Halofuginone suppresses gastric tumor growth by inducing ROS-dependent inhibition of the PI3K/AKT pathway

Halofuginone suppresses gastric tumor growth by inducing ROS-dependent inhibition of the PI3K/AKT pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Halofuginone suppresses gastric tumor growth by inducing ROS-dependent inhibition of the PI3K/AKT pathway Jing Jiang, PeiPei Liu, DaWei Tang, YueHua Han, YongPing Li, Lei Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8446424/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Despite advances in diagnosis and therapy, the survival rate for gastric cancer, particularly in advanced stages, remains exceedingly low. Current chemotherapy regimens frequently lead to treatment failure and recurrence due to severe drug resistance and toxicity, underscoring the urgent need for novel therapeutic strategies with new mechanisms of action capable of effectively overcoming resistance. Natural products and their derivatives have attracted significant attention as a rich source of anticancer agents. Halofuginone (HF), a plant-derived natural compound possessing multi-target antitumor properties, has emerged as a highly promising anticancer candidate. However, its antitumor efficacy against gastric cancer and the precise underlying molecular mechanisms remain largely unexplored. This study aimed to investigate the effects and mechanisms of HF in SGC7901 and MKN45 cell lines. Our experiments demonstrated that HF inhibited gastric cancer cell proliferation while inducing apoptosis and S-phase cell cycle arrest. Mechanistically, HF triggered excessive mitochondrial reactive oxygen species (ROS) production, subsequentlysuppressing the activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pro-survival pathway. Scavenging ROS with N-acetylcysteine (NAC) reversed these effects, confirming ROS as the central mediator of HF's antitumor activity. In a mouse xenograft model, HF administration inhibited primary tumor growth without inducing hepatorenal toxicity. These findings reveal a ROS-centric mechanism through which HF suppresses gastric cancer progression, positioning it as a promising therapeutic candidate to address unmet clinical needs in gastric cancer management. Gastric cancer Halofuginone Apoptosis Cell cycle ROS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Gastric cancer (GC) represents a malignancy with high global incidence and mortality rates, particularly in China and East Asia (Fan X et al. 2021 ). Patients with advanced gastric cancer face a poor prognosis, exhibiting a five-year survival rate of less than 10%, as most are diagnosed with locally advanced or metastatic disease (Tan Z et al. 2019). Although surgical resection combined with adjuvant chemoradiotherapy remains the primary treatment strategy, therapy failure and disease recurrence are common, largely attributable to chemotherapy resistance and severe systemic toxicity (Fujitani K et al. 2013). Molecularly, the pathogenesis and progression of gastric cancer involve aberrant activation of multiple signaling pathways, including the epidermal growth factor receptor (EGFR) (Ding Q et al. 2015 ), phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) (Zhou P et al. 2021 ), and vascular endothelial growth factor (VEGF) axes (Park DJ et al. 2015 ), as well as interactions between immune cells and cancer cells within the tumor microenvironment (Li Y et al. 2022 ). Furthermore, molecular features such as HER2 amplification (May M et al. 2021 ), microsatellite instability (MSI) (Yamamoto H et al. 2024 ), and Epstein-Barr virus (EBV) infection (Kerr JR et al. 2019) contribute to the heterogeneity of GC and pose challenges for personalized therapy. Currently, imaging and endoscopy remain the mainstay for GC diagnosis and staging, yet their sensitivity for detecting early minimal lesions and peritoneal metastases remains limited (Sakamoto E et al. 2023 ). In recent years, liquid biopsy techniques, such as circulating tumor DNA (ctDNA) analysis, have emerged as promising approaches for early detection and disease monitoring (Rodon et al. 2022). Regarding treatment, the median overall survival for advanced gastric cancer patients often falls below 12 months. Established chemotherapy regimens like platinum-based doublets (Zaanan A et al. 2022) and targeted agents such as trastuzumab (Lee J et al. 2024 ) demonstrate limited efficacy and are frequently associated with acquired resistance. Consequently, developing novel therapeutic agents with distinct mechanisms of action, capable of overcoming drug resistance and improving patient outcomes, represents a pressing and unmet need in current clinical and translational research for gastric cancer. HF a quinazolinone alkaloid derivative derived from the traditional Chinese medicine (Dichroa febrifuga), has recently garnered significant attention due to its remarkable antitumor activity demonstrated across various malignancies (Wang J et al. 2020 , Li H et al. 2021 ,Guo J et al. 2022 , de Figueiredo-Pontes LL et al. 2011 and Ni J et al. 2023 ). Research indicates that HF inhibits tumor cell proliferation, metastasis, and induces apoptosis through multiple pathways, showing significant efficacy in models of non-small cell lung cancer (NSCLC) (Han Y et al. 2024), prostate cancer (McLaughlin NP et al. 2014 ), colorectal cancer (Gong RH et al. 2022 ), breast cancer (Juárez P et al. 2017 ), and multiple myeloma (Leiba M et al. 2012 ). For instance, in NSCLC models, HF promotes apoptosis by upregulating the pro-apoptotic protein Bax, downregulating the anti-apoptotic protein Bcl-2, and activating the Caspase-3 pathway. Concurrently, it induces cell cycle arrest, such as at the G1 phase, through mechanisms involving the downregulation of Cyclin D1 and CDK2 expression (Xia X et al. 2019 ). Its mechanism of action involves multi-target regulation, primarily by inhibiting prolyl-tRNA synthetase, which triggers the integrated stress response and subsequently impacts multiple signaling pathways (Keller TL et al. 2012 ) Regarding the inhibition of tumor metastasis, HF significantly suppresses the epithelial-mesenchymal transition (EMT) process (Chen Y et al. 2018 ). It attenuates tumor cell migration and invasion capabilities by inhibiting the TGF-β/Smad signaling pathway, reducing the expression of mesenchymal markers like N-cadherin and Vimentin, and upregulating E-cadherin expression (Gnainsky Y et al. 2007 ). Notably, HF holds substantial potential in reversing tumor drug resistance. Studies demonstrate that it can restore chemosensitivity in resistant tumor cells by inhibiting the activity and expression of P-glycoprotein (P-gp) (Zuo R et al. 2022 ). In 5-fluorouracil-resistant colorectal cancer cells, HF effectively suppresses proliferation and induces apoptosis via regulating the miR-132-3p/TGF-β axis (Wang C et al. 2020 ). Furthermore, another crucial mechanism of HF, observed in NSCLC, involves triggering substantial intracellular accumulation of reactive oxygen species (ROS), leading to oxidative damage and activation of stress-related apoptotic pathways, thereby inhibiting NSCLC progression (Demiroglu-Zergeroglu A et al. 2020 ). This effect appears to be mediated through HF-induced ROS accumulation, which causes oxidative stress, further resulting in DNA damage and mitochondrial dysfunction, ultimately suppressing tumor growth (Xu F et al. 2024 ). Based on this evidence, our study rationally hypothesizes that HF may induce mitochondrial dysfunction and provoke intracellular ROS generation in gastric cancer cells. Reactive oxygen species (ROS) serve as critical signaling molecules that profoundly influence the fate decisions of tumor cells through their spatiotemporal regulation of various signaling pathways (Mittler R et al. 2022 ). Recent studies reveal that ROS dynamically regulate the phosphatidylinositol 3-kinase (PI3K)/serine-threonine kinase (AKT) pathway via redox modifications of key kinases or phosphatases, a process that is complex and exhibits significant tumor type-dependency (Pavlovic S et al. 2018 ). For example, in liver cancer, overexpression of PDHA1 (pyruvate dehydrogenase E1 alpha subunit) increases intracellular ROS levels, subsequently inhibiting PI3K/AKT pathway activation and inducing G0/G1 phase cell cycle arrest and apoptosis (Wang J et al. 2025 ). The inhibition of PI3K/AKT was alleviated upon ROS reduction using the antioxidant N-acetylcysteine (NAC), confirming the crucial mediating role of ROS (Pavlovic S et al. 2018 ). Additionally, ROS accumulation can lead to the collapse of mitochondrial membrane potential, promoting the release of cytochrome C into the cytoplasm and activating the Caspase cascade, ultimately inducing apoptosis (Bock FJ et al. 2020). In a breast cancer study, a certain inducer led to increased ROS levels accompanied by induced apoptosis and cell cycle arrest; this involved upregulation of Bax, downregulation of Bcl-2, consequently increasing the Bax/Bcl-2 ratio, promoting mitochondrial pathway apoptosis, and downregulation of CDK2 expression inducing G0/G1 phase arrest (Das U et al. 2025 ). In the present study, we demonstrate that the natural compound HF significantly inhibits the proliferation of human gastric cancer cells and induces apoptosis and cell cycle arrest. Further mechanistic investigation reveals that its antitumor effect is closely associated with the activation of reactive oxygen species (ROS) generation, which negatively regulates the PI3K/AKT signaling pathway, thereby inhibiting downstream pro-survival and proliferative signals. Notably, in a mouse xenograft model, HF treatment did not induce significant systemic toxicity or adverse reactions, indicating a favorable safety profile. This research unveils a novel redox regulatory mechanism based on the inhibition of the ROS-PI3K/AKT axis, not only providing fresh insights into the spatiotemporal regulation of kinase signaling pathways but also suggesting a potential target for overcoming drug resistance in gastric cancer and developing novel combination therapeutic strategies. 2. Materials and Methods 2.1 Cell Culture Human gastric cancer cell lines SGC7901 and MKN45 were purchased from Wuhan Procell Life Science & Technology Co., Ltd. Cells were cultured in RPMI 1640 medium (Gibco Life Technologies) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cultures were maintained at 37°C in a humidified incubator with 5% CO₂. 2.2 Reagents and Chemicals Tetrandrine and N-acetylcysteine (NAC) were both purchased from MedChem Express; the molecular formula of tetrandrine is C16H17BrClN3O3, with a purity of 99.28%, and the molecular formula of N-acetylcysteine is C5H9NO3S, with a purity of 99.86%. HF was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution, which was stored at − 80°C. Cell Counting Kit-8 (CCK-8) was purchased from Biosharp. Apoptosis and cell cycle detection kits were acquired from Beyotime Biotechnology. The EdU-488 Cell Proliferation Kit and Reactive Oxygen Species (ROS) Assay Kit were from Beyotime Institute of Biotechnology. RPMI 1640 medium was from Gibco. Phosphate-buffered saline (PBS) was sourced from Wuhan Servicebio Technology Co., Ltd.The following primary antibodies were used: PARP (#9542), Cleaved PARP (#94885), Caspase-3 (#9663), Cleaved Caspase-3 (#9661), Cyclin A2 (#4656), P21 ( #2947), Bax (50599-2-Ig, Proteintech), Bcl-2 (60178-1-Ig, Proteintech), CDK2 (10122-1-AP, Proteintech), Phospho-CDK2 (AF3237, Affinity), β-Actin (#3700), PI3K (#4257), Phospho-PI3K (#4228), AKT (#9272), Phospho-AKT (#4060) (all CST unless specified). 2.3 Animals Four- to five-week-old male BALB/c nude mice (N = 60, average body weight 20 ± 2 g) were purchased from Jiangsu Qionglongshan Biological Co., Ltd. (China). After one week of acclimatization, the mice were randomly assigned into groups (n = 8 per group). All animals were provided with food and water and maintained under standard specific pathogen-free (SPF) conditions. Following 3–5 days of additional acclimation post-grouping, subcutaneous tumor inoculation was performed. 2.4 Xenograft Tumor Model All animal experiments were approved by the Animal Experiment Ethics Committee of Bengbu Medical University (Approval No. #[2023]526) and conducted in accordance with the guidelines for the care and use of laboratory animals. The experimental procedure was as follows: Step 1: Grouping and Model Establishment. Mice were randomly assigned to groups following one week of acclimatization. A xenograft model was established. Step 2: Tumor Cell Inoculation. After the acclimatization period, MKN45 cells were resuspended in 0.8% sodium carboxymethyl cellulose (Na-CMC) at a density of 8 × 10⁶ cells/mL. Each mouse received a subcutaneous injection of 0.2 mL of this cell suspension in the shoulder region. Step 3: Group Allocation and Treatment Initiation. Tumor growth was monitored daily at the same time point, and volumes were measured using a caliper. When the measured tumor volumes reached 100–150 mm³, the mice were randomly divided into four groups (n = 8 per group): a saline control group, a low-dose HF group (0.10 mg/kg), a high-dose HF group (0.20 mg/kg), and a cisplatin group (2 mg/kg). Step 4: Drug Administration. The high- and low-dose HF groups received intraperitoneal (i.p.) injections every two days. The cisplatin group received i.p. injections at a dose of 2 mg/kg every three days. The control group received equivalent volumes of saline administered on a schedule matching the high-dose HF group. All treatments were administered via a 1mL syringe and continued for two weeks. Step 5: Sample Collection and Termination. After two weeks of treatment, blood samples were collected from the mouse eyes for biochemical analysis. Subsequently, the mice were euthanized by cervical dislocation. Tumors were excised, photographed, and fixed in 4% paraformaldehyde for subsequent hematoxylin and eosin (H&E) staining observation. 2.5 CCK-8 Assay SGC7901 and MKN45 cells were seeded in 96-well plates at a density of 8,000 cells per well and cultured until adherent. After 24 hours, when cell confluence reached approximately 70–80%, the culture medium was replaced with fresh medium containing varying concentrations of HF. SGC7901 cells were treated with HF at 0, 60, 120, 240, 480, and 960 nmol/L, while MKN45 cells were treated with 0, 30, 60, 120, 240, 480, and 960 nmol/L. Each treatment group consisted of six replicate wells. Following 24 or 48 hours of drug exposure, 10 µL of CCK-8 solution was added to each well under light-protected conditions. The plates were then incubated at 37°C for 30 minutes, and the absorbance at 450 nm was measured using a microplate reader to calculate cell viability. 2.6 Colony Formation Assay Cells were seeded in 6-well plates at a density of 8,000 cells per well. After 24 hours of attachment, the cells were washed twice with PBS and incubated with fresh medium containing different concentrations of HF. The medium was refreshed every 3–4 days. After 10–14 days, when visible colonies had formed, the cells were fixed with 1–2 mL of ice-cold 4% paraformaldehyde for 15 minutes and then stained with 2 mL of 0.1% crystal violet solution for 30 minutes at room temperature. Following staining, the plates were washed twice with PBS and air-dried. The dried plates were photographed, and the number of colonies was counted and analyzed using ImageJ software. 2.7 EdU-488 Cell Proliferation Assay Cell proliferation was further assessed using an EdU (5-ethynyl-2'-deoxyuridine) assay kit. Cells were seeded in confocal dishes at 5×10⁵ cells per dish and allowed to adhere until reaching approximately 70% confluence. They were then treated with different concentrations of HF for 24 hours. An equal volume of pre-warmed (37°C) 2×EdU working solution (20 µM) was added to each dish, and incubation continued for another 2 hours. Subsequently, the cells were fixed and permeabilized with 1 mL of permeabilization buffer (PBS containing 0.5% Triton X-100) for 15–20 minutes at room temperature in the dark. After removal of the permeabilization buffer, 1mL of 1×Hoechst 33342 solution was added to each dish and incubated for 30 minutes at room temperature in the dark. The cells were then washed three times with PBS (5 minutes per wash). An anti-fade mounting medium (1 µL) was applied, and the cells were imaged under a fluorescence microscope. 2.8 Flow Cytometry Analysis of Apoptosis The percentage of apoptotic cells was quantified using an Annexin V-FITC/PI apoptosis detection kit via flow cytometry. Cells in the logarithmic growth phase were harvested and seeded in 6-well plates at a density of 5×10⁵ cells per well. After adherence, the cells were treated with various concentrations of HF for 48 hours. Following treatment, both adherent and floating cells were collected, washed with PBS, and resuspended in 400 µL of 1× binding buffer. Then, 4 µL of Annexin V-FITC solution was added to the cell suspension, which was incubated for 15 minutes at room temperature in the dark. Subsequently, 5 µL of propidium iodide (PI) solution was added, and incubation continued for another 5 minutes in the dark. Apoptosis was immediately analyzed by flow cytometry following the incubation period. 2.9 Flow Cytometry Analysis of Cell Cycle Distribution Cell cycle distribution was analyzed using a cell cycle analysis kit via flow cytometry. Cells were seeded in 6-well plates at a density of 4×10⁵ cells per well. After adherence, the cells were treated with different concentrations of HF for 24 hours. Following treatment, the cells were harvested by trypsinization, collected in centrifuge tubes, and centrifuged. The cell pellet was washed with PBS and resuspended in 0.5 mL of PBS. Subsequently, the cells were fixed by adding pre-cooled 75% ethanol dropwise under gentle vortexing and incubated at -20°C for at least 1 hour. After fixation, the cells were centrifuged, and the ethanol was carefully aspirated, leaving approximately 50 µL to avoid disturbing the cell pellet. The cells were then washed twice with pre-cooled PBS. After a final centrifugation at 1200 rpm for 5 minutes, the supernatant was completely removed, and the cell pellet was resuspended in 480 µL of PBS. Then, 20 µL of RNase A (50×) was added, and the mixture was incubated in a 37°C water bath for 30 minutes. The cell suspension was filtered through a 400-mesh cell strainer, centrifuged, and the pellet was resuspended in 400 µL of 1× propidium iodide (PI) staining solution. The cells were incubated in the dark at 4°C for 1 hour before cell cycle distribution was analyzed by flow cytometry. 2.10 Western Blotting After 24 hours of drug treatment, cellular proteins were extracted. Total protein concentration was determined using a BCA protein assay kit. Briefly, cells were collected and centrifuged, and the supernatant was removed. The cell pellet was lysed on ice with freshly prepared RIPA lysis buffer (containing protease and phosphatase inhibitors) for 30 minutes. The lysate was then centrifuged at 12,000 rpm for 20 minutes at 4°C, and the supernatant was collected. According to the kit instructions, a working reagent was prepared by mixing Reagent A and Reagent B at a 50:1 ratio. In a 96-well plate, 200 µL of the working reagent, 19 µL of PBS, and 1 µL of protein sample were added to each well. Each sample was assayed in triplicate to minimize experimental error. The plate was covered with foil and incubated at 37°C for 30 minutes in the dark. The absorbance at 562 nm was measured using a microplate reader, and the protein concentration of each sample was calculated. Concentrated samples were diluted with RIPA lysis buffer to normalize concentrations across all samples. An appropriate volume of loading buffer was added to the protein lysates, which were then denatured by heating at 95°C for 5 minutes, followed by brief centrifugation at 1000 rpm for 2 minutes. The proteins were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk for 2–3 hours at room temperature and washed three times with PBST. Subsequently, the membranes were incubated with specific primary antibodies at 4°C overnight (~ 10 hours). After washing, the membranes were incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 1.5 hours at room temperature. Protein bands were visualized using a gel imaging system with an enhanced chemiluminescence (ECL) substrate. For blot reprobing, the membranes were stripped with stripping buffer for 3 minutes, washed three times with PBST, and then rapidly re-blocked with a fast-blocking buffer for 10 minutes before sequential incubation with primary and secondary antibodies against a loading control protein. The relative intensity of the target protein bands was quantified using ImageJ software. 2.11 Reactive Oxygen Species (ROS) Detection Cells were seeded in 6-well plates at a density of 4×10⁵ cells per well for the intracellular ROS assay. After 24 hours, the cells were subjected to various drug treatments. A positive control was established by treating cells with a reagent (e.g., Rosup) at a 1:1000 dilution in culture medium for 20 minutes, followed by a 3–4 hour incubation. At the end of the treatment period, the cells were incubated with 1 mL of serum-free medium containing 10 µM DCFH-DA (diluted 1:1000 from the stock) at 37°C for 30 minutes in the dark. Subsequently, the cells were harvested, washed twice with serum-free medium to remove excess probe, and resuspended in 0.5 mL of PBS. The intracellular ROS levels, as indicated by the fluorescence intensity of DCF, were immediately analyzed by flow cytometry within 30 minutes. 2.12 Network Pharmacology Potential targets of HF were retrieved from the STITCH and PharmMapper databases. Known therapeutic targets for gastric cancer (GC) were identified using TTD, OMIM, and GeneCards databases. Common targets between HF and GC were identified using an online Venn diagram tool (SRplot). Protein-protein interaction (PPI) networks of the common targets were constructed using the STRING database. Functional enrichment analysis (Gene Ontology - GO, and Kyoto Encyclopedia of Genes and Genomes - KEGG) of the common targets was performed using SRplot and Sangerbox platforms. 2.13 Statistical Analysis Data are presented as mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 8.0. Comparisons between two groups were performed using unpaired two-tailed Student's t-tests. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test. A P value < 0.05 was considered statistically significant. 3. Results 3.1. HF inhibits the viability and proliferation of gastric cancer cells To investigate the effect of HF on the proliferation of gastric cancer SGC7901 and MKN45 cells, cell viability was assessed using the CCK-8 assay following treatment with various concentrations of HF for 24 and 48 hours. The calculated IC₅₀ values for HF in SGC7901 cells were 1276 nmol/L at 24 hours and 397.6 nmol/L at 48 hours. In MKN45 cells, HF exhibited greater potency, with IC₅₀ values of 114.6 nmol/L at 24 hours and 72.52 nmol/L at 48 hours. These results demonstrate a concentration- and time-dependent inhibitory effect of HF on the viability of both gastric cancer cell lines 3.2. HF activates apoptotic programs and induces CDK2-dependent cell cycle arrest in gastric cancer cells To determine whether HF induces apoptosis, SGC7901 and MKN45 cells were treated with varying concentrations of HF for 48 hours. Apoptosis was subsequently assessed using flow cytometry with an Annexin V-FITC/PI apoptosis detection kit. Compared with the control group, HF treatment significantly increased the percentage of apoptotic cells in both SGC7901 and MKN45 cell lines in a concentration-dependent manner (Fig. 2 A-C). Western blot analysis was performed to examine the expression of apoptosis-related proteins (Fig. 2 G-I). The results showed that HF treatment for 48 hours increased the expression of Cleaved-PARP, Cleaved-caspase-3, and Bax, while it decreased the expression of full-length PARP and Bcl-2 in a dose-dependent fashion. These results collectively indicate that HF induces apoptosis in a dose-dependent manner. The effect of HF on cell cycle distribution in SGC7901 and MKN45 cells was investigated using flow cytometry. Compared with the control, HF treatment increased the proportion of cells in the G1 phase while decreasing the proportion in the S phase (Fig. 2 D-F), suggesting that HF induces G1 phase arrest. To further explore the underlying mechanism, the expression levels of the key cell cycle-related proteins CDK2 and p-CDK2 were analyzed by western blot after treatment with different concentrations of HF (Fig. 2 J-L). The results demonstrated that HF reduced the expression levels of both CDK2 and p-CDK2, consistent with the observed alteration in cell cycle phase distribution. 3.3. HF suppresses tumor growth in a mouse xenograft model To evaluate the antitumor efficacy of HF in vivo, a subcutaneous xenograft model was established in nude mice using MKN45 cells. The mice were assigned to the following treatment groups: high-dose HF (0.2 mg/kg), low-dose HF (0.1 mg/kg), cisplatin (2 mg/kg), and a control group receiving saline. Both HF and cisplatin treatment resulted in a significant reduction in tumor volume compared to the control group (Fig. 3 A, B). Consistently, the weight of excised tumors from the HF and cisplatin groups was significantly lower than that from the control group (Fig. 3 E). Throughout the experimental period, no significant difference in body weight was observed between the HF -treated groups and the control group. In contrast, the cisplatin group exhibited a decrease in body weight over time (Fig. 3 C), suggesting treatment-related toxicity. TUNEL staining of the excised tumor tissues revealed a more pronounced induction of apoptosis in the HF and cisplatin groups compared to the control (Fig. 3 D). Immunohistochemical staining of tumor tissues for Ki67 and Caspase-3 showed that HF treatment increased the expression level of Cleaved Caspase-3, indicating enhanced apoptosis. Conversely, the expression level of Ki67 was decreased in the HF and cisplatin groups compared to the control (Fig. 3 F, G), suggesting a suppression of tumor cell proliferation. Histopathological examination by H&E staining demonstrated intact architecture and no significant pathological alterations in the liver, spleen, heart, lungs, or kidneys (Fig. 3 J). Furthermore, analysis of blood biochemical markers, including aspartate aminotransferase (AST), alanine aminotransferase (ALT) for liver function, and blood urea nitrogen (BUN) and creatinine (CREA) for kidney function, confirmed that all measured parameters remained within normal ranges across all groups (Fig. 3 H). In summary, HF administration at a dose of 0.10 mg/kg significantly inhibited the growth of MKN45 xenograft tumors without inducing apparent systemic toxicity, as evidenced by stable body weight, normal organ histology, and unaltered serum biochemical indices. 3.4. HF promotes ROS generation in gastric cancer cells To investigate the connection between HF and gastric cancer, a Venn diagram was constructed using the SRplot platform to identify overlapping targets, resulting in 100 cross-target genes (Fig. 4 A). The top 10 Gene Ontology (GO) terms for biological process (BP), cellular component (CC), and molecular function (MF) are displayed in a bar plot (Fig. 4 B). The BP terms were predominantly associated with the response to oxidative stress, cellular response to oxidative stress, and response to reactive oxygen species. As ROS are typically generated and accumulated under oxidative stress and influence various biological processes, intracellular ROS levels in SGC7901 and MKN45 cells were detected using DCFH-DA staining followed by flow cytometric analysis (Fig. 4 C, D). HF treatment significantly increased ROS levels in both cell lines in a concentration-dependent manner. 3.5. Scavenging ROS rescues HF-induced apoptosis and cell cycle arrest To further verify whether ROS acts as a critical upstream mediator of HF-induced apoptosis and cell cycle arrest, cells were pretreated with the ROS inhibitor N-acetylcysteine (NAC, 5 mM). The combination of NAC and HF attenuated the rightward shift in the ROS fluorescence peak induced by HF alone (Fig. 5 A, B), indicating effective ROS scavenging. Cell cycle analysis demonstrated that co-treatment with NAC and HF reduced the extent of HF-induced S-phase arrest (Fig. 5 C, D). Similarly, apoptosis assays revealed that the combination of NAC and HF significantly decreased the percentage of apoptotic cells compared to HF treatment alone (Fig. 5 E, F). These results collectively indicate that the elevation of intracellular ROS levels by HF plays a pivotal role in mediating its effects on cell cycle progression and apoptosis. 3.6. HF exerts antitumor effects by modulating the PI3K/Akt pathway in gastric cancer cells To elucidate the potential mechanism underlying the antitumor effects of HF, RNA sequencing was performed. As described in Section 2.4 , we identified 100 overlapping targets between HF and gastric cancer. An interaction network for these overlapping targets was subsequently constructed using the STRING platform. Key genes, including HIF1A, TGFB1, HSP90AB1, NFKB1, and SMAD3, occupied central positions within this network (Fig. 6 A). KEGG pathway enrichment analysis of the protein-protein interaction (PPI) network revealed that the PI3K/Akt signaling pathway plays a significant role in the action of HF against gastric cancer (Fig. 6 B). Both the gene expression profile and KEGG analysis indicated the PI3K/Akt signaling pathway was among the top pathways affected by HF. The protein levels of key components of the PI3K/AKT pathway were assessed by Western blotting in HF-treated SGC7901 and MKN45 cells, using β-actin as a loading control. The results demonstrated that HF treatment significantly reduced the phosphorylation levels of AKT and PI3K (p-AKT and p-PI3K), while the levels of total AKT and PI3K remained largely unchanged (Fig. 6 C-E). 4. Discussion Gastric cancer (GC) remains one of the most common malignancies worldwide, characterized by high incidence and mortality rates, particularly in East Asia, which bears a substantial disease burden ( SIEGEL R L et al. 2016). Approximately 40–50% of patients with advanced GC develop distant metastases, frequently to the peritoneum, liver, and lymph nodes, leading to a dramatic deterioration in prognosis and a five-year survival rate of less than 10% (Smyth EC et al. 2020 ). The metastatic propensity of GC is closely associated with its highly heterogeneous tumor microenvironment, aberrant activation of epidermal growth factor receptor (EGFR) signaling (Zhang X et al. 2022 ), and VEGF-mediated angiogenesis and lymphatic invasion (Hamada Y et al. 2024 ). Despite advancements in systemic chemotherapy and molecular targeted therapies (e.g., anti-HER2 agents), the treatment of advanced GC continues to face significant clinical challenges due to high tumor heterogeneity, frequent primary or acquired resistance, and limited drug penetration, especially in peritoneal metastases (Cai X et al. 2024 ). Consequently, developing novel therapeutic strategies targeting the mechanisms of gastric cancer metastasis remains an urgent research priority. HF, an alkaloid derivative derived from the traditional Chinese medicine (Dichroa febrifuga), is a multi-target small-molecule inhibitor that has demonstrated significant antitumor potential across various malignancy models (Hei YY et al. 2022 ). Its primary mechanism of action involves inhibiting prolyl-tRNA synthetase, triggering an amino acid starvation response, thereby disrupting protein synthesis and tumor microenvironment remodeling (Wang J et al. 2024 ). Studies have shown that HF effectively inhibits tumor cell proliferation, induces apoptosis and cell cycle arrest, and significantly suppresses metastatic progression by modulating the TGF-β signaling pathway and epithelial-mesenchymal transition (EMT) (Wang J et al. 2025 ). Furthermore, this compound has exhibited promising antitumor activity with manageable toxicity in numerous preclinical models, including colorectal cancer (Gong RH et al. 2022 ), breast cancer (Xia X et al. 2019 ), and multiple myeloma ( Lamora A et al. 2015 ). Against this backdrop, we innovatively proposed that HF might hold substantial value in gastric cancer treatment. Despite its notable effects in various cancers, its efficacy and underlying mechanisms in GC, particularly in advanced stages, remained largely unexplored. This study aimed to systematically evaluate the inhibitory effects of HF on gastric cancer cells and explore its potential therapeutic application, while elucidating the underlying molecular mechanisms. In in vitro experiments, we first assessed the impact of HF on gastric cancer cell viability at different concentrations and exposure times. By calculating the half-maximal inhibitory concentration (IC₅₀), we found that HF significantly inhibited the survival of GC cell lines, such as SGC7901 and MKN45, at low nanomolar concentrations, demonstrating potent in vitro antitumor activity. Based on these findings, we established the concentration range for subsequent experiments. Our results clearly indicated that HF suppressed gastric cancer cell survival in a concentration- and time-dependent manner, consistent with its known multi-target mechanisms of action (Sun Y et al. 2025 , Liu K et al.1998 and Zcharia E et al. 2012 ). Furthermore, EdU incorporation and colony formation assays confirmed that HF markedly inhibited the proliferation and long-term clonogenic expansion of gastric cancer cells. These findings prompted a deeper investigation into the specific mechanisms underlying its growth-inhibitory effects, providing a theoretical foundation for advancing its development as a therapeutic strategy for GC. As demonstrated in our results, HF effectively induced S-phase cell cycle arrest and apoptosis in gastric cancer cells, highlighting its role as a multi-target natural compound in anti-GC therapy. Flow cytometric analysis revealed significant S-phase arrest accompanied by a concentration-dependent increase in the Sub-G1 apoptotic population, suggesting concurrent disruption of DNA replication and activation of programmed cell death pathways. These phenotypic observations align with HF's known mechanism of inhibiting prolyl-tRNA synthetase, triggering an amino acid stress response, and disrupting proteostasis (Tye MA et al. 2022 ). Mechanistically, S-phase arrest is closely associated with replication stress response. HF treatment suppressed CDK2 kinase activity, impeding DNA synthesis, a finding consistent with previous reports of HF causing replication fork stalling and genomic instability in other cancer types (Sun P et al. 2022). Additionally, HF exhibited significant pro-apoptotic capacity, experimentally confirmed by its induction of mitochondrial membrane potential loss, promotion of cytochrome c release, and marked increase in cleaved caspase-3 and cleaved PARP levels, indicating the initiation of apoptosis via the intrinsic mitochondrial pathway. This aligns with HF's ability to upregulate the pro-apoptotic protein Bax, downregulate the anti-apoptotic protein Bcl-2, thereby altering the Bcl-2 family balance (Wang H et al. 2024 ). Notably, our preliminary data suggested that this compound could induce substantial reactive oxygen species (ROS) generation, and this ROS burst might serve as an upstream event mediating mitochondrial pathway apoptosis and cell cycle arrest. The specific role and mechanism of ROS within the GC regulatory network became a key focus for further investigation in this study. To validate this hypothesis, we measured intracellular ROS levels following HF treatment. The experimental results demonstrated that HF induced significant ROS accumulation in gastric cancer cells, which subsequently inhibited the phosphorylation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT), ultimately repressing the activation of this pro-survival signaling pathway. This effect is consistent with the role of ROS as second messengers regulating kinase activity: excessive ROS can enhance the dephosphorylation activity of phosphatases like PTEN through oxidative modification of cysteine residues, thereby negatively regulating the PI3K/AKT pathway (Wang SQ et al. 2016 ). Conversely, ROS burst can also directly or indirectly disrupt AKT phosphorylation, leading to the failure of its downstream proliferation and anti-apoptotic signals (Lee EY et al. 2017 ). Mechanistically, the oxidative stress triggered by HF and the concomitant inhibition of PI3K/AKT signaling together constitute the core molecular basis of its anti-gastric cancer effects. This study further confirms that halofuginone, via a ROS-dependent mechanism, inhibits the PI3K/AKT pathway, significantly impedes gastric cancer cell proliferation, and promotes apoptosis, providing novel experimental evidence and mechanistic support for the translational development of this compound as a therapeutic strategy for gastric cancer. HF demonstrated significant antitumor activity and a favorable safety profile in preclinical gastric cancer models, positioning it as a highly promising therapeutic candidate for translational development. In human GC cell line-derived xenograft models, HF treatment resulted in a significant, dose-dependent reduction in tumor volume and weight compared to the control group. More importantly, this notable therapeutic efficacy was achieved without observed significant systemic toxicity: serum biochemical parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA), remained within physiological ranges, and histopathological analysis revealed no significant damage to vital organs such as the liver and kidneys. This superior safety profile contrasts sharply with the common hepatotoxicity and myelosuppression associated with conventional chemotherapeutic agents, highlighting the potential selective advantage of natural, multi-target agents. These findings suggest that HF offers a high clinical safety window while effectively suppressing GC progression, providing crucial preclinical evidence for its further development as an anti-gastric cancer drug. The findings of this study indicate that HF significantly inhibits the malignant progression of gastric cancer, primarily through a mechanism involving the induction of reactive oxygen species (ROS) generation and the subsequent suppression of PI3K/AKT signaling pathway phosphorylation (Fig. 7 ). In summary, our experiments confirm that HF effectively inhibits gastric cancer cell proliferation, induces apoptosis, and causes S-phase cell cycle arrest in vitro. In in vivo animal models, it similarly demonstrates potent tumor-suppressive effects and regulates the expression of apoptosis- and cell cycle-related proteins. Mechanistically, we further elucidated that the antitumor effect of this compound depends on its inhibition of PI3K and AKT phosphorylation, thereby blocking downstream pro-survival and proliferative signaling. However, this study has certain limitations. For instance, the biodistribution and metabolic properties of HF within the complex in vivo microenvironment, as well as its potential crosstalk with other signaling pathways (e.g., TGF-β or MAPK), warrant further investigation. Declarations CRediT authorship contribution statement Jing Jiang: Performed cellular and animal experiments. Data curation, Writing - original draft. PeiPei Liu and DaWei Tang: Methodology, Investigation, Validation, Data curation. YongPing Li: Validation, Software, Formal analysis. PeiPei Liu: Visualization, Writing - review & editing, Supervision. YueHua Han: Project administration.CongYan Yang: Conceptualization, Resource acquisitionh, Data curation, Formal analysis, Writing - review & editing. All authors have read and agreed to the published version of the manuscript. Ethical Statement All animal experiments were reviewed and approved by the Animal Ethics Committee of Bengbu Medical College (Approval No. [2025] 132). All procedures were conducted in accordance with institutional guidelines and international standards for the care and use of laboratory animals. Consent for publication All authors consent to publication of this article. Declaration of competing interest The authors declare no conflict of interest. Author Contribution Jing Jiang: Performed cellular and animal experiments. Data curation, Writing - original draft. PeiPei Liu and DaWei Tang: Methodology, Investigation, Validation, Data curation. YongPing Li: Validation, Software, Formal analysis. PeiPei Liu: Visualization, Writing - review & editing, Supervision. YueHua Han: Project administration.CongYan Yang: Conceptualization, Resource acquisitionh, Data curation, Formal analysis, Writing - review & editing. All authors have read and agreed to the published version of the manuscript. Acknowledgments This work was supported by: 1.The Anhui Provincial Outstanding Research and Innovation Team Project (2022AH010084). 2.The Anhui Provincial Engineering Technology Research Center for Biochemical Drugs (2023SYKFD06). 3.The Key Project of Anhui Provincial Outstanding Young Talent Support Program in Higher Education Institutions (YQZD2024029). 4.The Bengbu Medical College Graduate Research and Innovation Program (Byycx24064). Availability of data and materials The datasets supporting the conclusions of this article are available from the corresponding author on reasonable request. References Bock FJ, Tait SWG. Mitochondria as multifaceted regulators of cell death. Nat Rev Mol Cell Biol. 2020;21 (2):85–100. doi: 10.1038/s41580-019-0173-8 . Cai X, Cao M, Yang Q, et al. HER2-targeted ADC DX126-262 combined with chemotherapy demonstrates superior antitumor efficacy in HER2-positive gastric cancer. Am J Cancer Res. 2024;14 (12):5752–5768. doi: 10.62347/QCDR9612 . 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Orally Administered Halofuginone-Loaded TPGS Polymeric Micelles Against Triple-Negative Breast Cancer: Enhanced Absorption and Efficacy with Reduced Toxicity and Metastasis. Int J Nanomedicine. 2022;17:2475–2491. doi: 10.2147/IJN.S352538 . Additional Declarations No competing interests reported. Supplementary Files WBOriginalImage.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8446424","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":583168237,"identity":"62ed705f-27f3-42b1-a4f3-c11ce1a81bef","order_by":0,"name":"Jing Jiang","email":"","orcid":"","institution":"Bengbu Medical College","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Jiang","suffix":""},{"id":583168238,"identity":"d03419ec-cf9d-4a2b-972d-80047d030453","order_by":1,"name":"PeiPei Liu","email":"","orcid":"","institution":"Bengbu Medical College","correspondingAuthor":false,"prefix":"","firstName":"PeiPei","middleName":"","lastName":"Liu","suffix":""},{"id":583168239,"identity":"93d22018-aa92-4e3f-889f-c0475e1149b1","order_by":2,"name":"DaWei Tang","email":"","orcid":"","institution":"Bengbu Medical College","correspondingAuthor":false,"prefix":"","firstName":"DaWei","middleName":"","lastName":"Tang","suffix":""},{"id":583168240,"identity":"344a09ad-d24c-45c5-8bd5-f229607e081b","order_by":3,"name":"YueHua Han","email":"","orcid":"","institution":"Bengbu Medical College","correspondingAuthor":false,"prefix":"","firstName":"YueHua","middleName":"","lastName":"Han","suffix":""},{"id":583168241,"identity":"ddef2d4e-cfb9-48d6-bc7a-5aa79fc63733","order_by":4,"name":"YongPing Li","email":"","orcid":"","institution":"Bengbu Medical College","correspondingAuthor":false,"prefix":"","firstName":"YongPing","middleName":"","lastName":"Li","suffix":""},{"id":583168242,"identity":"612e3eaa-6ecf-425a-b395-268d2a58471a","order_by":5,"name":"Lei Li","email":"","orcid":"","institution":"Bengbu Medical College","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Li","suffix":""},{"id":583168243,"identity":"8712ef62-10c6-4ed0-b1d5-73e7deb7e58d","order_by":6,"name":"CongYan Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYBACNmbGxgcfKiR47I/3sEGEDhDQws/O3Gw444yNDMOZM0Rqkexnb5PmbUmzYbiRQ6QWg8OMQC0Nh3kYZ7499uhmG4Mc340Exs8F+LU0W87dcZiHWTov3Ti3jcFY8kYCs/QM/Foab7w9c5iHTTrHTBqoJXHDjQQ2Zh78WhokeNsO8/BIngFrqSeoRbKZsUmSty2NR0KCB6wlwYCQFn5mRnAg8xjwAP2Sc07CcOaZh83S+LSw8R9/CIpKewP2s8ce55TZyPMdTz74GZ8WdCABxIwNJGgYBaNgFIyCUYANAACqrEtwgeEu8wAAAABJRU5ErkJggg==","orcid":"","institution":"Bengbu Medical College","correspondingAuthor":true,"prefix":"","firstName":"CongYan","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-12-25 05:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8446424/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8446424/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101752925,"identity":"bfb22047-588c-4900-8e5f-363e72215f92","added_by":"auto","created_at":"2026-02-03 10:38:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":924954,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHF inhibits the proliferation of gastric cancer cells.\u003c/strong\u003e (A, B) Cell viability of SGC7901 and MKN45 cells treated with the indicated concentrations of HF for 24 h and 48 h, as assessed by the CCK-8 assay. (C) Representative images of colony formation in SGC7901 and MKN45 cells following treatment with different concentrations of HF. (D, E) Quantitative analysis of the colony formation assays shown in (C). (F, G) Representative images of EdU staining (red) demonstrating the effect of HF on cell proliferation after 24 h of treatment. Cell nuclei were counterstained with Hoechst 33342 (blue). (H, I) Quantitative analysis of the EdU-positive cells from (F, G). Data are presented as the mean ± SD from three independent experiments (n=3). \u003cem\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001\u003c/em\u003e versus the control group.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8446424/v1/96dbf1c4a6accba343c2a7fc.png"},{"id":101753070,"identity":"50be27e8-06ee-4af1-8b09-4ac44d2053d5","added_by":"auto","created_at":"2026-02-03 10:39:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":954455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHF induces apoptosis and cell cycle arrest in gastric cancer cells. \u003c/strong\u003e(A) Flow cytometric analysis of apoptosis in SGC7901 and MKN45 cells after treatment with HF for 48 h. (B, C) Quantitative analysis of the apoptosis rate in SGC7901 and MKN45 cells, respectively. (D) Cell cycle distribution analyzed by flow cytometry following treatment with different concentrations of HF. (E, F) Quantitative analysis of the percentage of cells in G1, S, and G2/M phases for SGC7901 and MKN45 cells, respectively. (G) Western blot analysis of apoptosis-related proteins (Cleaved-PARP, Cleaved-caspase-3, Bcl-2, Bax) after 48 h of HF treatment. (H, I) Quantitative analysis of the expression levels of apoptosis-related proteins. (J) Western blot analysis of cell cycle-related proteins (CDK2, p-CDK2, P21, Cyclin A2). (K, L) Quantitative analysis of the expression levels of cell cycle-related proteins. Data are presented as the mean±SD from three independent experiments (n=3). \u003cem\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 \u003c/em\u003eversus the control group.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8446424/v1/a915479daaf2e9c5afd367eb.png"},{"id":101646867,"identity":"fa94e209-6450-4a1a-b3ae-cd1c76580050","added_by":"auto","created_at":"2026-02-02 08:44:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1689143,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHF suppresses tumor growth in a mouse xenograft model in vivo.\u003c/strong\u003e (A) Representative images of subcutaneous tumors from the saline control, HF (0.10 mg/kg), HF (0.20 mg/kg), and cisplatin (2 mg/kg) treatment groups after the dosing period. (B) Tumor volume growth curves for the different treatment groups. (C) Body weight change curves of mice in the different treatment groups during the experiment. (D) TUNEL staining of tumor tissues from the different treatment groups. (E) Tumor weights measured at the endpoint for each treatment group. (F) Representative immunohistochemical staining images of Ki67 and Cleaved Caspase-3 in tumor tissues from different treatment groups. (G) Quantitative analysis of Ki67-positive cells (G) and Cleaved Caspase-3-positive cells (H) from (F). (J) H\u0026amp;E staining of major organs (heart, liver, spleen, lungs, kidneys) from the different treatment groups. (H) Blood biochemistry levels of aspartate aminotransferase (AST, J), alanine aminotransferase (ALT, K), blood urea nitrogen (BUN, L), and creatinine (CREA, M) in mice from the different treatment groups. Data are presented as the mean ± SD (n=8 mice per group). \u003cem\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 \u003c/em\u003eversus the control group.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8446424/v1/7a697c83029beb301d500b8c.png"},{"id":101646871,"identity":"c8ce19dc-189a-4b0a-9f03-12ed76f6c8cb","added_by":"auto","created_at":"2026-02-02 08:44:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":440493,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHF inhibits gastric cancer cells via ROS-mediated mechanisms and promotes intracellular ROS levels in SGC7901 and MKN45 cells.\u003c/strong\u003e (A) Venn diagram illustrating the overlapping targets between HF and gastric cancer. (B) GO enrichment analysis bar plot showing the top 10 enriched terms for the common targets in biological process (BP), cellular component (CC), and molecular function (MF) categories. (C) Representative flow cytometry histograms showing intracellular ROS levels detected by DCFH-DA fluorescence in SGC7901 and MKN45 cells treated with HF. (D) Quantitative analysis of the mean fluorescence intensity (MFI) of DCF representing ROS levels in SGC7901 and MKN45 cells. Data are presented as the mean ± SD from three independent experiments (n=3). \u003cem\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01\u003c/em\u003e versus the control group.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8446424/v1/f1d5a4aedf07cf237e9b806e.png"},{"id":101646872,"identity":"27fb8c14-7c85-4c6b-8348-1009abed9e90","added_by":"auto","created_at":"2026-02-02 08:44:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":684603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScavenging ROS rescues HF-induced apoptosis and cell cycle arrest. \u003c/strong\u003e(A, B) Intracellular ROS levels after combined treatment with HF (120 nM) and NAC (5 mM), shown by representative histograms (A) and quantitative analysis (B) for SGC7901 and MKN45 cells, respectively. (C, D) Cell cycle distribution analyzed by flow cytometry and quantitative analysis of the percentage of cells in G1, S, and G2/M phases for SGC7901 and MKN45 cells after combined treatment. (E, F) Apoptosis assessed by flow cytometry and quantitative analysis of the apoptosis rate for SGC7901 and MKN45 cells after combined treatment. Data are presented as the mean ±SD from three independent experiments (n=3). \u003cem\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01 \u003c/em\u003eversus the indicated groups.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8446424/v1/93672650d16cd11dd133b11d.png"},{"id":101753370,"identity":"41d872bb-d5a2-46fe-8e32-d238d89615e1","added_by":"auto","created_at":"2026-02-03 10:39:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":479101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHF affects the protein levels of PI3K/Akt pathway components in gastric cancer cells.\u003c/strong\u003e (A) Protein-protein interaction (PPI) network of common targets between HF and gastric cancer. Central nodes are highlighted. (B) Top 20 enriched KEGG pathways for HF treatment in gastric cancer. (C) Western blot analysis of PI3K, p-PI3K, AKT, and p-AKT protein levels in SGC7901 and MKN45 cells treated with HF. β-Actin was used as a loading control. (D, E) Quantitative analysis of the relative protein expression levels of p-PI3K/PI3K and p-AKT/AKT in SGC7901 and MKN45 cells. Data are presented as the mean ± SD from three independent experiments (n=3). \u003cem\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01\u003c/em\u003e versus the control group.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8446424/v1/e0bf9ff99cd3066421f2c0d9.png"},{"id":101646865,"identity":"5c1e233f-dee0-49d5-9292-86050ccd0c42","added_by":"auto","created_at":"2026-02-02 08:44:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":310882,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram illustrating the proposed mechanism by which HF inhibits gastric cancer cells.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8446424/v1/090eaae4dfa6457907ea35ae.png"},{"id":105773582,"identity":"6a6c8e32-9b7f-4e71-b069-528b3832e254","added_by":"auto","created_at":"2026-03-31 02:26:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6640089,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8446424/v1/2518df73-dbc5-4979-a696-8b45625ebc25.pdf"},{"id":101646869,"identity":"e2b26af7-269c-46c0-9cda-fd002e123fc7","added_by":"auto","created_at":"2026-02-02 08:44:38","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1638945,"visible":true,"origin":"","legend":"","description":"","filename":"WBOriginalImage.docx","url":"https://assets-eu.researchsquare.com/files/rs-8446424/v1/2caa447c02f7f0e9e96b07e1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Halofuginone suppresses gastric tumor growth by inducing ROS-dependent inhibition of the PI3K/AKT pathway","fulltext":[{"header":"1. Introduction","content":" \u003cp\u003eGastric cancer (GC) represents a malignancy with high global incidence and mortality rates, particularly in China and East Asia (Fan X et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Patients with advanced gastric cancer face a poor prognosis, exhibiting a five-year survival rate of less than 10%, as most are diagnosed with locally advanced or metastatic disease (Tan Z et al. 2019). Although surgical resection combined with adjuvant chemoradiotherapy remains the primary treatment strategy, therapy failure and disease recurrence are common, largely attributable to chemotherapy resistance and severe systemic toxicity (Fujitani K et al. 2013). Molecularly, the pathogenesis and progression of gastric cancer involve aberrant activation of multiple signaling pathways, including the epidermal growth factor receptor (EGFR) (Ding Q et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) (Zhou P et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and vascular endothelial growth factor (VEGF) axes (Park DJ et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), as well as interactions between immune cells and cancer cells within the tumor microenvironment (Li Y et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, molecular features such as HER2 amplification (May M et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), microsatellite instability (MSI) (Yamamoto H et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and Epstein-Barr virus (EBV) infection (Kerr JR et al. 2019) contribute to the heterogeneity of GC and pose challenges for personalized therapy. Currently, imaging and endoscopy remain the mainstay for GC diagnosis and staging, yet their sensitivity for detecting early minimal lesions and peritoneal metastases remains limited (Sakamoto E et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In recent years, liquid biopsy techniques, such as circulating tumor DNA (ctDNA) analysis, have emerged as promising approaches for early detection and disease monitoring (Rodon et al. 2022). Regarding treatment, the median overall survival for advanced gastric cancer patients often falls below 12 months. Established chemotherapy regimens like platinum-based doublets (Zaanan A et al. 2022) and targeted agents such as trastuzumab (Lee J et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) demonstrate limited efficacy and are frequently associated with acquired resistance. Consequently, developing novel therapeutic agents with distinct mechanisms of action, capable of overcoming drug resistance and improving patient outcomes, represents a pressing and unmet need in current clinical and translational research for gastric cancer.\u003c/p\u003e \u003cp\u003eHF a quinazolinone alkaloid derivative derived from the traditional Chinese medicine (Dichroa febrifuga), has recently garnered significant attention due to its remarkable antitumor activity demonstrated across various malignancies (Wang J et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Li H et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e,Guo J et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, de Figueiredo-Pontes LL et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e and Ni J et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Research indicates that HF inhibits tumor cell proliferation, metastasis, and induces apoptosis through multiple pathways, showing significant efficacy in models of non-small cell lung cancer (NSCLC) (Han Y et al. 2024), prostate cancer (McLaughlin NP et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), colorectal cancer (Gong RH et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), breast cancer (Ju\u0026aacute;rez P et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and multiple myeloma (Leiba M et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). For instance, in NSCLC models, HF promotes apoptosis by upregulating the pro-apoptotic protein Bax, downregulating the anti-apoptotic protein Bcl-2, and activating the Caspase-3 pathway. Concurrently, it induces cell cycle arrest, such as at the G1 phase, through mechanisms involving the downregulation of Cyclin D1 and CDK2 expression (Xia X et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Its mechanism of action involves multi-target regulation, primarily by inhibiting prolyl-tRNA synthetase, which triggers the integrated stress response and subsequently impacts multiple signaling pathways (Keller TL et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) Regarding the inhibition of tumor metastasis, HF significantly suppresses the epithelial-mesenchymal transition (EMT) process (Chen Y et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It attenuates tumor cell migration and invasion capabilities by inhibiting the TGF-β/Smad signaling pathway, reducing the expression of mesenchymal markers like N-cadherin and Vimentin, and upregulating E-cadherin expression (Gnainsky Y et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNotably, HF holds substantial potential in reversing tumor drug resistance. Studies demonstrate that it can restore chemosensitivity in resistant tumor cells by inhibiting the activity and expression of P-glycoprotein (P-gp) (Zuo R et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In 5-fluorouracil-resistant colorectal cancer cells, HF effectively suppresses proliferation and induces apoptosis via regulating the miR-132-3p/TGF-β axis (Wang C et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, another crucial mechanism of HF, observed in NSCLC, involves triggering substantial intracellular accumulation of reactive oxygen species (ROS), leading to oxidative damage and activation of stress-related apoptotic pathways, thereby inhibiting NSCLC progression (Demiroglu-Zergeroglu A et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This effect appears to be mediated through HF-induced ROS accumulation, which causes oxidative stress, further resulting in DNA damage and mitochondrial dysfunction, ultimately suppressing tumor growth (Xu F et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Based on this evidence, our study rationally hypothesizes that HF may induce mitochondrial dysfunction and provoke intracellular ROS generation in gastric cancer cells.\u003c/p\u003e \u003cp\u003eReactive oxygen species (ROS) serve as critical signaling molecules that profoundly influence the fate decisions of tumor cells through their spatiotemporal regulation of various signaling pathways (Mittler R et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recent studies reveal that ROS dynamically regulate the phosphatidylinositol 3-kinase (PI3K)/serine-threonine kinase (AKT) pathway via redox modifications of key kinases or phosphatases, a process that is complex and exhibits significant tumor type-dependency (Pavlovic S et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For example, in liver cancer, overexpression of PDHA1 (pyruvate dehydrogenase E1 alpha subunit) increases intracellular ROS levels, subsequently inhibiting PI3K/AKT pathway activation and inducing G0/G1 phase cell cycle arrest and apoptosis (Wang J et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The inhibition of PI3K/AKT was alleviated upon ROS reduction using the antioxidant N-acetylcysteine (NAC), confirming the crucial mediating role of ROS (Pavlovic S et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, ROS accumulation can lead to the collapse of mitochondrial membrane potential, promoting the release of cytochrome C into the cytoplasm and activating the Caspase cascade, ultimately inducing apoptosis (Bock FJ et al. 2020). In a breast cancer study, a certain inducer led to increased ROS levels accompanied by induced apoptosis and cell cycle arrest; this involved upregulation of Bax, downregulation of Bcl-2, consequently increasing the Bax/Bcl-2 ratio, promoting mitochondrial pathway apoptosis, and downregulation of CDK2 expression inducing G0/G1 phase arrest (Das U et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, we demonstrate that the natural compound HF significantly inhibits the proliferation of human gastric cancer cells and induces apoptosis and cell cycle arrest. Further mechanistic investigation reveals that its antitumor effect is closely associated with the activation of reactive oxygen species (ROS) generation, which negatively regulates the PI3K/AKT signaling pathway, thereby inhibiting downstream pro-survival and proliferative signals. Notably, in a mouse xenograft model, HF treatment did not induce significant systemic toxicity or adverse reactions, indicating a favorable safety profile. This research unveils a novel redox regulatory mechanism based on the inhibition of the ROS-PI3K/AKT axis, not only providing fresh insights into the spatiotemporal regulation of kinase signaling pathways but also suggesting a potential target for overcoming drug resistance in gastric cancer and developing novel combination therapeutic strategies.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell Culture\u003c/h2\u003e \u003cp\u003eHuman gastric cancer cell lines SGC7901 and MKN45 were purchased from Wuhan Procell Life Science \u0026amp; Technology Co., Ltd. Cells were cultured in RPMI 1640 medium (Gibco Life Technologies) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cultures were maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Reagents and Chemicals\u003c/h2\u003e \u003cp\u003eTetrandrine and N-acetylcysteine (NAC) were both purchased from MedChem Express; the molecular formula of tetrandrine is C16H17BrClN3O3, with a purity of 99.28%, and the molecular formula of N-acetylcysteine is C5H9NO3S, with a purity of 99.86%. HF was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution, which was stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Cell Counting Kit-8 (CCK-8) was purchased from Biosharp. Apoptosis and cell cycle detection kits were acquired from Beyotime Biotechnology. The EdU-488 Cell Proliferation Kit and Reactive Oxygen Species (ROS) Assay Kit were from Beyotime Institute of Biotechnology. RPMI 1640 medium was from Gibco. Phosphate-buffered saline (PBS) was sourced from Wuhan Servicebio Technology Co., Ltd.The following primary antibodies were used: PARP (#9542), Cleaved PARP (#94885), Caspase-3 (#9663), Cleaved Caspase-3 (#9661), Cyclin A2 (#4656), P21 ( #2947), Bax (50599-2-Ig, Proteintech), Bcl-2 (60178-1-Ig, Proteintech), CDK2 (10122-1-AP, Proteintech), Phospho-CDK2 (AF3237, Affinity), β-Actin (#3700), PI3K (#4257), Phospho-PI3K (#4228), AKT (#9272), Phospho-AKT (#4060) (all CST unless specified).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Animals\u003c/h2\u003e \u003cp\u003eFour- to five-week-old male BALB/c nude mice (N\u0026thinsp;=\u0026thinsp;60, average body weight 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g) were purchased from Jiangsu Qionglongshan Biological Co., Ltd. (China). After one week of acclimatization, the mice were randomly assigned into groups (n\u0026thinsp;=\u0026thinsp;8 per group). All animals were provided with food and water and maintained under standard specific pathogen-free (SPF) conditions. Following 3\u0026ndash;5 days of additional acclimation post-grouping, subcutaneous tumor inoculation was performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Xenograft Tumor Model\u003c/h2\u003e \u003cp\u003e All animal experiments were approved by the Animal Experiment Ethics Committee of Bengbu Medical University (Approval No. #[2023]526) and conducted in accordance with the guidelines for the care and use of laboratory animals.\u003c/p\u003e \u003cp\u003eThe experimental procedure was as follows:\u003c/p\u003e \u003cp\u003eStep 1: Grouping and Model Establishment. Mice were randomly assigned to groups following one week of acclimatization. A xenograft model was established.\u003c/p\u003e \u003cp\u003eStep 2: Tumor Cell Inoculation. After the acclimatization period, MKN45 cells were resuspended in 0.8% sodium carboxymethyl cellulose (Na-CMC) at a density of 8 \u0026times; 10⁶ cells/mL. Each mouse received a subcutaneous injection of 0.2 mL of this cell suspension in the shoulder region.\u003c/p\u003e \u003cp\u003eStep 3: Group Allocation and Treatment Initiation. Tumor growth was monitored daily at the same time point, and volumes were measured using a caliper. When the measured tumor volumes reached 100\u0026ndash;150 mm\u0026sup3;, the mice were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;8 per group): a saline control group, a low-dose HF group (0.10 mg/kg), a high-dose HF group (0.20 mg/kg), and a cisplatin group (2 mg/kg).\u003c/p\u003e \u003cp\u003eStep 4: Drug Administration. The high- and low-dose HF groups received intraperitoneal (i.p.) injections every two days. The cisplatin group received i.p. injections at a dose of 2 mg/kg every three days. The control group received equivalent volumes of saline administered on a schedule matching the high-dose HF group. All treatments were administered via a 1mL syringe and continued for two weeks.\u003c/p\u003e \u003cp\u003eStep 5: Sample Collection and Termination. After two weeks of treatment, blood samples were collected from the mouse eyes for biochemical analysis. Subsequently, the mice were euthanized by cervical dislocation. Tumors were excised, photographed, and fixed in 4% paraformaldehyde for subsequent hematoxylin and eosin (H\u0026amp;E) staining observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 CCK-8 Assay\u003c/h2\u003e \u003cp\u003eSGC7901 and MKN45 cells were seeded in 96-well plates at a density of 8,000 cells per well and cultured until adherent. After 24 hours, when cell confluence reached approximately 70\u0026ndash;80%, the culture medium was replaced with fresh medium containing varying concentrations of HF. SGC7901 cells were treated with HF at 0, 60, 120, 240, 480, and 960 nmol/L, while MKN45 cells were treated with 0, 30, 60, 120, 240, 480, and 960 nmol/L. Each treatment group consisted of six replicate wells. Following 24 or 48 hours of drug exposure, 10 \u0026micro;L of CCK-8 solution was added to each well under light-protected conditions. The plates were then incubated at 37\u0026deg;C for 30 minutes, and the absorbance at 450 nm was measured using a microplate reader to calculate cell viability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Colony Formation Assay\u003c/h2\u003e \u003cp\u003eCells were seeded in 6-well plates at a density of 8,000 cells per well. After 24 hours of attachment, the cells were washed twice with PBS and incubated with fresh medium containing different concentrations of HF. The medium was refreshed every 3\u0026ndash;4 days. After 10\u0026ndash;14 days, when visible colonies had formed, the cells were fixed with 1\u0026ndash;2 mL of ice-cold 4% paraformaldehyde for 15 minutes and then stained with 2 mL of 0.1% crystal violet solution for 30 minutes at room temperature. Following staining, the plates were washed twice with PBS and air-dried. The dried plates were photographed, and the number of colonies was counted and analyzed using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 EdU-488 Cell Proliferation Assay\u003c/h2\u003e \u003cp\u003eCell proliferation was further assessed using an EdU (5-ethynyl-2'-deoxyuridine) assay kit. Cells were seeded in confocal dishes at 5\u0026times;10⁵ cells per dish and allowed to adhere until reaching approximately 70% confluence. They were then treated with different concentrations of HF for 24 hours. An equal volume of pre-warmed (37\u0026deg;C) 2\u0026times;EdU working solution (20 \u0026micro;M) was added to each dish, and incubation continued for another 2 hours. Subsequently, the cells were fixed and permeabilized with 1 mL of permeabilization buffer (PBS containing 0.5% Triton X-100) for 15\u0026ndash;20 minutes at room temperature in the dark. After removal of the permeabilization buffer, 1mL of 1\u0026times;Hoechst 33342 solution was added to each dish and incubated for 30 minutes at room temperature in the dark. The cells were then washed three times with PBS (5 minutes per wash). An anti-fade mounting medium (1 \u0026micro;L) was applied, and the cells were imaged under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Flow Cytometry Analysis of Apoptosis\u003c/h2\u003e \u003cp\u003eThe percentage of apoptotic cells was quantified using an Annexin V-FITC/PI apoptosis detection kit via flow cytometry. Cells in the logarithmic growth phase were harvested and seeded in 6-well plates at a density of 5\u0026times;10⁵ cells per well. After adherence, the cells were treated with various concentrations of HF for 48 hours. Following treatment, both adherent and floating cells were collected, washed with PBS, and resuspended in 400 \u0026micro;L of 1\u0026times; binding buffer. Then, 4 \u0026micro;L of Annexin V-FITC solution was added to the cell suspension, which was incubated for 15 minutes at room temperature in the dark. Subsequently, 5 \u0026micro;L of propidium iodide (PI) solution was added, and incubation continued for another 5 minutes in the dark. Apoptosis was immediately analyzed by flow cytometry following the incubation period.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Flow Cytometry Analysis of Cell Cycle Distribution\u003c/h2\u003e \u003cp\u003eCell cycle distribution was analyzed using a cell cycle analysis kit via flow cytometry. Cells were seeded in 6-well plates at a density of 4\u0026times;10⁵ cells per well. After adherence, the cells were treated with different concentrations of HF for 24 hours. Following treatment, the cells were harvested by trypsinization, collected in centrifuge tubes, and centrifuged. The cell pellet was washed with PBS and resuspended in 0.5 mL of PBS. Subsequently, the cells were fixed by adding pre-cooled 75% ethanol dropwise under gentle vortexing and incubated at -20\u0026deg;C for at least 1 hour. After fixation, the cells were centrifuged, and the ethanol was carefully aspirated, leaving approximately 50 \u0026micro;L to avoid disturbing the cell pellet. The cells were then washed twice with pre-cooled PBS. After a final centrifugation at 1200 rpm for 5 minutes, the supernatant was completely removed, and the cell pellet was resuspended in 480 \u0026micro;L of PBS. Then, 20 \u0026micro;L of RNase A (50\u0026times;) was added, and the mixture was incubated in a 37\u0026deg;C water bath for 30 minutes. The cell suspension was filtered through a 400-mesh cell strainer, centrifuged, and the pellet was resuspended in 400 \u0026micro;L of 1\u0026times; propidium iodide (PI) staining solution. The cells were incubated in the dark at 4\u0026deg;C for 1 hour before cell cycle distribution was analyzed by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Western Blotting\u003c/h2\u003e \u003cp\u003eAfter 24 hours of drug treatment, cellular proteins were extracted. Total protein concentration was determined using a BCA protein assay kit. Briefly, cells were collected and centrifuged, and the supernatant was removed. The cell pellet was lysed on ice with freshly prepared RIPA lysis buffer (containing protease and phosphatase inhibitors) for 30 minutes. The lysate was then centrifuged at 12,000 rpm for 20 minutes at 4\u0026deg;C, and the supernatant was collected. According to the kit instructions, a working reagent was prepared by mixing Reagent A and Reagent B at a 50:1 ratio. In a 96-well plate, 200 \u0026micro;L of the working reagent, 19 \u0026micro;L of PBS, and 1 \u0026micro;L of protein sample were added to each well. Each sample was assayed in triplicate to minimize experimental error. The plate was covered with foil and incubated at 37\u0026deg;C for 30 minutes in the dark. The absorbance at 562 nm was measured using a microplate reader, and the protein concentration of each sample was calculated. Concentrated samples were diluted with RIPA lysis buffer to normalize concentrations across all samples. An appropriate volume of loading buffer was added to the protein lysates, which were then denatured by heating at 95\u0026deg;C for 5 minutes, followed by brief centrifugation at 1000 rpm for 2 minutes. The proteins were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk for 2\u0026ndash;3 hours at room temperature and washed three times with PBST. Subsequently, the membranes were incubated with specific primary antibodies at 4\u0026deg;C overnight (~\u0026thinsp;10 hours). After washing, the membranes were incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 1.5 hours at room temperature. Protein bands were visualized using a gel imaging system with an enhanced chemiluminescence (ECL) substrate. For blot reprobing, the membranes were stripped with stripping buffer for 3 minutes, washed three times with PBST, and then rapidly re-blocked with a fast-blocking buffer for 10 minutes before sequential incubation with primary and secondary antibodies against a loading control protein. The relative intensity of the target protein bands was quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Reactive Oxygen Species (ROS) Detection\u003c/h2\u003e \u003cp\u003eCells were seeded in 6-well plates at a density of 4\u0026times;10⁵ cells per well for the intracellular ROS assay. After 24 hours, the cells were subjected to various drug treatments. A positive control was established by treating cells with a reagent (e.g., Rosup) at a 1:1000 dilution in culture medium for 20 minutes, followed by a 3\u0026ndash;4 hour incubation. At the end of the treatment period, the cells were incubated with 1 mL of serum-free medium containing 10 \u0026micro;M DCFH-DA (diluted 1:1000 from the stock) at 37\u0026deg;C for 30 minutes in the dark. Subsequently, the cells were harvested, washed twice with serum-free medium to remove excess probe, and resuspended in 0.5 mL of PBS. The intracellular ROS levels, as indicated by the fluorescence intensity of DCF, were immediately analyzed by flow cytometry within 30 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Network Pharmacology\u003c/h2\u003e \u003cp\u003ePotential targets of HF were retrieved from the STITCH and PharmMapper databases. Known therapeutic targets for gastric cancer (GC) were identified using TTD, OMIM, and GeneCards databases. Common targets between HF and GC were identified using an online Venn diagram tool (SRplot). Protein-protein interaction (PPI) networks of the common targets were constructed using the STRING database. Functional enrichment analysis (Gene Ontology - GO, and Kyoto Encyclopedia of Genes and Genomes - KEGG) of the common targets was performed using SRplot and Sangerbox platforms.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.13 Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 8.0. Comparisons between two groups were performed using unpaired two-tailed Student's t-tests. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test. A P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1. HF inhibits the viability and proliferation of gastric cancer cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo investigate the effect of HF on the proliferation of gastric cancer SGC7901 and MKN45 cells, cell viability was assessed using the CCK-8 assay following treatment with various concentrations of HF for 24 and 48 hours. The calculated IC₅₀ values for HF in SGC7901 cells were 1276 nmol/L at 24 hours and 397.6 nmol/L at 48 hours. In MKN45 cells, HF exhibited greater potency, with IC₅₀ values of 114.6 nmol/L at 24 hours and 72.52 nmol/L at 48 hours. These results demonstrate a concentration- and time-dependent inhibitory effect of HF on the viability of both gastric cancer cell lines\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2. HF activates apoptotic programs and induces CDK2-dependent cell cycle arrest in gastric cancer cells\u003c/h2\u003e \u003cp\u003eTo determine whether HF induces apoptosis, SGC7901 and MKN45 cells were treated with varying concentrations of HF for 48 hours. Apoptosis was subsequently assessed using flow cytometry with an Annexin V-FITC/PI apoptosis detection kit. Compared with the control group, HF treatment significantly increased the percentage of apoptotic cells in both SGC7901 and MKN45 cell lines in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). Western blot analysis was performed to examine the expression of apoptosis-related proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-I). The results showed that HF treatment for 48 hours increased the expression of Cleaved-PARP, Cleaved-caspase-3, and Bax, while it decreased the expression of full-length PARP and Bcl-2 in a dose-dependent fashion. These results collectively indicate that HF induces apoptosis in a dose-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effect of HF on cell cycle distribution in SGC7901 and MKN45 cells was investigated using flow cytometry. Compared with the control, HF treatment increased the proportion of cells in the G1 phase while decreasing the proportion in the S phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F), suggesting that HF induces G1 phase arrest. To further explore the underlying mechanism, the expression levels of the key cell cycle-related proteins CDK2 and p-CDK2 were analyzed by western blot after treatment with different concentrations of HF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ-L). The results demonstrated that HF reduced the expression levels of both CDK2 and p-CDK2, consistent with the observed alteration in cell cycle phase distribution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3. HF suppresses tumor growth in a mouse xenograft model\u003c/h2\u003e \u003cp\u003eTo evaluate the antitumor efficacy of HF in vivo, a subcutaneous xenograft model was established in nude mice using MKN45 cells. The mice were assigned to the following treatment groups: high-dose HF (0.2 mg/kg), low-dose HF (0.1 mg/kg), cisplatin (2 mg/kg), and a control group receiving saline. Both HF and cisplatin treatment resulted in a significant reduction in tumor volume compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Consistently, the weight of excised tumors from the HF and cisplatin groups was significantly lower than that from the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Throughout the experimental period, no significant difference in body weight was observed between the HF -treated groups and the control group. In contrast, the cisplatin group exhibited a decrease in body weight over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), suggesting treatment-related toxicity. TUNEL staining of the excised tumor tissues revealed a more pronounced induction of apoptosis in the HF and cisplatin groups compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunohistochemical staining of tumor tissues for Ki67 and Caspase-3 showed that HF treatment increased the expression level of Cleaved Caspase-3, indicating enhanced apoptosis. Conversely, the expression level of Ki67 was decreased in the HF and cisplatin groups compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G), suggesting a suppression of tumor cell proliferation. Histopathological examination by H\u0026amp;E staining demonstrated intact architecture and no significant pathological alterations in the liver, spleen, heart, lungs, or kidneys (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Furthermore, analysis of blood biochemical markers, including aspartate aminotransferase (AST), alanine aminotransferase (ALT) for liver function, and blood urea nitrogen (BUN) and creatinine (CREA) for kidney function, confirmed that all measured parameters remained within normal ranges across all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eIn summary, HF administration at a dose of 0.10 mg/kg significantly inhibited the growth of MKN45 xenograft tumors without inducing apparent systemic toxicity, as evidenced by stable body weight, normal organ histology, and unaltered serum biochemical indices.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4. HF promotes ROS generation in gastric cancer cells\u003c/h2\u003e \u003cp\u003eTo investigate the connection between HF and gastric cancer, a Venn diagram was constructed using the SRplot platform to identify overlapping targets, resulting in 100 cross-target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The top 10 Gene Ontology (GO) terms for biological process (BP), cellular component (CC), and molecular function (MF) are displayed in a bar plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The BP terms were predominantly associated with the response to oxidative stress, cellular response to oxidative stress, and response to reactive oxygen species. As ROS are typically generated and accumulated under oxidative stress and influence various biological processes, intracellular ROS levels in SGC7901 and MKN45 cells were detected using DCFH-DA staining followed by flow cytometric analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). HF treatment significantly increased ROS levels in both cell lines in a concentration-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Scavenging ROS rescues HF-induced apoptosis and cell cycle arrest\u003c/h2\u003e \u003cp\u003eTo further verify whether ROS acts as a critical upstream mediator of HF-induced apoptosis and cell cycle arrest, cells were pretreated with the ROS inhibitor N-acetylcysteine (NAC, 5 mM). The combination of NAC and HF attenuated the rightward shift in the ROS fluorescence peak induced by HF alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B), indicating effective ROS scavenging. Cell cycle analysis demonstrated that co-treatment with NAC and HF reduced the extent of HF-induced S-phase arrest (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Similarly, apoptosis assays revealed that the combination of NAC and HF significantly decreased the percentage of apoptotic cells compared to HF treatment alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). These results collectively indicate that the elevation of intracellular ROS levels by HF plays a pivotal role in mediating its effects on cell cycle progression and apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.6. HF exerts antitumor effects by modulating the PI3K/Akt pathway in gastric cancer cells\u003c/h2\u003e \u003cp\u003eTo elucidate the potential mechanism underlying the antitumor effects of HF, RNA sequencing was performed. As described in Section \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003e2.4\u003c/span\u003e, we identified 100 overlapping targets between HF and gastric cancer. An interaction network for these overlapping targets was subsequently constructed using the STRING platform. Key genes, including HIF1A, TGFB1, HSP90AB1, NFKB1, and SMAD3, occupied central positions within this network (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). KEGG pathway enrichment analysis of the protein-protein interaction (PPI) network revealed that the PI3K/Akt signaling pathway plays a significant role in the action of HF against gastric cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Both the gene expression profile and KEGG analysis indicated the PI3K/Akt signaling pathway was among the top pathways affected by HF. The protein levels of key components of the PI3K/AKT pathway were assessed by Western blotting in HF-treated SGC7901 and MKN45 cells, using β-actin as a loading control. The results demonstrated that HF treatment significantly reduced the phosphorylation levels of AKT and PI3K (p-AKT and p-PI3K), while the levels of total AKT and PI3K remained largely unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eGastric cancer (GC) remains one of the most common malignancies worldwide, characterized by high incidence and mortality rates, particularly in East Asia, which bears a substantial disease burden ( SIEGEL R L et al. 2016). Approximately 40\u0026ndash;50% of patients with advanced GC develop distant metastases, frequently to the peritoneum, liver, and lymph nodes, leading to a dramatic deterioration in prognosis and a five-year survival rate of less than 10% (Smyth EC et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The metastatic propensity of GC is closely associated with its highly heterogeneous tumor microenvironment, aberrant activation of epidermal growth factor receptor (EGFR) signaling (Zhang X et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and VEGF-mediated angiogenesis and lymphatic invasion (Hamada Y et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Despite advancements in systemic chemotherapy and molecular targeted therapies (e.g., anti-HER2 agents), the treatment of advanced GC continues to face significant clinical challenges due to high tumor heterogeneity, frequent primary or acquired resistance, and limited drug penetration, especially in peritoneal metastases (Cai X et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consequently, developing novel therapeutic strategies targeting the mechanisms of gastric cancer metastasis remains an urgent research priority.\u003c/p\u003e \u003cp\u003eHF, an alkaloid derivative derived from the traditional Chinese medicine (Dichroa febrifuga), is a multi-target small-molecule inhibitor that has demonstrated significant antitumor potential across various malignancy models (Hei YY et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Its primary mechanism of action involves inhibiting prolyl-tRNA synthetase, triggering an amino acid starvation response, thereby disrupting protein synthesis and tumor microenvironment remodeling (Wang J et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Studies have shown that HF effectively inhibits tumor cell proliferation, induces apoptosis and cell cycle arrest, and significantly suppresses metastatic progression by modulating the TGF-β signaling pathway and epithelial-mesenchymal transition (EMT) (Wang J et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Furthermore, this compound has exhibited promising antitumor activity with manageable toxicity in numerous preclinical models, including colorectal cancer (Gong RH et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), breast cancer (Xia X et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and multiple myeloma ( Lamora A et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Against this backdrop, we innovatively proposed that HF might hold substantial value in gastric cancer treatment. Despite its notable effects in various cancers, its efficacy and underlying mechanisms in GC, particularly in advanced stages, remained largely unexplored.\u003c/p\u003e \u003cp\u003eThis study aimed to systematically evaluate the inhibitory effects of HF on gastric cancer cells and explore its potential therapeutic application, while elucidating the underlying molecular mechanisms. In in vitro experiments, we first assessed the impact of HF on gastric cancer cell viability at different concentrations and exposure times. By calculating the half-maximal inhibitory concentration (IC₅₀), we found that HF significantly inhibited the survival of GC cell lines, such as SGC7901 and MKN45, at low nanomolar concentrations, demonstrating potent in vitro antitumor activity. Based on these findings, we established the concentration range for subsequent experiments. Our results clearly indicated that HF suppressed gastric cancer cell survival in a concentration- and time-dependent manner, consistent with its known multi-target mechanisms of action (Sun Y et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, Liu K et al.1998 and Zcharia E et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, EdU incorporation and colony formation assays confirmed that HF markedly inhibited the proliferation and long-term clonogenic expansion of gastric cancer cells. These findings prompted a deeper investigation into the specific mechanisms underlying its growth-inhibitory effects, providing a theoretical foundation for advancing its development as a therapeutic strategy for GC.\u003c/p\u003e \u003cp\u003eAs demonstrated in our results, HF effectively induced S-phase cell cycle arrest and apoptosis in gastric cancer cells, highlighting its role as a multi-target natural compound in anti-GC therapy. Flow cytometric analysis revealed significant S-phase arrest accompanied by a concentration-dependent increase in the Sub-G1 apoptotic population, suggesting concurrent disruption of DNA replication and activation of programmed cell death pathways. These phenotypic observations align with HF's known mechanism of inhibiting prolyl-tRNA synthetase, triggering an amino acid stress response, and disrupting proteostasis (Tye MA et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Mechanistically, S-phase arrest is closely associated with replication stress response. HF treatment suppressed CDK2 kinase activity, impeding DNA synthesis, a finding consistent with previous reports of HF causing replication fork stalling and genomic instability in other cancer types (Sun P et al. 2022). Additionally, HF exhibited significant pro-apoptotic capacity, experimentally confirmed by its induction of mitochondrial membrane potential loss, promotion of cytochrome c release, and marked increase in cleaved caspase-3 and cleaved PARP levels, indicating the initiation of apoptosis via the intrinsic mitochondrial pathway. This aligns with HF's ability to upregulate the pro-apoptotic protein Bax, downregulate the anti-apoptotic protein Bcl-2, thereby altering the Bcl-2 family balance (Wang H et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Notably, our preliminary data suggested that this compound could induce substantial reactive oxygen species (ROS) generation, and this ROS burst might serve as an upstream event mediating mitochondrial pathway apoptosis and cell cycle arrest. The specific role and mechanism of ROS within the GC regulatory network became a key focus for further investigation in this study.\u003c/p\u003e \u003cp\u003eTo validate this hypothesis, we measured intracellular ROS levels following HF treatment. The experimental results demonstrated that HF induced significant ROS accumulation in gastric cancer cells, which subsequently inhibited the phosphorylation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT), ultimately repressing the activation of this pro-survival signaling pathway. This effect is consistent with the role of ROS as second messengers regulating kinase activity: excessive ROS can enhance the dephosphorylation activity of phosphatases like PTEN through oxidative modification of cysteine residues, thereby negatively regulating the PI3K/AKT pathway (Wang SQ et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Conversely, ROS burst can also directly or indirectly disrupt AKT phosphorylation, leading to the failure of its downstream proliferation and anti-apoptotic signals (Lee EY et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Mechanistically, the oxidative stress triggered by HF and the concomitant inhibition of PI3K/AKT signaling together constitute the core molecular basis of its anti-gastric cancer effects. This study further confirms that halofuginone, via a ROS-dependent mechanism, inhibits the PI3K/AKT pathway, significantly impedes gastric cancer cell proliferation, and promotes apoptosis, providing novel experimental evidence and mechanistic support for the translational development of this compound as a therapeutic strategy for gastric cancer.\u003c/p\u003e \u003cp\u003eHF demonstrated significant antitumor activity and a favorable safety profile in preclinical gastric cancer models, positioning it as a highly promising therapeutic candidate for translational development. In human GC cell line-derived xenograft models, HF treatment resulted in a significant, dose-dependent reduction in tumor volume and weight compared to the control group. More importantly, this notable therapeutic efficacy was achieved without observed significant systemic toxicity: serum biochemical parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA), remained within physiological ranges, and histopathological analysis revealed no significant damage to vital organs such as the liver and kidneys. This superior safety profile contrasts sharply with the common hepatotoxicity and myelosuppression associated with conventional chemotherapeutic agents, highlighting the potential selective advantage of natural, multi-target agents. These findings suggest that HF offers a high clinical safety window while effectively suppressing GC progression, providing crucial preclinical evidence for its further development as an anti-gastric cancer drug.\u003c/p\u003e \u003cp\u003eThe findings of this study indicate that HF significantly inhibits the malignant progression of gastric cancer, primarily through a mechanism involving the induction of reactive oxygen species (ROS) generation and the subsequent suppression of PI3K/AKT signaling pathway phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In summary, our experiments confirm that HF effectively inhibits gastric cancer cell proliferation, induces apoptosis, and causes S-phase cell cycle arrest in vitro. In in vivo animal models, it similarly demonstrates potent tumor-suppressive effects and regulates the expression of apoptosis- and cell cycle-related proteins. Mechanistically, we further elucidated that the antitumor effect of this compound depends on its inhibition of PI3K and AKT phosphorylation, thereby blocking downstream pro-survival and proliferative signaling. However, this study has certain limitations. For instance, the biodistribution and metabolic properties of HF within the complex in vivo microenvironment, as well as its potential crosstalk with other signaling pathways (e.g., TGF-β or MAPK), warrant further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCRediT authorship contribution statement\u003c/h2\u003e \u003cp\u003eJing Jiang: Performed cellular and animal experiments. Data curation, Writing - original draft. PeiPei Liu and DaWei Tang: Methodology, Investigation, Validation, Data curation. YongPing Li: Validation, Software, Formal analysis. PeiPei Liu: Visualization, Writing - review \u0026amp; editing, Supervision. YueHua Han: Project administration.CongYan Yang: Conceptualization, Resource acquisitionh, Data curation, Formal analysis, Writing - review \u0026amp; editing. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthical Statement\u003c/h2\u003e \u003cp\u003e All animal experiments were reviewed and approved by the Animal Ethics Committee of Bengbu Medical College (Approval No. [2025] 132). All procedures were conducted in accordance with institutional guidelines and international standards for the care and use of laboratory animals.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent for publication\u003c/h2\u003e \u003cp\u003eAll authors consent to publication of this article.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJing Jiang: Performed cellular and animal experiments. Data curation, Writing - original draft. PeiPei Liu and DaWei Tang: Methodology, Investigation, Validation, Data curation. YongPing Li: Validation, Software, Formal analysis. PeiPei Liu: Visualization, Writing - review \u0026amp; editing, Supervision. YueHua Han: Project administration.CongYan Yang: Conceptualization, Resource acquisitionh, Data curation, Formal analysis, Writing - review \u0026amp; editing. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by: 1.The Anhui Provincial Outstanding Research and Innovation Team Project (2022AH010084). 2.The Anhui Provincial Engineering Technology Research Center for Biochemical Drugs (2023SYKFD06). 3.The Key Project of Anhui Provincial Outstanding Young Talent Support Program in Higher Education Institutions (YQZD2024029). 4.The Bengbu Medical College Graduate Research and Innovation Program (Byycx24064).\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe datasets supporting the conclusions of this article are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBock FJ, Tait SWG. Mitochondria as multifaceted regulators of cell death. 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Orally Administered Halofuginone-Loaded TPGS Polymeric Micelles Against Triple-Negative Breast Cancer: Enhanced Absorption and Efficacy with Reduced Toxicity and Metastasis. Int J Nanomedicine. 2022;17:2475\u0026ndash;2491. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2147/IJN.S352538\u003c/span\u003e\u003cspan address=\"10.2147/IJN.S352538\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Gastric cancer, Halofuginone, Apoptosis, Cell cycle, ROS","lastPublishedDoi":"10.21203/rs.3.rs-8446424/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8446424/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDespite advances in diagnosis and therapy, the survival rate for gastric cancer, particularly in advanced stages, remains exceedingly low. Current chemotherapy regimens frequently lead to treatment failure and recurrence due to severe drug resistance and toxicity, underscoring the urgent need for novel therapeutic strategies with new mechanisms of action capable of effectively overcoming resistance. Natural products and their derivatives have attracted significant attention as a rich source of anticancer agents. Halofuginone (HF), a plant-derived natural compound possessing multi-target antitumor properties, has emerged as a highly promising anticancer candidate. However, its antitumor efficacy against gastric cancer and the precise underlying molecular mechanisms remain largely unexplored. This study aimed to investigate the effects and mechanisms of HF in SGC7901 and MKN45 cell lines. Our experiments demonstrated that HF inhibited gastric cancer cell proliferation while inducing apoptosis and S-phase cell cycle arrest. Mechanistically, HF triggered excessive mitochondrial reactive oxygen species (ROS) production, subsequentlysuppressing the activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pro-survival pathway. Scavenging ROS with N-acetylcysteine (NAC) reversed these effects, confirming ROS as the central mediator of HF's antitumor activity. In a mouse xenograft model, HF administration inhibited primary tumor growth without inducing hepatorenal toxicity. These findings reveal a ROS-centric mechanism through which HF suppresses gastric cancer progression, positioning it as a promising therapeutic candidate to address unmet clinical needs in gastric cancer management.\u003c/p\u003e","manuscriptTitle":"Halofuginone suppresses gastric tumor growth by inducing ROS-dependent inhibition of the PI3K/AKT pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-02 08:44:33","doi":"10.21203/rs.3.rs-8446424/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"19d0d7dd-1e43-4cf0-88b5-a5cacb3bbc6c","owner":[],"postedDate":"February 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-31T02:25:34+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-02 08:44:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8446424","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8446424","identity":"rs-8446424","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}