LTB4R inhibits apoptosis and induces radiosensitivity by promoting the PI3K/AKT pathway in esophageal squamous cell carcinoma

Introduction

Esophageal cancer (EC) is a major global health burden, ranking seventh for incidence and sixth for cancer-related mortality worldwide (1). It primarily manifests in two distinct pathological forms: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC). Alarmingly, it is concerning that approximately 50% of all diagnosed EC cases globally occur in China, where ESCC is the dominant subtype (2,3). Characterized by its aggressive nature, ESCC tends to proliferate rapidly and shows a strong tendency to spread to lymph nodes, typically impacting the upper two-thirds of the esophageal region (4). Signs such as difficulty swallowing and swelling of the neck lymph nodes are often present only in advanced stages of the disease (5), contributing to a dismal 5-year survival rate and prognosis.

Radiotherapy (RT) serves as a critical therapeutic modality for ESCC, having demonstrated efficacy in enhancing local cancer control and mitigating recurrence risk (6). Nevertheless, the intrinsic radioresistance of ESCC cells presents significant challenges, curtailing the clinical effectiveness of RT and often resulting in tumor recurrence and distant metastasis (7). Emerging evidence indicates that RT reshapes the tumor microenvironment (TME) and induces molecular alterations, which can be captured by sequencing to identify biomarkers and therapeutic targets associated with radiosensitivity (8). Thus, there exists a pressing necessity to clarify the molecular pathways that contribute to this resistance to radiation and to discover possible targets that could improve treatment results in ESCC.

Research indicates that the response to DNA damage caused by radiation (DDR) can instigate the release of various cytokines and chemokines (9), subsequently triggering inflammatory responses and alterations in the TME. A diverse set of immunosuppressive cells, including cancer-associated fibroblasts (CAFs), macrophages, myeloid-derived suppressor cells (MDSCs), and additional stromal cell populations, constitutes this TME (10). The marked presence of immature MDSCs is associated with impaired T cell immune function and enhanced tumor invasion and metastasis mediated by direct cell-cell interactions and cytokine secretion. Mechanisms contributing to radioresistance are diverse and may involve the presence of cancer stem cells (CSCs), enhanced DNA repair activity, efficient elimination of reactive oxygen species (ROS), epithelial-mesenchymal transition (EMT), and dysregulated programmed cell death (11,12). Consequently, the main objective of our study is to enhance the effectiveness of RT and improve treatment outcomes for patients with ESCC.

The leukotriene B4 receptor (LTB4R) serves as a receptor with a strong affinity for LTB4, mediating interactions that not only recruit various inflammatory cell types but also engage non-immune cells to initiate or amplify pathological inflammation across different tissues. Within tumor contexts, these interactions operate on distinct target cells, exhibiting complex effects that can manifest as either pro-cancer or anti-cancer influences (13). Tumor initiation, growth, invasion, and metastasis are profoundly shaped by the immune microenvironment, implying that immune regulation may provide a novel approach to cancer prevention and treatment (14). Notably, elevated LTB4R expression is observed in ESCC tissues compared with nearby normal tissues, and this elevation promotes cell proliferation, invasion, and metastasis (14). Nonetheless, the connection between LTB4R expression and radio-sensitivity in ESCC is yet to be investigated.

Concurrently, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is recognized for being constitutively activated across various cancer types and is essential in regulating tumor cell growth, apoptosis, metastasis, and other malignant phenotypes (15-18). Dysregulation of key components within this pathway has been implicated in aberrant activation of downstream signaling, ultimately facilitating cancer development (19,20). ESCC cell growth and progression are largely regulated by the PI3K/Akt pathway, which affects multiple stages of cellular development and differentiation and contributes to metastasis, proliferation, and apoptosis. Its role in promoting EMT is particularly noteworthy, as it allows tumor cells to acquire migratory and invasive characteristics through diminished expression of cell adhesion molecules and the adoption of mesenchymal traits (21).

EMT serves as an important process in cancer metastasis, allowing epithelial tumor cells to acquire characteristics typical of mesenchymal cells, thereby facilitating the dissemination of cancer cells (22). Typically, metastasis initiates when tumor cells undergo EMT, granting them the invasive and migratory properties necessary to detach from their primary tumor site and infiltrate surrounding tissues. Following intravasation, tumor cells may be transported via the bloodstream to distant organs, where they either initiate metastatic colonization or return to the primary site.

In this research, our main focus is to analyze the influence of LTB4R on the radiosensitivity of ESCC and delve into the underlying mechanisms that may contribute to this relationship. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0819/rc).

Methods

Collection and statistics of patient clinical data

Pathological data from patients who received neoadjuvant RT and chemotherapy, followed by surgical resection at Taizhou Hospital of Zhejiang Province from 2011 to 2019, were retrospectively collected. Basic patient information, including clinical and pathological characteristics like age, gender, weight, type of pathology, location, level of differentiation, and TNM stage, was gathered. The cohort comprised 11 individuals who achieved a pathological complete response (pCR) and 13 patients with non-pathological complete response (non-pCR) diagnosed with ESCC. mRNA expression profiling analysis was conducted on these 24 ESCC patients whose pathological results indicated either pCR or non-pCR following neoadjuvant RT. The gene expression profile data was utilized to establish a classification model. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Institutional Review Board (IRB) of Taizhou Hospital of Zhejiang Province (No. K20251201). The requirement for written informed consent was waived by the IRB because the study involved only retrospective analysis of anonymized clinical data and residual pathological specimens, which did not affect the rights or welfare of the participants.

Data collection preprocessing

The original mRNA intensity data from the GSE104958 dataset were retrieved from The Cancer Genome Atlas (TCGA) repository. This gene expression data facilitated the analysis of differential gene expression between ESCC patients with pCR and non-pCR following neoadjuvant RT. An intersection of differentially expressed genes was determined within the independent GSE104958 dataset, yielding 26 common differentially expressed genes associated with NACT-pCR and non-NACT-pCR.

Cellular models and cultivation techniques

The TE1 and ECA109 cell lines were purchased from Saibkang Biotechnology Co., Ltd. (Shanghai, China) and propagated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, while TE3, TE10 and TE12 were obtained from the same supplier and cultured in high-glucose DMEM containing the same concentrations of FBS and antibiotics. The cells were grown under regulated conditions at 37 ℃ with 5% CO₂ and routinely subcultured at 80–90% confluence.

Construction of lentivirus

In order to create cell lines with LTB4R knockdown and overexpression, lentiviral vectors were procured from Shanghai Jima Pharmaceutical Technology Co., Ltd. (Shanghai, China). The lentivirus was utilized to infect adherent cells following the protocols provided by the manufacturer, and reliable cell lines were established following selection in a medium containing 2 µg/L puromycin.

Real-time quantitative polymerase chain reaction (qRT-PCR)

Total RNA from ESCC cells was obtained with the help of Trizol reagent (Birgen Tian Biotechnology Co., Ltd., Shanghai, China). Reverse transcription was carried out in a 20 µL reaction using the PrimeScript™ RT reagent kit, as directed by the manufacturer. Gene expression levels were assessed employing the TB Green^® Premix Ex Taq™ II reagent kit, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) utilized as an internal reference. The levels of relative expression for LTB4R were assessed through the 2^−ΔΔCt approach.

Western blot experiment

After resolution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), total protein extracts were transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skim milk, the PVDF membrane underwent overnight incubation at 4 ℃ with the primary antibody, which was followed by a 1-hour room temperature incubation with the horseradish peroxidase (HRP)-linked secondary antibody. The protein bands that responded to the immune reaction were detected using the Odyssey infrared imaging system, and quantification was performed using ImageJ software. Antibody details are included in Table S1.

X-ray irradiation

ESCC cells were subjected to X-ray radiation delivered by a single-energy 6-MV VitalBeam™ linear accelerator, delivered at a rate of 3 Gy per minute. The device was adjusted to provide X-rays at a linear rate of 6 MV, outputting 300 cGy per minute.

Cell viability assay

A total of 2×10³ cells per well, either with LTB4R knockdown or overexpression, were plated into 96-well plates and later received radiation doses of 0, 2, 4, 6, and 8 Gy post-attachment. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) kit (Wuhan Saiwei Biotechnology Co., Ltd., Wuhan, China) at 0, 24, 48, and 72 hours post-culture. After a 2-hour incubation, absorbance at 450 nm for each well was measured with a microplate reader, and cell growth curves were generated from three independent replicates. The calculation for cell viability is performed using this formula: Cell viability (%) = [(A_treated − A_blank)/(A_{0 treated} − A_blank)] × 100%.

Experiment for colony development

ESCC cell lines (TE1 and ECA109) were seeded in 6-well plates at a concentration of 4×10³ cells for each well. After adherence, the cells received X-ray irradiation at doses of 0, 2, 4, 6 or 8 Gy. Following a 10-day incubation, cells were preserved in a 4% paraformaldehyde solution for a duration of 20 minutes, dyed with a 0.1% crystal violet solution for an additional 20 minutes, and then washed gently with water for colony counting. Colonies were digitized and quantified using ImageJ software (version 1.53t). To eliminate background noise and faint clusters, a ‘colony’ was strictly defined as a cluster containing at least 50 cells. The automated particle analysis parameters in ImageJ were set as follows: size (pixel²) = 10 − infinity (corresponding to an area >0.01 mm²) and circularity = 0–1.00. For wells with high colony density and overlapping (e.g., the 0 and 2 Gy groups), the ‘Watershed’ binary algorithm was applied to segment contiguous colonies to ensure accurate counting as much as possible. Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Šídák’s multiple comparisons test. The sensitiser enhancement ratio (SER) was calculated using the surviving fraction at 2 Gy (SF₂). SER =SF₂ (control group)/SF₂ (experimental group). A SER value greater than 1 indicates radiosensitization, whereas a value less than 1 indicates radioresistance.

Scratch cell migration assay

Cells were plated in 6-well plates, and a vertical scratch was created across the bottom of each well using a 200 µL pipette. The cells were incubated with 700 µL of complete medium supplemented with 10% FBS. Images were captured at 0, 12, and 24 hours using an optical microscope. The scraped area was measured with ImageJ and evaluated using GraphPad Prism 8 software.

Transwell cell invasion assay

A total of 3×10⁴ cells per well were seeded in the upper chamber, and 700 µL of complete medium containing 10% FBS was supplied to the lower chamber. After incubation at 37 ℃ and 5% CO₂ for 48 hours, cells underwent fixation with 4% paraformaldehyde for 15 minutes. Post-fixation, cells were treated with 0.5% crystal violet solution for a duration of 15 minutes for staining, and surplus cells were eliminated from the upper chamber. Cell invasion was examined with the help of an inverted light microscope.

Cell apoptosis detection

Cell apoptosis was assessed using the Annexin V-APC/7-AAD detection kit, following the manufacturer’s instructions. In summary, cells from each sample underwent digestion with 0.25% trypsin and were subsequently resuspended in PBS three times. The mixture was generated by combining 5 µL of membrane-associated protein V-APC and 5 µL of propidium iodide (PI) solution with 300 µL of binding buffer. The rates of apoptosis were evaluated using flow cytometry (Beckman, USA) and determined with the help of FlowJo software.

Xenograft tumor model

A total of thirty 4-week-old male BALB/C-nu/nu nude mice were sourced from Hangzhou Hangs Biotechnology Co., Ltd. (Hangzhou, China) and randomly allocated into ten groups: overexpression negative control (OE-NC), LTB4R overexpression (OE-LTB4R), OE-NC + irradiation (IR), OE-LTB4R + IR, shRNA negative control (SH-NC), LTB4R knockdown (SH-LTB4R), SH-NC + IR, SH-LTB4R + IR, blank control (NC), and NC + LTB4R inhibitor (inhibitor) + IR (n=3). Each mouse received a subcutaneous injection of 5×10⁶ resuspended cells in PBS (100 µL) into the right axilla. IR treatment commenced 7 days post-transplantation. Tumor size measurements (length ‘a’ and width ‘b’) were performed every three days, with volumes calculated using V = 1/2 ab². On day 31, the mice underwent euthanasia, and tumors were collected, weighed, photographed, and their dimensions noted. All animal experiments were completed at Taizhou Enze Medical Center (Group) and were performed under a project license (No. tzyy2025223) granted by ethics committee of Taizhou Enze Medical Center (Group), in compliance with institutional guidelines for the care and use of animals. For euthanasia: animals were first deeply anesthetized via intraperitoneal injection of sodium pentobarbital at a dose of 50 mg/kg body weight. After confirming unconsciousness via the absence of pedal withdrawal and corneal reflexes, euthanasia was performed by cervical dislocation or exsanguination. The rationale for this method was to ensure rapid and painless death, minimizing any potential distress to the animals.

Immunohistochemistry (IHC)

The formalin-fixed, paraffin-embedded tissue sections received deparaffinization and rehydration treatment, and antigen retrieval was achieved through microwaving in a sodium citrate buffer solution. The activity of endogenous peroxidase was neutralized by applying a 3% solution of hydrogen peroxide. The sections were treated with primary antibodies and left to incubate at 4 ℃ for an overnight period, including LTB4R monoclonal rabbit antibody (1:500, HA721994, Hangzhou Huaan Biotechnology Co., Ltd., Hangzhou, China). The secondary antibody used was a goat anti-rabbit IgG conjugated with HRP, sourced from Quanhui International under the product code RQ7025. Subsequent staining was performed using 3,3'-diaminobenzidine tetrachloride (DAB), with hematoxylin used for counterstaining. Following the processes of dehydration, clarification, and preparation for mounting, LTB4R expression was quantified through three randomly selected fields per specimen at 200× magnification, with intensity measured using ImageJ software. The mean optical density (OD) was calculated by normalizing the total staining intensity to the staining area across all three regions.

Hematoxylin-eosin (HE) staining

Deparaffinization and rehydration were conducted on formalin-fixed, paraffin-embedded tissue sections. Sequential staining with hematoxylin and eosin was applied, followed by graded ethanol dehydration (80%, 90%, 100%), xylene clearing, and mounting in neutral resin. Microscopic imaging was carried out using a Leica microscope (Germany).

Statistical analysis

Analysis of the data was carried out using GraphPad Prism 8.0 (GraphPad Software, Inc., CA, USA) or R software version 3.5.2. All experiments were performed in three independent biological replicates (n=3), and results are presented as mean ± standard deviation (SD). A two-tailed Student’s t-test was used for comparisons between two groups. One-way ANOVA was applied for multiple-group comparisons with a single factor. Two-way ANOVA followed by Tukey’s multiple comparisons test was used to assess the main and interaction effects of radiation dose and LTB4R expression. Exact P values are reported throughout the manuscript. Effect sizes were calculated using Cohen’s d for t-tests and partial eta-squared (η²ₚ) for ANOVA to quantify the magnitude of differences. A P value <0.05 was defined as statistically significant.

Results

LTB4R affects the therapeutic efficacy of neoadjuvant chemoradiotherapy (nCRT) in ESCC patients

A total of 24 patients with ESCC treated with nCRT and subsequent surgical resection were included in this study. Based on postoperative pathology, patients were categorized into the pCR group (11 cases) or the non-pCR group (13 cases) (Table S2). Through transcriptome sequencing and analysis of the TCGA database, it was found that the differentially expressed immune-related gene LTB4R exhibited sensitivity to chemoradiotherapy (Figure 1A-1D). Therefore, this study explored the sensitivity of LTB4R to nCRT for EC and investigated its related molecular mechanisms, which will help improve the sensitivity of patients to chemoradiotherapy, enhance clinical efficacy, and improve patient prognosis.

Figure 1 Identification of LTB4R as a candidate immune-related gene in ESCC. (A) Volcano plot showing the intersection of previously reported immune-related genes and our identified DEGs. A total of 245 novel immune-related DEGs associated with neoadjuvant chemotherapy response were obtained. (B,C) Differential gene analysis was performed using the GSE104958 and TCGA-ESCA datasets. Further intersection analysis identified LTB4R as a candidate immune-related gene linked to neoadjuvant chemotherapy efficacy. (D) Box plot of LTB4R expression in ESCA tumor tissues and paired adjacent normal tissues from the TCGA database. LTB4R was significantly upregulated in tumor tissues. (E) Western blot and qRT-PCR analysis of LTB4R expression in five ESCC cell lines. (F,G) Western blot and qRT-PCR validation of LTB4R overexpression and knockdown efficiency in TE1 and ECA109 cells. Mean ± SD, n=3. Two-tailed Student’s t-test. Exact P values and effect sizes are indicated. *, P<0.05; ***, P<0.001; ****, P<0.0001. DEG, differentially expressed gene; ESCA, esophageal carcinoma; ESCC, esophageal squamous cell carcinoma; FC, fold change; LTB4R, leukotriene B4 receptor; OE-LTB4R, LTB4R overexpression; OE-NC, overexpression negative control; qRT-PCR, real-time quantitative polymerase chain reaction; SD, standard deviation; SH-LTB4R, LTB4R knockdown; SH-NC, shRNA negative control; TCGA, The Cancer Genome Atlas.

First, the expression of the LTB4R protein was observed in five different cell lines, including TE1, TE3, TE10, TE12 and ECA109. This was verified through Western blot and qRT-PCR experiments (Figure 1E). Each of the five cell lines was subjected to chemotherapy or RT treatment.

The experimental results showed that LTB4R did not show a significant relationship with chemotherapy sensitivity, but was significantly correlated with radiosensitivity (Figures S1-S3). Therefore, this experiment mainly focused on the correlation between LTB4R and radiosensitivity, and the subsequent experiments were conducted accordingly.

The levels of LTB4R protein expression were analyzed across five ESCC cell lines. The findings indicated that the levels of LTB4R expression in TE1 and ECA109 cells were greater compared to those in the other ESCC cell lines (Figure 1E). These two cell lines were identified for use in the upcoming experiments.

To examine the link between LTB4R and radioresistance in ESCC cells, we developed stable lentiviral stable transfected cells with knockdown and overexpression of LTB4R in ECA109 and TE1 cell lines. The effectiveness of LTB4R knockdown and overexpression was confirmed using qRT-PCR and Western analyses (Figure 1F,1G).

LTB4R promotes the growth ability of cells and inhibits the radiosensitivity of ESCC cells

The proliferation capacity of cells subjected to different irradiation doses was evaluated through the CCK-8 assay. The CCK-8 assay results showed that under irradiation doses of 0, 2, 4, 6, and 8 Gy, the OE-LTB4R group exhibited a markedly higher growth rate at 24, 48, and 72 hours relative to the OE-NC group (Figure 2A,2B). Findings from the CCK-8 assay demonstrated that at radiation doses of 0, 2, 4, 6, and 8 Gy, the SH-LTB4R group exhibited significantly lower growth rates than the SH-NC group after 24, 48, and 72 hours (Figure 2C,2D).

Figure 2 LTB4R inhibits the radiosensitivity of ESCC cells. (A-D) Cell viability was measured via CCK-8 assay at 0, 24, 48 and 72 h after X-ray irradiation at doses of 0, 2, 4, 6 and 8 Gy. (E,G) Cell survival of LTB4R-overexpressing cells (E) and LTB4R-knockdown cells (G) assessed by colony formation assay following X-ray irradiation at 0, 2, 4, 6 and 8 Gy, stained with 0.1% crystal violet. (F,H) Clonogenic ability of LTB4R-overexpressing (F) and LTB4R-knockdown (H) ESCC cells upon X-ray irradiation. Mean ± SD, n=3. Two-way ANOVA with Tukey’s multiple comparisons test. Exact P-values and effect sizes are indicated. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. ANOVA, analysis of variance; CCK-8, Cell Counting Kit-8; ESCC, esophageal squamous cell carcinoma; LTB4R, leukotriene B4 receptor; NC, blank control; OE-LTB4R, LTB4R overexpression; OE-NC, overexpression negative control; SD, standard deviation; SH-LTB4R, LTB4R knockdown; SH-NC, shRNA negative control.

The colony formation assay was used to analyze variations in cell survival and colony-forming ability across irradiation treatments of 0, 2, 4, 6, and 8 Gy. Analysis indicated that, relative to the SH-NC group, the SH-LTB4R group formed significantly fewer colonies after irradiation, suggesting enhanced radiosensitivity in ESCC cells with LTB4R knockdown (Figure 2E,2F). Colony formation analysis demonstrated that the OE-LTB4R group, relative to OE-NC, significantly inhibited post-irradiation colony loss, reflecting decreased radiosensitivity in ESCC cells overexpressing LTB4R (Figure 2G,2H). To further quantify the effect of LTB4R on cellular radiosensitivity, we calculated the SER based on SF₂ values. In ECA109 cells, the SER was 0.76 for LTB4R overexpression and 1.23 for LTB4R knockdown. In TE1 cells, the SER was 0.81 in the overexpression group and 1.16 in the knockdown group (Table S3). Collectively, these data demonstrated that LTB4R overexpression conferred radioresistance, while LTB4R knockdown enhanced the radiosensitivity of ESCC cells.

LTB4R promotes cell migration and invasion while decreasing the radiosensitivity of ESCC cells

Cell scratch assays were conducted to evaluate the migration ability of cells subjected to irradiation doses of 0 and 6 Gy for 48 hours. Analysis indicated that the cell migration rate in OE-LTB4R cells was significantly elevated compared to OE-NC cells (Figure 3A), while migration in SH-LTB4R cells was significantly decreased relative to SH-NC cells (Figure 3B).

Figure 3 LTB4R reduces the radiosensitivity of ESCC cells and regulates cell migration and invasion. (A,B) Cell invasion of LTB4R-overexpressing cells (A) and LTB4R-knockdown cells (B) was assessed using Transwell assay 48 h after 0 or 6 Gy X-ray irradiation, stained with 0.1% crystal violet. (C,D) Cell migration of LTB4R-overexpressing cells (C) and LTB4R-knockdown cells (D) was detected by scratch assay 48 h after 0 or 6 Gy X-ray irradiation, stained with 0.1% crystal violet. Mean ± SD, n=3. Two-way ANOVA with Tukey’s multiple comparisons test. Exact P values and effect sizes are indicated. **, P<0.01; ***, P<0.001; ****, P<0.0001. ANOVA, analysis of variance; ESCC, esophageal squamous cell carcinoma; LTB4R, leukotriene B4 receptor; NC, blank control; OE-LTB4R, LTB4R overexpression; OE-NC, overexpression negative control; SD, standard deviation; SH-LTB4R, LTB4R knockdown; SH-NC, shRNA negative control.

Through transwell invasion assays, the capacity of cells under various radiation exposure levels (0 and 6 Gy) for 48 hours was analyzed. Analysis indicated that the OE-LTB4R group had a substantially elevated invasion rate versus OE-NC (Figure 3C), whereas SH-LTB4R cells exhibited a marked reduction relative to SH-NC (Figure 3D).

The findings from these tests suggest that an increased expression of LTB4R leads to a reduction in the radiosensitivity of ESCC cells, thereby triggering the growth, penetration, and movement of ESCC cells. Knockdown of LTB4R induces the radiosensitivity of ESCC cells, thereby inhibiting the growth, penetration, and movement of ESCC cells.

LTB4R stimulates the development of xenograft tumors in ESCC cells and reduces radiosensitivity

To assess the in vivo radiosensitivity of EC following LTB4R overexpression, a BALB/c-nu/nu nude mouse xenograft model of ESCC was established. The xenografts athymic mice received radiation from X-rays (single dose of 4 Gy) on the 7th, 11th, and 15th days after tumor cell injection (Figure 4A). Following treatment, tumor tissues from each group were excised and displayed a firm consistency. We found that tumors in the OE-LTB4R group were markedly larger than those in the OE-NC control group, while tumors in the SH-LTB4R group were notably smaller than those in the SH-NC control group. Importantly, following RT, the OE-LTB4R + IR group exhibited a markedly larger tumor volume than the OE-NC + IR group, whereas the SH-LTB4R + IR group showed a more pronounced reduction in volume relative to the SH-NC + IR group (Figure 4B). Subsequently, the measurement of tumor tissue weight and volume confirmed that LTB4R knockdown followed by RT significantly inhibited tumor growth (Figure 4C,4D). The results showed that the tumor inhibition rates (volume and weight) of the SH-LTB4R + IR group and the NC + inhibitor + IR group were more significant than those of the NC group. These findings suggest that LTB4R overexpression markedly reduces the radiosensitivity of tumor tissues, whereas LTB4R knockdown enhances it, providing evidence that LTB4R influences the RT response of ESCC tissues in vivo. Immunohistochemical analysis demonstrated that LTB4R expression was markedly elevated in the OE-LTB4R group compared with OE-NC, whereas it was notably reduced in the SH-LTB4R group relative to SH-NC. The expression level of LTB4R in the NC + inhibitor + IR group was lower than that in the NC group, which was positively correlated with the tumor growth curve results and the tumor weighing results (Figure 4E,4F).

Figure 4 The effects of different LTB4R expression levels on ESCC xenograft tumors in vivo. (A) Schematic diagram of the mouse xenograft model establishment. (B) Representative images of xenograft tumors. (C) Tumor growth curve. (D) Statistical analysis of tumor weight. (E,F) HE staining and IHC of subcutaneous tumor tissues. Corresponding staining scores were quantified and analyzed. Mean ± SD, n=3. Two-way ANOVA with Tukey’s multiple comparisons test. Exact P values and effect sizes are indicated. *, P<0.05; **, P<0.01; ****, P<0.0001. ANOVA, analysis of variance; ESCC, esophageal squamous cell carcinoma; HE, hematoxylin-eosin; IHC, immunohistochemistry; IR, irradiation; LTB4R, leukotriene B4 receptor; NC, blank control; OE-LTB4R, LTB4R overexpression; OE-NC, overexpression negative control; SD, standard deviation; SH-LTB4R, LTB4R knockdown; SH-NC, shRNA negative control.

LTB4R reduces RT-induced apoptosis of ESCC cells

Flow cytometry analysis demonstrated that, compared with the NC + IR treatment, LTB4R overexpression combined with IR in ESCC cell lines resulted in reduced cell apoptosis and enhanced RT resistance (Figure 5A,5B). Compared with the single treatment of LTB4R knockdown or IR, the combined method of LTB4R knockdown and IR in the ESCC cell lines induced a higher level of cell apoptosis (Figure 5C,5D).

Figure 5 LTB4R reduces the radiosensitivity of ESCC cells and inhibits cell apoptosis after radiotherapy. (A-D) Cell apoptosis detected by flow cytometry 48 h after (A,B) 0 or 8 Gy and (C,D) 0 or 6 Gy X-ray irradiation. (E-H) Western blot analysis of apoptosis-related protein expression in OE-NC, OE-LTB4R, OE-NC + IR and OE-LTB4R + IR groups 48 h after (E,F) 0 or 8 Gy and (G,H) 0 or 6 Gy X-ray irradiation. Mean ± SD, n=3. Two-way ANOVA with Tukey’s multiple comparisons test. Exact P values and effect sizes are indicated. ns, not significant; **, P<0.01; ****, P<0.0001. ANOVA, analysis of variance; ESCC, esophageal squamous cell carcinoma; IR, irradiation; LTB4R, leukotriene B4 receptor; NC, blank control; OE-LTB4R, LTB4R overexpression; OE-NC, overexpression negative control; SD, standard deviation; SH-LTB4R, LTB4R knockdown; SH-NC, shRNA negative control.

In the Western blot experiment, relative to the combined treatment of NC and IR, cells treated with LTB4R overexpression plus IR exhibited decreased Bax expression, indicative of reduced apoptotic capacity, and increased Bcl2 expression, reflecting enhanced anti-apoptotic potential. There were also significant downward trends in cleaved-caspase 9 and cleaved-caspase 3 (Figure 5E,5F). After LTB4R knockdown and IR combined treatment, Bax showed an increasing trend, Bcl2 protein expression showed a decreasing trend, and cleaved-caspase 9 and cleaved-caspase 3 showed an increasing trend (Figure 5G,5H).

These observations indicate that overexpression of LTB4R inhibits DNA apoptosis after RT, thereby inhibiting radiosensitivity; knockdown of LTB4R promotes DNA apoptosis after RT, thereby enhancing radiosensitivity.

LTB4R inhibits the radiosensitivity of EC cells by affecting the PI3K/AKT pathway

To explore through which downstream pathway LTB4R regulates ESCC cells to increase their radiosensitivity, we conducted transcriptome sequencing on TE1 cells that were stably transfected with lentivirus for RT. Sequencing analysis revealed that LTB4R overexpression influenced the PI3K/AKT pathway in cells relative to the NC group (Figure 6A). We verified the results through Western blot experiments. Since p-Pi3k and p-Akt are indicators of the Pi3k/Akt pathway for cell proliferation, we next detected the association between LTB4R expression and p-Pi3k and p-Akt expression in ESCC cells. By extracting proteins from cells treated with different irradiation doses (0 and 6 Gy) after 48 hours, we compared the downstream pathways at the protein level.

Figure 6 LTB4R attenuates the radiosensitivity of ESCC cells via activating the PI3K/AKT signaling pathway. (A) KEGG pathway enrichment analysis of differentially expressed genes. Left: top enriched pathways between OE-NC and OE-LTB4R groups. Right: top enriched pathways between SH-NC and SH-LTB4R groups. The PI3K-AKT signaling pathway was significantly enriched in both sets of comparisons. (B,C) Western blot analysis of pathway-related protein expression in OE-NC, OE-LTB4R, OE-NC + IR and OE-LTB4R + IR groups. Cells were lysed 48 h after 0 or 6 Gy X-ray irradiation. (D,E) Western blot detection of pathway-related proteins in SH-NC, SH-LTB4R, SH-NC + IR and SH-LTB4R + IR groups. Protein samples were collected 48 h after 0 or 6 Gy X-ray irradiation. (F-I) Detection of EMT-related phenotypes in tumor tissues 48 h after 0 or 6 Gy X-ray irradiation. Mean ± SD, n=3. Two-way ANOVA with Tukey’s multiple comparisons test. Exact P values and effect sizes are indicated. ns, not significant; ***, P<0.001; ****, P<0.0001. AKT, protein kinase B; ANOVA, analysis of variance; EMT, epithelial-mesenchymal transition; ESCC, esophageal squamous cell carcinoma; IR, irradiation; KEGG, Kyoto Encyclopedia of Genes and Genomes; LTB4R, leukotriene B4 receptor; NC, blank control; OE-LTB4R, LTB4R overexpression; OE-NC, overexpression negative control; PI3K, phosphoinositide 3-kinase; SD, standard deviation; SH-LTB4R, LTB4R knockdown; SH-NC, shRNA negative control.

The research findings showed that following 6 Gy X-ray irradiation, the OE-LTB4R group exhibited markedly higher p-PI3K and p-Akt levels than the OE-NC group, along with greater proliferation capacity (Figure 6B,6C). The findings indicate that LTB4R overexpression modulates radiosensitivity through the PI3K/AKT pathway.

Western blot analysis showed that, relative to the SH-NC group, the SH-LTB4R group exhibited reduced p-PI3K and p-Akt expression following 6 Gy X-ray irradiation, along with diminished proliferation capacity (Figure 6D,6E). The findings demonstrate that LTB4R knockdown enhances radiosensitivity via the PI3K/AKT pathway. To further verify the involvement of PI3K/AKT pathway, rescue experiments using LY294002 were performed. To further verify the involvement of PI3K/AKT pathway in LTB4R-mediated radioresistance, rescue assays using the PI3K-specific inhibitor LY294002 were performed. Four experimental groups were established: NC + DMSO, LTB4R-OE + DMSO, NC + LY294002, and LTB4R-OE + LY294002. After exposure to gradient radiation doses (0, 2, 4, 6 and 8 Gy), CCK-8 assay was used to evaluate cell viability (Figure S4). The detailed experimental procedure is described in Appendix 1. LTB4R overexpression significantly improved cell survival following irradiation relative to the NC + DMSO group, whereas blockade of PI3K/AKT signaling via LY294002 effectively reversed the radioresistant phenotype triggered by LTB4R overexpression. Collectively, these rescue findings confirm that LTB4R facilitates radioresistance in ESCC via activating the PI3K/AKT pathway.

EMT is the process by which epithelial cells acquire a mesenchymal phenotype (23). A key feature of EMT is the downregulation of E-cadherin and upregulation of N-cadherin, facilitating cancer cell metastasis and dissemination (24). Analysis revealed that N-cadherin expression increased and E-cadherin expression decreased in OE-LTB4R cells relative to OE-NC after 6 Gy X-ray exposure, accompanied by a pronounced increase in metastasis and dissemination ability (Figure 6F,6G). N-cadherin expression in the SH-LTB4R group was reduced compared with SH-NC, while E-cadherin levels were notably higher than in OE-NC, and the metastatic and invasive capacity was markedly diminished relative to SH-NC (Figure 6H,6I). The findings suggest that LTB4R modulates radiosensitivity in ESCC via EMT.

The research results show that LTB4R suppresses radiosensitivity in ESCC through activation of EMT and the PI3K/AKT pathway.

Discussion

ESCC represents a highly invasive malignant tumor within the digestive tract, characterized by rapid tumor cell proliferation and a dismal clinical prognosis, underscored by a low 5-year survival rate (4). In light of the pivotal role of RT in the local tumor control of EC, alongside the significant clinical challenges posed by intrinsic or acquired radioresistance (6,7), it becomes imperative to elucidate the molecular determinants influencing radio-sensitivity. This study identifies LTB4R as a candidate molecule associated with RT responses in EC through a comprehensive approach combining transcriptome sequencing with data analysis from TCGA. Subsequent in vitro and in vivo experiments were designed to confirm its regulatory function and elucidate underlying mechanisms.

Functional assays revealed that LTB4R expression promotes the development of multiple malignant phenotypes after RT, including enhanced proliferation, increased migration, and elevated invasion. Mechanistically, LTB4R overexpression correlates with elevated phosphorylation levels of PI3K and AKT, which are integral to the activation of pro-survival signaling cascades. These findings are accompanied by altered expressions of EMT markers typical of enhanced migratory abilities and a reduction in mitochondrial apoptotic signaling. Collectively, these molecular alterations provide a coherent framework for understanding how LTB4R facilitates ESCC cells in resisting RT-induced damage while preserving or reinstating invasive capabilities (Figure 7).

Figure 7 Schematic mechanism model illustrating how LTB4R regulates the radiosensitivity of ESCC cells via the PI3K/AKT and apoptotic signaling pathways. LTB4R is highly expressed in ESCC cells. Upon radiotherapy, activated LTB4R enhances the PI3K/AKT pathway, promotes EMT, suppresses radiation-induced cell apoptosis and strengthens anti-apoptotic activity. Collectively, these effects reduce the radiosensitivity of ESCC cells. AKT, protein kinase B; EMT, epithelial-mesenchymal transition; ESCC, esophageal squamous cell carcinoma; LTB4R, leukotriene B4 receptor; PI3K, phosphoinositide 3-kinase.

Verification of these mechanisms revealed that silencing LTB4R significantly diminishes the phosphorylation levels of PI3K and AKT in ESCC cells post-irradiation, thereby enhancing radio-sensitivity. These observations align with the well-established role of the PI3K/AKT pathway as a critical regulatory element governing essential cellular processes, including growth, proliferation, invasion, migration, and apoptosis (25,26). This pathway is integral to cell cycle regulation, metabolic reprogramming, and inflammatory recruitment within the cancer milieu (27,28). In the context of ESCC, PI3K expression correlates with clinical staging, distant metastasis, and histological differentiation (29). Thus, our findings support the notion that LTB4R promotes radioresistance through the reinforcement of PI3K/AKT signaling, which preserves proliferative signals and mitigates the cytotoxic effects of RT. Conversely, the downregulation of LTB4R could curtail PI3K/AKT activation, inhibit proliferation, and amplify the efficacy of RT.

EMT serves as a critical biological mechanism enabling epithelial tumor cells to gain mesenchymal characteristics, thereby promoting invasion and metastasis, and contributing to RT resistance (30). Characteristic alterations in EMT include the downregulation of E-cadherin alongside the upregulation of N-cadherin (31,32). Our empirical results showed that ESCC cells overexpressing LTB4R post-irradiation exhibited reduced E-cadherin expression alongside elevated N-cadherin levels; conversely, LTB4R silencing effectively reversed these molecular alterations. These outcomes are congruent with functional assessments of migration and invasion, suggesting that LTB4R is a key player in the EMT program associated with RT in ESCC. The inhibition of LTB4R alleviates EMT, which is consistent with a reduction in invasive capabilities and an increase in radio-sensitivity.

The induction of apoptosis is a principal mechanism for tumor cell clearance via RT (30). The Bax/Bcl2 balance is a vital determinant of susceptibility to mitochondrial apoptosis; where Bax catalyzes apoptotic pathways, Bcl2 functions to suppress them (33-35), with downstream caspase activation (notably cleaved caspase 9 and cleaved caspase 3) executing the apoptotic program (36-38). Flow cytometric analysis indicated that ESCC cells exhibiting high LTB4R levels demonstrated a reduced apoptosis rate following RT relative to control counterparts; conversely, silencing LTB4R resulted in an elevated apoptotic rate. These observations were substantiated by Western blotting, which showed that LTB4R overexpression led to a diminished Bax/Bcl2 ratio alongside lowered levels of cleaved caspase 9 and cleaved caspase 3, whereas LTB4R silencing elicited the opposite effects. Collectively, these findings underscore the role of LTB4R in inhibiting radiation-induced mitochondrial apoptosis and highlight the potential for restored apoptotic responsiveness following LTB4R downregulation, which in turn may enhance radiosensitivity.

To extend our in vitro findings, we examined the functional implications of LTB4R in xenograft models of ESCC. Tumors overexpressing LTB4R exhibited accelerated growth and were more resistant to RT compared to control tumors. In contrast, tumors in which LTB4R was knocked out or that received the LTB4R inhibitor Etalocib demonstrated slower growth post-irradiation; these observations were corroborated by consistent tumor volume and weight measurements. Immunohistochemical analysis verified effective knockdown and inhibition of LTB4R in treated tumors. Consequently, the in vivo findings support our in vitro observations, confirming that LTB4R promotes radioresistance, and that its inhibition-either genetically or pharmacologically-improves the effectiveness of RT in the xenograft model.

Etalocib is a well-characterized LTB4R inhibitor with a favorable safety profile in previous studies. Previous investigations have demonstrated that etalocib, in conjunction with chemotherapy, can induce apoptosis in leukemia cell lines (23). Furthermore, phase II clinical trials have assessed the efficacy of combining etalocib with chemotherapy across several types of malignant tumors, including pancreatic cancer and non-small cell lung cancer (24,39). Our in vivo data show that combining etalocib with RT effectively suppresses the growth of ESCC xenograft tumors. These results demonstrate that pharmacological inhibition of LTB4R enhances radiosensitivity in preclinical ESCC models, laying the groundwork for further mechanistic and preclinical studies.

Consistent with the concept of biology-driven surveillance models in ESCC, as highlighted by Klacking et al., our findings suggest that LTB4R could serve as a novel predictive biomarker for RT response and recurrence risk in ESCC patients, warranting further clinical validation (11).

This study, while revealing important insights, is accompanied by several limitations that warrant discussion. Firstly, the investigation did not address the upstream regulatory factors governing LTB4R (e.g., ligand availability such as LTB4 levels or upstream signal regulators), leaving questions regarding the regulatory mechanisms steering LTB4R expression and activity in the context of RT unexamined. Secondly, the reliance on subcutaneous xenograft models within the in vivo experiments limited the translational relevance, as more clinically applicable in situ or patient-derived models were not utilized. This limitation constrains the interpretation of findings regarding the TME and host factors at play in clinical scenarios. Furthermore, the study did not explore the clinical correlation between LTB4R expression and relevant clinicopathological features or RT outcomes in ESCC patients, leaving the prognostic or predictive utility of LTB4R to be determined.

Addressing these limitations is critical for advancing translational research. Future investigations should strive to elucidate the mechanisms underlying LTB4R activity and its upstream regulators within the LTB4/LTB4R axis amid the context of RT. Additionally, validating our findings using in situ and immunologically intact models will enhance the clinical relevance of the outcomes. Large-scale clinical cohort studies should be conducted to assess the relationship between LTB4R expression and responses to RT, progression-free survival, and overall survival. Furthermore, exploring the strategy of combining LTB4R inhibitors with additional targeted therapies (such as PI3K/AKT pathway inhibitors) could determine the potential of multimodal targeted therapies in enhancing the radiosensitizing effects observed in ESCC treatment paradigms.

Conclusions

This study demonstrates that LTB4R in ESCC can enhance radioresistance by activating PI3K/AKT, promoting EMT and inhibiting mitochondrial apoptosis. In vitro and in vivo experiments show that knockdown of LTB4R or the use of the inhibitor Etalocib can increase radiosensitivity, suggesting that LTB4R is a potential convertible target for RT sensitization, and it is worthy of further preclinical and clinical verification.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0819/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0819/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0819/prf

Funding: The present study was supposed by the Medical and Health Research Project of Zhejiang Province (No. 2023KY1311), the Medical and Health Research Project of Zhejiang Province (No. 2024KY523), Scientific Research Foundation of Taizhou Enze Medical Center (Group) (No. 20EZA01) and Medical and Health Science and Technology Project of Zhejiang Province (No. WKJ-ZJ-26101).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-0819/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Institutional Review Board (IRB) of Taizhou Hospital of Zhejiang Province (No. K20251201). The requirement for written informed consent was waived by the IRB because the study involved only retrospective analysis of anonymized clinical data and residual pathological specimens, which did not affect the rights or welfare of the participants. All animal experiments were completed at Taizhou Enze Medical Center (Group) and were performed under a project license (No. tzyy2025223) granted by ethics committee of Taizhou Enze Medical Center (Group), in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2024;74:229-63. [Crossref] [PubMed]
2. Zhu H, Ma X, Ye T, et al. Esophageal cancer in China: Practice and research in the new era. Int J Cancer 2023;152:1741-51. [Crossref] [PubMed]
3. Zhou M, Wang H, Zeng X, et al. Mortality, morbidity, and risk factors in China and its provinces, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet 2019;394:1145-58. [Crossref] [PubMed]
4. Morgan E, Soerjomataram I, Rumgay H, et al. The Global Landscape of Esophageal Squamous Cell Carcinoma and Esophageal Adenocarcinoma Incidence and Mortality in 2020 and Projections to 2040: New Estimates From GLOBOCAN 2020. Gastroenterology 2022;163:649-658.e2. [Crossref] [PubMed]
5. Zhang R, Lau LHS, Wu PIC, et al. Endoscopic Diagnosis and Treatment of Esophageal Squamous Cell Carcinoma. Methods Mol Biol 2020;2129:47-62. [Crossref] [PubMed]
6. Wang X, Yu N, Cheng G, et al. Prognostic value of circulating tumour DNA during post-radiotherapy surveillance in locally advanced esophageal squamous cell carcinoma. Clin Transl Med 2022;12:e1116. [Crossref] [PubMed]
7. van Hagen P, Hulshof MC, van Lanschot JJ, et al. Preoperative chemoradiotherapy for esophageal or junctional cancer. N Engl J Med 2012;366:2074-84. [Crossref] [PubMed]
8. Ma W, Baran N. Expanding horizons in esophageal squamous cell carcinoma: The promise of induction chemoimmunotherapy with radiotherapy. World J Clin Oncol 2025;16:104959. [Crossref] [PubMed]
9. Shevtsov M, Sato H, Multhoff G, et al. Novel Approaches to Improve the Efficacy of Immuno-Radiotherapy. Front Oncol 2019;9:156. [Crossref] [PubMed]
10. Jarosz-Biej M, Smolarczyk R, Cichoń T, et al. Tumor Microenvironment as A "Game Changer" in Cancer Radiotherapy. Int J Mol Sci 2019;20:3212. [Crossref] [PubMed]
11. Klacking E, Ma W. Perioperative circulating tumor DNA profiling for recurrence risk in esophageal squamous cell carcinoma: moving toward a biology-driven surveillance model. Ann Transl Med 2026;14:9. [Crossref] [PubMed]
12. An L, Li M, Jia Q. Mechanisms of radiotherapy resistance and radiosensitization strategies for esophageal squamous cell carcinoma. Mol Cancer 2023;22:140. [Crossref] [PubMed]
13. Qiu H, Johansson AS, Sjöström M, et al. Differential induction of BLT receptor expression on human endothelial cells by lipopolysaccharide, cytokines, and leukotriene B4. Proc Natl Acad Sci U S A 2006;103:6913-8. [Crossref] [PubMed]
14. Zhang X, Zhang X, Liu P, et al. Prognostic implications and functional enrichment analysis of LTB4R in patients with acute myeloid leukemia. Journal of Southern Medical University 2022;42:309-20.
15. Huo C, Wu D, Li X, et al. eIf3a mediates malignant biological behaviors in colorectal cancer through the PI3K/AKT signaling pathway. Cancer Biol Ther 2024;25:2355703. [Crossref] [PubMed]
16. Gu JX, Huang K, Zhao WL, et al. NCAPD2 augments the tumorigenesis and progression of human liver cancer via the PI3K‑Akt‑mTOR signaling pathway. Int J Mol Med 2024;54:84. [Crossref] [PubMed]
17. Liu Y, Li K, Zhang J, et al. Let-7 reduces the proliferation and migration of oral cancer cells via PI3K/AKT signaling pathway. J Biochem Mol Toxicol 2024;38:e23834. [Crossref] [PubMed]
18. Ma Y, Zhang Y, Jiang X, et al. KIFC3 promotes the proliferation, migration and invasion of non-small cell lung cancer through the PI3K/AKT signaling pathway. Sci Rep 2024;14:20471. [Crossref] [PubMed]
19. Chu Y, Zuo J, Zhang Y, et al. Co-culture with chorionic villous mesenchymal stem cells promotes endothelial cell proliferation and angiogenesis via ABCA9-AKT pathway. FASEB J 2022;36:e22568. [Crossref] [PubMed]
20. Liu X, Zhou C, Cheng B, et al. Genipin promotes the apoptosis and autophagy of neuroblastoma cells by suppressing the PI3K/AKT/mTOR pathway. Sci Rep 2024;14:20231. [Crossref] [PubMed]
21. Xu W, Yang Z, Lu N. A new role for the PI3K/Akt signaling pathway in the epithelial-mesenchymal transition. Cell Adh Migr 2015;9:317-24. [Crossref] [PubMed]
22. Wang R, Sun Y, Yu W, et al. Downregulation of miRNA-214 in cancer-associated fibroblasts contributes to migration and invasion of gastric cancer cells through targeting FGF9 and inducing EMT. J Exp Clin Cancer Res 2019;38:20. [Crossref] [PubMed]
23. Stranahan AW, Berezniuk I, Chakraborty S, et al. Leukotrienes promote stem cell self-renewal and chemoresistance in acute myeloid leukemia. Leukemia 2022;36:1575-84. [Crossref] [PubMed]
24. Saif MW, Oettle H, Vervenne WL, et al. Randomized double-blind phase II trial comparing gemcitabine plus LY293111 versus gemcitabine plus placebo in advanced adenocarcinoma of the pancreas. Cancer J 2009;15:339-43. [Crossref] [PubMed]
25. Liu L, Liu W, Deng W. Amylin inhibits gastric cancer progression by targeting CCN1 and affecting the PI3K/AKT signalling pathway. Ann Med 2025;57:2480754. [Crossref] [PubMed]
26. Duan Q, Wang M, Cui Z, et al. Saikosaponin D suppresses esophageal squamous cell carcinoma via the PI3K-AKT signaling pathway. Naunyn Schmiedebergs Arch Pharmacol 2025;398:6059-70. [Crossref] [PubMed]
27. Alzahrani AS. PI3K/Akt/mTOR inhibitors in cancer: At the bench and bedside. Semin Cancer Biol 2019;59:125-32. [Crossref] [PubMed]
28. Liu J, Yu Q, Yang X. Circ_0102231 inactivates the PI3K/AKT signaling pathway by regulating the miR-635/NOVA2 pathway to promote the progression of non-small cell lung cancer. Thorac Cancer 2023;14:3453-64. [Crossref] [PubMed]
29. Yang J, Nie J, Ma X, et al. Targeting PI3K in cancer: mechanisms and advances in clinical trials. Mol Cancer 2019;18:26. [Crossref] [PubMed]
30. Verheij M, Bartelink H. Radiation-induced apoptosis. Cell Tissue Res 2000;301:133-42. [Crossref] [PubMed]
31. Debaugnies M, Rodríguez-Acebes S, Blondeau J, et al. RHOJ controls EMT-associated resistance to chemotherapy. Nature 2023;616:168-75. [Crossref] [PubMed]
32. Manfioletti G, Fedele M. Epithelial-Mesenchymal Transition (EMT) 2021. Int J Mol Sci 2022;23:5848. [Crossref] [PubMed]
33. Czabotar PE, Lessene G, Strasser A, et al. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol 2014;15:49-63. [Crossref] [PubMed]
34. Llambi F, Green DR. Apoptosis and oncogenesis: give and take in the BCL-2 family. Curr Opin Genet Dev 2011;21:12-20. [Crossref] [PubMed]
35. Zhang YS, Shen Q, Li J. Traditional Chinese medicine targeting apoptotic mechanisms for esophageal cancer therapy. Acta Pharmacol Sin 2016;37:295-302. [Crossref] [PubMed]
36. Araya LE, Soni IV, Hardy JA, et al. Deorphanizing Caspase-3 and Caspase-9 Substrates In and Out of Apoptosis with Deep Substrate Profiling. ACS Chem Biol 2021;16:2280-96. [Crossref] [PubMed]
37. Allan LA, Clarke PR. Apoptosis and autophagy: Regulation of caspase-9 by phosphorylation. FEBS J 2009;276:6063-73. [Crossref] [PubMed]
38. Dehkordi MH, Munn RGK, Fearnhead HO. Non-Canonical Roles of Apoptotic Caspases in the Nervous System. Front Cell Dev Biol 2022;10:840023. [Crossref] [PubMed]
39. Jänne PA, Paz-Ares L, Oh Y, et al. Randomized, double-blind, phase II trial comparing gemcitabine-cisplatin plus the LTB4 antagonist LY293111 versus gemcitabine-cisplatin plus placebo in first-line non-small-cell lung cancer. J Thorac Oncol 2014;9:126-31. [Crossref] [PubMed]