Abstract
Background/Aim: Non-small cell lung cancer (NSCLC) is a type of lung cancer with a high mortality rate. Many molecular and biochemical mechanisms are involved in its development, among which the suppression of tumorigenicity-2 protein (ST2) signaling pathway. Soluble ST2 (sST2) competes with transmembrane ST2 (ST2 ligand) for IL-33 binding. Activation of IL-33/ST2 pathway leads to M2 macrophage polarization, which promotes tumor progression. Since Caspase-3 is implicated in the regulation of IL-33 activity, the present study aimed to analyze the serum levels of IL-33, sST2, and caspase-3 in non-small cell lung cancer patients, compare them with control samples, and simultaneously investigate their predictive capacity.
Materials and Methods: In this study, sST2 protein, IL-33, and caspase-3 levels were investigated using the ELISA method in serum samples collected from 25 patients diagnosed with non-small cell lung cancer and 25 completely healthy volunteer individuals. In the study, receiver operating characteristic (ROC) analysis was performed to evaluate the diagnostic power of the biomarkers.
Results: Serum IL-33 levels were found to be significantly elevated in patients compared to the control group (p<0.0001). Similarly, the patient group showed significantly higher caspase-3 serum levels than the control group (p<0.030). While sST2 serum levels were higher in the patient group, the difference did not reach statistical significance. The ROC analysis for IL-33 showed an area under the curve (AUC) value of 0.838 [95% confidence interval (CI)=0.725-0.952, p<0.05], indicating that IL-33 is good diagnostic capability for NSCLC. The AUC value for Caspase-3 was 0.678 (95% CI=0.525-0.832, p=0.023), while for sST2, the AUC value was 0.499 (95% CI=0.333-0.665, p>0.05).
Conclusion: NSCLC patients are characterized by increased IL-33 and caspase-3 serum levels. These findings suggest that these markers could serve as valuable diagnostic and prognostic indicators, in addition to being potential therapeutic targets.
Introduction
Lung cancer is the most common malignancy and the leading cause of cancer-related death in men worldwide. In women, it ranks third in incidence and second in cancer-related mortality (1). Lung cancer is characterized by its tendency to spread at an early stage and the potential to metastasize to organs with different anatomical-physiological structures. Since most patients are diagnosed at an advanced stage, the presence of multiple metastases is common, complicating targeted therapies and diminishing the effectiveness of systemic treatment (2). Based on histological characteristics, lung cancers are primarily categorized into two types: small cell lung cancer (SCLC), representing around 15% of all cases, and non-small cell lung cancer (NSCLC), which makes up approximately 85%. Among NSCLC subtypes, adenocarcinoma and squamous cell carcinoma are the most prevalent, while other histological variants are relatively uncommon (3). Although tobacco use and exposure to certain chemicals are major risk factors, disruptions in various intracellular signaling pathways are also believed to contribute to disease pathogenesis (4). These mechanisms include the soluble suppression of tumorigenicity-2 (sST2) protein, which is the receptor of interleukin 33 (IL-33), a member of the cytokine family known to be associated with inflammation (5).
ST2 belongs to the interleukin-1 (IL-1) receptor family and exists in two distinct isoforms: a membrane-bound version known as ST2L and a soluble variant called sST2. These isoforms are expressed in various cell types in response to stimuli such as inflammation and cellular stress. The natural ligand of ST2, IL-33, interacts specifically with the transmembrane ST2L, initiating immunoregulatory and protective signaling pathways in immune cells, heart muscle cells (cardiomyocytes), and certain types of cancer cells. However, the soluble form in circulation, sST2, prevents this interaction by binding IL-33, thus suppressing the beneficial ST2L/İL-33 signaling pathway. Recent studies have revealed that the ST2/IL-33 pathway, and particularly sST2 levels, play an important role in many clinical conditions such as cancer, inflammatory diseases, and cardiovascular diseases (5-7). In addition, emerging evidence supports the role of IL-33 in enhancing tumor growth and metastasis through mechanisms involving immune evasion (8). Nevertheless, altered expression levels of IL-33 have been observed in several inflammatory and non-inflammatory diseases, limiting its utility as a disease-specific biomarker (9).
It has been reported that activation of the IL-33/ST2 pathway induces glycolysis in NSCLC cells, thus increasing metastasis and cell growth (10). Moreover, a study highlighted that ST2, particularly its membrane-bound form ST2L, triggers signal transduction through interaction with IL-33, whereas the soluble variant sST2 inhibits this process by sequestering IL-33. It was also noted that sST2 levels were reduced in patients with lung cancer when compared to healthy controls (11).
ST2 has been reported to contribute to cancer progression within the inflammatory tumor micro-environment in cancers such as, NSCLC, gastric cancer, and colorectal cancer (12). In the literature, differing interpretations have been made regarding the role of sST2 in various cancers, including lung cancer (5). While several studies have demonstrated elevated sST2 levels in patients with poor prognosis (13-16), other studies have reported the opposite (17, 18). It has also been found that lower sST2 levels in lung cancer patients were associated with reduced survival (19).
Caspases are cysteine-aspartate proteases that play a role in the process of programmed cell death (apoptosis). Caspase-3 and -7 are the main caspases involved in the mechanism of cell death. Dysregulation in the apoptotic pathway has been reported to contribute to the development of many types of cancer (20-22). Specifically, low levels of caspase-3 have been reported to be associated with poor prognosis in lung cancers (23). Although IL-33 has been shown to be proteolytically processed by caspase-3 and caspase-7 during apoptosis, it has been shown that caspase-mediated processing of IL-33 is not required for binding to the ST2 receptor and ST2-mediated NF-κB activation. Therefore, IL-33 activation is not dependent on proteolytic processing; in fact, its biological activity is reduced by caspase-dependent cleavage during apoptosis. In this context, caspase-driven proteolysis is considered an important regulatory mechanism that limits the proinflammatory potential of IL-33 (24).
In the current literature there is no comprehensive study that simultaneously evaluated the serum levels of caspase-3, IL-33, and sST2 biomarkers in lung cancer. Thus, the present study was designed to simultaneously compare the serum levels of sST2, IL-33, and caspase-3 in patients with NSCLC with those of healthy volunteers.
Materials and Methods
Study population and clinical procedures. This study included 25 patients with lung cancer who underwent clinical evaluation and 25 healthy volunteer controls. Ethical approval for the study was obtained from the Ethics Committee of Hitit University on 31/10/2024, with file number 2024-303 and decision number 2024-22. The demographic and clinical characteristics of the patients were collected from their medical records.
ELISA analyses. Serum sST2 levels were measured using the sST2 ELISA Kit (ABT2802Hu, Human sST2, A.B.T., Ankara, Türkiye). IL-33 levels were measured using the Human IL-33 ELISA Kit (ABT2428Hu, A.B.T.) and caspase-3 levels were measured using the human caspase-3 (CASP3) ELISA Kit (ABT2850Hu-A.B.T.). Absorbance values of standards, patients and control samples were measured at 450 nm using a SYNERGY/HTX Multi-Mode Reader. Sample concentrations (pg/ml and ng/ml) were calculated from the standard curve. The ELISA protocols for sST2, IL-33, and Caspase-3 were performed according to the kit instructions. A standard curve was created using standard solutions with known concentrations and the concentrations of the target substances in the patient samples were calculated using the standard curve.
Statistical analysis. The statistical analyses of the data obtained in the study were conducted using the Statistical Analysis System (SAS) v9.4 software package (SAS Institute, Cary, NC, USA). Quantitative variables are presented as median (range), while qualitative variables are presented as counts and percentages. The Kolmogorov-Smirnov and skewness values were used to assess the normal distribution of the data. The Mann Whitney U test was applied for the comparisons between two independent groups. Spearman correlation analysis was conducted to determine whether there are mutual relationships among the IL-33, Caspase-3 and sST2 in the study, as well as to identify the direction and strength of these relationships. The data were analyzed for sensitivity and specificity for the detection of NSCLC using receiver operating characteristic (ROC) curve analysis. Cutoff values for IL-33, Caspase-3, and sST2 were determined using the Youden index (calculated as Sensitivity+Specificity-1), and the areas under the curve (AUCs) were compared. In the entire study, p-values of ≤0.05 were considered statistically significant.
Results
Demographic data. As shown in Table I, the proportion of male individuals was 80.0% in the patient group and 60.0% in the control group. The mean age of individuals in the patient group was 59.6±7.32 years, while it was 42.0±10.09 years in the control group. The mean height in the patient group was 172.2±8.49 cm, and the mean weight was 75.8±9.49 kg. In the control group, the mean height and weight were 170.1±9.94 cm and 72.3±14.56 kg, respectively. In the patient group, 48.0% of the patients had adenocarcinoma and 52.0% had squamous cell carcinoma. It was determined that 24.0% of the patients had a family history of cancer. Hypertension was found to be the most common other disease in (24.0%), Finaly it was found that 48.0% of the patients had COPD (Table II).
Demographic characteristics of the patient and control groups.
Clinical characteristics of the patients.
Descriptive statistics and comparison of IL-33, Caspase-3 and sST2 levels between the patient and control groups are presented Table III. Statistically significant differences were observed in IL-33 and Caspase-3 levels between the patient and control groups (p=0.0001 and p=0.0305, respectively). IL-33 and Caspase-3 levels were higher in the patient group compared to the control group (Figure 1A and B). On the other hand, no statistically significant difference was found in sST2 levels between the two groups (Table III, Figure 1). When the patients were stratified according to the tumor type, no statistically significant differences in IL-33, caspase-3, or sST2 levels between patients with adenocarcinoma and those with squamous cell carcinoma (Table IV).
Differences in IL-33, sST2, and Caspase-3 serum levels between patient and control groups.
Comparison of interleukin-33 (IL-33), Caspase-3, and soluble suppression of tumorigenicity 2 (sST2) protein levels between groups. Bar graphs show a comparative analysis of the levels of IL-33 (A), Caspase-3 (B), and sST2 (C) in serum samples from non-small cell lung cancer (NSCLC) patients and healthy controls. A statistically significant increase was observed in the levels of IL-33 and Caspase-3 in patients compared to the controls (p=0.0001 and p=0.0305, respectively), while sST2 levels were comparable between the 2 groups.
Descriptive statistics and comparison results of IL-33, Caspase-3, and sST2 levels in patients according to tumor type.
ROC analysis. In this study, sensitivity and specificity calculations were obtained for various threshold values regarding the IL-33, Caspase-3 and sST2 values in predicting disease status. The most suitable threshold value for IL-33, Caspase-3 and sST2, along with the sensitivity and specificity values, were presented. Additionally, the empirical ROC corresponding to these findings was generated using a non-parametric method in SAS software.
The ROC analysis for IL-33 resulted in an AUC value of 0.838 [95% confidence interval (CI)=0.725-0.952, p<0.05] (Table V, Figure 2A), indicating its predictive capacity to distinguish between NSCLC and healthy individuals. For Caspase-3, the AUC value was 0.678 (95% CI=0.525-0.832, p=0.023) (Table V, Figure 2B). Finally, for sST2 the AUC value was 0.499 (95% CI=0.333-0.665, p>0.05) (Table V, Figure 2C).
Results from the receiver operating characteristic (ROC) analysis.
Analysis of the receiver operating characteristic (ROC) curves of the three protein markers for distinguishing non-small cell lung cancer (NSCLC) from healthy individuals. Interleukin-33 (IL-33) had an area under the curve (AUC) value of 0.838 (p<0.05) (A), Caspase-3 had an AUC value of 0.678 (p=0.023) (B), and soluble suppression of tumorigenicity 2 (sST2) had limited diagnostic value (AUC=0.499, p>0.05) (C).
A correlation analysis was performed to determine the relationships among IL-33, Caspase-3, and sST2 levels in the patient group. The results shown a moderate, positive, statistically significant correlation between IL-33 and caspase-3 levels. However, no significant correlations were found between sST2 and either IL-33 or caspase-3 (Table VI, Figure 3).
The results of the correlation analysis between IL-33, caspase-3, and sST2 protein levels.
Spearman correlation of ınterleukin-33 (IL-33), caspase-3, and soluble suppression of tumorigenicity 2 (sST2) serum levels in non-small cell lung cancer (NSCLC) patients. The scatter plot matrix shows the correlations between pairs of the three markers, IL-33, Caspase-3, and sST2, as well as the corresponding Spearman coefficients (r) and p-values.
Discussion
Lung cancer is the most common type of cancer worldwide, and its prognosis is influenced by numerous biological mechanisms. IL-33, a member of the IL-1 cytokine family, functions as an alarmin cytokine involved in inflammatory and immune responses associated with tumor progression. It contributes to the pathogenesis of various diseases and can mediate both proinflammatory and immunoregulatory responses through interaction with its receptor, ST2 (25, 26). In literature it is reported that IL-33 may have either protumoral or antitumoral effects depending on the biological context, and it acts on various cells within the tumor microenvironment, including T cells, innate lymphoid cells (ILC2s), mast cells, and macrophages (27). IL-33/ST2 signaling is critical for macrophage polarization and it has been demonstrated that enhanced activation of this pathway causes M2 macrophage polarization that accelerates tumor growth (28). On the other hand, Rab protein-mediated exocytosis of sST2 induces a shift of macrophage polarization to anti-tumoral M1 type in lung cancer (19).
In the present study, the expression levels of IL-33, sST2, and Caspase-3 were compared between patients with NSCLC patients and healthy individuals, and their predictive capacity was evaluated. The findings demonstrated that serum IL-33 and Caspase-3 levels were significantly higher in NSCLC patients compared to healthy controls. In addition, IL-33 had good distinguishing ability between NSCLC and healthy individuals, supporting its potential as a diagnostic biomarker and a therapeutic target.
The IL-33/ST2 signaling pathway in lung cancer is known to be regulated in a complex manner, where sST2 serves as a negative regulator that can suppress IL-33-mediated immune responses in the tumor microenvironment (29). However, the exact mechanism varies depending on disease stage, histological subtype, and the surrounding microenvironment. The functional consequences of the IL-33/sST2 axis differ across various types of cancer. For instance, in breast cancer it was reported that high serum IL-33 levels were associated with poor prognosis; in the same study, sST2 levels were found to be high in estrogen receptor (ER) positive cases and low in ER-negative cases (16). In colorectal cancers, increased IL-33 levels have been observed; on the other hand, sST2 is suppressed in cells with high metastatic potential and an inverse correlation of sST2 with tumor growth has been reported (30). In gastric cancers, increased levels of IL-33 and its receptor ST2 have been reported (31). A study in head and neck squamous cell carcinomas highlighted a strong association between the expression of IL-33 and ST2 and tumor progression and prognosis (32). These findings underline the importance of the IL-33/ST2 signaling pathway in cancer biology.
Consistent with previous studies, our findings confirmed significantly elevated IL-33 serum levels in NSCLC patients, whereas sST2 levels showed a non-significant increase. This indicates that the IL-33/ST2 axis may be tightly regulated, with IL-33 exerting a more dominant biological influence in this context. The modest elevation in sST2 may reflect a compensatory response, potentially due to competitive binding with IL-33 to ST2. Previous studies also support elevated IL-33 levels in NSCLC patients (33).
It has been reported that Caspase-3, one of the effector caspases of the apoptotic pathway, causes the proteolysis of IL-33 (34). It has been emphasized that IL-33 is inactivated by caspase-3 and caspase-7 (35). In a study conducted with non-mucinous lung adenocarcinomas, strong Caspase-3 expression levels were detected in patients with lymph node metastasis, and a significant relationship was reported between Caspase-3 levels and lymph node metastasis and tumor stage (36). Our findings showed that IL-33 and Caspase-3 levels increase together in patients’ group. It is stated in the literature that IL-33 may contribute to caspase-3 activation by triggering proinflammatory responses in the tumor microenvironment or may interfere with processes related to cell death by triggering anti-apoptotic signals (26, 37). For example, IL-33 was shown to inhibit apoptosis by modulating BCL2 and BAX expression via the ERK1/2 signaling pathway (38). The concurrent increase in IL-33 and caspase-3 may reflect a dynamic equilibrium between survival and death signals in tumor cells (39). Therefore, co-assessment of IL-33 and caspase-3 may offer valuable insight into new molecular targets for lung cancer treatment. Particularly, modulation of the IL-33/ST2 axis may help develop novel therapeutic strategies by influencing both immune responses and apoptotic processes in the tumor microenvironment.
Study limitations. First, the relatively small sample size limits the generalizability of the findings. Second, only serum levels were evaluated, while tissue-level expression patterns were not analyzed. Additionally, the membrane-bound form of ST2 was not measured, making it difficult to fully elucidate the IL-33/ST2 signaling cascade. Future studies with larger cohorts, tissue-level analyses, and comprehensive evaluation of ST2 isoforms are needed to better understand the regulatory roles of these biomarkers in lung cancer biology.
Conclusion
Our study revealed that serum levels of IL-33 and Caspase-3 are significantly increased in NSCLC patients. IL-33 emerges as a potential biomarker that plays a critical role in both tumor development and immune responses. Due to the complex and tightly regulated nature of the IL-33/ST2 axis, the role of sST2 appears less prominent; however, therapeutic strategies developed through this pathway may target both the immune system and apoptotic mechanisms in lung cancer. Nevertheless, it is clear that further detailed studies involving larger patient cohorts are necessary to translate our findings into clinical practice. Additionally, investigating the roles of these biomarkers at the tissue level and studying different ST2 isoforms together will contribute to identifying new molecular targets for lung cancer treatment.
Footnotes
Authors’ Contributions
All Authors declare that they have participated in the design, execution and analysis of the study and that they have approved the final version.
Conflicts of Interest
The Authors have no conflicts of interest to declare.
Artificial Intelligence (AI) Disclosure
During the preparation of this manuscript, a large language model (Trinka Al) was used solely for language editing and stylistic improvements in select paragraphs.
- Received June 13, 2025.
- Revision received July 19, 2025.
- Accepted July 28, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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