Peroxidation Of Polyunsaturated Fatty Acids Research Articles

52 Ferroptosis Suppressor Protein 1 is a Potent and Targetable Inhibitor of Ferroptosis in Chromophobe Renal Cell

Abstract Background There are currently no proven therapies for metastatic or unresectable Chromophobe renal cell carcinoma (ChRCC). Ferroptosis is an iron-dependent form of cell death characterized by the peroxidation of membrane polyunsaturated fatty acids, leading to membrane damage and cell death. Glutathione, a potent cellular antioxidant, counters lipid peroxidation to prevent ferroptosis. Both reduced and oxidized glutathione are markedly elevated in ChRCC (PNAS, 2018). We have previously discovered that ChRCC is hypersensitive to ferroptotic cell death induced by the pharmacologic disruption of glutathione homeostasis via the cysteine transporter SLC7A11 or glutathione peroxidase (GPX4) inhibition (PNAS, 2022). Ferroptosis suppressor protein (FSP1) is a glutathione-independent suppressor of ferroptosis. FSP1’s role in ChRCC is unexplored. Methods Transcriptional data from DepMap (484 cell lines) and Cancer Therapeutics Response Portal (CTRP) (860 cell lines) were used to study genetic or pharmacologic inhibition of GPX4 and SLC7A11. Statistical analysis was performed in PRISM 10 using Mann-Whitney U and ANOVA tests. Statistical significance was defined as p < 0.05. Results DepMap data revealed that FSP1 is the top upregulated gene in cells resistant to RNAi inhibition of GPX4 (Achilles, DEMETER2) (Panel A). In CTRP data, FSP1 was the top upregulated gene in cells resistant to the GPX4 inhibitor RSL3 and the 19th most upregulated gene in cells resistant to the SCL7A11 inhibitor, Erastin (Panel B). In The Cancer Genomic Atlas (TCGA) KICH (ChRCC) dataset, FSP1 was 2-fold higher in ChRCC compared to normal kidney (mean RSEM = 818.9 vs 445.9, p-value <0.0001) (Panel C). In vitro studies of two ChRCC cell lines, UOK276 and RCJ-T2, revealed that while siRNA-mediated inhibition of GPX4 or FSP1 individually did not induce substantial cell death, their combination was synergistic and resulted in almost complete cell death (UOK276 mean viability 8% of siCTRL, p<0.0001; RCJ-T2 mean viability 13% of siCTRL, p<0.0001). Cell viability was completely rescued by the ferroptosis inhibitor ferrostatin-1 but not by the necroptosis inhibitor necrostatin-1 or the apoptosis inhibitor Z-Vad-FMK (Panel D). siRNA-mediated GPX4 knockdown combined with FSP1 pharmacologic inhibition (icFSP1 or FSEN1) induced extensive cell death (mean viability of siGPX4 + icFSP1 group = 21% of siCTRL + DMSO group, p<0.0001; mean viability of siGPX4 + FSEN1 group = 29% of siCTRL + DMSO group, p<0.0001) (Panel E). Combining RSL3 with FSEN1, or IKE with icFSP1, also demonstrated synergy, with the ChRCC cell lines showing increased overall sensitivity to these drug combinations compared to 786-0 cells (ccRCC-derived) and HeLa cells (Panel F). Snapshots from these experiments taken at [IKE] = 177nM and/or [icFSP1] = 20 mM for UOK276, and [RSL3] = 39.4 nM and/or [FSEN1] = 20 mM for RCJ-T2, show almost complete cell death when pharmacologic inhibitors of glutathione-dependent and -independent ferroptosis suppressors are combined, but no substantial cell death when each is used individually (7% and 10% viability in combination group compared to DMSO group for UOK276 and RCJ-T2 respectively, p<0.0001) (Panel G). Conclusions We show that in vitro genetic or pharmacologic inhibition of FSP1, a glutathione-independent inhibitor of ferroptosis, leads to ferroptosis in ChRCC-derived cells when combined with genetic or pharmacologic inhibition of the glutathione-dependent suppressors of ferroptosis GPX4 or SLC7A11.

Dietary Supplementation with n-3 Polyunsaturated Fatty Acids Delays the Phenotypic Manifestation of Krabbe Disease and Partially Restores Lipid Mediator Production in the Brain-Study in a Mouse Model of the Disease.

Lipid mediators from fatty acid oxidation have been shown to be associated with the severity of Krabbe disease (KD), a disorder linked to mutations in the galactosylceramidase (GALC) gene. This study aims to investigate the effects of n-3 polyunsaturated fatty acid (PUFA) supplementation on KD traits and fatty acid metabolism using Twitcher (Tw) animals as a natural model for KD. Wild-type (Wt), heterozygous (Ht), and affected Tw animals were treated orally with 36 mg n-3 PUFAs/kg body weight/day from 10 to 35 days of life. The end product of PUFA peroxidation (8-isoprostane), the lipid mediator involved in the resolution of inflammatory exudates (resolvin D1), and the total amount of n-3 PUFAs were analyzed in the brains of mice. In Tw mice, supplementation with n-3 PUFAs delayed the manifestation of disease symptoms (p < 0.0001), and in the bran, decreased 8-isoprostane amounts (p < 0.0001), increased resolvin D1 levels (p < 0.005) and increased quantity of total n-3 PUFAs (p < 0.05). Furthermore, total brain n-3 PUFA levels were associated with disease severity (r = -0.562, p = 0.0001), resolvin D1 (r = 0.712, p < 0.0001), and 8-isoprostane brain levels (r = -0.690, p < 0.0001). For the first time in a natural model of KD, brain levels of n-3 PUFAs are shown to determine disease severity and to be involved in the peroxidation of brain PUFAs as well as in the production of pro-resolving lipid mediators. It is also shown that dietary supplementation with n-3 PUFAs leads to a slowing of the phenotypic presentation of the disease and restoration of lipid mediator production.

#1955 Ferroptotic proteins ACSL4 and ALOX15 as therapeutic targets in AKI

Abstract Background and Aims Ferroptosis is a regulated form of necrosis which is dependent on cellular iron and is characterized by the accumulation of lipid peroxides and failure of cellular antioxidant defences. We have previously described in vivo that ferroptosis is the primary cause of folic acid-induced acute kidney injury (FA-AKI) and that necroinflammation secondary to ferroptosis may further worsen kidney injury, since ferroptosis inhibition improved kidney function and decreased tubular cell death and oxidative stress. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is involved in the incorporation of polyunsaturated fatty acid (PUFA) into membranes, while 15-Lipoxygenase (Alox15) catalyzes the regio- and enantioselective peroxidation of membrane-esterified PUFAs, forming the ultimate peroxidized species that induce ferroptosis. In the present work, we aim to explore the potential of both proteins as therapeutic targets in AKI, as well as the molecular characterization of kidney ferroptosis in cultured tubular cells. Method Animal model: Female 12- to 14-week-old C57BL/6J wild type mice received a single intraperitoneal (i.p) injection of folic acid or vehicle and were sacrificed 48 hours later. Troglitazone was intravenously administered for in vivo ACSL4 inhibition, and Compound 1 was i.p administered for in vivo ALOX15 inhibition. In vitro characterization of ferroptosis: HK2 human tubular kidney cells were incubated with arachidonic acid (AA) prior to sublethal RSL3 administration for sensitization to ferroptosis. We used Ferrostatin-1 (Fer-1) and Liproxstatin-1 (Lpx-1) as inhibitors of ferroptosis. The thiazolidinedione family members Rosiglitazone, Pioglitazone and Troglitazone were used as pharmacological inhibitors of ACSL4. We also performed transcriptional silencing of ACSL4 using a specific siRNA. Compound 1 and PD1646 were administered for in vitro Alox15 inhibition. Cellular lipid peroxidation was analyzed by flow cytometry with Bodipy 581/591 staining. To characterize the lipid signature of ferroptotic cells, supernatants were collected and 15-HETEs levels were measured by ELISA. Oxido-lipidomic analysis of ferroptotic cells was performed by LC-MS. Real Time PCR and Western Blot of whole kidneys and cultured cells were performed for the detection and quantification of ACSL4 and ALOX15. Cell death was assessed by measurement of cell viability (MTT assay) and cytotoxicity (LDH assay). Tissue cell death was assessed by TUNEL. Results Kidney transcriptomics identified Acsl4 as the most upregulated member of Acsl family during FA-AKI. This was validated at mRNA and protein levels. Supplementation with AA, the preferred ACSL4 substrate, sensitized HK2 tubular cells to ferroptosis under sublethal RSL3 conditions, and this was prevented with specific ferroptosis inhibitors Fer-1 and Lpx-1. Pharmacological inhibition of ACSL4 with Troglitazone and a specific siRNA protected from cell death and lipid peroxidation induced by co-stimulation of AA and RSL3 in HK2 cells. Likewise, ALOX15 proteins levels were also increased in FA-AKI at 48 hours. PD1646 and Compound 1, both used to inhibit ALOX15, protected from ferroptotic cell death and lipid peroxidation in HK2 cells. Lipidomic analysis on HK2 tubular cells stimulated with AA+RSL3 uncovered an increased content of key peroxidized lipid species involved in ferroptosis execution, which was alleviated by targeting ACSL4 or ALOX15. Additionally, renal function of FA-AKI in mice was improved by ACSL4 pharmacological inhibition with Troglitazone and with ALOX15 inhibition with Compound 1. Conclusion Our preliminary results suggest therapeutic potential of ACSL4 and ALOX15 as ferroptotic targes during AKI. High PUFA content sensitizes human tubular HK2 cells to cell death and lipid peroxidation in the presence of sublethal ferroptotic triggers, and this was prevented by targeting ACSL4 or ALOX15 both in vitro and in vivo in FA-AKI. Closely related lipid peroxide species were decreased by ACSL4 or ALOX15 inhibitors.

The efficacy of ferroptosis-inducing compounds IKE and RSL3 correlates with the expression of ferroptotic pathway regulators CD71 and SLC7A11 in biliary tract cancer cells.

Biliary tract cancer (BTC) is a lethal disease with a bad overall survivability, partly arising from inadequate therapeutic alternatives, detection at a belated stage, and a resistance to common therapeutic approaches. Ferroptosis is a form of programmed cell death that depends on reactive oxygen species (ROS) and iron, causing excessive peroxidation of polyunsaturated fatty acids (PUFAs). Therefore, the objective of this investigation is, whether ferroptosis can be induced in BTC in vitro and whether this induction is dependent on specific molecular markers. The study conducted resazurin assay and IC25/50 calculation to explore the possible cytotoxic outcomes of different classes of ferroptosis-inducing substances (FINs) on a comprehensive in vitro model of 11 BTC cell lines. Combinatory treatments with different cell death inhibitors were performed to evaluate the magnitude of ferroptosis induction. To ascertain whether ferroptotic cell death occurred, liperfluo and iron assay kits were employed to evaluate lipid ROS and intracellular iron abundance. Potential biomarkers of ferroptosis sensitivity were then assessed via western blot analysis, a rtPCR panel and functional assay kits. The study found that different FINs reduced cell viability in a cell line-dependent manner. In addition, we measured increased lipid ROS and intracellular Fe2+ levels upon exposure to FINs in BTC cells. Combining FINs with inhibitors of ferroptosis, necroptosis or apoptosis suggests the occurrence of ferroptotic events in BTC cell lines CCC-5, HuH-28 and KKU-055. Furthermore, we found that BTC cells display a heterogeneous profile regarding different molecular genes/markers of ferroptosis. Subsequent analysis revealed that sensitivity of BTC cells towards IKE and RSL3 positively correlated with CD71 and SLC7A11 protein expression. Our results demonstrate that induction of ferroptosis is a promising approach to inhibit BTC cell growth and that the sensitivity of BTC cells towards ferroptosis induction might be dependent on molecular markers such as CD71 and SLC7A11.

Abstract 5996: Genome-wide CRISPR screening reveals a central role for ferroptotic cell death in the anti-tumor response to mTOR inhibitors in HNSCC

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer globally, resulting in more than 300,000 deaths each year worldwide. Treatment options for HNSCC patients include surgery, radiation, chemotherapy and molecularly targeted therapies, and although immunotherapies have recently revolutionized the treatment landscape, &amp;lt;20% of HNSCC patients respond to immune check point blockade (ICB) therapies. The survival rate of HNSCC patients has changed only modestly over the past decades, and therefore HNSCC is a significant global health problem with high mortality and morbidity. Genomic alterations converging in the persistent activation of the PI3K/mTOR pathway (&amp;gt;80% cases) represent one of the most frequently altered signaling circuitries in HNSCC. This overreliance on PI3K/mTOR signaling for tumor growth may expose a cancer vulnerability that can be exploited therapeutically, as revealed in recent clinical trials using mTOR inhibitors (mTORi) in HNSCC in the adjuvant and neoadjuvant setting. Here, we took advantage of a whole-genome CRISPR screening approach to identify mechanisms of sensitivity and resistance to mTORi, aimed at increasing their clinical activity. We found that the activation of autophagy, a biological process that plays a paradoxical pro-survival or antitumoral role in a cancer specific fashion, is strictly required for mTORi cell growth inhibition in HNSCC. In depth analysis of our CRISPR screening also revealed multiple hits suggestive of a novel role for iron metabolism and ferroptosis downstream from mTORi. Ferroptosis is iron-dependent regulated cell death process caused by the peroxidation of polyunsaturated fatty acids. Indeed, we found that mTORi induces ferroptosis in HNSCC cells, and that inhibition of ferroptosis reduces the effect of mTORi. Evidence will be presented supporting a novel mechanism linking mTORi-triggered autophagy and the activation of ferroptotic death programs, as well as new synergistic combinations with mTORi by repurposing approved drugs that disable cellular ferroptotic defense mechanisms. Our studies uncovered how mTORi act in HNSCC, thereby revealing new multimodal precision therapies for HNSCC and many human malignancies displaying overactive PI3K/mTOR signaling. Citation Format: Keiichi Koshizuka, Xingyu Wu, Kuniaki Sato, Pham Thuy Vo, Gosia M. Murawska, Tomohiko Ishikawa, Zhiyong Wang, Alfredo A. Molinolo, Edward A. Dennis, Prashant Mali, J. Silvio Gutkind. Genome-wide CRISPR screening reveals a central role for ferroptotic cell death in the anti-tumor response to mTOR inhibitors in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5996.

Understanding the unique mechanism of ferroptosis: a promising therapeutic target.

Ferroptosis is an iron-dependent form of regulated cell death and is characterized by high concentrations of intracellular lipid peroxide and a redox imbalance in the cells. Ferroptosis shows distinct morphological and biological features compared with other prominent mechanisms of programmed cell death. The distinct characteristics of ferroptosis include the dysfunction of the lipid peroxide repair enzyme glutathione peroxidase 4, the presence of ferrous iron overload, and the lipid peroxidation of polyunsaturated fatty acids. Several other metabolic pathways (including iron, lipid, and amino acid metabolism) and ferritinophagy, as well as transcription factors, can modulate ferroptosis. However, to date, the molecular mechanism of ferroptosis has not been elucidated. This review outlines the discovery, characterization, regulatory mechanisms, and crosstalk of ferroptosis. Further, we have noted the controversial elements in the ferroptosis-related mechanisms. Our inferences may provide a partial reference for developing strategies to regulate ferroptosis.

From MS/MS library implementation to molecular networks: Exploring oxylipin diversity with NEO-MSMS

Oxylipins, small polar molecules derived from the peroxidation of polyunsaturated fatty acids (PUFAs), serve as biomarkers for many diseases and play crucial roles in human physiology and inflammation. Despite their significance, many non-enzymatic oxygenated metabolites of PUFAs (NEO-PUFAs) remain poorly reported, resulting in a lack of public datasets of experimental data and limiting their dereplication in further studies. To overcome this limitation, we constructed a high-resolution tandem mass spectrometry (MS/MS) dataset comprising pure NEO-PUFAs (both commercial and self-synthesized) and in vitro free radical-induced oxidation of diverse PUFAs. By employing molecular networking techniques with this dataset and the existent ones in public repositories, we successfully mapped a wide range of NEO-PUFAs, expanding the strategies for annotating oxylipins, and NEO-PUFAs and offering a novel workflow for profiling these molecules in biological samples.

Dysregulated palmitic acid metabolism promotes the formation of renal calcium-oxalate stones through ferroptosis induced by polyunsaturated fatty acids/phosphatidic acid

The pathogenesis of renal calcium-oxalate (CaOx) stones is complex and influenced by various metabolic factors. In parallel, palmitic acid (PA) has been identified as an upregulated lipid metabolite in the urine and serum of patients with renal CaOx stones via untargeted metabolomics. Thus, this study aimed to mechanistically assess whether PA is involved in stone formation. Lipidomics analysis of PA-treated renal tubular epithelial cells compared with the control samples revealed that α-linoleic acid and α-linolenic acid were desaturated and elongated, resulting in the formation of downstream polyunsaturated fatty acids (PUFAs). In correlation, the levels of fatty acid desaturase 1 and 2 (FADS1 and FADS2) and peroxisome proliferator-activated receptor α (PPARα) in these cells treated with PA were increased relative to the control levels, suggesting that PA-induced upregulation of PPARα, which in turn upregulated these two enzymes, forming the observed PUFAs. Lipid peroxidation occurred in these downstream PUFAs under oxidative stress and Fenton Reaction. Furthermore, transcriptomics analysis revealed significant changes in the expression levels of ferroptosis-related genes in PA-treated renal tubular epithelial cells, induced by PUFA peroxides. In addition, phosphatidyl ethanolamine binding protein 1 (PEBP1) formed a complex with 15-lipoxygenase (15-LO) to exacerbate PUFA peroxidation under protein kinase C ζ (PKC ζ) phosphorylation, and PKC ζ was activated by phosphatidic acid derived from PA. In conclusion, this study found that the formation of renal CaOx stones is promoted by ferroptosis of renal tubular epithelial cells resulting from PA-induced dysregulation of PUFA and phosphatidic acid metabolism, and PA can promote the renal adhesion and deposition of CaOx crystals by injuring renal tubular epithelial cells, consequently upregulating adhesion molecules. Accordingly, this study provides a new theoretical basis for understanding the correlation between fatty acid metabolism and the formation of renal CaOx stones, offering potential targets for clinical applications.

Gastric cancer secreted miR-214-3p inhibits the anti-angiogenesis effect of apatinib by suppressing ferroptosis in vascular endothelial cells.

Different from necrosis, apoptosis, autophagy and other forms of cell death, ferroptosis is a mechanism that catalyzes lipid peroxidation of polyunsaturated fatty acids under the action of iron divalent or lipoxygenase, leading to cell death. Apatinib is currently used in the third-line standard treatment of advanced gastric cancer, targeting the anti-angiogenesis pathway. However, Apatinib-mediated ferroptosis in vascular endothelial cells has not been reported yet. Tumor-secreted exosomes can be taken up into target cells to regulate tumor development, but the mechanism related to vascular endothelial cell ferroptosis has not yet been discovered. Here, we show that exosomes secreted by gastric cancer cells carry miR-214-3p into vascular endothelial cells and directly target zinc finger protein A20 to negatively regulate ACSL4, a key enzyme of lipid peroxidation during ferroptosis, thereby inhibiting ferroptosis in vascular endothelial cells and reducing the efficiency of Apatinib. In conclusion, inhibition of miR-214-3p can increase the sensitivity of vascular endothelial cells to Apatinib, thereby promoting the antiangiogenic effect of Apatinib, suggesting a potential combination therapy for advanced gastric cancer.

Malondialdehyde Assays in Higher Plants.

Malondialdehyde is a three-carbon dialdehyde produced as a byproduct of polyunsaturated fatty acid peroxidation widely used as a marker of the extent of lipid peroxidation in plants. There are several methodological approaches to quantify malondialdehyde contents in higher plants, ranging from the simplest, cheapest, and quickest spectrophotometric approaches to the more complex ones using tandem mass spectrometry. This chapter summarizes the advantages and limitations of approaches followed and provides brief protocols with some tips to facilitate the selection of the best method for each experimental condition and application.

Enhancing lipid peroxidation via radical chain transfer reaction for MRI guided and effective cancer therapy in mice

Lipid peroxidation (LPO), the process of membrane lipid oxidation, is a potential new form of cell death for cancer treatment. However, the radical chain reaction involved in LPO is comprised of the initiation, propagation (the slowest step), and termination stages, limiting its effectiveness in vivo. To address this limitation, we introduce the radical chain transfer reaction into the LPO process to target the propagation step and overcome the sluggish rate of lipid peroxidation, thereby promoting endogenous lipid peroxidation and enhancing therapeutic outcomes. Firstly, radical chain transfer agent (CTA-1)/Fe nanoparticles (CTA-Fe NPs-1) was synthesized. Notably, CTA-1 convert low activity peroxyl radicals (ROO·) into high activity alkoxyl radicals (RO·), creating the cycle of free radical oxidation and increasing the propagation of lipid peroxidation. Additionally, CTA-1/Fe ions enhance reactive oxygen species (ROS) generation, consume glutathione (GSH), and thereby inactivate GPX-4, promoting the initiation stage and reducing termination of free radical reaction. CTA-Fe NPs-1 induce a higher level of peroxidation of polyunsaturated fatty acids in lipid membranes, leading to highly effective treatment in cancer cells. In addition, CTA-Fe NPs-1 could be enriched in tumors inducing potent tumor inhibition and exhibit activatable T1-MRI contrast of magnetic resonance imaging (MRI). In summary, CTA-Fe NPs-1 can enhance intracellular lipid peroxidation by accelerating initiation, propagation, and inhibiting termination step, promoting the cycle of free radical reaction, resulting in effective anticancer outcomes in tumor-bearing mice.

Heat stress induces ferroptosis of porcine Sertoli cells by enhancing CYP2C9-Ras- JNK axis

Heat stress leads to the accumulation of lipid peroxides in Sertoli cells. Unrestricted lipid peroxidation of catalyzed polyunsaturated fatty acids by Cytochrome P450 (CYP) drive the ferroptosis. However, little is known about the role of CYP cyclooxygenase in heat stress-induced ferroptosis in Sertoli cells. In this study, we investigated the relationship between CYP cyclooxygenase and heat stress-induced ferroptosis in porcine Sertoli cells, as well as whether Ras-JNK signaling is involved in the process. The results showed that heat stress significantly increased the expression of cytochrome P450 cyclooxygenase 2C9 (CYP2C9) and the content of epoxyeicosatrienoic acids (EETs), although there are no significant effect on the expression of cytochrome P450 cyclooxygenase 2J2 (CYP2J2) and cytochrome P450 cyclooxygenase 2C8 (CYP2C8). In addition, heat stress reduced the cell viability, the protein expression level of glutathione peroxidase 4 (GPX4) and Ferritin (all P < 0.01) while increased the level of intracellular reactive oxygen species (ROS) and the protein level of Transferrin receptor 1(TFR1) (both P < 0.01), as well as activating the Ras-JNK signaling pathway. Ferrostatin-1, a ferroptosis-specific inhibitor, reduced ROS levels and the protein level of TFR1 (both P < 0.01), but elevated the cell viability, the protein level of GPX4, and Ferritin (all P < 0.01). Sulfaphenazole, a specific inhibitor of CYP2C9 or two small interfering RNAs targaring CYP2C9 enhanced the cell viability (all P < 0.01), while reduced the content of EETs (all P < 0.01) and inhibited the Ras-JNK signaling and ferroptosis under heat stress. Salirasib, a specific inhibitor of Ras, significantly elevated the cell viability, whereas reduced the level of intracellular ROS and inhibited the phosphorylation of JNK, and alleviated heat stress-induced ferroptosis in porcine Sertoli cells. Notably, there is no effect on the expression of CYP2C9 and the content of EETs. These results indicate that heat stress can induce ferroptosis in Sertoli cells by increasing the expression of CYP2C9 and the content of EETs, which in true activates the Ras-JNK signaling pathway, but there is no feedback from Ras-JNK signaling to the expression of CYP2C9. Our study finds a novel heat stress-induced cell death model of Sertoli cells as well as providing the therapeutic potential for anti-ferroptosis.

Inhibition of ACLY overcomes cancer immunotherapy resistance via polyunsaturated fatty acids peroxidation and cGAS-STING activation.

Adenosine 5'-triphosphate citrate lyase (ACLY) is a cytosolic enzyme that converts citrate into acetyl-coenzyme A for fatty acid and cholesterol biosynthesis. ACLY is up-regulated or activated in many cancers, and targeting ACLY by inhibitors holds promise as potential cancer therapy. However, the role of ACLY in cancer immunity regulation remains poorly understood. Here, we show that ACLY inhibition up-regulates PD-L1 immune checkpoint expression in cancer cells and induces T cell dysfunction to drive immunosuppression and compromise its antitumor effect in immunocompetent mice. Mechanistically, ACLY inhibition causes polyunsaturated fatty acid (PUFA) peroxidation and mitochondrial damage, which triggers mitochondrial DNA leakage to activate the cGAS-STING innate immune pathway. Pharmacological and genetic inhibition of ACLY overcomes cancer resistance to anti-PD-L1 therapy in a cGAS-dependent manner. Furthermore, dietary PUFA supplementation mirrors the enhanced efficacy of PD-L1 blockade by ACLY inhibition. These findings reveal an immunomodulatory role of ACLY and provide combinatorial strategies to overcome immunotherapy resistance in tumors.

A guide to ferroptosis, the biological rust of cellular membranes.

Unprotected iron can rust due to oxygen exposure. Similarly, in our body, oxidative stress can kill cells in an iron-dependent manner, which can give rise to devastating diseases. This type of cell death is referred to as ferroptosis. Generally, ferroptosis is defined as an iron-catalyzed form of regulated necrosis that occurs through excessive peroxidation of polyunsaturated fatty acids within cellular membranes. This review summarizes how ferroptosis is executed by a rather primitive biochemical process, under tight regulation of lipid, iron, and redox metabolic processes. An overview is given of major classes of ferroptosis inducers and inhibitors, and how to detect ferroptosis. Finally, its detrimental role in disease is briefly discussed.

Treatment of cardiomyocytes isolated from left ventricular myocardium of dogs with chronic heart failure with ALDA-1, an activator of aldehyde dehydrogenase-2, improves mitochondrial function

Abstract Background Reactive oxygen species (ROS) generated by dysfunctional mitochondria (MITO) of the failing heart plays an important role in lipid peroxidation of polyunsaturated fatty acids in cell and MITO membranes resulting in the production of aldehydes, such as 4-hydroxy-2-nonenal (4-HNE). MITO aldehyde dehydrogenase-2 (mALDH2) is a key enzyme for the detoxification of endogenous aldehyde substrates through NAD dependent oxidation. ALDH2 catalyzes toxic 4HNE to nontoxic 4HNA in cardiac cells. We previously showed that mALDH2 expression and activity are markedly reduced in MITO of failing LV cardiomyocytes (CM), a maladaptation that can lead to considerable accumulation of 4-HNE in MITO and ultimately to progressive LV systolic and diastolic dysfunction. In this study, we tested the hypothesis that exposure of failing cardiomyocytes to ALDA-1, an activator of mALDH2, improves MITO function. Methods Freshly isolated CM from LV myocardium of dogs (n = 7) with heart failure (HF, LV ejection fraction ≤35%) and of normal (NL) dogs (n = 4) were incubated for 1 hour in absence (baseline) and presence of Alda-1 at concentration of 10 µM. After incubation, ADP-stimulated respiration (ADPresp) and maximal rate of ATP synthesis (ATPmax) were measured using Strathklein respirometer and bioluminescence ATP assay kit. Results Treatment of isolated CM from HF dogs with 10 µM Alda-1 significantly increased ADPresp (277 ± 18 vs. 189 ± 12 nAtom O/min/mg, p&amp;lt;0.05) and ATPmax (30 ± 3 vs. 16 ± 3 nmoles/min/mg, p&amp;lt;0.05) compared to untreated CM of HF dogs. Treatment of CM from NL dogs with 10 µM Alda-1 had no effect on ADPresp (488 ± 18 vs. 484 ± 19 nAtom O/min/mg) or ATPmax (54 ± 3 vs. 52 ± 2 nmoles/min/mg) compared to untreated NL CM. Conclusions Treatment of CM from HF dogs with Alda-1 improved overall MITO function. Activation of mALDH2 represents a potential viable therapeutic target for treating HF.

Hypoxia and lactate influence VOC production in A549 lung cancer cells.

Introduction: Cancer cells emit characteristic volatile organic compounds (VOCs), which are potentially generated from ROS-based lipid peroxidation of polyunsaturated fatty acids. The metabolism of such VOCs and their regulation remain to be fully investigated. In fact, the enzymes involved in the synthesis of these VOCs have not been described yet. Methods: In this study, we firstly conducted in vitro enzyme assays and demonstrated that recombinant alcohol dehydrogenase (ADH) converted Trans 2-hexenal into Trans 2-hexenol. The latter has previously been reported as a cancer VOC. To study VOC metabolism, 14 different culture conditions were compared in view of Trans 2-hexenol production. Results and discussion: The data indicate that hypoxia and the addition of lactate positively influenced Trans 2-hexenol production in A549 cancer cells. The RNAseq data suggested certain gene expressions in the VOC pathway and in lactate signaling, parallel to VOC production. This implies that hypoxia and lactate signaling with a VOC production can be characteristic for cancer in vitro.

Malondialdehyde as a Potential Oxidative Stress Marker for Allergy-Oriented Diseases: An Update.

Malondialdehyde (MDA) is a compound that is derived from the peroxidation of polyunsaturated fatty acids. It has been used as a biomarker to measure oxidative stress in various biological samples in patients who are affected by a wide range of diseases. The aim of our work is to provide an updated overview of the role of MDA as a marker of oxidative stress in allergy-related diseases. We considered studies involving both paediatric and adult patients affected by rhinitis, asthma, urticaria and atopic dermatitis. The measurement of MDA was performed on different types of samples. The reported data highlight the role of serum MDA in inflammatory airway diseases. According to the literature review, the oxidative stress status in asthmatic patients, assessed via MDA determination, appears to worsen in the presence of other allergic airway diseases and in relation to the disease severity. This suggests that MDA can be a suitable marker for monitoring the disease status. However, there are several limitations in the considered studies due to the different samples used and the lack of phenotyping and description of the clinical period of patients examined. In cutaneous allergic diseases, the role of MDA is controversial because of the smallness of the studies and the heterogeneity of the samples and patients.

Abstract P3173: Exposure Of Mitochondria Isolated From Left Ventricular Myocardium Of Dogs With Moderate Heart Failure To 4-hydroxy-2-nonenal (4-hne) Worsens Existing Mitochondrial Dysfunction

Background: Reactive oxygen species (ROS) generated by dysfunction of mitochondria (MITO) of the failing heart plays an important role in lipid peroxidation of polyunsaturated fatty acids in cell and MITO membranes resulting in the production of aldehydes, such as malondialdehyde and 4-hydroxy-2-nonenal (4-HNE). MITO aldehyde dehydrogenase-2 (mALDH2) is a key enzyme for the detoxification of endogenous aldehyde substrates through NAD dependent oxidation. We previously showed that mALDH2 expression and activity are markedly reduced in MITO of failing LV cardiomyocytes, a maladaptation that can lead to considerable accumulation of 4-HNE in MITO. It is unclear whether such an accumulation of 4-HNE can further exacerbate MITO dysfunction resulting in further limitation to ATP formation via oxidative phosphorylation. Hypothesis: This study tested the hypothesis that exposure of MITO isolated from failing hearts to exogenous 4-HNE can result in further deterioration of MITO function. Methods: Freshly isolated MITO from LV myocardium of dogs with moderate heart failure (HF, LV ejection fraction ~40%) were incubated for 1 hour in absence (baseline) and presence of 4-HNE at concentrations of 3 and 30 μM. MITO energetic parameters namely ADP-stimulated respiration (ADPresp), maximal respiratory capacity (MRC) and ATP-linked respiration (ATPresp) were evaluated using an XFe96 analyzer (Seahorse Biosciences). Results: Compared to baseline, exposure of MITO to 3 μM 4-HNE has no significant effects on any of the MITO energetic measures. However, exposure of MITO to 30 μM 4-HNE resulted in significant reduction of ADPresp (490 ± 37 vs. 179 ± 7 OCR pmols/O 2 /min, p&lt;0.05), significant reduction of MRC (334 ± 56 vs. 124 ± 18 OCR pmols/O 2 /min, p&lt;0.05), and significant reduction of ATPresp (48 ± 10 vs. 20 ± 12 OCR pmols/O 2 /min, p&lt;0.05). Conclusions: Exposure of MITO from failing myocardium to elevated levels of 4-HNE can lead to marked deterioration of MITO function that can result in a further reduction of ATP leading to progression of LV dysfunction. Limiting the accumulation of 4-HNE through pharmacologic normalization of ALDH2 activity can potentially represent a therapeutic target for retarding the progression of LV dysfunction toward intractable HF.

Dietary docosahexaenoic acid plays an opposed role in ferroptotic and non-ferroptotic acute kidney injury

Ferroptosis due to polyunsaturated fatty acid (PUFA) peroxidation has been implicated in the pathogenesis of acute kidney injury (AKI), suggesting the risk of dietary intake of PUFA for people susceptible to AKI. Clinically, however, in addition to ferroptosis, other mechanisms also contribute to different types of AKI such as inflammation associated necroptosis and pyroptosis. Therefore, the role of PUFA, especially ω3 PUFA which is a common food supplement, in various AKIs deserves further evaluation. In this study, rhabdomyolysis- and folic acid-induced AKI (Rha-AKI and FA-AKI) were established in mice fed with different fatty acids Histology of kidney, blood urea nitrogen and creatinine, lipid peroxidation, and inflammatory factors were examined. Results showed that these two types of AKIs had diametrically different pathogenesis indicated by that ferrostatin-1 (Fer-1), a lipid antioxidant, can attenuate FA-AKI rather than Rha-AKI. Further, dietary DHA (provided by fish oil) reduced tubular injury and renal lesion by inhibiting peroxidation and inflammation in mice with Rha-AKI while increasing cell death, tissue damage, peroxidation and inflammation in mice with FA-AKI. In human renal tubular epithelial cell line HK-2, MTT assay and DHE staining showed that both myoglobin and ferroptosis inducers can cause cell death and oxidative stress. Ferroptosis inducer-induced cell death was promoted by DHA, while such result was not observed in myoglobin-induced cell death when adding DHA. This study illustrates that the mechanisms of AKI might be either ferroptosis dependent or -independent and the deterioration effect of dietary DHA depends on whether ferroptosis is involved.

Detoxification of the Lipid Peroxidation Aldehyde, 4-Hydroxynonenal, by Apple Phloretin In Vitro and in Mice.

4-Hydroxy-2-nonenal (4-HNE) is a secondary cytotoxic product generated from lipid peroxidation of polyunsaturated fatty acids (PUFAs). The accumulation of 4-HNE can covalently modify biomolecules, such as DNA and proteins, leading to various pathological conditions. Apple phloretin has been shown to be able to trap 4-HNE in vitro, but the trapping mechanisms of 4-HNE by phloretin are not fully understood. Moreover, whether the in vitro trapping efficacy of phloretin toward 4-HNE could be transferred into in vivo environments has never been investigated. In the present study, we observed the formation of 4-HNE conjugates of phloretin increased as phloretin decreased during the in vitro incubation. We then purified and characterized three mono-4-HNE-conjugates of phloretin using NMR and LC-MS/MS techniques. We thereafter demonstrated that apple phloretin could scavenge in vivo 4-HNE via the formation of at least three mono-4-HNE-conjugates of phloretin in a dose-dependent manner in mice after oral administration of three doses of phloretin (25, 100, and 400 mg/kg). The findings from this study pave the way to understanding how dihydrochalcones could act as effective scavengers of 4-HNE by working as sacrificial nucleophiles in vivo, thereby preventing or reducing the risk of 4-HNE-associated chronic diseases.