Network Pharmacology Reveals SFI Blocks Glioma via SRC/PI3K/AKT Pathway

Study Background and Research Question

Gliomas, accounting for up to half of all primary brain tumors, are notorious for their aggressive proliferation, invasiveness, and resistance to standard treatments. Therapy options, including surgery, radiotherapy, and chemotherapy, offer limited survival benefits, with median survival often below 17 months. There is an urgent need to better understand the molecular mechanisms underpinning glioma progression and to identify novel targets for anti-angiogenic compound development. Traditional Chinese medicine formulations such as Shenqi Fuzheng injection (SFI), which combines Codonopsis pilosula and Astragalus membranaceus, have seen widespread clinical use as adjuvants in cancer therapy, but their precise molecular effects on glioma cells remain largely undefined.

Key Innovation from the Reference Study

The central innovation of the study by Li et al. (2024) lies in its integrated use of network pharmacology with experimental validation to unravel the molecular basis of SFI’s anti-glioma actions. For the first time, the research systematically mapped the potential targets of SFI in glioma, revealing the SRC/PI3K/AKT signaling pathway as a principal mediator. This pathway is well known for its role in cell proliferation, survival, and migration, and its dysregulation is implicated in both tumor growth and angiogenesis. By pinpointing this axis, the study provides a mechanistic rationale for SFI’s observed inhibition of glioma progression and supplies a template for rational design of future angiogenesis inhibitor research.

Methods and Experimental Design Insights

The study followed a two-pronged approach:

• Network Pharmacology: Researchers first curated the active components of SFI using databases and literature, identifying 26 major constituents. They then mapped potential protein targets of these compounds and cross-referenced them with glioma-associated genes. This intersection yielded 79 shared targets, which were subjected to pathway enrichment analysis.
• Experimental Validation: In vitro assays included CCK-8 for cell viability, EdU and plate colony formation for proliferation, scratch and Transwell assays for migration, flow cytometry for cell cycle analysis, and Western blot/immunofluorescence for protein expression. U87 and T98G human glioma cell lines were used. In vivo, a subcutaneous GL261 mouse glioma model assessed tumor growth and response to SFI, with histological and immunohistochemical analyses corroborating the cellular findings.

This robust combination of computational and experimental methods ensured that the predicted pathways were not merely theoretical but also functionally relevant in biological systems.

Core Findings and Why They Matter

The study’s primary findings include:

• SFI significantly inhibited proliferation and migration of U87 and T98G glioma cells in vitro, with clear evidence of S-phase cell cycle arrest (Li et al., 2024).
• SFI treatment downregulated epithelial-mesenchymal transition (EMT) markers, indicating suppression of migratory and invasive phenotypes.
• In the GL261 mouse model, SFI administration resulted in marked reduction of tumor volume and weight, with histological analysis confirming decreased proliferation indices and altered expression of key signaling proteins.
• Network enrichment highlighted the SRC/PI3K/AKT pathway as a convergence point for SFI’s action. Western blot and immunofluorescence showed reduced phosphorylation of SRC and downstream PI3K/AKT components in SFI-treated tumors and cell lines.

These findings matter because the SRC/PI3K/AKT pathway is a well-validated driver of tumor angiogenesis and growth, and its pharmacological inhibition is a cornerstone of anti-angiogenic therapy. By demonstrating that a multi-component herbal injection can target this pathway, the study opens routes for both mechanistic exploration and the rational design of combinatorial anti-angiogenic strategies. It also bridges traditional ethnomedicine with modern molecular oncology.

Comparison with Existing Internal Articles

A review of internal resources provides additional context for the current findings:

• The article "SFI Inhibits Glioma via SRC/PI3K/AKT: Network Pharmacology Insights" summarizes how SFI disrupts glioma progression via the same signaling axis, supporting the reference study’s conclusion that targeting SRC/PI3K/AKT is effective for both anti-proliferative and anti-migratory outcomes in glioma models.
• In the context of anti-angiogenic compound research, internal articles such as "AAL-993: VEGF Receptor Inhibitor for Tumor Angiogenesis Modeling" discuss the role of selective VEGF receptor inhibitors for probing angiogenic mechanisms in tumors. While AAL-993 (SKU C3730) directly targets VEGF receptor kinases, the reference paper’s approach targets an upstream regulatory pathway, illustrating complementary strategies for tumor angiogenesis research.

This comparison highlights the value of pathway-centric versus receptor-targeted inhibition, with each strategy offering distinct mechanistic insights and potential for combination in preclinical studies.

Limitations and Transferability

Despite its strengths, the study has certain limitations:

• The primary data are derived from in vitro cell lines and a subcutaneous mouse model; orthotopic brain tumor models may better recapitulate the glioma microenvironment.
• The multi-component nature of SFI complicates attribution of specific effects to individual phytochemicals, although network pharmacology helps narrow down key contributors.
• The findings are most readily transferable to preclinical research settings, and clinical translation will require further toxicological and pharmacokinetic evaluation.

Nonetheless, the experimental workflow—combining network pharmacology with targeted assays—serves as a robust template for dissecting the action of other complex anti-angiogenic agents.

Protocol Parameters

• SFI treatment in vitro: Apply SFI at concentrations determined by dose-response CCK-8 assays (e.g., 0.5–2 mg/mL) for 24–48 hours; assess proliferation and migration endpoints.
• Cell cycle analysis: After SFI exposure, fix cells and stain with PI; analyze S-phase fraction by flow cytometry to quantify arrest.
• In vivo tumor model: Inject GL261 cells subcutaneously into C57BL/6 mice; treat with SFI via intraperitoneal injection at 10 mL/kg, daily or every other day, for 2–3 weeks; monitor tumor volume with calipers.
• Pathway validation: Use Western blot or immunofluorescence to assess SRC, PI3K, and AKT phosphorylation in both cell and tumor lysates.

Research Support Resources

For investigators studying tumor angiogenesis or evaluating anti-angiogenic compounds, precision kinase inhibitors such as AAL-993 (SKU C3730) offer robust selectivity for VEGFR-1, VEGFR-2, and VEGFR-3, enabling high-fidelity modeling of VEGF-driven pathways. According to the product information, AAL-993 demonstrates potent inhibition of VEGFR-2 and VEGFR-3 kinases, supporting its use in both in vitro and in vivo angiogenesis studies. When designing experiments that parallel the SRC/PI3K/AKT axis or probe direct VEGF receptor blockade, AAL-993 from APExBIO can complement pathway-based approaches exemplified by SFI, facilitating more comprehensive analysis of tumor angiogenesis and metastasis inhibition.