The inhibitory effects of modified HSJZ decoction on NSCLC by regulating regulatory T cells via downregulation of EZH2 and PI3K/AKT pathway

Huangqi Si Jun Zi decoction (HSJZ), a modified traditional Chinese medicine formula, is known to enhance immunity. However, its immunomodulatory effects in non-small cell lung cancer (NSCLC) remain poorly understood. This study aimed to investigate the anti-tumor potential of HSJZ in NSCLC and elucidate its mechanisms of action. In vitro studies assessed HSJZ cytotoxicity in 4 tumor cell lines and its ability to activate immune cells from NSCLC patients, followed by evaluating the cytotoxicity of these activated immune cells against NSCLC cell lines. An orthotopic lung cancer model in mice evaluated in vivo anti-tumor activity. Flow cytometry, immunohistochemistry, and Western blot analysis were conducted to analyze immune cell populations, cytokine production, and protein expression levels, including Tregs, CD8 + T cells, EZH2, and PI3K/AKT signaling pathways. While HSJZ alone exhibited no direct cytotoxicity on NSCLC cells in vitro , it significantly enhanced immune cell-mediated killing when co-cultured with patient-derived peripheral blood mononuclear cells (PBMCs), accompanied with increased IFN-γ and TNF-α production. In vivo , HSJZ (1 g/kg) treatment in a murine orthotopic lung cancer model resulted in a 54.2 % reduction in tumor burden, as assessed by IVIS spectrum imaging. Mechanistically, HSJZ suppressed regulatory T cells (Tregs) both in vivo and in vitro , as demonstrated by decreased Treg frequency and downregulation of phosphorylated PI3K/AKT signaling in mice bearing Lewis tumor. Moreover, HSJZ significantly inhibited the expression of EZH2, an epigenetic regulator crucial for Treg differentiation. These findings provide compelling evidence that HSJZ exerts anti-tumor effects in NSCLC by modulating the immune microenvironment, particularly through the inhibition of Tregs activity, as assessed by in vitro co-culture system, in vivo mouse orthotopic Lewis cancer model, immunohistochemistry, etc. Our results support the potential clinical application of HSJZ as an adjunct therapy for NSCLC patients.