Evodiamine Exhibits Anti-Bladder Cancer Activity by Suppression of Glutathione Peroxidase 4 and Induction of Ferroptosis

Abstract

Evodiamine (EVO) exhibits anti-cancer activity through the inhibition of cell proliferation; however, little is known about its underlying mechanism. To determine whether ferroptosis is involved in the therapeutic effects of EVO, we investigated critical factors, such as lipid peroxidation levels and glutathione peroxidase 4 (GPX4) expression, under EVO treatment. Our results showed that EVO inhibited the cell proliferation of poorly differentiated, high-grade bladder cancer TCCSUP cells in a dose- and time-dependent manner. Lipid peroxides were detected by fluorescence microscopy after cancer cell exposure to EVO. GPX4, which catalyzes the conversion of lipid peroxides to prevent cells from undergoing ferroptosis, was decreased dose-dependently by EVO treatment. Given the features of iron dependency and lipid-peroxidation-driven death in ferroptosis, the iron chelator deferoxamine (DFO) was used to suppress EVO-induced ferroptosis. The lipid peroxide level significantly decreased when cells were treated with DFO prior to EVO treatment. DFO also attenuated EVO-induced cell death. Co-treatment with a pan-caspase inhibitor or necroptosis inhibitor with EVO did not alleviate cancer cell death. These results indicate that EVO induces ferroptosis rather than apoptosis or necroptosis. Furthermore, EVO suppressed the migratory ability, decreased the expression of mesenchymal markers, and increased epithelial marker expression, determined by a transwell migration assay and Western blotting. The TCCSUP bladder tumor xenograft tumor model confirmed the effects of EVO on the inhibition of tumor growth and EMT. In conclusion, EVO is a novel inducer for activating the ferroptosis of bladder cancer cells and may be a potential therapeutic agent for bladder cancer.

Keywords: bladder cancer; evodiamine; ferroptosis; glutathione peroxidase 4; lipid peroxidation.

Figure 1

EVO induces cytotoxicity and G2/M cell cycle arrest in TCCSUP cells. (A) TCCSUP cells were treated with different concentrations of EVO for 24 h, and cell viability was detected by the MTS assay (n = 8). (B) Time course of cell viability in response to 15 μM EVO, analyzed by the MTS assay (n = 8). (C) Representative phase-contrast microscopic images of EVO-induced cell death are shown (Scale bar = 100 μm). (D) Cell cycle analysis through PI staining, followed by flow cytometric analysis for the cells after EVO treatment for 24 h. DNA histograms of cells exposed to various concentrations of EVO are shown. (E) Quantitative measurement of the cell population at different phases of the cell cycle (n = 3 for each group). Percentages of cell population at G1, S, and G2/M phases are shown. Data are shown as the mean ± SEM. *** p < 0.001 as compared with the control group.

Figure 2

EVO decreases GPX4 expression to facilitate lipid peroxidation in TCCSUP cells. (A) TCCSUP cells were treated with 100 µM of the iron chelator DFO or the vehicle (DMSO) for 1 h, followed by treatment with 15 µM EVO or the vehicle (DMSO) for 24 h. The cells were incubated with the ROS-sensitive fluorometric probe DCFDA (10 μM) for 30 min in the dark, and the content of ROS was detected with fluorescence microscopy. Nuclei were stained with DAPI (blue). Representative fluorescent and bright-field microscopic images are shown (scale bar = 20 μm). (B,C) TCCSUP cells were treated with DFO or DMSO for 1 h, followed by treatment with EVO or DMSO for 24 h as described in A. The cells were then stained with the lipid peroxidation reporter probe C11-BODIPY (1 μM) at 37 °C for 30 min in the dark. The amount of lipid peroxides (green fluorescence) was measured by flow cytometry (n = 4 for each group). Fluorescence histograms (B) and mean fluorescence intensity (MFI) values (C) are shown. Data are mean ± SEM. CTL—control; ns—non-significant. (D,E) Detection (D) and quantitation (E) of the anti-oxidant GPX4 protein in TCCSUP cells after treatment with EVO for 24 h by Western blot analysis (D) and densitometric analysis using the ImageJ software (n = 6 for each group) (E). Expression of β-actin served as the loading control. The horizontal bars shown in F denote the mean value of each group. * p < 0.05, ** p < 0.01, *** p < 0.001 as compared with the control group.

Figure 3

EVO induces cell death and cell cycle arrest through ferroptosis in TCCSUP cells. (A) TCCSUP cells were treated with 100 µM DFO or the vehicle (DMSO) for 1 h, followed by treatment with 15 µM EVO or the vehicle (DMSO) for 24 h. Representative phase-contrast microscopic images of EVO-induced cell death are shown (Scale bar = 100 μm). (B) TCCSUP cells were treated with DFO (100 µM), Z-VAD-FMK (20 µM), necrostatin-1 (Nec-1, 50 µM), or the vehicle (DMSO) for 1 h, followed by treatment with 15 µM EVO or the vehicle (DMSO) for 24 h. Cell viability was analyzed by the MTS assay (n = 8 for each group). (C,D) Cell cycle analysis with PI staining and flow cytometry after EVO treatment for 24 h with or without prior DFO treatment for 1 h. DNA content histograms (C) and quantitation (D) of different cell cycle phases (n = 6 for each group) are shown. Data are mean ± SEM. ** p < 0.01, *** p < 0.001 as compared with the control group.

Figure 4

EVO inhibits cell migration and EMT in TCCSUP cells. (A,B) TCCSUP cells that were treated with indicated concentrations of EVO for 24 h were seeded (1 × 10⁴ cells) in the upper chamber of the transwell and cultured for 24 h. Cells that migrated through the membrane to the lower surface were stained with Giemsa and quantified. Representative microscopic images (magnification × 100; scale bar = 100 μm) (A) and quantitation (n = 8 for each group) (B) of the migratory cells are shown. (C–G) Detection (C) and quantitation of E-cadherin (D), N-cadherin (E), vimentin (F), and Snail (G) proteins in TCCSUP cells after treatment with indicated concentrations of EVO for 24 h by Western blot analysis (C) and densitometric analysis using the ImageJ software (n = 6 for each group) (D–G). Expression of β-actin served as the loading control. Data are mean ± SEM. The horizontal bars shown in (D–G) denote the mean value of each group. * p < 0.05, ** p < 0.01, *** p < 0.001 as compared with the control group.

Figure 5

EVO inhibits tumor growth and induces MET in NOD-SCID mice bearing TCCSUP tumor xenografts. (A–G) Groups of 14 NOD/SCID mice were inoculated subcutaneously with TCCSUP cells (1 × 10⁶) at day 0, followed by intraperitoneal injection of EVO (20 mg/kg) or PBS for 35 consecutive days. Representative images of gross appearance of tumors excised at day 35 (A). Tumor volumes were measured weekly for 5 weeks and expressed as a percentage of the tumor volume at day 7 in each group (B). Detection (C) and quantitation of E-cadherin (D), N-cadherin (E), vimentin (F), and Snail (G) proteins in day 35 tumor tissues by Western blot analysis (C) and densitometric analysis using the ImageJ software (n = 5 for each group) (D–G). Expression of β-actin served as the loading control. Data are mean ± SEM. * p < 0.05, ** p < 0.01 as compared with the control group.

Figure 6

A schematic illustration of evodiamine (EVO)-induced ferroptosis through suppression of GPX4 in human bladder cancer cells. In TCCSUP human bladder cancer cells, EVO induces cell death and G2/M cell cycle arrest and inhibits cell migration and EMT. Furthermore, EVO suppresses the expression of GPX4, an antioxidant enzyme serving as a crucial negative regulator of ferroptosis, leading to the induction of ferroptosis through accumulation of lipid ROS. The iron chelator deferoxamine (DFO) alleviates EVO-induced cell death, G2/M cell cycle arrest, and lipid peroxidation via chelation of intracellular iron, which is essential for the execution of ferroptosis. In the TCCSUP tumor xenograft model, EVO inhibits tumor growth and suppresses EMT.