The oxidized phospholipid PGPC impairs endothelial function by promoting endothelial cell ferroptosis via FABP3.

Ferroptosis is a novel cell death mechanism that is mediated by iron-dependent lipid peroxidation. It may be involved in atherosclerosis development. Products of phospholipid oxidation play a key role in atherosclerosis. 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) is a phospholipid oxidation product present in atherosclerotic lesions. It remains unclear whether PGPC causes atherosclerosis by inducing endothelial cell ferroptosis. In this study, human umbilical vein endothelial cells (HUVECs) were treated with PGPC. Intracellular levels of ferrous iron, lipid peroxidation, superoxide anions (O2•-), and glutathione were detected, and expression of fatty acid binding protein-3 (FABP3), glutathione peroxidase 4 (GPX4), and CD36 were measured. Additionally, the mitochondrial membrane potential (MMP) was determined. Aortas from C57BL6 mice were isolated for vasodilation testing. Results showed that PGPC increased ferrous iron levels, the production of lipid peroxidation and O2•-, and FABP3 expression. However, PGPC inhibited the expression of GPX4 and glutathione production and destroyed normal MMP. These effects were also blocked by ferrostatin-1, an inhibitor of ferroptosis. FABP3 silencing significantly reversed the effect of PGPC. Furthermore, PGPC stimulated CD36 expression. Conversely, CD36 silencing reversed the effects of PGPC, including PGPC-induced FABP3 expression. Importantly, E06, a direct inhibitor of the oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine IgM natural antibody, inhibited the effects of PGPC. Finally, PGPC impaired endothelium-dependent vasodilation, ferrostatin-1 or FABP3 inhibitors inhibited this impairment. Our data demonstrate that PGPC impairs endothelial function by inducing endothelial cell ferroptosis through the CD36 receptor to increase FABP3 expression. Our findings provide new insights into the mechanisms of atherosclerosis and a therapeutic target for atherosclerosis.

Suppression of metastatic organ colonization and antiangiogenic activity of the orally bioavailable lipid raft-targeted alkylphospholipid edelfosine.

Metastasis is the leading cause of cancer mortality. Metastatic cancer is notoriously difficult to treat, and it accounts for the majority of cancer-related deaths. The ether lipid edelfosine is the prototype of a family of synthetic antitumor compounds collectively known as alkylphospholipid analogs, and its antitumor activity involves lipid raft reorganization. In this study, we examined the effect of edelfosine on metastatic colonization and angiogenesis. Using non-invasive bioluminescence imaging and histological examination, we found that oral administration of edelfosine in nude mice significantly inhibited the lung and brain colonization of luciferase-expressing 435-Lung-eGFP-CMV/Luc metastatic cells, resulting in prolonged survival. In metastatic 435-Lung and MDA-MB-231 breast cancer cells, we found that edelfosine also inhibited cell adhesion to collagen-I and laminin-I substrates, cell migration in chemotaxis and wound-healing assays, as well as cancer cell invasion. In 435-Lung and other MDA-MB-435-derived sublines with different organotropism, edelfosine induced G2/M cell cycle accumulation and apoptosis in a concentration- and time-dependent manner. Edelfosine also inhibited in vitro angiogenesis in human and mouse endothelial cell tube formation assays. The antimetastatic properties were specific to cancer cells, as edelfosine had no effects on viability in non-cancerous cells. Edelfosine accumulated in membrane rafts and endoplasmic reticulum of cancer cells, and membrane raft-located CD44 was downregulated upon drug treatment. Taken together, this study highlights the potential of edelfosine as an attractive drug to prevent metastatic growth and organ colonization in cancer therapy. The raft-targeted drug edelfosine displays a potent activity against metastatic organ colonization and angiogenesis, two major hallmarks of tumor malignancy.

TMEM164 is an acyltransferase that forms ferroptotic C20:4 ether phospholipids.

Ferroptosis is an iron-dependent form of cell death driven by oxidation of polyunsaturated fatty acid (PUFA) phospholipids. Large-scale genetic screens have uncovered a specialized role for PUFA ether phospholipids (ePLs) in promoting ferroptosis. Understanding of the enzymes involved in PUFA-ePL production, however, remains incomplete. Here we show, using a combination of pathway mining of genetic dependency maps, AlphaFold-guided structure predictions and targeted lipidomics, that the uncharacterized transmembrane protein TMEM164-the genetic ablation of which has been shown to protect cells from ferroptosis-is a cysteine active center enzyme that selectively transfers C20:4 acyl chains from phosphatidylcholine to lyso-ePLs to produce PUFA ePLs. Genetic deletion of TMEM164 across a set of ferroptosis-sensitive cancer cell lines caused selective reductions in C20:4 ePLs with minimal effects on C20:4 diacyl PLs, and this lipid profile produced a variable range of protection from ferroptosis, supportive of an important but contextualized role for C20:4 ePLs in this form of cell death.

Serum metabolomics analysis reveals amelioration effects of sea cucumber ether phospholipids on oxidative stress and inflammation in high-fat diet-fed mice.

Emerging evidence suggests that sea cucumber ether phospholipids (ether-PLs) can modulate high-fat diet (HFD)-induced metabolic disorders. However, whether this modulation is associated with metabolic pathways related to oxidative stress and inflammation remains unclear. This study aimed to investigate the antioxidative and anti-inflammatory effects on HFD-fed mice and the associated metabolism pathways in response to administration with sea cucumber ether-PLs using integrated biochemistry and a metabolomics approach. Biochemistry analysis and histological examinations showed that sea cucumber ether-PLs significantly decreased body weight gain and fat deposition in tissues. PE-P was superior to PC-O in alleviating reactive oxygen species (ROS), malondialdehyde (MDA) and inflammatory responses (IL-6, TNF-α and MCP-1) in the HFD-induced mouse model. Serum metabolomics analysis revealed that it upregulated four metabolites and downregulated twenty-four metabolites compared to those in HFD mice after ether-PL administration. Pathway analysis indicated that sea cucumber ether-PLs alleviate the HFD-induced inflammation and oxidative stress by three main metabolic pathways, namely fatty acid metabolism, branched-chain amino acid (BCAA) metabolism, and trichloroacetic acid (TCA) metabolism. Taken together, sea cucumber ether-PLs showed great potential to become a natural functional food against oxidative stress and inflammation caused by HFD.

Sea cucumber ether-phospholipids improve hepatic steatosis and enhance hypothalamic autophagy in high-fat diet-fed mice.

As a promising group of natural bioactive lipids, ether-phospholipids (ether-PLs), exhibit the ability to attenuate high-fat diet (HFD)-induced lipid accumulation and atherosclerosis. However, the underlying mechanism is unclear. Autophagy has been implicated in the regulation of obesity. Therefore, we investigated the effects of dietary ether-PLs on hepatic steatosis and the activation of hypothalamic autophagy. HFD-fed C57BL/6J mice were orally administered with ether-PLs (150 mg/kg body weight) including plasmenyl phosphatidylethanolamine (PE-P) and plasmanyl phosphatidylcholine (PC-O) for three days or eight weeks. Ether-PLs supplementation relieved diet-induced hepatic lipid accumulation and regulated the hypothalamic peroxisome proliferator-activated receptor gamma (PPARγ) and CD36. Notably, PE-P activated hypothalamic autophagy more strongly than PC-O, with an increased ratio of microtubule-associated protein light chain 3 II/I (LC3II/I) and reduced p62/sequestosome-1 (p62) accumulation by rescuing the HFD-impaired autophagy-lysosome fusion. The phosphorylation of ULK1 mediated by Akt-mTOR and AMPK, was involved in ether-PLs activated autophagy. Furthermore, the enhanced hypothalamic autophagy promoted the production of α-melanocyte-stimulating hormone (α-MSH), which has been reported to maintain energy balance. It is concluded that ether-PLs ameliorated HFD-induced hypothalamic autophagy and ameliorated hepatic steatosis. Ether-PLs could thus be an attractive autophagy-enhancers against chronic HFD-induced obesity.

Hepatoprotective effects of sea cucumber ether-phospholipids against alcohol-induced lipid metabolic dysregulation and oxidative stress in mice.

Sea cucumber is widely consumed as food and folk medicine in Asia, and its phospholipids are rich sources of dietary eicosapentaenoic acid enriched ether-phospholipids (ether-PLs). Emerging evidence suggests that ether-PLs are associated with neurodegenerative disease and steatohepatitis. However, the function and mechanism of ether-PLs in alcoholic liver disease (ALD) are not well understood. To this end, the present study sought to investigate the hepatoprotective effects of sea cucumber ether-PLs, including plasmenyl phosphatidylethanolamine (PlsEtn) and plasmanyl phosphatidylcholine (PlsCho), and their underlying mechanisms. Our results showed that compared with EtOH-induced mice, ether-PL treated mice showed improved liver histology, decreased serum ALT and AST levels, and reduced alcohol metabolic enzyme (ALDH2 and ADH1) expressions. Mechanistic studies showed that ether-PLs attenuated "first-hit" hepatic steatosis and lipid accumulation evoked by alcohol administration. Moreover, PlsEtn more effectively restored endogenous plasmalogen levels than PlsCho, thereby enhancing hepatic antioxidation against "second-hit" reactive oxygen species (ROS) due to the damaged mitochondria and abnormal ethanol metabolism. Taken together, sea cucumber ether-PLs show great potential to become a natural functional food against chronic alcohol-induced hepatic steatosis and lipid metabolic dysregulation.

Oral batyl alcohol supplementation rescues decreased cardiac conduction in ether phospholipid-deficient mice.

Plasmalogens (Pls) are a class of membrane phospholipids which serve a number of essential biological functions. Deficiency of Pls is associated with common disorders such as Alzheimer's disease or ischemic heart disease. A complete lack of Pls due to genetically determined defective biosynthesis gives rise to rhizomelic chondrodysplasia punctata (RCDP), characterized by a number of severe disabling pathologic features and death in early childhood. Frequent cardiac manifestations of RCDP include septal defects, mitral valve prolapse, and patent ductus arteriosus. In a mouse model of RCDP, reduced nerve conduction velocity was partially rescued by dietary oral supplementation of the Pls precursor batyl alcohol (BA). Here, we examine the impact of Pls deficiency on cardiac impulse conduction in a similar mouse model (Gnpat KO). In-vivo electrocardiographic recordings showed that the duration of the QRS complex was significantly longer in Gnpat KO mice than in age- and sex-matched wild-type animals, indicative of reduced cardiac conduction velocity. Oral supplementation of BA for 2 months resulted in normalization of cardiac Pls levels and of the QRS duration in Gnpat KO mice but not in untreated animals. BA treatment had no effect on the QRS duration in age-matched wild-type mice. These data suggest that Pls deficiency is associated with increased ventricular conduction time which can be rescued by oral BA supplementation.

Squalenoyl-gemcitabine/edelfosine nanoassemblies: Anticancer activity in pediatric cancer cells and pharmacokinetic profile in mice.

Despite the great advances accomplished in the treatment of pediatric cancers, recurrences and metastases still exacerbate prognosis in some aggressive solid tumors such as neuroblastoma and osteosarcoma. In view of the poor efficacy and toxicity of current chemotherapeutic treatments, we propose a single multitherapeutic nanotechnology-based strategy by co-assembling in the same nanodevice two amphiphilic antitumor agents: squalenoyl-gemcitabine and edelfosine. Homogeneous batches of nanoassemblies were easily formulated by the nanoprecipitation method. Their anticancer activity was tested in pediatric cancer cell lines and pharmacokinetic studies were performed in mice. In vitro assays revealed a synergistic effect when gemcitabine was co-administered with edelfosine. Squalenoyl-gemcitabine/edelfosine nanoassemblies were found to be capable of intracellular translocation in patient-derived metastatic pediatric osteosarcoma cells and showed a better antitumor profile than squalenoyl-gemcitabine nanoassemblies alone. The intravenous administration of this combinatorial nanomedicine in mice exhibited a controlled release behavior of gemcitabine and diminished edelfosine plasma peak concentrations. These findings make it a suitable pre-clinical candidate for childhood cancer therapy.

Role of lipids in pathophysiology, diagnosis and therapy of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive and widespread cancer. Patients with liver cirrhosis of different aetiologies are at a risk to develop HCC. It is important to know that in approximately 20% of cases primary liver tumors arise in a non-cirrhotic liver. Lipid metabolism is variable in patients with chronic liver diseases, and lipid metabolites involved therein do play a role in the development of HCC. Of note, lipid composition of carcinogenic tissues differs from non-affected liver tissues. High cholesterol and low ceramide levels in the tumors protect the cells from oxidative stress and apoptosis, and do also promote cell proliferation. So far, detailed characterization of the mechanisms by which lipids enable the development of HCC has received little attention. Evaluation of the complex roles of lipids in HCC is needed to better understand the pathophysiology of HCC, the later being of paramount importance for the development of urgently needed therapeutic interventions. Disturbed hepatic lipid homeostasis has systemic consequences and lipid species may emerge as promising biomarkers for early diagnosis of HCC. The challenge is to distinguish lipids specifically related to HCC from changes simply related to the underlying liver disease. This review article discusses aberrant lipid metabolism in patients with HCC.

The effect of edelfosine on GRA1 and MIC3 expressions in acute toxoplasmosis.

Phosphoinositide-dependent phospholipase-C (PI-PLC) triggers the calcium signaling pathway which plays an important role in dense granule and microneme secretion and pathogenesis of Toxoplasma gondii (T. gondii). There are limited data about the effects of phospholipid analogues against T. gondii. The current study assessed the effect of edelfosine, as a phospholipid analogue, on GRA1 and MIC3 expressions using in vitro and in vivo models of acute toxoplasmosis. Infected Vero cells were treated by edelfosine in two subgroups: 24 h following the cell infection and treatment at the same time of cell infection. Animal study was performed on forty mice in four groups including non-infected, infected untreated, infected edelfosine-treated, and infected pyrimethamine-treated. Gene and protein expression analyses were done using quantitative real-time PCR and western blot, respectively. Edelfosine significantly reduced the GRA1 (P < 0.01) and MIC3 (P < 0.01) mRNA and protein expressions in 24 h following the cell infection and at the same time of cell infection groups. In vivo study showed that the edelfosine significantly reduced the GRA1 expression in eye, and MIC3 expression in brain and liver. Moreover, the edelfosine-treated infected mice had significant higher survival rate compared with uninfected mice. The reducing effect of edelfosine on GRA1 and MIC3 mRNA and protein levels 24 h following the cell infection was more than treatment at the same time of cell infection group. Moreover, the effect of edelfosine on GRA1 and MIC3 expression in animal tissues was variable. These data showed that the edelfosine may decrease the T. gondii excretory/secretory antigens through inhibition of PI-PLC.

Flavonoid-alkylphospholipid conjugates elicit dual inhibition of cancer cell growth and lipid accumulation.

Cancer development is often associated with lipid metabolic reprogramming, including aberrant lipid accumulation. We create novel paradigms endowed with dual functions of anticancer activity and inhibition of lipid accumulation by conjugating the natural product quercetin and synthetic alkylphospholipid drugs, and harnessing the biomedical effects of both. These conjugates offer fresh perspectives in the search for anticancer candidates.

Arachidonic acid and lysophosphatidylcholine inhibit multiple late steps of regulated exocytosis.

The canonical Phospholipase A2 (PLA2) metabolites lysophosphatidylcholine (LPC) and arachidonic acid (ARA) affect regulated exocytosis in a wide variety of cells and are proposed to directly influence membrane merger owing to their respective spontaneous curvatures. According to the Stalk-pore hypothesis, negative curvature ARA inhibits and promotes bilayer merger upon introduction into the distal or proximal monolayers, respectively; in contrast, with positive curvature, LPC has the opposite effects. Using fully primed, release-ready native cortical secretory vesicles (CV), well-established fusion assays and standardized lipid analyses, we show that exogenous ARA and LPC, as well as their non-metabolizable analogous, ETYA and ET-18-OCH3, inhibit the docking/priming and membrane merger steps, respectively, of regulated exocytosis.

Preclinical Pharmacokinetics and Dosimetry Studies of 124I/131I-CLR1404 for Treatment of Pediatric Solid Tumors in Murine Xenograft Models.

Cancer is the second leading cause of death for children between the ages of 5 and 14 y. For children diagnosed with metastatic or recurrent solid tumors, for which the utility of external-beam radiotherapy is limited, the prognosis is particularly poor. The availability of tumor-targeting radiopharmaceuticals for molecular radiotherapy (MRT) has demonstrated improved outcomes in these patient populations, but options are nonexistent or limited for most pediatric solid tumors. 18-(p-iodophenyl)octadecylphosphocholine (CLR1404) is a novel antitumor alkyl phospholipid ether analog that broadly targets cancer cells. In this study, we evaluated the in vivo pharmacokinetics of 124I-CLR1404 (CLR 124) and estimated theranostic dosimetry for 131I-CLR1404 (CLR 131) MRT in murine xenograft models of the pediatric solid tumors neuroblastoma, rhabdomyosarcoma, and Ewing sarcoma. Methods: Tumor-bearing mice were imaged with small-animal PET/CT to evaluate the whole-body distribution of CLR 124 and, correcting for differences in radioactive decay, predict that of CLR 131. Image volumes representing CLR 131 provided input for Geant4 Monte Carlo simulations to calculate subject-specific tumor dosimetry for CLR 131 MRT. Pharmacokinetics for CLR 131 were extrapolated to adult and pediatric humans to estimate normal-tissue dosimetry. In neuroblastoma, a direct comparison of CLR 124 with 124I-metaiodobenzylguanidine (124I-MIBG) in an MIBG-avid model was performed. Results: In vivo pharmacokinetics of CLR 124 showed selective uptake and prolonged retention across all pediatric solid tumor models investigated. Subject-specific tumor dosimetry for CLR 131 MRT presents a correlative relationship with tumor-growth delay after CLR 131 MRT. Peak uptake of CLR 124 was, on average, 22% higher than that of 124I-MIBG in an MIBG-avid neuroblastoma model. Conclusion: CLR1404 is a suitable theranostic scaffold for dosimetry and therapy with potentially broad applicability in pediatric oncology. Given the ongoing clinical trials for CLR 131 in adults, these data support the development of pediatric clinical trials and provide detailed dosimetry that may lead to improved MRT treatment planning.

Enhanced Radiosensitivity in Solid Tumors using a Tumor-selective Alkyl Phospholipid Ether Analog.

Antitumor alkyl phospholipid (APL) analogs comprise a group of structurally related molecules with remarkable tumor selectivity. Some of these compounds have shown radiosensitizing capabilities. CLR127 is a novel, clinical-grade antitumor APL ether analog, a subtype of synthetic APL broadly targeting cancer cells with limited uptake in normal tissues. The purpose of this study was to investigate the effect of CLR127 to modulate radiation response across several adult and pediatric cancer types in vitro as well as in murine xenograft models of human prostate adenocarcinoma, neuroblastoma, Ewing sarcoma, and rhabdomyosarcoma. In vitro, CLR127 demonstrated selective uptake in cancer cells compared to normal cells. In cancer cells, CLR127 treatment prior to radiation significantly decreased clonogenic survival in vitro, and led to increased radiation-induced double-stranded DNA (dsDNA) breakage compared with radiation alone, which was not observed in normal controls. In animal models, CLR127 effectively increased the antitumor response to fractionated radiotherapy and led to delayed tumor regrowth at potentially clinically achievable doses. In conclusion, our study highlights the ability of CLR127 to increase radiation response in several cancer types. Given almost universal uptake of CLR127 in malignant cells, future research should test whether the observed effects can be extended to other tumor types. Our data provide a strong rationale for clinical testing of CLR127 as a tumor-targeted radiosensitizing agent. Mol Cancer Ther; 17(11); 2320-8. ©2018 AACR.

Anticancer Agent Edelfosine Exhibits a High Affinity for Cholesterol and Disorganizes Liquid-Ordered Membrane Structures.

Edelfosine is an anticancer drug with an asymmetric structure because, being a derivative of glycerol, it possesses two hydrophobic substituents of very different lengths. We showed that edelfosine destabilizes liquid-ordered membranes formed by either 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine, sphingomyelin (SM), and cholesterol (1:1:1 molar ratio) or SM and cholesterol (2:1 molar ratio). This was observed by differential scanning calorimetry in which phase transition arises from either of these membrane systems after the addition of edelfosine. The alteration in the liquid-ordered domains was characterized by using a small-angle X-ray diffraction that revealed the formation of gel phases as a consequence of the addition of edelfosine at low temperatures and by a wide-angle X-ray diffraction that confirmed changes in the membranes, indicating the formation of these gel phases. The increase in phase transition derived by the edelfosine addition was further confirmed by Fourier-transform infrared spectroscopy. The effect of edelfosine was compared with that of structurally analogue lipids: platelet-activating factor and 1-palmitoyl-2-acetyl- sn-glycero-3-phosphocholine, which also have the capacity of destabilizing liquid-ordered domains, although they are less potent than edelfosine for this activity, and lysophosphatidylcholine, which lacks this capacity. It was concluded that edelfosine may be associated with cholesterol favorably competing with sphingomyelin, and that this sets sphingomyelin free to undergo a phase transition. Finally, the experimental observations can be described by molecular dynamics calculations in terms of intermolecular interaction energies in phospholipid-cholesterol membranes. Higher interaction energies between asymmetric phospholipids and cholesterol than between sphingomyelin and cholesterol were obtained. These results are interesting because they biophysically characterize one of the main molecular mechanisms to trigger apoptosis of the cancer cells.

Targeting Brain-Adaptive Cancer Stem Cells Prohibits Brain Metastatic Colonization of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) exhibits more traits possessed by cancer stem cells (CSC) than other breast cancer subtypes and is more likely to develop brain metastases. TNBC patients usually have shorter survival time after diagnosis of brain metastasis, suggesting an innate ability of TNBC tumor cells in adapting to the brain. In this study, we establish novel animal models to investigate early tumor adaptation in brain metastases by introducing both patient-derived and cell line-derived CSC-enriched brain metastasis tumorsphere cells into mice. We discovered astrocyte-involved tumor activation of protocadherin 7 (PCDH7)-PLCß-Ca2+-CaMKII/S100A4 signaling as a mediator of brain metastatic tumor outgrowth. We further identified and evaluated the efficacy of a known drug, the selective PLC inhibitor edelfosine, in suppressing the PCDH7 signaling pathway to prohibit brain metastases in the animal models. The results of this study reveal a novel signaling pathway for brain metastases in TNBC and indicate a promising strategy of metastatic breast cancer prevention and treatment by targeting organ-adaptive cancer stem cells.Significance: These findings identify a compound to block adaptive signaling between cancer stem cells and brain astrocytes. Cancer Res; 78(8); 2052-64. ©2018 AACR.

Plasmalogen lipids: functional mechanism and their involvement in gastrointestinal cancer.

The plasmalogens are a class of glycerophospholipids which contain a vinyl-ether and an ester bond at the sn-1 and sn-2 positions, respectively, in the glycerol backbone. They constitute 10 mol% of the total mass of phospholipids in humans, mainly as membrane structure components. Plasmalogens are important for the organization and stability of lipid raft microdomains and cholesterol-rich membrane regions involved in cellular signaling. In addition to their structural roles, a subset of ether lipids are thought to function as endogenous antioxidants and emerging studies suggest that they are involved in cell differentiation and signaling pathways. Although the clinical significance of plasmalogens is linked to peroxisomal disorders, the pathophysiological roles and their possible metabolic pathways are not fully understood since they present unique structural attributes for the different tissue types. Studies suggest that changes in plasmalogen metabolism may contribute to the development of various types of cancer. Here, we review the molecular characteristics of plasmalogens in order to significantly increase our understanding of the plasmalogen molecule and its involvement in gastrointestinal cancers as well as other types of cancers.

Edelfosine: An Antitumor Drug Prototype.

BACKGROUND: Lung cancer is the most prevalent cancer and a high fatality disease. Despite of all available therapeutic approaches, drug resistance of chemotherapy agents for patients remain as an obstacle. New drugs integrating immunotherapeutic and conventional cytotoxic effects is a powerful strategy for the treatment of cancer to overcome this limitation. Antineoplastic phospholipids combine both of these activities by affecting lipid metabolism and signaling through lipid rafts. Therefore, they emerge as interesting scaffolds for designing new drugs. OBJECTIVE: We aimed to evaluate antineoplastic phospholipids as scaffolds for designing new drugs for lung cancer treatment. METHODS: The initial screening in A549 cells was performed by MTT assay. Others cytotoxic effects were evaluated in A549 cells by clonogenic assay, Matrigel 3D culture and flow cytometry analyses of cell cycle, apoptosis, mitochondrial membrane electronic potential and superoxide production. Immunological effects of ED were accessed on dendritic cells (DCs) and the expression of some markers were evaluated by flow cytometry. In vivo lung colonization analysis was performed after intravenously injection of A549 cells and daily treatment with ED. RESULTS: Herein, ED showed to be the most efficient compound concerning cytotoxic, thereby, ED was selected for following tests. ED showed a cytotoxic profile in both monolayer and 3D culture and also in vivo models using A549 cells. This profile is due to G0/G1 phase cellular arrest and apoptosis drove by mitochondrial membrane depolarization and superoxide overproduction. Moreover, ED modulated DCs toward an activated pattern by the increased expression of CD83 and a remarkable decreased expression of PD-L1/CD274 on DCs membrane. CONCLUSIONS: Thus, ED is an interesting antitumor drug prototype due to not only its direct cellular cytotoxicity but also given its immunological features.

The oxidized phospholipid POVPC impairs endothelial function and vasodilation via uncoupling endothelial nitric oxide synthase.

Endothelial dysfunction is an early stage of atherosclerosis. We recently have shown that 25-hydroxycholesterol found in atherosclerotic lesions could impair endothelial function and vasodilation by uncoupling and inhibiting endothelial nitric oxide synthase (eNOS). 1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), the oxidation product of oxidized low-density lipoprotein, is another proinflammatory lipid and has also been found in atherosclerotic lesions. However, whether POVPC promotes atherosclerosis like 25-hydroxycholesterol remains unclear. The purpose of this study was to explore the effects of POVPC on endothelial function and vasodilation. Human umbilical vein endothelial cells (HUVECs) were incubated with POVPC. Endothelial cell proliferation, migration and tube formation were measured. Nitric oxide (NO) production and superoxide anion generation (O2-) were determined. The expression and phosphorylation of endothelial nitric oxide synthase (eNOS), AKT, PKC-ßII and P70S6K as well as the association of eNOS and heat shock protein 90 (HSP90) were detected by immunoblotting and immunoprecipitation. Endothelial cell apoptosis was monitored by TUNEL staining. The expression of Bcl-2, Bax, and Cleaved Caspase 3 were detected by immunoblotting. Finally, aortic ring from C57BL6 mice were isolated and treated with POVPC and the endothelium-dependent vasodilation was evaluated. POVPC significantly inhibited HUVECs proliferation, migration, tube formation, decreased NO production but increased O2- generation. POVPC inhibited the phosphorylation of Akt and eNOS at Ser1177, increased activation of PKC-ßII, P70S6K and the phosphorylation of eNOS at Thr495, reduced the association of HSP90 with eNOS. Meanwhile, POVPC induced endothelial cell apoptosis by inhibiting Bcl-2 expression, increasing Bax and cleaved caspase-3 expressions as well as caspase-3 activity and impaired endothelium-dependent vasodilation. These data demonstrated that POVPC impaired endothelial function by uncoupling and inhibiting eNOS as well as by inducing endothelial cell apoptosis. Therefore, POVPC may play an important role in the development of atherosclerosis and may be considered as a potential therapeutic target for atherosclerosis.

Mitochondria and lipid raft-located FOF1-ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine.

BACKGROUND: Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs) are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine) for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast. CONCLUSIONS/SIGNIFICANCE: The present study shows that the antileishmanial and anticancer actions of edelfosine share some common signaling processes, with mitochondria and raft-located FOF1-ATP synthase being critical in the killing process, thus identifying novel druggable targets for the treatment of leishmaniasis.