Differences in cell death in methionine versus cysteine depletion.

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.