Ferroptosis in Neurons and Cancer Cells Is Similar But Differentially Regulated by Histone Deacetylase Inhibitors

Models of cysteine deprivation. A, The ferroptosis inhibitor erastin is a selective inhibitor of the X_c− transporter. Similarly, in cells devoid of ionotropic glutamate receptors, such as tumor cells and immature neurons, glutamate and its analog HCA work as nonspecific inhibitors of the System X_c− transporter by counteracting the glutamate gradient resulting in reduced uptake of cystine into the cells. This leads to a decrease in glutathione synthesis, which is essential for the endogenous antioxidant defense. Ferroptosis inhibitors are indicated in green. NAC, N-acetylcysteine. B, HT1080 fibrosarcoma cells were treated with increasing dose of erastin at previously described density (left; 100,000 cells/ml) and adjusted density to reach 70% confluency before treatment (right; 25,000 cells/ml) to determine the toxicologically meaningful dose of erastin. Representative live/dead staining are shown, green indicating live cells (calcein AM) and red indicating dead cells (ethidium homodimer-1). Scale bar, 50 µm. C, Dose–response of erastin (left) and HCA (right) in primary cortical neurons. Representative live/dead staining are shown. Scale bar, 50 µm. D, Dose–response of erastin in DRG neurons and representative live/dead staining. Scale bar, 100 µm.

Ferroptosis inhibitors abrogate ferroptosis in cancer cells (HT1080) and primary cortical neurons (PCNs). A, HT1080 cells were treated with 1 µM erastin, PCNs with 5 µM erastin or 5 mM glutamate analog HCA (all LD₅₀) glutamate analog HCA and chemical inhibitors effective in ferroptosis were examined. Numbers show mean ± SD at representative concentration in brackets. Grayscale coding indicates the continuum from no protection in the presence of erastin (black) to maximal cell viability (white). *p < 0.05 versus erastin or glutamate analog (HCA), #p < 0.05 versus inactive analog U0124. B, C, Representative live/dead staining in HT1080 cells (B) and PCN (C) are shown, green indicating live cells (calcein AM) and red indicating dead cells (ethidium homodimer-1). Images for cycloheximide are shown in Figure 8 because it is also a criterion for apoptosis. Scale bar, 50 µm.

Dose–responses of ferroptosis inhibitors in cancer cells (HT1080) and primary cortical neurons (PCNs). HT1080 cells were treated with 1 µM erastin, PCN with 5 µM erastin or 5 mM glutamate analog HCA and chemical inhibitors effective in ferroptosis were examined. Dose–response for cycloheximide is shown in Figure 9 because it is also a criterion for apoptosis. Values represent mean ± SD, except for actinomycin D and Trolox in HT1080 cells, Ferrostatin-1, Deferoxamine, N-acetylcysteine, Trolox, and U0126 in PCN treated with erastin as well as Ferrostatin-1, Deferoxamine, N-acetylcysteine, and U0126 in PCNs treated with glutamate analog (HCA) where medians are given. *p < 0.05 versus erastin or glutamate analog (HCA), #p < 0.05 versus U0124. For exact p values refer to Figure 3-1.

Apoptosis inhibitors do not inhibit ferroptosis in cancer cells (HT1080) and primary cortical neurons (PCNs). A, HT1080 cells were treated with 1 µM erastin, PCN with 5 µM erastin or 5 mM glutamate analog HCA and chemical inhibitors effective in apoptosis were examined. Numbers show mean ± SD at representative concentration in brackets. Grayscale coding indicates the continuum from no protection in the presence of erastin (black) to maximal cell viability (white). *p < 0.05 versus erastin or glutamate analog (HCA). B, C, Representative live/dead staining in HT1080 cells (B) and PCNs (C) are shown, green indicating live cells (calcein AM) and red indicating dead cells (ethidium homodimer-1). Scale bar, 50 µm.

Dose–responses of apoptosis inhibitors in cancer cells (HT1080) and primary cortical neurons (PCNs). HT1080 cells were treated with 1 µM erastin, PCN with 5 µM erastin or 5 mM glutamate analog HCA and chemical inhibitors effective in apoptosis were examined. Values represent mean ± SD, except for cyclosporin A, SP600125, cycloheximide in HT1080 cells, cyclosporin A in PCNs treated with erastin as well as z-VAD-fmk, cyclosporin A, SB203580, SP600125 in PCNs treated with glutamate analog (HCA) where medians are given. *p < 0.05 versus erastin or glutamate analog (HCA). For exact p values refer to Figure 5-1.

Necroptosis inhibitors, but not parthanatos inhibitors, inhibit ferroptosis in cancer cells (HT1080) and primary cortical neurons (PCNs). A, HT1080 cells were treated with 1 µM erastin, PCNs with 5 µM erastin or 5 mM glutamate analog HCA and chemical inhibitors effective in necroptosis and parthanatos were examined. Numbers show mean ± SD at representative concentration in brackets. Grayscale coding indicates the continuum from no protection in the presence of erastin (black) to maximal cell viability (white). *p < 0.05 versus erastin or glutamate analog (HCA), #p < 0.05 versus Necrostatin-1i. B, C, Representative live/dead staining in HT1080 cells (B) and PCN (C) are shown, green indicating live cells (calcein AM) and red indicating dead cells (ethidium homodimer-1). Scale bar, 50 µm.

Dose–responses of parthanatos and necroptosis inhibitors in cancer cells (HT1080) and primary cortical neurons (PCNs). HT1080 cells were treated with 1 µM erastin, PCN with 5 µM erastin or 5 mM glutamate analog HCA and chemical inhibitors effective in necroptosis and parthanatos were examined. Values represent mean ± SD, except for Necrostatin-1 and necrosulfonamide in HT1080 cells, necrosulfonamide in PCNs treated with erastin as well as Olaparib, GSK872, and necrosulfonamide in PCNs treated with glutamate analog (HCA) where medians are given. *p < 0.05 versus erastin or glutamate analog (HCA), #p < 0.05 versus Necrostatin-1i. For exact p values refer to Figure 7-1.

Autophagy inhibitors inhibit ferroptosis in cancer cells (HT1080) and primary cortical neurons (PCNs). A, HT1080 cells were treated with 1 µM erastin, PCN with 5 µM erastin or 5 mM glutamate analog HCA and chemical inhibitors effective in autophagy were examined. Numbers show mean ± SD at representative concentration in brackets. Grayscale coding indicates the continuum from no protection in the presence of erastin (black) to maximal cell viability (white). *p < 0.05 versus erastin or glutamate analog (HCA). B, C, Representative live/dead staining in HT1080 cells (B) and PCN (C) are shown, green indicating live cells (calcein AM) and red indicating dead cells (ethidium homodimer-1). Scale bar, 50 µm.

Dose–responses of autophagy inhibitors in cancer cells (HT1080) and primary cortical neurons (PCNs). HT1080 cells were treated with 1 µM erastin, PCNs with 5 µM erastin or 5 mM glutamate analog HCA and chemical inhibitors effective in autophagy were examined. Values represent mean ± SD, except for 3-methyladenine, Bafilomycin A1, and Chloroquine in PCNs treated with erastin as well as all inhibitors in PCNs treated with glutamate analog (HCA) where medians are given. *p < 0.05 versus erastin or glutamate analog (HCA). For exact p values refer to Figure 9-1.

Ferroptosis inducer erastin and glutamate analog HCA activate necroptotic machinery. Levels of phospho-RIP1 (normalized to β-actin) were measured in HT22 cells exposed to erastin (A), glutamate analog (HCA; B) or 100 ng/ml TNFα + 5 µM z-VAD-fmk for 8 h (positive control in A and B). Necrostatin-1 served as confirmation for specificity of RIP1 kinase activity. Values represent median. *p < 0.05 versus 0 h erastin or glutamate analog (HCA), ‡p < 0.05 versus 8 h TNFα/zVAD. For exact p values refer to Figure 10-1.

Systematic pharmacologic characterization reveals inhibitors of autophagy and necroptosis abrogate ferroptosis in cancer cells (HT1080) and primary cortical neurons (PCNs). A, Comparison of protection profile of chemical inhibitors between operationally defined ferroptosis (Dixon et al., 2012, 2014), erastin-induced toxicity in HT1080 cells at toxicologically meaningful dose, erastin and glutamate analog (HCA) in PCNs and previously published hemin toxicity in PCNs (Zille et al., 2017). B, Statistical analysis of profile of chemical inhibitors between operationally defined ferroptosis and erastin-induced toxicity in HT1080 revealed that they are statistically similar, but major differences (i.e., protection by inhibitors of autophagy and necroptosis) exist. Statistical analysis showed no difference between erastin treatment in PCNs and HT1080 cells, erastin- and glutamate analog (HCA)-treated PCNs or erastin in HT1080 cells and HCA in PCNs.

Do inhibitors of HCA-induced death in neurons prevent ferroptosis in cancer cells? A, HT1080 cells were treated with 1 µM erastin (LD₅₀) and chemical inhibitors effective in glutamate analog (HCA)-induced neuronal toxicity (values from published work and references indicated in last 2 columns) were examined. Numbers show mean ± SD at representative concentration in brackets. Grayscale coding indicates the continuum from no protection in the presence of erastin (black) to maximal cell viability (white). *p < 0.05 versus erastin, #p < 0.05 versus Nullscript (negative control). B, Representative live/dead staining are shown, green indicating live cells (calcein AM) and red indicating dead cells (ethidium homodimer-1). Scale bar, 50 µm.

Protective effect of inhibitors of neuronal toxicity of glutamate analog (HCA) in erastin-induced death in HT1080 cells. A, Dose–responses of chemical inhibitors effective in HCA-induced neuronal toxicity. Values represent mean ± SD, except for Adaptaquin, Apicidin, and Scriptaid, where medians are given. *p < 0.05 versus erastin. B, Gene expression levels of Mithramycin target genes c-Myc and p21 were assessed in HT1080 cells exposed to 50 or 300 nm Mithramycin. Values represent medians. *p = 0.009 versus vehicle. C, Dose–responses of Scriptaid and Nullscript (negative control) in erastin-induced cell death in primary cortical neurons (PCNs). Values represent mean ± SD, *p < 0.05 versus erastin, #p < 0.05 versus Nullscript (negative control). D, Protein levels of acetylated and total histone H4 were assessed in HT1080 cells exposed to Scriptaid or its inactive analog Nullscript. E, Gene expression of HDACs from class I in PCNs versus HT1080 cells. Values represent medians. *p < 0.0125 versus PCNs. For exact p values refer to Figures 13-1, 13-2, 13-3, and 13-4.