Ellagic acid activates p38 and upregulates Keap1 to suppress Nrf2/HO-1, markedly enhancing RSL3-induced ferroptosis in pancreatic ductal adenocarcinoma

EA plus RSL3 synergistically enhances ferroptosis and suppresses tumor growth in PDAC cells and PANC-1 xenografts. EA was previously known in PDAC to induce apoptosis, G1 arrest, and suppress NF-κB/COX-2, but its role in modulating ferroptosis was unexplored; this study positions EA as a ferroptosis sensitizer. In vitro CCK-8 showed the combination significantly reduced viability versus monotherapy (p<0.001); in vivo tumor weight was 1.22±0.10 mg and tumor size 312.2±18.3 mm³, both significantly lower than control and RSL3 alone (p<0.001), with n=6 per group.

The combination amplified ferroptotic markers and downregulated GPX4, and Fer-1 rescued viability while inhibitors of other death pathways did not, supporting ferroptosis as the dominant death mode. Beyond reporting elevated ferroptotic markers, the study used Fer-1, Z-VAD-FMK, necrostatin-1, and 3-MA controls to exclude apoptosis, necroptosis, and autophagy as the primary mechanism. Fer-1 raised PANC-1 viability from 41.37%±1.89% to 71.43%±2.15% and BxPC-3 from 29.28%±1.76% to 72.55%±2.34% (p<0.001), whereas the other inhibitors produced no rescue (p>0.05).

EA suppresses the Nrf2/HO-1 axis by activating p38 MAPK and upregulating Keap1, constituting its ferroptosis-sensitizing mechanism. Prior work linking Nrf2/HO-1 suppression to ferroptosis sensitization largely used monoclonal antibodies or synthetic inhibitors; this study proposes a natural polyphenol acting through the p38–Keap1–Nrf2–HO-1 axis and adds a p38 upstream mechanism. EA treatment increased phospho-Thr180 p38; the p38 inhibitor SB202190 reversed this increase and attenuated EA-mediated Nrf2/HO-1 suppression and cytotoxicity; siRNA knockdown of Nrf2 enhanced combination sensitivity and raised MDA and lipid ROS; overexpression of Nrf2 or HO-1, or activation of either with t-BHQ/hemin, partially counteracted the combination's growth inhibition.

No obvious systemic toxicity was observed in vivo, and in vivo results were consistent with in vitro findings. The study measured both ferroptosis parameters and the p38/Nrf2/HO-1 axis in xenografts, showing in vitro and in vivo mechanistic consistency. Body weight did not differ significantly across groups (p>0.05) and no animal reached predefined humane endpoints; in vivo total iron, chelatable iron, MDA, and lipid ROS increased while GPX4 decreased (p<0.001), with Nrf2/HO-1 reduced and Keap1 and phospho-p38 elevated.

The results apply to KRAS-mutant and wild-type PDAC cell lines (PANC-1, BxPC-3) and a PANC-1 subcutaneous xenograft model, suggesting EA can serve as a ferroptosis sensitizer combined with a GPX4 inhibitor; for follow-up work, the authors recommend validation in orthotopic PDAC models or patient-derived organoids, assessment of synergy with gemcitabine/nab-paclitaxel, interrogation of stromal crosstalk using co-culture models with cancer-associated fibroblasts, and exploration of p38–Keap1–Nrf2–HO-1 axis components or circulating lipid peroxides as predictive biomarkers to identify patients likely to benefit.

EA has limited systemic bioavailability in humans and is converted by gut microbiota into urolithin derivatives that may contribute substantially to in vivo activity, so whether the effects seen in animal models will reproduce clinically remains an open question; moreover, subcutaneous xenografts do not fully reflect PDAC stroma and anatomical context, and this study did not evaluate pharmacokinetic parameters, so the dosing regimen and exposure-response relationship for EA+RSL3 remain to be defined; whether p38–Keap1–Nrf2–HO-1 axis components can serve as predictive biomarkers also requires prospective validation.