YiQiChuTan formula (YQCTF) inhibit the progression of non-small cell lung cancer via down-regulating EGFR/ITGB2 signaling: Triangulated evidence from network pharmacology, proteomic profiling, and experimental validation

Background: YiQiChuTan Formula (YQCTF) is a traditional Chinese medicine formula composed of eight carefully selected herbal Materials. which therapeutic efficacy has been clinically verified as it inhibits the progression of non-small cell lung Cancer (NSCLC), prolongs the overall survival, and improves the life quality of NSCLC patients. However, the specific active components and precise mechanistic basis of YQCTF remain unveiled.

Purpose: This study aims to investigate the therapeutic effects of YQCTF on NSCLC, identify its active components, and clarify which molecular mechanisms through an integrated approach combining network pharmacology, proteomic profiling, and experimental validation.

Methods: The therapeutic efficacy of YQCTF against NSCLC was evaluated in a syngeneic C57BL/6 mice model established by subcutaneous inoculation of Lewis lung carcinoma (LLC) cells. The potential mechanisms were investigated through network pharmacology-based predictions integrated with proteomic profiling. Furthermore, the anti-neoplastic effects and underlying mechanisms of Thunberg Fritillary Bulb, a major herbal component of YQCTF, and its active ingredient Peimisine, were systematically explored by combining in vivo studies in LLC subcutaneous transplanted mice with in vitro validation on wild-type and EGFR-knockout (KO) A549 cell lines via Western blot (WB), flow cytometry, immunohistochemistry (IHC), Transwell migration assays, and scratch wound-healing assays.

Results: Both the water extract (WE) and water extract-alcohol precipitation (WEAP) of YQCTF demonstrated significant efficacy in suppressing the growth of subcutaneously transplanted tumor in mice. Proteomics analysis indicates that YQCTF may inhibit the progression of NSCLC by targeting the ITGB2/EGFR signaling pathway. UPLC-MS/MS analysis identified Alkaloids from Thunberg Fritillary Bulb as active components of YQCTF, with peimisine being a major active ingredient in the Thunberg Fritillary Bulb Extract (TFBE). Consistent with the YQCTF's effect, both TFBE and Peimisine effectively inhibited subcutaneous tumor growth in mice and regulated EMT-related proteins in tumor tissue. Specifically, they up-regulated the EMT-inhibitory protein E-cadherin, while down-regulating EMT-promoting proteins, including N-Cadherin, Vimentin, Snail1, Slug, MMP-2 and MMP-9. In vitro screening in A549 cells confirmed that peimisine as the major active ingredient in TFBE. In vitro, peimisine suppressed the migration of A549, reduced the protein expression of mesenchymal markers (N-Cadherin and Vimentin), EMT transcription factors (Snail1 and Slug) and invasive proteins (MMP-9 and MMP-2), but up-regulated epithelial marker E-cadherin. Notably, Peimisine inhibited cell migration, ITGB2 and EMT signals on wild-type A549 cells, these effects however were evidently attenuated in EGFR-KO A549 cells.

Conclusion: YQCTF significantly suppressed the progression of NSCLC in murine model. Peimisine, a steroidal alkaloid derived from the herbal material Thunberg Fritillary Bulb, plays a crucial role in YQCTF's mechanism of action by inhibiting the ITGB2/EGFR signaling pathway, suppressing EMT, thereby impeding tumor growth.

EGFR; EMT; Lung cancer; Peimisine; Thunberg Fritillary Bulb Extract (TFBE); YQCTF.