Total flavonoids from Ageratum conyzoides synergize with docetaxel to induce ferroptosis in prostate cancer cells via the NRF2/SLC7A11/GPX4 signaling pathway.

Abstract

Prostate cancer is one of the most common malignant tumors in men, and its treatment faces numerous challenges. This study aims to investigate whether the combined treatment of Total Flavonoids from Ageratum conyzoides (TFG) and Docetaxel (DTX) can enhance the antitumor effect of DTX on prostate cancer cells by regulating the NRF2/SLC7A11/GPX4 signaling axis and inducing mitochondria-related ferroptosis. Cell viability was detected using the CCK-8 assay. Plate colony formation, scratch, and Transwell assays were used to evaluate proliferation, migration, and invasion, respectively. Flow cytometry was employed to analyze apoptosis and cell cycle distribution. PCR-Array was used to screen differentially expressed genes. RT-PCR and Western blot were performed to measure ferroptosis-related gene and protein expression. Reactive oxygen species (ROS) levels were detected using DCFH-DA, mitochondrial membrane potential using JC-1, lipid peroxidation using BODIPY-C11, and cellular ultrastructure by transmission electron microscopy. To validate the mechanistic involvement of ferroptosis, rescue experiments were performed using Ferrostatin-1 (Fer-1, a ferroptosis inhibitor) and Z-VAD-FMK (a pan-caspase inhibitor). Pharmacological inhibitors ML385 (NRF2 inhibitor) and RSL3 (GPX4 inhibitor) were used to probe pathway causality. Key findings were further validated in LNCaP (AR-positive) prostate cancer cells. TFG and DTX dose-dependently inhibited PC3 cell viability and showed synergistic effects. The combination significantly suppressed colony formation, migration, and invasion, induced late apoptosis, and arrested the cell cycle in S phase. Mechanistically, the combination upregulated TF and HMOX1 while downregulating NRF2, SLC7A11, and GPX4 expression. Fer-1 significantly rescued combination-induced cell death (~ 70% recovery), while Z-VAD-FMK showed partial rescue (~ 30% recovery), confirming ferroptosis as the dominant mechanism with secondary apoptosis involvement. ML385 + DTX and RSL3 + DTX phenocopied the TFG + DTX effect, supporting that TFG acts upstream of NRF2 and GPX4 is a key downstream effector. BODIPY-C11 staining confirmed increased lipid peroxidation, which was blocked by Fer-1. These findings were validated in LNCaP cells, showing consistent synergistic inhibition and downregulation of GPX4 and NRF2. TFG enhances the antitumor effect of DTX in prostate cancer cells by inhibiting the NRF2/SLC7A11/GPX4 axis, inducing lipid peroxidation, and triggering mitochondria-associated ferroptosis. Pharmacological rescue and inhibitor studies confirm ferroptosis as the primary mechanism, with apoptosis playing a secondary role. These findings provide a strong mechanistic rationale for developing TFG as a natural chemosensitizer for prostate cancer therapy, particularly for overcoming DTX resistance.