfection

Control siRNA and ST2 siRNA were constructed in Shanghai Genechem Co., Ltd. A total of 5 × 105 A549 cells were seeded into 6-well plates (Nest, China) and incubated overnight. Cells were then transfected with control siRNA or ST2 siRNA using Lipofectamine 2000 (Thermo Fisher Scientific, MA, US) following the manufacturer’s instructions. Knockdown efficiency was assessed 48 h post-transfection by Western blot analysis. The sequence of the ST2 siRNA was as follows: 5’-GCCCTGAATTTGCATGGCTTG-3’.

Experimental grouping

To study the effects of IL-33 on apoptosis in lung cancer cells, lentivirus was transfected into A549 and LLC1 cells. To investigate the role of ST2, ST2 siRNA or Control siRNA (Ctrl siRNA) was transfected into A549 cells. After 48 h of transfection, ST2 siRNA (+) or Ctrl siRNA (-) were stimulated with the conditioned medium (CM) from the supernatants of Ctrl (-) and cIL33 (+) cells for another 48 h. To explore the role of ERK1/2, Ctrl, flIL33, and cIL33 cells were treated with an ERK inhibitor (FR 180204, 10 µM, Beyotime, China) or Dimethylsulfoxide (DMSO, Solarbio, China) for 48 h.

Immunofluorescence (IF)

A total of 1 × 105 cells were seeded onto 24-well plates containing 14 mm slides (Biosharp, China) and incubated overnight. The slides were washed twice with PBS (Bioss, China) and then fixed with 4% paraformaldehyde (Biosharp, China) for 15 minutes. After fixation, the slides were washed again with PBS and incubated in permeabilisation solution (0.5% Triton-X 100 in PBS) on ice for 5 minutes. Subsequently, the slides were blocked with PBS containing 3% bovine serum albumin (BSA; Solarbio, China) for 1 hour. The slides were then incubated overnight at 4°C in a humidified chamber with HA antibody (Cell Signaling Technology, MA, US). Following incubation, the slides were washed three times with PBS and incubated with a secondary antibody (Cell Signaling Technology, MA, US) for 1 hour at room temperature in the dark. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; Beyotime, China) for 5 min, followed by two washes with PBS. Finally, the slides were mounted with coverslips before imaging with a microscope.

Cell proliferation

Cells were inoculated on 96-well plates (3 × 103/well), and starvated overnight in serum-free DMEM. Cell Counting Kit-8 (CCK-8, Bioss, China) was used to detect the absorbance values at 0, 24, 48, and 72 h following the manufacturer’s protocol.

Migration analysis

Cells were inoculated on 12-well plates (3 × 105/well) and starved overnight in serum-free DMEM to reach a confluent monolayer. Using a sterile micropipette tip, a straight, uniform scratch was created across the cell monolayer. The area of the scratch was marked with a marking pen for easy identification during imaging. Images of the wound-healing assay were captured at 0 and 72 h using a microscope. The cell migration distance was subsequently measured using ImageJ software (version 1.53, National Institutes of Health, USA).

Invasion assay

The invasion assay was conducted using Transwell inserts containing 8 μm membrane filters (LABSELECT, China). After overnight starvation in serum-free DMEM, cells were harvested by trypsinisation. Equal numbers of cells (2 × 10⁴/well) in serum-free medium were plated into the upper chamber, which was coated with Matrigel (BD Biosciences, USA). DMEM with 10% FBS was added to the lower chamber. After incubation for 24 h, the upper chamber was washed with PBS, fixed with 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet for 15 min. Non-invasive cells were carefully scraped from the upper chamber with cotton tips, and the invasive cells were counted under a microscope.

Quantification of apoptotic cell

The apoptosis assay was performed according to the instructions provided in the Apoptosis Assay Kit (Sungene Biotech, China). Briefly, cells were washed twice with cold PBS and then suspended in 1 mL of 1× Binding Buffer. The suspension was centrifuged at 300 g for 10 min. Cells were resuspended in 1 mL of 1 × Binding Buffer, and 100 µL of the cell suspension (1 × 105 cells) was added to each labelled tube. To the appropriate tubes, 5 µL of Annexin V-FITC was added. Each tube was gently vortexed and incubated for 10 min at room temperature, protected from light. Subsequently, 5 µL of 7-AAD solution was added and incubated for 5 min at room temperature, protected from light. PBS was then added to bring the volume to 500 µL, and the tubes were gently vortexed. The samples were analysed using a Guava easyCyte flow cytometer (Luminex, TX, US), and the results were analysed using FlowJo v10 (FlowJo software, OR, US).

Enzyme-linked immunosorbent assay (ELISA)

A total of 5 × 105 cells were inoculated on 6-well plates for 48 h, and the cell supernatant was collected. IL-33 in the supernatant from cells was quantitatively determined using ELISA following the manufacturer’s instructions (R&D Systems, US). The absorbance of each well was measured at 450 nm using a microplate reader (EPOCH2; BioTek, VT, US).

Western blot analysis

The total protein from cells was collected using RIPA buffer (Beyotime, China) and protease inhibitor (Roche, USA) at 4 °C for 10 min, followed by centrifugation at 10,000 g for 20 min. The supernatant was collected. The protein concentration was determined using a BCA Protein Quantification Kit (Biomed, China). All samples were separated using SDS-PAGE and then transferred to a PVDF membrane (Merck Millipore, Germany). The membranes were blocked with 5% skimmed milk (Yeasen, China) in Tris-buffered saline with Tween 20 (TBST, Solarbio, China) at room temperature for 2 h. The membranes were then incubated overnight at 4 °C with primary antibodies against ST2 (Novus, USA), ERK1/2 (Cell Signaling Technology, MA, US), p-ERK1/2 (Cell Signaling Technology, MA, US), JNK (ZenBio Science, China), p-JNK (Cell Signaling Technology, MA, US), P38 (ZenBio Science, China), p-P38 (Abcam, UK), BCL2 (Abcam, UK), BAX (ZenBio Science, China), and GAPDH (Proteintech, China). Afterwards, the PVDF membranes were washed three times with TBST for 10 min each, incubated with HRP-conjugated secondary antibody (Bioss, China) for 40 min at room temperature, and washed three more times with TBST on a shaker. Specific bands were detected using a chemiluminescence assay with ECL detection reagents (ZenBio Science, China). GAPDH was used as the loading control. The intensity of the Western blot signals was analysed using ImageJ software (version 1.53, National Institutes of Health, USA).

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

Total RNA was extracted using a Trizol reagent (Takara, Japan), and reverse-transcribed into cDNA using a cDNA Synthesis Kit (Tolobio, China). Real-time PCR was performed using an Agilent Mx3000P machine by mixing SYBR Green Master Mix (Takara, Japan). The human forward and reverse primer sequences for the test genes are shown below, GAPDH (forward 5’-GCACCGTCAAGGCTGAGAAC-3’ reverse 5’-GGATCTCGCTCCT GGAAGATG-3’), BCL2 (forward 5’-ATCGCCCTGTGGATGACTGA-3’), reverse 5’-GACAGCCAGGAGAAATCAAACAG-3’); BAX (forward 5’-TGACGGCAACTTCAACTGGG-3’, reverse 5’-AGCACTCCCGCCACAAAGA-3’); flIL33 (forward 5’-GCCTTGTGTTTCAAGCTGGG-3’, reverse 5’-CCAAAGGCAAAGCACTCCAC-3’); cIL33 (forward 5’-AGGTGACGGTGTTGATGGT-3’, reverse 5’-CCTGGTCTGGCAGTGGTTT-3’). The mouse forward and reverse primer sequences for the test genes are shown below, Gapdh (forward 5’-GACTTCAACAGCAACTCCCACTC-3’ reverse 5’-TAGCCGTATTCATTGTCATACCAG-3’), flIl33 (forward 5’-ACTATGAGTCTCCCTGTCCTG-3’, reverse 5’- ACGTCACCCCTTTGAAGC-3’); cIl33 (forward 5’- AGGTGCTACTACGCTACTATGA-3’, reverse 5’-TCTCATCTTTCTCCTCCACT-3’). The relative expression of the target mRNAs was calculated relative to the amount of GAPDH and Gapdh using the 2−ΔΔCt method.

RNA sequencing analysis

RNA sequencing analysis was performed by Personalbio co. ltd. In brief, the total RNA of Ctrl, flIL33, and cIL33 cells were extracted using Trizol Reagent (Takara, Japan). RNA integrity was first assessed by using the Agilent Bioanalyzer 2100. Only samples with clean rRNA profiles were used for subsequent steps. Paired-end sequencing was performed using the Illumina HighSeq 4000 instrument (Illumina Inc. CA. US).

Statistical analysis

All results were obtained from at least three replications. Statistical significance was tested using an unpaired two-tailed t-test. Results are presented as the mean ± standard deviation (SD). Analyses were performed with GraphPad Prism 8.0 statistical software (GraphPad Software, CA, US). A p-value less than 0.05 was considered statistically significant.

Results

Endogenous IL-33 inhibits apoptosis in lung cancer cells

To investigate the effect of IL-33 on the apoptosis in lung cancer cells and to eliminate potential ST2-independent functions of full-length IL-33 (flIL-33), we constructed lentiviruses encoding human full-length IL33 (flIL33) and a C-terminal domain of IL-33, devoid of the nuclear localisation domain (cIL33), as an artificial replicate for mIL-33. A549 cells were transfected with control (Ctrl), flIL33 or cIL33 lentiviruses. qPCR analysis confirmed that flIL33 and cIL33 were highly expressed in the corresponding transfected cells compared to the Ctrl group (Fig. 1A). Immunofluorescence staining using an HA antibody revealed that flIL-33 was localised in both the cytoplasm and nucleus of A549 cells, with predominant nuclear expression, while cIL-33 was restricted to the cytoplasm (Fig. 1B). These results confirm the successful establishment of stable overexpression of flIL-33 and cIL-33 in A549 cells.

Fig. 1.

Fig. 1

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Endogenous IL-33 inhibited the apoptosis of lung cancer cells. Transfecting control vector (Ctrl), flIL33 and cIL33 lentivirus into A549 cells. (A) qPCR detected the expression of flIL33 in Ctrl and flIL33 cells (left), and the expression of cIL33 in Ctrl and cIL33 cells (right); (B) Immunofluorescence detected the localisation of IL-33 expression constructs with HA tag in flIL33 and cIL33 cells. Representative images for HA-tagged proteins are shown in red, with DAPI in blue. Scale bar: 10 μm; (C-E) Ctrl, flIL33 and cIL33 cells were cultured for 48 h, and apoptosis of Ctrl, flIL33 and cIL33 cells was assessed; (C) Representative dot plots of Ctrl, flIL33 and cIL33 cells stained with annexin V/7-AAD were analysed by flow cytometry. Bar graphs show the mean ± SD of the percentage of apoptotic (Q2 + Q3) cells; (D) The expression of BCL2 and BAX in Ctrl, flIL33 and cIL33 cells was detected by qPCR; (E) The protein levels of BCL2 and BAX in Ctrl, flIL33 and cIL33 cells were detected by Western blot analysis; (F) The quantification of Fig. 1E was analysed by ImageJ. The grouping of blots was cropped from different gels. Uncropped blots are available in Supplementary Fig. 4A. The arrow indicates the location of the marker in the Western blot figure. Data are shown as mean ± SD and were analysed by unpaired two-tailed t-test. * P < 0.05, ** P < 0.01, *** P < 0.001.

To assess the impact of flIL-33 and cIL-33 on apoptosis, cells were stained with annexin V/7-AAD and analysed by flow cytometry. Flow cytometry results indicated a reduced apoptosis rate in both the flIL33 and cIL33 groups compared to the Ctrl group (Fig. 1C). Although the apoptosis rate in the cIL33 group was lower than in the flIL33 group, the difference was not statistically significant (Fig. 1C). Apoptosis can be influenced by the expression of BCL2 and BAX29. To further investigate the role of IL-33 in the apoptosis of A549 cells, we measured the expression of apoptosis-related proteins BCL2 and BAX in Ctrl, flIL33 and cIL33 cells. qPCR analysis showed that overexpression of flIL33 and cIL33 increased BCL2 expression and inhibited BAX expression in A549 cells. BCL2 expression was higher and BAX expression lower in the cIL33 group compared to the flIL33 group (Fig. 1D). Similarly, Western blot analysis revealed elevated BCL2 levels and reduced BAX levels in both the flIL33 and cIL33 groups compared to the Ctrl group (Fig. 1E-F), no significant changes in BCL-xL or BAD were observed (Supplementary Fig. 1). These findings suggest that endogenous IL-33 inhibits apoptosis in NSCLC cells by regulating the expression of BCL2 and BAX.

Endogenous IL-33 enhances proliferation and invasion of lung cancer cells

To further investigate the role of endogenous IL-33 in lung cancer, we assessed its effects on cell proliferation, migration, and invasion. The results indicated that both flIL33 and cIL33 groups significantly promoted the proliferation (Fig. 2A) and invasive capacity (Fig. 2C) of A549 cells compared with the Ctrl group. However, no significant difference in migration ability was observed among the groups (Fig. 2B). To further explore these findings, we generated mouse lung cancer cell lines overexpressing flIl33 and cIl33 (Supplementary Fig. 2A). The flIl33 and cIl33 groups demonstrated enhanced proliferation (Supplementary Fig. 2B) and invasion (Supplementary Fig. 2C) in LLC1 cells. Flow cytometry analysis revealed a reduced apoptosis rate in both the flIl33 and cIl33 groups compared to the Ctrl group (Supplementary Fig. 2D). These results suggest that endogenous IL-33 promotes cell proliferation and invasion in lung cancer cells.

Fig. 2.

Fig. 2

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Endogenous IL-33 Increased the Cell Proliferation and Invasion of Lung Cancer Cells. The proliferation, invasion, and migration of Ctrl, flIL33 and cIL33 A549 cells were assessed. (A) CCK-8 assay was used to measure the absorbance values of Ctrl, flIL33 and cIL33 cells at 0, 24, 48, and 72 h; (B) Scratch assay was conducted to evaluate the migration of Ctrl, flIL33 and cIL33 cells, with images captured of the wound-healing assay at 0 and 72 h using a microscope; (C) Transwell inserts with 8 μm membrane filters were employed to determine the invasion of Ctrl, flIL33 and cIL33 cells. Scale bar: 100 μm. Data are shown as mean ± SD and were analysed by unpaired two-tailed t-test. * P < 0.05, ** P < 0.01.

Endogenous IL-33 inhibits apoptosis of lung cancer cells via the ST2 receptor

We observed that the overexpression of flIL33 and cIL33 in A549 cells led to the secretion of IL-33, which was not detectable in the Ctrl group (Fig. 3A). Furthermore, the cIL33 group secreted more IL-33 compared to the flIL33 group, a finding consistent with that in LLC1 cells (Supplementary Fig. 2E). To determine whether IL-33 secreted by A549 cells inhibits apoptosis via activation of the ST2 receptor, we first knocked down ST2 in A549 cells using ST2 siRNA. Western blot analysis confirmed a marked reduction in ST2 expression in cells transfected with ST2 siRNA compared to those transfected with control siRNA (Ctrl siRNA) (Fig. 3B).

Fig. 3.

Fig. 3

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Endogenous IL-33 Inhibited the Apoptosis of Lung Cancer Cells via the ST2 Receptor. (A) Ctrl, flIL33 and cIL33 A549 cells were cultured for 48 h, and cell supernatants were collected. ELISA was performed to detect the secretion of IL-33. UD: Undetected; (B) A549 cells were transfected with control siRNA (Ctrl siRNA) or ST2 siRNA. Western blot analysis detected the protein level of ST2 in Ctrl siRNA and ST2 siRNA cells (left), and the quantification was analysed by ImageJ (right). Uncropped blots are available in Supplementary Fig. 4C; (C-E) Stimulation of Ctrl siRNA (-) and ST2 siRNA (+) cells with the supernatants (CM) from Ctrl (-) and cIL33 (+) cells for 48 h; (C) Representative dot plots of cells stained with annexin V/7-AAD were analysed by flow cytometry. Bar graphs show the mean ± SD of the percentage of apoptotic (Q2 + Q3) cells; (D) The expression of BCL2 and BAX was detected by qPCR; (E) The protein levels of BCL2 and BAX were detected by Western blot analysis; (F) The quantification of Fig. 3E was analysed by ImageJ. The grouping of blots was cropped from different gels. Uncropped blots are available in Supplementary Fig. 4D. The arrow indicates the location of the marker in the Western blot figure. Data are shown as mean ± SD and were analysed by unpaired two-tailed t-test. * P < 0.05, ** P < 0.01.

Next, to investigate the role of IL-33 in apoptosis inhibition via the ST2 receptor, we stimulated Ctrl siRNA (-) and ST2 siRNA (+) cells with conditioned medium (CM) from the supernatants of Ctrl (-) and cIL33 (+) cells. Flow cytometry analysis showed that the apoptosis rate in the Ctrl siRNA group was reduced by CM from cIL33-expressing cells compared to CM from Ctrl cells (Fig. 3C). However, this anti-apoptotic effect was abolished in the ST2 siRNA group (Fig. 3C), which indicating that the inhibition of apoptosis by the CM of cIL33 was dependent on ST2 expression.

Additionally, the expression of BCL2 and BAX was assessed at both mRNA and protein levels. In the Ctrl siRNA group, CM from cIL33 cells upregulated BCL2 and downregulated BAX expression (Fig. 3D-F). However, in the ST2 siRNA group, the increase in BCL2 and the decrease in BAX were inhibited (Fig. 3D-F). These findings suggest that endogenous IL-33 in lung cancer cells can be secreted and inhibit apoptosis through activation of the ST2 receptor in an autocrine manner.

IL-33 activates the ERK1/2 pathway via the ST2 receptor

To further investigate the mechanism by which IL-33 inhibits apoptosis in lung cancer cells, we performed transcriptome sequencing to identify differentially enriched pathways in Ctrl, flIL33 and cIL33 cells. Using the KEGG database to analyse the enrichment of signalling pathways by differential gene expression, we found that comparisons between Ctrl and flIL33, as well as Ctrl and cIL33 were enriched in the MAPK signalling pathway. However, comparisons between flIL33 and cIL33 did not show enrichment in the MAPK pathway (Fig. 4A).

Fig. 4.

Fig. 4

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IL-33 Induces the Activation of ERK1/2 through the ST2 Receptor. (A) RNA-seq of Ctrl, flIL33 and cIL33 A549 cells was conducted, and KEGG enrichment analysis was performed for signalling pathways in Ctrl vs. flIL33, Ctrl vs. cIL33, and flIL33 vs. cIL33 comparisons. Red rectangles highlight the MAPK signalling pathway, the KEGG pathway map was reproduced from Kanehisa Laboratories (https://www.kegg.jp/)30; (B) Ctrl, flIL33, and cIL33 cells were cultured for 48 h, and the protein levels of ERK1/2, p-ERK1/2 was detected by Western blot; (C) The quantification of Fig. 4B was analysed by ImageJ; (D) Ctrl, flIL33 and cIL33 cells were cultured for 48 h, and the protein levels of P38, p-P38, JNK, and p-JNK, was detected by Western blot; (E) The quantification of Fig. 4D was analysed by ImageJ; (F) Ctrl siRNA (-) and ST2 siRNA (+) cells were stimulated with supernatants (CM) from Ctrl (-) and cIL33 (+) cells for 48 h, and the protein levels of ERK1/2 and p-ERK1/2 were detected by Western blot analysis; (G) The quantification of Fig. 4F was analysed by ImageJ. The grouping of blots was cropped from different gels. Uncropped blots are available in Supplementary Fig. 4E-G and Supplementary Fig. 5A. The arrow indicates the location of the marker in the Western blot figure. Data are shown as mean ± SD and were analysed by unpaired two-tailed t-test. * P < 0.05.

To explore the specific mechanism, we examined the activation of P-38, JNK, and ERK1/2 pathways in Ctrl, flIL33 and cIL33 cells by Western blot. The results revealed that p-ERK1/2 expression was significantly increased in both the flIL33 and cIL33 groups compared with the Ctrl group (Fig. 4B, C). No significant differences were observed in the expression of p-P38 and p-JNK (Fig. 4D, E).

To further elucidate the role of the ST2 receptor in ERK pathway activation, we examined the expression of ERK1/2 and p-ERK1/2 in Ctrl siRNA and ST2 siRNA cells treated with conditioned medium (CM) from Ctrl and cIL33 cells. Western blot analysis showed that CM from cIL33 cells significantly promoted p-ERK1/2 expression in Ctrl siRNA cells. However, p-ERK1/2 expression induced by CM from cIL33 cells was notably lower in the ST2 siRNA group compared to the Ctrl siRNA group (Fig. 4F, G). These results suggest that the ST2 receptor is involved in IL-33-mediated activation of the ERK1/2 pathway.

IL-33 inhibits apoptosis in lung cancer cells via activation of the ERK1/2 pathway

To investigate the role of the ERK1/2 signalling pathway in IL-33-mediated apoptosis inhibition, we treated Ctrl, flIL33 and cIL33 cells with either DMSO or the ERK inhibitor FR 108,024. The results of the Western blot analysis demonstrated that ERK inhibitors significantly inhibited the activation of ERK1/2 (Supplementary Fig. 3A). Flow cytometry results demonstrated that the inhibitory effect of supernatants from cIL33 cells on A549 cell apoptosis was attenuated in the FR 108,024-treated group compared to the DMSO-treated group (Fig. 5A).

Fig. 5.

Fig. 5

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The Inhibition of Apoptosis by IL-33 is Attenuated by ERK Inhibitor. (A-C) Ctrl, flIL33 and cIL33 cells were treated with DMSO or FR 180,204 (ERK inhibitor); (A) Representative dot plots of cells stained with annexin V/7-AAD were analysed by flow cytometry. Bar graphs show the mean ± SD of the percentage of apoptotic (Q2 + Q3) cells; (B) The expression of BCL2 and BAX was detected by qPCR; (C) The protein levels of BCL2 and BAX were detected by Western blot analysis; (D) The quantification of Fig. 5C was analysed by ImageJ. The grouping of blots was cropped from different gels. Uncropped blots are available in Supplementary Fig. 5B. The arrow indicates the location of the marker in the Western blot figure. Data are shown as mean ± SD and were analysed by unpaired two-tailed t-test. * P < 0.05, ** P < 0.01.

Additionally, qPCR and Western blot analysis revealed that treatment with FR 108,024 decreased BCL2 expression induced by supernatants from cIL33 cells and reversed the inhibitory effect of these supernatants on BAX expression (Fig. 5B-D). Additionally, we investigated the effects of ERK inhibitors in LLC cells overexpressing IL-33. Western blot analysis revealed consistent effects of ERK inhibitors in both LLC and A549 cells overexpressing IL-33 (Supplementary Fig. 3B). These findings suggested that the ERK1/2 pathway plays a critical role in IL-33-mediated regulation of BCL2 and BAX expression, thereby inhibiting apoptosis in lung cancer cells.

Discussion

In this study, we investigated the effect of endogenous IL-33 on the apoptosis of lung cancer cells by transfecting flIL33 and cIL33 constructs, thereby exploring the cytokine function of IL-33. Our findings indicate that endogenous IL-33 inhibits apoptosis in NSCLC cells by regulating BCL2 and BAX expression through the ERK1/2 pathway in an autocrine and ST2-dependent manner.

A study demonstrated that IL-33 is highly expressed in lung cancer tissues, with abundant expression found in cancer cells31. The role of IL-33 in lung cancer remains complex, as diverse studies have reported varying serum IL-33 levels in lung cancer patients compared to healthy individuals, with levels being similar, higher, or lower in different study32,33,34. IL-33 protein is unstable and prone to proteolytic cleavage12, highlighting the need for further verification of the effect of secreted IL-33 on lung cancer. Our work demonstrated that both flIL-33 and cIL-33 suppressed apoptosis in lung cancer cells, and these constructs enhanced cell proliferation and invasion, consistent with previous findings28. However, unlike earlier studies which showed that IL-33 did not affect the migration of lung cancer cells, potentially due to low IL-33 concentrations in the cell supernatants, insufficient to influence A549 cell migration. Our study suggest that IL-33 primarily enhances the survival and proliferative capacity of lung cancer cells.

IL-33’s anti-apoptotic properties have been reported in other contexts as well, such as its inhibition of Con A-induced apoptosis in hepatocytes via BCL2 upregulation and BAX downregulation35. Our results similarly showed that IL-33 from lung cancer cells increased BCL2 expression and decreased BAX expression, but no significant changes in BCL-xL or BAD were observed. While there were some differences between flIL33 and cIL33 in terms of BCL2 and BAX expression, these did not translate into significant differences in apoptosis rates. This may be due to the secreted IL-33 concentrations in both groups being insufficient to manifest phenotypic differences. IL-33 interacts with its receptor ST2 to activate downstream signalling36, and our results demonstrated that silencing ST2 significantly abolished IL-33-mediated regulation of BCL2 and BAX, as well as its anti-apoptotic effects. This suggests that IL-33 exerts its apoptosis-inhibiting effects through an ST2-dependent mechanism.

Most studies targeting IL-33 have focused on its role in inflammatory diseases37, though recent research has underscored the involvement of the ST2 axis in the development and progression of various cancers24, 28, 32, 38, 39. IL-33 has been shown to promote NSCLC cell migration28, but little is known regarding the effect of IL-33 on the apoptosis of NSCLC. Previously studies have shown that IL-33 binds to ST2 and activates downstream pathways such as MAPK, NF-κB, or PI3K/AKT40–42. Our data revealed that IL-33 enhances ERK1/2 phosphorylation in lung cancer cells via an autocrine manner. Moreover, inhibiting ERK1/2 with an inhibitor impaired IL-33’s anti-apoptotic effects and altered BCL2 and BAX expression. Thus, our findings suggest that IL-33 inhibits apoptosis in lung cancer cells by regulating BCL2 and BAX via the ST2 receptor and that the ERK1/2 signalling pathway mediates this process. Since the cells used in this study predominantly harbour KRAS mutations, further investigation is required to determine whether IL-33 exerts similar effects in non-KRAS mutant cells and whether its mechanism of action remains consistent.

This study demonstrates that endogenous IL-33 acts as a pro-tumour factor in lung cancer, functioning through an autocrine ST2-dependent pathway. It also highlights that tumour-derived IL-33, in addition to IL-33 from immune cells in the tumour microenvironment, may contribute to tumour progression. These findings underscore the necessity to consider IL-33 and its receptor, ST2, as therapeutic targets in cancer treatment.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (455.8KB, pdf)

Acknowledgements

Not applicable.

Author contributions

LL conceived and carried out experiments, analysed data, and generated figures; HL performed soft analysis and contributed essential resources; YX and YW carried out experiments; SR and HS were involved in data analysis and discussions; DL and ZX designed and supervised the study; DL, ZX and LL wrote the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (No. 32101193; 81501423).

Data availability

The data that support the findings of this study is available in this study. The original raw datasets generated during the current study are available from the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

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Zhuoyuan Xin, Email: xinzy@jlu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (455.8KB, pdf)

Data Availability Statement

The data that support the findings of this study is available in this study. The original raw datasets generated during the current study are available from the corresponding author.

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