Ponicidin suppresses pancreatic cancer growth by inducing ferroptosis: Insight gained by mass spectrometry-based metabolomics

•Ponicidin had an inhibitory effect on the proliferation of pancreatic cancer SW1990 cells.•Ponicidin could form a Pon-GSH conjugate with glutathione through Michael addition in the gamma-glutamyl cycle.•Ponicidin could bind to GPX4 to reduce the content of the activity of GPX4.•Ponicidin could induce cell ferroptosis by affecting the gamma-glutamyl cycle. Pancreatic cancer is one of the most common malignant tumors of the digestive tract. Ponicidin, a tetracyclic diterpenoid active ingredient extracted from the traditional phytomedicine Rubescens, has high safety and great inhibitory effect on the proliferation of a variety of cancer cells, especially malignant tumor cells of the digestive tract. However, the inhibitory effect and mechanism of ponicidin on pancreatic cancer cells is still unclear. Our study aimed to use metabonomics technology to analyze and explore the suppressive effect of ponidicin against pancreatic cancer cells. MTT and flow cytometry were conducted to study the potential effect of ponicidin on SW1990 cells. Secondly, UPLC-MS/MS was used to analyze the small molecule metabolites and relevant differential metabolic pathways induced by ponicidin treatment. Furthermore, through the determination of glutathione peroxidase 4 (GPX4) activity and molecular docking simulation experiments, the effects of intracellular GPX4 activity and GSH/GSSG ratio after ponicidin were evaluated. Finally, the determination of the content of iron ions and malondialdehyde in cells, and the experiment of the effect of ferroptosis inhibitors on cell viability, the effect of ponicidin on the induction of ferroptosis in SW1990 cells was also detected. The IC50 of ponicidin on SW1990 cells was 20 μM, which could significantly induce cell apoptosis and arrest the cells in G2/M phase. Metabolomics results showed that the contents of endogenous small molecules such as gamma-glutamylcysteine, 5-oxoproline, glutamic acid, reduced glutathione (GSH), oxidized glutathione (GSSG) and arachidonic acid have changed significantly. Main differential compounds were involved in the gamma-glutamyl cycle and polyunsaturated fatty acid metabolism of pancreatic cancer cell lines. Additionally, ponicidin could covalently bind to GSH in SW1990 cells to form a conjugate Pon-GSH, which further reduced the content of free GSH and GPX4 activity in cells. Notably, ponicidin dose-dependently increased levels of iron ions, malondialdehyde and reactive oxygen species in SW1990 cells, and the