The reagents used are as follows: IVM (purity ≥98%) purchased from MilliporeSigma; MET (purity ≥98%), obtained from Macklin Inc., 3-Methyladenine (3-MA; purity ≥98%; MilliporeSigma); N-acetylcysteine (NAC; Macklin Inc.), matrix adhesive (Hangzhou Lianke Meixun Biomedical Technology Co., Ltd.) and crystal violet (Biosharp Life Sciences). p62/SQSTM Rabbit Ab (cat. no. 118420-1-AP; Proteintech Group Inc.); Phospho-AKT (cat. no. WLP001; Wanleibio Co., Ltd.); THBS1 (cat. no. A75291; Nature Biosciences LtD.); β-actin (cat. no. AC038; ABclonal Biotech Co., Ltd.); LC3B (cat. no. A19665; ABclonal Biotech Co., Ltd.); Beclin1 (cat. no. A21191; ABclonal Biotech Co., Ltd.); Bcl-2 (cat. no. A19693; ABclonal Biotech Co., Ltd.); PI3 Kinase p85 (cat. no. A4992; ABclonal Biotech Co., Ltd.); Phospho-PI3KP85α (cat. no. AP0854; ABclonal Biotech Co., Ltd.); AKT (cat. no. A20779; ABclonal Biotech Co., Ltd.); mTOR (cat. no. A11345; ABclonal Biotech Co., Ltd.); Phospho-mTOR (cat. no. AP0115; ABclonal Biotech Co., Ltd.). For Western blotting and immunofluorescence, HRP-conjugated Goat Rabbit IgG (H+L) (cat. no. AS014; ABclonal Biotech Co., Ltd.) and Alexa Flour 594-Goat Anti-Rabbit IgG (AD9279; cat. no. ABbox) were used as secondary antibodies. All antibodies were stored at −20°C in accordance with the conditions recommended in the instructions.

The MCF-7 human breast cancer cell line used in the present study was kindly provided by Professor Dong Zhiqiang's laboratory at Huazhong Agricultural University, Wuhan, China. The cells were cultured in high-glucose DMEM medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (BIOVISTECH PTY. LTD) and 1% penicillin-streptomycin dual antibiotic solution (MilliporeSigma) and maintained in a constant-temperature incubator at 37°C, 5% CO₂ and 95% humidity. When the cell confluence reached 80–90%, they were passaged using 0.25% trypsin (EDTA-containing) for 1 min at 37°C, then divided into culture flasks at a 1:3 ratio. All experiments strictly limited cell passage to ≤15 generations, with regular mycoplasma detection and morphological observation to ensure cell viability.

MCF-7 cells in the logarithmic growth phase were prepared as single-cell suspensions (1×10⁴ cells/ml) and inoculated into 96-well culture plates (100 µl per well), with three replicates per group. After 12 h of cell adhesion (37°C, 5% CO₂) to achieve 50% confluency, the following treatments were administered: Blank control (with PBS only), IVM monotherapy (5–45 µmol/l), MET monotherapy (5–50 mmol/l) and combined treatment (IVM + MET; aforementioned concentrations). Treatment durations were set at 12, 24 and 48 h at 37°C in a 5% CO₂ incubator. For mechanistic studies, cells were pre-treated with 3-MA (5 mM; 2 h) or NAC (10 mM; 2 h) at 37°C in a 5% CO₂ incubator before receiving the final drug at 24 h. After culture termination, 100 µl working solution was added per well according to the Cell Counting Kit-8 (CCK-8; Wuhan Huiyucheng Biotechnology Co., Ltd.) protocol. The mixture was incubated at 37°C for 30 min, followed by absorbance measurement using a Bio-Rad Laboratories, Inc. microplate reader at a wavelength of 450 nm (OD value) to assess cell viability. Three independent replicates were performed throughout the experiment to ensure data reliability.

The present study employed the Transwell invasion assay to evaluate the combined effects of IVM and MET on the invasion of MCF-7 cells. A 100 µl Matrigel matrix gel (Xi'an Zhongtuan Biotechnology Co., Ltd.) was uniformly coated on the upper chamber of Transwell chambers (8 µM pore size; Corning, Inc.). After 4 h of polymerization at 37°C, 100 µl of cell suspension (in serum-free DMEM) containing different treatment groups (density 5×10⁴ cells/ml) was inoculated into the upper chamber. By contrast, 800 µl DMEM medium with 10% fetal bovine serum served as a chemotactic inducer in the lower chamber. The cultures were incubated at 37°C with 5% CO₂ for 24 h. Following medium removal, cells were washed with PBS, fixed with 100% methanol for 30 min at 37°C, stained with 0.1% crystal violet for 30 min at 37°C and membrane-penetrating cell counts were quantified by randomly selecting 5 fields under an inverted Olympus microscope (Olympus Corporation).

The present study evaluated the combined effects of IVM and MET on MCF-7 cell migration using a scratch assay. The protocol involved seeding MCF-7 cells at 2×10⁵ cells per well in 6-well plates in DMEM supplemented with 10% fetal bovine serum, incubating them at 37°C with 5% CO₂ for 80% fusion. A 200 µl sterile pipette was used to create vertical scratch lines perpendicular to the plate surface, followed by gentle washing with PBS to remove detached cells. Four experimental groups were established: Serum-free DMEM control, 6 µM IVM monotherapy, 6 mM MET monotherapy and IVM (6 µM) + MET (6 mM) combined intervention. All groups were cultured under identical conditions for 24 h, with the progress of scratch closure monitored every 8 h using an inverted microscope (100×; Olympus CKX41; Olympus Corporation). Migration area changes were quantified using ImageJ software (version 1.53e; National Institutes of Health) to evaluate treatment effects.

The ROS levels in MCF-7 cells were detected using the 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence probe method. The experimental protocol was as follows: MCF-7 cells were seeded at 2×10⁵ cells per well and cultured in 6-well plates until 70% confluency, then subjected to the following treatments: i) Different drug groups (PBS, IVM, MET and combination therapy) for 12 or 24 h at 37°C in a 5% CO₂ incubator; ii) drug administration after 2 h pretreatment with 10 mM NAC; iii) positive control [Ros up (a compound mixture that induces ROS production); 50 µg/ml stimulation for 20 min at 37°C; Beyotime Biotechnology]. Cells were collected and adjusted to a density of 1×10⁹ cells/ml. Then, they were incubated with a 10 µM DCFH-DA probe (Wuhan Huiyucheng Biotechnology Co., Ltd.) at 37°C in the dark for 30 min. After washing 3× with PBS and resuspension, the fluorescence intensity was measured using a flow cytometer (CytoFLEX; Beckman Coulter) with excitation/emission wavelengths of 488 nm and 530 nm, respectively. Each group had 3 duplicate wells, and the experiment was independently repeated 3 times. Data were expressed as mean fluorescence intensity ± SD.

The present study employed a transmission electron microscope (accelerated voltage 100 kV; Hitachi H7650; Hitachi, Ltd.) to investigate the effects of IVM, MET and their combination on the ultrastructure of MCF-7 cells. Cells were inoculated into 6-well plates (1×10⁵ cells per well). After achieving 60–70% fusion, they were treated with the respective drugs (PBS, IVM, MET or IVM + MET) for 24 h at 37°C in a 5% CO₂ incubator. After fixation with 2.5% glutaraldehyde for 2 h at 4°C, the samples underwent an ethanol gradient dehydration process (50, 70, 90 and 100%) followed by epoxy resin embedding and polymerization at 60°C for 48 h and ultra-thin sectioning (80 nm, using a Leica UC6 microtome; Leica Microsystems). Uranium acetate staining for 30 min at room temperature in the dark was applied to enhance contrast. A total of 10 random fields from each group were systematically observed, with a particular focus on analyzing autophagosome formation and morphological changes in organelles, such as mitochondria and the endoplasmic reticulum.

The present study systematically evaluated the effects of various treatment protocols on protein expression profiles in MCF-7 cells using western blot analysis. The experimental protocol was as follows: MCF-7 cells were cultured in 6-well plates until 80% confluency, after which the following groups were established: i) PBS control group, IVM single drug group (6 µM), MET single drug group (6 mM) and combined treatment group, all treated for 24 h at 37°C in a 5% CO₂ incubator; ii) Autophagy inhibitor pretreatment group (5mM 3-MA administered 2 h prior to treatment at 37°C in a 5% CO₂ incubator). Experimental procedures included collecting cells, washing them 3 times with pre-chilled PBS, adding RIPA lysis buffer (containing protease inhibitors; cat. no. P0013B; Beyotime Biotechnology) for lysing on ice for 30 min. Protein concentrations were measured using the BCA Protein Quantification Kit (Thermo Fisher Scientific Inc.) and adjusted to a uniform concentration of 15 µg/well. Protein samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane using the gel-membrane sandwich that was submerged in transfer buffer and electrophoresed at constant voltage. Subsequent steps included blocking (5% skim milk; room temperature for 2 h), primary antibody incubation at 4°C overnight with the following dilutions: p62/SQSTM (1:1,000), Phospho-AKT (1:500), THBS1 (1:500), β-actin (1:2,000), LC3B (1:1,000), Beclin1 (1:1,000), Bcl-2 (1:1,000), PI3K p85 (1:1,000), Phospho-PI3K p85 (1:500), AKT (1:1,000), mTOR (1:1,000) and Phospho-mTOR (1:500) secondary antibody incubation (HRP-labelled; 1:5,000 dilution; room temperature for 2 h). Finally, signals were detected using a Fusion FX7 chemiluminescence imaging system and quantified with ImageJ software (version 1.53e; National Institutes of Health) to analyze the expression ratio of the target protein to the internal control β-actin. To ensure experimental reliability, all experiments included three independent biological replicates.

The present study employed molecular cloning techniques to construct an overexpression vector for the THBS1 gene. Specific primers based on the mouse THBS1 coding sequence (accession no. NP_035710.2) from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) were first designed to amplify the target gene. The primer sequences used for amplifying the mouse THBS1 CDS were as follows: Forward, 5′-CTATAGGGAGACCCAAGCTGGCTAGCGCCACCATGGAGCTCCTGCGGGGACTAGGTGTCCTGTTCCTGTTGCATATG-3′; reverse, 5′-GTTTAAACGGGCCCTCTAGACTCGAGTTAAGCGTAGTCTGGGACGTCGTATGGGTAGGAATCTCGACACTCGTATTTC-3′. The reverse primer sequence is presented in its full-length synthesized form, which includes a 5′ vector homology arm (for In-Fusion cloning) and a sequence encoding a C-terminal HA tag (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) immediately upstream of the THBS1 gene-specific region. For sequence verification purposes, only the gene-specific portion should be used for BLAST alignment against the mouse THBS1 reference sequence. Using MCF-7 cell cDNA as a template, PCR amplification was performed with PrimeSTAR HS high-fidelity DNA polymerase (Takara Bio, Inc.) to ensure genetic accuracy. The amplified products were digested with NheI/XhoI restriction endonucleases (Thermo Fisher Scientific, Inc.) and ligated to the homologous pcDNA3.1 (+) eukaryotic expression vector. The recombinant plasmid was transformed into DH5α competent cells (Beijing Quanshijin Biotechnology Co., Ltd.) via the heat shock method, followed by ampicillin resistance screening to identify positive clones. After Sanger sequencing (Shenzhen BGI Genomics Co., Ltd.) confirmed the correct insertion sequences, the vector (2 µg per well of a 6-well plate) was introduced into MCF-7 cells using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) via liposome transfection at 37°C in a 5% CO₂ incubator for 6 h, after which the medium was replaced with fresh complete medium. Western blotting was used to verify the expression levels of THBS1 protein. In these experiments, cells transfected with the empty pcDNA3.1(+) vector served as the negative control.

The present study utilized immunofluorescence technology to assess the effects of various treatments on MCF-7 cells. MCF-7 cells were cultured at a density of 1×10⁴ cells per well in 24-well plates until they reached 80% confluency. A total of four treatment groups were established: Blank control (PBS), 6 µM IVM (IVM monotherapy), 6 mM MET (MET monotherapy) and combined treatment group (6 µM IVM + 6 mM MET). After 24-h treatment, cells were washed with PBS, fixed in 4% polyformaldehyde (Biosharp Life Sciences) for 30 min at room temperature, permeabilized with 0.2% Triton X-100 for 15 min at room temperature and blocked with 5% goat serum for 2 h at room temperature. Samples were incubated with specific primary antibodies at the following dilutions: Anti-LC3B (1:200) and anti-p62 (1:200) overnight at 4°C, followed by a 2-h incubation at room temperature in the dark with a FITC-labelled secondary antibody (1:500). DAPI staining (Biosharp Life Sciences) was performed for 5 min at room temperature. The cells were observed under a fluorescence microscope (Olympus IX73; Olympus Corporation).

This study employed standardized statistical methods to process and analyze the experimental data. All quantitative data were derived from three independent biological replicate experiments, presented as mean ± SD. Data analysis was conducted using GraphPad Prism 10.1 (Dotmatics) professional statistical software, with one-way ANOVA primarily used for intergroup comparisons, supplemented by Tukey's post hoc tests for multiple comparisons. Statistical significance thresholds were defined as follows: P<0.05 indicates a statistically significant difference, P<0.01 denotes highly significant differences, P<0.001 signifies extremely significant differences, while P>0.05 (marked as ns) indicates no statistically significant difference.

Results of the present study demonstrated that both IVM and MET monotherapy reduced MCF-7 cell viability, exhibiting typical concentration-dependent and time-dependent characteristics (Fig. 1A and B). Through the calculation of half-inhibitory concentrations (I) and orthogonal experimental analysis, the optimal combination concentration was determined to be 6 µM IVM combined with 6 mM MET, which confirmed the enhanced inhibitory effect of the combination (Fig. 1C). Notably, the combined treatment group exhibited a significant synergistic effect, demonstrating markedly improved inhibitory efficacy when compared with either monotherapy (Fig. 1D). This optimized formulation was subsequently employed in subsequent experimental studies.

The combination of IVM and MET demonstrated significant inhibitory effects on the malignant phenotype of MCF-7 cells. Transwell invasion assays revealed that, compared with the control group and single-drug treatment groups, the combined treatment group exhibited a markedly reduced cell invasion capacity (Fig. 2A and B). The scratch-healing assay further indicated that the drug-coated treatment group exhibited significantly slower cell migration rates compared with both the control and single-drug treatment groups (Fig. 2C and D). These data collectively demonstrate that the synergistic application of IVM and MET effectively suppresses the invasive migration capabilities of breast cancer cells.

During the development of breast tumors, various cytokines and protein molecules carry out key roles. THBS1, a key extracellular matrix protein, is associated with tumor cell proliferation, migration and invasion (24). Western blot analysis revealed that, compared with the control group and single-drug treatment groups, the IVM combined with MET treatment significantly reduced THBS1 expression in MCF-7 cells, with statistically significant difference; (Fig. 3A and B). These results demonstrate that the IVM combined with MET effectively suppresses THBS1 expression in breast cancer cells.

The PI3K/AKT/mTOR signaling pathway plays a key role in the development of tumors (25). The present study employed western blotting to evaluate the effects of different drug combinations on phosphorylation levels within this pathway. Results demonstrated that when IVM was combined with MET treatment, intracellular phosphorylation levels of phosphorylated (p)-PI3K, p-AKT and p-mTOR were significantly reduced in MCF-7 cells compared with control groups and single-drug treatments (Fig. 4A and B). These findings suggest that the IVM-MET combination may target the PI3K/AKT/mTOR pathway and induce tumor cell death.

Studies have demonstrated that the expression of THBS1 is regulated by the PI3K/AKT/mTOR signaling pathway (21,26). To investigate whether THBS1 exerts feedback regulation on this pathway, the present study utilized transcriptome data (NP_035710.2; NCBI database). The present study identified the CDS region of THBS1 from the National Centre for Biotechnology Information (NCBI) database. Using pcDNA3.1 (+) as the vector, an overexpression plasmid for THBS1 was constructed (Fig. 5A). Agarose gel electrophoresis revealed amplified gene fragments from the THBS1 CDS region, measuring 3558 bp in length (Fig. 5B).

The THBS1 overexpression construct used in this study was originally generated for parallel investigations in mouse and canine mammary tumor models, leveraging the high cross-species conservation of THBS1. Its application in human MCF-7 cells is supported by the >97% amino acid sequence identity between mouse and human THBS1, ensuring functional relevance (27).

Western blotting was used to detect the overexpression efficiency of THBS1 in MCF-7 cells, transfected with the empty pcDNA3.1(+) vector serving as the negative control. The results showed that, compared with the Ctrl group and the negative control [pcDNA3.1(+)] group, the overexpression efficiency of THBS1 was significantly increased (Fig. 5C and D).

Disruption of autophagy is associated with the development and progression of various diseases. To investigate the effects of IVM combined with MET on autophagy in breast tumors, the present study employed cellular immunofluorescence to detect autophagy-related proteins LC3B and p62 in MCF-7 cells. The results showed that compared with the control group, the combined treatment group exhibited elevated LC3B expression (Fig. 6A) and decreased p62 levels (Fig. 6B), indicating that IVM combined with MET can induce excessive autophagy in breast tumor cells. Excessive autophagy leads to over-degradation of multiple cellular components. Transmission electron microscopy analysis revealed that, compared with the control group and single-drug treatment groups, the IVM + MET group contained numerous double-membrane-structured autophagosomes and an increased number of autophagic lysosomes (Fig. 6C). Western blot analysis revealed that, compared with the PBS control group, LC3B and Beclin1 protein expressions were upregulated in MCF-7 cells after IVM, MET and IVM + MET treatments, while p62 and Bcl2 were downregulated (Fig. 6D and E). These results demonstrate that IVM, when combined with MET, can promote autophagy in MCF-7 cells.3-MA is an early-stage autophagy inhibitor (28). To investigate how autophagy inhibition affects cytotoxicity caused by IVM combined with MET, the present used the CCK-8 assay to evaluate the effects of co-treatment with 3-MA and/or IVM + MET on cell viability. The results showed that the co-treated group exhibited a significant rebound in cell activity compared with the IVM + MET-only group (Fig. 6F).

Flow cytometry analysis of ROS in MCF-7 cells revealed significantly elevated intracellular ROS levels following IVM and MET treatments compared with the control group, with the combined IVM + MET treatment showing even higher ROS levels (P<0.05; Fig. 7A and B). This suggests that the IVM + MET regimen may induce oxidative stress by increasing intracellular ROS, thereby affecting cell survival and function. NAC, an antioxidant that reduces intracellular ROS (29).

MCF-7 cells were pre-treated with 10 mM NAC in the present study (NAC group). Subsequent experimental groups included Ctrl, IVM + MET and IVM + MET + NAC. After 24 h of stimulation, ROS levels were measured using flow cytometry. Results demonstrated a partial reduction in ROS levels in the IVM + MET + NAC group compared with the IVM + MET group (Fig. 7C and D). To investigate whether IVM + MET affects PI3K/AKT/mTOR pathway phosphorylation through ROS, western blotting was employed to detect p-PI3K, p-AKT and p-mTOR protein expression in MCF-7 cells. The results demonstrated that IVM + MET inhibited phosphorylation activity in the PI3K/AKT/mTOR signaling pathway compared with Ctrl and single-drug treatments, and this inhibition was associated with elevated intracellular ROS levels. Furthermore, compared with the IVM + MET group, the IVM + MET + NAC group showed a partial elevation in protein expression levels of p-PI3K, p-AKT and p-mTOR (Fig. 7E and F). This suggests that the combination of IVM and MET may suppress phosphorylation levels in the PI3K/AKT/mTOR signaling pathway by inducing ROS accumulation. When intracellular ROS levels increase, key proteins in the PI3K/AKT/mTOR signaling pathway undergo oxidative modifications, resulting in reduced activity (30). To investigate whether the IVM + MET combination could alter autophagy-related proteins via ROS, western blot analysis was performed on MCF-7 cells for LC3B, Beclin1, p62 and Bcl-2. Results revealed that, compared with the IVM + MET group, the IVM + MET + NAC group exhibited partial decreases in LC3B and Beclin1 protein expression, while showing partial increases in p62 and Bcl-2 expression (Fig. 7G and H). These findings suggest that the IVM + MET combination inhibits the phosphorylation of the PI3K/AKT/mTOR signaling pathway by inducing excessive ROS accumulation within cells, thereby triggering tumor cell autophagy and exerting antitumor effects.