Antitumor effects of Mzs-1 from Chinese Artium lappa L. on HGC-27 cells via the PI3K/AKT/mTOR pathway in vitro and in vivo.

Antitumor effects of Mzs-1 from Chinese Artium lappa L. on HGC-27 cells via the PI3K/AKT/mTOR pathway in vitro and in vivo. | 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 Article Antitumor effects of Mzs-1 from Chinese Artium lappa L. on HGC-27 cells via the PI3K/AKT/mTOR pathway in vitro and in vivo. Yi Sun, Lei Yang, Xiao-Feng Yu, Han Yao, Chen Chai This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6972121/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 Background: Gastric cancer (GC), particularly at advanced stages, often results in therapeutic failure due to drug resistance and adverse effects. Traditional Chinese medicine (TCM), with its multi-target actions and favorable safety profile, presents a compelling alternative. Purpose: To evaluate the anti-tumor efficacy of Mzs-1, a synthetic analogue derived from Arctium lappa L. , and elucidate its mechanism of action in gastric cancer. Study design: This study integrated computational prediction and experimental validation to evaluate the anti-tumor effects of Mzs-1 in vitro and in vivo. Methods: Network pharmacology analysis was conducted to identify the potential targets and pathways of Mzs-1, highlighting the PI3K/AKT/mTOR pathway. Molecular docking was performed to predict the binding affinity of Mzs-1 to key proteins in this pathway. Functional assays, including cell proliferation, apoptosis, and invasion, were performed in HGC-27 cells. Western blotting was used to examine the expression of PI3K/AKT/mTOR pathway components. In vivo efficacy was assessed in xenograft mouse models following Mzs-1 treatment. Results: In vitro, Mzs-1 suppressed HGC-27 cell proliferation and invasion while inducing apoptosis. These effects were enhanced by PI3K inhibition and attenuated by AKT activation. In vivo, Mzs-1 inhibited tumor growth and downregulated PI3K/AKT/mTOR signaling in xenograft tissues. Conclusion: Mzs-1 exerts anti-GC activity by modulating the PI3K/AKT/mTOR pathway, supporting its potential as a therapeutic candidate for gastric cancer. Biological sciences/Cancer Biological sciences/Drug discovery Biological sciences/Molecular biology Biological sciences/Plant sciences Gastric cancer Traditional Chinese medicine Mzs-1 Apoptosis PI3K/AKT/mTOR pathway Figures Figure 1 Figure 2 Figure 3 1. Introduction Gastric cancer (GC) ranks as the fifth most common malignancy worldwide and accounts for approximately one in every 13 deaths(Sung et al., 2021 ). Due to its asymptomatic onset, most patients are diagnosed at an advanced stage with local or distant metastases, leading to poor prognosis(Guan et al., 2023 ). Standard therapies, including surgery and chemotherapy, are hampered by substantial toxicity and limited efficacy(Janjigian et al., 2021 ; Zhu et al., 2022 ). Moreover, effective treatments for advanced gastric cancer remain inadequate, primarily due to tumor heterogeneity and acquired resistance(Kim et al., 2022 ). Thus, elucidating the molecular mechanisms underlying disease progression and identifying robust therapeutic targets are imperative for improving clinical outcomes and reducing the healthcare burden. Natural products have long served as a rich source of anticancer agents, with over 50% of approved drugs derived from compounds of natural origin(Muhammad et al., 2022 ). These agents are valued for their structural diversity, high target affinity, and multitarget potential, making them attractive candidates for novel therapeutic development(Gupta et al., 2022 ; Naeem et al., 2022 ). Consequently, natural product–based strategies are increasingly investigated for gastric cancer (GC) therapy. Arctium lappa L.(commonly known as burdock) , a plant in the Asteraceae family, has traditionally been used for its anti-inflammatory, antiviral, and anticancer properties(Li et al., 2024 ). Its pharmacological effects have been documented in a range of diseases, including liver fibrosis(Xiang et al., 2024 ), hepatic steatosis(Ma et al., 2022 ) and cerebral ischemia(Yang et al., 2021 ). However, existing antitumor studies have primarily focused on lung, cervical, breast, and colorectal cancers(Kim et al., 2020 ; Lee et al., 2022 ; Shi et al., 2020 ; Yang et al., 2023 ), with limited investigation in GC. Given these findings, it is essential to determine whether compounds derived from A. lappa, such as Mzs‑1, also exert antitumor activity in gastric cancer. In our previous work, we synthesized Mzs-1, a lignan precursor derived from Arctium lappa L. (Xia et al., 2015 ). Thus far, no studies have investigated the therapeutic potential or molecular mechanisms of A. lappa–derived synthetic compounds in gastric cancer. To fill this gap, we assessed the effects of Mzs-1 both in vitro and in vivo and investigated its underlying mechanisms. 2. Materials and Methods 2.1 Network pharmacology analysis To predict potential protein targets of Mzs-1, reverse pharmacophore mapping was performed using PharmMapper ( http://www.lilab-ecust.cn/pharmmapper/ ). The 3D structure of Mzs-1 (mol2 format) was screened against the Human Protein Target Database (top 300 targets, default settings). Targets were ranked by pharmacophore fit scores and normalized similarity values. Candidate targets were ranked based on pharmacophore fit scores and normalized similarity values. Top hits were subsequently evaluated by molecular docking to assess structural feasibility of interaction. 2.2 Pharmaceuticals and cell culture Mzs-1, a synthetic derivative of compounds extracted from Arctium lappa L. components, was kindly provided by Professor Yamu Xia(State Key Laboratory Base of Eco-chemical Engineering, Qingdao University of Science and Technology). LY294002 (a PI3K inhibitor) and SC79 (an Akt activator) were purchased from MedChemExpress (MCE). HGC-27 human gastric cancer cells were obtained from Haixing Bioscience Co., Ltd. (China) and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Corning, USA) supplemented with 10% fetal bovine serum (FBS; BI, Israel) and 1% penicillin-streptomycin (Corning, USA). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ and passaged at 80–90% confluence using 0.25% trypsin-EDTA (Corning, USA). The Cell Counting Kit-8 (CCK-8) was purchased from Biosharp (China). Annexin V-FITC/PI Apoptosis Detection Kit and AO/EB Double Staining Kit were obtained from Elabscience and Shanghai Maokang Biotechnology Co., respectively. Paraformaldehyde solution was from Wuhan Servicebio Technology Co. Matrigel and crystal violet staining solution were purchased from Beyotime Biotechnology (China). Primary antibodies against PI3K, phospho-PI3K, mTOR, phospho-mTOR, Bcl-2, Bax, caspase-3, and β-actin were supplied by Boaoson Biotechnology (Beijing, China). Unless otherwise stated, all reagents were of analytical grade. 2.3 Clonogenic Assay HGC-27 cells in the logarithmic growth phase were seeded into 6-well plates (1,000 cells/well). After adherence, cells were treated with the indicated compounds for 24 h, followed by incubation in drug-free medium for 14 days, with medium refreshed every 3 days. Colonies were fixed with 4% paraformaldehyde for 30 min, stained with 0.5% crystal violet for 20 min, washed, air-dried, and imaged. 2.4 Transwell Invasion Assay Inserts with 8-µm pores (Corning) were pre-coated with 1 mg/mL Matrigel (BD Biosciences) and incubated at 37°C for 3 h. HGC-27 cells, serum-starved for 24 h, were seeded into the upper chamber (1×10⁵ cells/well) in serum-free medium containing the indicated compounds. Medium supplemented with 10% FBS was added to the lower chamber as a chemoattractant. After 24 h, non-invading cells were removed, and membranes were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. Invaded cells on the lower surface were imaged and counted in five randomly selected fields. Migration assays were conducted under the same conditions without Matrigel coating. 2.5 Western Blot Analysis Cell lysates were centrifuged at 12,000 g for 10 min at 4°C, and supernatants were collected. Protein concentrations were quantified using the BCA assay (Thermo Fisher). Equal protein amounts (20 µg) were resolved on 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 2 h at room temperature, then incubated overnight at 4°C with primary antibodies against PI3K, p-PI3K, mTOR, p-mTOR, BCL-2, BAX, caspase-3 and β-actin. After washing, membranes were incubated with HRP-conjugated secondary antibodies for 2 h at 37°C. Signals were detected using an enhanced chemiluminescence kit. 2.6 Cell Viability Assay (CCK-8) HGC-27 cells were seeded in 96-well plates at 1×10⁵ cells/mL and incubated overnight. After treatment for 24 h, 10 µL of CCK-8 reagent (Dojindo, Japan) was added to each well and incubated for 2 h at 37°C. Absorbance was measured at 450 nm using a microplate reader. 2.7 Wound Healing Assay HGC-27 cells were plated in 6-well plates (1×10⁶ cells/mL) and cultured overnight. Linear scratches were made using a 200 µL pipette tip, and detached cells were removed by PBS(phosphate-buffered saline) washing. Cells were then incubated in drug-containing medium. Scratch closure was imaged at 0 and 24 h using an inverted microscope. Wound width was quantified using ImageJ, and migration rates were calculated. 2.8 Flow Cytometric Analysis of Apoptosis HGC-27 cells (1×10⁶/mL) were treated with compounds for 24 h. Cells were collected, washed with PBS, and resuspended in 500 µL Annexin V binding buffer. Apoptotic cells were stained with 5 µL Annexin V-FITC and 5 µL propidium iodide (50 µg/mL), vortexed gently, and incubated in the dark for 20 min at room temperature. Samples were analyzed by flow cytometry. 2.9 AO/EB Double Staining AO/EB staining solution was prepared by mixing AO, EB, and buffer at a 1:1:8 ratio. HGC-27 cells were seeded on glass coverslips in 6-well plates (1×10⁶ cells/well) and treated for 24 h. Cells were washed with PBS and stained with 1 mL AO/EB for 10 min in the dark, then visualized using a confocal laser scanning microscope. 2.10 In Vivo Tumor Xenograft Model HGC-27 cells (4 × 10⁶) suspended in PBS were subcutaneously injected into the inguinal region of 4-week-old female BALB/c nude mice. The mice were purchased from Shaanxi Shiyao Yike Biotechnology Co., Ltd. (Xi’an, China), and housed under specific pathogen-free (SPF) conditions. After tumors became palpable, the mice were randomly divided into three groups (n = 5 per group): Mzs-1, Mzs-1 + SC79 (a PI3K activator), and NC (normal saline control). Treatments were administered once daily for 7 consecutive days. Tumor volumes were measured weekly using a digital caliper and calculated with the formula: V = ½ × a × b² (a = length, b = width). One mouse from each group was euthanized at weeks 0, 1, 2, 3, and 4, respectively. Prior to euthanasia, mice were anesthetized with 2–3% isoflurane until complete loss of righting reflex, followed by cervical dislocation. Tumors were excised, photographed, and processed for analysis. Western blotting was performed on tumor tissues to assess the expression levels of PI3K, p-PI3K, and caspase-3. All animal procedures complied with the institutional and national regulations on the ethical use of animals in research. The study protocol was reviewed and approved by the Ethics Committee of The People’s Hospital of Suzhou New District(2020-002). All procedures were carried out in accordance with relevant guidelines and regulations, and the study is reported in accordance with the ARRIVE guidelines. 2.11 Statistical Analysis All data are presented as mean ± standard error of the mean (SEM) unless otherwise stated. Statistical analyses were performed using GraphPad Prism 8.0 and SPSS 20.0. For comparisons between two groups, paired or unpaired Student’s t-test, Mann–Whitney test, or two-tailed χ² test was used, as appropriate. One-way ANOVA followed by Bonferroni’s multiple comparisons test was applied for multi-group comparisons. A P value < 0.05 was considered statistically significant. 3. Results 3.1 Target Prediction and Prognostic Significance of Key Genes Pharmacophore-based inverse docking identified ten potential protein targets of Mzs-1 (Fig. 1 A, B). Among the top candidates, several—GSK3β (6th), MAPK14 (3rd), JAK2 (4th), and the glucocorticoid receptor (GR; 8th)—are known to regulate or be regulated by the PI3K/AKT cascade. To validate these putative interactions, molecular docking was conducted between Mzs-1 and representative PI3K/AKT pathway components (Fig. 1 C). Mzs-1 exhibited favorable binding affinities to PI3Kα (–7.846 kcal/mol), PI3Kγ (–7.525 kcal/mol), AKT2 (–6.990 kcal/mol), and ERK2 (–7.072 kcal/mol), indicating potential direct interaction with key nodes of the signaling network. Figure 1 D illustrates the binding modes of Mzs-1 to PI3Kα (left panel) and PI3Kγ (right panel). Mzs-1 engages the ATP-binding pocket through hydrogen bonding, electrostatic forces, π–π stacking, and hydrophobic interactions with critical amino acid residues. These interactions support its high-affinity binding and provide a structural basis for the proposed PI3K-related activity of Mzs-1. To investigate the expression patterns of this signaling pathway in gastric adenocarcinoma, we analyzed RNA-seq data from the TCGA-STAD cohort ( https://portal.gdc.cancer.gov ), processed via the STAR pipeline and quantified in TPM. After log2 transformation [log2(TPM + 1)] , we compared gene expression between tumor (n = 375) and adjacent normal tissues (n = 32). As shown in Fig. 1 E, expression levels of PIK3CA, AKT1, AKT2, MAPK14, and MAPK1 were significantly higher in tumors compared to normal tissues (Wilcoxon rank-sum test, p < 0.05 ), suggesting the involvement of PI3K/AKT and MAPK signaling in gastric cancer. To explore their prognostic implications, Kaplan–Meier survival analysis was conducted by stratifying patients based on median gene expression. Notably, elevated expression of PIK3CA and AKT1 correlated with poorer overall survival (Fig. 1 F, p = 0.046 ). These results suggested that dysregulation of PI3K/AKT pathway may drive disease progression and impact patient outcomes. 3.2 In Vitro Validation of PI3K/AKT/mTOR Pathway Involvement To elucidate the anti-tumor mechanism of Mzs-1, we assessed its effects on HGC-27 cells function and associated signaling pathways in vitro. As shown by the CCK-8 assay, Mzs-1 significantly impaired cell viability (Fig. 2 A, [0.52 ± 0.02, P < 0.0001 ]). Co-treatment with LY294002 slightly inhibited cell viability further, but co-treatment with SC79 could significantly restored cell viability, suggesting PI3K/AKT pathway involvement (Mzs-1 vs. Mzs-1 + SC79: 0.52 ± 0.02 vs. 1.14 ± 0.07, P < 0.01 ). In wound healing assays (Fig. 2 B), Mzs-1 or co-treatment with LY294002 markedly inhibited migration compared to the NC group ([quantified% wound closure at 24 h, 0.24: 0.22 vs 0.7, P < 0.01 ]), while the effect was slightly alleviated by SC79 when compared with the treatment group. Flow cytometry (Fig. 2 C) revealed that Mzs‑1 induced apoptosis in nearly 50% of cells, which was further supported by AO/EB staining (Fig. 2 D), showing characteristic apoptotic morphology including cell shrinkage and orange-red nuclear fluorescence. Consistent with these findings, Mzs‑1 significantly suppressed cell invasion in Transwell assays (Fig. 2 E). Colony formation assays (Fig. 2 F) further demonstrated a marked decrease in both the number and size of colonies after Mzs-1 treatment. To elucidate the molecular mechanism underlying Mzs-1–mediated tumor suppression, we examined the expression of key components in the PI3K/AKT/mTOR pathway and apoptosis-related proteins by Western blotting under different treatment conditions. As shown in Fig. 2 G, Mzs-1 treatment led to decreased phosphorylation of PI3K and mTOR, suggesting pathway inhibition. The addition of LY294002 significantly reduced the phosphorylation levels, confirming effective inhibition of PI3K signaling. In contrast, co-treatment with SC79 could increase the expression of p-AKT and p-mTOR, validating the responsiveness of the pathway. Regarding apoptotic markers, Mzs-1 treatment decreased anti-apoptotic BCL-2 while increasing pro-apoptotic BAX and caspase-3. These effects were further enhanced by LY294002 (statistical quantification, n = 3, P < 0.05 ; BCL‑2: NC 1.00, Mzs‑1 0.46, Mzs‑1 + LY294002 0.33; BAX: NC 1.00, Mzs‑1 2.45, Mzs‑1 + LY294002 3.23; caspase‑3: NC 1.00, Mzs‑1 1.90, Mzs‑1 + LY294002 2.38), whereas SC79 partially reversed these effects (BCL‑2: Mzs‑1 0.46, Mzs‑1 + SC79 0.74; BAX: Mzs‑1 2.45, Mzs‑1 + SC79 1.92; caspase‑3: Mzs‑1 1.90, Mzs‑1 + SC79 1.63). These findings underscore the involvement of the PI3K/AKT/mTOR pathway in Mzs‑1-induced apoptosis. 3.3 In Vivo Confirmation of PI3K/AKT Pathway Activation As shown in the flow chart (Fig. 3 A), gastric cancer tumor volumes were monitored weekly over four weeks in three groups: negative control (normal saline gastric lavage), Mzs‑1 (gastric lavage), and Mzs‑1 combined with SC79 (gastric lavage). With the exception of week 3, Mzs‑1 treatment significantly suppressed tumor growth compared to the control group. Specifically, tumor volumes remained relatively stable in the Mzs‑1 group, while those in the control group increased progressively. Co-administration of SC79 markedly reversed the inhibitory effect of Mzs‑1, resulting in a significant increase in tumor volume from week 3 onward (Fig. 3 B–C). The expression levels of phosphorylated PI3K relative to total PI3K (P-PI3K/PI3K) and Caspase-3 were analyzed at weeks 0, 1, 2, 3, and 4 (Fig. 3 D). Mzs-1 treatment significantly reduced P-PI3K/PI3K levels from week 1, suggesting inhibition of the PI3K pathway as a potential mechanism of tumor suppression. Concurrently, Caspase-3 expression was elevated, indicating enhanced apoptosis. In contrast, combination treatment with SC79 partially reversed these effects, increasing P-PI3K/PI3K expression from week 2 and reducing Caspase-3 expression before week 4. Figure 3 E illustrated the potential mechanisms of Mzs-1’s effect on HGC-27 cells. 4. Discussion Our results demonstrated that Mzs-1, a synthetic analogue of Arctium lappa L. constituents, induces apoptosis in HGC-27 cells via inhibition of the PI3K/AKT/mTOR pathway. While A. lappa L. has long been recognized for its anti-inflammatory and immunomodulatory properties, its anti-tumor potential, particularly in GC, remains underexplored(Gao et al., 2018 ; Lee et al., 2022 ; Sandberg et al., 2018 ; Sun et al., 2021 ). Although several bioactive components have been isolated from A. lappa L. (Chan et al., 2011 ), few efforts have focused on their chemical synthesis or mechanistic characterization. Our previous work successfully synthesized Mzs-1, a chemical analogue of the active components found in burdock seeds(Xia et al., 2015 ). To elucidate its potential mechanism in gastric cancer, we carried out this study. Mechanistically, Mzs-1-induced effects were enhanced by the PI3K inhibitor LY294002 and reversed by the AKT activator SC79, suggesting that Mzs-1 acts upstream of AKT and modulates downstream signaling. Overactivation of the PI3K/AKT/mTOR pathway is a key driver of GC cell proliferation, invasion, and chemoresistance, and is closely associated with poor clinical outcomes(Fattahi et al., 2020 ; Ma et al., 2023 ). Our findings partially align with those of Meng et al., who showed that PI3K/AKT/mTOR signaling mediates the anti-tumor activity of Fructus Arctii in triple-negative breast cancer (TNBC)(Meng et al., 2025 ). Similarly, the same pathway has been implicated in the anti-prostate cancer effects of Arctium lappa L. in vitro(Sun et al., 2021 ). Other studies have demonstrated that Arctium lappa L. induces S-phase arrest via CDKN1C/p57 activation in colorectal cancer cells(Yang et al., 2023 ), and inhibits YAP signaling both transcriptionally and post-translationally in HeLa, MDA-MB-231, SW480, and PC3 cells(Li et al., 2021 ). In addition, it has been shown to enhance the therapeutic efficacy of azithromycin in breast cancer(Lee et al., 2020 ). The CEA–KRT1–PI3K/AKT axis has also been implicated in modulating oxaliplatin sensitivity in GC cells(Chen et al., 2025 ). Notably, the upregulation of caspase-3 and BAX, along with suppression of BCL-2, correlated with reduced PI3K/AKT activity, confirming that Mzs-1 induces apoptosis at least in part through inhibition of this survival pathway. Furthermore, restoration of anti-apoptotic markers by SC79 supports a feedback loop wherein reactivation of AKT mitigates Mzs-1-induced cytotoxicity. This is consistent with prior reports showing that PI3K/AKT inhibition sensitizes cancer cells to apoptosis(Han et al., 2022 ; Wang et al., 2023 ). Collectively, our study is the first to demonstrate that Mzs-1—a synthetic derivative of Arctium lappa L. —exerts potent anti-tumor effects in gastric cancer by modulating the PI3K/AKT/mTOR pathway and promoting apoptosis. These findings identify Mzs-1 as a promising therapeutic candidate or adjuvant to conventional chemotherapy. By disrupting survival signaling, Mzs‑1 may reduce the required dose of conventional drugs, improve efficacy, and help overcome resistance. However, further preclinical investigations—including pharmacokinetics, toxicity assessment, and validation in orthotopic or genetically engineered mouse models—are necessary to advance clinical translation. Despite these encouraging findings, several limitations should be acknowledged. First, although A. lappa L. is also known for its immunoregulatory and anti-inflammatory properties, our study focused solely on tumor cell–intrinsic mechanisms. Given the critical interplay between immunity, inflammation, and tumor survival, future studies should explore whether Mzs-1 affects the tumor microenvironment or immune responses. Second, while we confirmed involvement of the PI3K/AKT/mTOR pathway, the precise regulatory mechanisms remain unclear. Future investigations should examine whether Mzs-1 alters the balance between apoptosis and autophagy, and determine the roles of specific PI3K isoforms and mTOR complexes (mTORC1 vs. mTORC2). Third, feedback activation of compensatory pathways such as MAPK/ERK could attenuate the durability of response, a common challenge in targeted therapy. Advanced techniques such as isoform-selective inhibition, CRISPR-based gene editing, and phosphoproteomic profiling will be pivotal in elucidating the broader signaling networks regulated by Mzs-1. To extend our findings, future studies should prioritize: (1) the development of xenograft or genetically engineered gastric cancer models to evaluate the antitumor efficacy, pharmacokinetics, and safety profile of Mzs-1; (2) the use of gene editing to confirm pathway dependency and uncover potential resistance mechanisms; and (3) exploration of synergistic interactions between Mzs-1 and standard chemotherapeutics or targeted agents in combination regimens. 5. Conclusion Mzs-1 has demonstrated promising anti-gastric cancer activity, potentially addressing the urgent need for more effective treatment strategies in GC. However, further mechanistic and preclinical investigations are essential to validate these findings and support the therapeutic development of Mzs-1. Abbreviations AKT, protein kinase B; BAX, Bcl-2-associated X protein; CCK-8, Cell Counting Kit-8; ERK2, extracellular signal-regulated kinase 2; GC, gastric cancer; GR, glucocorticoid receptor; HGC-27, human gastric carcinoma cell line; JAK2, Janus kinase 2; MAPK14, mitogen-activated protein kinase 14; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; SC79, AKT activator; TCGA, The Cancer Genome Atlas; TCM, Traditional Chinese Medicine; TNBC, triple-negative breast cancer; TPM, transcripts per million. Declarations Conflicts of interest statement : The authors declare no conflicts of interest that pertain to this work. Funding: Suzhou High tech Zone People's Hospital Scientific Development Innovation Fund Project (SGY2024B04); Technology Development Project of Suzhou City (SYSD2020084); Science and Technology Project of Suzhou city (SKYD2023089, SKYD2023033); Diagnosis and Treatment Technology Project of Suzhou Clinical Key Disease(LCZX202354). Author Contribution Y. Sun and C. Chai conceptualized the study. L. Yang was responsible for data acquisition. Data analysis was conducted by X.F. Yu and H. Yao. Y. Sun and C. Chai contributed to writing the original draft, review, and editing. All authors reviewed and approved the final manuscript. Data Availability The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. All relevant data supporting the findings of this study are included in the article and its supplementary materials. References Chan, Y. S. et al. A review of the pharmacological effects of Arctium lappa (burdock). 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Arctium lappa L. roots ameliorates cerebral ischemia through inhibiting neuronal apoptosis and suppressing AMPK/mTOR-mediated autophagy. Phytomedicine 85 , 153526 (2021). Zhu, X. D. et al. XELOX doublet regimen versus EOX triplet regimen as first-line treatment for advanced gastric cancer: An open-labeled, multicenter, randomized, prospective phase III trial (EXELOX). Cancer Commun. (Lond) . 42 (4), 314–326 (2022). Additional Declarations No competing interests reported. Supplementary Files Highlights.docx SupplementaryWesternBlots.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. 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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-6972121","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":500214865,"identity":"88882a4f-3ead-438b-876d-ce8d4dde99a7","order_by":0,"name":"Yi Sun","email":"","orcid":"","institution":"The People’s Hospital of Suzhou New District","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Sun","suffix":""},{"id":500214866,"identity":"5be2fd4e-8c9a-4284-87a8-9e4f0ede4309","order_by":1,"name":"Lei Yang","email":"","orcid":"","institution":"First Hospital of Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Yang","suffix":""},{"id":500214867,"identity":"717dace1-4fa7-4cc2-b02d-a220fc967035","order_by":2,"name":"Xiao-Feng Yu","email":"","orcid":"","institution":"The People’s Hospital of Suzhou New District","correspondingAuthor":false,"prefix":"","firstName":"Xiao-Feng","middleName":"","lastName":"Yu","suffix":""},{"id":500214868,"identity":"a7083292-39ab-4824-9096-fbf5a3d4c11f","order_by":3,"name":"Han Yao","email":"","orcid":"","institution":"The People’s Hospital of Suzhou New District","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Yao","suffix":""},{"id":500214869,"identity":"01c0cd97-755a-4ade-9f88-8eb4d8f04664","order_by":4,"name":"Chen Chai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYDACCQaGA4wNNjJgDg8JWtJ4SNPCwNhwmAQt/LObHx74ueM8j/yMBMYHb9sY5M0JWnLnmMHB3jO3eRhnJDAbzm1jMNzZQECLgUSCwWHGtts8zBIJbNK8bQwJBgcIakn/ANRyjodNIoH9N5FackC2HODhAdrCTJQWiRs5BQd725J5JHgeNkvOOSdhuIGQFv4Z6Zs//Gyzk5NvTz744U2ZjTxBW5AAYwMDJJpGwSgYBaNgFFAMAK1ZPECwyRN5AAAAAElFTkSuQmCC","orcid":"","institution":"The People’s Hospital of Suzhou New District","correspondingAuthor":true,"prefix":"","firstName":"Chen","middleName":"","lastName":"Chai","suffix":""}],"badges":[],"createdAt":"2025-06-25 08:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6972121/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6972121/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89491727,"identity":"bd8db85b-d26e-4c0f-9a8f-d651e9ff3303","added_by":"auto","created_at":"2025-08-20 14:03:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":181433,"visible":true,"origin":"","legend":"\u003cp\u003eMzs-1 Target Prediction and Prognostic Analysis of Candidate Genes. (A) Chemical structure of Mzs-1. (B) Scatter plot showing similarity scores of genes (e.g., 5-LOX, PGHS1, PGHS2) to the reference molecule. Point size indicates score magnitude; darker colors represent higher similarity. (C) Bar chart displaying docking scores (kcal/mol) for proteins (PI3Ka, PI3Ky, ERK2, AKT2, MAPK14, AKT1). Negative values indicate lower binding energies and more stable interactions. (D) Mzs-1 binds to ATP sites of PI3Kα and PI3Kγ via hydrogen bonds, electrostatic interactions, π-π stacking, and hydrophobic interactions with key residues. (E) Violin plot showing expression levels of genes (PIK3CA, PIK3CG, AKT1, AKT2, MAPK14, MAPK1) in normal and tumor tissues. Asterisks indicate significant differences (**\u003cem\u003eP\u0026lt;\u003c/em\u003e0.01, ***\u003cem\u003eP\u0026lt;\u003c/em\u003e0.001). (F) Kaplan-Meier curves showing survival probability over time for high (red) and low (blue) expression groups. P=0.046 indicates a significant difference.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6972121/v1/953b7aa2c0de39a6266644a6.png"},{"id":89491714,"identity":"1d096927-be23-45dc-b5f5-220ef5322e07","added_by":"auto","created_at":"2025-08-20 14:03:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":347698,"visible":true,"origin":"","legend":"\u003cp\u003eMzs-1 inhibits tumor progression via the PI3K/AKT/mTOR pathway. (A) CCK-8 assay showing cell viability in the NC, Mzs-1, Mzs-1+LY294002, and Mzs-1+SC79 groups (all compared with the NC group). (B) Wound healing assays at 0 h and 24 h. (C) Flow cytometry analysis of apoptotic cell populations. (D) AO/EB dual fluorescence staining for apoptosis. (E) Transwell invasion assay quantifying invaded cells. (F) Colony formation assays after treatment. (G) Western blot analysis of PI3K/AKT/mTOR pathway-related proteins and apoptotic markers. All quantitative data are presented as mean ± SD. Statistical comparisons were made using one-way ANOVA with Tukey’s post hoc test; ****\u003cem\u003eP \u0026lt; 0.0001\u003c/em\u003e, **\u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6972121/v1/100b7586a28f619e0aa7ccd1.png"},{"id":89491713,"identity":"0e41478b-9e7e-4928-8ebb-6dc9e9b6b2c8","added_by":"auto","created_at":"2025-08-20 14:03:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":130964,"visible":true,"origin":"","legend":"\u003cp\u003eMzs-1 suppresses tumor growth via activation of apoptosis through the PI3K/AKT/mTOR pathway in vivo. (A) Schematic diagram of the in vivo experimental design. Mice bearing gastric tumors were treated with normal saline, Mzs 1, or Mzs 1 combined with SC79 via gastric irrigation. (B) Representative images of tumors harvested at different timepoints(week 1-4) (C) Tumor growth curve over four weeks in each treatment group. (D) Western blot quantification of p-PI3K/PI3K and caspase-3 protein levels at indicated time points. (E) Schematic model illustrating the proposed mechanism by which Mzs 1 induces apoptosis through PI3K/AKT/mTOR pathway inhibition, leading to increased BAX and caspase 3, and decreased BCL 2 expression.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6972121/v1/dc7e0a2ae23a3c7195201401.png"},{"id":104436815,"identity":"d575ed7c-eb6a-4dc4-8404-4020585ad559","added_by":"auto","created_at":"2026-03-11 16:55:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1639244,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6972121/v1/38de0c51-8739-4895-acf9-4e7c375c7261.pdf"},{"id":89492619,"identity":"c29825ae-114b-4763-9b8b-d47bf852fb10","added_by":"auto","created_at":"2025-08-20 14:11:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14549,"visible":true,"origin":"","legend":"","description":"","filename":"Highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-6972121/v1/ff79e3945f07da4c41df6294.docx"},{"id":89492621,"identity":"b7484dd9-6468-4a9e-aadf-5b12de4847a0","added_by":"auto","created_at":"2025-08-20 14:11:34","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1371010,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryWesternBlots.docx","url":"https://assets-eu.researchsquare.com/files/rs-6972121/v1/0a762cdb3ededf18b0029988.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAntitumor effects of Mzs-1 from Chinese \u003cem\u003eArtium lappa L.\u003c/em\u003e on HGC-27 cells via the PI3K/AKT/mTOR pathway in vitro and in vivo. \u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGastric cancer (GC) ranks as the fifth most common malignancy worldwide and accounts for approximately one in every 13 deaths(Sung et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Due to its asymptomatic onset, most patients are diagnosed at an advanced stage with local or distant metastases, leading to poor prognosis(Guan et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Standard therapies, including surgery and chemotherapy, are hampered by substantial toxicity and limited efficacy(Janjigian et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, effective treatments for advanced gastric cancer remain inadequate, primarily due to tumor heterogeneity and acquired resistance(Kim et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Thus, elucidating the molecular mechanisms underlying disease progression and identifying robust therapeutic targets are imperative for improving clinical outcomes and reducing the healthcare burden.\u003c/p\u003e\u003cp\u003eNatural products have long served as a rich source of anticancer agents, with over 50% of approved drugs derived from compounds of natural origin(Muhammad et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These agents are valued for their structural diversity, high target affinity, and multitarget potential, making them attractive candidates for novel therapeutic development(Gupta et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Naeem et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Consequently, natural product\u0026ndash;based strategies are increasingly investigated for gastric cancer (GC) therapy. \u003cem\u003eArctium lappa L.(commonly known as burdock)\u003c/em\u003e, a plant in the Asteraceae family, has traditionally been used for its anti-inflammatory, antiviral, and anticancer properties(Li et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Its pharmacological effects have been documented in a range of diseases, including liver fibrosis(Xiang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), hepatic steatosis(Ma et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and cerebral ischemia(Yang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, existing antitumor studies have primarily focused on lung, cervical, breast, and colorectal cancers(Kim et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), with limited investigation in GC. Given these findings, it is essential to determine whether compounds derived from A. lappa, such as Mzs‑1, also exert antitumor activity in gastric cancer.\u003c/p\u003e\u003cp\u003eIn our previous work, we synthesized Mzs-1, a lignan precursor derived from \u003cem\u003eArctium lappa L.\u003c/em\u003e(Xia et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Thus far, no studies have investigated the therapeutic potential or molecular mechanisms of A. lappa\u0026ndash;derived synthetic compounds in gastric cancer. To fill this gap, we assessed the effects of Mzs-1 both in vitro and in vivo and investigated its underlying mechanisms.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Network pharmacology analysis\u003c/h2\u003e\u003cp\u003eTo predict potential protein targets of Mzs-1, reverse pharmacophore mapping was performed using PharmMapper (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.lilab-ecust.cn/pharmmapper/\u003c/span\u003e\u003cspan address=\"http://www.lilab-ecust.cn/pharmmapper/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The 3D structure of Mzs-1 (mol2 format) was screened against the Human Protein Target Database (top 300 targets, default settings). Targets were ranked by pharmacophore fit scores and normalized similarity values. Candidate targets were ranked based on pharmacophore fit scores and normalized similarity values. Top hits were subsequently evaluated by molecular docking to assess structural feasibility of interaction.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Pharmaceuticals and cell culture\u003c/h2\u003e\u003cp\u003eMzs-1, a synthetic derivative of compounds extracted from \u003cem\u003eArctium lappa L.\u003c/em\u003e components, was kindly provided by Professor Yamu Xia(State Key Laboratory Base of Eco-chemical Engineering, Qingdao University of Science and Technology). LY294002 (a PI3K inhibitor) and SC79 (an Akt activator) were purchased from MedChemExpress (MCE). HGC-27 human gastric cancer cells were obtained from Haixing Bioscience Co., Ltd. (China) and cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Corning, USA) supplemented with 10% fetal bovine serum (FBS; BI, Israel) and 1% penicillin-streptomycin (Corning, USA). Cells were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂ and passaged at 80\u0026ndash;90% confluence using 0.25% trypsin-EDTA (Corning, USA). The Cell Counting Kit-8 (CCK-8) was purchased from Biosharp (China). Annexin V-FITC/PI Apoptosis Detection Kit and AO/EB Double Staining Kit were obtained from Elabscience and Shanghai Maokang Biotechnology Co., respectively. Paraformaldehyde solution was from Wuhan Servicebio Technology Co. Matrigel and crystal violet staining solution were purchased from Beyotime Biotechnology (China). Primary antibodies against PI3K, phospho-PI3K, mTOR, phospho-mTOR, Bcl-2, Bax, caspase-3, and β-actin were supplied by Boaoson Biotechnology (Beijing, China). Unless otherwise stated, all reagents were of analytical grade.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Clonogenic Assay\u003c/h2\u003e\u003cp\u003eHGC-27 cells in the logarithmic growth phase were seeded into 6-well plates (1,000 cells/well). After adherence, cells were treated with the indicated compounds for 24 h, followed by incubation in drug-free medium for 14 days, with medium refreshed every 3 days. Colonies were fixed with 4% paraformaldehyde for 30 min, stained with 0.5% crystal violet for 20 min, washed, air-dried, and imaged.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Transwell Invasion Assay\u003c/h2\u003e\u003cp\u003eInserts with 8-\u0026micro;m pores (Corning) were pre-coated with 1 mg/mL Matrigel (BD Biosciences) and incubated at 37\u0026deg;C for 3 h. HGC-27 cells, serum-starved for 24 h, were seeded into the upper chamber (1\u0026times;10⁵ cells/well) in serum-free medium containing the indicated compounds. Medium supplemented with 10% FBS was added to the lower chamber as a chemoattractant. After 24 h, non-invading cells were removed, and membranes were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. Invaded cells on the lower surface were imaged and counted in five randomly selected fields. Migration assays were conducted under the same conditions without Matrigel coating.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Western Blot Analysis\u003c/h2\u003e\u003cp\u003eCell lysates were centrifuged at 12,000 g for 10 min at 4\u0026deg;C, and supernatants were collected. Protein concentrations were quantified using the BCA assay (Thermo Fisher). Equal protein amounts (20 \u0026micro;g) were resolved on 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 2 h at room temperature, then incubated overnight at 4\u0026deg;C with primary antibodies against PI3K, p-PI3K, mTOR, p-mTOR, BCL-2, BAX, caspase-3 and β-actin. After washing, membranes were incubated with HRP-conjugated secondary antibodies for 2 h at 37\u0026deg;C. Signals were detected using an enhanced chemiluminescence kit.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Cell Viability Assay (CCK-8)\u003c/h2\u003e\u003cp\u003eHGC-27 cells were seeded in 96-well plates at 1\u0026times;10⁵ cells/mL and incubated overnight. After treatment for 24 h, 10 \u0026micro;L of CCK-8 reagent (Dojindo, Japan) was added to each well and incubated for 2 h at 37\u0026deg;C. Absorbance was measured at 450 nm using a microplate reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Wound Healing Assay\u003c/h2\u003e\u003cp\u003eHGC-27 cells were plated in 6-well plates (1\u0026times;10⁶ cells/mL) and cultured overnight. Linear scratches were made using a 200 \u0026micro;L pipette tip, and detached cells were removed by PBS(phosphate-buffered saline) washing. Cells were then incubated in drug-containing medium. Scratch closure was imaged at 0 and 24 h using an inverted microscope. Wound width was quantified using ImageJ, and migration rates were calculated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Flow Cytometric Analysis of Apoptosis\u003c/h2\u003e\u003cp\u003eHGC-27 cells (1\u0026times;10⁶/mL) were treated with compounds for 24 h. Cells were collected, washed with PBS, and resuspended in 500 \u0026micro;L Annexin V binding buffer. Apoptotic cells were stained with 5 \u0026micro;L Annexin V-FITC and 5 \u0026micro;L propidium iodide (50 \u0026micro;g/mL), vortexed gently, and incubated in the dark for 20 min at room temperature. Samples were analyzed by flow cytometry.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 AO/EB Double Staining\u003c/h2\u003e\u003cp\u003eAO/EB staining solution was prepared by mixing AO, EB, and buffer at a 1:1:8 ratio. HGC-27 cells were seeded on glass coverslips in 6-well plates (1\u0026times;10⁶ cells/well) and treated for 24 h. Cells were washed with PBS and stained with 1 mL AO/EB for 10 min in the dark, then visualized using a confocal laser scanning microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 In Vivo Tumor Xenograft Model\u003c/h2\u003e\u003cp\u003eHGC-27 cells (4 \u0026times; 10⁶) suspended in PBS were subcutaneously injected into the inguinal region of 4-week-old female BALB/c nude mice. \u003cb\u003eThe mice were purchased from Shaanxi Shiyao Yike Biotechnology Co., Ltd. (Xi\u0026rsquo;an, China), and housed under specific pathogen-free (SPF) conditions.\u003c/b\u003e After tumors became palpable, the mice were randomly divided into three groups (n\u0026thinsp;=\u0026thinsp;5 per group): Mzs-1, Mzs-1\u0026thinsp;+\u0026thinsp;SC79 (a PI3K activator), and NC (normal saline control). Treatments were administered once daily for 7 consecutive days. Tumor volumes were measured weekly using a digital caliper and calculated with the formula: V = \u0026frac12; \u0026times; a \u0026times; b\u0026sup2; (a\u0026thinsp;=\u0026thinsp;length, b\u0026thinsp;=\u0026thinsp;width). \u003cb\u003eOne mouse from each group was euthanized at weeks 0, 1, 2, 3, and 4, respectively. Prior to euthanasia, mice were anesthetized with 2\u0026ndash;3% isoflurane until complete loss of righting reflex, followed by cervical dislocation.\u003c/b\u003e Tumors were excised, photographed, and processed for analysis. Western blotting was performed on tumor tissues to assess the expression levels of PI3K, p-PI3K, and caspase-3. \u003cb\u003e All animal procedures complied with the institutional and national regulations on the ethical use of animals in research. The study protocol was reviewed and approved by the Ethics Committee of The People\u0026rsquo;s Hospital of Suzhou New District(2020-002). All procedures were carried out in accordance with relevant guidelines and regulations, and the study is reported in accordance with the ARRIVE guidelines.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) unless otherwise stated. Statistical analyses were performed using GraphPad Prism 8.0 and SPSS 20.0. For comparisons between two groups, paired or unpaired Student\u0026rsquo;s t-test, Mann\u0026ndash;Whitney test, or two-tailed χ\u0026sup2; test was used, as appropriate. One-way ANOVA followed by Bonferroni\u0026rsquo;s multiple comparisons test was applied for multi-group comparisons. A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Target Prediction and Prognostic Significance of Key Genes\u003c/h2\u003e\u003cp\u003ePharmacophore-based inverse docking identified ten potential protein targets of Mzs-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Among the top candidates, several\u0026mdash;GSK3β (6th), MAPK14 (3rd), JAK2 (4th), and the glucocorticoid receptor (GR; 8th)\u0026mdash;are known to regulate or be regulated by the PI3K/AKT cascade. To validate these putative interactions, molecular docking was conducted between Mzs-1 and representative PI3K/AKT pathway components (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Mzs-1 exhibited favorable binding affinities to PI3Kα (\u0026ndash;7.846 kcal/mol), PI3Kγ (\u0026ndash;7.525 kcal/mol), AKT2 (\u0026ndash;6.990 kcal/mol), and ERK2 (\u0026ndash;7.072 kcal/mol), indicating potential direct interaction with key nodes of the signaling network. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD illustrates the binding modes of Mzs-1 to PI3Kα (left panel) and PI3Kγ (right panel). Mzs-1 engages the ATP-binding pocket through hydrogen bonding, electrostatic forces, π\u0026ndash;π stacking, and hydrophobic interactions with critical amino acid residues. These interactions support its high-affinity binding and provide a structural basis for the proposed PI3K-related activity of Mzs-1.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate the expression patterns of this signaling pathway in gastric adenocarcinoma, we analyzed RNA-seq data from the TCGA-STAD cohort (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), processed via the STAR pipeline and quantified in TPM. After log2 transformation\u003cem\u003e[log2(TPM\u0026thinsp;+\u0026thinsp;1)]\u003c/em\u003e, we compared gene expression between tumor (n\u0026thinsp;=\u0026thinsp;375) and adjacent normal tissues (n\u0026thinsp;=\u0026thinsp;32). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, expression levels of PIK3CA, AKT1, AKT2, MAPK14, and MAPK1 were significantly higher in tumors compared to normal tissues (Wilcoxon rank-sum test, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), suggesting the involvement of PI3K/AKT and MAPK signaling in gastric cancer. To explore their prognostic implications, Kaplan\u0026ndash;Meier survival analysis was conducted by stratifying patients based on median gene expression. Notably, elevated expression of PIK3CA and AKT1 correlated with poorer overall survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, p\u0026thinsp;\u003cem\u003e=\u0026thinsp;0.046\u003c/em\u003e). These results suggested that dysregulation of PI3K/AKT pathway may drive disease progression and impact patient outcomes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 In Vitro Validation of PI3K/AKT/mTOR Pathway Involvement\u003c/h2\u003e\u003cp\u003eTo elucidate the anti-tumor mechanism of Mzs-1, we assessed its effects on HGC-27 cells function and associated signaling pathways in vitro. As shown by the CCK-8 assay, Mzs-1 significantly impaired cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, [0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/em\u003e]). Co-treatment with LY294002 slightly inhibited cell viability further, but co-treatment with SC79 could significantly restored cell viability, suggesting PI3K/AKT pathway involvement (Mzs-1 vs. Mzs-1\u0026thinsp;+\u0026thinsp;SC79: 0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 vs. 1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). In wound healing assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), Mzs-1 or co-treatment with LY294002 markedly inhibited migration compared to the NC group ([quantified% wound closure at 24 h, 0.24: 0.22 vs 0.7, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e]), while the effect was slightly alleviated by SC79 when compared with the treatment group. Flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) revealed that Mzs‑1 induced apoptosis in nearly 50% of cells, which was further supported by AO/EB staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), showing characteristic apoptotic morphology including cell shrinkage and orange-red nuclear fluorescence. Consistent with these findings, Mzs‑1 significantly suppressed cell invasion in Transwell assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Colony formation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) further demonstrated a marked decrease in both the number and size of colonies after Mzs-1 treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the molecular mechanism underlying Mzs-1\u0026ndash;mediated tumor suppression, we examined the expression of key components in the PI3K/AKT/mTOR pathway and apoptosis-related proteins by Western blotting under different treatment conditions. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, Mzs-1 treatment led to decreased phosphorylation of PI3K and mTOR, suggesting pathway inhibition. The addition of LY294002 significantly reduced the phosphorylation levels, confirming effective inhibition of PI3K signaling. In contrast, co-treatment with SC79 could increase the expression of p-AKT and p-mTOR, validating the responsiveness of the pathway. Regarding apoptotic markers, Mzs-1 treatment decreased anti-apoptotic BCL-2 while increasing pro-apoptotic BAX and caspase-3. These effects were further enhanced by LY294002 (statistical quantification, n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; BCL‑2: NC 1.00, Mzs‑1 0.46, Mzs‑1\u0026thinsp;+\u0026thinsp;LY294002 0.33; BAX: NC 1.00, Mzs‑1 2.45, Mzs‑1\u0026thinsp;+\u0026thinsp;LY294002 3.23; caspase‑3: NC 1.00, Mzs‑1 1.90, Mzs‑1\u0026thinsp;+\u0026thinsp;LY294002 2.38), whereas SC79 partially reversed these effects (BCL‑2: Mzs‑1 0.46, Mzs‑1\u0026thinsp;+\u0026thinsp;SC79 0.74; BAX: Mzs‑1 2.45, Mzs‑1\u0026thinsp;+\u0026thinsp;SC79 1.92; caspase‑3: Mzs‑1 1.90, Mzs‑1\u0026thinsp;+\u0026thinsp;SC79 1.63). These findings underscore the involvement of the PI3K/AKT/mTOR pathway in Mzs‑1-induced apoptosis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 In Vivo Confirmation of PI3K/AKT Pathway Activation\u003c/h2\u003e\u003cp\u003eAs shown in the flow chart (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), gastric cancer tumor volumes were monitored weekly over four weeks in three groups: negative control (normal saline gastric lavage), Mzs‑1 (gastric lavage), and Mzs‑1 combined with SC79 (gastric lavage). With the exception of week 3, Mzs‑1 treatment significantly suppressed tumor growth compared to the control group. Specifically, tumor volumes remained relatively stable in the Mzs‑1 group, while those in the control group increased progressively. Co-administration of SC79 markedly reversed the inhibitory effect of Mzs‑1, resulting in a significant increase in tumor volume from week 3 onward (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe expression levels of phosphorylated PI3K relative to total PI3K (P-PI3K/PI3K) and Caspase-3 were analyzed at weeks 0, 1, 2, 3, and 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Mzs-1 treatment significantly reduced P-PI3K/PI3K levels from week 1, suggesting inhibition of the PI3K pathway as a potential mechanism of tumor suppression. Concurrently, Caspase-3 expression was elevated, indicating enhanced apoptosis. In contrast, combination treatment with SC79 partially reversed these effects, increasing P-PI3K/PI3K expression from week 2 and reducing Caspase-3 expression before week 4. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE illustrated the potential mechanisms of Mzs-1\u0026rsquo;s effect on HGC-27 cells.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur results demonstrated that Mzs-1, a synthetic analogue of \u003cem\u003eArctium lappa L.\u003c/em\u003e constituents, induces apoptosis in HGC-27 cells via inhibition of the PI3K/AKT/mTOR pathway. While \u003cem\u003eA. lappa L.\u003c/em\u003e has long been recognized for its anti-inflammatory and immunomodulatory properties, its anti-tumor potential, particularly in GC, remains underexplored(Gao et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sandberg et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although several bioactive components have been isolated from \u003cem\u003eA. lappa L.\u003c/em\u003e (Chan et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), few efforts have focused on their chemical synthesis or mechanistic characterization. Our previous work successfully synthesized Mzs-1, a chemical analogue of the active components found in burdock seeds(Xia et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). To elucidate its potential mechanism in gastric cancer, we carried out this study.\u003c/p\u003e\u003cp\u003eMechanistically, Mzs-1-induced effects were enhanced by the PI3K inhibitor LY294002 and reversed by the AKT activator SC79, suggesting that Mzs-1 acts upstream of AKT and modulates downstream signaling. Overactivation of the PI3K/AKT/mTOR pathway is a key driver of GC cell proliferation, invasion, and chemoresistance, and is closely associated with poor clinical outcomes(Fattahi et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ma et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our findings partially align with those of Meng et al., who showed that PI3K/AKT/mTOR signaling mediates the anti-tumor activity of Fructus Arctii in triple-negative breast cancer (TNBC)(Meng et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Similarly, the same pathway has been implicated in the anti-prostate cancer effects of \u003cem\u003eArctium lappa L.\u003c/em\u003e in vitro(Sun et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Other studies have demonstrated that \u003cem\u003eArctium lappa L.\u003c/em\u003e induces S-phase arrest via CDKN1C/p57 activation in colorectal cancer cells(Yang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and inhibits YAP signaling both transcriptionally and post-translationally in HeLa, MDA-MB-231, SW480, and PC3 cells(Li et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, it has been shown to enhance the therapeutic efficacy of azithromycin in breast cancer(Lee et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The CEA\u0026ndash;KRT1\u0026ndash;PI3K/AKT axis has also been implicated in modulating oxaliplatin sensitivity in GC cells(Chen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Notably, the upregulation of caspase-3 and BAX, along with suppression of BCL-2, correlated with reduced PI3K/AKT activity, confirming that Mzs-1 induces apoptosis at least in part through inhibition of this survival pathway. Furthermore, restoration of anti-apoptotic markers by SC79 supports a feedback loop wherein reactivation of AKT mitigates Mzs-1-induced cytotoxicity. This is consistent with prior reports showing that PI3K/AKT inhibition sensitizes cancer cells to apoptosis(Han et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCollectively, our study is the first to demonstrate that Mzs-1\u0026mdash;a synthetic derivative of \u003cem\u003eArctium lappa L.\u003c/em\u003e\u0026mdash;exerts potent anti-tumor effects in gastric cancer by modulating the PI3K/AKT/mTOR pathway and promoting apoptosis. These findings identify Mzs-1 as a promising therapeutic candidate or adjuvant to conventional chemotherapy. By disrupting survival signaling, Mzs‑1 may reduce the required dose of conventional drugs, improve efficacy, and help overcome resistance. However, further preclinical investigations\u0026mdash;including pharmacokinetics, toxicity assessment, and validation in orthotopic or genetically engineered mouse models\u0026mdash;are necessary to advance clinical translation.\u003c/p\u003e\u003cp\u003eDespite these encouraging findings, several limitations should be acknowledged. First, although \u003cem\u003eA. lappa L.\u003c/em\u003e is also known for its immunoregulatory and anti-inflammatory properties, our study focused solely on tumor cell\u0026ndash;intrinsic mechanisms. Given the critical interplay between immunity, inflammation, and tumor survival, future studies should explore whether Mzs-1 affects the tumor microenvironment or immune responses. Second, while we confirmed involvement of the PI3K/AKT/mTOR pathway, the precise regulatory mechanisms remain unclear. Future investigations should examine whether Mzs-1 alters the balance between apoptosis and autophagy, and determine the roles of specific PI3K isoforms and mTOR complexes (mTORC1 vs. mTORC2). Third, feedback activation of compensatory pathways such as MAPK/ERK could attenuate the durability of response, a common challenge in targeted therapy. Advanced techniques such as isoform-selective inhibition, CRISPR-based gene editing, and phosphoproteomic profiling will be pivotal in elucidating the broader signaling networks regulated by Mzs-1. To extend our findings, future studies should prioritize: (1) the development of xenograft or genetically engineered gastric cancer models to evaluate the antitumor efficacy, pharmacokinetics, and safety profile of Mzs-1; (2) the use of gene editing to confirm pathway dependency and uncover potential resistance mechanisms; and (3) exploration of synergistic interactions between Mzs-1 and standard chemotherapeutics or targeted agents in combination regimens.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eMzs-1 has demonstrated promising anti-gastric cancer activity, potentially addressing the urgent need for more effective treatment strategies in GC. However, further mechanistic and preclinical investigations are essential to validate these findings and support the therapeutic development of Mzs-1.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAKT, protein kinase B; BAX, Bcl-2-associated X protein; CCK-8, Cell Counting Kit-8; ERK2, extracellular signal-regulated kinase 2; GC, gastric cancer; GR, glucocorticoid receptor; HGC-27, human gastric carcinoma cell line; JAK2, Janus kinase 2; MAPK14, mitogen-activated protein kinase 14; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; SC79, AKT activator; TCGA, The Cancer Genome Atlas; TCM, Traditional Chinese Medicine; TNBC, triple-negative breast cancer; TPM, transcripts per million.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of interest statement\u003c/strong\u003e: The authors declare no conflicts of interest that pertain to this work.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eSuzhou High tech Zone People's Hospital Scientific Development Innovation Fund Project (SGY2024B04); Technology Development Project of Suzhou City (SYSD2020084); Science and Technology Project of Suzhou city (SKYD2023089, SKYD2023033); Diagnosis and Treatment Technology Project of Suzhou Clinical Key Disease(LCZX202354).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY. Sun and C. Chai conceptualized the study. L. Yang was responsible for data acquisition. Data analysis was conducted by X.F. Yu and H. Yao. Y. Sun and C. Chai contributed to writing the original draft, review, and editing. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. All relevant data supporting the findings of this study are included in the article and its supplementary materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChan, Y. S. et al. A review of the pharmacological effects of Arctium lappa (burdock). \u003cem\u003eInflammopharmacology\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e (5), 245\u0026ndash;254 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen, Y. et al. 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(Lond)\u003c/em\u003e. \u003cb\u003e42\u003c/b\u003e (4), 314\u0026ndash;326 (2022).\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, Traditional Chinese medicine, Mzs-1, Apoptosis, PI3K/AKT/mTOR pathway","lastPublishedDoi":"10.21203/rs.3.rs-6972121/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6972121/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eGastric cancer (GC), particularly at advanced stages, often results in therapeutic failure due to drug resistance and adverse effects. Traditional Chinese medicine (TCM), with its multi-target actions and favorable safety profile, presents a compelling alternative.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e To evaluate the anti-tumor efficacy of Mzs-1, a synthetic analogue derived from \u003cem\u003eArctium lappa L.\u003c/em\u003e, and elucidate its mechanism of action in gastric cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy design: \u003c/strong\u003eThis study integrated computational prediction and experimental validation to evaluate the anti-tumor effects of Mzs-1 in vitro and in vivo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Network pharmacology analysis was conducted to identify the potential targets and pathways of Mzs-1, highlighting the PI3K/AKT/mTOR pathway. Molecular docking was performed to predict the binding affinity of Mzs-1 to key proteins in this pathway. Functional assays, including cell proliferation, apoptosis, and invasion, were performed in HGC-27 cells. Western blotting was used to examine the expression of PI3K/AKT/mTOR pathway components. In vivo efficacy was assessed in xenograft mouse models following Mzs-1 treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eIn vitro, Mzs-1 suppressed HGC-27 cell proliferation and invasion while inducing apoptosis. These effects were enhanced by PI3K inhibition and attenuated by AKT activation. In vivo, Mzs-1 inhibited tumor growth and downregulated PI3K/AKT/mTOR signaling in xenograft tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eMzs-1 exerts anti-GC activity by modulating the PI3K/AKT/mTOR pathway, supporting its potential as a therapeutic candidate for gastric cancer.\u003c/p\u003e","manuscriptTitle":"Antitumor effects of Mzs-1 from Chinese Artium lappa L. on HGC-27 cells via the PI3K/AKT/mTOR pathway in vitro and in vivo.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-20 14:03:29","doi":"10.21203/rs.3.rs-6972121/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":"d72c325d-8db3-4199-a0ad-51c910fcdd74","owner":[],"postedDate":"August 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53454080,"name":"Biological sciences/Cancer"},{"id":53454081,"name":"Biological sciences/Drug discovery"},{"id":53454082,"name":"Biological sciences/Molecular biology"},{"id":53454083,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-03-11T16:55:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-20 14:03:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6972121","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6972121","identity":"rs-6972121","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}