Sesquiterpene lactones, derived from plants, are widely employed in TCM for their anti-inflammatory and anticancer properties[34]. These compounds exhibit reactivity with functional groups, notably the thiol group on proteins and enzymes. They demonstrate selectivity towards tumor and cancer stem cells by targeting specific signaling pathways, making them noteworthy agents in cancer clinical trials[35, 36]. Previous studies from our research group have established the neuroprotective effects of Brevilin A against lipopolysaccharide-induced neuroinflammation both in vitro and in vivo. In the present study, we elucidate the therapeutic efficacy of Brevilin A in the context of lung cancer, operating through multi-target, multi-biological processes, and multi-pathway mechanisms.
Our results highlight STAT3, TNF, HIF1A, PTEN, ESR1, and MTOR as potential therapeutic targets for Brevilin A's anticancer activity. STAT3, a transcription factor integral to diverse biological processes, including cell proliferation, survival, differentiation, and angiogenesis[37], has been implicated in various human cancers, such as head and neck tumors, cervical cancer, gastric carcinoma, and colon cancer[38–41]. Notably, exosome-mediated transfer of specific microRNAs has been associated with the activation of STAT3 signaling-induced epithelial-mesenchymal transition in lung cancer cells[42]. TNF-α, a member of the tumor necrosis factor superfamily, exhibits a spectrum of biological activities[43] and has been implicated in numerous human cancers, influencing processes such as growth, invasion, and metastasis[44, 45]. In NSCLC patients, elevated levels of IL-1, IL-6, and TNF-α have been linked to cancer pain and prognosis[46]. HIF (hypoxia-inducible factor)[47], a transcription factor crucial for tumor angiogenesis, cell survival, proliferation, apoptosis, metastasis, infiltration, and metabolism[48], plays a pivotal role in promoting lung cancer cell proliferation under conditions of chronic intermittent hypoxia[49]. Studies also demonstrate that certain formulations in TCM can inhibit NSCLC cell proliferation by downregulating HIF-1α expression[50]. Phosphatase and tensin homolog deleted on chromosome ten (PTEN), encoding the classical PTEN protein with phosphatase activity, acts as a tumor suppressor by antagonizing the activity of tyrosine kinases and other phosphorylases. Meta-analyses indicate a correlation between PTEN and poor prognosis in lung cancer[51], and clinical studies confirm abnormal expression of EGFR, TGF-α, P-AKT, and PTEN in NSCLC patients[52], potentially contributing to NSCLC pathogenesis. In the present study, we utilized AutoDock Vina and PyMOL software to investigate Brevilin A's potential binding sites with these proteins, revealing robust binding activity. GSEA analysis further validated the strong association of these targets with lung cancer.
The GO and KEGG analyses revealed 2893 enriched GO terms and 157 enriched KEGG pathways, encompassing notable pathways such as the PI3K-Akt signaling pathway, FoxO signaling pathway, and HIF-1 signaling pathway. The PI3K-Akt signaling pathway, governing various cellular functions including growth, differentiation, proliferation, survival, motility, invasion, and intracellular trafficking, plays a pivotal role in tumorigenesis[53]. Studies have reported the induction of apoptosis and inhibition of invasion in NSCLC through the PI3K/Akt/mTOR signaling pathway by compounds like Aloperine[54]. Additionally, CAF-derived exosomes have been identified to promote NSCLC cellular proliferation and chemoresistance through regulation of the PTEN/PI3K-AKT signaling axis[55]. The FOXO signaling pathway, triggered by the PI3K/AKT pathway, is instrumental in mediating cell proliferation, differentiation, and tumorigenesis[56–58]. Inhibition of CCCTC-binding factor (CTCF) has been shown to regulate the FoxO signaling pathway, impeding tumor growth in vivo[59]. Notably, our study is the first to unveil that Brevilin A exerts anti-lung cancer effects by targeting the HIF-1 signaling pathway. Gene-pathway correlation analysis further revealed significant associations between most hub genes and the cellular response to hypoxia pathway. Hypoxia, influencing tumor signaling pathways through hypoxia-inducible factors (HIFs) and reducing free radical production, holds significance in tumor progression. Studies have demonstrated the role of hypoxia in activating EGFR and inducing resistance to gefitinib in EGFR-mutant non-small cell lung cancer[61]. Silencing HIF-1α expression has been shown to significantly reduce the invasive ability of lung cancer cells under hypoxic conditions[62]. Molecular docking analysis and molecular dynamics simulation affirmed the robust interaction of Brevilin A with HIF1A and PTEN, respectively. In vitro experiments demonstrated that Brevilin A induces dose- and time-dependent cell death in A549 cells, concomitant with decreased HIF-1α mRNA expression and increased PTEN mRNA levels. These results suggest the potential of the HIF-1 signaling pathway as a therapeutic target for Brevilin A in lung cancer treatment.
In summary, our study delineated the core targets and key pathways of Brevilin A in lung cancer through an integrated approach involving network pharmacology, molecular docking analysis, and experimental validation. The therapeutic effects of Brevilin A in lung cancer were demonstrated to involve a multi-target, multi-biological process, and multi-pathway mechanism, with noteworthy inhibition of the HIF-1 signaling pathway. These results lay a theoretical foundation for the prospective clinical application of Brevilin A. Nevertheless, it is imperative to acknowledge certain limitations in this study. Firstly, the utilization of more comprehensive databases would enhance the reliability of results. Secondly, further experimental validations are imperative to consolidate the findings derived from network pharmacology. Therefore, additional investigations are warranted to unravel the anti-lung cancer molecular mechanisms of Brevilin A.