Unlocking the potential of Traditional Chinese Medicine (TCM): Shipi Xiaoji formula (SPXJF) as a novel ferroptosis inducer in hepatocellular carcinoma.

Ethnopharmacological Relevance: Hepatocellular Carcinoma (HCC) is a major health concern with limited treatment options. Traditional Chinese Medicine (TCM) offers potential therapeutic approaches for HCC, and SPXJF, a TCM formula, has shown promise in clinical observations for prolonging the survival of liver cancer patients.

Aim Of The Study: To investigate the anti-tumor effects of SPXJF on HCC cells and explore its potential mechanism, focusing on ferroptosis induction.

Materials And Methods: LC/Q-TOF-MS was used for compound identification. Cell viability assays, EdU proliferation assay, colony formation assay, wound healing assay, Transwell assay, and Western-blotting were conducted to evaluate the effects of SPXJF on HCC cell proliferation, migration, and invasion. Bioinformatics analysis and RT-PCR were employed to identify potential ferroptosis-related genes and validate the results. Ferroptosis induction was investigated using ferroptosis inhibitors, ROS and lipid peroxidation detection, and TEM. In vivo experiments using a subcutaneous xenograft tumor model confirmed the anti-tumor effects of SPXJF and its ability to induce ferroptosis in HCC.

Results: SPXJF effectively inhibited the proliferation, migration, and invasion of HCC cells in vitro. The mechanism of action was found to be related to the induction of ferroptosis, as evidenced by increased intracellular Fe and ROS levels, decreased GSH levels, altered mitochondrial morphology, and upregulation of ferroptosis-inducing proteins ACSL4 and LPCAT3, along with downregulation of ferroptosis-inhibiting proteins xCT and GPX4. Bioinformatics analysis and RT-PCR further identified GSTZ1, CDC25A, AURKA, NOX4, and CAPG as potential ferroptosis-related genes regulated by SPXJF. In vivo experiments confirmed the anti-tumor effects of SPXJF and its ability to induce ferroptosis in HCC.

Conclusions: SPXJF exerts anti-tumor effects on HCC cells by inducing ferroptosis, and its mechanism of action involves the regulation of ferroptosis-related genes and proteins. This study provides a theoretical basis for the clinical treatment of HCC and the development of new anti-cancer drugs, offering a valuable contribution to the field of ethnopharmacology.