Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

Abstract

Inhibition of complex I (CI) of the mitochondrial respiratory chain by BAY 87-2243 ('BAY') triggers death of BRAF V600E melanoma cell lines and inhibits in vivo tumor growth. Here we studied the mechanism by which this inhibition induces melanoma cell death. BAY treatment depolarized the mitochondrial membrane potential (Δψ), increased cellular ROS levels, stimulated lipid peroxidation and reduced glutathione levels. These effects were paralleled by increased opening of the mitochondrial permeability transition pore (mPTP) and stimulation of autophagosome formation and mitophagy. BAY-induced cell death was not due to glucose shortage and inhibited by the antioxidant α-tocopherol and the mPTP inhibitor cyclosporin A. Tumor necrosis factor receptor-associated protein 1 (TRAP1) overexpression in BAY-treated cells lowered ROS levels and inhibited mPTP opening and cell death, whereas the latter was potentiated by TRAP1 knockdown. Knockdown of autophagy-related 5 (ATG5) inhibited the BAY-stimulated autophagosome formation, cellular ROS increase and cell death. Knockdown of phosphatase and tensin homologinduced putative kinase 1 (PINK1) inhibited the BAY-induced Δψ depolarization, mitophagy stimulation, ROS increase and cell death. Dynamin-related protein 1 (Drp1) knockdown induced mitochondrial filamentation and inhibited BAY-induced cell death. The latter was insensitive to the pancaspase inhibitor z-VAD-FMK, but reduced by necroptosis inhibitors (necrostatin-1, necrostatin-1s)) and knockdown of key necroptosis proteins (receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and mixed lineage kinase domain-like (MLKL)). BAY-induced cell death was also reduced by the ferroptosis inhibitor ferrostatin-1 and overexpression of the ferroptosis-inhibiting protein glutathione peroxidase 4 (GPX4). This overexpression also inhibited the BAYinduced ROS increase and lipid peroxidation. Conversely, GPX4 knockdown potentiated BAY-induced cell death. We propose a chain of events in which: (i) CI inhibition induces mPTP opening and Δψ depolarization, that (ii) stimulate autophagosome formation, mitophagy and an associated ROS increase, leading to (iii) activation of combined necroptotic/ferroptotic cell death.

TRAP1 regulates autophagy in lung cancer cells

“…TRAP1, along with HSP90 mitochondrial pool and HSP60, has been described to physically interact with matrix CypD modulating its regulatory activity and resulting in mPTP inhibition . Surprisingly, TRAP1 depletion resulted in a more closed mPTP conformation under basal conditions, which was the opposite effect to that observed in melanoma . Our result was not due to loss of CypD expression or activity as CypD expression was intact and addition of cyclosporin A resulted in an increase in mPTP closing.…”

“…3,8 Surprisingly, TRAP1 depletion resulted in a more closed mPTP conformation under basal conditions, which was the opposite effect to that observed in melanoma. 38 Our result was not due to loss of CypD expression or activity as CypD expression was intact and addition of cyclosporin A resulted in an increase in mPTP closing. The discrepancy with previous works may reflect that these studies relied on extrapolation of mPTP status from analysis of DΨ m alterations 8 or on evaluation of cell viability in the presence or absence of CsA.…”

Pseudomonas Quinolone Signal molecule PQS behaves like a B Class inhibitor at the I Q site of mitochondrial complex I

“…Elevated ROS values can induce mitophagy . To investigate whether PQS treatment would modulate the degree of mitophagy in HeLa cells, the distribution of the fusion protein LC3::mCherry after PQS treatment was compared with the control.…”

“…Elevated ROS values can induce mitophagy. 46 To investigate whether PQS treatment would modulate the degree of mitophagy in HeLa cells, the distribution of the fusion protein LC3::mCherry after PQS treatment was compared with the control. During the activation of autophagy pathways, 22 amino acids are cleaved from the C-terminus of LC3 to form LC3-II which selectively localizes in newly formed autophagosomes.…”

Emerging Strategies of Cancer Therapy Based on Ferroptosis

“…[37] Schockel et al recently demonstrated that BAY 87–2243, a potent inhibitor of NADH-coenzyme Q oxidoreductase (complex I), induced dose-dependent ferroptosis on a panel of BRaf V600E melanoma cell lines. [38,39] Summarily, the experimental compounds including erastin, 1,2-dioxolane, siramesine, lapatinib, and BAY 87–2243 (Figure 1), are promising as potential clinical drugs for ferroptosis-based cancer therapy.…”