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Breast Cancer : Targets and Therapy logoLink to Breast Cancer : Targets and Therapy
. 2026 Mar 4;18:580570. doi: 10.2147/BCTT.S580570

Combined Treatment with Shuyu Pills and Everolimus Suppresses Triple-Negative Breast Cancer Growth via the PI3K/AKT/mTOR Pathway

Jingzhe Zhao 1,*, Xiaoshan Luo 2,*, Huan Li 1, Yu Cheng 1, Xiangping Lin 3,*,✉, Su Xie 4,*,✉
PMCID: PMC12968039  PMID: 41809619

Abstract

Background

Triple-negative breast cancer (TNBC) is clinically characterized by the absence of therapeutic targets and limited treatment options after surgery, leading to poor therapeutic outcomes and prognosis. Shuyu Pills (SYP), a classical traditional Chinese medicine (TCM) formula for treating deficiency syndromes with pathogenic excess, has been widely applied in the treatment of TNBC. However, its mechanisms of action against TNBC remain unclear.

Purpose

This study aimed to investigate the anti-tumor effect and the underlying mechanisms of SYP in combination with the mTOR inhibitor everolimus in TNBC both in vitro and in vivo experiments.

Methods

Network pharmacology was used to predict the potential signaling pathways targeted by SYP. The pharmacological effects of SYP and its combination with everolimus were evaluated through in vitro and in vivo experiments, including CCK-8 assay, transwell migration, wound healing assay, flow cytometry, Immunohistochemistry staining and Western blot analysis.

Results

The combination of SYP and everolimus exhibits synergistic effects in inhibiting TNBC. Synergistic therapy suppressed TNBC cell proliferation and colony formation, cell migration and invasion, induced cell cycle arrest and apoptosis and reduced tumor growth in TNBC-bearing mice. The addition of SYP significantly promoted apoptosis of TNBC cells via regulated apoptosis-related protein including Bax, Caspase3, Bcl-2, CDK1 and Cyclin B. Further, synergistic treatment reduced TNBC tumor growth and Ki67 expression. Furthermore, the combined therapy effectively suppresses the expression of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR proteins.

Conclusion

The present study aimed to investigate the pharmacological mechanism of the SYP. SYP can inhibit the proliferation, migration, and invasion of TNBC cells by arresting the cell cycle and inducing apoptosis. This mechanism may involve downregulation of the PI3K/AKT/mTOR signaling pathway. SYP can cooperate with everolimus to inhibit TNBC. Overall, these findings provide valuable evidence for the potential clinical application of SYP.

Keywords: triple-negative breast cancer, cell cycle, apoptosis, pi3k/akt/mtor signaling pathway, shuyu pills, everolimus

Introduction

Breast cancer is the most common malignancy among women worldwide with an increasing incidence in recent years.1,2 Approximately 10–15% of cases are TNBC, which lacks expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor.3,4 As a result, TNBC does not respond to endocrine or HER2-targeted therapies. Despite standard treatment with surgery and chemoradiotherapy, TNBC remains associated with high recurrence rates and poor survival outcomes.5 There is an urgent need to identify new therapeutic targets and effective treatment strategies for TNBC.

TNBC is a malignant tumor characterized by the uncontrolled proliferation of mammary epithelial cells, where aberrant proliferation, differentiation, and evasion of apoptosis serve as the core driving factors for disease initiation and progression.6,7 The PI3K/AKT/mTOR signaling pathway is frequently aberrantly activated in TNBC and participates in multiple oncogenic processes, including regulation of cell proliferation, apoptosis, cell cycle progression, and metastasis.8,9 Hyperactivation of this pathway confers sustained growth advantages and resistance to apoptotic signaling in TNBC cells. As a key downstream kinase of PI3K/AKT, mTOR plays a central role in tumor cell growth and survival, and its overactivation is associated with poor prognosis in TNBC. Accordingly, mTOR has emerged as a potential therapeutic target in this disease.10,11 Everolimus is a clinically approved mTOR inhibitor for breast cancer treatment; however, monotherapy with everolimus shows limited efficacy in TNBC.12 Current research therefore focuses on combining mTOR inhibitors with other therapeutic modalities to enhance antitumor activity.13 Integrating targeted therapy with TCM has recently gained increasing attention as a novel strategy in oncology, with the potential to achieve synergistic antitumor effects and improve therapeutic outcomes in TNBC.

In recent years, the value of TCM in the comprehensive treatment of tumors has gradually gained attention. Existing studies have systematically summarized the potential of TCM in improving patients’ physical constitution, alleviating tumor progression, and relieving uncomfortable symptoms during treatment, while also pointing out that its mechanism of action and evidence-based system still need to be further clarified.14 Therefore, combining TCM with modern targeted therapy is regarded as a feasible approach to enhance anti-tumor efficacy, but in-depth research on specific regimens and molecular mechanisms is still lacking. In modern clinical practice, SYP has been widely used in the treatment of tumors, cerebrovascular diseases, and autoimmune disorders.15–18 In the context of cancer, SYP has demonstrated notable benefits in improving patients’ physical condition, enhancing quality of life, and prolonging survival, thereby playing an important role in both cancer prevention and therapy.19 Based on the clinical characteristics of TNBC patients—namely, physical debilitation and accumulation of cancer-related toxins following surgery and adjuvant chemoradiotherapy—our research team modified the original SYP formula by incorporating Hedyotis diffusa and Scutellaria barbata, two herbs with well-documented antitumor properties, to enhance its anticancer efficacy.Clinical investigations conducted by our team have shown that SYP can alleviate chemotherapy-induced adverse effects and reduce the expression of the proliferation marker Ki67 in TNBC patients.20,21 Further, SYP was found to suppress tumor growth in breast cancer-bearing mice and improve immune function following chemotherapy.22,23 However, the effects of SYP on TNBC cell proliferation and apoptosis-related mechanisms remain unclear. Therefore, in this study, we first employed network pharmacology to predict the potential mechanisms of SYP against TNBC. We then performed in vitro and in vivo experiments to elucidate the antitumor effects of SYP combined with everolimus and to investigate their regulatory roles in the PI3K/AKT/mTOR pathway, cell proliferation, and apoptosis. This study aims to elucidate the molecular basis of the antitumor activity of the combination regimen as a preliminary mechanistic exploration of a potential therapeutic direction for TNBC.

Materials and Methods

Reagents

The DMEM culture medium was purchased from Gibco (USA); fetal bovine serum (FBS) was obtained from Biological Industries (BI, Israel); penicillin-streptomycin solution was purchased from HyClone (USA). The CCK-8 assay kit and Annexin V-FITC/PI apoptosis detection kit were obtained from MeilunBio®(China), Ltd. High-efficiency RIPA lysis buffer, dimethyl sulfoxide (DMSO), and SDS-PAGE gel rapid preparation kit were purchased from Beijing Solarbio Science & Technology Co, Ltd. Primary antibodies including β-actin (81115-1-RR), Bax (50599-2-Ig), Bcl-2 (80,313-1-RR), Caspase-3 (82,202-1-RR), Caspase-8 (40,930), Cyclin B (55004-1-AP), and CDK1 (19,532-1-AP) were obtained from Proteintech Group, Inc(China), Ltd. Antibodies against PI3K (ab40776), mTOR (ab32028), and AKT (ab38449) were purchased from Abcam (UK), while the phosphorylated forms p-PI3K (341468), p-mTOR (381557), and p-AKT (381555) were provided by Chengdu Zenon Biotechnology Co., Ltd. Matrigel (Cat. No. 356234) was purchased from BD Biosciences (USA).

Drug Preparation

The herbal formulation SYP was used in a modified form based on the original prescription. The granules were supplied by Guangdong Yifang Pharmaceutical Co., Ltd., and were prepared according to the equivalent dose conversion from crude herbal slices. The composition per dose was as follows: Dioscorea opposita (5 g), Angelica sinensis (6.67 g), Rehmannia glutinosa (7.69 g), Endothelium corneum gigeriae galli (0.48 g), Ligusticum chuanxiong (2 g), Colla corii asini (3 g), Cinnamomum cassia (1.09 g), Bupleurum chinense (1.50 g), Ligustrum lucidum (1.92 g), Paeonia lactiflora (1.11 g), Atractylodes macrocephala (3.84 g), Prunus armeniaca (0.83 g), Panax ginseng (2 g), Platycodon grandiflorus (3.33 g), Poria cocos (0.40 g), Saposhnikovia divaricata (2.50 g), Zingiber officinale (0.24 g), Ampelopsis japonica (0.75 g), Ziziphus jujuba (2.50 g), Glycyrrhiza uralensis (6 g), Curcuma zedoaria (1.87 g), and Hedyotis diffusa (3.77 g). Based on the “ body surface area conversion table between humans and animals”, the corresponding mouse dose was calculated as: SYP in adults × mouse equivalence coefficient (0.0026) = 10.04 g/kg/day. Previous experiments have confirmed that the optimal dose of SYP in mice is the medium dose of 20.08 g/kg/day, which corresponds to twice the clinical equivalent dose in humans.24 Everolimus (HY-10218/CS-0064) was purchased from MedChemExpress (USA).

Network Pharmacological Analysis

The main chemical components of SYP were collected by TCM System Pharmacology (TCMSP) (https://www.tcmsp-e.com/#/database), and HERB (https://drug.ac.cn/). Oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 were set as the screening conditions. The corresponding targets were analyzed from the UniProt database (https://www.uniprot.org/). Using databases including Online Men-delian Inheritance in Man (OMIM) (https://omim.org/), Gene-Cards (http://www.genecards.org/), and Therapeutic Target Database (TTD) (http://db-idrblab.net/ttd/) as search keywords, we identified TNBC targets under the term “triple-negative breast cancer”. After compiling these targets, duplicates were systematically removed to ensure data accuracy.

To screen for key targets, the intersection targets of SYP bioactive components and TNBC were subjected to protein-protein interaction (PPI) analysis using the STRING database. The intersecting targets were imported into STRING (https://string-db.org/), with the interaction score set to >0.9, and isolated nodes were removed to construct the PPI network. The network was further analyzed using the CytoNCA plugin in Cytoscape 3.9.1 to identify core targets involved in disease regulation. Subsequently, the intersection targets were uploaded to the Metascape database (https://metascape.org/gp/index.html#/main/step1) with Homo sapiens selected as the research subject for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The molecular docking analysis was performed using the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) to download the structure files of the key active compounds in SDF format, which were used as ligands. The three-dimensional structures of the core target proteins were obtained from the Protein Data Bank (PDB; https://www.rcsb.org/) in PDB format and used as receptors. Molecular docking and binding energy calculations were conducted using the CB-Dock2 online platform.25

Preparation of SYP-Containing Serum and Control Serum

Forty female Sprague-Dawley rats (200±20 g) were purchased and randomly assigned to either the control group or the SYP group (n=20 per group). Rats in the control group received normal saline by gavage at a dose of 2 mL per administration, twice daily. Rats in the SYP group were gavaged with a prepared SYP decoction at 2 mL per administration (equivalent to 72.50 g/kg crude drug), based on our previous studies selecting a high-dose regimen corresponding to four times the human clinical equivalent dose. Both groups were treated twice daily for 7 consecutive days. At 1.5 h after the final administration, rats were anesthetized and blood was collected from the abdominal aorta. Serum was separated under sterile conditions, inactivated in a 56 °C water bath for 30 min, and filtered through a 0.22 μm sterile microporous membrane to obtain control serum and SYP-containing serum.

Cell Treatment Protocol

Cells were treated as follows:Model group: 10% control serum + DMEM; SYP group: 10% SYP-containing serum + DMEM; Everolimus group: 10% control serum+everolimus (IC50) + DMEM; SYP+Everolimus group: 10% SYP-containing serum+everolimus (IC50) + DMEM.

Cell Lines and Animals

The human TNBC cell line MDA-MB-231 (Batch No. ZQ0118) and the murine 4T1 cell line (Batch No. ZQ0201) were obtained from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. A total of 40 female SPF-grade Sprague-Dawley rats (180–200 g) were purchased from Guangdong Vtlong Laboratory Animal Technology Co., Ltd. [Production license: SCXK (Yue) 2022–0063], and housed in the SPF-grade animal facility of the Clinical Research Center at the Affiliated Hospital of Guizhou Medical University, under controlled conditions (temperature 22–24°C, humidity 45%–50%) with access to standard chow and distilled water. All animal procedures were approved by the Animal Ethics Committee of Guizhou Medical University (Approval No. 1900650). During the experiment, all procedures involving animals were strictly conducted in accordance with GB/T 35892–2018 Standard for Laboratory Animal Welfare.

CCK-8 Assay

Logarithmic-phase 4T1 and MDA-MB-231 cells were seeded in 96-well plates at a density of 5 × 103 cells per well, with three replicate wells per group. After cell adherence, the indicated drugs were added according to groups 1, 2, and 3. Following the desired treatment duration, 10 μL of CCK-8 solution was added to each well and incubated for 1 h at 37°C in the dark. Absorbance was measured at 450 nm using a multifunctional microplate reader. Cell viability and proliferation inhibition rates were calculated. Experiments were repeated three times.

Colony Formation Assay

Log-phase 4T1 and MDA-MB-231 cells were seeded in 6-well plates at 500 cells/well. After 24 h of adherence, drug interventions were applied for 48 h. The medium was then replaced with fresh complete medium and cultured for 14 days. Cell morphology and colony formation were observed under an inverted microscope, with medium replaced as needed. After discarding the medium, cells were washed twice with PBS, fixed with 600 μL of paraformaldehyde for 15 min, stained with crystal violet for 15 min, rinsed twice with PBS, and air-dried for imaging. The experiment was repeated three times.

Wound Healing Assay

Log-phase 4T1 and MDA-MB-231 cells were seeded in 6-well plates at a density of 1 × 106 cells/well. After 24 h of incubation to allow adherence, a vertical scratch was made using a sterile 200 μL pipette tip. The medium was replaced with drug-containing serum-free medium according to treatment groups 1, 2, 3, and 4. Images were captured at 0 h and 24 h using an inverted microscope. Wound areas were measured using ImageJ software to calculate cell migration rates. Experiments were performed in triplicate.

Transwell Invasion Assay

Matrigel was pre-cooled and diluted with serum-free medium at a 1:8 ratio, then added to the upper chamber of the Transwell insert and incubated at 37°C until solidified. Each upper chamber was seeded with 200 μL of cell suspension (3 × 105 drug-treated cells), and the lower chamber was filled with 600 μL of complete medium. After 24 h incubation, non-invading cells in the upper chamber were removed using cotton swabs. Cells were washed twice with PBS, fixed with paraformaldehyde for 15 min, and stained with crystal violet for 10 min. Excess dye was washed off with PBS, and cells were imaged under an inverted microscope. The number of invasive cells was quantified using ImageJ software. Experiments were repeated three times.

Flow Cytometry

Cells were collected and digested with EDTA-free trypsin, followed by centrifugation and washing with PBS. After gentle resuspension using a pipette, cells were suspended in 1× Binding Buffer and adjusted to a final concentration of 1 × 106 cells/mL. A 100 μL aliquot (containing approximately 1 × 105 cells) of the cell suspension was transferred into a flow cytometry tube. Then, 5 μL of Annexin V-FITC and 10 μL of PI were added. The samples were incubated at room temperature in the dark for 15 min, followed by flow cytometric analysis. All experiments were performed in triplicate.

Immunohistochemistry

Tumor tissues fixed in 4% paraformaldehyde for 48 h were dehydrated in an ethanol gradient, embedded in paraffin, and sectioned at a thickness of 4 μm. After deparaffinization with xylene and rehydration through graded ethanol, sections were processed using the SP two-step immunohistochemistry method. Antigen retrieval was performed using microwave heating, followed by endogenous peroxidase blocking with 3% H2O2, primary and secondary antibody incubation, and DAB staining. Protein expression was observed under a light microscope.

Western Blot Analysis

Cells or tissue proteins were lysed on ice for 30 min using RIPA lysis buffer. Protein concentrations were determined by the BCA assay. Equal amounts of protein samples were subjected to SDS-PAGE and transferred to PVDF membranes using a wet transfer method. The membranes were blocked with 5% non-fat milk for 1.5 h at room temperature, followed by overnight incubation with primary antibodies at 4°C on a shaker. The next day, membranes were incubated with HRP-conjugated secondary antibodies (1:1000) at room temperature for 1 h. After washing, protein bands were visualized using a chemiluminescence imaging system. β-actin was used as a loading control, and the relative protein expression levels were quantified using ImageJ software.

Statistical Analysis

All experimental data were analyzed using SPSS 26.0 software. Graphs were generated using GraphPad Prism 9.0. Data were tested for normality. If the data followed a normal distribution, a chi-square test for homogeneity of variance was performed. When variances were unequal, the Wilcoxon signed-rank test was applied. If the data were normally distributed with homogeneous variances, an independent-samples t test was used, whereas the Mann–Whitney U-test was employed when the data were normally distributed but variances were unequal. A p-value of <0.05 was considered statistically significant.

Results

Network Pharmacology Screening of Potential Targets of SYP

Based on the TCMSP, Herb and Swiss Target Prediction database, we collected 565 potential targets related to 294 ingredients of SYP. Further, a total of 2231 TNBC targets were obtained using the GeneCards, TTD and OMIM databases. The Venn diagram of targets for TNBC and SYP were plotted in the venny 2.1 server. As shown in Figure 1A, there are 229 common targets between SYP and TNBC, which were considered as possible targets for the treatment of TNBC (Supplementary Figure 1). To further explore the mechanisms underlying the effects of SYP on TNBC, we constructed a PPI network to investigate and visualized the interaction among the targets, which were determined according to the degree of the target in the network by Cytoscape (top 50 targets) (Figure 1B). Finally, TP53, AKT1, EGFR, MYC, and TNF were regarded as critical effector targets of SYP against TNBC in the PPI network.

Figure 1.

Figure 1

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Potential mechanisms of SYP in the treatment of TNBC predicted by network pharmacology analysis. (A) Venn diagram of 294 active compounds of SYP obtained from TCMSP, Herb, Swiss Target Prediction databases and TNBC-related targets retrieved from GeneCards and OMIM databases. (B) Potential targets of SYP against TNBC in the PPI network using the STRING database. (C) Top 10 enriched terms in Gene Ontology (GO) analysis, including biological processes (BP), molecular functions (MF), and cellular components (CC). (D) KEGG pathway enrichment analysis showing the most significantly involved signaling pathways of SYP.

GO and KEGG pathway enrichment analysis was utilized to excavate the biological functions and potential signaling pathways of the 226 overlapping targets against TNBC via the Metascape online database. The top 10 significantly enriched terms were in cellular components, molecular functions, and biological processes (Figure 1C), which indicated that SYP may regulate response to hormone, positive regulation of phosphorus metabolic process, transcription regulator complex, membrane raft, kinase binding and protein kinase activity to exert its therapeutic effects on TNBC. After performing KEGG pathway annotation, the underlying pathways of SYP against TNBC was analyzed about the top 20 significantly enriched pathways (Figure 1D). The potential targeted pathways were mainly enriched in the PI3K-Akt, AGE-RAGE, FoxO and P53 signaling pathways.

In breast cancer, the PI3K/AKT/mTOR signaling pathway is one of the most frequently dysregulated pathways associated with tumor progression and plays a crucial regulatory role in cancer cell growth, proliferation, apoptosis, and other biological processes. Based on the PPI network analysis, AKT1, mTOR, Caspase-3, and Bcl-2 were identified as core targets with high rankings. KEGG pathway enrichment analysis revealed that the PI3K/AKT signaling pathway ranked first, while GO analysis indicated that regulation of apoptotic signaling pathway, protein kinase activity, and membrane raft were significantly enriched, suggesting that SYP may inhibit the progression of TNBC through this pathway. Accordingly, the active components of SYP were ranked, and the top five compounds with the highest DRGEE values (quercetin, β-sitosterol, stigmasterol, kaempferol, and wogonin) were selected for molecular docking analysis with key proteins in the pathway (PI3K, AKT, and mTOR) (Figure 2). The molecular docking results are presented in Supplementary Table 1, with all binding energies less than-6.0 kcal·mol, indicating strong binding affinities. These findings suggest that the PI3K/AKT/mTOR signaling pathway is a key mechanism underlying the anti-TNBC effects of SYP.

Figure 2.

Figure 2

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Molecular docking results of the core active components of SYP with key proteins in the PI3K/AKT/mTOR signaling pathway.

Treatment with SYP and Everolimus Synergistically Suppresses the Viability of 4T1 and MDA-MB-231 Cells

SYP-containing serum was used to evaluate its antiproliferative activity in TNBC cells. 4T1 and MDA-MB-231 cells were treated with increasing concentrations of SYP-containing serum (1.25%, 2.5%, 5%, 10%, and 20%) for 24 h (Figure 3A and B), 48 h, and 72 h to assess antitumor efficacy. At concentrations ranging from 1.25% to 10%, SYP-containing serum significantly suppressed TNBC cell viability in both a dose- and time-dependent manner (Supplementary Table 2). Notably, serum containing 10% SYP exhibited the most appropriate inhibitory effect in both 4T1 cells and MDA-MB-231 cells, while maintaining cell viability above 50% at 24, 48, and 72 h. For everolimus, the half-maximal inhibitory concentration (IC50) values were determined separately as 12.08 μM for 4T1 cells and 24.28 μM for MDA-MB-231 cells (Figure 3C and D). We then assessed the viability of 4T1 (Figure 3E) and MDA-MB-231 (Figure 3F) cells treated with different doses of SYP-containing serum in combination with everolimus. The results showed that the combined treatment inhibited cell survival in a concentration-dependent manner. Combination index (CI) analysis based on the Chou-Talalay method revealed that CI values were <1 across most Fa ranges, indicating a synergistic inhibitory effect of SYP-containing serum combined with everolimus on both 4T1 (Figure 3G) and MDA-MB-231 cells (Figure 3H) (CI<1, synergy; CI=1, additive; CI>1, antagonism). In 4T1 cells, the combination of 10% SYP-containing serum with 12.08 μM everolimus showed strong synergistic activity. Similarly, in MDA-MB-231 cells, 10% SYP-containing serum combined with 24.28 μM everolimus also exhibited marked synergy. These combinations were therefore selected for subsequent mechanistic studies to further explore the basis of their synergistic effects.

Figure 3.

Figure 3

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Effects of SYP and everolimus on the viability of 4T1 and MDA-MB-231 cells. (A) Cell viability of 4T1 and MDA-MB-231 (B) cells treated with different concentrations of SYP-containing serum for 24 hours. (C) Dose-response curves of 4T1 and MDA-MB-231 (D) cells treated with everolimus alone. (E) Cell viability of 4T1 and MDA-MB-231 (F) cells after treatment with different combinations of SYP-containing serum and everolimus. (G) Synergistic effect analysis in 4T1 and MDA-MB-231 (H) cells.

The Anti-Tumor Effects of SYP Combined with Everolimus in Vitro

To further investigate the effects on TNBC cell proliferation, colony formation assays were performed. A colony formation assay demonstrated that the proliferation rate and colony numbers of the 4T1 and MDA-MB-231 cells treated with SYP and everolimus were significantly decreased compared with the model group (Figure 4A and B). And the inhibitory rate of the combined treatment group was significantly higher compared with that of the SYP or everolimus treatment group. Wound healing and Transwell invasion assays were conducted to evaluate whether the treatments affected cell motility and invasiveness. In the wound healing assay (Figure 4C), wound closure was markedly reduced in both 4T1 (Figure 4D) and MDA-MB-231 (Figure 4E) cells following treatment. In contrast, the scratch was still largely uncovered at 24 h in model cells. As shown in Figure 4F and G, in comparison with untreated model cells, SYP and/or everolimus significantly reduced the invasion of TNBC cells through the matrigel membrane. Collectively, these results demonstrate that SYP-containing serum combined with everolimus effectively inhibits the proliferation, migration, and invasion of TNBC cells in vitro.

Figure 4.

Figure 4

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Anti-proliferative and anti-migratory effects of SYP combined with everolimus in vitro. (A) Colony formation assays were performed to assess the proliferation capacity of TNBC cells under various treatments, with corresponding quantitative analysis (B). (C) Wound healing assays were conducted to evaluate the migration abilities of 4T1 (D) and MDA-MB-231 (E) cells after 24 h of treatment. (F) Representative images of the transwell assay of TNBC cells following treatment with SYP and/or everolimus for 24 h. (G) Number of invaded of TNBC cells calculated from the transwell assay. Data are presented as mean ± SD. Compared with the model group: *P< 0.05. Compared with the everolimus group: #P < 0.05, *#P < 0.01.

The SYP Combined with Everolimus Induced Apoptosis and Cell Cycle Arrest via Inhibiting the PI3K/AKT/mTOR Signaling Pathway in TNBC Cells

Disruption of apoptosis and dysregulation of the cell cycle are critical contributors to cell proliferation of TNBC. The results of flow cytometry revealed that SYP significantly increased the apoptosis rate in both MDA-MB-231 and 4T1 cells (Figure 5A–C). Further, the treatment with SYP, everolimus, and their combined group upregulated protein expression of pro-apoptotic proteins Bax, Caspase-3, and Caspase-8 (Figure 5D and E and and Supplementary Figure 2). In contrast, the level of anti-apoptotic protein Bcl-2 and cell cycle-related proteins CDK1 and Cyclin B were downregulated significantly. These findings suggested that SYP can inhibit TNBC progression by inducing apoptosis and causing cell cycle arrest, thereby contributing to its antitumor effects.

Figure 5.

Figure 5

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The mechanism of SYP combined with everolimus induces apoptosis and cell cycle arrest in TNBC cells. (A) Flow cytometric analysis of apoptosis in TNBC cells under different treatment conditions, along with corresponding quantitative results (B and C). Western blot analysis of the expression levels of Bax, Bcl-2, CDK1, Cyclin B, Caspase-3, and Caspase-8 proteins in 4T1 (D) and MDA-MB-231 (E) cells. (F and G) Western blot analysis of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR protein expression levels in 4T1 (F), MDA-MB-231 (G). Data are presented as mean ± SD. Compared with the model group: *P< 0.05. Compared with the everolimus group: #P < 0.05.

The PI3K/AKT/mTOR signaling pathway was significantly enriched in the network pharmacology analysis and is known to play a pivotal role in the progression of TNBC. The protein expression levels of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR were downregulated following treatment with SYP, everolimus, and their combination in 4T1 cells and MDA-MB-231 cells (Figure 5F and G). Notably, the combined treatment group exhibited the most pronounced inhibitory effect. These findings suggest that the antitumor activity of SYP, particularly when combined with everolimus, was attributed to the suppression of the PI3K/AKT/mTOR signaling pathway.

The Anti-Tumor Activity of SYP Combined with Everolimus Against TNBC in Vivo

To evaluate the efficacy of the treatment in vivo the BALB/c mice were successfully established with orthotopic TNBC tumor models to simulate TNBC. After tumor implantation, mice were orally administered with SYP, everolimus, or their combination until day 15 (Figure 6A). Following tumor establishment, tumor volume and body weight of the mice were monitored every three days. There were no significant differences in body weight among the groups (Figure 6B). In contrast, tumor tissues of the model group showed a continuous upward trend, while tumor sizes in the SYP group, everolimus group, and especially the SYP+everolimus combination group were markedly reduced (Figure 6C and D). Tumor weights were also significantly decreased in these treatment groups (Figure 6E). Notably, the combination treatment group (SYP+everolimus) exhibited the most pronounced inhibitory effect on tumor volume progression. Collectively, these findings confirm that SYP, everolimus, and particularly their combination, possessed significant antitumor efficacy against TNBC in vivo.

Figure 6.

Figure 6

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Evaluation of the anti-tumor effect of SYP combined with everolimus in vivo. (A) Animal experiment scheme. (B) During the treatment period, body weight and tumor volume (C) were measured at regular intervals. (D) Representative images of excised tumors and statistical analysis of tumor weights (E) at the end of the experiment. Data are presented as mean ± SD. Compared with the model group: *P< 0.05, **P < 0.01. Compared with the everolimus group: #P < 0.05, *#P < 0.01.

Antitumor Mechanism of SYP Combined with Everolimus in Vivo

To elucidate the mechanisms by which SYP, everolimus and their combination suppress TNBC progression in vivo, we performed IHC and WB analyses to assess tumor cell proliferation and apoptosis in tumor tissues. Compared to the model group, Ki67 expression was significantly reduced in the SYP, everolimus and SYP+ everolimus groups (Figure 7A and B), indicating suppressed tumor cell proliferation. WB analysis further confirmed that the expression levels of pro-apoptotic proteins Bax, Caspase-3, and Caspase-8 were upregulated, while the expression of anti-apoptotic protein Bcl-2 and cell cycle-related proteins CDK1 and Cyclin B were downregulated in all treatment groups compared to the model group (Figure 7C and Supplementary Figure 3). These results suggest that SYP, everolimus and their combination can inhibit TNBC tumor growth by inducing apoptosis and arresting the cell cycle, with the combination treatment demonstrating the most potent antitumor effect. Western blot results demonstrated that the expression levels of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR were reduced following treatment with SYP, everolimus, or their combination in tumor tissues (Figure 7D). We hypothesized that medicines might exert therapeutic effects by affecting the expression levels of PI3K- AKT-mTOR pathway to trigger apoptosis.

Figure 7.

Figure 7

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In vivo evaluation of the effects of SYP combined with everolimus on tumor proliferation and apoptosis. (A and B) Immunohistochemical staining and quantitative analysis of Ki67 expression in tumor tissues from different treatment groups. (C) Western blot analysis of Bax, Bcl-2, CDK1, CyclinB, Caspase-3 and Caspase-8 protein expression levels in tumor tissues. (D) Western blot analysis of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR protein expression levels in tumor tissues. Data are presented as mean ± SD. Compared with the model group: *P< 0.05. Compared with the everolimus group: *#P < 0.01.

Discussion

Breast cancer is the second most commonly diagnosed malignancy worldwide and the leading cancer among women. Its persistently high incidence and mortality rates pose a serious threat to women’s physical and psychological health and represent a major global public health challenge.26 Among the various breast cancer subtypes, TNBC is one of the most aggressive forms, characterized by high malignancy, lack of defined therapeutic targets, and significantly shortened patient survival, making it a major clinical challenge. In response to this therapeutic dilemma, the antitumor potential of TCM has attracted increasing attention from both basic researchers and clinicians, and accumulating evidence highlights its unique advantages in cancer treatment.27–29 From the perspective of TCM theory, patients with TNBC often present, after surgery and chemotherapy, with a pattern of deficiency of vital qi, imbalance of yin and yang, and concurrent pathological factors such as qi stagnation, phlegm accumulation, residual toxin, and blood stasis. SYP is formulated based on the principles of strengthening the spleen and harmonizing the middle, tonifying qi and nourishing blood, and restoring the balance of yin and yang, thereby achieving a therapeutic balance between reinforcing the healthy qi and eliminating pathogenic factors. The formula integrates warming, harmonizing, clearing, tonifying, and resolving strategies, and is designed to inhibit tumor progression in a holistic manner. Based on this rationale, the present study investigated the antitumor effects and potential mechanisms of SYP in combination with the mTOR inhibitor everolimus in the treatment of TNBC, aiming to provide experimental evidence for an integrative Chinese and Western medicine–based targeted therapeutic strategy for TNBC.

Uncontrolled cellular proliferation is a central hallmark of TNBC progression. Ki67, a proliferation-specific nuclear antigen, directly reflects tumor cell proliferative activity and invasive potential.30 In the present study, SYP not only markedly reduced Ki67 expression in tumor tissues but also effectively suppressed the in vitro proliferative capacity of TNBC cell lines. Cell cycle regulation critically governs tumor cell proliferation, and dysfunction of the G2/M checkpoint is a key event driving uncontrolled cell division. The Cyclin B/CDK1 complex is the core regulator of this checkpoint, and its overexpression strongly promotes entry into mitosis.31,32 Our data demonstrate that SYP significantly downregulated Cyclin B and CDK1 expression in MDA-MB-231 and 4T1 cells as well as in tumor tissues from tumor-bearing mice, indicating that SYP induces cell-cycle arrest by targeting the G2/M checkpoint, thereby exerting anti-TNBC effects. Cancer is characterized by aberrant expression of apoptosis-regulating proteins, with excessive anti-apoptotic factors and suppressed pro-apoptotic signals. The caspase-8/caspase-3 proteolytic cascade and the imbalance between Bcl-2 and Bax constitute key regulatory nodes of tumor cell apoptosis.33,34 Flow cytometry and protein analyses in this study confirmed that SYP significantly promoted apoptosis in TNBC cells, accompanied by upregulation of caspase-3, caspase-8, and Bax, and downregulation of Bcl-2. These findings indicate that induction of tumor cell apoptosis is a major mechanism underlying the antitumor activity of SYP.

The PI3K/AKT/mTOR signaling pathway is commonly overexpressed in TNBC and plays a pivotal role in mediating a range of malignant biological behaviors, including tumor cell proliferation, apoptosis inhibition, and metastasis promotion. In TNBC, approximately 30% of patients exhibit hyperactivation of this pathway. Among its core components, PI3K is considered one of the most critical proteins, as it regulates tumor cell growth, proliferation, and metabolism. Mutations in the PIK3CA gene are a primary cause of PI3K/AKT/mTOR pathway hyperactivation and serve as major drivers of aberrant tumor cell proliferation, metastasis, and resistance to apoptosis.35 AKT functions as a central intracellular effector in this pathway regulates a range of downstream proteins, including Bax, and Bcl-2, thereby modulating both cell proliferation and apoptosis.36 As a key downstream target of the PI3K/AKT axis, mTOR exists in two functionally distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2).37 mTORC1 activates p70 S6 kinase 1 (S6K1), which promotes the proliferation, invasion, and migration of tumor cells.38 TNBC exhibits marked molecular heterogeneity. In this study, the 4T1 and MDA-MB-231 cell lines were used, and they belong to the mesenchymal (MES)39 and basal-like immunosuppressed (BLIS)40 subtypes of TNBC, respectively. Jiang et al41 performed comprehensive genomic and transcriptomic profiling in a large Chinese TNBC cohort and reported that mutations in key components of the PI3K/AKT/mTOR pathway-including PIK3CA, PTEN, PIK3R1, and AKT1-occur at relatively high frequencies in both MES and BLIS subtypes. Specifically, mutation rates were 18% for PIK3CA, 6% for PTEN, and 3% for both PIK3R1 and AKT1. Previous studies have demonstrated that MES and BLIS TNBC cell lines are more sensitive to PI3K/mTOR inhibitors,42 suggesting that patients with these subtypes may particularly benefit from therapies targeting the PI3K/AKT/mTOR pathway. Therefore, targeting the PI3K/AKT/mTOR signaling axis may hold greater therapeutic value for the MES and BLIS molecular subtypes of TNBC. However, no inhibitors targeting this pathway have been approved for clinical use in patients with TNBC. Everolimus, an mTOR inhibitor approved by the FDA for the treatment of hormone receptor-positive, HER2-negative advanced breast cancer, exerts its antitumor effects by suppressing tumor cell growth, nutrient metabolism, and angiogenesis, whose monotherapy has limited efficacy in TNBC. Numerous studies have demonstrated that combining everolimus with other therapeutic modalities can effectively prolong patient survival.43,44 The synergistic effects of combining TCM with modern medical therapies in breast cancer treatment have gained increasing attention. Identifying Chinese herbal medicines that target this pathway and cooperate with molecular inhibitors to enhance antitumor efficacy may exert beneficial effects on the treatment of patients with TNBC.

Research on the integration of TCM with modern targeted therapies in breast cancer treatment has attracted growing attention, as their combination is expected to produce synergistic antitumor effects. Therefore, identifying Chinese herbal formulations that can modulate the PI3K/AKT/mTOR signaling pathway and enhance the efficacy of pathway inhibitors is of considerable value for the treatment of triple-negative breast cancer. In the present study, network pharmacology analysis suggested that the PI3K/AKT/mTOR signaling pathway may represent a core therapeutic target of SYP in TNBC. Both in vitro and in vivo experiments further confirmed that SYP, either alone or in combination with everolimus, effectively downregulated the protein expression levels of PI3K, AKT, and mTOR in TNBC cells and tumor tissues. In summary, this study demonstrates that SYP exhibits therapeutic efficacy against MES and BLIS subtypes of TNBC, and its combination with the targeted inhibitor everolimus produces potential synergistic antitumor effects.

However, several limitations of this study should be acknowledged. First, although SYP was shown to modulate the PI3K/AKT/mTOR pathway in TNBC cells, we did not employ models with enforced overexpression of this pathway for comparative analysis. Therefore, it cannot be concluded that the antitumor effects of SYP are exclusively or primarily mediated through PI3K/AKT/mTOR signaling, nor can compensatory regulation by parallel pathways be excluded. Second, SYP is a multi-component TCM formula with a complex chemical composition, and this study did not perform component-level characterization or identification of the key active constituents. Third, the experimental design and models also have inherent limitations. The relatively short observation period precluded a comprehensive evaluation of the long-term effects of SYP, alone or in combination with everolimus, on metastasis, and overall prognosis in vivo.

Conclusion

In summary, combined treatment with SYP and everolimus inhibits proliferation and invasion of MES and BLIS subtype TNBC, and is associated with cell cycle arrest and induction of apoptosis. These effects are likely mediated through the regulation of the PI3K/AKT/mTOR signaling pathway (Figure 8). This research provides experimental evidence supporting the development of a new anti-cancer strategy and therapy that integrate TCM with pathway-targeted therapies.

Figure 8.

Figure 8

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Mechanistic diagram of this study. Upward arrows (↑) are associated with gain-of-function mutations, whereas downward arrows (↓) indicate loss-of-function mutations. The T-shaped line indicates inhibition by SYP and Everolimus. In TNBC, the PI3K/AKT/mTOR pathway is frequently hyperactivated, which disrupts the balance between apoptosis and cell-cycle regulation. This aberrant activation increases the expression of the anti-apoptotic protein Bcl-2, decreases the expression of the pro-apoptotic protein BAX, and suppresses the activity of the executioner caspases Caspase-8 and Caspase-3, allowing tumor cells to evade apoptosis. In parallel, upregulation of key cell-cycle regulators Cyclin B and CDK1 drives uncontrolled cell-cycle progression, ultimately leading to malignant proliferation. This study demonstrates that SYP exerts anti-tumor effects in TNBC by modulating the PI3K/AKT/mTOR signaling pathway, leading to cell cycle arrest and apoptosis, thereby inhibiting tumor progression. Moreover, its combination with the targeted inhibitor everolimus produces a synergistic therapeutic effect.

Funding Statement

National Natural Science Foundation of China (Grant No. 82460883). Ling Xiangli National Famous Traditional Chinese Medicine Practitioner Inheritance Workshop Construction Project (State Office of Traditional Chinese Medicine Human Education Letter [2022] No. 245), Guizhou Provincial Science and Technology Programme Project (Qiankehe Foundation-ZK [2022] General 442), Guizhou Provincial Science and Technology Programme Project (Qiankeheji-ZK[2024] General 204). The Youth Guidance Project of the Basic Research Program of the Science and Technology Department of Guizhou Province (Qiankehe Foundation QN [2025]127).

Data Sharing Statement

The datasets generated and analyzed in this study are not publicly available but can be obtained from the corresponding author Su Xie upon reasonable request, Email xiesutcm@163.com.

Ethics Statement

All animal experiments were approved by the Animal Care Welfare Committee of Guizhou Medical University (No. 1900650, GB/T35892-2018).

Disclosure

All the authors have no known competing financial interests or personal relationships that could influence the work reported in this article.

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Associated Data

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

Data Availability Statement

The datasets generated and analyzed in this study are not publicly available but can be obtained from the corresponding author Su Xie upon reasonable request, Email xiesutcm@163.com.

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