Inhibition of gastric adenocarcinoma proliferation by WSGC@MS: Role of KEAP1/NRF2 signaling pathway and autophagy regulation

Dongjian Song; Qiuliang Liu; Zechen Yan; Qi Wang; Meng Su; Hui Zhang; Longyan Shi; Yingzhong Fan; Qian Zhang; Heying Yang; Da Zhang

doi:10.1016/j.mtbio.2025.101995

. 2025 Jun 16;33:101995. doi: 10.1016/j.mtbio.2025.101995

Inhibition of gastric adenocarcinoma proliferation by WSGC@MS: Role of KEAP1/NRF2 signaling pathway and autophagy regulation

Dongjian Song ^a,^b,^⁎,¹, Qiuliang Liu ^a,¹, Zechen Yan ^b,^c,¹, Qi Wang ^a,¹, Meng Su ^a, Hui Zhang ^a, Longyan Shi ^a, Yingzhong Fan ^a, Qian Zhang ^a, Heying Yang ^a, Da Zhang ^a

PMCID: PMC12272921 PMID: 40688680

Abstract

Porous Magnetic Chitosan Microspheres (MS) serve as effective carriers for targeted drug delivery due to their magnetic properties and biocompatibility. This study investigates the therapeutic potential of WSGC Peptide-loaded porous MS (WSGC@MS) in treating gastric adenocarcinoma by elucidating its mechanism of action in inhibiting cancer cell proliferation. The prepared porous MS was characterized using various techniques, and the physicochemical properties of the nanocomposites were evaluated. In vitro experiments demonstrated that WSGC@MS effectively suppressed the proliferation of gastric adenocarcinoma cells, enhanced apoptosis, and increased cellular uptake and reactive oxygen species production under magnetic field exposure. In vivo studies in a gastric adenocarcinoma mouse model revealed significant accumulation of WSGC@MS at tumor sites, resulting in substantial inhibition of tumor growth and metastasis. Transcriptomic analysis uncovered the downregulation of FAM117B, inhibition of the KEAP1/NRF2 pathway, and activation of autophagy mediated by ROS/AMPK/mTOR. Overall, WSGC@MS shows great promise for targeted therapy in gastric adenocarcinoma by degrading NRF2 ubiquitination, regulating the KEAP1/NRF2 signaling pathway, and inducing autophagy, offering a novel nanotechnology-based treatment strategy.

Keywords: Magnetic chitosan microspheres, WSGC peptide, Gastric adenocarcinoma, Kelch-like ECH-Associated protein 1/nuclear factor erythroid 2-related factor 2 signaling pathway, Autophagy, Nanotechnology treatment

Graphical abstract

The molecular mechanism by which the WSGC@MS/FAM117B/NRF2 axis inhibits gastric adenocarcinoma progression and drug resistance via the ROS/AMPK/mTOR/Autophagy signaling pathway.

1. Introduction

Gastric adenocarcinoma is one of the most common malignancies worldwide, with incidence and mortality rates among the highest of all cancers [1,2]. It ranks third globally as a cause of cancer-related death [[3], [4], [5]]. Often asymptomatic in its early stages, most patients are diagnosed at an advanced stage, significantly reducing the effectiveness of treatments and patient survival rates. Traditional treatments for gastric adenocarcinoma, including surgical resection, chemotherapy, radiotherapy, and targeted drug therapies, are often limited by cancer recurrence, metastasis, and damage to healthy tissues [6]. Consequently, there is an urgent need to develop new therapeutic strategies to improve the prognosis for patients with advanced gastric adenocarcinoma.

Targeted drug delivery systems represent a novel cancer treatment strategy, offering the principal advantage of delivering drugs directly to tumor cells, thus maximizing therapeutic efficacy while minimizing toxicity to normal tissues [[7], [8], [9]]. However, despite their theoretical advantages, practical application faces numerous challenges [[10], [11], [12]]. These include ensuring the stability and bioavailability of the drug within the body, avoiding immune system clearance, and precisely controlling the timing and location of drug release. Moreover, existing drug delivery systems often struggle to balance efficient drug loading with precise targeting, limiting their widespread clinical use [[13], [14], [15]].

The Kelch-like ECH-associated protein 1 (KEAP1)/Nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway is one of the primary defense mechanisms against oxidative stress in cells [16,17]. Normally, this pathway regulates antioxidative responses and cellular metabolism to maintain physiological stability [[18], [19], [20]]. In addition to its antioxidant functions, the KEAP1/NRF2 pathway also regulates the expression of key genes involved in apoptosis and cell proliferation [21]. Tossetta et al. have reported that this pathway also plays a critical role in inflammatory diseases such as periodontitis [22]. In malignant diseases, the accumulation of oxidative damage often leads to malignant transformation and tumor development. Therefore, activation of KEAP1/NRF2 in the early stages of tumorigenesis is considered beneficial [23]. However, in the later stages of cancers such as gastric adenocarcinoma, excessive activation of NRF2 can promote tumor cell growth and survival and even enhance their resistance to chemotherapy [19,20,24]. Consequently, targeting the NRF2 signaling pathway to inhibit its abnormal tumor activation has emerged as a potential therapeutic strategy [[25], [26], [27]]. Research indicates that specific inhibitors or small molecule drugs can effectively modulate this signaling pathway, inhibiting tumor growth and metastasis [28,29].

Autophagy, as an intracellular cleaning mechanism, helps cells remove damaged proteins and organelles, maintaining stability within the cellular environment [[30], [31], [32]]. In cancer therapy, both activation and inhibition of autophagy have shown potential therapeutic value [[33], [34], [35]]. On the one hand, activating autophagy can promote cancer cell death, especially under conditions of nutrient or oxygen deprivation; on the other hand, under certain conditions, inhibiting autophagy can increase tumor cell sensitivity to chemotherapy or radiotherapy [36,37]. Currently, precisely controlling the autophagy process to achieve optimal therapeutic effects is a focal point of research [[38], [39], [40]].

This study aims to develop a novel type of Porous Magnetic Chitosan Microspheres (MS) as carriers for WSGC peptide, specifically targeting the KEAP1/NRF2 signaling pathway and activating autophagy in gastric adenocarcinoma to inhibit tumor proliferation and metastasis. Through detailed physicochemical characterization and biological evaluation, we have validated the potential of these nanocarriers to enhance the efficiency and specificity of drug delivery. We anticipate that this research will not only provide a new effective strategy for treating gastric adenocarcinoma but also serve as a reference for the treatment of other types of cancer. Moreover, the outcomes of this study are expected to improve drug bio-distribution and mechanisms of action, reduce side effects during treatment, and provide more precise and personalized therapy options for gastric adenocarcinoma patients, significantly improving their quality of life and overall survival rates.

2. Materials and methods

2.1. Materials and reagents

(C₆H₁₁NO₄)_n (69047438), FeCl₃·6H₂O (XW011002577102), Na₃C₆H₅O₇·2H₂O (C446332500), CH₂O (C119690010), NaH₂PO₄·H₂O (C447740010), and Na₂HPO₄·12H₂O (C447250025) were procured from Sinopharm Chemical Reagent Co., Ltd. 5-Fluorouracil (F6627) and oxaliplatin (PHR1528) were sourced from Sigma. All other chemical reagents were of analytical grade and required no further purification. Ultrapure water (18.2 MΩ × cm) was obtained from the Milli-Q Integral 5 system (Merck Millipore, USA).

2.2. Porous magnetic chitosan microspheres loaded with WSGC peptide

2.2.1. Synthesis and source of WSGC peptide

Previous studies identified a specific non-inflammatory biomarker in the serum of gastric adenocarcinoma patients, a protein peptide with a mass-to-charge ratio (M/z) of 6449 Da and sequence: MQPRVLALLASADASLLSFMYMKHATKTAKDVQVAQQARGWVTDGFSSL, derived from apoC-III [68]. The main active site was found to be a 40-amino acid peptide, WSGC (sequence: SEAEDASLLSFMQGYMKHATKTAKDALSSVQESQVAQQAR, molecular weight 4344 g/mol). Both WSGC and control peptides were synthesized by Beijing LifeTein Co., Ltd.

2.2.2. Synthesis of Fe₃O₄ magnetic nanoparticles

Fe₃O₄ Magnetite nanoparticles were synthesized via an improved hydrothermal method. A solution containing 2 mmol of FeCl₃·6H₂O, 8 mmol of Na₃C₆H₅O₇·2H₂O, and 12 mmol of urea (U5128, Sigma, USA) was prepared in 80 mL of deionized water. After adding 0.3 g of polyacrylamide (PAM) (738743, Sigma, USA) and ensuring complete dissolution, the mixture was transferred to a Teflon-lined stainless steel autoclave. The autoclave was sealed and reacted at 473 K for 12 h. The product was collected using a magnet, washed sequentially with deionized water and ethanol, and then vacuum dried at 333 K for 10 h to yield the Fe₃O₄ magnetite nanoparticles.

2.2.3. Synthesis of porous MS

Porous MS were fabricated using a water-in-oil microemulsion technique. Chitosan (0.8 g) was dissolved in 40 mL of 2 % acetic acid solution (45754, Sigma, USA) and mixed with Fe₃O₄ magnetic nanoparticles followed by ultrasonication for 30 min. This solution was gradually added to a microemulsion containing 16 mL of Span-80 (85548, Sigma, USA) and 320 mL of liquid paraffin (76233, Sigma, USA), and stirred for 30 min. Subsequently, 16 mL of formaldehyde solution (47608, Sigma, USA) was introduced, and the mixture was vigorously stirred for 1 h. The pH was adjusted to 9–10, and the reaction was allowed to proceed for 4 h. The product was collected using a magnet, washed sequentially with petroleum ether (PX0424, Sigma, USA), deionized water, and ethanol (1.00967, Sigma, USA), and finally vacuum dried at 333 K to yield the MS.

2.2.4. Characterization of porous MS

The crystalline structure of the samples was identified by X-ray diffraction (XRD) using an Empyrean system (PANalytical B.V., Netherlands, Cu Kα radiation). Functional groups on the surface of the samples were analyzed by Fourier-transform infrared spectroscopy (FTIR) using a Vertex 70 (Bruker, Germany, KBr). The morphology and particle diameter were examined via scanning electron microscopy (SEM) on a Nova 400 (FEI, USA) and transmission electron microscopy (TEM) on a Tecnai G2 F20 (FEI, USA). The magnetic properties were studied using a vibrating sample magnetometer (VSM) on a PPMS-9 (Quantum Design, USA).

2.2.5. Protein loading and In vitro release experiments

Following the methods reported in the literature, the loading and release characteristics of WSGC peptide on MS were investigated. Fifty milligrams of the sample was added to 50 mL of WSGC peptide phosphate-buffered saline (PBS, 0.01 mol/L) and agitated at 310 K. The equilibrium state was studied by varying the loading time and solution pH. The residual protein concentration in the filtrate was measured at a wavelength of 280 nm using a UV–visible spectrophotometer to calculate the loading capacity of the WSGC peptide. For in vitro release studies, 50 mg of the dried WSGC peptide-loaded sample was suspended in 5 mL of PBS (0.02 mol/L, pH 7.4) and agitated at 310 K at 150 rpm. Samples were periodically collected and replaced with an equal volume of fresh PBS. The content of WSGC peptide in the release buffer was determined using a UV–visible spectrophotometer [41].

2.3. Cell-related experiments

2.3.1. Cell culture

Human gastric adenocarcinoma cells KATO III (HTB-103) were obtained from ATCC, MKN-45 cells (CL-0292) from Pricella, and normal gastric mucosal epithelial cells GES-1 from Beyotime. KATO III and MKN-45 cells were maintained in RPMI-1640 medium containing 10 % FBS, and GES-1 cells were grown in Complete Gastric Mucosal Epithelial Cell Medium (CM-H048, Pricella). All cells were incubated at 37 °C in a 5 % CO₂ incubator.

2.3.2. Lentivirus construction

Two shRNA sequences targeting FAM117B and KEAP1 were designed, with a non-targeting shRNA sequence serving as a negative control (shNC). Primer sequences are as follows: shNC: GGGTGAACTCACGTCAGAA; shFAM117B-1: TGGCAACCATGCAGCTATTAA; shFAM117B-2: TGAATCAGGAGATTGAAATAA; shKEAP1-1: GCGAATGATCACAGCAATGAA; shKEAP1-2: GCCTTAATTCAGCTGAGTGTT. These oligonucleotides were synthesized by GenePharma®. Lentiviral overexpression vectors pCDH-CMV-MCS-EF1α-copGFP (System Biosciences, USA) were used to construct overexpression vectors for FAM117B and NRF2 genes. Lentiviral particles were packaged into HEK-293T cells using the Lentivirus Packaging Kit (A35684CN, Invitrogen, USA), and viral supernatants were collected after 48 h. Transfection experiments employed Lipofectamine 3000 (L3000150, Thermo Fisher).

2.3.3. Construction of drug-resistant gastric adenocarcinoma cell lines

Following Coley's method, resistance to 5-fluorouracil and oxaliplatin (F6627, Sigma, USA) was induced incrementally. Parental cells were exposed to drug solutions starting at 20 % of the EC50, with concentrations gradually increased over 30 passages to establish resistance. The development of the drug-resistant model took 12 months [42,43].

2.3.4. Labeling and monitoring the uptake of Nanocomplexes

KATO III R-5-Fu and MKN-45 R-5-Fu cells were seeded into confocal dishes at a density of 1 × 10⁵ cells per dish. Cy5.5 (SA1018, Invitrogen) -labeled WSGC@MS (1.5 mg/mL) was added and incubated for 1–6 h. Cells were then washed and fluorescent images captured using a confocal laser scanning microscope (CLSM) (Leica Microsystems, Germany). In targeted studies, GES-1, KATO III, and MKN-45 cells were incubated in confocal dishes for 24 h, followed by the addition of Cy5.5-labeled WSGC@MS and a further 6 h of incubation before fluorescence imaging.

2.3.5. Lysosomal escape

KATO III and MKN-45 cells were cultured in confocal dishes and after 24 h, Cy5.5-labeled WSGC@MS (1.5 mg/mL) was added. Following incubation for a predetermined period, lysosomes were stained with Lysotracker red (L12492, Thermofisher) and observed via CLSM.

2.3.6. Cell viability Assessment

Cell viability was assessed using a CCK-8 kit (96992, Sigma, USA). Cells were seeded in 96-well plates and treated with 0–2 mg/mL of MS and WSGC@MS for 24 h. After adding CCK-8 solution and incubating for 1 h, absorbance was measured at 450 nm to calculate cell survival rates. Chemotherapy sensitivity studies were conducted on KATO III and MKN-45 cells using the CCK-8 method to evaluate cell viability.

2.3.7. Plate cloning experiment

After cell culture, cells were washed twice with PBS, fixed in methanol (34860, Sigma, USA) for 15 min, and stained with 0.1 % crystal violet (C0775, Sigma, USA) for 20 min before rinsing. Colonies formed in each well or dish were counted.

2.3.8. Reactive oxygen species (ROS) detection

Intracellular ROS levels were detected using the DCFH-DA probe (D6883, Sigma-Aldrich, USA). After treatment, cells were incubated with 10 μM DCFH-DA for 30 min, followed by fluorescence imaging with CLSM or analysis using flow cytometry.

2.3.9. Live/dead cell Identification

Cells were seeded into confocal dishes and treated with MS for 24 h. Following treatment, cells were stained for 20 min using Calcein-AM/PI working solution (CBA415, Sigma-Aldrich) and examined via CLSM.

2.3.10. Detection of actin filament distribution

Cells were cultured on coverslips until 80 % confluency, fixed with 4 % paraformaldehyde (158127, Sigma-Aldrich) for 10 min, permeabilized with 0.1 % Triton X-100 (T8787, Sigma-Aldrich) for 5 min, and blocked. Alexa Fluor 488-labeled phalloidin (A12379, Thermo Fisher Scientific) was added and incubated for 30 min. Nuclei were counterstained with DAPI for 5 min, and the samples were observed using a confocal microscope.

2.3.11. Flow cytometric analysis of cell apoptosis

Cells were seeded in 6-well plates and treated, followed by staining with PI and FITC-Annexin V. Apoptosis (APOAF, Sigma-Aldrich) was analyzed using a FACS Calibur flow cytometer (BD Biosciences).

2.3.12. Nucleocytoplasmic fractionation experiment

A nucleocytoplasmic fractionation kit (NUC201, Sigma-Aldrich) was used to separate cytoplasmic and nuclear fractions. Protein concentrations were measured, and NRF2 protein levels were detected via Western Blot.

2.3.13. RT-qPCR analysis of target gene expression

Total RNA was extracted using Trizol (Thermo, 16096020, USA), and cDNA was synthesized. RT-qPCR was performed using SYBR Green PCR Master Mix (RR037Q, TAKARA) on an ABI PRISM 7500 system. GAPDH served as the internal control. The relative expression of target genes was analyzed using the 2^−ΔΔCt method, and primer sequences are listed in Table S1.

2.3.14. Western Blot

Tissues or cells were collected and lysed using RIPA lysis buffer (P0013B). Protein concentrations were determined using a BCA Protein Assay Kit (P0012). Proteins were separated by 10 % SDS-PAGE and transferred to a PVDF membrane (IPVH00010). Information on the primary antibodies used is provided in Table S2. After washing, HRP-conjugated secondary antibodies, goat anti-rabbit (ab6721, 1:2000, Abcam) or goat anti-mouse (ab6785, 1:1000, Abcam), were added and incubated at room temperature. Detection was performed using Pierce™ ECL Western Blot Substrate (32209) and quantified with a Bio-Rad imaging system.

Although the theoretical molecular weight of NRF2 is approximately 68 kDa, it is well-documented that NRF2 frequently migrates at ∼100 kDa in SDS-PAGE due to post-translational modifications (e.g., phosphorylation, ubiquitination) and its acidic amino acid composition. This observation is consistent with the Abcam datasheet of the antibody used (ab62352) and has been reported in previous studies [[44], [45], [46]]. All other antibodies showed band positions consistent with their expected theoretical weights.

2.3.15. Prussian blue staining

Tumor tissues were fixed, dehydrated, cleared, and sectioned. Sections were stained with Prussian blue to identify iron deposits, and results were documented through microscopic observation.

2.3.16. CHX-chase analysis

Cells co-incubated with or without WSGC@MS were treated with 25 μM cycloheximide (C7698, Sigma-Aldrich) to inhibit protein synthesis. Cell lysates were collected at 0, 15, 30, 45, and 60 min after CHX treatment. Western Blot analysis used antibodies against FAM117 B (PA5-101425, 1:1000, Thermo Fisher Scientific) and NRF2 (ab62352, 1:1000, Abcam).

2.3.17. Immunoprecipitation assay

Experimental groups studying the interaction between FAM117B and KEAP1 in KATO III R-5-Fu and MKN-45 R-5-Fu cells are as follows: Control group (Input) containing untreated cell lysates; IgG group, where nonspecific IgG antibodies were used for immunoprecipitation as a negative control; KEAP1 group, where anti-KEAP1 antibodies were used to immunoprecipitate and detect the binding of FAM117B with KEAP1.

The experimental groups for verifying the interaction between HA-tagged FAM117B and Flag-tagged KEAP1 in HEK293T cells include: IgG control group; Flag control group; co-transfection group with HA-FAM117B-WT and Flag-Keap1-WT; HA-FAM117B-WT only transfection group; Flag-Keap1-WT only transfection group.

2.3.18. GST pull-down assay

500 ng of glutathione (Y0000517, Sigma-Aldrich, USA) was bound to GST (SRP5348, Sigma-Aldrich, USA) or GST-KEAP1-WT (E3-310-050, Biotechne) proteins coupled to agarose beads (G0924, Sigma-Aldrich, USA). This was incubated with 200 ng of purified FAM117B (507759, Novopro) in binding buffer at 4 °C for 24 h. Western Blot then analyzed the samples using antibodies specific for FAM117B (PA5-101425, 1:1000, Thermo Fisher Scientific) and GST (ab138491, 1:1000, Abcam).

2.4. Bioinformatics-related experiments

2.4.1. High-throughput transcriptome sequencing, data quality control, and differential gene analysis

Total RNA was extracted from PBS-treated (n = 3) and WSGC@MS-treated KATO III R-5-Fu cell samples (n = 3) using Trizol reagent (Thermo, 16096020, USA). cDNA libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit and sequenced on the Illumina HiSeq 550 platform. Differential gene expression was identified using the "Limma" package in R software. Heatmaps of differentially expressed genes (DEGs) were created using R's "heatmap" package, and volcano plots were generated with the "ggplot2" package. DAVID v6.8 was used for GO and KEGG pathway enrichment analyses of DEGs, with results visualized using R software.

2.4.2. Pan-cancer analysis

RNA sequencing data were obtained from the TCGA database and processed in R software to compute TPM values. Expression differences of FAM117B across various cancer types were visualized using Wilcoxon rank-sum tests to assess expression disparities.

2.4.3. Receiver operating characteristic (ROC) analysis

ROC analysis of FAM117B expression data was conducted using the pROC package, with results visualized through ggplot2.

2.5. Mouse-related experiments

2.5.1. Construction of mouse model

The animal study was conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee. Female BALB/c nude mice, weighing approximately 18–20 g (strain code: 401, Vital River), were subcutaneously inoculated on the left flank with human gastric adenocarcinoma cells KATO III R-5-Fu and MKN-45 R-5-Fu, which are resistant to 5-Fu (5 × 10⁶ tumor cells per mouse). Once the mouse models were established and the tumors reached approximately 50 mm³ in volume (about two weeks later), the mice were randomly divided into groups.

Pharmacological Evaluation Grouping: Mice were divided into four groups (n = 5 each): Control group received saline injections; 1.5 mg/ml WSGC group; 1.5 mg/ml WSGC@MS group; 1.5 mg/ml WSGC@MS + MF group: WSGC@MS at 1.5 mg/ml was administered via tail vein injection followed immediately by the placement of a 0.15 T Nb-Fe-B disc-shaped magnet (10 mm diameter × 2 mm thickness) over the tumor for 2 h. Treatments were administered every 2 days for 21 days. Tumor length (a) and width (b) were measured every 3 days until day 36 to calculate tumor volume (v = a × b²/2) [47].

Gastric Cancer Xenograft Model: Mice were inoculated subcutaneously with 0.2 mL of cell suspension containing 2 × 10⁷ KATO III R-5-Fu/MKN-45 R-5-Fu or KATO III R-Oxa/MKN-45 R-Oxa cells into the dorsum of nude mice. The animals were grouped as follows: Control group; WSGC@MS + oeNC group; WSGC@MS + oeNRF2 group, with treatments administered every 2 days. After 20 days, mice were euthanized by cervical dislocation, and tumor tissues were harvested for further analysis.

Nude Mouse Liver Metastasis Model: Cells (2 × 10⁷ KATO III R-5-Fu/MKN-45 R-5-Fu or KATO III R-Oxa/MKN-45 R-Oxa) were injected via tail vein into nude mice in a volume of 0.2 mL. The mice were divided into three groups (n = 5 each): Control group; WSGC + oeNC group; WSGC@MS + oeNRF2 group. Treatments were administered every 2 days. After 36 days, mice were euthanized by cervical dislocation, liver tissues were harvested, and Hematoxylin and Eosin (H&E) staining was performed to count the average number of liver metastases [48].

2.5.2. Systemic toxicity Assessment

Fourteen days post-treatment, mice were euthanized, and comprehensive samples, including whole blood, serum, major organs, and tumors, were collected for complete blood analysis, blood chemistry, and histological examination. Additionally, major organs such as the liver, kidneys, heart, and spleen were subjected to H&E staining. Blood samples were analyzed for hematotoxicity and hepatic and renal function using a hematology analyzer (Beckman Coulter, USA) and a biochemical analyzer (Siemens, Germany).

2.5.3. In vivo targeting studies

Tumor-bearing nude mice were divided into Control, WSGC@MS, and WSGC@MS + MF. Each group received an intravenous injection of either PBS or Cy5.5-labeled WSGC@MS (80 nmol/g). Post-injection, optical fluorescence imaging was performed at specified time points using a 3D optical real-time imaging system (IVIS Spectrum, USA). Seventy-two hours after intratumoral drug administration, the mice were euthanized, and both tumors and organs (liver, heart, lungs, spleen, and kidneys) were dissected for in vivo imaging.

2.5.4. Immunohistochemical staining

Tissue samples were fixed, dewaxed, and underwent antigen retrieval before being stained with primary antibodies: Ki67 (ab16667, 1:200, Cell Signaling Technology), Bcl-2 (ab182858, 1:500, Abcam), Bax (ab32503, 1:250, Abcam), Cleaved Caspase-3 (9661S, 1:200, Abcam), and PCNA (ab29, 1:10000, Abcam). Immunohistochemical staining was performed, followed by optical microscopy to observe and quantify protein expression.

2.5.5. H&E staining

H&E staining was performed using an H&E staining kit (PT001, purchased from Shanghai Bogoo Biotech, China). Liver tissue sections were stained to observe morphological changes across different groups.

2.5.6. Immunofluorescence and Terminal Deoxynucleotidyl Transferase dUTP Nick end labeling (TUNEL) assay

For cell immunofluorescence detection, cells were fixed with 4 % paraformaldehyde for 20 min and washed with PBS three times. Subsequently, the cells were incubated with Triton X-100 (X100PC, Sigma-Aldrich, USA) for 5 min, blocked with 3 % BSA (B2064, Sigma-Aldrich, USA) at 37 °C for 1 h, and then incubated overnight at 4 °C with primary antibodies FAM117B (PA5-101425, 1:200, Thermo Fisher), NRF2 (ab62352, 1:1000, Abcam), and KEAP1 (ab218815, 1:200, Abcam). After washing the cells with PBS three times, they were incubated with secondary antibodies: keyFluor 488 goat anti-rabbit, keyFluor 488 goat anti-mouse, Cy3-conjugated goat anti-rabbit IgG, or Cy3-conjugated goat anti-mouse IgG for 1 h. After another three washes with PBS, the cells were stained with DAPI (D9542, Sigma-Aldrich, USA).

In tissue immunofluorescence detection, 5 μm-thick paraffin sections were deparaffinized, rehydrated, and washed with PBS three times. Antigens in the tissue were fixed using an antigen retrieval solution (C9999, Sigma-Aldrich, USA). Subsequently, the tissue sections were incubated with Triton X-100 for 5 min, treated with 3 % hydrogen peroxide at 37 °C for 25 min, blocked with 3 % BSA at 37 °C for 1 h, and then incubated overnight at 4 °C with primary antibodies FAM117B (PA5-101425, 1:200, Thermo Fisher), NRF2 (ab62352, 1:200, Abcam), and KEAP1 (ab218815, 1:200, Abcam). After washing the tissue sections with PBS three times, they were incubated with Cy3-conjugated goat anti-rabbit IgG antibody for 1 h, followed by another three washes with PBS.

The TUNEL assay was performed using the TUNEL apoptosis detection kit (S7100, Sigma-Aldrich, USA) following the manufacturer's instructions to confirm in situ cell apoptosis on 5 μm-thick paraffin sections. All images were captured using an inverted microscope (Nikon, Japan).

2.5.7. Autophagosome detection

Autophagosomes were evaluated using the GFP-mRFP-LC3 lentivirus (HB-LP2100001, Hanbio, China) to assess autophagosomes. Subsequently, the localization and quantification of autophagosomes were observed under a confocal microscope (LSM710, Zeiss, Germany). Autophagic bodies were visualized in red and green (yellow fluorescence), while autolysosomes were labeled in red. TEM was employed to capture images using an electron microscope (FEI Tecnai, USA) for further analysis.

2.5.8. Measurement of oxidative phosphorylation and glycolysis

The Seahorse XF96 metabolic flux analyzer (Agilent Seahorse, USA) was utilized to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of cells in the specified groups following the manufacturer's instructions. Data was quantified using the XFe Wave software (Agilent Seahorse) following the manufacturer's protocol.

2.5.9. Evaluation of mitochondrial membrane potential

The JC-1 assay kit (C2006, Beyotime, China) was employed to assess the mitochondrial membrane potential, and quantification was based on the red-to-green fluorescence intensity ratio.

2.5.10. Detection of GSH/GSSG ratio and GPX activity

The ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) was determined using the GSH and GSSG assay kit (S0053, Beyotime) following the manufacturer's instructions. According to the manufacturer's protocol, the glutathione peroxidase activity (GPX) was evaluated in the glutathione peroxidase assay kit (S0056, Beyotime).

2.5.11. In vivo ubiquitination analysis

Endogenous NRF2 was immunoprecipitated using anti-NRF2 antibody, followed by Western Blot with ubiquitin antibody to assess endogenous NRF2 ubiquitination. Flag-tagged FAM117B, MYC-tagged NRF2, and HA-tagged ubiquitin plasmid construction services were outsourced to GenScript company. Transfection of Flag-tagged FAM117B, MYC-tagged NRF2, and HA-tagged ubiquitin into HEK 293T cells was carried out using Lipofectamine 3000 (L3000150, Thermo Fisher) to evaluate the ubiquitination of exogenous NRF2. Western Blot was performed on the lysates with antibodies against FAM117B (NBP2-47192, Novus Biologicals), NRF2 (ab62352, Abcam), and Ubiquitin (3933S, Cell Signaling Technology) following protein precipitation.

2.6. Statistical analysis

Data were derived from at least three independent experiments and are presented as mean ± standard deviation (Mean ± SD). Statistical analyses were conducted using GraphPad Prism 9 and R software. A p-value of less than 0.05 was considered statistically significant.

3. Results

3.1. Preparation of porous MS

Pure chitosan microspheres have shown broad application prospects in targeted drug delivery, gene therapy, tissue engineering, controlled release systems, and immunotherapy. However, their inability to target lesions specifically has significantly limited their use in delivery systems [49]. Gastric cancer, a common malignant tumor with high incidence and mortality rates, has driven researchers to explore effective treatment modalities continuously. Recent studies have demonstrated that incorporating magnetic nanoparticles into microsphere delivery systems enables selective drug targeting to lesion sites, offering high efficiency and low toxicity [50]. These MS particles enhance the targeting of gastric cancer cells and allow for precise positioning and controlled drug release through an external MF, significantly improving therapeutic outcomes.

Initially, the crystalline structures and compositions of Fe₃O₄ magnetite nanoparticles and MS particles were characterized using XRD patterns (Fig. 1A). The XRD pattern of Fe₃O₄ magnetite nanoparticles (a) displayed six characteristic diffraction peaks at 2θ values of 30.08°, 35.44°, 43.12°, 53.42°, 57.13°, and 62.71°, corresponding to the crystal planes (220), (311), (400), (422), (511), and (440), respectively. These peaks match the standard spinel structure of Fe₃O₄ (JCPDS card: 19–0629), confirming that the nanoparticles are pure magnetite with a spinel structure. In contrast, the XRD pattern of MS (b) showed the same diffraction pattern as that of the Fe₃O₄ magnetite nanoparticles (Fig. 1A), indicating that the encapsulation process in chitosan did not alter the phase of the Fe₃O₄ magnetite nanoparticles. The absence of characteristic diffraction peaks in chitosan (c) also confirms the successful encapsulation of Fe₃O₄ magnetite nanoparticles within the chitosan microspheres. Therefore, the Fe₃O₄ magnetite nanoparticles retain their magnetic properties and are effectively incorporated into the chitosan matrix, making them suitable for targeted drug delivery systems.

The FTIR spectroscopy results for chitosan, Fe₃O₄ magnetite nanoparticles, and MS particles are presented in Fig. 1B. In the FTIR spectrum of chitosan (a), a broad band at 3440 cm-1 is associated with the stretching vibrations of the hydroxyl (O-H) groups. The stretching vibrations of the C-H bonds in the pyranose rings are indicated by two characteristic absorption peaks at 2925 cm-1 and 2860 cm-1, and the peak at 1085 cm-1 corresponds to the C-O stretching vibrations of glycosidic linkages. Compared to the spectrum of chitosan, the spectrum of MS (b) exhibits an enhanced peak at 1640 cm-1. The peak intensity at 3440 cm-1 in the MS (b) is also enhanced, indicating the exposure of more active hydroxyl groups during the microsphere formation process. Similarly, the spectrum of MS also shows a characteristic absorption peak at 580 cm-1, analogous to the FT-IR spectrum of Fe₃O₄ magnetite nanoparticles (c), due to the Fe-O stretching vibrations. Thus, it is inferred that the Fe₃O₄ magnetite nanoparticles have been successfully encapsulated within the chitosan matrix.

As depicted in Fig. 1C, both chitosan microspheres and MS underwent progressive weight loss across different temperature ranges. The first stage, below 120 °C, corresponds to a weight loss of 10–12 %, attributed to the desorption of physically adsorbed water. For MS, degradation observed between 120 and 220 °C is negligible, with a significant 30 % weight loss occurring near 350 °C. Conversely, chitosan microspheres reached a 30 % weight loss before 200 °C, with the peak degradation rate at 180 °C corresponding to a 45 % weight loss and a subsequent reduction in degradation rate, achieving a 70 % weight loss by 400 °C. The weight loss between 200 and 400 °C is due to depolymerization occurring through the degradation of glycosidic bonds in the polymer chains. The results indicate that chitosan microspheres exhibit lower thermal stability below 400 °C compared to MS. The weight loss in chitosan microspheres between 120 and 220 °C is 35 % higher than in MS. The chitosan microspheres experience more weight loss in the 120–220 °C range, indicating lower crystallinity than MS. Introducing magnetic nanoparticles enhances chitosan's crystallinity, thereby improving its thermal stability. This results in reduced weight loss for MS within this temperature range, as confirmed by XRD analysis.

Furthermore, studies have shown that besides targeting capabilities, the distribution of magnetic nanoparticles within the polymer matrix provides appropriate texture and increased surface roughness, thereby enhancing the surface area to accommodate more drug molecules within the system [51].

Next, we examined the morphology and microstructure of the MS using SEM. As shown in Fig. 1D, the prepared MS displayed a typical spherical shape with diameters ranging from 200 to 300 μm and featured a rough surface texture with numerous pores. The internal structure of Fe₃O₄ magnetite nanoparticles within the microspheres was analyzed using high-resolution TEM (HRTEM) (Fig. 1D), and lattice spacings of 0.295 nm (c) and 0.483 nm (d) were calculated. These measurements correspond to the (220) and (111) crystal planes of standard Fe₃O₄ magnetite, confirming that the Fe₃O₄ magnetite nanoparticles are well encapsulated within the chitosan microspheres.

The magnetic hysteresis loops (Fig. 1E) indicate that the MS and the Fe₃O₄ magnetite nanoparticles exhibit similar superparamagnetic properties. The saturation magnetizations of the Fe₃O₄ magnetite nanoparticles (a1) and MS (a2) were measured at 70.364 emu/g and 11.069 emu/g, respectively. Although the saturation magnetization of the MS is significantly reduced due to the presence of non-magnetic components (chitosan), their magnetization strength is sufficient for effective separation from aqueous solutions under an external MF (Fig. 1F). This excellent magnetic responsiveness ensures that the MS can be effectively used as drug carriers for targeted therapy.

We studied porous MS' drug loading capacity and release characteristics. Under pH 7.4 and 310 K conditions, the loading kinetics of bovine serum albumin (BSA) were investigated. The experimental results, as shown in Fig. S1A, indicate that the slope of the kinetic curve was highest between 0 and 1 h, suggesting a rapid initial loading rate. During this period, WSGC molecules, due to their lower mass transfer resistance, could easily approach the MS. From 1 to 24 h, the slope gradually decreased, indicating a slowdown in the loading rate until saturation was reached, a trend attributed to the increased spatial hindrance caused by the accumulation of WSGC molecules bound to the microspheres. After 24 h, the slope approached zero, suggesting that the loading of WSGC had nearly reached equilibrium.

Further, we explored the impact of pH on the WSGC@MS. The experiments were conducted using a 1.0 mg/mL solution of WSGC in phosphate buffer solutions of varying pH levels. It was observed that pH did not significantly affect the loading of WSGC into the microspheres. The maximum loading of WSGC was observed at a pH of 6.0; however, the loading significantly decreased as the pH increased from 6.0 to 8.0. Similarly, a decrease in pH from 6.0 to 3.0 also reduced WSGC loading. Overall, the optimal loading of WSGC (947.01 mg/g at pH 6.0) was achieved within a pH range of 5.0–6.8 (Fig. S1B).

Protein loading is a complex phenomenon [52]. WSGC molecules are thought to primarily bind to MS via ionic electrostatic attraction, supplemented by van der Waals forces, hydrogen bonding, and hydrophobic interactions, with ionic electrostatic attraction playing a major role in WSGC loading (Fig. S1C). As previously mentioned, the Fe₃O₄ magnetite nanoparticles are well-encapsulated within the chitosan; thus, the effect of pH on the WSGC@MS is likely linked to the net charge of chitosan, Fe₃O₄ magnetite nanoparticles, and WSGC molecules.

The acid dissociation constant (pKa) of chitosan is 6.5, and the isoelectric points (pI) of Fe₃O₄ magnetite nanoparticles and WSGC are 6.8 and 5.4, respectively [53,54]. As shown in Fig. S1C, the positive charge of chitosan and the NH3+ content of WSGC increase with decreasing pH (NH2 + H+ ⇌ NH3+). Conversely, as the pH increases, the negative charge (COO⁻) content of WSGC increases due to the dissociation of carboxyl groups (COOH) into carboxylate (COO⁻) and hydrogen ions (H⁺). A similar mechanism applies to magnetite nanoparticles, whose isoelectric point (pI 6.8) denotes a neutral charge point.

The results indicate that at pH < 4.7, chitosan, Fe₃O₄ magnetite nanoparticles, and WSGC all carry positive charges, leading to electrostatic repulsion between them, which is unfavorable for WSGC loading onto MS. Similarly, at pH > 6.8, chitosan, Fe₃O₄ magnetite nanoparticles, and WSGC all carry negative charges, leading to a decrease in the loading of WSGC onto the MS. WSGC carries a negative charge, whereas chitosan and Fe₃O₄ magnetite nanoparticles exhibit positive charges within a pH range of 4.7–6.8. Consequently, the substantial electrostatic attraction between them can enhance the loading of WSGC onto the MS (Fig. S1C).

The in vitro release behavior of WSGC from MS is illustrated by the cumulative release curve (Fig. S1D). An initial burst release of 44.6 % was observed within the first 15 h, followed by a steady sustained release from 15 to 120 h. After 120 h, the release rate of WSGC slowed until equilibrium was reached, with a total cumulative release of 87.4 %. These results suggest that the initial burst effect can be attributed to the weaker adsorption forces of WSGC. However, after the initial burst, the release rate of WSGC decreased due to the strong ionic electrostatic attraction between WSGC and the MS. These experimental findings demonstrate the favorable drug release characteristics of the WSGC@MS.

To assess the in vitro stability of WSGC@MS, the nanoparticles were incubated with 0.01 mol/L PBS solution, PBS solution containing 10 % FBS, and complete cell culture medium (with 10 % FBS) at 37 °C for 72 h or at 4 °C for 28 days. The results showed a slight increase in aggregation of WSGC@MS at 37 °C over 72 h, indicating nanoparticle aggregation (Fig. S1E). However, at 4 °C, the particle size remained relatively stable over 28 days, suggesting no significant aggregation occurred (Fig. S1F).

When drug carriers possess biocompatibility, their appropriate degradation characteristics become highly effective. The partial disintegration of MS after one month of degradation in PBS is depicted in Fig. S1G. The formation of cavities or channels within the microspheres led to structural instability and the emergence of cracks. The loss of structural integrity facilitates matrix disintegration, thereby enhancing the release of the encapsulated drug molecules.

No magnetic particles leaching from the MS were observed after months of swelling studies in PBS. Without binding between the magnetic particles and the chitosan, all magnetic particles would have leached out upon immediate contact with the medium. Instead, the lack of any leaching after prolonged degradation suggests a strong bond between the magnetic particles and the polymer matrix, enhancing physiological functionality (Fig. S1G).

In conclusion, this study successfully prepared and characterized porous MS (Fig. 1G). The characterization results confirm that the Fe₃O₄ magnetite nanoparticles were successfully encapsulated within the chitosan, endowing the microspheres with significant magnetic properties. This magnetic feature represents a crucial advantage for drug delivery systems compared to non-magnetic drug carriers. Moreover, the prepared MS exhibited excellent drug loading capacity, achieving a 947.01 mg/g drug load. Additionally, these microspheres demonstrated a high drug release rate (87.4 %) and significant sustained release properties in vitro. Consequently, WSGC@MS emerge as a promising drug carrier nanoplatform with broad applications in the biomedical field.

3.2. WSGC@MS exhibits high intracellular uptake, potent cytotoxicity, and targeting ability

Fluorescence imaging from live/dead assays was used to evaluate the viability of human gastric adenocarcinoma cells (KATO III and MKN-45) and normal gastric epithelial cells (GES-1) treated with WSGC@MS at concentrations of 0.5, 1.5, and 2 mg/ml for 24 h. A high proportion of live cells was observed in all samples. Fig. S2A shows the quantified percentage of viable cells, providing a comparative study of the biocompatibility of MS at different concentrations. The results, along with MTT assays (Fig. S2A–B), confirm the good biocompatibility of the microspheres.

The unique filamentous cytoskeleton of cells, which participates in various cytoplasmic and nuclear functions, makes the distribution of actin filaments post-treatment significant [55]. It provides direct information about the actin structure and overall cell morphology, reflecting the interactions between the cells and the material. Cytoskeletal images (Fig. S2C) indicate no alterations in the actin microfilaments/nuclear structures or cytoskeletal organization across all samples, comparable to untreated control cells, further validating the microspheres' cellular compatibility.

Subsequently, we assessed the impact of graded concentrations of WSGC@MS on the viability of KATO III and MKN-45 cells over 24, 48, and 72 h. The results demonstrate that the half-maximal inhibitory concentrations (IC50) of WSGC@MS for KATO III cells were 1.5 mg/mL at 24 h, 1.2 mg/mL at 48 h, and 1.0 mg/mL at 72 h. For MKN-45 cells, the IC50 values were 1.4 mg/mL at 24 h, 1.2 mg/mL at 48 h, and 1.0 mg/mL at 72 h, as shown in Fig. 2A.

Fig. 2 — *In vitro* cytotoxicity and cellular uptake tests of WSGC@MS. Note: (A) Effects of gradient concentrations of WSGC@MS on the viability of human gastric adenocarcinoma cells KATO III (left) and MKN-45 (right) after 24 h, 48 h, and 72 h of treatment. (B) Cell viability of human gastric adenocarcinoma cells (KATO III and MKN-45) incubated with concentrations of 0–2 mg/mL MS (left) and WSGC@MS (right) for 24 h, measured using the MTT assay. (C–E) Evaluation of *in vitro* cytotoxicity in human gastric adenocarcinoma cells (KATO III and MKN-45) treated with PBS (Control), WSGC, 1.5 mg/mL WSGC@MS, and 1.5 mg/mL WSGC@MS + MF (0.15T) for 24 h (C), 48 h (D), and 72 h (E). (F) Colony formation assay results show the clonogenic capacity of various groups. (G–H) Fluorescence imaging of human gastric adenocarcinoma cells KATO III (G) and MKN-45 (H) incubated with WSGC@MS, and 1.5 mg/mL WSGC, 1.5 mg/mL WSGC@MS + MF (0.15T) for 1 h; images display WSGC fluorescence (red), nuclei stained with DAPI (blue), and overlaid images (scale bar: 15 μm). (I–J) TEM images of human gastric adenocarcinoma cells KATO III (I) and MKN-45 (J) incubated with WSGC@MS (scale bar: 1 μm ). ∗Significant differences between two groups, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; all cell experiments were repeated three times. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

We utilized the colorimetric CCK-8 assay to quantitatively assess the cytotoxicity of MS and WSGC@MS on human gastric adenocarcinoma cells, KATO III and MKN-45. Cells were incubated with concentrations of 0–2 mg/mL of MS and WSGC@MS for 24 h. As shown in Fig. 2B, after 24 h of incubation with MS, the survival rates of KATO III and MKN-45 cells were over 90 %. In contrast, WSGC@MS exhibited significant cytotoxic effects, particularly at concentrations greater than 1.0 mg/mL, where it inhibited more than 50 % of KATO III and MKN-45 cells. Therefore, while MS alone shows high biocompatibility, WSGC@MS demonstrates potent cytotoxic effects on human gastric adenocarcinoma cells KATO III and MKN-45.

Subsequently, we investigated the cytotoxic effects of 1.5 mg/mL WSGC@MS on KATO III and MKN-45 cells under a 0.15T MF in vitro. After treating the human gastric adenocarcinoma cells for 24, 48, or 72 h, the results indicated that the cytotoxicity of WSGC@MS was enhanced in the presence of a MF, surpassing that of free WSGC. Notably, the application of the MF increased the cytotoxicity of WSGC@MS, likely due to the enhanced contact between WSGC@MS and the gastric adenocarcinoma cells (Fig. 2C–E). These findings are further supported by clonogenic assay results (Fig. 2F).

At the same concentration, cells treated with WSGC@MS displayed brighter red fluorescence than those treated with free WSGC (Fig. 2G and H), suggesting that WSGC@MS enters cells effectively through a receptor-mediated endocytosis mechanism, while free WSGC diffuses passively into cells. Moreover, under the influence of a MF, the fluorescence intensity of WSGC in human gastric adenocarcinoma cells was notably brighter compared to conditions without MF application (Fig. 2G and H). We infer that the MF may enhance the internalization of WSGC by concentrating and directing WSGC@MS toward the cells. Furthermore, in the absence of MF, incubation with WSGC@MS revealed many electron-dense magnetic nanoparticles within the cytoplasm and endosomes/lysosomes of human gastric adenocarcinoma cells through TEM observations (Fig. 2I and J), indicating effective cellular uptake and specific intracellular localization of WSGC@MS. This demonstrates the excellent cellular uptake and stability of MS, enabling effective drug delivery and release, thereby enhancing cytotoxic effects on cancer cells. In summary, WSGC@MS exhibits significant cytotoxic effects on human gastric adenocarcinoma cells, with MF further enhancing its cytotoxicity.

To further evaluate the cellular targeting behavior of WSGC@MS, we assessed GES-1, KATO III, and MKN-45 cells using CLSM. As shown in Fig. S3A, the red fluorescence signals in KATO III and MKN-45 cells were significantly stronger than those in GES-1 cells after 6 h of incubation, attributing this difference to the specific binding of Cy5.5-labeled WSGC@MS with human gastric adenocarcinoma cells.

To further assess the contribution of MF response to the targeted cellular uptake efficiency of WSGC@MS, the microspheres were labeled with Cy5.5. Confocal imaging revealed a significantly stronger red fluorescence signal in cells co-cultured with WSGC@MS under MF conditions compared to those without a MF (Fig. S3B), indicating that MF substantially enhances the efficiency of targeted cellular uptake of WSGC@MS.

Research indicates that lysosomal escape is crucial for maintaining the pharmacological activity of endocytosed nanomedicines, as it prevents drug degradation, ensures effective drug delivery, enhances therapeutic efficacy, reduces toxicity to normal cells, and promotes specific cellular responses, thereby maximizing the therapeutic potential of nanomedicines [56]. To evaluate the lysosomal escape behavior of Cy5.5-labeled WSGC@MS, lysosomes in KATO III and MKN-45 cells were stained with Lysotracker and visualized using CLSM (Fig. S4A–B). Pearson correlation analysis quantified the escape rate; Fig. S4C–D illustrates the changes in the Pearson correlation coefficient between the green signal of Cy5.5 and the red signal of Lysotracker over incubation time. The coefficient after 4 h exceeded that at 2 h, indicating that WSGC@MS was not fully internalized earlier. As the incubation time extended from 4 to 12 h, the coefficient decreased from 0.61 to 0.28, suggesting that WSGC@MS achieves lysosomal escape post-endocytosis and its capability to escape lysosomes increases with prolonged incubation.

Next, we assessed apoptosis in KATO III and MKN-45 cells following various treatments using flow cytometry. The results indicated a certain degree of cell death in both the WSGC and WSGC@MS groups; however, the highest level of cell death was induced in the WSGC@MS + MF group (Fig. S3C and S3E). These findings suggest that the cytotoxicity of WSGC@MS is significantly enhanced under MF response. Additionally, we used the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe to analyze the generation of ROS in different treatment groups. CLSM images revealed significant green fluorescence in the WSGC@MS + MF group, whereas the PBS-treated control group exhibited the lowest fluorescence (Fig. S3D and S3F). These results confirm that WSGC@MS significantly induces ROS generation under MF response, further supported by Prussian blue staining results (Fig. S3G).

In summary, WSGC@MS demonstrates excellent targeting and cytotoxicity under MF response.

3.3. Effective inhibition of human gastric adenocarcinoma cell proliferation by WSGC@MS In vivo

Before conducting in vivo antitumor therapy, we verified the targeting capability of WSGC@MS. Mice were intravenously injected with either PBS or WSGC@MS, with the latter group divided into non-MF and MF conditions, followed by in vivo fluorescence imaging.

Compared to the control group (PBS injection) and the WSGC@MS group (non-MF), the WSGC@MS + MF group exhibited significantly enhanced fluorescence signals at the tumor site from 0 to 24 h post-injection, peaking at 6 h (Fig. 3B, S6A). Twenty-four hours after injection, tumors and major organs were collected for analysis, and notably, the tumor fluorescence in the WSGC@MS + MF group was significantly stronger than that in the WSGC@MS group (Fig. 3C–S6B), indicating that under MF response, WSGC@MS actively targets human gastric adenocarcinoma cells, resulting in enhanced accumulation and prolonged retention at the tumor site.

Subsequently, we established a mouse gastric adenocarcinoma model using human gastric adenocarcinoma cell lines KATO III and MKN-45 (Fig. 3A). Tumor-bearing mice were randomly divided into four groups and treated respectively with PBS, WSGC, WSGC@MS, and WSGC@MS + MF to assess the effects on tumor growth inhibition. The results showed that the WSGC@MS + MF treatment group exhibited the slowest tumor growth rate and the smallest tumor volume and weight (Fig. 3D–E, S6C-D). Further histological examination of the excised tumors with H&E, Ki-67 antibody, and TUNEL staining revealed that, compared to other groups, the WSGC@MS + MF group displayed the most significant apoptosis and the greatest reduction in proliferation rates (Fig. 3F–S6E).

Immunohistochemical analysis of tumor tissues in a mouse model revealed that the expression levels of Bax and Cleaved Caspase-3 were highest in the WSGC@MS treatment group with MF application, while the expression of Bcl-2, Ki67, and PCNA proteins was the lowest. Compared to the non-MF response group, the changes in WSGC@MS with MF response were the most pronounced (Fig. S6F).

Regarding the toxicity of the nanoparticles, routine blood and biochemical tests conducted after intravenous injection of WSGC@MS showed no significant abnormal findings compared to the control group (Fig. S5A), indicating good hemocompatibility of the nanoparticles. The major organs of the mice, including the heart, liver, spleen, lungs, and kidneys, were subjected to H&E staining (Fig. S5B). The results indicated that the impact of WSGC@MS on tissue structure and integrity was negligible compared to the control, demonstrating the nanoparticles' good biocompatibility in vivo applications.

3.4. Exploring the downstream molecular mechanisms of WSGC@MS therapy for gastric adenocarcinoma

To further investigate the potential mechanisms through which WSGC@MS inhibits human gastric adenocarcinoma, we performed RNA sequencing on KATO III cells treated with WSGC@MS. As illustrated in the volcano plot, 263 genes were upregulated (red) and 128 genes downregulated (blue) in the WSGC@MS group, indicating significant changes in gene expression triggered by the treatment. Notably, the expression of FAM117B was significantly reduced in the WSGC@MS treatment group (Fig. 4A). In gastric cancer research, FAM117B has been found to promote gastric cancer cells' growth and drug resistance by targeting the KEAP1/NRF2 signaling pathway [57]. We hypothesize that WSGC@MS may inhibit gastric adenocarcinoma by modulating the expression of FAM117B.

Fig. 4 — Transcriptomic analysis of gastric adenocarcinoma treated with WSGC@MS. Note: (A) DEGs analysis showing gene expression changes in the Treat group compared to the Control group. Red dots represent upregulated genes, and green dots represent downregulated genes (Control = 3, WSGC@MS = 3). (B) Expression level differences of FAM17B between tumor and normal samples across 33 types of tumors based on TCGA RNAseq data. Blue box plots represent normal tissues, and red box plots represent tumor tissues. (C) ROC curve assessing the accuracy of FAM117B as a prognostic marker. (D–E) Functional enrichment analysis showing significantly enriched GO and KEGG pathways among DEGs. Important enriched pathways include autophagy regulation, oxidative stress response, TOR complex, and AMPK signaling pathway. Blue indicates biological processes (BP), red indicates cellular components (CC), purple indicates molecular functions, and yellow indicates KEGG pathways. ∗Indicates p < 0.05, ∗∗indicates p < 0.01, ∗∗∗indicates p < 0.001 between the two groups. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

The pan-cancer analysis further demonstrated high expression of FAM117B across multiple cancers, particularly in gastric adenocarcinoma, indicating that high expression of FAM117B is associated with poorer prognosis (Fig. 4B). The AUC of FAM117B is 0.860, indicating high predictive accuracy (Fig. 4C).

Subsequent GO and KEGG pathway analysis indicated significant enrichment of genes in the WSGC@MS group within pathways related to autophagy regulation, oxidative stress response, the TOR complex, and the AMPK signaling pathway (Fig. 4D and E). These results suggest that WSGC@MS may exert its inhibitory effects on the development of gastric adenocarcinoma by regulating processes such as autophagy and oxidative stress.

Due to the complex molecular mechanisms involved, gastric adenocarcinoma often manifests as malignant proliferation and chemoresistance, leading to poor prognosis for patients [58]. Previous research has shown that FAM117B can inhibit the growth and chemoresistance of human gastric cancer cells HGC-27, KATO III, and SNU-668 [57].

Transcriptomic analysis from this study revealed that the expression of FAM117B in human gastric adenocarcinoma cells treated with WSGC@MS was significantly lower than in untreated cells. Consequently, we investigated whether WSGC@MS exerts its effects by modulating FAM117B in chemoresistant gastric adenocarcinoma cells. We initially overexpressed FAM117B in KATO III and MKN-45 cells using a FAM117B overexpression plasmid, demonstrating high overexpression efficiency (Fig. S7A).

Subsequently, we examined the role of WSGC@MS and FAM117B in regulating the proliferation and chemoresistance of gastric adenocarcinoma cells. Results showed that compared to the control group, both the growth and colony formation abilities of cells treated with WSGC@MS and 5-Fu were significantly reduced, with the combined WSGC@MS+5-Fu treatment group showing the most notable changes, indicating a synergistic effect in inhibiting tumor cell growth and enhancing chemosensitivity. Furthermore, WSGC@MS treatment significantly reduced the resistance of gastric adenocarcinoma cells to 5-Fu, further supporting its potential as an anticancer therapy. Restoring FAM117B expression mitigated the growth inhibition and chemosensitization effects of WSGC@MS treatment (Fig. S7B–E).

To investigate the impact of WSGC@MS treatment on the resistance of gastric adenocarcinoma cells to 5-Fu and its synergy with oxaliplatin (Oxa), we developed cell lines resistant to both drugs and compared them to their respective parent cell lines. We then determined the dose-response curves using cell viability assays (Figs. S8A and S9A). The extent of induced resistance was estimated by comparing the EC50 values of the resistant cell lines to those of their parent lines. The results indicated that the 5-Fu and Oxa-resistant cell lines exhibited significant tolerance to higher concentrations of these drugs than their parent counterparts.

Studies have identified ABCC2, ATP7A, and CTR1 as key molecular markers associated with resistance to cisplatin (CDDP). Expression levels of ABCC2 and ATP7A were significantly higher in the 5-Fu and Oxa-resistant cell lines (KATO III/MKN-45 R-5-Fu; KATO III/MKN-45 R-Oxa) than in the wild-type cells (KATO III/MKN-45 WT), while CTR1 expression was significantly reduced in the resistant strains [59] (Figs. S8B and S9B).

We analyzed cell death using Annexin V and PI staining through flow cytometry to investigate the response to 5-Fu and Oxa-induced cell death in parental and resistant gastric adenocarcinoma cell lines. The results indicated that the death rate in resistant cells was significantly lower than in the parental cell lines. When comparing the overall percentage of cell death (Annexin V positive/PI negative (Q4), Annexin V/PI double-positive cells (Q2), and Annexin V negative/PI positive (Q1)), the percentage of cell death in resistant cells was significantly reduced compared to the parental cells (p < 0.001; Fig. S8C and S8H; Fig. S9C and S9H). Based on the representative flow cytometry plots depicting cell death, compared to drug-resistant cells (Fig. S8D and Fig. S8I; Fig. S9D and Fig. S9I), WT cells exhibited a significant increase in cell death and a decrease in the population of viable cells (Fig. S8E and Fig. S8J; Fig. S9E and Fig. S9J). Representative dot plots of WT and resistant group cells are displayed in Fig. S8F and Fig. S8K, as well as Fig. S9F and Fig. S9K. Fig. S8G and S8L, along with Fig. S9G and S9L, indicate that under conditions without 5-Fu/Oxa exposure, most cells remained viable, demonstrating high drug resistance and low rates of apoptosis or necrosis. In conclusion, we have successfully established drug-resistant gastric adenocarcinoma cell lines KATO III/MKN-45 R-5-Fu and KATO III/MKN-45 R-Oxa for subsequent experiments.

We overexpressed FAM117B in resistant KATO III and MKN-45 cells using a FAM117B overexpression plasmid, demonstrating high overexpression efficiency (Fig. S8M; S9M). Compared to the control group, the growth and colony formation capabilities of the resistant cells treated with WSGC@MS and chemotherapy drugs (5-Fu/Oxa) were significantly reduced. Notably, the combination treatment group of WSGC@MS+5-Fu/Oxa showed the most significant changes, indicating a synergistic effect in inhibiting tumor cell growth and reducing drug resistance (Fig. S8N–O; S9N-O).

These data suggest that WSGC@MS inhibits the growth of gastric adenocarcinoma cells and enhances chemosensitivity by downregulating FAM117B.

3.5. WSGC@MS inhibits the KEAP1/NRF2 signaling pathway by reducing NRF2 ubiquitin-proteasome degradation in gastric adenocarcinoma cells

Previous research has shown that FAM117B activates the KEAP1/NRF2 signaling pathway in gastric adenocarcinoma cells, promoting chemoresistance [57]. We investigated whether WSGC@MS also counteracts chemoresistance in gastric adenocarcinoma cells through the KEAP1/NRF2 pathway.

Results from Fig. 5A and B and Fig. S10A–D show that NRF2 protein levels were downregulated in the WSGC@MS treatment group while restoring FAM117B expression reversed the suppression of NRF2 in gastric cancer cells. KEAP1 protein levels remained consistent across different treatment groups. Nuclear-cytoplasmic fractionation experiments revealed that WSGC@MS reduced NRF2 protein levels in both cytoplasm and nucleus and restoring FAM117B expression could upregulate NRF2 protein levels (Fig. 5C and D).

Additionally, in the WSGC@MS and WSGC@MS + FAM117B overexpression groups in KATO III R-5-Fu cells, the mRNA levels of NRF2 target genes were downregulated and upregulated (Fig. 5E). These data suggest that WSGC@MS suppresses the KEAP1/NRF2 signaling pathway in resistant gastric cancer cells by downregulating the FAM117B gene.

Further, we explored how WSGC@MS regulates NRF2 protein levels in gastric cancer cells. Quantitative PCR (qPCR) measured NRF2 mRNA levels in KATO III/MKN-45 R-5-Fu cells. As shown in Fig. 6A, changes due to WSGC@MS treatment and FAM117B protein overexpression did not affect NRF2 mRNA expression, indicating that WSGC@MS and FAM117B do not alter NRF2 protein levels through transcriptional regulation. Thus, we hypothesize that WSGC@MS influences NRF2 protein levels through the ubiquitination degradation pathway.

Fig. 6B shows that MG132, a proteasome inhibitor, prevents WSGC@MS from reducing NRF2 protein levels in cells. Cycloheximide chase experiments indicate that WSGC@MS or overexpression of FAM117B respectively shortens or extends the half-life of NRF2 protein degradation (Fig. 6C and D). We also examined the effects of FAM117B knockdown on NRF2 ubiquitination in KATO III/MKN-45 R-5-Fu cells; as shown in Fig. 6E and F, knocking down FAM117B significantly increased the ubiquitination levels of NRF2, while overexpression of FAM117B reversed this effect.

Research suggests that KEAP1 regulates NRF2 ubiquitin-mediated degradation. Therefore, we further investigated whether the upregulation of NRF2 induced by FAM117B in resistant gastric adenocarcinoma cells depends on KEAP1 [57]. Subsequently, we explored the impact of WSGC@MS on the interaction between NRF2 and KEAP1. As depicted in Fig. S11A–B, the WSGC@MS treatment group lost the ability to downregulate NRF2 protein levels in cells with silenced KEAP1, indicating that WSGC@MS regulates NRF2 in a KEAP1-dependent manner. Immunofluorescence results show FAM117B and KEAP1 co-localization in the cytoplasm of KATO III and MKN-45 R-5-Fu cells (Fig. S11C). Co-immunoprecipitation experiments reveal that endogenous FAM117B can interact with endogenous KEAP1 (Fig. S11D–E).

Furthermore, in HEK293T cells, HA-tagged FAM117B interacts with flag-tagged KEAP1 (Fig. S11F). We conducted GST pull-down assays to prove that FAM117B directly binds to KEAP1. As illustrated in Fig. S11G, GST-KEAP1 protein could pull down purified FAM117B protein but not GST protein, confirming that FAM117B directly binds to KEAP1. Subsequently, we studied whether FAM117B could interfere with the interaction between NRF2 and KEAP1 in KATO III/MKN-45 R-5-Fu cells. Fig. S11H–I shows that knockdown or overexpression of FAM117B respectively promoted or inhibited the binding between KEAP1 and NRF2. These results indicate that in resistant gastric cancer cells, FAM117B competes with NRF2 for binding to KEAP1.

These findings demonstrate that WSGC@MS inhibits the KEAP1/NRF2 signaling pathway by modulating FAM117B expression, thereby reducing the protein stability of NRF2.

3.6. WSGC@MS inhibits proliferation of gastric adenocarcinoma cells by suppressing NRF2 levels

We further developed a gastric cancer xenograft model in nude mice to investigate whether NRF2 is a critical downstream substrate in inhibiting human gastric adenocarcinoma progression by WSGC@MS (Fig. 7A). We found that overexpression of NRF2 in KATO III/MKN-45 R-5-Fu or KATO III/MKN-45 R-Oxa cells counteracted the reduction in malignant proliferation observed in the WSGC@MS treatment group (Fig. 7B and D; Fig. S12A and S12C). Results from Ki67 and Cleaved caspase-3 staining supported these findings (Fig. 7C and E; Fig. S12B and S12D). In a liver metastasis model, overexpression of NRF2 significantly negated the inhibitory effects of the WSGC@MS treatment group on liver metastases (Fig. 7F and G; Fig. S12E–F).

Fig. 7 — WSGC@MS inhibits the proliferation of gastric adenocarcinoma cells *in vivo* through ubiquitination-mediated degradation of NRF2. Note: (A) Schematic of the animal experiment protocol. (B) Representative images of tumors from nude mice carrying KATO III R-5-Fu cells in the designated groups, with quantification of tumor volume and weight. (C) IHC staining of Ki67 and Cleaved caspase-3 in tumor sections from each group (scale bar = 100 μm). (D) Representative images of tumors from nude mice carrying MKN-45 R-5-Fu cells in the designated groups, quantifying tumor volume and weight. (E) IHC staining of Ki67 and Caspase-3 in tumor sections from each group (scale bar = 100 μm). (F–G) H&E staining and quantification of liver metastatic nodules in the indicated groups (scale bar = 100 μm). Black arrows indicate the location of tumor metastasis in the liver. ∗Indicates p < 0.05, ∗∗Indicates p < 0.01, ∗∗∗Indicates p < 0.001 between the two groups; n = 6 animals per group.

These findings demonstrate that WSGC@MS inhibits the proliferation of gastric adenocarcinoma cells by suppressing NRF2 levels.

3.7. WSGC@MS inhibits gastric adenocarcinoma progression and the Warburg effect through ROS/AMPK/mTOR-mediated autophagy activation

To further explore the mechanisms by which WSGC@MS inhibits the progression of gastric adenocarcinoma, we conducted RNA-Seq transcriptome analysis on KATO III R-5-Fu cells treated with WSGC@MS. As shown in Fig. 8A, gene set enrichment analysis (GSEA) revealed significant associations between WSGC@MS treatment and pathways, including KEAP1/NRF2 signaling, autophagy, and nanoparticle-triggered autophagic cell death. Research indicates that autophagy and the NRF2 signaling pathway are interregulated [60]. p62/SQSTM1, an autophagy receptor, can directly bind to KEAP1, thereby competitively inhibiting the interaction between NRF2 and KEAP1 and protecting NRF2 from KEAP1-Cul3-RBX1 mediated degradation, suggesting that WSGC@MS may regulate both the KEAP1/NRF2 pathway and autophagy [61].

Furthermore, NRF2 is widely recognized for regulating redox biology and suppressing ROS production [62]. Studies suggest that modulating ROS levels in the tumor microenvironment could be a potential therapeutic approach, as low ROS levels can promote the malignant proliferation of tumors [63]. Given this, we hypothesize that WSGC@MS may induce ROS accumulation, inhibiting gastric adenocarcinoma progression by reducing NRF2 stability. Initially, the mitochondrial potential was assessed using JC-1 staining. The staining results showed that WSGC@MS treatment significantly reduced mitochondrial potential in KATO III and MKN-45 R-5-Fu cells (Fig. 8B and C).

Flow cytometry analysis revealed that WSGC@MS treatment elevated intracellular ROS levels in KATO III and MKN-45 R-5-Fu cells (Fig. 8D and E). Additionally, WSGC@MS treatment significantly reduced the GSH to GSSG ratio and markedly decreased GPX activity (Fig. 8F and G). The AMPK signaling pathway is highly sensitive to oxidative stress and can be activated by ROS [64]. AMPK, a metabolic sensor that maintains cellular energy homeostasis, is considered a key regulator of the Warburg effect, where cancer cells predominantly produce energy through aerobic glycolysis rather than oxidative phosphorylation, aiding their survival and proliferation [64,65]. Our studies show that WSGC@MS significantly reduced the glycolytic rate and increased mitochondrial respiration in KATO III and MKN-45 R-5-Fu cells (Fig. 8H), indicating that WSGC@MS inhibits the Warburg effect in gastric adenocarcinoma cells.

The activation of AMPK can inhibit mTORC1, thereby activating autophagy. GSEA and KEGG analysis also suggest significant correlations between WSGC@MS and autophagy (Fig. 4D and E; Fig. 8A). Western Blot analysis confirmed that compared to the control group, the WSGC@MS group showed a significant increase in phosphorylated AMPK levels and a significant decrease in phosphorylated mTOR levels, along with an increased LC3 II/I ratio and a marked reduction in p62 protein expression. These results suggest that WSGC@MS promotes autophagy by activating the AMPK and inhibiting the mTOR signaling pathways (Fig. 8I).

We studied KATO III, and MKN-45 R-5-Fu cells transfected with GFP-mRFP-LC3 to assess autophagy flux. The results indicated that WSGC@MS treatment significantly increased the accumulation of GFP-mRFP-LC3 puncta; however, overexpression of NRF2 reduced the number of puncta (Fig. S13A; S13C). As confirmed by TEM, autophagosome formation corroborated these findings (Fig. S13B; S13D).

Furthermore, TEM images (Fig. S13E) showed that the number of autophagosomes in KATO III and MKN-45 R-5-Fu cells treated with WSGC@MS was significantly elevated, while overexpression of NRF2 significantly decreased the number of autophagosomes. Immunofluorescence was used to directly assess LC3 expression. Further analysis revealed that WSGC@MS treatment significantly increased LC3 expression in KATO III and MKN-45 R-5-Fu cells, whereas overexpression of NRF2 significantly reduced the percentage of LC3-positive cells (Fig. S13F). Fig. S13G shows WSGC@MS significantly elevated ROS levels in KATO III and MKN-45 R-5-Fu cells. Interestingly, overexpression of NRF2 effectively attenuated ROS production.

In summary, WSGC@MS promotes ROS/AMPK/mTOR-dependent autophagy in KATO III/MKN-45 R-5-Fu cells by activating the NRF2 ubiquitination degradation pathway.

4. Discussion

The high incidence and mortality rates of gastric adenocarcinoma pose a significant challenge to global public health. Traditional treatment methods, such as surgery, chemotherapy, and radiotherapy, have made some progress but still fail to substantially improve the long-term survival rates of patients with gastric adenocarcinoma [6]. Thus, developing new therapeutic strategies that can precisely target tumor cells while minimizing damage to normal cells is crucial. In this study, we explored the potential of WSGC@MS in treating gastric adenocarcinoma, particularly through mechanisms that inhibit tumor proliferation via the KEAP1/NRF2 signaling pathway and autophagy.

Our research indicates that WSGC@MS not only effectively loads and releases WSGC peptides but also demonstrates significant anti-tumor effects both in vitro and in vivo. This efficacy is partly due to the efficient uptake of WSGC@MS by gastric adenocarcinoma cells and enhanced drug accumulation and ROS generation mediated by an external magnetic field. Particularly in vitro, WSGC@MS significantly reduced the viability of gastric adenocarcinoma cells by promoting autophagy and inhibiting the NRF2 signaling pathway, thereby enhancing chemosensitivity. These results not only confirm the efficiency of WSGC@MS as a drug carrier but also highlight its potential application in the treatment of gastric adenocarcinoma.

Further mechanistic studies revealed that WSGC@MS plays a crucial role in suppressing the KEAP1/NRF2 signaling pathway by downregulating FAM117B gene expression. This mechanism is vital in inhibiting the proliferation and drug resistance of gastric adenocarcinoma cells. Specifically, by downregulating NRF2 protein levels, WSGC@MS significantly impacts the ROS/AMPK/mTOR signaling pathway, leading to the activation of autophagy. These findings provide new insights into the anti-tumor mechanisms of WSGC@MS in gastric adenocarcinoma cells, demonstrating that by regulating FAM117B and NRF2, WSGC@MS can effectively inhibit tumor progression and enhance therapeutic efficacy. In vivo experiments further support these findings, showing that the significant inhibitory effect of WSGC@MS on tumor growth is notably reversed after overexpression of NRF2, indicating that NRF2 plays a key role in the anti-tumor effects mediated by WSGC@MS and confirming the potential of targeting NRF2 and its downstream signaling pathways to suppress tumors. FAM117B is a highly conserved protein composed of 589 amino acids in animals. Previous studies have linked FAM117B to the development of lacunar stroke and sarcoidosis through sequencing analysis [66,67]. Zhou et al. demonstrated that FAM117B can promote gastric cancer growth and drug resistance by reducing the ubiquitination-mediated degradation of NRF2 [57]. However, research on FAM117B remains limited, and the upstream regulatory signals affecting FAM117B expression are still unclear. In our study, we confirmed that WSGC@MS significantly downregulated the expression of the FAM117B gene, although the direct mechanism by which WSGC@MS targets FAM117B has not yet been elucidated. We will focus on this aspect in future studies.

Compared to other nanomedicine delivery systems, WSGC@MS stands out not only for its efficient drug-loading performance and magnetic responsiveness but also for its ability to inhibit tumor growth through multiple mechanisms. Specifically, WSGC@MS exhibits extensive anti-tumor effects by activating mitochondrial autophagy, reducing NRF2 stability, and inhibiting the Warburg effect among other pathways. This integration of multiple mechanisms not only enhances therapeutic effectiveness but may also reduce the likelihood of cancer recurrence by eliminating supportive factors within the tumor microenvironment, thereby offering a more comprehensive and effective treatment option for patients with gastric adenocarcinoma.

Although this study demonstrates the significant potential of WSGC@MS in the treatment of gastric adenocarcinoma, several limitations remain. Firstly, despite showing excellent targeting ability and marked antitumor effects in both in vitro and in vivo models, the long-term biodistribution, metabolic pathways, and potential chronic toxicity of WSGC@MS have not been thoroughly evaluated. These factors may impact its clinical translation and warrant systematic validation through long-term toxicological studies. Secondly, while this study focused on gastric adenocarcinoma, the applicability and therapeutic efficacy of WSGC@MS in other types of solid tumors remain to be investigated. Moreover, a dose-gradient experiment was not conducted, leaving the dose–response relationship, minimal effective concentration, and safety window of WSGC@MS undefined. These aspects are essential for future dose optimization and translational development. Thirdly, although transcriptomic analysis confirmed that WSGC@MS significantly downregulates FAM117B, thereby destabilizing NRF2 protein and inducing autophagy, the direct targeting mechanism of WSGC@MS on FAM117B remains unclear. Whether this modulation is dependent on upstream signaling pathways also needs further investigation. Future studies should elucidate the detailed molecular mechanisms underlying the WSGC@MS–FAM117B interaction.

In summary, this study successfully developed a novel type of porous MS, WSGC@MS, and demonstrated its effectiveness and potential mechanisms in treating gastric adenocarcinoma. WSGC@MS significantly inhibits the proliferation and drug resistance of gastric adenocarcinoma cells by regulating the FAM117B/NRF2 signaling pathway. Future research should further explore the long-term biocompatibility and safety of WSGC@MS and assess its potential application in other cancer types to facilitate its clinical translation.

CRediT authorship contribution statement

Dongjian Song: Writing – review & editing, Validation, Supervision, Software, Project administration, Methodology, Funding acquisition, Conceptualization. Qiuliang Liu: Writing – original draft, Visualization, Software, Resources, Investigation, Formal analysis, Data curation. Zechen Yan: Writing – original draft, Validation, Software, Investigation, Formal analysis, Conceptualization. Qi Wang: Writing – original draft, Validation, Methodology, Formal analysis. Meng Su: Writing – original draft, Validation, Resources, Methodology, Investigation, Formal analysis, Data curation. Hui Zhang: Writing – review & editing, Validation, Software, Resources, Investigation, Formal analysis, Data curation. Longyan Shi: Writing – review & editing, Visualization, Validation, Methodology, Formal analysis, Data curation. Yingzhong Fan: Writing – review & editing, Visualization, Validation, Software, Methodology, Investigation, Formal analysis, Data curation. Qian Zhang: Writing – review & editing, Validation, Software, Resources, Methodology, Formal analysis, Data curation. Heying Yang: Writing – review & editing, Visualization, Supervision, Methodology, Investigation, Formal analysis, Data curation. Da Zhang: Writing – review & editing, Software, Methodology, Investigation, Formal analysis, Data curation.

Ethics approval and consent to participate

All animal experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Approval no. 2019-KY-200).

Consent for publication

Not applicable.

Availability of data and materials

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

This study was supported by National Natural Science Foundation of China (81902471), Henan Province Young and Middle-aged Health Science and Technology Innovation Talents Outstanding Youth Project (YXKC2021060), Key Scientific Research Projects of Higher Education Institutions in Henan Province (20B320023), Joint Co-construction Project of Henan Provincial Medical Science and Technology Research Plan (LHGJ20200323), Provincial and Ministry Co-construction Project of Henan Provincial Medical Science and Technology Research Plan (SB201904003), Young and Middle-Aged Discipline Leaders of Henan Health Commission(HNSWJW-2020021).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

Not applicable.

Footnotes

^{Appendix A}

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtbio.2025.101995.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1

mmc1.docx^{(3.6MB, docx)}

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia component 1

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Data Availability Statement

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Data will be made available on request.

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PERMALINK

Inhibition of gastric adenocarcinoma proliferation by WSGC@MS: Role of KEAP1/NRF2 signaling pathway and autophagy regulation

Dongjian Song

Qiuliang Liu

Zechen Yan

Qi Wang

Meng Su

Hui Zhang

Longyan Shi

Yingzhong Fan

Qian Zhang

Heying Yang

Da Zhang

Abstract

Graphical abstract

1. Introduction

2. Materials and methods

2.1. Materials and reagents

2.2. Porous magnetic chitosan microspheres loaded with WSGC peptide

2.2.1. Synthesis and source of WSGC peptide

2.2.2. Synthesis of Fe3O4 magnetic nanoparticles

2.2.3. Synthesis of porous MS

2.2.4. Characterization of porous MS

2.2.5. Protein loading and In vitro release experiments

2.3. Cell-related experiments

2.3.1. Cell culture

2.3.2. Lentivirus construction

2.3.3. Construction of drug-resistant gastric adenocarcinoma cell lines

2.3.4. Labeling and monitoring the uptake of Nanocomplexes

2.3.5. Lysosomal escape

2.3.6. Cell viability Assessment

2.3.7. Plate cloning experiment

2.3.8. Reactive oxygen species (ROS) detection

2.3.9. Live/dead cell Identification

2.3.10. Detection of actin filament distribution

2.3.11. Flow cytometric analysis of cell apoptosis

2.3.12. Nucleocytoplasmic fractionation experiment

2.3.13. RT-qPCR analysis of target gene expression

2.3.14. Western Blot

2.3.15. Prussian blue staining

2.3.16. CHX-chase analysis

2.3.17. Immunoprecipitation assay

2.3.18. GST pull-down assay

2.4. Bioinformatics-related experiments

2.4.1. High-throughput transcriptome sequencing, data quality control, and differential gene analysis

2.4.2. Pan-cancer analysis

2.4.3. Receiver operating characteristic (ROC) analysis

2.5. Mouse-related experiments

2.5.1. Construction of mouse model

2.5.2. Systemic toxicity Assessment

2.5.3. In vivo targeting studies

2.5.4. Immunohistochemical staining

2.5.5. H&E staining

2.5.6. Immunofluorescence and Terminal Deoxynucleotidyl Transferase dUTP Nick end labeling (TUNEL) assay

2.5.7. Autophagosome detection

2.5.8. Measurement of oxidative phosphorylation and glycolysis

2.5.9. Evaluation of mitochondrial membrane potential

2.5.10. Detection of GSH/GSSG ratio and GPX activity

2.5.11. In vivo ubiquitination analysis

2.6. Statistical analysis

3. Results

3.1. Preparation of porous MS

Fig. 1.

3.2. WSGC@MS exhibits high intracellular uptake, potent cytotoxicity, and targeting ability

Fig. 2.

3.3. Effective inhibition of human gastric adenocarcinoma cell proliferation by WSGC@MS In vivo

Fig. 3.

3.4. Exploring the downstream molecular mechanisms of WSGC@MS therapy for gastric adenocarcinoma

Fig. 4.

3.5. WSGC@MS inhibits the KEAP1/NRF2 signaling pathway by reducing NRF2 ubiquitin-proteasome degradation in gastric adenocarcinoma cells

Fig. 5.

Fig. 6.

3.6. WSGC@MS inhibits proliferation of gastric adenocarcinoma cells by suppressing NRF2 levels

Fig. 7.

3.7. WSGC@MS inhibits gastric adenocarcinoma progression and the Warburg effect through ROS/AMPK/mTOR-mediated autophagy activation

Fig. 8.

4. Discussion

CRediT authorship contribution statement

Ethics approval and consent to participate

Consent for publication

2.2.2. Synthesis of Fe₃O₄ magnetic nanoparticles