Norcantharidin induces apoptosis in breast cancer cells by regulating the TNF-α/NF-κB/BCL-2 pathway

Norcantharidin induces apoptosis in breast cancer cells by regulating the TNF-α/NF-κB/BCL-2 pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Norcantharidin induces apoptosis in breast cancer cells by regulating the TNF-α/NF-κB/BCL-2 pathway YuMei Jia, Yang Li, XinYu Jiang, Bo Zhang, Pei Yi Dai, Kai Yang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9539541/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Breast cancer is one of the most common malignant tumors in women worldwide and poses a serious threat to women’s health. Norcantharidin (NCTD), derived from the traditional Chinese medicine Mylabris, has low toxicity and anti-cancer potential; however, its mechanism of action in breast cancer remains unclear. Objective To investigate whether NCTD induces apoptosis in breast cancer cells by regulating the TNF-α/NF-κB/BCL-2 axis. Methods Network pharmacology was used to predict NCTD targets and pathways, and molecular docking was employed to validate binding to key proteins. The proliferation and apoptosis of MCF-7 and MDA-MB-231 cells were assessed using CCK-8 and flow cytometry, respectively, while western blot analysis was performed to detect the expression of TNF-α, IκBα, total p65, nuclear p65, BCL-2, cleaved caspase-3, and c-PARP. Results NCTD shares 20 common targets with breast cancer, which are enriched in the apoptosis and TNF pathways. Molecular docking studies suggest that NCTD binds to TNFR1, IKKα, PARP, and Caspase-3. NCTD inhibits cell proliferation and induces apoptosis; it upregulates TNF-α, IκBα, and the apoptotic executor proteins Caspase-3 and c-PARP, while downregulating nuclear p65 and BCL2, with total p65 remaining unchanged. Conclusion NCTD induced apoptosis in breast cancer cells by regulating the TNF-α/NF-κB/BCL-2 axis, thereby activating apoptotic signals, inhibiting NF-κB activity, and downregulating BCL-2. Norcantharidin (NCTD) breast cancer TNF-α/NF-κB/BCL-2 axis apoptosis signaling pathway molecular dockin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1.Introduction Breast cancer (BC) is the most common malignant tumor in women worldwide [ 1 ] . According to data from the 2022 Global Cancer Statistics Report, there are approximately 2.309 million new cases of breast cancer and approximately 666,000 deaths worldwide, making it the leading cause of cancer-related deaths among women [ 2 ] . Currently, clinical treatment methods for breast cancer primarily include surgery, radiotherapy, chemotherapy, molecularly targeted therapy, immunotherapy, and traditional Chinese medicine [ 3 ] . Among these, surgery combined with chemotherapy serves as the cornerstone of comprehensive breast cancer treatment, and the use of chemotherapeutic agents such as anthracyclines and taxanes has significantly prolonged patient survival [ 4 ] . Radiation therapy, as a key component of local treatment for breast cancer, plays an irreplaceable role in the post-breast-conserving surgery setting and in locally advanced breast cancer [ 5 ] . Targeted and immunotherapy have advanced rapidly, with various targeted drugs,_including PI3K/AKT/mTOR inhibitors and PRAP inhibitors,—having been approved for clinical use by the U.S. Food and Drug Administration (FDA) [ 6 ] . In recent years, breast cancer mortality rates have declined owing to increasingly refined molecular subtyping, widespread adoption of early screening, and continuous advances in precision therapy [ 7 ] . Currently, the 10-year overall survival rate for non-selective breast cancer ranges from 70% to 85% across different countries. However, the clinical management of breast cancer still has numerous limitations and challenges. For example, postoperative flap necrosis remains a common clinical challenge following surgical treatment for breast cancer [ 8 ] ; while CDK4/6 inhibitors combined with endocrine therapy can synergistically inhibit tumor cell proliferation through mechanisms such as inhibiting the G1/S cell cycle transition, activating antitumor immunity, and modulating the tumor microenvironment and metabolism, the adverse reactions they induce—such as cytopenia and cardiotoxicity—cannot be overlooked [ 9 ] . The mechanism of action of immunotherapy accounts for its different safety profile compared to traditional cytotoxic drugs; however, an overactivated immune system can sometimes damage normal cells, leading to various immune-related adverse events [ 10 ] . Overall, issues such as drug resistance, risk of recurrence, and severe side effects remain key factors limiting clinical efficacy [ 11 ] , posing a serious challenge to public health [ 12 , 13 ] . Given the limitations of current clinical treatments, such as drug resistance and adverse reactions, the search for novel, highly effective, and low-toxicity therapeutic agents has become a key focus in breast cancer research. Traditional Chinese medicine (TCM) has demonstrated unique advantages in adjuvant treatment of cancer. TCM and its active ingredients are characterized by multi-targeted, multi-pathway, and low-toxicity properties [ 14 , 15 ] . They exert antitumor effects through various mechanisms, including regulation of cell proliferation, apoptosis, autophagy, and the tumor microenvironment [ 16 ] , and show promising prospects for improving patients’quality of life, alleviating the side effects of radiotherapy and chemotherapy, and delaying the onset of drug resistance. Among these, norcantharidin (NCTD) has attracted the attention of researchers due to its antitumor properties [ 17 ] and has gradually emerged as a potential candidate drug in breast cancer treatment research [ 18 ] . NCTD can inhibit the proliferation, migration, and invasion of breast cancer cells, arrest the cell cycle, and promote apoptosis [ 19 ] . Although NCTD has demonstrated the potential to inhibit tumor growth in various cancer models, its specific mechanisms of action and target networks in breast cancer have not yet been systematically investigated [ 20 ] . Furthermore, given NCTD’s multi-target nature of NCTD, investigating its antitumor mechanisms not only holds academic value, but also offers new possibilities for clinical applications. Currently, preliminary research suggests that NCTD may inhibit tumor cell growth in breast cancer by regulating apoptosis, suppressing angiogenesis, and modulating stress signaling pathways [ 21 , 22 ] . However, research on the specific molecular targets and mechanisms of action of NCTD remain scarce, leaving gaps in our understanding. This study aimed to analyze the potential mechanisms of action of NCTD in breast cancer treatment and to explore the feasibility of its further translation into clinical practice. Our findings indicate that NCTD significantly inhibited the proliferation of breast cancer cells. Mechanistically, NCTD promoted apoptosis by regulating the TNF-α/NF-κB/BCL-2 axis, activating death signals, inhibiting NF-κB activity, and downregulating BCL-2,thereby inducing apoptosis in breast cancer cells. Tumor necrosis factor-α (TNF-α) is a multifunctional pro-inflammatory cytokine that plays a key role in apoptosis, inflammation, and immune regulation [ 23 ] . Studies have shown that TNF-α levels are elevated in the serum of patients with breast cancer and are positively correlated with the number and size of metastatic lesions [ 23 ] . TNF-α initiates downstream signal transduction by binding to the TNFR1 receptor on the cell membrane [ 24 ] , and its upregulation promotes tumor cell proliferation, migration, and invasiveness [ 25 ] . Nuclear factor-kappa B (NF-κB) is a key transcription factor that regulates inflammation, immunity, and cell survival [ 26 ] . Under resting conditions, NF-κB (most commonly a p50/p65 heterodimer) binds to its repressor protein IκBα and remains in the cytoplasm in an inactive form [ 27 ] . When cells receive stimulatory signals such as TNF-α, the IκB kinase (IκK) complex is activated, leading to IκBα phosphorylation. This triggers ubiquitination and degradation, releasing NF-κB into the cell nucleus to initiate the transcription of target genes [ 24 ] . TNF-α is a key activator of the NF-κB pathway [ 28 ] . Concurrently, upon activation, NF-κB regulates the expression of various target genes, including the NFKBIA gene encoding IκBα, thereby establishing a negative feedback regulatory mechanism [ 28 ] . Additionally, NF-κB regulates the expression of anti-apoptotic protein B-cell lymphoma-2 (BCL-2) [ 25 ] . BCL-2 is localized to the mitochondrial membrane and exerts its anti-apoptotic function by inhibiting the release of cytochromec [ 29 ] . In breast cancer tissue, NF-κB is in a state of persistent activation, and its activation level is positively correlated with tumor grade, TNM staging, and lymph node metastasis [ 23 ] . Studies have shown that the expression of TNF-α and NF-κB in breast cancer is correlated, and both are associated with the expression of estrogen receptor (ER) and progesterone receptor (PR), suggesting that the TNF-α/NF-κB signaling axis may play a significant role in the progression of breast cancer [ 23 ] . However, there is currently a lack of systematic research on whether demethylaster induces apoptosis in breast cancer cells by regulating the TNF-α/NF-κB/BCL-2 signaling axis. 2.Materials and Methods 2.1Cell culture Human breast cancer cell lines (MDA-MB-231 and MCF-7) obtained from Wuhan Savier Biotechnology Co., Ltd., and cultured in complete medium at 37°C and 5% CO₂. The breast cancer cell lines used in the experiment (MDA-MB-231 and MCF-7) were between 5 and 7 passages. When the cells reached approximately 80% confluence, MDA-MB-231 and MCF-7 cells were treated with NCTD-containing medium at different concentrations, whereas the control group was cultured in fresh complete medium. 2.2Laboratory reagents NCTD (≥ 98%, catalog number N159736-250mg) were purchased from Aladdin Mall. The MDA-MB-231 culture medium (Cat. No. GZ10503-500M00), and MCF-7 culture medium (Cat. No. GZ10504-500M), trypsin (trypsin-EDTA) (Cat. No. G4001-100ML), Cell Counting Kit-8 (CCK-8) (Cat. No. G4103-5ML), and the Annexin V-FITC Apoptosis Detection Kit (Cat. No. G1511-100T), and RIPA lysis buffer (Cat. No. G2002-100ML), PMSF (Cat. No. G2008-1ML), cocktail (Cat. No. G2006-250UL), Phosphorylation Inhibitor (Cat. No. G2007-1ML), BCA Protein Quantification (Cat. No. G2026-200T), Electrophoresis Buffer (Cat. No. G2081-1L), Transfer Buffer (Cat. No. G2028-1L), and Gel Kit (10%) (Cat. No. G2177-50T), and coagulants (Cat. No. G5036-5ML), Rapid Blocking Solution (Cat. No. G2052-500ML), and ECL Development Reagent (Cat. No. G2014-100ML00) was purchased from Wuhan Savier Biotechnology Co., Ltd. Protein markers, TNF-α antibody (3707), IκBα antibody (4812), total p65 antibody (8248), nuclear p65 antibody (8248), BCL-2 antibody (3498), Caspase-3 antibody, cleaved-PARP antibody, and rabbit secondary antibodies were purchased from Cell Signaling Technology (CST). 2.3Laboratory equipment Laminar flow hood (Model HR1500-IIA2), CO₂ incubator (Model 371GPCM), ultra-low temperature freezer (Model TDE50086FV-ULTS), inverted imaging system (Model CKX41), refrigerator (Model BCD-245TMGH), Freeze Centrifuge (Model FRESC017), Decolorization Shaker (Model TS-300T), Electrophoresis Unit, microplate reader (Model MUITISKAN FU), FongCyte™ research-grade flow cytometer (3 lasers, 8 channels), cell counter, mini chemiluminescence imaging system (Model MiniChemi910), electric heating water bath (Model HWS-12), and cell counter (Model HD-8FL). 2.4Network Pharmacology Research The molecular structures of NCTDs were imported into the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Thresholds were set based on oral bioavailability (OB) and drug-like (DL) parameters to conduct preliminary screening of potential targets. Subsequently, disease-related targets associated with breast cancer (BC) were retrieved from the DisGeNET database. After merging the data and removing duplicates, a set of breast cancer targets was obtained. The screened potential NCTD targets and the breast cancer disease target set were imported into the Venny 2.1.0 online tool to generate a Venn diagram and identify overlapping targets, thereby identifying common targets between NCTD and breast cancer. The overlapping targets were submitted to the STRING database to construct a protein-protein interaction (PPI) network. To elucidate the biological functions and pathways associated with the core targets, GO functional enrichment analysis and KEGG pathway enrichment analysis were performed using the Bioconductor platform, with significance thresholds set at P < 0.05 and Q < 0.05. The top 10 most significantly enriched GO terms (including biological processes, cellular components, and molecular functions) and KEGG pathways were selected, and a bubble plot was generated to visualize the results. 2.5Molecular docking Retrieve protein information from UniProt and protein structures from the SCSB PDB database. The obtained structures were imported into Discovery Studio 2019 for protein structure optimization. This process primarily involves dewatering, hydrogenation, charge balancing, amino acid completion, and side-chain completion. Finally, the optimized protein structure was obtained and exported as a PDB file. Small-molecule structures were obtained from the PubChem website and energy minimization using Discovery Studio 2019, saving the results as a PDB file. Use AutoDock 4.0 to prepare the protein and small-molecule PDBQT files, and docking was performed using AutoDock VINA 1.2.6. Finally, visualization analysis was performed using PyMOL 3.1 and Discovery Studio 2019. 2.6CCK-8 Test The CCK-8 assay was used to evaluate the proliferation-inhibitory effects of NCTD on MDA-MB-231 and MCF-7 cells: Cells in the logarithmic growth phase were digested and counted, then seeded into a 96-well plate at a density of 3 × 10⁴ cells per well and cultured overnight at 37°C and 5% CO₂. When the cells reached approximately 80% confluency, the original culture medium was discarded. For the experimental groups, the medium was replaced with drug-containing medium containing NCTD (5, 10, 20, 40, and 80 µmol/L); for the control group, fresh complete medium without the drug. After 24, 48, and 72 h of treatment, 10 µL of CCK-8 reagent was added to each well, gently mixed by shaking, and incubated for an additional 40 min to 1 hour. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. All experiments were performed in triplicate and independently repeated three times. Cell viability was calculated using the following formula: cell viability (%) = [(experimental group OD value – blank group OD value) / (control group OD value – blank group OD value)] × 100%. Results are expressed as the mean ± standard deviation. 2.7Apoptosis Assay Flow cytometry was used to measure the effects of NCTD on cell cycle distribution and apoptosis of breast cancer cells. MDA-MB-231 and MCF-7 cells (5 × 10⁴ cells per well) were seeded in 6-well plates. Once the cells had adhered and reached approximately 80% confluence, the original culture medium was discarded, and the wells were washed twice with PBS. The experimental group was treated with medium containing different concentrations of NCTD (20, 40, 60, and 80 µmol/L), whereas the control group was cultured in fresh medium. After 48 h, cells from the experimental and control groups were harvested, stained, and analyzed using a flow cytometer to measure PI fluorescence intensity. Statistical analysis and graph generation were performed to determine the cell cycle distribution. 2.8Western blot assay Changes in protein expression in NCTD-treated breast cancer cells were evaluated using by western blot analysis. MDA-MB-231 and MCF-7 cells were treated with NCTD for 48 hours. The cells were lysed on ice for 30 min using RIPA buffer containing protease and phosphatase inhibitors. The lysates were centrifuged at 12,000 rpm for 10 min to collect the supernatants. The protein expression levels were quantified using the BCA method (Thermo Fisher Scientific). 2.9Statistical Analysis Statistical analysis of the data was performed using the GraphPad Prism software (version 10.0). Data are expressed as mean ± standard deviation (SD). Data were analyzed using unpaired Student’s t-test and one-way analysis of variance (ANOVA). The threshold for statistical significance was set at P < 0.05. 3.Result 3.1NCTD and the Prediction of Key Targets in Breast Cancer To systematically elucidate the molecular mechanisms by which NCTD inhibits the malignant progression of breast cancer, we conducted a bioinformatics analysis. We identified 25 NCTD targets from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and 1,844 breast cancer-associated targets from the DisGeNET database. Subsequently, we used the Venny 2.1 tool to generate a Venn diagram, which revealed that NCTD and breast cancer shared 20 overlapping targets (Fig. 1 ). These overlapping targets were further investigated in using Gene Ontology (GO) and KEGG pathway enrichment analysis. Figure 1 Venn diagram showing NCTD and BC targets (blue represents NCTD targets; yellow represents BC targets). The overlapping area indicates common targets 3.2Enrichment Analysis of Overlapping Targets GO enrichment analysis revealed that the molecular functions of the common targets are concentrated in protein-protein interaction interfaces such as cytokine receptor binding, TNF receptor superfamily binding, and BH3 domain binding, which primarily involve the extrinsic apoptosis signaling pathway, NIK/NF-κB signaling, oxidative stress, and cellular responses to TNF (Fig. 2 a). KEGG pathway enrichment analysis indicated that the aforementioned targets were significantly enriched in disease-related pathways such as “Apoptosis”, “TNF Signaling” and “Lipids and Atherosclerosis” (Fig. 2 b). Figure 2 Functional enrichment analysis of common targets shows that BP, CC, and MF are significantly enriched among the top 10 in the GO analysis(Fig. 2 a). KEGG pathway enrichment analysis reveals the top 10 significantly enriched pathways(Fig. 2 b) 3.3Construction of Protein Interaction Networks and Screening of Key Targets Using the STRING database, we analyzed 20 common targets of NCTD and BC to generate a protein-protein interaction (PPI) network (Fig. 3 ). Eight core targets were identified, including: CASP3, TNF, BCL2, NFKBIA, and IL6. Figure 3 The darker the color, the higher the connectivity 3.4Molecular docking simulation To investigate the potential binding patterns of NCTD with key proteins in the TNF-α/NF-κB pathway and apoptotic execution proteins, we performed a molecular docking analysis using AutoDock Vina software and visualized the results using PyMOL software. The results showed that NCTD binds to Caspase-3 and C-PARP, with minimum binding free energies of -4.9 kcal/mol and − 6.4 kcal/mol, respectively, forming hydrogen bonds with TRP-214 and ASN-208 of Caspase-3 (Fig. 4 a); and with HIS-909, ARG-865, and ASN-868 (Fig. 4 b), indicating robust docking of NCTD with both Caspase-3 and C-PARP. Notably, the binding energy for Caspase-3 was weaker than that for C-PARP, suggesting that NCTD exerts a stronger direct effect on C-PARP than on Caspase-3 protein. Furthermore, NCTD exhibited potential binding to TNFR1 and IKKα, with minimum binding free energies of − 5.7 kcal/mol and − 6.5 kcal/mol, respectively. Figure 4 (a–b) Molecular docking of NCTD (yellow) and CASP3 (blue). (c–d) Molecular docking results of NCTD (yellow) and CPARP (blue). (e–f) Molecular docking results of NCTD (yellow) and IKKB (blue). (g–h) Molecular docking results of NCTD (yellow) and TNFR1 (blue) 3.5NCTD inhibited the proliferation of MCF-7 and MDA-MB-231 cells in vitro The experimental results demonstrated dose and time-dependent inhibitory effects of NCTD at different concentrations (5, 10, 20, 40, and 80 µmol/L) on the viability of MCF-7 and MDA-MB-231 cells (Fig. 5 ). Figure 5 NCTD inhibited the proliferation of breast cancer cells in vitro. MCF-7 and MDA-MB-231 cells were treated with different concentrations of NCTD for 24, 72, and 48 hours, and cell viability was then assessed using the CCK-8 assay. Figures A and B show the statistical analysis of changes in cell viability for MCF-7 and MDA-MB-231 cells, respectively, * P < 0.05, ** P < 0.01, *** P < 0.001,**** P < 0.0001.The x-axis represents drug concentration, and the y-axis represents cell viability. Blue indicates 24 hours, purple indicates 48 hours, and red indicates 72 hours. Cell viability in both cell lines showed a decreasing trend with increasing time and drug concentration 3.6NCTD induces early apoptosis in cells NCTD reduces cell viability in a dose- and time-dependent manner. Flow cytometry analysis showed that after 48 h of treatment with NCTD at different concentrations (0, 20, 40, and 60 µmol/L), the early apoptosis rate in MCF-7 and MDA-MB-231 cells gradually increased (Fig. 6 ). Combined with previous KEGG and GO enrichment analyses, these results further support the hypothesis that the mechanism by which NCTD inhibits cell viability may be related to the induction of apoptosis. Figure 6 Assessment of apoptosis and quantitative analysis via flow cytometry in MCF-7 (A) and MDA-MB-231 (B) cells treated with specific concentrations of NCTD. Con, Low, Medium, and High represent the control group, low-concentration group, medium-concentration group, and high-concentration group, respectively; * P < 0.05, ** P < 0.01, *** P < 0.001,**** P < 0.0001 3.7In vitro, NCTD increased the expression levels of the apoptotic proteins Caspase-3 and C-PARP in breast cancer cells To further investigate the inhibitory effects of NCTD on breast cancer (BC), we performed Western blot analyses to detect key apoptotic proteins. As shown in Figs. 7 a–c, NCTD induced a dose-dependent increase in the levels of apoptotic proteins Caspase-3 and C-PARP in MCF-7 and MDA-MB-231 cells. Notably, Caspase-3 levels in MDA-MB-231 cells were slightly higher than those in MCF-7 cells. Combined with the results of the CCK-8 assay, it was speculated that the drug-treated MDA-MB-231 cell group was more sensitive due to the drug-induced upregulation of Caspase-3. This finding was consistent with the results of the CCK-8 assay. Combined with molecular docking experiments, it was hypothesized that NCTD promotes apoptosis by regulating the expression of Caspase-3 and C-PARP through direct or indirect mechanisms. Figure 7 NCTD increased the expression levels of the apoptotic proteins Caspase-3 and C-PARP in vitro(a/d). Western blotting was used to detect changes in Caspase-3 and C-PARP protein levels in MCF-7(e/f)and MDA-MB-231(b/c)cells treated with NCTD versus untreated cells, followed by statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 3.8NCTD regulates the expression of proteins associated with the TNF-α/NF-κB/BCL-2 axis in vitro To further investigate the pro-apoptotic effects of NCTD on breast cancer cells, we focused on the TNF-α/NF-κB/BCL-2 axis and examined changes in the expression of key proteins in this pathway. Western blotting results (Fig. 8 ) showed that TNF-α protein levels increased in a concentration-dependent manner following NCTD treatment, suggesting that the apoptotic signaling pathway was activated. At the same time, there was a significant accumulation of IκBα protein, while total p65 protein levels remained largely unchanged. However, nuclear p65 protein levels decreased in a concentration-dependent manner. In addition, BCL-2 protein expression was downregulated in a concentration-dependent manner. Figure 8 Column b shows the protein expression levels of BCL2, IκBα, nuclear p65, TNF-α, and total p65 in MDA-MB-231 cells from the control group and the NCTD-treated group; Column a displays the corresponding statistical bar charts; Column c shows the expression of BCL2, IκBα, nuclear p65, TNF-α, and total p65 proteins in MCF-7 cells from the blank control and NCTD-treated groups; Column d shows the corresponding statistical bar charts. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. 4.Discussion Breast cancer is one of the most common malignancies among women worldwide, with its incidence rising annually; in most developed countries, it has become the leading cause of cancer in women [ 30 ] . Although existing treatments (such as surgery, radiation therapy, and chemotherapy) can yield favorable outcomes in certain cases, they still face challenges such as drug resistance and severe side effects [ 13 , 20 ] . Therefore, there is an urgent need to explore new therapeutic strategies to improve the survival rates and quality of life of patients with breast cancer. Through modern medical research on traditional Chinese medicine, we have found that various intrinsic bioactive components can achieve significant antitumor effects by interfering with tumor cell signaling pathways and regulating protein expression [ 31 , 32 ] . NCTD, derived from cantharidin, is a synthetic compound that has garnered significant attention owing to its unique antitumor activity. It plays a significant role in reversing tumor drug resistance and has dose-sparing and synergistic effects [ 19 , 33 , 34 ] . However, its role in breast cancer treatment has not yet been thoroughly investigated, and there is an urgent need to elucidate its specific mechanisms of action and target networks. This study aimed to investigate the antitumor activity of NCTD in breast cancer and the underlying molecular mechanisms by combining network pharmacology analysis with in vitro experiments. The results indicate that NCTD shares multiple targets with breast cancer, including IL-6, BCL-2, and CASP3. Based on the results of GO and KEGG pathway analyses, these functional modules further converged on core disease-related pathways such as “apoptosis,” “TNF signaling,” and “lipids and atherosclerosis.” This suggests that the mechanism of action of the drug may be closely related to direct intervention in the initiation of apoptosis, as well as the regulation of inflammatory and stress response signaling. This indicates that NCTD may exert its antitumor effects by modulating the apoptosis and inflammatory signaling pathways. This study provideds a theoretical foundation for the clinical application of NCTD and offers scientific evidence for the development of novel targeted therapeutic strategies for breast cancer. Eight core targets of NCTD and BC were identified by constructing a PPI network. Among them, Caspase-3, encoded by CASP3, is a key protease involved in apoptosis and plays a crucial role in cancer development and progression [ 1 ] . TNF-α, encoded by TNF, initiates downstream signaling through its receptor TNFR1; IκBα, encoded by NFKBIA, is an inhibitor of NF-κB; and BCL2, encoded by BCL2, is an anti-apoptotic molecule downstream of NF-κB. Furthermore, KEGG pathway enrichment analysis revealed significant enrichment of the NF-κB pathway with key genes in this pathway including CHUK (encoding IKKα). IKKα is the catalytic subunit of the IKK that phosphorylates IκBα and serves as a key regulatory node for NF-κB activation. NCTD may inhibit IKKα activity by directly binding to it (as the upstream kinase of IκBα), thereby blocking the phosphorylation and degradation of IκBα and leading to IκBα accumulation and inhibition of NF-κB nuclear translocation. Together, these genes constitute the TNF-α/NF-κB/BCL2 signaling axis, suggesting that they may play a central role in NCTD-induced apoptosis in breast cancer cells. This hypothesis was supported by subsequent molecular docking, CCK-8, flow cytometry, and western blotting experiments. We investigated the potential molecular mechanisms underlying NCTD’s role of NCTD in breast cancer treatment by using molecular docking. These results suggest that NCTD may regulate the TNF-α/NF-κB signaling pathway by directly binding to TNFR1 and IKKα and promoting the cleavage and activation of PARP through direct binding. These findings are consistent with the predictions from network pharmacology. Subsequent Western blot experiments further validated NCTD’s regulatory effects of NCTD on key proteins in the pathway and apoptotic effector proteins. The induction of apoptosis is one of the key mechanisms underlying the antitumor activity of NCTD. For example, it may induce apoptosis in breast cancer cells via the Wnt/β-catenin pathway and promote apoptosis in SMMC-7721 liver cancer cells by activating the JNK signaling pathway and inhibiting NF-κB activity [ 35 , 36 ] . Through bioinformatics analysis, we determined that NCTD acts via a multi-target mechanism, influencing apoptosis and related signaling pathways by upregulating proteins such as C-PARP and CASP3. These findings support the potential of NCTD as a novel antitumor agent, particularly for breast cancer treatment. By activating extrinsic apoptosis pathways and regulating the TNF signaling pathway, NCTD may play a key role in the onset and progression of cancer, providing a theoretical basis for the development of new targeted therapeutic strategies [ 11 , 20 ] . Regarding the regulatory role of NCTD in key proteins in the pathway, as demonstrated by the second western blot experiment, NCTD treatment led to elevated TNF-α levels, which initiated the death signal. Concurrently, the accumulation of IκBα blocks the nuclear translocation of p65 and reduces NF-κB activity, thereby revoking the transcriptional activation of BCL-2. The reduction in BCL-2 protein weakens the cell’s anti-apoptotic capacity, ultimately leading to the activation of caspase-3 and cleavage of PARP, causing the cell to undergo apoptosis. IκBα is an inhibitor of NF-κB, and its accumulation typically results from impaired degradation; while total p65 remains unchanged. A decrease in nuclear p65 indicates that p65 nuclear translocation is inhibited, rather than a decline in p65 expression itself. Together, these findings confirm that NCTD inhibits activation of the NF-κB pathway. BCL-2 is a key anti-apoptotic protein downstream of NF-κB, and its downregulation implies that the cell’s anti-apoptotic barrier is lifted. The activation of death signals coupled with lifting of the anti-apoptotic barrier is the key mechanism by which NCTD induces apoptosis. In summary, NCTD treatment resulted in elevated TNF-α levels, suppressed NF-κB activity, and downregulation of BCL-2. These changes were consistent with the upward trends in C-PARP and cleaved caspase-3 levels, indicating that NCTD induced apoptosis in breast cancer cells by regulating the TNF-α/NF-κB/BCL-2 axis. Building on previous studies that have investigated how NCTD promotes apoptosis in MDA-MB-231 breast cancer cells via the intrinsic mitochondrial pathway [37], this study further demonstrated that NCTD can also activate the extrinsic apoptosis pathway by regulating the TNF-α/NF-κB/BCL-2 axis. These two mechanisms synergistically promoted breast cancer cell apoptosis, providing a new perspective for further exploration of the anti-breast cancer mechanisms of NCTD. Finally, the implications of these findings on the immune mechanisms should not be overlooked. Our study suggests that NCTD may influence immune responses by regulating inflammatory cytokines (such as IL-6) and their roles in the tumor microenvironment. This regulation may have significant implications for immune evasion and drug resistance in breast cancer, suggesting that future research should focus on the potential application of NCTD in immunotherapy. This provides a foundation for the development of new immunotherapies and further advances in-depth research on breast cancer treatment [ 38 , 39 ] . The limitations of this study are reflected in several aspects. First, although the anti-breast cancer activity of NCTD has been validated through in vitro experiments, the lack of in vivo experimental support limits a comprehensive assessment of its efficacy and toxicity under physiological conditions. Second, target prediction relies primarily on public databases, which may introduce batch-to-batch variability in the data and affect the reliability of the results. Furthermore, the study did not validate the findings using clinical samples, and the lack of empirical evidence supporting the correlation between target expression and therapeutic efficacy may weaken the potential for clinical translation. Finally, this study has not yet conducted mRNA-level assays; subsequent studies should validate the transcriptional changes in relevant genes via qPCR to further confirm the regulation of the NF-κB pathway at the transcriptional level. Therefore, future research should delve into the molecular mechanisms underlying the pro-apoptotic effects of the drug and design appropriate in vivo experiments combined with clinical samples for in-depth validation to further confirm the therapeutic efficacy and safety of NCTD. In summary, this study revealed a multi-target mechanism by which NCTD inhibits breast cancer through systematic bioinformatics analysis and cellular experiments. NCTD has potential as an anti-breast cancer drug by regulating apoptosis and inflammatory signaling pathways, particularly through the expression of proteins associated with the TNF-α/NF-κB/BCL-2 axis. However, further in vivo experiments and preclinical studies are required to advance its application in breast cancer treatment. Declarations Acknowledgments The authors would like to thankthe staff at the Clinical Research Center of Hunan Cancer Hospital for their technical and equipment support in the cell culture and western blot experiments. This work was supported by a Hunan Provincial Natural Science Foundation Grant (20250507). The authors declare that the funders have no role in the study design, data collection, analysis, or decision to publish. Funding Sources: This study was funded by a Hunan Provincial Natural Science Foundation Grant (20250507). Apart from providing research funding, the funding source did not participate in the study design and implementation, data collection, analysis, and interpretation; or in the writing and publication of this paper. This study did not receive any financial, service, or in-kind support from a third party other than the aforementioned funding source. Interests: None of the authors has any potential conflicts of interest with any individuals or institutions related to this study. None of the authors have any employment, collaboration, part-time work, consulting, equity ownership, sponsorship, remuneration, or patent licensing relationships with any individual or institution that may benefit directly or indirectly from this study. None of the authors have any potential conflicts of interest that could influence the study design and implementation, data collection, data analysis and interpretation, or manuscript preparation and publication. Data Availability: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Author contributions: Conceptualization and Funding acquisition: [Yang Li]; Methodology ,Formal analysis and investigation: [YuMei Jia] ,[XinYu Jiang]; Writing - original draft preparation: [YuMei Jia]; Writing - review and editing: [YuMei Jia],[XinYu Jiang],[Bo Zhang],[Kai Yang],[PeiYi Dai],[YaQi Li]. All authors contributed to the study conception and design. Cell lines: Human breast cancer MDA-MB-231 cells were purchased from Wuhan Saiver Co., Ltd., lot number: CVCL_0062 MDA-MB-231; human breast cancer MCF-7 cells were purchased from Wuhan Saiver Co., Ltd., lot number: CVCL_0031 MCF-7 Ethics Statement：The cell lines used in this study were purchased from [Wuhan Savier Co., Ltd.]. As no human or animal subjects were involved, no ethical approval was required. References Tang Y, Zhu J, Liu Z (2025) Trends of Female Breast Cancer Burden in China over 25 Years: A Join Point Regression and Age-Period-Cohort Analysis Based on the GBD (1997–2021). Cancer Invest 43(8):610–622. https://doi.org/10.1080/07357907.2025.2554631 Bray F, Laversanne M, Sung H et al (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74(3):229–263. https://doi.org/10.3322/caac.21834 Chen Chao Y, Ping L, Lumin et al (2026) Recent Advances in the Use of Moxibustion for Treating Bone Marrow Suppression Following Chemotherapy for Breast Cancer [J]. J New Chin Med 58(03):193–199. https://doi.org/10.13457/j.cnki.jncm.2026.03.031 Liu Qinqiang C, Peng, Wang Y et al (2026) The Predictive Value of AAPR, Q-Td, and Serum Gal-9 for Cardiotoxicity During Chemotherapy in Breast Cancer Patients [J]. Shaanxi Med J 55(02):239–243 Chen Rongyao L, Yuting (2026) Shi Junyue. Advances in the Application of Artificial Intelligence in Breast Cancer Radiotherapy [J]. Chin J Med Instrum 50(01):7–14 Hu Y, Wang C, Liang H, Li J, Yang Q (2024) The treatment landscape of triple-negative breast cancer. Med Oncol 41(10):236 Published 2024 Aug 29. 10.1007/s12032-024-02456-9 Taylor R, Davis P, Boyages J (2003) Long-term survival of women with breast cancer in New South Wales. Eur J Cancer 39(2):215–222. https://doi.org/10.1016/s0959-8049(02)00486-0 Zhuo Qiuyue L, Yuling Z, Yonglei et al (2026) A Study on the Efficacy of Topical Application of Taohong Siwu Decoction Combined with Rheum and Mirabilite in the Treatment of Postoperative Flap Necrosis in Breast Cancer Patients [J]. Clin Traditional Med Pharmacol 38(01):175–179. https://doi.org/10.16448/j.cjtcm.2026.0137 Wang X (2026) Recent Advances in Research on Cardiotoxicity Induced by Cyclin-Dependent Kinase 4/6 Inhibitors [J]. Chin J Hosp Pharm 46(02):221–227. https://doi.org/10.13286/j.1001-5213.2026.02.15 Lu Xunxi L, Fengling B (2026) Recent Advances in Research on Biomarkers for Predicting the Efficacy of Immune Checkpoint Inhibitors in Breast Cancer [J]. Chin J Clin Experimental Pathol 42(02):212–218. https://doi.org/10.13315/j.cnki.cjcep.2026.02.011 Kageyama S, Ii H, Taniguchi K et al (2018) Mechanisms of Tumor Growth Inhibition by Depletion of γ-Glutamylcyclotransferase (GGCT): A Novel Molecular Target for Anticancer Therapy. Int J Mol Sci. ;19(7):2054. Published 2018 Jul 14. https://doi.org/10.3390/ijms19072054 Xiong X, Zheng LW, Ding Y et al (2025) Breast cancer: pathogenesis and treatments. Signal Transduct Target Ther 10(1):49 Published 2025 Feb 19. 10.1038/s41392-024-02108-4 Shetti D, Lee BH (2021) Pristane-induced mammary carcinomas. Methods Cell Biol 163:187–195. https://doi.org/10.1016/bs.mcb.2020.10.016 Lei Yiming C, Shuoying W, Linlin et al Recent Advances in the Pharmacological Activity and Mechanisms of Action of Glycyrrhizic Acid in Antitumor Therapy [J/OL]. China J Chin Materia Med, 1–16 [2026-04-02]. https://doi.org/10.19540/j.cnki.cjcmm.20260305.706 Lin Yajie, Li Dongdong C (2026) Recent Advances in the Use of Traditional Chinese Medicine to Modulate Tumor-Associated Immune Cells for the Treatment of Colorectal Cancer [J]. J Drug Evaluation Res 49(03):1042–1057 Yu Lirui W, Qi H, Junjie et al (2026) Mechanism of Action of Ethyl Acetate Extract of Wintergreen in Inhibiting Hepatocellular Carcinoma Based on Metabolomics Combined with Network Pharmacology [J]. Chin J Hosp Pharm 46(06):623–630. https://doi.org/10.13286/j.1001-5213.2026.06.03 Yu G, Peng J, Li L, Yu W, He B, Xie B (2024) The role and mechanisms of cordycepin in inhibiting cancer cells. Braz J Med Biol Res 57 e13889. Published 2024 Aug 23. https://doi.org/10.1590/1414-431X2024e13889 Wang Qingqing G, Song L, Zixu et al (2024) Mechanisms of Antitumor Activity and Potential for Combination Therapy of NCTD [J]. J Shenyang Pharm Univ 41(05):598–609. https://doi.org/10.14066/j.cnki.cn21-1349/r.2022.0865 Zhang, Yuanding (2024) A Study on the Antitumor Effects and Mechanisms of NCTD Combined with Cisplatin in Breast Cancer [D]. Jilin Univ. 10.27162/d.cnki.gjlin.2024.007614 Zhong G, Yang C, Liu S et al (2019) Polymers with distinctive anticancer mechanism that kills MDR cancer cells and inhibits tumor metastasis. Biomaterials 199:76–87. https://doi.org/10.1016/j.biomaterials.2019.01.036 Liu, Weiwan (2025) Ren Cui'ai. Investigation of the Effects of Arsenic Trioxide Combined with NCTD on Jurkat Cell Proliferation and Apoptosis via the Wnt/β-catenin Pathway [J]. J Experimental Hematol 33(03):640–647. https://doi.org/10.19746/j.cnki.issn1009-2137.2025.03.003 Du Xiangqing (2015) Study on the Inhibition of Angiogenesis in MCF-7 Breast Cancer Cells by NCTD [J]. Chin Med Nat Prod 30(01):23–24. https://doi.org/10.16368/j.issn.1674-8999.2015.01.008 Zhou XL, Fan W, Yang G, Yu MX (2014) The clinical significance of PR, ER, NF- κ B, and TNF- α in breast cancer. Dis Markers 2014:494581. https://doi.org/10.1155/2014/494581 Chen W, Li Z, Bai L, Lin Y (2011) NF-kappaB in lung cancer, a carcinogenesis mediator and a prevention and therapy target. Front Biosci (Landmark Ed) 16(3):1172–1185 Published 2011 Jan 1. https://doi.org/10.2741/3782 Yang F, Duan S, Liu J et al (2025) Antitumor effects of cannabidiol (CBD) on osteosarcoma by targeting TNF-α/NF-κB/CCL5 signaling axis. Phytomedicine 145:157066. https://doi.org/10.1016/j.phymed.2025.157066 Mukherjee S, Pati S, Dhar S et al (2025) The Role of Nuclear Factor-κB in Cancer Immunity and Immunoediting. Cancer Control 32:10732748251406677. https://doi.org/10.1177/10732748251406677 Zhou H, Liu Y, Cheung LH et al (2010) Characterization and mechanistic studies of a novel melanoma-targeting construct containing IκBa for specific inhibition of nuclear factor-κB activity. Neoplasia 12(10):766–777. https://doi.org/10.1593/neo.10214 Ben-Neriah Y, Karin M (2011) Inflammation meets cancer, with NF-κB as the matchmaker. Nat Immunol 12(8):715–723 Published 2011 Jul 19. https://doi.org/10.1038/ni.2060 Ma W, Hu J (2023) The linear ANRIL transcript P14AS regulates the NF-κB signaling to promote colon cancer progression. Mol Med. ;29(1):162. Published 2023 Dec 1. https://doi.org/10.1186/s10020-023-00761-z Wang Yiwén G, Qingqi Z, Kangyu et al (2026) Recent Advances in the Mechanisms of Traditional Chinese Medicine in Modulating the OPG/RANK/RANKL Signaling Pathway to Intervene in Bone Metastasis of Breast Cancer [J]. Chin Traditional Herb Drugs 57(04):1492–1505 Gao Zongyue X, Zuowu Q, Hewei et al (2025) Recent Advances in the Use of Bioactive Compounds from Traditional Chinese Medicine to Induce Cellular Autophagy for the Treatment of Colorectal Cancer [J]. World J Traditional Chin Med 20(15):2752–2759 Gao Yue S, Baichao Y, Jiannan et al (2025) Recent Advances in the Study of the Anticancer Effects of Traditional Chinese Medicine Polysaccharides on Gynecological Malignancies [J]. J Int Obstet Gynecol 52(06):685–689 Wang Ning X, Xinyu F, Xisheng et al (2025) Recent Advances in the Mechanisms of action of nor-tetramethoxypsilidin in reversing tumor drug resistance [J]. Translational Med J 14(02):255–261 Ma Xiaoyu. A Study on the Attenuation of Cyclophosphamide-Induced Immunosuppression in Mice by NCTD via TRAF6 [D]. Guizhou University (2023) https://doi.org/10.27047/d.cnki.ggudu.2023.003934 Yin Shiliang Z, Lina HJ et al (2019) NCTD Induces Apoptosis in MCF-7 Breast Cancer Cells via the Wnt/β-catenin Signaling Pathway [J]. J Shenyang Med Coll 21(06):500–504. https://doi.org/10.16753/j.cnki.1008-2344.2019.06.003 Cai Huiyun (2013) The Role of the JNK Signaling Pathway in NCTD-Induced Apoptosis of Hepatocellular Carcinoma Cells [D]. Hebei Medical University Yuan X, Qingling J, Xiaoting W et al (2023) NCTD promotes apoptosis in MDA-MB-231 breast cancer cells by inducing autophagosome aggregation [J]. J China Pharm Univ 54(06):757–768 Zhu Q, Zhang K, Cao Y, Hu Y (2024) Adipose stem cell exosomes, stimulated by pro-inflammatory factors, enhance immune evasion in triple-negative breast cancer by modulating the HDAC6/STAT3/PD-L1 pathway through the transporter UCHL1. Cancer Cell Int 24(1):385. https://doi.org/10.1186/s12935-024-03557-1 Coker-Gurkan A, Ozakaltun B, Akdeniz BS et al (2020) Proinflammatory cytokine profile is critical in autocrine GH-triggered curcumin resistance engulf by atiprimod cotreatment in MCF-7 and MDA-MB-231 breast cancer cells. Mol Biol Rep 47(11):8797–8808. 10.1007/s11033-020-05928-z Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9539541","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":632891952,"identity":"8a7952a9-6dd9-4b93-a524-7978388a2c3c","order_by":0,"name":"YuMei Jia","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"YuMei","middleName":"","lastName":"Jia","suffix":""},{"id":632891953,"identity":"cdde8b19-5d2f-47f6-84b8-d5c37e0e8bbe","order_by":1,"name":"Yang Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYBACPmYwZZNAvBY2iJY0UrRAqMOkaGHnMfxc8Ot8njn74QcMH/fUMvDPbiDkMB5j6Zl9t4ste9IMGGc8O84gcecAQS0G0rw9txM33GAwYOY5cIzBQIKAI0G2/ObtOQfUwv6BaC1m0jw/DgC18IBsqSFGC1uZNW9DcuKGMzkFB2ccOMAjcYOAFn7+w5tv8/yxS9xw/PjGBx8O1MnxzyCghYGBw4CBsQ3CPACMIB5C6oGA/QEDwx84r44IHaNgFIyCUTDSAAD9UkCkTLp/2gAAAABJRU5ErkJggg==","orcid":"","institution":"Hunan Cancer Hospital / Xiangya School of Medicine, Central South University Affiliated Cancer Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yang","middleName":"","lastName":"Li","suffix":""},{"id":632891954,"identity":"15108713-4391-49b4-a4ac-4bc54f46135b","order_by":2,"name":"XinYu Jiang","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"XinYu","middleName":"","lastName":"Jiang","suffix":""},{"id":632891955,"identity":"ec694ea1-2eca-42bf-be15-00f7d493477e","order_by":3,"name":"Bo Zhang","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Zhang","suffix":""},{"id":632891956,"identity":"b5bf0cc3-803e-48f1-9124-eb9009e5eb2f","order_by":4,"name":"Pei Yi Dai","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Pei","middleName":"Yi","lastName":"Dai","suffix":""},{"id":632891957,"identity":"ecfbd121-4e7e-4a15-8e1f-b5f88b3015ce","order_by":5,"name":"Kai Yang","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Yang","suffix":""},{"id":632891958,"identity":"8d05af4b-8e6d-40a7-969e-ee2abe216f9b","order_by":6,"name":"YaQi Li","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"YaQi","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-04-27 09:39:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9539541/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9539541/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108829493,"identity":"48d366e1-ace0-4b3a-baf6-9972e93f0930","added_by":"auto","created_at":"2026-05-08 19:01:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":422258,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVenn diagram of NCTD and BC targets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 1\u003c/strong\u003e Venn diagram showing NCTD and BC targets (blue represents NCTD targets; yellow represents BC targets). The overlapping area indicates common targets\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9539541/v1/5f2210110c3944ae6e06aa53.png"},{"id":108977374,"identity":"9f210d93-442b-40e5-8ce7-5e4f8fb018ee","added_by":"auto","created_at":"2026-05-11 11:31:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":663465,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGO and KEGG analysis diagram\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 2\u003c/strong\u003e Functional enrichment analysis of common targets shows that BP, CC, and MF are significantly enriched among the top 10 in the GO analysis(Figure 2a). KEGG pathway enrichment analysis reveals the top 10 significantly enriched pathways(Figure 2b)\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9539541/v1/960127e52a27ea6b558c976e.png"},{"id":108976611,"identity":"b6633bbc-aa90-4a75-ad81-1e5be531b18f","added_by":"auto","created_at":"2026-05-11 11:26:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2207221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePPI network of potential targets for NCTD and BC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 3 The darker the color, the higher the connectivity\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9539541/v1/93a0932ce54b6a1d8c36e437.png"},{"id":108976829,"identity":"a017e9ac-ec9d-4e08-b78e-d32d4bb0f568","added_by":"auto","created_at":"2026-05-11 11:29:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1120471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDocking of NCTD with selected protein molecules\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 4 (a–b) Molecular docking of NCTD (yellow) and CASP3 (blue). (c–d) Molecular docking results of NCTD (yellow) and CPARP (blue). (e–f) Molecular docking results of NCTD (yellow) and IKKB (blue). (g–h) Molecular docking results of NCTD (yellow) and TNFR1 (blue)\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9539541/v1/89c0cff70581d1642349cc6e.png"},{"id":108976819,"identity":"a960f185-cc82-4ba2-af68-172742230941","added_by":"auto","created_at":"2026-05-11 11:28:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":386502,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStatistical graph showing changes in cell viability in MCF-7 (a) and MDA-MB-231 (b) cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 5 NCTD inhibited the proliferation of breast cancer cells in vitro. MCF-7 and MDA-MB-231 cells were treated with different concentrations of NCTD for 24, 72, and 48 hours, and cell viability was then assessed using the CCK-8 assay. Figures A and B show the statistical analysis of changes in cell viability for MCF-7 and MDA-MB-231 cells, respectively, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001,****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.The x-axis represents drug concentration, and the y-axis represents cell viability. Blue indicates 24 hours, purple indicates 48 hours, and red indicates 72 hours. Cell viability in both cell lines showed a decreasing trend with increasing time and drug concentration\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9539541/v1/6a5a58959aa760ed63cbda65.png"},{"id":108976779,"identity":"f73f669b-98de-4886-8cf5-dd755d6750ca","added_by":"auto","created_at":"2026-05-11 11:28:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2135996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNCTD induces early apoptosis in cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure6 Assessment of apoptosis and quantitative analysis via flow cytometry in MCF-7 (A) and MDA-MB-231 (B) cells treated with specific concentrations of NCTD. Con, Low, Medium, and High represent the control group, low-concentration group, medium-concentration group, and high-concentration group, respectively; *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001,****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9539541/v1/f7571f3cbeee60ce924c4c94.png"},{"id":108829496,"identity":"862a8dff-99f2-4051-ad40-2233bda89b00","added_by":"auto","created_at":"2026-05-08 19:01:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":984260,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of Caspase-3 and C-PARP protein levels and grayscale values in breast cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure7 NCTD increased the expression levels of the apoptotic proteins Caspase-3 and C-PARP in vitro(a/d). Western blotting was used to detect changes in Caspase-3 and C-PARP protein levels in MCF-7(e/f)and MDA-MB-231(b/c)cells treated with NCTD versus untreated cells, followed by statistical analysis. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9539541/v1/419ccb4ea91fd604c1a3b9a8.png"},{"id":108829499,"identity":"75371e79-ebc6-461c-8174-d53fcf531a0b","added_by":"auto","created_at":"2026-05-08 19:01:59","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1884002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of BCL2, IκBα, nuclear p65, TNF-α, and total p65 protein levels and grayscale values in breast cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 8 Column b shows the protein expression levels of BCL2, IκBα, nuclear p65, TNF-α, and total p65 in MDA-MB-231 cells from the control group and the NCTD-treated group; Column a displays the corresponding statistical bar charts; Column c shows the expression of BCL2, IκBα, nuclear p65, TNF-α, and total p65 proteins in MCF-7 cells from the blank control and NCTD-treated groups; Column d shows the corresponding statistical bar charts. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-9539541/v1/aa0b6ada0cfd1877effed0c5.png"},{"id":109081340,"identity":"63298d57-ea30-4ae9-9987-d6ee50d3c8b7","added_by":"auto","created_at":"2026-05-12 12:17:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9388257,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9539541/v1/f2a06191-aa96-4900-a2bf-170014ce5a4e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Norcantharidin induces apoptosis in breast cancer cells by regulating the TNF-α/NF-κB/BCL-2 pathway","fulltext":[{"header":"1.Introduction","content":"\u003cp\u003eBreast cancer (BC) is the most common malignant tumor in women worldwide\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. According to data from the 2022 Global Cancer Statistics Report, there are approximately 2.309\u0026nbsp;million new cases of breast cancer and approximately 666,000 deaths worldwide, making it the leading cause of cancer-related deaths among women\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrently, clinical treatment methods for breast cancer primarily include surgery, radiotherapy, chemotherapy, molecularly targeted therapy, immunotherapy, and traditional Chinese medicine\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Among these, surgery combined with chemotherapy serves as the cornerstone of comprehensive breast cancer treatment, and the use of chemotherapeutic agents such as anthracyclines and taxanes has significantly prolonged patient survival\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Radiation therapy, as a key component of local treatment for breast cancer, plays an irreplaceable role in the post-breast-conserving surgery setting and in locally advanced breast cancer\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Targeted and immunotherapy have advanced rapidly, with various targeted drugs,_including PI3K/AKT/mTOR inhibitors and PRAP inhibitors,\u0026mdash;having been approved for clinical use by the U.S. Food and Drug Administration (FDA)\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn recent years, breast cancer mortality rates have declined owing to increasingly refined molecular subtyping, widespread adoption of early screening, and continuous advances in precision therapy\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Currently, the 10-year overall survival rate for non-selective breast cancer ranges from 70% to 85% across different countries. However, the clinical management of breast cancer still has numerous limitations and challenges. For example, postoperative flap necrosis remains a common clinical challenge following surgical treatment for breast cancer\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e; while CDK4/6 inhibitors combined with endocrine therapy can synergistically inhibit tumor cell proliferation through mechanisms such as inhibiting the G1/S cell cycle transition, activating antitumor immunity, and modulating the tumor microenvironment and metabolism, the adverse reactions they induce\u0026mdash;such as cytopenia and cardiotoxicity\u0026mdash;cannot be overlooked\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. The mechanism of action of immunotherapy accounts for its different safety profile compared to traditional cytotoxic drugs; however, an overactivated immune system can sometimes damage normal cells, leading to various immune-related adverse events\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Overall, issues such as drug resistance, risk of recurrence, and severe side effects remain key factors limiting clinical efficacy\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, posing a serious challenge to public health\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven the limitations of current clinical treatments, such as drug resistance and adverse reactions, the search for novel, highly effective, and low-toxicity therapeutic agents has become a key focus in breast cancer research. Traditional Chinese medicine (TCM) has demonstrated unique advantages in adjuvant treatment of cancer. TCM and its active ingredients are characterized by multi-targeted, multi-pathway, and low-toxicity properties\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. They exert antitumor effects through various mechanisms, including regulation of cell proliferation, apoptosis, autophagy, and the tumor microenvironment\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, and show promising prospects for improving patients\u0026rsquo;quality of life, alleviating the side effects of radiotherapy and chemotherapy, and delaying the onset of drug resistance.\u003c/p\u003e \u003cp\u003eAmong these, norcantharidin (NCTD) has attracted the attention of researchers due to its antitumor properties\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e and has gradually emerged as a potential candidate drug in breast cancer treatment research\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNCTD can inhibit the proliferation, migration, and invasion of breast cancer cells, arrest the cell cycle, and promote apoptosis\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Although NCTD has demonstrated the potential to inhibit tumor growth in various cancer models, its specific mechanisms of action and target networks in breast cancer have not yet been systematically investigated\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Furthermore, given NCTD\u0026rsquo;s multi-target nature of NCTD, investigating its antitumor mechanisms not only holds academic value, but also offers new possibilities for clinical applications.\u003c/p\u003e \u003cp\u003eCurrently, preliminary research suggests that NCTD may inhibit tumor cell growth in breast cancer by regulating apoptosis, suppressing angiogenesis, and modulating stress signaling pathways\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. However, research on the specific molecular targets and mechanisms of action of NCTD remain scarce, leaving gaps in our understanding. This study aimed to analyze the potential mechanisms of action of NCTD in breast cancer treatment and to explore the feasibility of its further translation into clinical practice. Our findings indicate that NCTD significantly inhibited the proliferation of breast cancer cells. Mechanistically, NCTD promoted apoptosis by regulating the TNF-α/NF-κB/BCL-2 axis, activating death signals, inhibiting NF-κB activity, and downregulating BCL-2,thereby inducing apoptosis in breast cancer cells.\u003c/p\u003e \u003cp\u003eTumor necrosis factor-α (TNF-α) is a multifunctional pro-inflammatory cytokine that plays a key role in apoptosis, inflammation, and immune regulation\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that TNF-α levels are elevated in the serum of patients with breast cancer and are positively correlated with the number and size of metastatic lesions\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. TNF-α initiates downstream signal transduction by binding to the TNFR1 receptor on the cell membrane\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, and its upregulation promotes tumor cell proliferation, migration, and invasiveness\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNuclear factor-kappa B (NF-κB) is a key transcription factor that regulates inflammation, immunity, and cell survival\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Under resting conditions, NF-κB (most commonly a p50/p65 heterodimer) binds to its repressor protein IκBα and remains in the cytoplasm in an inactive form\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. When cells receive stimulatory signals such as TNF-α, the IκB kinase (IκK) complex is activated, leading to IκBα phosphorylation. This triggers ubiquitination and degradation, releasing NF-κB into the cell nucleus to initiate the transcription of target genes\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. TNF-α is a key activator of the NF-κB pathway\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Concurrently, upon activation, NF-κB regulates the expression of various target genes, including the NFKBIA gene encoding IκBα, thereby establishing a negative feedback regulatory mechanism\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Additionally, NF-κB regulates the expression of anti-apoptotic protein B-cell lymphoma-2 (BCL-2)\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. BCL-2 is localized to the mitochondrial membrane and exerts its anti-apoptotic function by inhibiting the release of cytochromec\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn breast cancer tissue, NF-κB is in a state of persistent activation, and its activation level is positively correlated with tumor grade, TNM staging, and lymph node metastasis\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that the expression of TNF-α and NF-κB in breast cancer is correlated, and both are associated with the expression of estrogen receptor (ER) and progesterone receptor (PR), suggesting that the TNF-α/NF-κB signaling axis may play a significant role in the progression of breast cancer\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. However, there is currently a lack of systematic research on whether demethylaster induces apoptosis in breast cancer cells by regulating the TNF-α/NF-κB/BCL-2 signaling axis.\u003c/p\u003e"},{"header":"2.Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1Cell culture\u003c/h2\u003e \u003cp\u003eHuman breast cancer cell lines (MDA-MB-231 and MCF-7) obtained from Wuhan Savier Biotechnology Co., Ltd., and cultured in complete medium at 37\u0026deg;C and 5% CO₂. The breast cancer cell lines used in the experiment (MDA-MB-231 and MCF-7) were between 5 and 7 passages. When the cells reached approximately 80% confluence, MDA-MB-231 and MCF-7 cells were treated with NCTD-containing medium at different concentrations, whereas the control group was cultured in fresh complete medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2Laboratory reagents\u003c/h2\u003e \u003cp\u003eNCTD (\u0026ge;\u0026thinsp;98%, catalog number N159736-250mg) were purchased from Aladdin Mall. The MDA-MB-231 culture medium (Cat. No. GZ10503-500M00), and MCF-7 culture medium (Cat. No. GZ10504-500M), trypsin (trypsin-EDTA) (Cat. No. G4001-100ML), Cell Counting Kit-8 (CCK-8) (Cat. No. G4103-5ML), and the Annexin V-FITC Apoptosis Detection Kit (Cat. No. G1511-100T), and RIPA lysis buffer (Cat. No. G2002-100ML), PMSF (Cat. No. G2008-1ML), cocktail (Cat. No. G2006-250UL), Phosphorylation Inhibitor (Cat. No. G2007-1ML), BCA Protein Quantification (Cat. No. G2026-200T), Electrophoresis Buffer (Cat. No. G2081-1L), Transfer Buffer (Cat. No. G2028-1L), and Gel Kit (10%) (Cat. No. G2177-50T), and coagulants (Cat. No. G5036-5ML), Rapid Blocking Solution (Cat. No. G2052-500ML), and ECL Development Reagent (Cat. No. G2014-100ML00) was purchased from Wuhan Savier Biotechnology Co., Ltd. Protein markers, TNF-α antibody (3707), IκBα antibody (4812), total p65 antibody (8248), nuclear p65 antibody (8248), BCL-2 antibody (3498), Caspase-3 antibody, cleaved-PARP antibody, and rabbit secondary antibodies were purchased from Cell Signaling Technology (CST).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3Laboratory equipment\u003c/h2\u003e \u003cp\u003eLaminar flow hood (Model HR1500-IIA2), CO₂ incubator (Model 371GPCM), ultra-low temperature freezer (Model TDE50086FV-ULTS), inverted imaging system (Model CKX41), refrigerator (Model BCD-245TMGH), Freeze Centrifuge (Model FRESC017), Decolorization Shaker (Model TS-300T), Electrophoresis Unit, microplate reader (Model MUITISKAN FU), FongCyte\u0026trade; research-grade flow cytometer (3 lasers, 8 channels), cell counter, mini chemiluminescence imaging system (Model MiniChemi910), electric heating water bath (Model HWS-12), and cell counter (Model HD-8FL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4Network Pharmacology Research\u003c/h2\u003e \u003cp\u003eThe molecular structures of NCTDs were imported into the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Thresholds were set based on oral bioavailability (OB) and drug-like (DL) parameters to conduct preliminary screening of potential targets. Subsequently, disease-related targets associated with breast cancer (BC) were retrieved from the DisGeNET database. After merging the data and removing duplicates, a set of breast cancer targets was obtained. The screened potential NCTD targets and the breast cancer disease target set were imported into the Venny 2.1.0 online tool to generate a Venn diagram and identify overlapping targets, thereby identifying common targets between NCTD and breast cancer. The overlapping targets were submitted to the STRING database to construct a protein-protein interaction (PPI) network. To elucidate the biological functions and pathways associated with the core targets, GO functional enrichment analysis and KEGG pathway enrichment analysis were performed using the Bioconductor platform, with significance thresholds set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and Q\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The top 10 most significantly enriched GO terms (including biological processes, cellular components, and molecular functions) and KEGG pathways were selected, and a bubble plot was generated to visualize the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5Molecular docking\u003c/h2\u003e \u003cp\u003eRetrieve protein information from UniProt and protein structures from the SCSB PDB database. The obtained structures were imported into Discovery Studio 2019 for protein structure optimization. This process primarily involves dewatering, hydrogenation, charge balancing, amino acid completion, and side-chain completion. Finally, the optimized protein structure was obtained and exported as a PDB file. Small-molecule structures were obtained from the PubChem website and energy minimization using Discovery Studio 2019, saving the results as a PDB file. Use AutoDock 4.0 to prepare the protein and small-molecule PDBQT files, and docking was performed using AutoDock VINA 1.2.6. Finally, visualization analysis was performed using PyMOL 3.1 and Discovery Studio 2019.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6CCK-8 Test\u003c/h2\u003e \u003cp\u003eThe CCK-8 assay was used to evaluate the proliferation-inhibitory effects of NCTD on MDA-MB-231 and MCF-7 cells: Cells in the logarithmic growth phase were digested and counted, then seeded into a 96-well plate at a density of 3 \u0026times; 10⁴ cells per well and cultured overnight at 37\u0026deg;C and 5% CO₂. When the cells reached approximately 80% confluency, the original culture medium was discarded. For the experimental groups, the medium was replaced with drug-containing medium containing NCTD (5, 10, 20, 40, and 80 \u0026micro;mol/L); for the control group, fresh complete medium without the drug. After 24, 48, and 72 h of treatment, 10 \u0026micro;L of CCK-8 reagent was added to each well, gently mixed by shaking, and incubated for an additional 40 min to 1 hour. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. All experiments were performed in triplicate and independently repeated three times. Cell viability was calculated using the following formula: cell viability (%) = [(experimental group OD value \u0026ndash; blank group OD value) / (control group OD value \u0026ndash; blank group OD value)] \u0026times; 100%. Results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7Apoptosis Assay\u003c/h2\u003e \u003cp\u003eFlow cytometry was used to measure the effects of NCTD on cell cycle distribution and apoptosis of breast cancer cells. MDA-MB-231 and MCF-7 cells (5 \u0026times; 10⁴ cells per well) were seeded in 6-well plates. Once the cells had adhered and reached approximately 80% confluence, the original culture medium was discarded, and the wells were washed twice with PBS. The experimental group was treated with medium containing different concentrations of NCTD (20, 40, 60, and 80 \u0026micro;mol/L), whereas the control group was cultured in fresh medium. After 48 h, cells from the experimental and control groups were harvested, stained, and analyzed using a flow cytometer to measure PI fluorescence intensity. Statistical analysis and graph generation were performed to determine the cell cycle distribution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8Western blot assay\u003c/h2\u003e \u003cp\u003eChanges in protein expression in NCTD-treated breast cancer cells were evaluated using by western blot analysis. MDA-MB-231 and MCF-7 cells were treated with NCTD for 48 hours. The cells were lysed on ice for 30 min using RIPA buffer containing protease and phosphatase inhibitors. The lysates were centrifuged at 12,000 rpm for 10 min to collect the supernatants. The protein expression levels were quantified using the BCA method (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of the data was performed using the GraphPad Prism software (version 10.0). Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Data were analyzed using unpaired Student\u0026rsquo;s t-test and one-way analysis of variance (ANOVA). The threshold for statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3.Result","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1NCTD and the Prediction of Key Targets in Breast Cancer\u003c/h2\u003e \u003cp\u003eTo systematically elucidate the molecular mechanisms by which NCTD inhibits the malignant progression of breast cancer, we conducted a bioinformatics analysis. We identified 25 NCTD targets from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and 1,844 breast cancer-associated targets from the DisGeNET database. Subsequently, we used the Venny 2.1 tool to generate a Venn diagram, which revealed that NCTD and breast cancer shared 20 overlapping targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These overlapping targets were further investigated in using Gene Ontology (GO) and KEGG pathway enrichment analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e Venn diagram showing NCTD and BC targets (blue represents NCTD targets; yellow represents BC targets). The overlapping area indicates common targets\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2Enrichment Analysis of Overlapping Targets\u003c/h2\u003e \u003cp\u003eGO enrichment analysis revealed that the molecular functions of the common targets are concentrated in protein-protein interaction interfaces such as cytokine receptor binding, TNF receptor superfamily binding, and BH3 domain binding, which primarily involve the extrinsic apoptosis signaling pathway, NIK/NF-κB signaling, oxidative stress, and cellular responses to TNF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). KEGG pathway enrichment analysis indicated that the aforementioned targets were significantly enriched in disease-related pathways such as \u0026ldquo;Apoptosis\u0026rdquo;, \u0026ldquo;TNF Signaling\u0026rdquo; and \u0026ldquo;Lipids and Atherosclerosis\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e Functional enrichment analysis of common targets shows that BP, CC, and MF are significantly enriched among the top 10 in the GO analysis(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). KEGG pathway enrichment analysis reveals the top 10 significantly enriched pathways(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3Construction of Protein Interaction Networks and Screening of Key Targets\u003c/h2\u003e \u003cp\u003eUsing the STRING database, we analyzed 20 common targets of NCTD and BC to generate a protein-protein interaction (PPI) network (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Eight core targets were identified, including: CASP3, TNF, BCL2, NFKBIA, and IL6.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e The darker the color, the higher the connectivity\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4Molecular docking simulation\u003c/h2\u003e \u003cp\u003eTo investigate the potential binding patterns of NCTD with key proteins in the TNF-α/NF-κB pathway and apoptotic execution proteins, we performed a molecular docking analysis using AutoDock Vina software and visualized the results using PyMOL software. The results showed that NCTD binds to Caspase-3 and C-PARP, with minimum binding free energies of -4.9 kcal/mol and \u0026minus;\u0026thinsp;6.4 kcal/mol, respectively, forming hydrogen bonds with TRP-214 and ASN-208 of Caspase-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea); and with HIS-909, ARG-865, and ASN-868 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), indicating robust docking of NCTD with both Caspase-3 and C-PARP. Notably, the binding energy for Caspase-3 was weaker than that for C-PARP, suggesting that NCTD exerts a stronger direct effect on C-PARP than on Caspase-3 protein. Furthermore, NCTD exhibited potential binding to TNFR1 and IKKα, with minimum binding free energies of \u0026minus;\u0026thinsp;5.7 kcal/mol and \u0026minus;\u0026thinsp;6.5 kcal/mol, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (a\u0026ndash;b) Molecular docking of NCTD (yellow) and CASP3 (blue). (c\u0026ndash;d) Molecular docking results of NCTD (yellow) and CPARP (blue). (e\u0026ndash;f) Molecular docking results of NCTD (yellow) and IKKB (blue). (g\u0026ndash;h) Molecular docking results of NCTD (yellow) and TNFR1 (blue)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5NCTD inhibited the proliferation of MCF-7 and MDA-MB-231 cells in vitro\u003c/h2\u003e \u003cp\u003eThe experimental results demonstrated dose and time-dependent inhibitory effects of NCTD at different concentrations (5, 10, 20, 40, and 80 \u0026micro;mol/L) on the viability of MCF-7 and MDA-MB-231 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e NCTD inhibited the proliferation of breast cancer cells in vitro. MCF-7 and MDA-MB-231 cells were treated with different concentrations of NCTD for 24, 72, and 48 hours, and cell viability was then assessed using the CCK-8 assay. Figures A and B show the statistical analysis of changes in cell viability for MCF-7 and MDA-MB-231 cells, respectively, *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001,****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.The x-axis represents drug concentration, and the y-axis represents cell viability. Blue indicates 24 hours, purple indicates 48 hours, and red indicates 72 hours. Cell viability in both cell lines showed a decreasing trend with increasing time and drug concentration\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6NCTD induces early apoptosis in cells\u003c/h2\u003e \u003cp\u003eNCTD reduces cell viability in a dose- and time-dependent manner. Flow cytometry analysis showed that after 48 h of treatment with NCTD at different concentrations (0, 20, 40, and 60 \u0026micro;mol/L), the early apoptosis rate in MCF-7 and MDA-MB-231 cells gradually increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Combined with previous KEGG and GO enrichment analyses, these results further support the hypothesis that the mechanism by which NCTD inhibits cell viability may be related to the induction of apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e Assessment of apoptosis and quantitative analysis via flow cytometry in MCF-7 (A) and MDA-MB-231 (B) cells treated with specific concentrations of NCTD. Con, Low, Medium, and High represent the control group, low-concentration group, medium-concentration group, and high-concentration group, respectively; *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001,****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.7In vitro, NCTD increased the expression levels of the apoptotic proteins Caspase-3 and C-PARP in breast cancer cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the inhibitory effects of NCTD on breast cancer (BC), we performed Western blot analyses to detect key apoptotic proteins. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea\u0026ndash;c, NCTD induced a dose-dependent increase in the levels of apoptotic proteins Caspase-3 and C-PARP in MCF-7 and MDA-MB-231 cells. Notably, Caspase-3 levels in MDA-MB-231 cells were slightly higher than those in MCF-7 cells. Combined with the results of the CCK-8 assay, it was speculated that the drug-treated MDA-MB-231 cell group was more sensitive due to the drug-induced upregulation of Caspase-3. This finding was consistent with the results of the CCK-8 assay. Combined with molecular docking experiments, it was hypothesized that NCTD promotes apoptosis by regulating the expression of Caspase-3 and C-PARP through direct or indirect mechanisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e NCTD increased the expression levels of the apoptotic proteins Caspase-3 and C-PARP in vitro(a/d). Western blotting was used to detect changes in Caspase-3 and C-PARP protein levels in MCF-7(e/f)and MDA-MB-231(b/c)cells treated with NCTD versus untreated cells, followed by statistical analysis. *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.8NCTD regulates the expression of proteins associated with the TNF-α/NF-κB/BCL-2 axis in vitro\u003c/h2\u003e \u003cp\u003eTo further investigate the pro-apoptotic effects of NCTD on breast cancer cells, we focused on the TNF-α/NF-κB/BCL-2 axis and examined changes in the expression of key proteins in this pathway. Western blotting results (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) showed that TNF-α protein levels increased in a concentration-dependent manner following NCTD treatment, suggesting that the apoptotic signaling pathway was activated.\u003c/p\u003e \u003cp\u003eAt the same time, there was a significant accumulation of IκBα protein, while total p65 protein levels remained largely unchanged. However, nuclear p65 protein levels decreased in a concentration-dependent manner. In addition, BCL-2 protein expression was downregulated in a concentration-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e Column b shows the protein expression levels of BCL2, IκBα, nuclear p65, TNF-α, and total p65 in MDA-MB-231 cells from the control group and the NCTD-treated group; Column a displays the corresponding statistical bar charts; Column c shows the expression of BCL2, IκBα, nuclear p65, TNF-α, and total p65 proteins in MCF-7 cells from the blank control and NCTD-treated groups; Column d shows the corresponding statistical bar charts. *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"4.Discussion","content":"\u003cp\u003eBreast cancer is one of the most common malignancies among women worldwide, with its incidence rising annually; in most developed countries, it has become the leading cause of cancer in women\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Although existing treatments (such as surgery, radiation therapy, and chemotherapy) can yield favorable outcomes in certain cases, they still face challenges such as drug resistance and severe side effects\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Therefore, there is an urgent need to explore new therapeutic strategies to improve the survival rates and quality of life of patients with breast cancer.\u003c/p\u003e \u003cp\u003eThrough modern medical research on traditional Chinese medicine, we have found that various intrinsic bioactive components can achieve significant antitumor effects by interfering with tumor cell signaling pathways and regulating protein expression\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. NCTD, derived from cantharidin, is a synthetic compound that has garnered significant attention owing to its unique antitumor activity. It plays a significant role in reversing tumor drug resistance and has dose-sparing and synergistic effects\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. However, its role in breast cancer treatment has not yet been thoroughly investigated, and there is an urgent need to elucidate its specific mechanisms of action and target networks.\u003c/p\u003e \u003cp\u003eThis study aimed to investigate the antitumor activity of NCTD in breast cancer and the underlying molecular mechanisms by combining network pharmacology analysis with in vitro experiments. The results indicate that NCTD shares multiple targets with breast cancer, including IL-6, BCL-2, and CASP3. Based on the results of GO and KEGG pathway analyses, these functional modules further converged on core disease-related pathways such as \u0026ldquo;apoptosis,\u0026rdquo; \u0026ldquo;TNF signaling,\u0026rdquo; and \u0026ldquo;lipids and atherosclerosis.\u0026rdquo; This suggests that the mechanism of action of the drug may be closely related to direct intervention in the initiation of apoptosis, as well as the regulation of inflammatory and stress response signaling. This indicates that NCTD may exert its antitumor effects by modulating the apoptosis and inflammatory signaling pathways. This study provideds a theoretical foundation for the clinical application of NCTD and offers scientific evidence for the development of novel targeted therapeutic strategies for breast cancer.\u003c/p\u003e \u003cp\u003eEight core targets of NCTD and BC were identified by constructing a PPI network. Among them, Caspase-3, encoded by CASP3, is a key protease involved in apoptosis and plays a crucial role in cancer development and progression\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. TNF-α, encoded by TNF, initiates downstream signaling through its receptor TNFR1; IκBα, encoded by NFKBIA, is an inhibitor of NF-κB; and BCL2, encoded by BCL2, is an anti-apoptotic molecule downstream of NF-κB. Furthermore, KEGG pathway enrichment analysis revealed significant enrichment of the NF-κB pathway with key genes in this pathway including CHUK (encoding IKKα). IKKα is the catalytic subunit of the IKK that phosphorylates IκBα and serves as a key regulatory node for NF-κB activation. NCTD may inhibit IKKα activity by directly binding to it (as the upstream kinase of IκBα), thereby blocking the phosphorylation and degradation of IκBα and leading to IκBα accumulation and inhibition of NF-κB nuclear translocation. Together, these genes constitute the TNF-α/NF-κB/BCL2 signaling axis, suggesting that they may play a central role in NCTD-induced apoptosis in breast cancer cells. This hypothesis was supported by subsequent molecular docking, CCK-8, flow cytometry, and western blotting experiments.\u003c/p\u003e \u003cp\u003eWe investigated the potential molecular mechanisms underlying NCTD\u0026rsquo;s role of NCTD in breast cancer treatment by using molecular docking. These results suggest that NCTD may regulate the TNF-α/NF-κB signaling pathway by directly binding to TNFR1 and IKKα and promoting the cleavage and activation of PARP through direct binding. These findings are consistent with the predictions from network pharmacology. Subsequent Western blot experiments further validated NCTD\u0026rsquo;s regulatory effects of NCTD on key proteins in the pathway and apoptotic effector proteins.\u003c/p\u003e \u003cp\u003eThe induction of apoptosis is one of the key mechanisms underlying the antitumor activity of NCTD. For example, it may induce apoptosis in breast cancer cells via the Wnt/β-catenin pathway and promote apoptosis in SMMC-7721 liver cancer cells by activating the JNK signaling pathway and inhibiting NF-κB activity\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Through bioinformatics analysis, we determined that NCTD acts via a multi-target mechanism, influencing apoptosis and related signaling pathways by upregulating proteins such as C-PARP and CASP3. These findings support the potential of NCTD as a novel antitumor agent, particularly for breast cancer treatment. By activating extrinsic apoptosis pathways and regulating the TNF signaling pathway, NCTD may play a key role in the onset and progression of cancer, providing a theoretical basis for the development of new targeted therapeutic strategies\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRegarding the regulatory role of NCTD in key proteins in the pathway, as demonstrated by the second western blot experiment, NCTD treatment led to elevated TNF-α levels, which initiated the death signal. Concurrently, the accumulation of IκBα blocks the nuclear translocation of p65 and reduces NF-κB activity, thereby revoking the transcriptional activation of BCL-2. The reduction in BCL-2 protein weakens the cell\u0026rsquo;s anti-apoptotic capacity, ultimately leading to the activation of caspase-3 and cleavage of PARP, causing the cell to undergo apoptosis.\u003c/p\u003e \u003cp\u003eIκBα is an inhibitor of NF-κB, and its accumulation typically results from impaired degradation; while total p65 remains unchanged. A decrease in nuclear p65 indicates that p65 nuclear translocation is inhibited, rather than a decline in p65 expression itself. Together, these findings confirm that NCTD inhibits activation of the NF-κB pathway. BCL-2 is a key anti-apoptotic protein downstream of NF-κB, and its downregulation implies that the cell\u0026rsquo;s anti-apoptotic barrier is lifted. The activation of death signals coupled with lifting of the anti-apoptotic barrier is the key mechanism by which NCTD induces apoptosis.\u003c/p\u003e \u003cp\u003eIn summary, NCTD treatment resulted in elevated TNF-α levels, suppressed NF-κB activity, and downregulation of BCL-2. These changes were consistent with the upward trends in C-PARP and cleaved caspase-3 levels, indicating that NCTD induced apoptosis in breast cancer cells by regulating the TNF-α/NF-κB/BCL-2 axis. Building on previous studies that have investigated how NCTD promotes apoptosis in MDA-MB-231 breast cancer cells via the intrinsic mitochondrial pathway [37], this study further demonstrated that NCTD can also activate the extrinsic apoptosis pathway by regulating the TNF-α/NF-κB/BCL-2 axis. These two mechanisms synergistically promoted breast cancer cell apoptosis, providing a new perspective for further exploration of the anti-breast cancer mechanisms of NCTD.\u003c/p\u003e \u003cp\u003eFinally, the implications of these findings on the immune mechanisms should not be overlooked. Our study suggests that NCTD may influence immune responses by regulating inflammatory cytokines (such as IL-6) and their roles in the tumor microenvironment. This regulation may have significant implications for immune evasion and drug resistance in breast cancer, suggesting that future research should focus on the potential application of NCTD in immunotherapy. This provides a foundation for the development of new immunotherapies and further advances in-depth research on breast cancer treatment\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe limitations of this study are reflected in several aspects. First, although the anti-breast cancer activity of NCTD has been validated through in vitro experiments, the lack of in vivo experimental support limits a comprehensive assessment of its efficacy and toxicity under physiological conditions. Second, target prediction relies primarily on public databases, which may introduce batch-to-batch variability in the data and affect the reliability of the results. Furthermore, the study did not validate the findings using clinical samples, and the lack of empirical evidence supporting the correlation between target expression and therapeutic efficacy may weaken the potential for clinical translation. Finally, this study has not yet conducted mRNA-level assays; subsequent studies should validate the transcriptional changes in relevant genes via qPCR to further confirm the regulation of the NF-κB pathway at the transcriptional level. Therefore, future research should delve into the molecular mechanisms underlying the pro-apoptotic effects of the drug and design appropriate in vivo experiments combined with clinical samples for in-depth validation to further confirm the therapeutic efficacy and safety of NCTD.\u003c/p\u003e \u003cp\u003eIn summary, this study revealed a multi-target mechanism by which NCTD inhibits breast cancer through systematic bioinformatics analysis and cellular experiments. NCTD has potential as an anti-breast cancer drug by regulating apoptosis and inflammatory signaling pathways, particularly through the expression of proteins associated with the TNF-α/NF-κB/BCL-2 axis. However, further in vivo experiments and preclinical studies are required to advance its application in breast cancer treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thankthe staff at the Clinical Research Center of Hunan Cancer Hospital for their technical and equipment support in the cell culture and western blot experiments.\u003c/p\u003e\n\u003cp\u003eThis work was supported by a Hunan Provincial Natural Science Foundation Grant (20250507). The authors declare that the funders have no role in the study design, data collection, analysis, or decision to publish.\u003c/p\u003e\n\u003cp\u003eFunding Sources:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was funded by a Hunan Provincial Natural Science Foundation Grant (20250507). Apart from providing research funding, the funding source did not participate in the study design and implementation, data collection, analysis, and interpretation; or in the writing and publication of this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study did not receive any financial, service, or in-kind support from a third party other than the aforementioned funding source.\u003c/p\u003e\n\u003cp\u003eInterests:\u003c/p\u003e\n\u003cp\u003eNone of the authors has any potential conflicts of interest with any individuals or institutions related to this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNone of the authors have any employment, collaboration, part-time work, consulting, equity ownership, sponsorship, remuneration, or patent licensing relationships with any individual or institution that may benefit directly or indirectly from this study. None of the authors have any potential conflicts of interest that could influence the study design and implementation, data collection, data analysis and interpretation, or manuscript preparation and publication.\u003c/p\u003e\n\u003cp\u003eData Availability:\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eAuthor contributions: Conceptualization and Funding acquisition: [Yang Li]; Methodology ,Formal analysis and investigation: [YuMei Jia] ,[XinYu Jiang]; Writing - original draft preparation: [YuMei Jia]; Writing - review and editing: [YuMei Jia],[XinYu Jiang],[Bo Zhang],[Kai Yang],[PeiYi Dai],[YaQi Li]. All authors contributed to the study conception and design.\u003c/p\u003e\n\u003cp\u003eCell lines: Human breast cancer MDA-MB-231 cells were purchased from Wuhan Saiver Co., Ltd., lot number: CVCL_0062 MDA-MB-231; human breast cancer MCF-7 cells were purchased from Wuhan Saiver Co., Ltd., lot number: CVCL_0031 MCF-7\u003c/p\u003e\n\u003cp\u003eEthics Statement：The cell lines used in this study were purchased from [Wuhan Savier Co., Ltd.]. As no human or animal subjects were involved, no ethical approval was required.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTang Y, Zhu J, Liu Z (2025) Trends of Female Breast Cancer Burden in China over 25 Years: A Join Point Regression and Age-Period-Cohort Analysis Based on the GBD (1997\u0026ndash;2021). Cancer Invest 43(8):610\u0026ndash;622. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/07357907.2025.2554631\u003c/span\u003e\u003cspan address=\"10.1080/07357907.2025.2554631\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBray F, Laversanne M, Sung H et al (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74(3):229\u0026ndash;263. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3322/caac.21834\u003c/span\u003e\u003cspan address=\"10.3322/caac.21834\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Chao Y, Ping L, Lumin et al (2026) Recent Advances in the Use of Moxibustion for Treating Bone Marrow Suppression Following Chemotherapy for Breast Cancer [J]. J New Chin Med 58(03):193\u0026ndash;199. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.13457/j.cnki.jncm.2026.03.031\u003c/span\u003e\u003cspan address=\"10.13457/j.cnki.jncm.2026.03.031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Qinqiang C, Peng, Wang Y et al (2026) The Predictive Value of AAPR, Q-Td, and Serum Gal-9 for Cardiotoxicity During Chemotherapy in Breast Cancer Patients [J]. Shaanxi Med J 55(02):239\u0026ndash;243\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Rongyao L, Yuting (2026) Shi Junyue. Advances in the Application of Artificial Intelligence in Breast Cancer Radiotherapy [J]. Chin J Med Instrum 50(01):7\u0026ndash;14\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu Y, Wang C, Liang H, Li J, Yang Q (2024) The treatment landscape of triple-negative breast cancer. Med Oncol 41(10):236 Published 2024 Aug 29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12032-024-02456-9\u003c/span\u003e\u003cspan address=\"10.1007/s12032-024-02456-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaylor R, Davis P, Boyages J (2003) Long-term survival of women with breast cancer in New South Wales. Eur J Cancer 39(2):215\u0026ndash;222. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0959-8049(02)00486-0\u003c/span\u003e\u003cspan address=\"10.1016/s0959-8049(02)00486-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhuo Qiuyue L, Yuling Z, Yonglei et al (2026) A Study on the Efficacy of Topical Application of Taohong Siwu Decoction Combined with Rheum and Mirabilite in the Treatment of Postoperative Flap Necrosis in Breast Cancer Patients [J]. Clin Traditional Med Pharmacol 38(01):175\u0026ndash;179. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.16448/j.cjtcm.2026.0137\u003c/span\u003e\u003cspan address=\"10.16448/j.cjtcm.2026.0137\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X (2026) Recent Advances in Research on Cardiotoxicity Induced by Cyclin-Dependent Kinase 4/6 Inhibitors [J]. Chin J Hosp Pharm 46(02):221\u0026ndash;227. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.13286/j.1001-5213.2026.02.15\u003c/span\u003e\u003cspan address=\"10.13286/j.1001-5213.2026.02.15\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu Xunxi L, Fengling B (2026) Recent Advances in Research on Biomarkers for Predicting the Efficacy of Immune Checkpoint Inhibitors in Breast Cancer [J]. Chin J Clin Experimental Pathol 42(02):212\u0026ndash;218. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.13315/j.cnki.cjcep.2026.02.011\u003c/span\u003e\u003cspan address=\"10.13315/j.cnki.cjcep.2026.02.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKageyama S, Ii H, Taniguchi K et al (2018) Mechanisms of Tumor Growth Inhibition by Depletion of γ-Glutamylcyclotransferase (GGCT): A Novel Molecular Target for Anticancer Therapy. Int J Mol Sci. ;19(7):2054. Published 2018 Jul 14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms19072054\u003c/span\u003e\u003cspan address=\"10.3390/ijms19072054\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiong X, Zheng LW, Ding Y et al (2025) Breast cancer: pathogenesis and treatments. Signal Transduct Target Ther 10(1):49 Published 2025 Feb 19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41392-024-02108-4\u003c/span\u003e\u003cspan address=\"10.1038/s41392-024-02108-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShetti D, Lee BH (2021) Pristane-induced mammary carcinomas. Methods Cell Biol 163:187\u0026ndash;195. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/bs.mcb.2020.10.016\u003c/span\u003e\u003cspan address=\"10.1016/bs.mcb.2020.10.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLei Yiming C, Shuoying W, Linlin et al Recent Advances in the Pharmacological Activity and Mechanisms of Action of Glycyrrhizic Acid in Antitumor Therapy [J/OL]. China J Chin Materia Med, 1\u0026ndash;16 [2026-04-02]. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.19540/j.cnki.cjcmm.20260305.706\u003c/span\u003e\u003cspan address=\"10.19540/j.cnki.cjcmm.20260305.706\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin Yajie, Li Dongdong C (2026) Recent Advances in the Use of Traditional Chinese Medicine to Modulate Tumor-Associated Immune Cells for the Treatment of Colorectal Cancer [J]. J Drug Evaluation Res 49(03):1042\u0026ndash;1057\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu Lirui W, Qi H, Junjie et al (2026) Mechanism of Action of Ethyl Acetate Extract of Wintergreen in Inhibiting Hepatocellular Carcinoma Based on Metabolomics Combined with Network Pharmacology [J]. Chin J Hosp Pharm 46(06):623\u0026ndash;630. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.13286/j.1001-5213.2026.06.03\u003c/span\u003e\u003cspan address=\"10.13286/j.1001-5213.2026.06.03\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu G, Peng J, Li L, Yu W, He B, Xie B (2024) The role and mechanisms of cordycepin in inhibiting cancer cells. Braz J Med Biol Res 57 e13889. Published 2024 Aug 23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/1414-431X2024e13889\u003c/span\u003e\u003cspan address=\"10.1590/1414-431X2024e13889\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Qingqing G, Song L, Zixu et al (2024) Mechanisms of Antitumor Activity and Potential for Combination Therapy of NCTD [J]. J Shenyang Pharm Univ 41(05):598\u0026ndash;609. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.14066/j.cnki.cn21-1349/r.2022.0865\u003c/span\u003e\u003cspan address=\"10.14066/j.cnki.cn21-1349/r.2022.0865\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Yuanding (2024) A Study on the Antitumor Effects and Mechanisms of NCTD Combined with Cisplatin in Breast Cancer [D]. Jilin Univ. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.27162/d.cnki.gjlin.2024.007614\u003c/span\u003e\u003cspan address=\"10.27162/d.cnki.gjlin.2024.007614\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhong G, Yang C, Liu S et al (2019) Polymers with distinctive anticancer mechanism that kills MDR cancer cells and inhibits tumor metastasis. Biomaterials 199:76\u0026ndash;87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biomaterials.2019.01.036\u003c/span\u003e\u003cspan address=\"10.1016/j.biomaterials.2019.01.036\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Weiwan (2025) Ren Cui'ai. Investigation of the Effects of Arsenic Trioxide Combined with NCTD on Jurkat Cell Proliferation and Apoptosis via the Wnt/β-catenin Pathway [J]. J Experimental Hematol 33(03):640\u0026ndash;647. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.19746/j.cnki.issn1009-2137.2025.03.003\u003c/span\u003e\u003cspan address=\"10.19746/j.cnki.issn1009-2137.2025.03.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu Xiangqing (2015) Study on the Inhibition of Angiogenesis in MCF-7 Breast Cancer Cells by NCTD [J]. Chin Med Nat Prod 30(01):23\u0026ndash;24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.16368/j.issn.1674-8999.2015.01.008\u003c/span\u003e\u003cspan address=\"10.16368/j.issn.1674-8999.2015.01.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou XL, Fan W, Yang G, Yu MX (2014) The clinical significance of PR, ER, NF- κ B, and TNF- α in breast cancer. Dis Markers 2014:494581. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2014/494581\u003c/span\u003e\u003cspan address=\"10.1155/2014/494581\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen W, Li Z, Bai L, Lin Y (2011) NF-kappaB in lung cancer, a carcinogenesis mediator and a prevention and therapy target. Front Biosci (Landmark Ed) 16(3):1172\u0026ndash;1185 Published 2011 Jan 1. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2741/3782\u003c/span\u003e\u003cspan address=\"10.2741/3782\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang F, Duan S, Liu J et al (2025) Antitumor effects of cannabidiol (CBD) on osteosarcoma by targeting TNF-α/NF-κB/CCL5 signaling axis. Phytomedicine 145:157066. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.phymed.2025.157066\u003c/span\u003e\u003cspan address=\"10.1016/j.phymed.2025.157066\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMukherjee S, Pati S, Dhar S et al (2025) The Role of Nuclear Factor-κB in Cancer Immunity and Immunoediting. Cancer Control 32:10732748251406677. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/10732748251406677\u003c/span\u003e\u003cspan address=\"10.1177/10732748251406677\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou H, Liu Y, Cheung LH et al (2010) Characterization and mechanistic studies of a novel melanoma-targeting construct containing IκBa for specific inhibition of nuclear factor-κB activity. Neoplasia 12(10):766\u0026ndash;777. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1593/neo.10214\u003c/span\u003e\u003cspan address=\"10.1593/neo.10214\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBen-Neriah Y, Karin M (2011) Inflammation meets cancer, with NF-κB as the matchmaker. Nat Immunol 12(8):715\u0026ndash;723 Published 2011 Jul 19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ni.2060\u003c/span\u003e\u003cspan address=\"10.1038/ni.2060\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa W, Hu J (2023) The linear ANRIL transcript P14AS regulates the NF-κB signaling to promote colon cancer progression. Mol Med. ;29(1):162. Published 2023 Dec 1. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s10020-023-00761-z\u003c/span\u003e\u003cspan address=\"10.1186/s10020-023-00761-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Yiw\u0026eacute;n G, Qingqi Z, Kangyu et al (2026) Recent Advances in the Mechanisms of Traditional Chinese Medicine in Modulating the OPG/RANK/RANKL Signaling Pathway to Intervene in Bone Metastasis of Breast Cancer [J]. Chin Traditional Herb Drugs 57(04):1492\u0026ndash;1505\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao Zongyue X, Zuowu Q, Hewei et al (2025) Recent Advances in the Use of Bioactive Compounds from Traditional Chinese Medicine to Induce Cellular Autophagy for the Treatment of Colorectal Cancer [J]. World J Traditional Chin Med 20(15):2752\u0026ndash;2759\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao Yue S, Baichao Y, Jiannan et al (2025) Recent Advances in the Study of the Anticancer Effects of Traditional Chinese Medicine Polysaccharides on Gynecological Malignancies [J]. J Int Obstet Gynecol 52(06):685\u0026ndash;689\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Ning X, Xinyu F, Xisheng et al (2025) Recent Advances in the Mechanisms of action of nor-tetramethoxypsilidin in reversing tumor drug resistance [J]. Translational Med J 14(02):255\u0026ndash;261\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa Xiaoyu. A Study on the Attenuation of Cyclophosphamide-Induced Immunosuppression in Mice by NCTD via TRAF6 [D]. Guizhou University (2023) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.27047/d.cnki.ggudu.2023.003934\u003c/span\u003e\u003cspan address=\"10.27047/d.cnki.ggudu.2023.003934\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin Shiliang Z, Lina HJ et al (2019) NCTD Induces Apoptosis in MCF-7 Breast Cancer Cells via the Wnt/β-catenin Signaling Pathway [J]. J Shenyang Med Coll 21(06):500\u0026ndash;504. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.16753/j.cnki.1008-2344.2019.06.003\u003c/span\u003e\u003cspan address=\"10.16753/j.cnki.1008-2344.2019.06.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai Huiyun (2013) The Role of the JNK Signaling Pathway in NCTD-Induced Apoptosis of Hepatocellular Carcinoma Cells [D]. Hebei Medical University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan X, Qingling J, Xiaoting W et al (2023) NCTD promotes apoptosis in MDA-MB-231 breast cancer cells by inducing autophagosome aggregation [J]. J China Pharm Univ 54(06):757\u0026ndash;768\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Q, Zhang K, Cao Y, Hu Y (2024) Adipose stem cell exosomes, stimulated by pro-inflammatory factors, enhance immune evasion in triple-negative breast cancer by modulating the HDAC6/STAT3/PD-L1 pathway through the transporter UCHL1. Cancer Cell Int 24(1):385. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12935-024-03557-1\u003c/span\u003e\u003cspan address=\"10.1186/s12935-024-03557-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoker-Gurkan A, Ozakaltun B, Akdeniz BS et al (2020) Proinflammatory cytokine profile is critical in autocrine GH-triggered curcumin resistance engulf by atiprimod cotreatment in MCF-7 and MDA-MB-231 breast cancer cells. Mol Biol Rep 47(11):8797\u0026ndash;8808. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11033-020-05928-z\u003c/span\u003e\u003cspan address=\"10.1007/s11033-020-05928-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Norcantharidin (NCTD), breast cancer, TNF-α/NF-κB/BCL-2 axis, apoptosis signaling pathway, molecular dockin","lastPublishedDoi":"10.21203/rs.3.rs-9539541/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9539541/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBreast cancer is one of the most common malignant tumors in women worldwide and poses a serious threat to women\u0026rsquo;s health. Norcantharidin (NCTD), derived from the traditional Chinese medicine Mylabris, has low toxicity and anti-cancer potential; however, its mechanism of action in breast cancer remains unclear.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo investigate whether NCTD induces apoptosis in breast cancer cells by regulating the TNF-α/NF-κB/BCL-2 axis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eNetwork pharmacology was used to predict NCTD targets and pathways, and molecular docking was employed to validate binding to key proteins. The proliferation and apoptosis of MCF-7 and MDA-MB-231 cells were assessed using CCK-8 and flow cytometry, respectively, while western blot analysis was performed to detect the expression of TNF-α, IκBα, total p65, nuclear p65, BCL-2, cleaved caspase-3, and c-PARP.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eNCTD shares 20 common targets with breast cancer, which are enriched in the apoptosis and TNF pathways. Molecular docking studies suggest that NCTD binds to TNFR1, IKKα, PARP, and Caspase-3. NCTD inhibits cell proliferation and induces apoptosis; it upregulates TNF-α, IκBα, and the apoptotic executor proteins Caspase-3 and c-PARP, while downregulating nuclear p65 and BCL2, with total p65 remaining unchanged.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eNCTD induced apoptosis in breast cancer cells by regulating the TNF-α/NF-κB/BCL-2 axis, thereby activating apoptotic signals, inhibiting NF-κB activity, and downregulating BCL-2.\u003c/p\u003e","manuscriptTitle":"Norcantharidin induces apoptosis in breast cancer cells by regulating the TNF-α/NF-κB/BCL-2 pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-08 19:01:54","doi":"10.21203/rs.3.rs-9539541/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5b3298fd-0ef4-463b-8734-3c88a5aea599","owner":[],"postedDate":"May 8th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-09T19:20:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T19:32:13+00:00","index":27,"fulltext":""},{"type":"reviewerAgreed","content":"326460479523539324616224542965230173871","date":"2026-05-01T09:24:05+00:00","index":26,"fulltext":""},{"type":"reviewersInvited","content":"20","date":"2026-04-30T16:13:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T06:06:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T06:05:58+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-09T19:24:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-08 19:01:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9539541","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9539541","identity":"rs-9539541","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}