Differences in cell death in methionine versus cysteine depletion

Abstract

Restriction of the sulfur amino acids methionine and cysteine has recently been proposed as potential adjuvant therapy in cancer. While cysteine depletion has been associated with ferroptotic cell death, methionine depletion has not. We hypothesized that comparing the response of melanoma cell lines to depletion of the amino acids methionine and cysteine would give us insight into the critical role in cancer of these two closely related amino acids. We analyzed the response to three conditions: methionine depletion, methionine replacement with homocysteine, and cysteine depletion. In cancer cells, the transcription factor ATF4 was induced by all three tested conditions. The replacement of methionine with homocysteine produced a strong ferroptotic gene signature. We also detected an activation of the NRF2 antioxidant pathway by both methionine and cysteine depletion. Total glutathione levels were decreased by 42% in melanoma cells grown without methionine, and by 95% in cells grown without cysteine. Lipid peroxidation was increased in cells grown without cysteine, but not in cells grown without methionine. Despite the large degree of overlap in gene expression between methionine and cysteine depletion, methionine depletion and replacement of methionine with homocysteine was associated with apoptosis while cysteine depletion was associated with ferroptosis. Glutamine depletion produced comparable gene expression patterns and was associated with a 28% decrease in glutathione. Apoptosis was detected in these cells. In this experiment, a strong ATF4-driven ferroptotic gene signature was insufficient to induce ferroptosis without a concomitant profound decrease in glutathione levels.

Keywords: cell death; cysteine; glutathione; methionine.

FIGURE 1

Proliferation and viability in methionine- and cysteine-depleted cells at 72 hr. (a) Proliferation assessed by Hoechst staining for DNA, expressed as fluorescence relative to control treated cells. (b) Proliferation/viability assessed by fluorescent detection of the resorufin metabolite of resazurin. CTL, control media; CYS-, cysteine-free media; HCY, methionine-free, homocysteine-supplemented media; MET-, methionine-free media

FIGURE 2

Activation of the ATF4 integrated stress response at 24 h. Gene expression of ATF4 (a and b) and its downstream target SESN2 (c and d) in A101D human melanoma cells (a and c) and CCL-75 human normal fibroblasts (b and d). CTL, control media; CYS-, cysteine-free media; HCY, methionine-free, homocysteine-supplemented media; MET-, Methionine-free media

FIGURE 3

Gene expression patterns in A101D human melanoma cells. Expression at 24 h for genes (a) TRIB3, (b) FGF21, (c) SLC7A11, (d) SLC7A5, (e) NFE2L2, (f) HMOX1, (g) GCLM, and (h) CHAC1. CTL, control media; CYS-, cysteine-free media; HCY, methionine-free, homocysteine-supplemented media; MET-, methionine-free media

FIGURE 4

Confirmation analysis. Western blot analysis of the protein ATF4 in a nuclear extract of the cell line A101D, with amido black staining of the same membrane for reference, as well as CHAC1 in the cytoplasm compared with the loading control β-Actin (a). siRNA knockdown of ATF4 (si-ATF4) and NRF2 (si-NRF2) followed by gene expression analysis. CTL, control media; CYS-, cysteine-free media; HCY, methionine-free, homocysteine-supplemented media; MET-, methionine-free media

FIGURE 5

Glutathione and cell death in A101D human melanoma cells. (a) Total glutathione (GSH + GSSG). (b) Rescue of cell proliferation (Hoechst assay) by reduced ethyl ester glutathione (GSH). (c) Flow cytometry quantification of oxidized to reduced BODIPY. (d) Rescue of cell proliferation (Hoechst assay) by ferrostatin (Fer). (e) Flow cytometry quantification of Annexin V-positive cells. (f) Rescue of cell proliferation (Hoechst assay) by the caspase inhibitor Z-VAD-FMK (Z-VADCTL, control media; CYS-, cysteine-free media; HCY, methionine-free, homocysteine-supplemented media; MET-, methionine-free media

FIGURE 6

Methionine and cysteine depletion in normal human CCL-75 fibroblasts. Gene expression of (a) TRIB3, (b) HMOX1, and (c) CHAC1. (d) Total glutathione levels. (e) Proliferation and (f) viability rescue with Hoechst DNA staining. CTL, control media; CYS-, cysteine-free media; HCY, methionine-free, homocysteine-supplemented media; MET-, methionine-free media

FIGURE 7

Glutamine depletion in human melanoma cells. Gene expression (a), total glutathione levels (b), and apoptosis analysis by Annexin V flow cytometry (c). CTL, control media; GLN-, glutamine-free media