Fatty acid called DGLA can kill human cancer cells


Researchers have demonstrated that a fatty acid called dihomogamma-linolenic acid, or DGLA, can kill human cancer cells.

The study, published in Developmental Cell on July 10, found that DGLA can induce ferroptosis in an animal model and in actual human cancer cells.

Ferroptosis is an iron-dependent type of cell death that was discovered in recent years and has become a focal point for disease research as it is closely related to many disease processes.

Jennifer Watts, a Washington State University associate professor and corresponding author on the paper, said this discovery has many implications, including a step toward a potential treatment for cancer.

“If you could deliver DGLA precisely to a cancer cell, it could promote ferroptosis and lead to tumor cell death,” Watts said.

“Also, just knowing that this fat promotes ferroptosis might also affect how we think about conditions such as kidney disease and neurodegeneration where we want to prevent this type of cell death.”

DGLA is a polyunsaturated fatty acid found in small amounts in the human body, though rarely in the human diet. Compared to other fatty acids, such as those found in fish oil, DGLA is relatively understudied.

Watts has been researching dietary fats including DGLA for nearly twenty years, using the nematode Caenorhabditis elegans as an animal model.

A microscopic worm, C. elegans is often used in molecular research because it is transparent and allows scientists to easily study cell-level activity in a whole animal over its relatively short lifespan. Results found in the C. elegans cells are also often transferable to human cells.

Watts’ research team discovered that feeding nematodes a diet of DGLA-laden bacteria killed all the germ cells in the worms as well as the stem cells that make the germ cells. The way the cells died carried many signs of ferroptosis.

“Many of the mechanisms we saw in the nematodes were consistent with the hallmarks of ferroptosis in mammalian systems, including the presence of redox-active iron and the inability to repair oxidized lipids, which are like molecular executioners,” said Marcos Perez, a WSU doctoral student and first author on the paper.

To see if the results would translate to human cells, Watts and Perez collaborated with Scott Dixon of Stanford University, who has been studying ferroptosis and its potential for battling cancer for many years.

Taking what they had learned from the nematode work, the researchers showed that DGLA could induce ferroptosis in human cancer cells. They also found an interaction with another fatty acid class, called an ether lipid, that had a protective effect against DGLA. When they took out the ether lipids, the cells died faster in the presence of DGLA.

In addition to this new knowledge, the study also demonstrated that C. elegans can be a useful animal research model in the study of ferroptosis, a field that has had to rely mostly on cell cultures.

To take this research further, Watts’ team recently received a $1.4 million grant from the National Institutes of Health to investigate what makes the nematode germ cells so susceptible to DGLA and explore the role of mitochondria, the cell organelles involved in burning fat and regulating metabolism, in ferroptosis.

Ferroptosis is a non-apoptotic, iron-dependent form of regulated cell death (RCD) occurring when the intracellular levels of lipid reactive oxygen species (L-ROS) exceed the antioxidant activity of glutathione-dependent peroxidase (GPX4) thus leading to the collapse of cellular redox homeostasis [1].

Ferroptosis is defined by three essential hallmarks:

(i) oxidation of polyunsaturated fatty acid (PUFA)-containing membrane phospholipids;

(ii) availability of redox-active iron; and

(iii) loss of lipid hydroperoxide (LOOH) repair capacity [2].

The physiological function of ferroptosis as well as its involvement in multiple human diseases, such as ischemic organ injury, neurodegeneration, and cancer, have been established [3–6].

Unlike other RCDs, ferroptosis appears more like a cellular “sabotage” than a pro-active “suicide” mechanism [7]. While the “suicide” pathway (i.e., apoptosis, necroptosis, and pyroptosis) is actively triggered by a dedicated pro-death molecular machinery, the “sabotage” mechanism occurs when either inactivation or hyper activation of physiological processes causes a lethal metabolic imbalance with a so far undefined involvement of dedicated pro-death proteins [8].

During ferroptosis, cells are “sabotaged” by their own ongoing metabolism [9]. In cancer, such metabolic imbalance fosters the removal of tumor cells, thus suggesting ferroptosis is a sort of adaptive response which exerts a tumor suppressive function [10].

In this perspective, modulation of ferroptosis might represent a potential therapeutic approach for the so-called “persister” cancer cells, resistant to either standard chemotherapy or molecular-targeted therapies [11].

The essential role of cellular metabolism in ferroptosis is currently widely investigated. Mounting experimental evidence has demonstrated that numerous metabolic pathways, including cellular respiration (i.e., mitochondrial tricarboxylic acid (TCA) cycle and electron transport chain (ETC)), lipid metabolism, and amino acid metabolism contribute to ferroptosis through the generation of L-ROS [12,13].

Of note, iron metabolism may also induce ferroptosis through the lipid peroxide-generating Fenton Reaction [14]. As master regulators of oxidative phosphorylation (OXPHOS), mitochondria are the main intracellular producers of ROS [15].

Mitochondria are also focal hubs in iron metabolism and homeostasis [16]. The assessment of mitochondrial iron by using mitochondrion-selective fluorescent iron indicators or by using electron paramagnetic resonance revealed that, depending on the cell type, these organelles contain up to 20–50% of the total intracellular iron [17,18].

Mitochondrial iron mainly participates in iron–sulfur (Fe–S) cluster biogenesis and heme synthesis [19]; however, there also exists a free and redox active iron pool [20] which actively participates in the accumulation of mitochondrial ROS (mitoROS) [21].

In cancer cells, mitoROS act as second messengers in oncogenic signal transduction cascades, including those driven by mitogen-activated protein kinase (MAPK) and by the transcription factor NF-kB [22].

Upon accumulation, mitoROS can react with PUFAs in mitochondrial membranes leading to lipid peroxidation, mitochondrial DNA (mtDNA) damage, and subsequent defects in mtDNA-encoded subunits of the ETC complexes [23].

Such modifications have been observed not only in cancer cells but also in those diseases in which oxidative stress is increased such as chronic inflammations and neurodegenerative diseases [24,25].

All these observations are consistent with the potential involvement of mitochondria in ferroptosis. A series of molecular, pharmacological, and metabolomic analyses highlight that the metabolic activity of mitochondria, including both TCA cycle and ETC, is required for the generation of sufficient L-ROS to initiate ferroptosis [26].

Indeed, pharmacological induction of ferroptosis leads to mitoROS accumulation, mitochondrial fragmentation, alteration of the mitochondrial membrane potential (∆Ψm), and ATP depletion [27–29].

Recent studies have also shown that a dysregulation of mitochondrial iron is typical of some neurological diseases, including Alzheimer’s disease, Huntington’s disease, Friedreich’s ataxia, and Parkinson’s disease which are all linked to ferroptosis [30–33].

Despite the involvement of mitochondria in ferroptosis being clearly defined, a comprehensive characterization of the underlying molecular mechanisms is still missing. Moreover, whether the involvement of mitochondria in ferroptosis is context-dependent or rather a general phenomenon is still unclear.

In this review, we summarize recent advances in our understanding of mitochondrial involvement in ferroptosis, and we discuss the potential opportunity to use mitochondria-mediated ferroptosis as a new strategy for cancer therapy.

Mitochondria at the Crossroad of Ferroptosis and Cancer Suppression
Mitochondria play a pivotal role in metabolic plasticity in malignant cells, as well as in the regulation of many RCD processes, and ferroptosis is no exception [140].

Mitochondria seem to be involved in ferroptosis induced by cystine deprivation (CDI) which, indeed, is associated with mitochondrial membrane hyperpolarization and lipid peroxide accumulation [26].

In agreement, erastin treatment boosts the production of mitoROS [26] which, in turn, cause opening of mitochondrial permeability transition pore (mPTP), dissipation of ∆Ψm and ATP depletion [141]. Cells undergoing ferroptosis exhibit mitochondria fragmentation and specific changes in mitochondrial morphology such as reduction of mitochondrial cristae and decrease in mitochondrial size [142].

However, some questions remain controversial. Whether mitochondrial dysregulation is able, per se, to initiate this type of cell death or it is just a consequence of the metabolic imbalance is unclear.

Based on Gaschler et al. [134], cells lacking mitochondria are still sensitive to ferroptosis. Conversely, according to Gao et al. [26], inhibition of TCA cycle and mitochondrial ETC can rescue cells from mitochondrial membrane hyperpolarization, lipid peroxide accumulation, and ferroptosis.

Mitochondrial role in ferroptosis seems context dependent. Upon cystine deprivation, mitochondria contribute to reducing GSH and to promoting ROS production. Glutaminolysis is required for CDI ferroptosis.

Of note, in the absence of Gln, neither cystine starvation nor erastin inhibition of system xc− can induce ferroptosis [26]. Mitochondrial free iron accumulation exacerbates erastin-mediated ferroptosis [143].

Alternatively, sequestering iron within mitochondria via overexpression of mitochondrial ferritin (FtMt) can counteract erastin-induced cell death, both in vitro and in vivo [76]. Supporting this last observation, impaired mitochondrial iron metabolism is a common feature of many neurodegenerative diseases (i.e., Alzheimer’s, Parkinson’s, Huntington’s diseases) [144–147], all linked to ferroptosis.

Morphologically, mitochondria in brains isolated from mice models of these diseases exhibit disrupted cristae [148] that are reminiscent of those observed in ferroptosis. Upon GPX4 inhibition, ferroptosis appears, instead, independent of mitochondria [26].

In the following sections, we review the morphological, metabolic, and energetic features that closely relate mitochondria to ferroptotic cell death (Figure 3).

Figure 3. Mitochondrial metabolic processes in ferroptosis. (1) Iron uptake via Mfrn1/2 increases LIP amount, promoting mitoROS generation through Fenton Reaction. (2) BID triggers ferroptosis through BAX and BAK activation and the consequent dysregulation of ∆Ψm. (3) ACSF2 regulates activation of fatty acids derived from carnitine shuttle mechanism, providing the specific lipid precursor for β-oxidation. (4) VDAC2/3 imports Fe2+ into mitochondria. Fe2+  contributes to enhance LIP which,  in turn, generates mitoROS. (5) MnSOD converts superoxide anion (O2−) from ETC to hydrogen peroxide (H2O2) which takes part into Fenton Reaction, thus promoting ferroptosis. (6) CISD1 regulates mitochondrial iron export acting as ferroptosis suppressor. (7) FtMt prevents Fenton Reaction through iron-storage and ferroxidase activities. (8) LONP1 maintains mitochondrial integrity, preventing ferroptosis induction. (9) ACSL4, LPCAT, and LOXs activate lipid peroxidation, driving ferroptosis.
(10) CS regulates fatty acid synthesis through the release of CoA, a precursor for β-oxidation, thus inducing ferroptosis. (11) Glutamine is converted to glutamate by the mitochondrial isoform GLS2. Glutamate is converted in α-KG by GDH and GOT/GPT enzymes,  thus providing fuel for TCA  cycle and lipid biosynthesis. Abbreviations used: Mfrn1/2, mitoferrin 1/2; LIP, labile iron pool; mitoROS, mitochondrial reactive oxygen species; BID, BH3 interacting-domain death agonist; BAX, Bcl-2-associated X protein (also known as bcl-2-like protein 4); BAK, Bcl-2 homologous antagonist killer; ACSF2, acyl-CoA synthetase family member 2; VDAC2/3, voltage-dependent anion channels 2/3; MnSOD, mitochondrial superoxide dismutase; ETC, electron transport chain; CISD1, CDGSH Iron Sulfur Domain 1; FtMt, mitochondrial ferritin; LONP1, lon peptidase 1; ACSL4, long-chain-fatty-acid—CoA ligase 4; LPCAT, lyso-phosphatidylcholine acyltransferase; LOXs, lipoxygenase; AA, arachidonic acid; PE, phosphatidylethanolamine; CPT1/2, carnitine palmitoyltransferase 1/2;CS, citrate synthase; CoA, coenzyme A; GLS1/2, glutaminase 1/2; α-KG: alpha-ketoglutarate; GDH, glutamate dehydrogenase; GOT, glutamic oxaloacetic transaminase; GTP, glutamic pyruvic transaminase; TCA cycle (tricarboxylic acid cycle); AOA, amino-oxyacetic acid.

Ferroptosis occurs when lipid hydroperoxide detoxification mediated by GPX4 activity is reduced to such an extent that it becomes insufficient to restrain iron-dependent membrane PUFA oxidation and toxic ROS accumulation [2].

As a main source of cellular ROS, mitochondrial metabolism is likely to play a pivotal role in the execution of ferroptosis [26]. A survey of the literature clearly highlights that ferroptosis is accompanied by severe morphological and functional mitochondrial damages and that, at the same time, a proper function of mitochondrial bioenergetic metabolism is mandatory for the initiation and the accomplishment of this new type of cell death [35,45,99].

Interference of key regulators of mitochondrial lipid metabolism (i.e., ASCF2 and CS), glutamine metabolism (i.e., GLS2), TCA cycle (i.e., FH) and other signaling pathways consistently enhance sensitivity to ferroptosis [26,34]. Nonetheless, our knowledge of the molecular mechanisms underlying these events are still limited and additional studies are warranted.

Suggestive evidence of the ferroptosis/mitochondria crosstalk is represented by the strong
iron dependency of this RCD [26,38]. Intracellular iron accumulation can generate ROS and cause oxidative stress via Fenton Reaction, thereby promoting lipid peroxidation [61].

Mitochondrial iron homeostasis is altered to satisfy the redox active iron demands for propagating ferroptosis [26,99]. A direct in vivo evidence for the involvement of mitochondrial iron metabolism in ferroptosis is represented by neurodegenerative diseases, whose pathogenetic mechanisms have been recently linked to ferroptosis [167].

Whether mitochondrial iron crosstalk with cytosolic iron or, otherwise, mitochondrial iron metabolism is independent, to a certain extent, from cytosolic iron metabolism is still under debate [16]. For instance, when heme synthesis is inhibited in the mitochondrion, iron continues to enter these organelles [192].

This finding may suggest the lack of communication between cytoplasm and mitochondrion, as iron continues to be transported into this organelle irrespective of heme synthesis inhibition. Otherwise, it can suggest that iron continues to enter the mitochondrion in
an effort to rescue heme synthesis.

A recent work by Li et al. [193], highlighted that fibroblasts and lymphoblasts from Friedreich’s ataxia (FA) patients display cytosolic iron-deficiency. Overexpression of mitochondrial ferritin (FtMt) in the mitochondrion leads to mitochondrial iron-loading and cytosolic iron deprivation [194]. Collectively, these data suggest that mitochondrial iron metabolism can mediate ferroptosis by modulating whole-cell iron processing.

Metabolic plasticity is a critical property that gives cancer cells the edge for expanding, persisting after therapeutic hits and evading immune surveillance [195]. Recently, metabolic reprogramming has been associated with acquired sensitivity to ferroptosis, thus opening up new opportunities to treat therapy-insensitive tumors [1].

Of note, either the genetic manipulation or the pharmacological targeting of proteins involved in ferroptosis have been found to induce cell death in a wide range of cancer cells [11]. The susceptibility of different types of cancer cells to ferroptosis is though significantly variable [155].

Based on some recent studies, the different sensitivity of cancer cells to ferroptosis depends on their basic metabolic status [78]. Considering the pivotal role of mitochondria in tumor cell metabolic rewiring, it is possible that modulation of the mitochondrial metabolic pathways might reshape the tumor microenvironment thus leading to ferroptosis-mediated tumor suppression.

To make some examples, cancer stem cells frequently present a mitochondrial metabolic shift from glycolysis to OXPHOS [196], that can be exploited to make these cells vulnerable to ferroptosis. Glutaminolysis is used by the majority of cancer cells to satisfy their bioenergetic requirements [197].

Since its role in promoting ferroptosis, glutaminolysis may represent a nodal point of vulnerability for cancer cells and a potential target for novel anti-tumor strategies [198]. Iron addiction is a characteristic of cancer cells [199].

Modulation of both mitochondrial FXN and NEET proteins has been associated with CDI ferroptosis in cancer cells.
Overall, these findings provide a clear support for the potential use of mitochondria-mediated ferroptosis in cancer treatment. Future studies exploring the effects of mitochondrial metabolic rewiring in in vivo models of ferroptosis would be necessary to confirm the role of this cell death as new exciting frontier in cancer biology.

Reference link

More information: Developmental Cell (2020). DOI: 10.1016/j.devcel.2020.06.019


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