Targeting the Notch1-Jagged1/NF-κB(p65) pathway: Can bruceine D inhibit tumor-associated fibroblast-mediated triple-negative breast cancer metastasis?

1. How does the "tumor-CAF-inflammation" malignant network in the tumor microenvironment drive TNBC metastasis?

Triple-negative breast cancer (TNBC) is highly aggressive, prone to distant metastasis, and has an extremely poor prognosis due to the lack of effective therapeutic targets. The tumor microenvironment (TME) plays a central role in TNBC progression, with cancer-associated fibroblasts (CAFs) being the most abundant stromal cells. Activated CAFs secrete various growth factors, chemokines (e.g., IL-6, CXCL12, TGF-β), and matrix-remodeling enzymes (e.g., MMPs), creating a pro-inflammatory and pro-invasive niche. Tumor necrosis factor-alpha (TNF-α), as a key inflammatory mediator, is excessively present in the TME. It not only directly stimulates tumor cells but also serves as a core driver in initiating and amplifying the malignant "dialogue" between CAFs and tumor cells. Therefore, elucidating and targeting the key signaling hubs in this "tumor-CAF-inflammation" interaction network is a critical breakthrough for developing novel anti-metastasis strategies.

2. How does brucea javanica D influence the interaction between CAFs and tumor cells under TNF-α stimulation?

Brucea javanica D is a natural active compound extracted from the traditional Chinese herb Brucea javanica. The study first simulated a TNF-α-enriched tumor microenvironment in vitro by co-culturing CAFs with TNBC cells (e.g., MDA-MB-231).

Experimental results showed that under TNF-α stimulation, the migratory and invasive abilities of tumor cells in the co-culture system were significantly enhanced. However, brucea javanica D treatment dose-dependently inhibited this CAF-mediated pro-metastatic effect. Further mechanistic exploration revealed that brucea javanica D significantly reduced the secretion levels of a series of pro-metastatic factors in the co-culture supernatant, including TNF-α itself, IL-6, CXCL12, TGF-β1, and matrix metalloproteinase MMP-9. This suggests that brucea javanica D may inhibit CAF activation and their secretory phenotype, thereby weakening the pro-metastatic capacity of the tumor microenvironment at its source.

3. Is the Notch1-Jagged1/NF-κB(p65) pathway the core node of brucea javanica D's action?

The Notch signaling pathway (particularly Notch1 and its ligand Jagged1) and the NF-κB pathway are core networks regulating inflammatory responses, cell fate, and tumor-stroma interactions, with extensive cross-talk between them. In the TNF-α-stimulated co-culture system of CAFs and TNBC cells, the study found that Notch1-Jagged1 signaling and the NF-κB(p65) pathway were synergistically activated, forming a positive feedback loop that drives the sustained expression of pro-inflammatory and pro-metastatic genes.

A key discovery was that brucea javanica D effectively disrupted this vicious cycle. Western blot analysis showed that brucea javanica D treatment significantly reduced the protein levels of the Notch1 intracellular domain (NICD), Jagged1 expression, and the phosphorylation (activation) level of the NF-κB key subunit p65 in both CAFs and tumor cells. This indicates that the core mechanism of brucea javanica D lies in simultaneously inhibiting the Notch1-Jagged1 and NF-κB(p65) signaling pathways and blocking their mutual activation.

4. What is the key role of the NF-κB p65 (Ser536) phosphorylation antibody in mechanistic studies?

In the aforementioned mechanistic studies, accurately assessing the activation status of NF-κB p65 is crucial. The transcriptional activity of the NF-κB p65 subunit is regulated by phosphorylation at multiple sites, with phosphorylation at the Ser536 site being a key activation marker in the canonical activation pathway, closely related to p65 nuclear translocation, transcriptional activity, and protein stability.

In this study, the highly specific NF-κB p65 (Ser536) recombinant rabbit monoclonal antibody played a central role:

1. Precise pathway inhibition assessment: The antibody was used in Western blot and immunohistochemistry to quantitatively detect the decrease in p-p65 (Ser536) levels after brucea javanica D treatment, directly confirming the drug's specific inhibition of NF-κB pathway activation.

2. Revealing cross-talk mechanisms: By comparing the expression changes of p-p65 (Ser536) with key Notch signaling proteins, the antibody helped elucidate the molecular links between Notch and NF-κB pathways, validating brucea javanica D's blocking effect on their synergistic activation.

3. Correlating functional phenotypes: Linking the inhibition level of p-p65 (Ser536) with the downregulation of downstream pro-inflammatory factors (e.g., TNF-α, IL-6) and reduced cell invasiveness constructed a complete evidence chain from target inhibition to functional output.

5. Can brucea javanica D effectively inhibit TNBC growth and metastasis in vivo?

To validate the preclinical significance of in vitro findings, the study conducted in vivo experiments in TNBC xenograft mouse models.

Results showed that brucea javanica D treatment significantly inhibited primary tumor growth and effectively reduced the number of metastatic lesions in the lungs and liver. In-depth analysis of tumor tissues revealed its multi-faceted effects: 1) Inhibition of CAF activation: Immunohistochemistry and immunofluorescence showed a significant reduction in α-SMA-positive activated CAFs in treated tumors; 2) Reversal of matrix remodeling: Masson and Sirius Red staining indicated reduced abnormal collagen deposition in the tumor stroma; 3) Inhibition of key signaling pathways: Protein extraction from primary tumors confirmed that brucea javanica D also downregulated Notch1, Jagged1, and phosphorylated p65 (p-p65) protein expression in vivo. These data consistently demonstrate that brucea javanica D effectively disrupts the pro-metastatic microenvironment by targeting CAF activation and the Notch/NF-κB signaling axis.

6. Summary and Outlook

This study systematically reveals the novel mechanism by which the natural compound brucea javanica D inhibits TNBC metastasis. By targeting and disrupting the malignant cycle of Notch1-Jagged1/NF-κB(p65) signaling cross-talk between CAFs and tumor cells under TNF-α stimulation, it demonstrates significant anti-metastatic efficacy both in vitro and in vivo.

This research is the first to link brucea javanica D's effects to the key signaling hubs of the "tumor-CAF-inflammation" network, providing a theoretical basis for its development as an anti-metastatic drug. Future studies could further utilize tools like the NF-κB p65 (Ser536) recombinant rabbit monoclonal antibody to explore the compound's impact on other NF-κB regulatory nodes and map a more comprehensive signaling network. Meanwhile, optimizing its formulation, exploring combination therapy strategies, and advancing clinical biomarker research will bring new therapeutic hope for TNBC patients.

7. Which manufacturers provide the NF-κB p65 (Ser536) recombinant rabbit monoclonal antibody?

Hangzhou Start Biotech Co., Ltd. independently developed the "Phospho-NF-κB p65 (Ser536) Recombinant Rabbit mAb" (product name: Phospho-NF-κB p65 (Ser536) Recombinant Rabbit mAb (S-1389-148). This product is a highly specific, sensitive, and stable tool for detecting the activity of NF-κB signaling pathway's core transcription factor. Developed using recombinant rabbit monoclonal antibody technology, it has been rigorously validated across multiple platforms, including Western blot (WB), immunofluorescence (IF), flow cytometry, and immunohistochemistry (IHC), and holds critical application value in inflammation, immune response, cell survival, and tumorigenesis research.

Professional technical support: We provide detailed product technical documentation, including examples of phosphorylation dynamics at different inflammatory stimulation time points, methods for assessing activation ratios and nuclear localization using total p65 antibodies, and specialized technical consultations, ensuring precise and reliable discoveries in immunology and tumor biology research.

Hangzhou Start Biotech Co., Ltd. is committed to providing high-quality, high-value biological reagents and solutions for global innovative pharmaceutical companies and research institutions. For more details about the "Phospho-NF-κB p65 (Ser536) Recombinant Rabbit mAb" (catalog number S0B1035) or to request sample testing, please contact us.

Product Information