Mitochondrial event as an ultimate step in ferroptosis

In ferroptosis, the roles of mitochondria have been controversial. To explore the role of mitochondrial events in ferroptosis, we employed mitochondrial DNA-depleted ρ0 cells that are resistant to cell death due to enhanced expression of antioxidant enzymes. Expression of mitochondrial-type GPx4 (mGPx4) but no other forms of GPx4 was increased in SK-Hep1 ρ0 cells. Likely due to high mGPx4 expression, SK-Hep1 ρ0 cells were resistant to ferroptosis by erastin inhibiting xCT channel. In contrast, SK-Hep1 ρ0 cells were susceptible to cell death by a high concentration of RSL3 imposing ferroptosis by GPx4 inhibition. Accumulation of cellular ROS and oxidized lipids was observed in erastin- or RSL3-treated SK-Hep1 ρ+ cells but not in erastin-treated SK-Hep1 ρ0 cells. Mitochondrial ROS and mitochondrial peroxidized lipids accumulated in SK-Hep1 ρ+ cells not only by RSL3 but also by erastin acting on xCT on the plasma membrane. Mitochondrial ROS quenching inhibited SK-Hep1 ρ+ cell death by erastin or a high dose of RSL3, suggesting a critical role of mitochondrial ROS in ferroptosis. Ferroptosis by erastin or RSL3 was inhibited by a more than 20-fold lower concentration of MitoQ, a mitochondrial ROS quencher, compared to DecylQ, a non-targeting counterpart. Ferroptosis of SK-Hep1 ρ+ cells by erastin or RSL3 was markedly inhibited by a VDAC inhibitor, accompanied by significantly reduced accumulation of mitochondria ROS, total peroxidized lipids, and mitochondrial peroxidized lipids, strongly supporting the role of mitochondrial events in ferroptotic death and that of VDAC in mitochondrial steps of ferroptosis induced by erastin or RSL3. SK-Hep1 ρ+ cell ferroptosis by sorafenib was also suppressed by mitochondrial ROS quenchers, accompanied by abrogation of sorafenib-induced mitochondrial ROS and mitochondrial peroxidized lipid accumulation. These results suggest that SK-Hep1 ρ0 cells are resistant to ferroptosis due to upregulation of mGPx4 expression and mitochondrial events could be the ultimate step in determining final cell fate.