Effect of on biological behavior of small cell lung cancer and its mechanism of action.

Introduction
Lung cancer is a prevalent malignancy worldwide, characterized by high and increasing morbidity and mortality rates (1,2). Small cell lung cancer (SCLC), constituting approximately 15% of all lung cancer cases (3,4), represents an advanced neuroendocrine tumor subtype with high malignancy and poor differentiation (5,6). The majority of patients are diagnosed at an advanced stage, resulting in a dismal 5-year survival rate below 5%. Despite the partial efficacy of conventional radiotherapy observed in recent years, there remains a dearth of effective drug targets for SCLC treatment.
At present, it is believed that lung cancer is a “metabolic” disease driven by changes in lipid metabolism and lipid signaling (7,8), and tumor cells can increase de novo adipogenesis and regulate nutrient metabolism to obtain energy. For example, acetyl-CoA thioesterase (ACOT), an enzyme involved in the de novo production of fat in cancer cells, may be overexpressed in lung cancer cases with poor prognosis (9). Fatty acid synthase (FAS) plays an important role in adipogenesis. Studies have shown that lung cancer patients with high FAS expression have a high recurrence rate and poor prognosis (10). In addition, a number of clinical studies have shown a close relationship between increased plasma cholesterol levels and high incidence of lung cancer (11). Lipid metabolism and its associated signal transduction can affect the proliferation, metastasis and invasion of tumor cells. In a number of preclinical studies, inhibitors targeting various lipid metabolizing enzymes can interfere with lipid metabolism in cancer at different levels to exert their anticancer effects. Therefore, in SCLC, screening various lipidases and lipid metabolism regulatory enzymes that have the potential to be therapeutic targets at biochemical and physiological levels will contribute to the early diagnosis, accurate typing and individualized targeted therapy of SCLC, thereby improving the quality of life and prognosis of patients and bringing new hope to cancer patients.
The alpha/beta hydrolase fold domain (ABHD) family was first identified in 1992. This family includes more than 50 enzymes, which is considered to be one of the most diverse and extensive protein families (12), with roles in the regulation of lipid metabolism, signaling and inflammatory responses. In recent years, a large number of studies have been devoted to exploring the potential mechanisms of the ABHD family in tumorigenesis and development, among which the isoforms of ABHD5, ABHD6, ABHD12 have been widely reported. ABHD5 is a cofactor of adipose triglyceride lipase, which is involved in the first step of lipolysis and is able to break down and convert triglyceride into diacylglycerol (13). Several studies have reported that ABHD5 is involved in the development of rectal and colorectal cancer as a cancer inhibiting factor. However, in endometrial cancer, ABHD5 is highly expressed and promotes cancer progression, suggesting that ABHD5 plays different functions in different cells (14). ABHD6 is a hydrolyzing enzyme of monoacylglycerol (MAG) in non-SCLC (NSCLC). High expression of ABHD6 is often associated with the malignant phenotype and poor prognosis of NSCLC (15). ABHD12 as a serine hydrolase hydrolyzes the endocannabinoid 2-alkyl polyglycerol (2-AG). It has been shown that ABHD12 expression is higher in human breast cancer tissues than in paraneoplastic tissues and it has been found in vivo that the knockdown of ABHD12 gene inhibits cell growth, proliferation, and migration of breast cancer cells (16).
ABHD4, a lateral homolog of ABHD5 (the two share 50–55% sequence identification), is a phospholipase/lyolipase B that catalyzes the deacylation of N-acylphosphatidyl ethanolamine (NAPE) and lysol-Nape (17). ABHD4 is involved in multiple lipid metabolism pathways, including adipocyte differentiation, N-acylphospholipid metabolism in mammalian nervous system, regulation of neurosensitivity genes and occurrence and development of unstable angina pectoris (18). Previous studies have indicated that the abnormal expression of ABHD4 is associated with various cancers, including prostate epithelial cells, nasopharyngeal carcinoma cells, ovarian cancer cells and others, thereby influencing tumor initiation and progression (19). A study on esophageal cancer have revealed that ABHD4 is highly expressed in esophageal cancer tissues, and patients with high expression of this gene exhibit significantly reduced survival rates. In vivo experiments have further confirmed that silencing the ABHD4 gene can markedly suppress the malignant biological behavior of esophageal cancer cell lines. These findings suggest that ABHD4 has potential as an oncogene promoting tumor progression; however, its biological role and molecular mechanism in SCLC remain poorly understood.
In this study, we initially confirmed the high expression of ABHD4 in both SCLC cell lines and tissues. Subsequent in vitro and in vivo experiments demonstrated that ABHD4 overexpression was associated with significantly enhanced SCLC cell proliferation, migration, invasion, tumor formation, and growth; conversely, knockdown of ABHD4 produced opposing phenotypic effects. Moreover, overexpression of ABHD4 attenuated apoptosis by inhibiting Bcl2 associated X (Bax) and promoting B-cell lymphoma-2 (Bcl2) expression, while interference with ABHD4 led to a substantial increase in apoptosis. Importantly, metabolomics analysis showed that ABHD4 was associated with lipid metabolism. Curcumin could inhibit the promotion of proliferation, migration, invasion, and anti-apoptotic ability of H69 transfected with oeABHD4 (overexpression ABHD) and could inhibit the activation of PI3K/AKT/mTOR signaling pathway in H69 transfected with oeABHD4. Collectively, these findings underscore the potential carcinogenic role of ABHD4 in SCLC progression and highlight its promise as a therapeutic target. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1854/rc).

Methods

Cell culture
The normal bronchial epithelial cell line BEAS-2B and SCLC cell lines H446 and H69 were obtained from the Shanghai Cell Bank of Chinese Academy of Sciences and Wuhan Prolife Bioscience Technology Co., Ltd., respectively, all with Short Tandem Repeats (STR) identification certificates. All cells were cultured in RPMI-1640 basic medium supplemented with 10% fetal bovine serum and 0.5 mL penicillin/streptomycin. The cells were maintained in a humidified incubator at 37 ℃ with 5% carbon dioxide. Curcumin was dissolved in dimethyl sulfoxide (DMSO) and diluted to concentrations of 1.25, 2.5, 5, 10, 20, and 40 µM using RPMI-1640 basic medium. Subsequently, the overexpressed stable H69 ABHD4 grafts were treated with various concentrations of curcumin and cultured for a duration of 24 h in a constant temperature incubator.

Tissue sample collection
The inclusion criteria were as follows: (I) patients diagnosed and treated with lung surgery at the Affiliated Hospital of Nantong University between December 2019 and December 2021; (II) no neoadjuvant therapy (e.g., radiotherapy, chemotherapy, or immunotherapy) was administered before surgery, and postoperative pathology confirmed SCLC. The paraffin sections were provided by the Department of Pathology at the Affiliated Hospital of Nantong University. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Human Trial Ethics Committee of the Affiliated Hospital of Nantong University (No. 2018-L100) and informed consent was taken from all the patients.

Immunohistochemistry
The collected cancer tissues and adjacent tissues were subjected to immunohistochemical experiments to determine the expression of ABHD4 in SCLC tissues and corresponding adjacent tissues. The tissue sections were sequentially dewaxed, rehydrated, and antigen-repaired before being treated with a 3% hydrogen peroxide solution to block endogenous peroxidase activity. Subsequently, the sections were incubated overnight at room temperature with primary antibody followed by incubation with secondary antibody on the next day.
Color development was achieved using 3, 3'-diaminobenzidine (DAB), while hematoxylin was employed for nuclear counterstaining. After dehydration and sealing, staining patterns were observed and images captured using an orthogonal microscope. Scoring of staining intensity was performed independently by two experienced pathologists from the Department of Pathology at the Affiliated Hospital of Nantong University. The scoring criteria used were as follows: staining intensity assessment—<5%: no staining (0 points); 5–25%: weak positive (1 point); 26–50%: moderate positivity (2 points); >50%: strong positivity (3 points). The immunoreactive score (IS) was calculated as the product of percentage of positive cells scored multiplied by staining intensity score.

Western-blot analysis
The cell proteins were extracted from radioimmunoprecipitation assay (RIPA) lysate using a serine protease inhibitor and phosphatase inhibitor. Protein concentrations were determined using the bicinchoninic acid assay (BCA) detection kit, and protein loading was adjusted based on the measured concentrations. Proteins with varying concentrations were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto a polyvinylidene fluoride (PVDF) membrane that was subsequently blocked with 5% skim milk powder at room temperature. The PVDF membrane was incubated overnight at 4 ℃ with primary antibodies (ABHD4, GAPDH, AKT, p-AKT, PI3K, p-PI3K, mTOR, p-mTOR, Bax, Bcl-2). Afterward, the membrane was incubated for 2 hours at room temperature with secondary antibodies [anti-rabbit immunoglobulin G (IgG) and anti-mouse IgG], followed by washing on a TBST shaker for 30 minutes. Subsequently, the developer solution was applied to the PVDF membrane before exposure and capture using a gel imaging apparatus. Image analysis was performed using ImageJ software. High molecular weight protein markers were obtained from Bioengineering while low molecular weight protein markers were purchased from Thermo Fisher Scientific.

Lentiviral transfection
The expression of ABHD4 in SCLC cells was downregulated using a GV493 shRNA lentiviral vector (Gikai Genetics, Shanghai, China) and upregulated using a GV493 RNA lentiviral vector (Gikai Genetics, Shanghai, China). SCLC cells were seeded in six-well plates and transfected with different multiplicity of infection (MOI) values [5, 10, 20] for preliminary experiments. After 2 days of transfection, fluorescence expression was observed under a fluorescence microscope to determine the percentage of transfected cells. Stable transfection strains were selected using puromycin at a concentration of 2 µg/mL, and the optimal MOI value was determined for subsequent experiments.

Cell proliferation assay
The 96-well plate was seeded with 1×104 cells per well in 100 µL of medium, followed by the addition of 10 µL of ccck8 solution at 0, 24, 48, 72, and 96 h time points. Subsequently, the cells were incubated under controlled temperature and light conditions for a duration of two hours before being analyzed at a wavelength of 450 nm using an enzyme labeling instrument.

Cell migration assay
Cells were collected and resuspended in RPMI-1640 basal medium. A total of 1×105 cells were inoculated into the upper chamber of a transwell system, while 600 µL of medium containing 20% serum was added to the lower chamber. After incubation for 24 hours, the upper chamber was carefully removed and washed with phosphate buffered saline (PBS) three times to eliminate non-migrating cells. The remaining cells were fixed with formaldehyde at room temperature for 20 minutes, stained with crystal violet for 10 minutes, and then washed again with PBS. Following air-drying, cell migration on the surface of the lower chamber was observed using an inverted microscope. At least three fields of view were captured for each experimental group, and statistical analysis was performed using ImageJ software. H69 cells were utilized to compare the migratory abilities among different groups by collecting and quantifying the cells present in the lower chamber.

Cell invasion assay
The BD Matrigel matrix gel was refrigerated at 4 ℃ overnight for solidification. After liquefaction, the base medium was pre-cooled on ice and mixed with BD Matrigel at a ratio of 4:1 to prepare the matrix gel. The gel was carefully encapsulated in the chamber membrane (100 µL per hole) to prevent bubble formation. Subsequently, it was incubated in a 37 ℃ incubator for 4 hours until complete solidification of the matrix gel occurred. The remaining steps followed those of the cell migration experiment, with an extended cell culture time of 48 hours.

Apoptosis assay
The cells to be detected were digested with trypsin without ethylenediaminetetraaceticacid (EDTA) and washed with PBS solution 2–3 times. Subsequently, the cell count in each group was adjusted to 5×105. For this experiment, the Annexin V-kFluor647 kit was employed. A centrifuge tube containing the cell sediment was supplemented with 500 µL of Binding Buffer, Annexin V-kFluor647 (5 µL), and PI (5 µL). Gentle shaking ensured thorough mixing of the contents within the tube. The reaction took place at room temperature for a duration of 5–15 minutes under light-free conditions, followed by flow cytometry analysis within an hour. This experimental procedure was repeated at least three times to calculate the apoptosis rate for each group and analyze intergroup differences.

Experimental studies on animals
A protocol was prepared before the study without registration. Experiments were performed under a project license (No. S20210703-011) granted by the Experimental Animal Ethics Committee of Nantong University Experimental Animal Center, in compliance with the national and institutional guidelines for the care and use of animals. Balb/C-nu/nu athymic male nude mice (5 weeks old, 16–18g) were procured from Nantong University Laboratory Animal Center. To further investigate the role of ABHD4 in SCLC development, a stable cell line expressing shABHD4 was subcutaneously injected into the dorsal region of nude mice. Each group of mice received a subcutaneous injection of 1×107 cells. There were four mice in each group. Throughout the experiments, animals were housed in the Barrier Environment Animal Room at Nantong University Experimental Animal Center. Mice activity and subcutaneous tumor growth were monitored every other day. Tumor length and width measurements were recorded to calculate subcutaneous tumor volume using the formula V= width2 × length × 0.52. After 28 days post-tumor cell inoculation, the mice were anaesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg) and subsequently cervical dislocation. And then tumors were excised, photographed, and stored at −80 ℃ for freezing and storage purposes. All animal experiments strictly adhered to the principles and procedures outlined by the Ethics Committee for Animal Experiments at Nantong University.

Metabolite analysis
After slow thawing of the samples at 4 ℃, an appropriate volume of samples was added to a pre-cooled solution of methanol/acetonitrile/water. The mixture was vortexed, sonicated at low temperature for 30 minutes, and then incubated at −20 ℃ for 10 minutes. Subsequently, centrifugation was performed at 14,000 g and 4 ℃ for 20 minutes. The resulting supernatant was dried under vacuum conditions and re-dissolved in 100 µL acetonitrile aqueous solution (acetonitrile: water =1:1, v/v) for mass spectrometry (MS) analysis. After vortexing and centrifugation for another 15 minutes, the supernatant was injected into the sample. Separation was carried out on an Agilent 1290 Infinity LC HILIC column with a flow rate of 0.5 mL/min and an injection volume of 2 µL. The mobile phase consisted of A: water +25 mM ammonium acetate +25 mM ammonia; B: acetonitrile. The gradient elution program used was as follows: from t=0 to t=0.5 min, B maintained at a constant level of 95%; from t=0.5 to t=7 min, B linearly decreased from 95% to 65%; from t=7 to t=8 min, B linearly decreased from 65% to 40%; from t=8 to t=9 min, B maintained at a constant level of 40%; from t=9 to t=9.1 min, B linearly increased from 40% to 95%, and then B was kept constant at 95% until t=12 min. The entire analytical process was carried out with the samples placed in an auto sample rheld at 4 ℃ in order to minimize any potential influence caused by instrument signal fluctuations. Furthermore, the samples were analyzed randomly in order to ensure unbiased results. The stability of the system and the reliability of the experimental data were monitored and evaluated by inserting quality control (QC) samples into the sample queue. Primary and secondary spectra of the samples were collected using an AB Triple TOF 6600 mass spectrometer. The samples were separated by Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC) system and analyzed using a Triple TOF 6600 mass spectrometer (AB SCIEX) with electrospray ionization (ESI) in positive and negative ion modes, respectively. The ESI source was configured with the following parameters—atomization gas auxiliary heating gas 1 (Gas1): 60; auxiliary heating gas 2 (Gas2): 60; curtain gas (CUR): 30 psi; ion source temperature: 600 ℃; IonSpray Voltage Floating (ISVF) ±5,500 V for both positive and negative modes. The first-stage mass-to-charge ratio detection range was set at 60–1,000 Da, while the second-stage daughter ions detection range was set at 25–1,000 Da. Primary MS had a detection range of 25–1,000 Da with a primary MS scanning accumulation time of 0.20 s/spectra, whereas secondary MS scanning accumulation time was set at 0.05 s/spectra in information dependent acquisition (IDA). Peak intensity value screening mode was adopted during acquisition with declustering potential (DP) set to ±60 V for both positive and negative modes, collision energy at a range of ±15 eV around a central value of 35 eV. The IDA settings included dynamic exclusion voltage (ISVF): ±5,500 V for both positive and negative modes, and dynamic exclusion of isotopeions was set at 4 Da for each scan with 10 fragments per scan.

Molecular docking experiments
In this study, the binding ability, binding mode and potential interaction between curcumin and ABHD4 were investigated through molecular docking using AutoDock and Pymol.

Statistical analysis
The statistical analysis was conducted using GraphPad Prism software (version 9.5.1). All in vitro experiments were independently replicated at least three times. Differences between two groups were assessed using t-tests, while differences among multiple groups were evaluated using analysis of variance. Data are presented as mean ± standard deviation (SD). Statistical significance was defined as P<0.05.

Results

ABHD4 is overexpressed in SCLC and low expression of ABHD4 inhibits tumor growth in vivo
The immunohistochemical staining of paraffin-embedded tissue sections from patients demonstrated predominant nuclear localization of ABHD4 protein, which exhibited increased levels in SCLC tissues when compared to adjacent normal lung tissues (Figure 1A). Moreover, the Western blot analysis revealed differential expression of ABHD4 in the normal bronchial epithelial cell line BEAS-2B and two SCLC cell lines, namely H446 and H69. Notably, ABHD4 expression was higher in both SCLC cell lines compared to the normal bronchial epithelial cell line BEAS-2B, with a further elevation observed in H446 as compared to H69 (Figure 1B). Collectively, these findings strongly supported the presence and upregulation of ABHD4 in SCLC. To elucidate the impact of ABHD4 in vivo tumor growth, we initially established stable transfections of shABHD4 in H446 cells using lentiviral transfection. The Western blot assay was performed to confirm the efficiency of transfection which demonstrated a reduction in ABHD4 expression in the shABHD4 stable transfection strain compared to the control group (Figure 1C). We assessed xenograft tumor development in nude mice through subcutaneous injection of stable shABHD4 implants and control cells. Our findings demonstrated a significant reduction in both tumor size and weight within the shABHD4 group compared to the control group after 28 days post-transplantation (Figure 1D,1E).

ABHD4 is associated with lipid metabolism in vivo
We employed UHPLC-quadrupole time-of-flight MS (UHPLC-Q-TOF MS) to detect 5 shABHD4 groups and 5 control groups, classifying and quantifying all identified metabolites in both positive and negative ion modes. Figure 2A illustrates that lipid metabolites related to ABHD4 accounted for 25.523%. Based on univariate analysis, all metabolites detected in negative ion mode (including unidentified ones) were screened based on a fold change (FC) difference >1.5 or FC <0.67 with a P<0.05 criteria, visualized using a volcano plot as depicted in the (Figure 2B). Correlation analysis can help to measure the degree of metabolic closeness among metabolites with significant differences [variable importance for the projection (VIP) >1, P<0.05], which is conducive to further understanding the mutual regulatory relationship between metabolites in the process of biological status change. Based on the correlation analysis method, the correlation between metabolites with significant differences was analyzed. And the results were visualized in the form of correlation heat maps, as shown in Figure 2C. In order to more intuitively reveal the co-regulatory relationship between various metabolites, the correlation matrix was converted into a chord diagram and a network diagram (Figure 2D,2E). We found that the metabolites with the most obvious changes after ABHD4 knockdown are lipid metabolites, suggesting that ABHD4 may play a role in SCLC through lipid metabolism.

ABHD4 expression is associated with aggressive behavior in SCLC cells, and curcumin counteracts phenotypes induced by ABHD4 overexpression
We established stable transfections of oeABHD4 in H69 cells using lentiviral transfection. The Western blot assay was performed to confirm the efficiency of transfection which demonstrated an elevation in ABHD4 expression in the oeABHD4 stable transfection strain compared to the control group (Figure 3A). In order to verify the relationship between curcumin and ABHD4, various concentrations of curcumin were added to H69 oeABHD4 stable grafts to determine its half-maximal drug inhibitory concentration (IC50) (Figure 3B).
Cell Counting Kit-8 (CCK8) analysis revealed that low expression of ABHD4 inhibited proliferation of H446 cells, whereas overexpression promoted proliferation of H69 cells (Figure 3C). Transwell invasion and migration assays showed diminished invasion and migration capabilities after low expression of ABHD4 (Figure 3D), while overexpression had an opposite effect (Figure 3E). To investigate the impact of ABHD4 expression on apoptosis in SCLC cells, we employed flow cytometry to assess the proportion of apoptotic cells. Our findings demonstrated that decreased ABHD4 expression elevated the percentage of apoptotic cells, whereas overexpression of ABHD4 diminishes this proportion (Figure 3F). These results indicated that ABHD4 plays a crucial role in affecting the apoptotic capacity of SCLC cells. In order to further elucidate the relationship between ABHD4 and apoptosis, we validated the expression levels of apoptosis-related genes Bax and Bcl2 through western blotting analysis (Figure 3G). We observed that knockdown of ABHD4 upregulates Bax gene expression while substantially inhibiting Bcl2 gene expression; conversely, overexpression of ABHD4 exhibits an opposite effect. Our findings unveiled that ABHD4 modulates apoptosis by regulating Bax and Bcl2 genes.
Then we further explored the relationship between curcumin and ABHD4. CCK8 assays showed that 8.737 µM curcumin could reverse the viability of H69 cells after overexpression of ABHD4 (Figure 3H). Transwell assays demonstrated that curcumin abolished overexpression of ABHD4 induced activation of cell migration and invasion (Figure 3I). Additionally, as demonstrated in Figure 3J, the apoptosis-inhibition role of overexpression of ABHD4 in H69 cells was also blocked by pretreatment with 8.737 µM of curcumin.

ABHD4 overexpression is associated with activation of the PI3K/AKT/mTOR pathway in SCLC cells
To explore the potential molecular mechanisms by which ABHD4 contributes to SCLC, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The results indicated a significant enrichment of genes associated with the mTOR signaling pathway (Figure 4). This finding suggests a potential link between ABHD4 expression and the activity of the PI3K/AKT/mTOR signaling axis in SCLC.

Curcumin attenuates the increased PI3K/AKT/mTOR signaling associated with ABHD4 overexpression in SCLC cells
Western blot analysis indicated that ABHD4 overexpression correlated with elevated phosphorylation levels of PI3K, AKT, and mTOR, whereas ABHD4 knockdown was associated with their reduction (Figure 5A). Given prior evidence implicating the PI3K/AKT/mTOR pathway in curcumin’s anti-tumor effects, we investigated whether curcumin could influence the signaling changes linked to ABHD4. Western blot analysis showed that curcumin treatment reduced the levels of p-PI3K, p-AKT, and p-mTOR in ABHD4-overexpressing H69 cells (Figure 5B). These results suggest that curcumin can attenuate the enhanced PI3K/AKT/mTOR signaling observed upon ABHD4 overexpression.

Discussion
In this study, we initiated our investigation by focusing on ABHD4, a gene associated with SCLC. Through preliminary experimental studies, we discovered that ABHD4 plays a crucial role in regulating the proliferation, aggressive migration, and apoptosis of SCLC cells. Furthermore, metabolomics sequencing revealed that ABHD4 is associated with lipid metabolism. Analysis using the KEGG database indicated that the regulatory mechanism primarily involves the mTOR signaling pathway, suggesting a potential connection between ABHD4 and the PI3K/AKT/mTOR signaling axis in SCLC. These findings highlight the significant impact of ABHD4 on driving malignant progression in SCLC and provide valuable markers and novel targets for future development of adjuvant therapies.
ABHD proteins belong to the α/β hydrolase folding superfamily. In addition to their hydrolase activity, most ABHD proteins also possess acyltransferase activity. This enzymatic capability allows them to regulate lipid metabolism and signal transduction. Cytological studies on various malignant tumors such as NSCLC, breast cancer, prostate cancer, colorectal cancer have revealed that the regulation of different molecular subtypes within the ABHD family (e.g., ABHD5, ABHD6, ABHD12) can impact tumor cell behaviors including proliferation, migration, invasion and apoptosis. Herein we report an upregulation of ABHD4 at the protein level in SCLC cell lines. Subsequent immunohistochemical analysis further demonstrated an overexpression of ABHD4 in SCLC tissue samples. Our subsequent in vivo and in vitro experiments elucidated an important role for ABHD4 in regulating the proliferation, migration, invasion and carcinogenesis of SCLC cells. MS analysis revealed that ABHD4 is associated with lipid metabolism. Based on these findings, we propose that ABHD4 may play a pivotal role in promoting carcinogenesis in SCLC.
Curcumin, a natural polyphenolic compound derived from the turmeric plant, exhibits diverse biological activities including anti-inflammatory, antibacterial, antiviral effects (20). Notably, its anticancer potential has garnered significant attention in recent years (21). Emerging evidence suggests that curcumin modulates various signaling pathways and molecular targets implicated in different types of cancer (22). In breast cancer, curcumin has been demonstrated to induce apoptosis and autophagy by inhibiting the PI3K/AKT pathway (23). Moreover, it hampers the growth and proliferation of breast cancer cells by interfering with epidermal growth factor receptor (EGFR) signaling mediated by tyrosine kinase receptors (24). In hematological malignancies, curcumin exerts tumor-suppressive effects through distinct signaling cascades both in vivo and in vitro settings (25). For instance, in non-Hodgkin lymphoma Burkitt lymphoma cases (26), curcumin impedes the PI3K/AKT dependent NF-κB pathway thereby augmenting radiotherapy-induced apoptosis of malignant cells (27). Additionally, curcumin induces downstream FoxO1 expression via modulation of the PI3K/AKT signaling axis leading to cell cycle arrest and apoptosis specifically within pancreatic cancer cells (28).
In NSCLC, curcumin exhibits anti-tumor activity by downregulating NF-κB, inhibiting JAK2 activity, and further suppressing the JAK2/STAT3 signaling pathway (29). Additionally, curcumin hinders lung tumor cell growth and induces apoptosis by inhibiting the PI3K/AKT signaling pathway (30). Despite demonstrating promising anti-cancer biological activity in various cancer types, the clinical application of curcumin remains limited due to its low bioavailability and solubility, which has been extensively investigated worldwide (31). To enhance the utilization and solubility of curcumin, two popular approaches involve developing novel derivatives through chemical structure modification and synthesis as well as innovating new transport systems to improve its pharmacokinetics (32). Both methods have been validated in animal studies with favorable outcomes (33); however, larger-scale preclinical investigations are required to explore human biological responses. In this study, cell detection revealed that curcumin partially counteracted the effects of ABHD4 overexpression on promoting cell proliferation, migration and invasion. Currently, the PI3K/AKT/mTOR signaling pathway is widely recognized as one of the most altered molecular pathways in malignant tumors (34). It plays a crucial role in various cellular processes including survival, proliferation, migration, metastasis, angiogenesis, cell metabolism, cell aging, genomic integrity and stem cell self-renewal. In numerous cancer types, the dysregulation of PI3K/AKT/mTOR signaling is closely associated with tumor initiation and progression. Notably, activated PI3K/AKT/mTOR has been detected in 40% of bladder cancer cases (35). Further investigations conducted by Ross et al. demonstrated that inhibition of PI3K had a certain suppressive effect on bladder cancer cells harboring PIK3CA mutation (36). In breast cancer research studies have shown that targeting this pathway hampers cell proliferation and invasion while sensitizing cancer cells to radiotherapy treatment (37). Moreover, for lung cancer therapy development purposes targeting the PI3K/AKT/mTOR pathway has long been acknowledged as an important strategy with related inhibitors currently undergoing clinical trials. We anticipate its potential application in clinical practice to exert anti-cancer effects in the future (38). Our study revealed through KEGG pathway enrichment analysis that ABHD4 expression is linked to the activity of the PI3K/AKT/mTOR signaling pathway which was further confirmed by western blotting experiments demonstrating knockdown or overexpression of ABHD4 respectively inhibiting or activating this pathway’s components expression levels accordingly. Additionally, rescue experiments indicated that curcumin could attenuate activation of the signaling pathway induced by ABHD4 overexpression. However, several limitations should be noted. The study relied on a limited number of clinical samples and cell lines, which may not fully capture the heterogeneity of SCLC. Validation in larger, independent cohorts is needed. Also, while ABHD4’s link to PI3K/AKT/mTOR was proposed, the precise molecular interactions remain unclear. Further biochemical studies are warranted. And the lipidomic alterations were not exhaustively characterized; targeted lipid profiling or isotope tracing could clarify ABHD4’s metabolic substrates.

Conclusions
Collectively, our findings highlight ABHD4 as a potential regulator of SCLC progression via lipid metabolism and PI3K/AKT/mTOR signaling, offering a preliminary framework for future therapeutic exploration.

Supplementary
The article’s supplementary files as