Ferroptosis: A Novel Anti-tumor Action for Cisplatin

Abstract

Purpose: Ferroptosis is a new mode of regulated cell death, which is completely distinct from other cell death modes based on morphological, biochemical, and genetic criteria. This study evaluated the therapeutic role of ferroptosis in classic chemotherapy drugs, including the underlying mechanism.

Materials and methods: Cell viabilitywas detected by using the methylthiazoltetrazlium dye uptake method. RNAiwas used to knockout iron-responsive element binding protein 2, and polymerase chain reaction, western blot was used to evaluate the efficiency. Intracellular reduced glutathione level and glutathione peroxidases activitywere determined by related assay kit. Intracellularreactive oxygen species levelswere determined by flowcytometry. Electron microscopywas used to observe ultrastructure changes in cell.

Results: Among five chemotherapeutic drugs screened in this study, cisplatin was found to be an inducer for both ferroptosis and apoptosis in A549 and HCT116 cells. The depletion of reduced glutathione caused by cisplatin and the inactivation of glutathione peroxidase played the vital role in the underlying mechanism. Besides, combination therapy of cisplatin and erastin showed significant synergistic effect on their anti-tumor activity.

Conclusion: Ferroptosis had great potential to become a new approach in anti-tumor therapies and make up for some classic drugs, which open up a new way for their utility in clinic.

Keywords: Cisplatin; Erastin; Ferroptosis; Glutathione; Glutathione peroxidase.

Fig. 1.

Cisplatin induced both apoptosis and ferroptosis on A549 and HCT116 cells. (A) A549, NCIH358, NCIH460, Calu-1, HCT116, and HT-1080 cells received treatments of cisplatin (5 μg/mL), fluorouracil (5-FU, 20 μg/mL), adriamycin (10 μg/mL), paclitaxel (10 μmol/L), and sulfasalazine (0.8 mmol/L), respectively in the absence or presence of ferrostatin-1 (Fer-1, 0.5 μmol/L) for 48 hours. (B) A549 and HCT116 cells were under the treatment of cisplatin (5 μg/mL) for 48 hours with different cell death inhibitors as described. C, cisplatin; Z, z-vad-fmk (20 μmol/L); N, necrostatin-1 (30 μmol/L); Ch, chloroquine (5 μg/mL); F, ferrostatin-1 (Fer-1, 0.5 μmol/L); D, deferoxamine, DFO (50 μmol/L); E, erastin (10 μmol/L); n.s., not significant. (C) A549 and HCT116 cells received treatment of cisplatin in different concentrations with or without Fer-1 (0.5 μmol/L) as displayed. Cell viabilities were analyzed by MTT. Standard error represents three independent experiments (n=3). ^*p < 0.05, ^**p < 0.01, Student’s t test.

Fig. 2.

Microscopy images of cisplatin treated cells. (A) Optical microscopy images of A549 cells under the treatment of cisplatin (5 μg/mL) with or without ferrostatin-1 (Fer-1, 0.5 μmol/L) for indicated time (×100, scale bars=100 μm). (B) Transmission electron microscopy images for HCT116 cells which were treated with cisplatin (5 μg/mL) and erastin (10 μmol/L), respectively for 48 hours. Arrows indicate mitochondria in cells (left: ×6,000, scale bars=2 μm; right: ×15,000, scale bars=1 μm).

Fig. 3.

Cisplatin induced ferroptosis in A549 and HCT116 cells. (A) Polymerase chain reaction and western blot analysis of HCT116 cells transferred with small interfering RNAs (siRNAs) targeting IREB2 (siIERB2). HCT116 cells were transferred with siIERB2 or siNeg 24 hours in advance, then, treated with cisplatin (5 μg/mL) or normal saline for 48 hours. Cell viabilities were analyzed by MTT. (B) A549 and HCT116 cells were under the treatment of cisplatin (5 μg/mL) for 48 hours with different specific ferroptosis inhibitors. Cell viabilities were analyzed by MTT. Standard error represents three independent experiments (n=3). ^*p < 0.05, Student’s t test. C, cisplatin; F, ferrostatin-1 (Fer-1, 0.5 μmol/L); β, β-mercaptoethanol (β-ME,=50 μmol/L); D, deferoxamine (DFO, 50 μmol/L); E, erastin (10 μmol/L). (C) A549 cells were under the treatment of cisplatin (5 μg/mL), erastin (10 μmol/L), or Fer-1 (0.5 μmol/L) as demonstrated for 48 hours. Reactive oxygen species (ROS) levels in cells were evaluated and exhibited. (D) HCT116 cells were under the treatment of cisplatin (5 μg/mL), erastin (10 μmol/L), or Fer-1 (0.5 μmol/L) as demonstrated for 48 hours. ROS levels in cells were evaluated and exhibited.

Fig. 4.

Cisplatin led to intracellular glutathione (GSH) depletion and glutathione peroxidases (GPXs) inactivation in A549 and HCT116 cells. (A) GSH level analysis of A549 and HCT116 cells. (B) GPXs activity analysis of A549 and HCT116 cells. Cells were treated with cisplatin (5 μg/mL) or erastin (10 μmol/L, as a positive control) for 48 hours, respectively. DMSO, dimethyl sulfoxide. Standard error represents three independent experiments (n=3). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, Student’s t test.

Fig. 5.

Improved anti-tumor activity was observed in combination of cisplatin and erastin. (A) A549 cells were treated with cisplatin and erastin of different concentrations as demonstrated for 48 hours. (B) HCT116 cells were treated with cisplatin and erastin of different concentrations as demonstrated for 48 hours. (C) A549 and HCT116 cells were under the treatment of cisplatin (5 μg/mL) or erastin (10 μmol/L) for 48 hours, together with z-vad-fmk (20 μmol/L) or ferrostatin-1 (Fer-1, 0.5 μmol/L), respectively. Cell viabilities were analyzed by MTT. C, cisplatin; E, erastin; F, ferrostatin-1 (Fer-1); Z, z-vad-fmk; β-ME, β-mercaptoethanol; n.s., not significant. Standard error represents three independent experiments (n=3). ^**p < 0.01, ^***p < 0.001, Student’s t test. (D) Optical microscopy images of A549 cells under the treatment of cisplatin (5 μg/mL), erastin (10 μmol/L) with or without β-ME (50 μmol/L) for indicated time (×100, scale bars=100 μm). (E) HCT116 cells were under the treatment of cisplatin (5 μg/mL), erastin (10 μmol/L) with or without β-ME (50 μmol/L) as demonstrated for 48 hours. Reactive oxygen species levels in cells were evaluated and exhibited.