Toosendanin induces ferroptosis in gastrointestinal stromal tumor cells through the regulation of the NCOA4 ferritinophagy pathway: implications for tumor proliferation, migration, and invasion

Introduction

Gastrointestinal stromal tumor (GIST) is the most common malignant tumors of mesenchymal origin in the gastrointestinal tract (1,2). These tumors primarily arise from interstitial cells of Cajal or precursor cells within the myenteric plexus and are characterized by the expression of the CD117 antigen, a receptor for the tyrosine kinase known as KIT (3,4). Surgical resection continues to be the primary treatment modality for localized GIST (5,6). In contrast, the management of metastatic GIST presents greater complexity, frequently requiring the use of targeted therapies such as imatinib (7). Nonetheless, the development of resistance to imatinib remains a significant challenge, underscoring the need for ongoing research into alternative therapeutic strategies and the potential application of additional targeted agents (8).

Ferroptosis, an iron-dependent and non-apoptotic form of cell death relying on glutathione peroxidase 4 (GPX4), has been demonstrated to play a role in the progression and therapeutic response of various cancer types (9,10). Research has indicated that iron-mediated cell death exerts significant regulatory effects on multiple cancers, particularly by inhibiting tumor cell proliferation, migration, and invasion (11,12). Recent research has highlighted the significance of ferroptosis in GIST. Imatinib was shown to induce ferroptosis in GIST cells by promoting the ubiquitination of GPX4 (13). It was reported that ANO6 (TMEM16F) inhibited GIST growth and induced ferroptosis, suggesting that targeting this pathway could be beneficial in managing GIST (14). The emerging concept of nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy, which integrates iron metabolism with autophagy mechanisms, may serve as a crucial pathway for regulating iron-induced cell death (15,16). Ferritinophagy not only directly impacts cellular iron homeostasis but also indirectly influences cell viability through lipid metabolism and oxidative stress (17). These findings provide potential targets for the development of new anti-GIST strategies.

Toosendanin (TSN) is an alkaloid compound derived from Toona sinensis, a traditional Chinese medicinal herb renowned for its diverse pharmacological properties and potential therapeutic applications in treating various diseases (18-20). Research has demonstrated that TSN exhibits anti-cancer properties across various tumor cell types, including the inhibition of cell proliferation, the promotion of apoptosis, and the induction of cell cycle arrest (18,21-23). Nevertheless, the precise mechanisms underlying its effects in GIST remain insufficiently understood.

NCOA4 is a selective cargo receptor that mediates ferritinophagy, a process by which ferritin, an iron-storage protein, is degraded in lysosomes to release iron (24). Dysregulation of NCOA4-mediated ferritinophagy has been implicated in ferroptosis and various diseases, including cancer (25-27). However, the role of the NCOA4 ferritinophagy pathway in TSN-induced ferroptosis in GISTs has not been investigated.

This study sought to elucidate the mechanism by which TSN induced ferroptosis in GIST through the NCOA4-mediated ferritinophagy pathway. By analyzing the synergistic anticancer effects of TSN and the function of NCOA4 in the regulation of ferritinophagy, we investigated whether TSN promoted ferroptosis in GIST-T1 cells by modulating NCOA4. We present this article in accordance with the MDAR reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2024-1002/rc).

Methods

Drug

The TSN powder (20 mg) was dissolved in 348 µL of dimethyl sulfoxide (DMSO), resulting in a final drug concentration of approximately 100 mmol/L. The bacteria were filtered using a 0.22 µm filter membrane and the flow rate was measured by drip method. Finally, the solution was packed, sealed, and stored in a refrigerator at 4 ℃ away from light.

Cell culture and treatment

The human GIST cell line GIST-T1 were obtained from Saibaikang Biotechnology (MIC- iCell -l004, Shanghai, China). These cells were cultured in DMEM high glucose medium (#PM150210, Procell, Wuhan, China) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA), and 100 µg/mL penicillin-streptomycin. Prior to conducting the formal assay, the the half-maximal inhibitory concentration (IC₅₀) of TSN on GIST-T1 cells was ascertained by exposing the cells to a concentration gradient of TSN (5, 10, 20, 40, 60, 80, 120, 160, and 240 nM) for a duration of 48 h. The cells were grouped using a random number table method. The cells in each group had 3 complex holes. At the beginning of the experiment, the DMEM medium was re-suspended and the cell concentration was adjusted to 2×10³ cells/mL inoculated into 96-well plates at 100 µL per well culture plates were placed in a 5% CO₂ incubator at 37 ℃ until the cells were attached to the wall. The TSN treatment group was provided with 6 multiple holes, and the zeroing hole and DMSO control were provided. After the intervention of different concentrations of TSN (40, 80, and 160 nM) for 48 h. For some experiments, the apoptosis inhibitor Z-VAD-FMK (25 µM, MedChemExpress, Shanghai, China, #HY-16658B), necrosis inhibitor necrostatin-1 (20 µM, MedChemExpress, #HY-15760), ferroptosis inhibitor ferrostatin-1 (10 µM, MedChemExpress, #HY-100579), pyroptosis inhibitor disulfiram (1 µM, MedChemExpress, #HY-B0240), or autophagy inhibitor 3-methyladenine (10 µM, MedChemExpress, #HY-19312) was used to pretreat cells for 2 h. Then, the cell morphology and density were observed under the microscope, and Cell Counting Kit-8 (CCK-8) was detected.

Cell proliferation assay

The viability of GIST-T1 cells was assessed by employing a CCK-8 (Thermo Fisher Scientific, Waltham, MA, USA) as per the guidelines provided by the manufacturer. The cellular viability was determined using an enzyme-linked immunosorbent assay at a wavelength of 450 nm.

Clone formation assay

The long-term effects of TSN on GIST-T1 cells were evaluated by clonal formation assay. GIST-T1 cells were digested, counted, and 1,000 cells were seeded per well in a 6-well plate. Once attached, the medium was replaced with 130 nM TSN, with DMSO as a negative control. Each group had three replicates. After a 2-week incubation, the supernatant was discarded, cells were stained with crystal violet for 30 min, washed with phosphate-buffered saline (PBS) three times, and clones were counted.

Transwell assay

The chamber for cell migration and invasion was established by filling Transwell filters (BD Biosciences, Franklin Lakes, NJ, USA) with serum-free medium at 4 ℃. GIST-T1 cells were then seeded into the upper chamber in serum-free medium (3.0×10⁵/mL). After incubating the chamber at 37 ℃ for 24 h, cell migration was assessed using a transwell without Matrigel, while cell invasion was evaluated using a transwell with Matrigel. The transwell cultures were subsequently rinsed twice with sterile PBS and fixed with methanol for 30 minutes. To visualize the cells, they were stained with 0.1% crystal violet at room temperature for another 30 min. Finally, an Olympus X51 inverted microscope (Tokyo, Japan) was used to observe both cell migration and invasion.

Cell survival analysis

For imaging flow cytometry analysis, Annxin V-APC/7-AAD viability dye was purchased from Lianke Biotechnology Co., Ltd. (Hangzhou, China). Following digestion with ethylenediaminetetra-acetic acid (EDTA), the cells in each group were subjected to centrifugation at 250 g for 5 min. The supernatant was carefully aspirated, and the cell pellet was washed with an appropriate volume of PBS. The resulting suspension was transferred to a 1.5 mL conical-bottomed Eppendorf tube and centrifuged again at 250 g for 5 min. The supernatant was removed to isolate the cell pellet. Subsequently, the cells were resuspended in 500 µL of Binding Buffer. To this suspension, 5 µL of Annexin V was gently added, followed by the addition of 10 µL of 7-AAD. The mixture was allowed to react for 5 minutes at room temperature, after which the analysis was performed using a Cytoflex flow cytometer (Beckman Coulter, Brea, CA, USA).

Lipid reactive oxygen species (ROS) production evaluation

Cells seeded in 6-well plates were incubated with 2 µM BODIPY 581/591 C11 (HY-D1301, MedChemExpress) at 37 ℃ for 30 min. The cells were then washed with PBS and resuspended. Lipid ROS levels were assessed using a Cytoflex flow cytometer.

Biochemical detection

The concentrations of malondialdehyde (MDA; Nanjing Jiancheng Bioengineering Institute, Nanjing, China, #A003-1-1), glutathione (GSH; Nanjing Jiancheng Bioengineering Institute, #A006-2-1), lactate dehydrogenase (LDH; Nanjing Jiancheng Bioengineering Institute, #A020-2-1), and Fe²⁺ (Solarbio, Beijing, China, #BC5415) in GIST-T1 cells were quantified using biochemical detection kits, following the manufacturer’s protocols. Optical density for these assays was also measured using the SpectraMAX Plus 384 microplate reader (Molecular Devices, San Jose, CA, USA).

Transmission electron microscopy (TEM)

Cell samples were initially fixed in a 3% glutaraldehyde solution, followed by post-fixation in a 1% osmium tetroxide solution for 2 hours. The samples were then dehydrated through a graded series of acetone solutions and embedded in Epon812 resin. Semi-thin sections, approximately 1 µm thick, were prepared using an ultramicrotome and stained with toluidine blue for preliminary analysis. Ultrathin sections, with a thickness of 60–90 nm, were obtained using a diamond knife. Following staining with uranyl acetate and lead citrate, the samples were analyzed using a TEM instrument (JEM-1400Flash, JEOL, Akishima, Japan).

Western blot analysis

The lysis solution for GIST-T1 cells was prepared using the RIPA buffer (Santa Cruz Biotechnology, Dallas, TX, USA) to extract total proteins. The concentration of proteins was determined utilizing the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). For SDS-PAGE analysis, an electrophoresis technique was employed to load and transfer 40 µg of total protein onto nitrocellulose membranes. The membranes were then subjected to blocking with 5% nonfat milk and incubation with specific protein antibodies or a rabbit anti-β-actin monoclonal antibody. Subsequently, the membranes were exposed to a horseradish peroxidase (HRP) Goat anti-Rabbit IgG (1:20,000; Boster, Wuhan, China; #BA1054). The ECL system (Amersham, Piscataway, NJ, USA) was utilized for signal development. Densitometric analysis of the results was conducted using Scion Image data analysis software (Scion Corporation, Frederick, MD, USA).

Immunofluorescence (IF) staining

Cell IF staining was performed by washing the cultured cells three times with PBS. Following fixation in 4% polyformaldehyde for 15 minutes, the cells were again washed three times with PBS. Subsequently, permeabilization of the cells was carried out using 0.5% Triton X-100 for a duration of 20 min, followed by another round of PBS washes. To block nonspecific binding sites, a solution containing 5% bovine serum albumin (BSA) was applied to the cells and incubated at a temperature of 37 ℃ for one hour. The primary antibodies used in this study were anti-ferritin (ab75973, Abcam, Cambridge, UK; diluted at 1:100) and anti-NCOA4 (A5695, Abclonal, Woburn, MA, USA; diluted at 1:200), which were incubated overnight at 4 ℃. HRP-labeled goat anti-rabbit secondary antibodies (GB22303; Servicebio, Wuhan, China; diluted at 1:100) were then employed. Finally, sections were stained using DAPI (G1012; Servicebio) and visualized through confocal fluorescence microscopy (Carl Zeiss, Thornwood, NY, USA).

Statistical analysis

All tests were repeated 3 times. Mean and standard deviation were presented for the results. Experimental data were analyzed using SPSS 22.0 software (IBM Corp., Armonk, NY, USA). The data were analyzed by the method of one-way analysis of variance (ANOVA), and post-hoc comparisons were performed using the least significant difference method. The level of statistical significance was established at P<0.05.

Results

The mode of cell death in GIST cells induced by TSN was associated with ferroptosis and autophagy

Firstly, the mechanism by which TSN induces GIST-T1 cell death was investigated, with a focus on various cell death pathways, including ferroptosis and autophagy. As shown in Figure 1A, the IC₅₀ of TSN on GIST-T1 cells was ascertained by exposing the cells to a concentration gradient of TSN for a duration of 48 h. Subsequently, cell proliferation was assessed using the CCK-8 assay, resulting in the determination of the IC₅₀ value as 131 nM. The results of the CCK-8 assay demonstrated that TSN treatment alone significantly decreased cell proliferation activity compared to the control group (Figure 1B). In the TSN-treated groups supplemented with different inhibitors, each inhibitor markedly enhanced cell proliferation activity (Figure 1B). Notably, the TSN-treated group supplemented with ferrostatin-1, a ferroptosis inhibitor, exhibited the most pronounced increase in cell proliferation activity (Figure 1B).

Figure 1 TSN induced ferroptosis in gastrointestinal stromal tumor cells and its regulatory effect on NCOA4-mediated ferritinophagy pathway. GIST-T1 cells were treated with increasing concentrations of TSN. In some experiments, GIST-T1 cells were pretreated with the apoptosis inhibitor Z-VAD-FMK (25 µM), necrostatin-1 (20 µM), ferroptosis inhibitor ferrostatin-1 (10 µM), pyroptosis inhibitor disulfiram (1 µM), or the autophagy inhibitor 3-methyladenine (10 µM) for 2 h. (A,B) Cell proliferation was measured using CCK-8. (C,D) Flow cytometry analysis using Annexin V/7-AAD demonstrated the effect of TSN on GIST-T1 cell mortality. (E-G) TSN treatment of GIST-T1 cells results in a substantial elevation in lipid-ROS levels and ferrous content. (H) Transmission electron microscopy images of GIST-T1 cells treated with TSN. Red arrow: mitochondria shriveled. (I) The levels of MDA and GSH were quantified by biochemical detection. (J-L) Western blot analysis of ferroptosis-related markers (GPX4 and SLC7A11) in GIST-T1 cells treated with TSN. β-actin was used as internal control. (M-P) Western blot analysis of autophagy-related markers (LC3, NCOA4, and ferritin) in GIST-T1 cells treated with TSN. β-actin was used as internal control. Data are presented as mean ± SD. *, P<0.05; **, P<0.01 vs. control. ^##, P<0.05 vs. TSN. ^&&, P<0.05 vs. Z-VAD-FMK/necrostatin-1/ferrostatin-1/disulfiram/3-methyladenine. CCK-8, Cell Counting Kit-8; GIST, gastrointestinal stromal tumor; GSH, glutathione; MDA, malondialdehyde; OD, optical density; ROS, reactive oxygen species; SD, standard deviation; TSN, toosendanin.

TSN induced ferroptosis in GIST cells and its regulatory effect on NCOA4-mediated ferritinophagy pathway

Flow cytometry analysis using Annexin V/7-AAD demonstrated a significant increase in cell mortality with increasing TSN concentration (Figure 1C,1D). Moreover, treatment of GIST-T1 cells with TSN resulted in a substantial elevation in lipid-ROS levels and ferrous content (Figure 1E-1G). TEM revealed notable alterations in mitochondria of TSN-treated GIST-T1 cells, including mitochondrial membrane shrinkage, reduction of mitochondrial crests, and overflow of mitochondrial contents (Figure 1H). The levels of MDA and GSH were quantified by biochemical detection, revealing a significant increase in MDA content and a marked decrease in GSH content upon TSN treatment of GIST-T1 cells (Figure 1I). Western blot analysis indicated a significant downregulation of ferroptosis-related markers GPX4 and SLC7A11 expression following TSN treatment in GIST-T1 cells (Figure 1J-1L). Additionally, the expression levels of LC3 II/I and NCOA4 were increased while ferritin expression exhibited dose-dependent reduction across low, medium, and high doses of TSN compared to the control group (Figure 1M-1P).

TSN inhibited the proliferation, migration and invasion of GIST cells by regulating NCOA4-mediated ferritinophagy pathway

The results of CCK-8 and clone formation assay demonstrated that both siRNA-NCOA4 and ferrostatin-1, a ferroptosis inhibitor, significantly enhanced cellular proliferation of GIST-T1 cells. However, upon co-treatment with TSN, there was a significant decrease in the level of cell proliferation observed (Figure 2A-2C). Furthermore, the heightened levels of cell migration (Figure 2D,2E) and invasion induced by NCOA4 silencing and ferrostatin-1 were effectively reversed through co-treatment with TSN (Figure 2F,2G).

Figure 2 TSN inhibited the proliferation, migration and invasion of gastrointestinal stromal tumor cells by regulating NCOA4-mediated ferritinophagy pathway. (A-C) CCK-8 assay and clone formation assay results showing the effect of siRNA-NCOA4 and ferrostatin-1, a ferroptosis inhibitor, on GIST-T1 cell proliferation. The cells in the clone formation assay were stained with crystal violet. (D,E) Cell migration assays using transwell chambers. The cells in the transwell chambers were fixed, stained with crystal violet, and subsequently examined under a microscope at a magnification of 10×. (F,G) Cell invasion assays using Matrigel-coated transwell chambers. The cells in the transwell chambers were stained with crystal violet and observed under a microscope at a magnification of 10×. Data are presented as mean ± SD. *, P<0.05; **, P<0.01 vs. siRNA-NC. ^##, P<0.01 vs. TSN-high + siRNA-NC. ^&&, P<0.01 vs. siRNA-NCOA4. ^$$, P<0.01 vs. siRNA-NC + ferrostatin-1. CCK-8, Cell Counting Kit-8; GIST, gastrointestinal stromal tumor; OD, optical density; SD, standard deviation; siRNA, small interfering RNA; TSN, toosendanin.

TSN promoted ferroptosis in GIST cells by regulating NCOA4-mediated ferritinophagy pathway

The results of Annexin V/7-AAD flow cytometry demonstrated that TSN intervention significantly enhanced the apoptosis of GIST-T1 cells (Figure 3A,3B). Furthermore, co-treatment with siRNA-NCOA4 and ferrostatin-1 further reduced the level of apoptosis in GIST-T1 cells induced by TSN (Figure 3A,3B). Additionally, TSN intervention markedly increased lipid-ROS and ferrous ion levels, which could be reversed by siRNA-NCOA4 and ferrostatin-1 (Figure 3C-3E). The TEM results demonstrated that TSN intervention significantly promoted mitochondrial damage in GIST-T1 cells, which could be reversed by siRNA-NCOA4 and ferrostatin-1 (Figure 3F). Moreover, the biochemical detection results demonstrated that treatment with TSN led to a significant increase in MDA content and a significant decrease in GSH content in GIST-T1 cells, both of which could be attenuated by siRNA-NCOA4 and ferrostatin-1 intervention (Figure 3G,3H). TSN decreased the expression of GPX4 and SLC7A11 in GIST-T1 cells (Figure 3I-3K). Moreover, siRNA-NCOA4 and ferrostatin-1 treatment remarkably rescued the expression of GPX4 and SLC7A11 (Figure 3I-3K).

Figure 3 TSN promoted ferroptosis in gastrointestinal stromal tumor cells by regulating NCOA4-mediated ferritinophagy pathway. (A,B) Annexin V/7-AAD flow cytometry analysis was performed to evaluate apoptosis in GIST-T1 cells. (C-E) Lipid-ROS and ferrous ion levels were measured in GIST-T1 cells. (F) Transmission electron microscopy images of GIST-T1 cells were obtained. (G,H) The levels of MDA and GSH were quantified using biochemical detection. (I-K) Western blot analysis was conducted to assess the expression of ferroptosis-related markers, GPX4 and SLC7A11, in GIST-T1 cells with β-actin serving as an internal control. Data are presented as mean ± SD. *, P<0.05; **, P<0.01 vs. siRNA-NC. ^#, P<0.05; ^##, P<0.01 vs. TSN-high + siRNA-NC. ^&, P<0.05, ^&&, P<0.01 vs. siRNA-NCOA4. ^$, P<0.05; ^$$, P<0.01 vs. siRNA-NC + ferrostatin-1. GIST, gastrointestinal stromal tumor; GSH, glutathione; MDA, malondialdehyde; ROS, reactive oxygen species; SD, standard deviation; siRNA, small interfering RNA; TSN, toosendanin.

In addition, TSN treatment resulted in a significant increase in the expression levels of LC3II/I and NCOA4, while causing a decrease in ferritin expression when compared to the siRNA-NC group (Figure 4A-4G). These protein expression changes were effectively reversed by siRNA-NCOA4 (Figure 4A-4G). However, co-treatment with TSN and ferrostatin-1 did not have a significant impact on the expression of these proteins when compared to the TSN treatment group (Figure 4E-4G).

Figure 4 TSN promoted ferritinophagy in gastrointestinal stromal tumor cells by regulating NCOA4 signaling pathway. (A-D) Western blot analysis of autophagy-related markers (LC3, NCOA4, and ferritin) in GIST-T1 cells treated with TSN. β-actin was used as internal control. (E-G) Immunofluorescence detection of protein expression of NCOA4 and ferritin, scale, 100 µm. Data are presented as mean ± SD. *, P<0.05; **, P<0.01 vs. siRNA-NC. ^##, P<0.01 vs. TSN-high + siRNA-NC. ^&&, P<0.01 vs. siRNA-NCOA4. ^$, P<0.05 vs. siRNA-NC + ferrostatin-1. GIST, gastrointestinal stromal tumor; SD, standard deviation; siRNA, small interfering RNA; TSN, toosendanin.

Discussion

Studies have shown that ferroptosis has significant antitumor effects in a variety of cancer types. This novel form of regulated cell death is characterized by iron-dependent lipid peroxidation and the accumulation of ROS (28,29). It was indicated that certain compounds, such as metformin and ursolic acid, can induce ferroptosis in cancer cells, thereby enhancing their antitumor effects when used in combination with other treatments (30,31). In GIST, ferroptosis has emerged as a critical mechanism that can be targeted to regulate tumor progression and improve treatment outcomes (13,32). The interplay between ferroptosis and various signaling pathways has been shown to affect the proliferation and metastasis of GIST. Previous research suggested that GIST cells exhibited a pronounced sensitivity to ferroptosis induction by RSL3, an effect that was mitigated by the application of liproxstatin and deferoxamine (32). The processes of lipid peroxidation and ferroptosis in GIST cells were facilitated by verteporfin and CA3, primarily through a marked reduction in antioxidant defenses (32). Furthermore, imatinib was found to induce ferroptosis in GIST by enhancing STUB1-mediated ubiquitination of GPX4 (13). Natural products have been identified as potential inducers of ferroptosis, offering new avenues for cancer treatment by leveraging their ability to trigger this cell death pathway (28,31). Our results suggested that TSN significantly induced ferroptosis in GIST cells, which was manifested by an increase in the content of ferrous iron in cells and a decrease in the expression of ferroptosis related proteins GPX4 and SLC7A11.

Alterations in NCOA4 expression are linked to a range of pathological conditions, especially concerning iron homeostasis and cellular responses to oxidative stress (33,34). NCOA4 is pivotal in the selective autophagic degradation of ferritin, a process crucial for regulating intracellular iron levels and mitigating oxidative damage (35). Beyond its involvement in iron metabolism, NCOA4 has been associated with various malignancies, where its expression levels may affect tumor progression and therapeutic responses (36). TRIM7 was shown to directly bind to and ubiquitinate NCOA4 through K48-linked chains, consequently attenuating NCOA4-mediated ferritinophagy and ferroptosis in human glioblastoma cells (37). The inhibition of NCOA4 resulted in a delay in the growth of pancreatic ductal adenocarcinomas (PDAC) and extended survival; however, this was accompanied by the emergence of compensatory iron acquisition pathways (38). Furthermore, increased ferritinophagy accelerated PDAC tumorigenesis, and a heightened ferritinophagy expression signature was associated with a poor prognosis in PDAC patients (38). Emodin was demonstrated to induce ferroptosis in colorectal cancer through NCOA4-mediated ferritinophagy and the inhibition of the NF-κB pathway (27). Similarly, salidroside augmented the susceptibility of triple-negative breast cancer to ferroptosis via SCD1-mediated lipogenesis and NCOA4-dependent ferritinophagy (26). The significance of elucidating the molecular mechanisms underlying NCOA4-mediated ferritinophagy is underscored by these findings. Our findings demonstrated that treatment with TSN exerted a significant inhibitory effect on the proliferation, migration, and invasion of GIST cells. However, the antitumor efficacy of TSN was notably reversed with NCOA4 silencing or ferrostatin-1 co-treatment. Thus, TSN has the potential to provide valuable insights into novel therapeutic targets for GIST treatment.

Limitations of the study

All experiments in this study were conducted in vitro, which may differ from the in vivo environment. Further in vivo experiments are needed to verify the therapeutic effect of TSN and its regulatory effect on the NCOA4 ferritinophagy metabolic pathway.

Conclusions

This study unveiled a potential mechanism through which TSN induced ferroptosis in GIST cells by modulating the NCOA4-mediated ferritinophagy metabolic pathway. The findings demonstrated that TSN significantly impeded the proliferation, migration, and invasion of GIST-T1 cells while effectively triggering apoptosis via upregulation of lipid ROS and iron ions, as well as downregulation of GPX4 and SLC7A11 expression. Furthermore, TSN enhanced LC3II/I and NCOA4 expression while reducing ferritin levels, underscoring its pivotal role in autophagy and iron metabolism regulation. Although these results highlight the promising therapeutic potential of TSN for GIST cells in vitro, further validation through in vivo experiments and preclinical studies is warranted.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2024-1002/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2024-1002/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2024-1002/prf

Funding: This study was supported by the Key Project of the Science and Technology Department of Sichuan Province (No. 2022YFS0440), the Key Project of Sichuan Traditional Chinese Medicine Administration (No. 2021ZD014), and Central Guidance Special Subject for Local Science and Technology Development of Sichuan Province (No. 2022ZYD0063).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2024-1002/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.

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/.

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