Obovatol induces apoptosis in breast cancer by downregulating the PI3K/Akt pathway.

Introduction
Breast cancer (BC) is the most common malignant neoplasm among women worldwide, and accounts for more than one-third of all female cancer cases (1). Tobacco use, obesity, and poor eating habits are all well-known risk factors for BC (2). The high heterogeneity of BC, in terms of its molecular subtypes, etiology, and clinical presentation, poses major challenges for its clinical management and prevention (3). The current therapeutic arsenal for BC includes surgical intervention, chemotherapy, radiotherapy, endocrine therapy, immunotherapy, and targeted molecular agents (4). Systemic chemotherapy is a cornerstone adjuvant treatment; however, its efficacy is often compromised by dose-limiting toxicities and drug resistance (5). Both neoadjuvant and adjuvant strategies for BC fundamentally rely on doxorubicin-based regimens (6). However, doxorubicin administration causes significant toxicities, such as cardiotoxicity, bone marrow suppression, and alopecia, posing serious threats to the long-term health of patients and survivors (7).
Advancements in molecular biology have elucidated essential mechanisms that promote BC cell proliferation and invasion (8). This recognition has enabled the development of small-molecule targeted therapies designed to selectively induce apoptosis in tumor cells while sparing normal tissues (9). Such findings facilitate the development of innovative therapies in BC. Emerging evidence demonstrates the potential of natural bioactive compounds, particularly phytochemicals, in BC management (10). With their ability to inhibit tumor growth, natural compounds offer a complementary strategy to conventional chemotherapy (11). Obovatol (Ob), a benzene ester lignan derived from Magnolia obovata leaf tissue, exerts multiple anti-inflammatory, antioxidant, antibacterial, antiplatelet, anxiolytic, antifungal, and neuroprotective effects (12-14). Previous research has shown the anti-cancer efficacy of Ob in the treatment of a range of tumors, including liver tumor, colorectal tumor, lung tumor, prostate tumor, and squamous cell tumor of the tongue, as evidenced by in vitro and in vivo assays (15). Ob exerts its anti-cancer effects by regulating the Janus kinase/signal transducer and activator of transcription (JAK/STAT) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways, thus triggering apoptosis, and suppressing proliferation and invasion in malignant cells (15,16). To date, the effects of Ob and its underlying mechanisms in BC are yet to be fully elucidated.
Using both in vitro BC cell lines and in vivo xenograft murine models, this study evaluated the effects of Ob to establish a novel theoretical and experimental basis for its potential use in BC therapy. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2627/rc).

Methods

Chemicals and cell cultures
Ob (Cat No. HY-N8259), doxorubicin hydrochloride (ADR; Cat No. HY-15142), and 740Y-P (Cat No. HY-P0175) were sourced from Med Chem Express (Shanghai, China). Figure 1A,1B presents the chemical structures of Ob and ADR. All the chemicals were dissolved in dimethyl sulfoxide. MDA-MB-468 cells (Cat No. CL0211), MDA-MB-231 cells (Cat No. CL0208), and the normal human breast cell line MCF-10A (Cat No. CL0205) were acquired from Fenghui (Changsha, China). The MCF-10A cell line was cultivated using cell-specific solution (Procell, Wuhan, China), and Dulbecco’s Modified Eagle Medium (DMEM; Procell, Wuhan, China) augmented with 10% fetal bovine serum (FBS; Procell, Wuhan, China) and 1% penicillin/streptomycin (Biosharp, Hefei, China) was used to maintain the two BC cell lines. The cells were grown under 5% carbon dioxide at 37 ℃ in a humidified environment.

Cell counting kit-8 (CCK-8)
The MDA-MB-231, MDA-MB-468, and MCF-10A cells were resuspended in culture medium and plated in a 96-well plate at a density of 5×103 cells/well. The culture cells in the plates were subsequently exposed to Ob (0, 25, 50, 75, and 100 µM) and ADR (0, 0.25, 0.5, 0.75, and 1 µM) for 48 h to assess cell vitality. CCK-8 solution (Solarbio, Beijing, China) was introduced to each well at a dilution ratio of 1:10 and incubated for at least 2.5 h. The microplate reader (BioTek, Vermont, USA) was used to measure the optical density (OD) values of cells at 450 nm. The half maximum inhibitory concentration (IC50) values of Ob and ADR on the BC cells were calculated using GraphPad Prism 8.0 (GraphPad Software, San Diego, USA). To assess the anti-proliferative effects of both Ob and ADR, the cells were inoculated onto plates at 3×103 cells per well and subjected to drug exposure for designated durations (0–96 h). Cell proliferation was then quantified by assessing the OD values. For the rescue experiments, parallel cultures were treated with 20 µM of 740Y-P (17,18).

Colony formation assays
The cells were planted at a density of 1.2×103 BC cells per well in 6-well plates. After cell adhesion, the cells were treated with Ob for 48 h. The supernatant was discarded subsequently, and fresh DMEM medium was added and replaced regularly to maintain cell growth until the visible generation of clones. After fixation for 20 min with 4% paraformaldehyde (Solarbio, Beijing, China), the cell clones were stained with 1% crystal violet and ammonium oxalate (Biosharp, Hefei, China) for 25 min, gently rinsed with running water, and then quantified using ImageJ software (Maryland, USA).

Wound-healing assays
After establishing a confluent monolayer (90–100%) in 6-well plates, scratch wounds were inflicted on the BC cell cultures using a 200-µL pipette tip. The cultures were grown in serum-free DMEM with 50 µM of Ob for 48 h after being cleaned with phosphate-buffered saline (PBS; Biosharp, Hefei, China). Cell migration was observed and captured at 0, 24, and 48 h using a microscope (Olympus, Tokyo, Japan).

Transwell invasion assays
Matrigel (Corning, New York, USA) diluted in 100 µL of DMEM was introduced to the top chambers, which were then placed into a cell incubator to create a Matrigel layer. Cell cultures (2×104 cells per chamber) were plated in the top chambers and subsequently treated with serum-free DMEM containing 50 µM of Ob. The bottom chambers contained 600 µL of DMEM enriched with 20% FBS to provide a chemical gradient for cellular invasion. To quantify the invaded cells, the upper chambers membranes were swabbed after 48 h to remove the non-migratory cells. After fixation and staining, the quantity of invading cells on the lower membrane surface was measured using a microscope. For the rescue experiments, the cells were treated with 20 µM of 740Y-P.

Flow cytometry assays
To assess apoptosis, cells were cultured on a plate (2×104 cells/well) and then exposed to 50 µM of Ob for 48 h. After washing with PBS, the cells were detached with an ethylenediaminetetraacetic acid-free trypsin solution. The cells were stained with a mixed solution (5 µL of propidium iodide, 5 µL of Annexin V-allophycocyanin conjugate and 500 µL of binding buffer) (KGI Biotechnology, Nanjing, China), incubated for 8 min in the darkness, and detected by flow cytometry (ACEA Biosciences, Hangzhou, China). The data analysis was conducted using NovoExpress software (ACEA Biosciences, Hangzhou, China). For the rescue experiments, the cells were treated with 20 µM of 740Y-P.

Western blotting (WB) assays
Upon reaching 80% confluence, the cells were treated with Ob, ADR, or 740Y-P for 48 hours. Radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitor cocktails (Beyotime, Shanghai, China) was used to lyse the cells and separate the total proteins. Protein concentrations were measured using the reagents provided in the enhanced Bicinchoninic Acid Protein Assay Kit (Beyotime, Shanghai, China). Proteins (40 µg) were electrophoresed using 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Yeasen Biotechnology, Shanghai, China) and then transferred onto polyvinylidene fluoride membranes. After blocking the membranes for 1.5 h at room temperature with 5% bovine serum albumin (BSA) blocking solution (Solarbio, Beijing, China), the membranes were incubated with specific protein antibodies for a whole night at 4 ℃. After washing with Tris-buffered saline Tween, the membranes were coated with the appropriate secondary antibodies and incubated, and the BeyoECL Plus kit (Beyotime, Shanghai, China) was used to visualize the protein bands. The protein bands in the membranes were observed with Image Lab software (Bio-Rad Laboratories, California, USA) and quantified with ImageJ software. The following relative protein antibodies were used: E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), vimentin (10366-1-AP), B-cell lymphoma 2 (Bcl-2; 68103-1-Ig), Bcl-2-associated X protein (Bax; 50599-2-Ig), caspase-3 (19677-1-AP), and cleaved caspase-3 (25128-1-AP) were purchased from Proteintech (Hubei, China). Protein kinase B (Akt; ab179463) and phosphorylated Akt (p-Akt; ab38449) were provided by Abcam (England, UK). Phosphoinositide 3-kinase (PI3K; bs-10657R), phosphorylated PI3K (p-PI3K; bs-6417R), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; bs-33033M), mouse secondary antibody (bs-0296G-HRP), and rabbit secondary antibody (bs-0295G-HRP) were supplied by Bioss (Beijing, China). β-tubulin (A12289) was acquired from ABclonal (Wuhan, China). β-tubulin and GAPDH were used as loading controls.

Mouse tumor xenograft assays
Female Bagg Albino/c (BALB/c) nude mice (3 weeks old, 14–15 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice were housed in a specific-pathogen-free laboratory, at a temperature of 21–25 ℃, under a 12-h light/12-h dark cycle, and granted unfettered access to food and drink. After acclimatization for a week, the mice were subcutaneously injected in the right flank with 100 µL of a mixture containing PBS, Matrigel, and 1×107 MDA-MB-231 cells (19). Upon the tumor volume reaching approximately 100 mm3, 36 mice were distributed at random into three groups: the control group (saline as a vehicle), the Ob group (6 mg/kg), and the ADR group (2.5 mg/kg) (20). The dose of Ob (6 mg/kg) was selected for the formal study based on a preliminary pilot experiment that evaluated both efficacy and tolerability (Figure S1). The mice received intraperitoneal injections every two days for a total of six treatments, according to their assigned experimental groups. Body weight and tumor volume were recorded bi-daily. Tumor volume was calculated using the following formula: Tumor volume = 1/2 × length × width2. On day 28, 18 mice (n=6 per group) were euthanized. The tumors were excised, weighed, and photographed. Samples from the tumors and main organs (kidney, lung, heart, liver, and spleen) were collected for immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining to evaluate the anti-cancer efficacy of the treatments and systemic toxicity. The remaining 18 mice (n=6 per group) were maintained for survival analysis, and their health status was monitored daily. To evaluate the long-term effects of Ob on survival, the mice were humanely euthanized when the tumor volume surpassed 1,000 mm³ or if severe morbidity was observed.

IHC and H&E staining
Following fixation in 4% paraformaldehyde, tumor and visceral tissues from each group were rinsed with PBS, encased in paraffin, and sliced to a thickness of 5 µm for IHC and H&E staining. For the IHC analysis, after dewaxing and hydrating the tumor tissue slices according to established techniques, the tumor slices were submerged in a citric acid buffer and heated at 100 ℃ for 15 min to promote antigen retrieval. The slices were left to stand for 2 h to reach room temperature. To inhibit intrinsic endogenous peroxidase activity, the slices were submerged in 3% hydrogen peroxide (Solarbio, Beijing, China) for 30 min after being washed with PBS. The slices were incubated with 5% BSA for 45 min to prevent nonspecific binding and were then treated with diluted relative protein antibodies at 4 ℃ overnight. After rinsing three times with PBS, the slices were treated with diluted enzyme-conjugated secondary antibodies for 1 h. Color development was carried out with diaminobenzidine substrate, followed by hematoxylin counterstaining. The slides were then dehydrated with graded ethanol, cleaned with xylene, and mounted with neutral gum. The slides were photographed using a microscope. The following primary antibodies were used: proliferating cell nuclear antigen (PCNA; bs-2007R), Ki67 (bs-52455R), Bax (bs-52316R), and rabbit secondary antibody (bs-0295G-HRP) were obtained from Bioss (Beijing, China). Bcl-2 (ab182858) was provided by Abcam (England, UK). For the H&E staining, the paraffin-embedded tissue slices were first deparaffinized in xylene, and then rehydrated in a succession of ethanol solutions. The tissue sections subsequently underwent H&E staining, dehydration, removal, mounting and light microscopy examination.

Transcriptome sequencing (RNA-seq) analysis
Following treatment with 50 µM of Ob for 48 h, total RNA was extracted and enriched from the MDA-MB-231 cells using an extraction kit. The transcriptome sequencing and analysis were conducted by Novogene (Beijing, China) following conventional protocols. DESeq2 software (Bioconductor Project, Baden-Württemberg, Germany) was used to examine the differentially expressed genes (DEGs). Genes with a fold change greater than 1 and a P value less than 0.05 were defined as statistically significant DEGs.

Real-time quantitative polymerase chain reaction (RT-qPCR) assays
The cells were harvested at 80–90% confluence after treatment with Ob, ADR, or 740Y-P. Total RNA was extracted from the cells using an extraction kit in accordance with the manufacturer’s instructions. A Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Delaware, USA) was used to assess the RNA purity and concentration by measuring ultraviolet absorbance. In accordance with the manufacturer’s instructions, 1000 nanograms of complete RNA were reverse transcribed by applying cDNA Synthesis SuperMix for qPCR. To assess the levels of mature mRNA, RT-qPCR was performed with PerfectStart® Green qPCR SuperMix (TransGen Biotech, Beijing, China) using the CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, California, USA). The cycling settings were set as follows: 94 ℃ for 30 s, followed by 45 cycles at 94 ℃ for 5 s, and 60 ℃ for 30 s. All primers were acquired from Sangon Biotech (Shanghai, China). Each individual gene was examined in triplicate across all samples. To evaluate the relative transcript levels, all the gene Ct values were adjusted with GAPDH and calculated using the ΔΔCt method. The primer sequences used for the experiments are set out in Table 1.

Statistical analysis
The test results were evaluated using GraphPad Prism 8.0. The results are presented as the mean ± standard deviation. Differences among the experimental groups were assessed by one-way analysis of variance, and significant differences among the means were identified using Duncan’s multiple-range test. All the trials were executed at least three times. A P value <0.05 was considered statistically significant.

Ethical statement
Animal experiments were performed under a project license (No. 2024179) granted by the Chengdu University of Traditional Chinese Medicine Laboratory Animal Ethics Committee, in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Results

Ob suppressed the proliferation of BC cells
To assess the anti-tumor efficacy of Ob (Figure 1C), the CCK-8 assay was used to evaluate the cell viability of two human BC cell lines (MDA-MB-231 and MDA-MB-468), with ADR serving as a positive control (Figure 1D). After 48 h of treatment, Ob suppressed the viability of both the MDA-MB-231 (IC50 =50.97 µM) and MDA-MB-468 (IC50 =48.29 µM) cells in a dose-dependent manner. Given that the IC50 values of Ob and ADR on the BC cells were approximately 50 µM and 0.5 µM, respectively, concentrations of 50 µM for Ob and 0.5 µM for ADR were used for the subsequent experiments. Further, the MCF-10A cells were used to evaluate the cytotoxicity of Ob and ADR by CCK-8 assay. While treatment with 75 µM of Ob or 0.5 µM of ADR significantly reduced MCF-10A cell viability (P=0.021), concentrations of up to 50 µM of Ob had no discernible effect compared to the untreated control (Figure 1E). Further assessments of OD values at different times revealed that Ob also inhibited BC cell proliferation in a time-dependent manner (Figure 1F). Additionally, colony formation assays demonstrated that the Ob treatment substantially reduced the development of colonies in the BC cells (P<0.001, Figure 1G). These analyses showed the anti-proliferation function of Ob in BC cells.

Ob inhibited the migration and invasion of BC cells
The wound-healing assays revealed that the wound areas of the BC cells were considerably increased in size compared with those of the control group following treatment of Ob for 24 and 48 h, indicating that Ob treatment independently and significantly hindered the migratory abilities of breast tumor cells (P<0.001, Figure 2A). Similarly, the Transwell invasion assays revealed that Ob exposure for 48 h significantly decreased the number of invading cells (P<0.001, Figure 2B). At the molecular level, the WB analysis revealed that the Ob treatment increased the expression of E-cadherin, while decreasing the expression of N-cadherin and vimentin (Figure 2C). This suggests that the epithelial-mesenchymal transition (EMT) process may be reversed by Ob in BC cells. Collectively, these findings demonstrated that Ob effectively suppressed both the migration and invasion capabilities of the BC cells, possibly via the modulation of key EMT-related proteins.

Ob induced the apoptosis of BC cells
Apoptosis assays by flow cytometry revealed that the proportion of apoptosis in the BC cells was significantly increased in the Ob group compared with the control group (P<0.001, Figure 3A). Moreover, Ob upregulated the expression of cleaved caspase-3 and Bax while downregulating the expression of Bcl-2 in the BC cells (Figure 3B). Together, these findings showed that Ob effectively triggered apoptosis in the BC cells, likely by mediating the regulation of the Bcl-2 family and the activation of caspase-3 proteins.

Ob inhibited the growth of breast tumors in the xenograft mice
Xenograft tumor models were established by injecting MDA-MB-231 cells beneath the skin of BALB/c nude female mice to examine the effect of Ob (Figure 4A). The results revealed that the volume and weight of the subcutaneous tumors were significantly decreased in the Ob group compared with the control group (Figure 4B-4D). The survival status and tumor volume of the mice were monitored to assess the effect of Ob on survival time. Ob administration significantly extended the survival time of the mice (Figure 4E). Notably, no significant body weight loss was observed in the Ob-treated mice. Conversely, the mice receiving ADR exhibited marked weight reduction and lethargy, indicating a more favorable safety profile for Ob (Figure 4F).
The IHC analysis revealed that Ob treatment downregulated the expression of proliferation markers (Ki67 and PCNA) and Bcl-2, while upregulating the expression of Bax (Figure 4G). The H&E staining of the tumor sections showed that the Ob-treated tumors exhibited irregular morphology, increased mitosis, and nuclear heterogeneity (Figure 4H). To further evaluate the safety of Ob, major organs were examined histologically. H&E staining demonstrated that tissue structures of the heart, liver, spleen, lung, and kidney remained intact in the Ob group, with no significant inflammatory infiltration or pathological alterations observed (Figure 4H). Conversely, the ADR group displayed severe cardiotoxicity, characterized by disordered myocardial fiber arrangement, striation loss, and cytoplasmic vacuolation (Figure 4H). In summary, Ob effectively inhibited breast tumor growth in vivo, prolonged survival, and demonstrated a favorable safety profile with no substantial toxicity in major organs.

Ob downregulated the PI3K/Akt pathway in BC cells
To examine the mechanisms by which OB inhibited BC progression, total RNA of the MDA-MB-231 cells from both the control and Ob groups was harvested for sequencing. Based on a threshold of |log2 fold change| >1 and P<0.05, 1,641 DEGs were identified in the Ob-treated cells compared with the controls. Among these, 1,016 genes were found to be more active, and 625 genes were found to be less active (Figure 5A). The Gene Ontology enrichment analysis indicated that the DEGs were significantly linked with transporter activity, signaling receptor binding, extracellular regions, and passive transmembrane transporter activity (Figure 5B). Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the DEGs were primarily enriched in the PI3K/Akt pathway, hematopoietic cell lineage, cytokine-cytokine receptor interactions, rheumatoid arthritis, neuroactive ligand-receptor interactions, and mineral absorption in the Ob groups (Figure 5C). A heatmap of the DEGs related to the PI3K/Akt pathway showed distinct expression patterns between the groups (Figure 5D).
Consistent with the transcriptomic findings, the WB analysis confirmed that Ob downregulated the PI3K/Akt pathway, as shown by reduced p-PI3K/PI3K and p-Akt/Akt ratios in the BC cells (Figure 5E). To validate the specific role of the PI3K/Akt pathway, 740Y-P, an agonist of this pathway, was used to conduct rescue experiments in the BC cells. As expected, the 740Y-P treatment reversed the Ob-induced suppression of the p-PI3K/PI3K and p-Akt/Akt ratios (Figure 5F). Together, these findings demonstrated that Ob suppressed the expression of the PI3K/Akt pathway in BC cells.
Ob reduced proliferation and invasion and triggered apoptosis in BC cells by downregulating the PI3K/Akt pathway.
Based on the established association between PI3K/Akt pathway dysregulation and BC progression, rescue experiments were performed to examine whether this pathway mediated the anti-tumor effects of Ob. Treatment with the PI3K/Akt agonist 740Y-P significantly reversed the suppressive effect of Ob on BC cell growth (Figure 6A). Similarly, 740Y-P attenuated the Ob-induced inhibition of cellular invasion (Figure 6B). At the molecular level, 740Y-P counteracted the Ob-mediated regulation of EMT markers, altering the mRNA expression of E-cadherin, N-cadherin, and vimentin (Figure 6C). Further, in the BC cells, 740Y-P partially rescued Ob-induced apoptosis (Figure 6D), while also reversing the Ob-mediated regulation of Bax and Bcl-2 (Figure 6E). Collectively, these findings demonstrated that Ob reduced proliferation and invasion while promoting apoptosis in the BC cells primarily via the inhibition of the PI3K/Akt pathway.

Discussion
BC patients are commonly treated with chemotherapeutic agents such as doxorubicin, epirubicin, and paclitaxel (21,22). However, approximately 50% of patients develop drug resistance or experience severe adverse effects; thus, better-tolerated and more effective therapies for BC need to be established (23).
Ob, a bioactive compound extracted from Magnolia obovata Thunb., has shown promising anti-tumor activity across multiple cancer types. A thorough understanding of the mechanism underlying Ob is essential for its potential pharmaceutical development. Previous studies have shown that Ob inhibits the proliferation of tumor cells and promotes apoptosis by suppressing the NF-κB and JAK/STAT pathways (15,24). The PI3K/Akt pathway is vital in the regulation of fundamental biological processes, including survival, proliferation, metabolism, protein synthesis, and apoptosis (25,26). Aberrant stimulation of the PI3K/Akt pathway, often seen in multiple carcinomas, is associated with carcinogenesis, progression, treatment resistance, and prognosis (27,28). The hyperactivation of the PI3K/Akt pathway enables tumor cells to evade apoptosis and sustain the proliferative pathway (29). In BC, aberrant PI3K/Akt pathway activation is a key contributor to endocrine therapy resistance, significantly complicating its clinical management (30).
Our findings showed that Ob effectively suppresses the proliferation, migration, and invasion of BC cells in vitro and inhibits the growth of xenograft tumors in vivo. Subsequent mechanistic investigations identified the suppression of PI3K/Akt pathway activation as a key underlying event. Consistent with this, the anti-tumor effects of Ob on cell proliferation and invasion were significantly attenuated by the PI3K/Akt pathway agonist 740Y-P. Collectively, these results suggest that Ob primarily exerted an anti-tumor effect in BC cells by downregulating the PI3K/Akt pathway.
Uncontrolled proliferation is a hallmark of cancer, driving tumorigenesis and metastasis (31). Consequently, targeting dysregulated proliferation has become a cornerstone of BC treatment (32). Ob has previously exhibited anti-proliferative effects across various disease types, including acute myeloid leukemia, colorectal cancer, prostate cancer, and lung cancer (15,33). Consistent with these reports, our study demonstrated that Ob significantly reduced the viability and clonogenic capacity of the BC cells, exerting potent growth-inhibitory effects.
As tumors expand, accelerated cell division often leads to hypoxic and nutrient-deprived microenvironments, activating pro-invasive signaling pathways such as EMT (34). EMT confers migratory and invasive properties on BC cells, promoting distant metastasis and therapy resistance (35). Targeting EMT is therefore crucial for metastasis prevention and treatment optimization (36). The EMT process is characterized by the upregulation of N-cadherin and vimentin, coupled with the loss of E-cadherin function (37). Previous studies have reported that Ob suppresses invasion and migration in liver cancer and tongue squamous cell carcinoma (15). Similarly, we found that Ob delayed wound closure, reduced invasive cell numbers, upregulated E-cadherin, and downregulated N-cadherin and vimentin in the BC cells, collectively indicating the suppression of their migratory and invasive capabilities.
Apoptosis, a tightly regulated physiological process essential for tissue homeostasis, represents a key target for anti-cancer drug development (38). Apoptosis assays by flow cytometry confirmed that Ob significantly increased the apoptotic rates of the BC cells. Caspase-3 activation is a critical early event in apoptosis execution, while Bcl-2 and Bax regulate the mitochondrial apoptotic pathway (39). These results demonstrated that Ob upregulated Bax and cleaved caspase-3 while downregulating Bcl-2, thereby promoting apoptosis in the BC cells.
In vivo, Ob exerted powerful anti-tumor effects. In the MDA-MB-231 cell xenograft mouse models, the intraperitoneal injection of Ob greatly decreased the tumor volume and weight while prolonging the survival time of the mice. IHC analyses of the tumor revealed Ob-reduced expression of Ki67, PCNA, and Bcl-2, along with the increased expression of Bax, suggesting that Ob inhibited tumor development and facilitated apoptosis in BC.
The Ob treatment did not cause significant adverse effects, unlike ADR, which caused significant weight loss and systemic toxicity. H&E staining revealed maintained tissue structures in the heart, liver, spleen, lungs, and kidneys, supporting the favorable biosafety profile of Ob. Despite its widespread use in BC chemotherapy, cumulative cardiotoxicity and the emergence of drug resistance limit the clinical efficacy of ADR. In this study, ADR-induced cardiotoxicity was characterized by structural damage, myofibrillar disarray, myolysis, and cytoplasmic vacuolation. It should be noted that while the 6 mg/kg dose of Ob used in this study was effective in the mice, further dose translation studies are required to establish the equivalent human dosage. To ultimately define the safety and efficacy profile of Ob, future studies incorporating broader dose ranges and larger cohorts are necessary to conclusively determine its dose-response relationship and identify the optimal dose.
RNA-seq analysis was performed to clarify the molecular mechanisms underlying the anti-cancer activity of Ob. The KEGG enrichment analysis revealed a significant association between the DEGs and PI3K/Akt pathway. The WB analysis confirmed that Ob reduced the p-PI3K/PI3K and p-Akt/Akt ratios, while the PI3K/Akt pathway agonist 740Y-P partially restored these ratios, supporting the Ob-mediated suppression of this pathway. Rescue experiments using 740Y-P demonstrated a partial reversal of the inhibitory effects of Ob on proliferation, invasion, and apoptosis, further supporting the involvement of the PI3K/Akt pathway.
Overall, our results indicated that Ob exerts anti-proliferative, anti-invasive, and pro-apoptotic effects in BC, likely through the downregulation of the PI3K/Akt pathway.

Conclusions
In conclusion, this study provides compelling evidence that Ob possesses significant anti-tumor activity against BC in vitro and in vivo. Mechanistically, we demonstrate that Ob exerts its effects primarily by inhibiting the PI3K/Akt pathway, leading to curtailed cellular proliferation, inhibited invasion and metastasis via reversal of the EMT, and promoted mitochondrial-dependent apoptosis through modulation of Bcl-2 family proteins. Taken together, our results elucidate the molecular basis for the anti-cancer activity of Ob and establish its potential as a viable candidate for developing complementary or alternative therapeutic approaches for BC. Nevertheless, future studies are needed to define the optimal in vivo dosing of Ob, evaluate its efficacy in combination with standard chemotherapy, and explore its impact on other resistance-related pathways in BC. Resolving these questions is critical for the clinical translation of these preclinical findings.

Supplementary
The article’s supplementary files as