In 2012, a team of researchers at Columbia led by Professor Brent R. Stockwell announced a new discovery; A novel kind of cell death that they named “Ferroptosis.” When cells undergo ferroptosis, their inner and outer membranes degrade, springing leaks that eventually cause the cell to die.

A decade after that initial discovery, Professor Daoling Tang published an article in Nat Rev Clin Oncol that explained the key molecular mechanisms of ferroptosis.

In this review, the authors proposed several points about ferroptosis:

1. Ferroptosis is a regulated cell death that depends on iron-mediated oxidative damage.

2. Ferroptosis can occur through two main pathways: the exogenous (transporter-dependent) pathway and the endogenous (enzyme-regulated) pathway.

3. Increased iron accumulation, production of free radicals, fatty acid supply and increased lipid peroxides are the keys to induce ferroptosis^[1].

Exogenous (transporter-dependent) pathway

The exogenous pathway is initiated by inhibiting cell membrane transporters such as cystine/glutamate antiporter (System xc-), or by activating the iron transporters serotransferrin and lactoferrin^[1].

1.Inhibition of cystine/glutamate antiporter to promote ferroptosis

System xc- is an important part of the antioxidant system in cells. System xc− consists of two subunits, SLC7A11 and SLC3A2. SLC7A11 is responsible for the main transport activity and is highly specific for cystine and glutamate, while SLC3A2 acts as a chaperone. System xc− imports cystine into cells with a 1:1 counter-transport of glutamate in exchange for extracellular cystine (Cys2). Once in cells, cystine (Cys2) can be oxidized to cysteine (Cys), which is used to synthesize glutathione (GSH) in a reaction catalysed by glutamate–cysteine ligase (GCL) and glutathione synthetase (GSS)^[1].

GSH is a reducing cofactor for glutathione peroxidase 4 (GPX4). GPX4 is a membrane lipid repair enzyme. The inhibition of System xc- influences the absorption of cystine and affects the synthesis of GSH, which in turn leads to a decrease in the activity of GPX4, and a decrease in the antioxidant capacity of cells, thereby promoting ferroptosis^[1].

2.Iron transporters and overloaded iron levels

Increasing iron absorption, reducing iron storage and limiting iron efflux all lead to increased iron accumulation, which promotes ferroptosis through a series of signaling pathways. Transferrin (serum transferrin or lactoferrin) mediates iron uptake through the transferrin receptor (TFRC) and FTH1/FTL (ferritin assembly) increases iron levels through autophagic degradation, which promote ferroptosis. In contrast, SLC40A1-mediated iron efflux and exosome-mediated ferritin export inhibits ferroptosis^[1].

Endogenous (enzyme-regulated) pathway

The endogenous pathway is activated by blocking intracellular antioxidant enzymes such as GPX4.

1.The inhibition of GPX4-induced ferroptosis

Lipid peroxide accumulation is a hallmark of ferroptosis. GPX4 can reduce cytotoxic lipid peroxides (L-OOH) to the corresponding alcohols (L-OH). The inhibition of GPX4 activity results in the accumulation of lipid peroxides in cell membranes.

Direct inhibition: For example, as an inducer of ferroptosis, RSL3 can directly act on GPX4 and inhibit its activity, thus reducing the antioxidant capacity of cells and accumulating ROS, leading to ferroptosis.

Indirect inhibition: The inhibition of GSH synthesis: Selenocysteine is one of the essential amino acids for the active group of GPX4. The inhibition of the mevalonate (MVA) pathway can down-regulate the maturation of selenocysteinetRNA to affect the growth of GPX4. Thus, the inhibition of GSH synthesis can affect the activity of GPX4 and induce ferroptosis^[2].

2.Regulatory pathways of other enzymes and fatty acid accumulation

Acetyl-CoA carboxylase (ACAC)-mediated fatty acid synthesis or lipophagy-mediated fatty acid release induces intracellular free fatty acid accumulation and fuels ferroptosis.

Long-chain fatty acid coenzyme A ligase 4 (ACSL4) and lysophospholipid acyltransferase 3 (LPCAT3) facilitate the incorporation of polyunsaturated fatty acids (PUFAs) into phospholipids to form polyunsaturated fatty acid phospholipids (PUFA-PL). PUFA-PLs are vulnerable to free radical-induced oxidation mediated by lipoxygenases (ALOXs). This oxidation ultimately leads to disruption of the lipid bilayer and affects membrane function, thereby promoting ferroptosis^[1].

After understanding the basic pathways of ferroptosis, how to effectively develop research strategies for ferroptosis has become the focus of everyone's attention. Now, we will explain in detail by taking a study, “Energy-stress-mediated AMPK activation inhibits ferroptosis” as an example.

In this article, Hyemin et al. established a cell model of ferroptosis: they used a variety of inducers and inhibitors, and a variety of detection methods were used to prove the regulatory relationship between ferroptosis and AMPK. They also established an AMPKα1/α2 knockout cell line (AMPK DKO) to verify the mechanism of AMPK deletion on ferroptosis sensitivity^[3].

1.Energy stress inhibits ferroptotic cell death.

First, Hyemin et al. explored the effect of glucose starvation on erastin-induced ferroptosis in immortalized mouse embryonic fibroblasts (MEFs). It was demonstrated that Erastin induces ferroptosis. Neither Caspase-3 nor PARP cleavage (a hallmark of apoptosis) was down-regulated. However, ferroptosis inhibitor Ferrostatin-1 could reverse erastin-induced cell death. Initially, they expected that conditions of glucose starvation would enhance erastin-induced ferroptosis, yet the results were quite the opposite: glucose starvation largely reversed ferroptosis induced in MEFs^[3].

Hyemin et al. further selected other compounds that can induce or mimic energy stress, including 2-deoxyglucose (2-DG), acadisine (AICAR), A769662. These compounds also significantly inhibited lipid peroxidation and ferroptosis induced by Erastin treatment. To sum up, energy stress inhibits ferroptosis^[3].

2.The establishment of AMPKα1/α2 DKO.

Next, Hyemin et al. validated the correlation between basal AMPK activation status (p-AMPK Thr172 as an activation marker) and ferroptosis (expression level of SLC7A11) in a panel of cell lines as shown in Figure 5c.

SLC7A11 high expressing cells were more resistant to ferroptosis relative to the low expressing cells as depicted in Fig. 5c-d. It was worth noting that although AMPK activation status in SLC7A11 high expressing cells was not related to ferroptosis sensitivity, AMPK activation in SLC7A11-low expressing cells was negatively correlated with ferroptosis sensitivity. The above data suggested that energy stress inhibits ferroptotic cell death partly through AMPK^[3].

3.AMPK inactivation sensitizes cells to ferroptotic cell death.

Hyemin et al. further investigated whether AMPK promotes ferroptosis resistance in cancer cell lines with high basal AMPK phosphorylation levels.

It was found that the AMPK inhibitor Compound C down-regulated AMPK activation, as shown in Figure 6a-b, Compound C sensitized ACHN cells (a ferritin-resistant cell line with high basal AMPK phosphorylation) to Erastin or cystine depletion. Transmission electron microscopy (TEM) results also showed that co-treatment of Compound C with Erastin or cystine depletion in ACHN cells resulted in mitochondrial shrinkage and increased membrane density, but no apparent DNA fragmentation in the nucleus (a characteristic morphology of ferroptotic cells)^[3]. This suggests that inhibition of AMPK sensitizes cancer cells to ferroptosis.

In conclusion, the inhibitory effect of energy stress on ferroptosis is achieved in part through the activation of AMPK.

Summary:

1. The GSH-GPX4 antioxidant system plays an important role in the ferroptosis pathway. Increased lipid peroxides, transferrin-mediated iron accumulation, and intracellular free fatty acid accumulation can induce ferroptosis.

2. Hyemin et al. used ferroptosis related inhibitors/inducers and established AMPK knockout cell lines to demonstrate the regulatory relationship between ferroptosis and AMPK.

3. Common experimental methods to detect ferroptosis are ferroptosis-related cell survival analysis, such as CCK8 (other cell viability detection methods include MTT method, trypan blue staining, etc.). Besides cell viability assay, lipid oxidation level determination (C11 BODIPY 581/591 staining), GSH assay, mitochondrial ROS assay, and GPX4 activity assay, monitoring of mitochondrial changes under electron microscope, and analysis of specific target molecules (WB, IHC, IF, etc.) are also commonly used as detection methods.

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