Tetrandrine Citrate Suppresses Breast Cancer via Depletion of Glutathione Peroxidase 4 and Activation of Nuclear Receptor Coactivator 4-Mediated Ferritinophagy

Abstract

Tetrandrine citrate (TetC), a novel tetrandrine salt with high water solubility, demonstrates a potent antitumor activity in chronic myeloid leukemia. Studies have indicated an important role of ferroptosis in breast cancer (BC). However, whether TetC inhibits BC progression via ferroptosis has never been explored. In the present study, we showed that TetC had a significant inhibitory effect on the proliferation and migration of MCF7 and MDA-MB-231 cells. Then, we combined TetC with different inhibitors to determine which form of cell death could be driven by TetC. MTT assay showed that ferrostatin (Fer-1) demonstrated the most potent effect on improving TetC-induced cell death in contrast to other inhibitors. TetC was also shown to significantly increase the mRNA level of prostaglandin-endoperoxide synthase 2 (Ptgs2), a ferroptosis marker. Further studies showed that TetC significantly suppressed the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1) but increased the expression of nuclear receptor coactivator 4 (NCOA4) in MCF7 and MDA-MB-231 cells even in the presence of erastin or Ras-selective lethal 3 (RSL3). Collectively, we showed novel data that ferroptosis was a major form of TetC-induced cell death. Moreover, TetC-induced ferroptotic cell death was achieved via suppressing GPX4 expression and activating NCOA4-mediated ferritinophagy in BC cells.

Keywords: breast cancer; ferroptosis; glutathione peroxidase 4; nuclear receptor coactivator 4; tetrandrine citrate.

FIGURE 1

MCF7 and MDA-MB-231 cells were sensitive to erastin- and RSL3-induced ferroptosis. MCF7 and MDA-MB-231 cells were preincubated with or without 1 μM Fer-1 for 1 h. After that, the cells were treated with erastin (10 μM) or RSL3 (1 μM) for 24 h. MTT assay showed that erastin or RSL3 significantly reduced MCF7 and MDA-MB-231 cell viability. (A) Erastin or RSL3 reduced GSH (B) levels but increased intracellular MDA (C) and free iron (D) levels in MCF7 and MDA-MB-231 cells (n = 3 independent repeats). ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001.

FIGURE 2

TetC exerts dose- and time-dependent proliferation inhibitory effects on BC cells. (A,B) IC50 of TetC in MCF7 and MDA-MB-231 cells was determined using MTT assay. (C,D) MCF7 and MDA-MB-231 cells were treated with 20 and 10 μM of TetC for 0, 12, 24, and 48 h. MTT assay showed that TetC exerted dose- and time-dependent proliferation inhibitory effects on MCF7 and MDA-MB-231 cells. (E) Transwell assay showed that TetC significantly suppressed BC cell migration. (F) Flow cytometry assay indicated that TetC induced MCF7 and MDA-MB-231 cell death (n = 3 independent repeats). ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001.

FIGURE 3

TetC drives ferroptosis in BC cells. MCF7 and MDA-MB-231 cells were preincubated with or without 1 μM Fer-1, 20 μM Z-VAD-FMK, 20 μM Nec-1, and 20 μM 3-MA for 1 h. Then, MCF7 and MDA-MB-231 cells were treated with 20 μM or 10 μM TetC for 24 h. MTT assay showed that compared with z-VAD-FMK, Nec-1, and 3-MA, Fer-1 demonstrated the potent cell death inhibitory effects in MCF7 (A) and MDA-MB-231 (B) cells. (C) RT-PCR analysis showed that TetC significantly upregulated the mRNA levels of Ptgs2 and Chac1 in BC cells but reduced GPX4 mRNA levels compared with those of control. TetC elevated the intracellular contents of Fe²⁺ (D) and MDA (E) but decreased the contents of GSH (F) in MCF7 and MDA-MB-231 cells. MCF7 and MDA-MB-231 cells were preincubated with 10 μM NAC or 10 μM DFO for 2h, followed by TetC treatement for another 24 h. DCFH-DA staining showed that TetC-induced accumulation of ROS could be largely attenuated by preincubation with NAC in both MCF7 and MDA-MB-231 cells; scale bar, 100 µm. (G) Upregulation of Fe²⁺ induced by TetC was also reversed by NAC preincubation in MCF7 and MDA-MB-231 cells (H). (I) MTT assay showed that DFO significantly reversed TetC-induced cell death (n = 3 independent repeats). ^$$$ p < 0.001 vs. DMSO; ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001 vs. TetC; ^# p < 0.05; ^## p < 0.01 vs. TetC + Fer-1.

FIGURE 4

TetC enhances erastin- and RSL3-induced ferroptosis in BC cells. (A–C) DCFH-DA staining showed that a combination of TetC and erastin or RSL3 enhanced ROS generation in MCF7 and MDA-MB-231 cells, compared with those of erastin or RSL3 alone; scale bar, 100 µm. (D–F) Annexin V/7-AAD assay showed that erastin- and RSL3-induced ferroptotic damage could be further enhanced after combination with TetC in BC cells (n = 3 independent repeats). ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001 vs DMSO; ^## p < 0.01; ^### p < 0.001 vs erastin or RSL3 alone.

FIGURE 5

TetC activates NCOA4-mediated ferritinophagy and inhibits GPX4 in BC cells. Western blot assay was performed to analyze the effects of TetC on the genes related to ferroptosis compared with erastin (A,B) or RLS3 (C,D) alone in MCF7 and MDA-MB-231 cells (n = 3 independent repeats). *p < 0.05; **p < 0.01; ***p < 0.001 vs. DMSO; ^# p < 0.05; ^## p < 0.01 vs. erastin or RSL3 alone.

FIGURE 6

FTH-1 degradation is achieved by TetC-induced ferritophagy in BC cells. MCF7 and MDA-MB-231 cells were treated 20 and 10 μM TetC for 24 h in the presence or absence of 10 μM chloroquine (CQ). Western blot assay showed that TetC significantly elevated the expression of ATG5, ATG7, and NCOA4 but decreased FTH1 and TfR protein levels in MCF7 (A) and MDA-MB-231 cells (B). TetC-induced degradation of FTH-1 expression was obviously rescued by blocking the lysosome function with CQ in MCF7 (C) and MDA-MB-231 (D) cells. ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001 vs DMSO.

FIGURE 7

Fer-1 alleviates TetC-induced ferroptosis in BC cells. Preincubation with Fer-1 significantly reduced the intracellular ROS (A) and Fe²⁺ (B) in both MCF7 and MDA-MB-231 cells treated with TetC; scale bar, 100 µm. (C) There was an increase in GSH in the TetC-treated group, but addition of Fer-1 obviously reversed such an effect. Fer-1 treatment significantly antagonized TetC-induced elevation of NCOA4 and downregulation of GPX4 and FTH1 in MCF7 (D) and MDA-MB-231 (E) cells. *p < 0.05; **p < 0.01; ***p < 0.001 vs DMSO; ^# p < 0.05; ^### p < 0.001 vs TetC alone.

FIGURE 8

Mechanism by which TetC suppresses ferroptosis in BC cells.