Lysosomal Integrity Research Articles

Cytotoxicity Assays with Zebrafish Cell Lines.

Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute toxicity. Thus, this protocol presents cytotoxicity assays modified to evaluate cell viability in zebrafish (Danio rerio) embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates. The cytotoxicity endpoints evaluated are mitochondrial integrity (Alamar Blue [AB] and MTT assays), membrane integrity via esterase activity (CFDA-AM assay), and lysosomal membrane integrity (Neutral Red [NR] assay). After the exposure of the test substances in a 96-well plate, the cytotoxicity assays are performed; here, AB and CFDA-AM are carried out simultaneously, followed by NR on the same plate, while the MTT assay is performed on a separate plate. The readouts for these assays are taken by fluorescence for AB and CFDA-AM, and absorbance for MTT and NR. The cytotoxicity assays performed with these fish cell lines can be used to study the acute toxicity of chemical substances on fish.

Monitoring Lysosome Function in Ferroptosis.

Ferroptosis is a type of regulated cell death that occurs due to iron-induced membrane lipid peroxidation. Lysosomes, which are acidic, membrane-bound organelles containing various hydrolases, play a vital role in ferroptosis. They not only aid in the degradation of autophagic substrates, but also serve as signaling hubs in cell death. Specifically, lysosomes are involved in the induction and execution of ferroptosis through autophagy-mediated degradation of anti-ferroptotic proteins, lysosomal membrane permeability-mediated release of cathepsins, and iron-induced lysosomal membrane lipid peroxidation. Therefore, it is essential to have reliable methods for monitoring lysosomal functions, including lysosomal activity, pH, and membrane integrity, as well as iron accumulation and lipid peroxidation, to understand ferroptosis. This chapter introduces several protocols, such as western blotting, immunofluorescence, lysosomal probes, and lipid peroxidation assay kits, for monitoring the process of lysosome-related ferroptosis.

Ursolic Acid Impairs Cellular Lipid Homeostasis and Lysosomal Membrane Integrity in Breast Carcinoma Cells.

Cancer is one of the leading causes of death worldwide, thus the search for new cancer therapies is of utmost importance. Ursolic acid is a naturally occurring pentacyclic triterpene with a wide range of pharmacological activities including anti-inflammatory and anti-neoplastic effects. The latter has been assigned to its ability to promote apoptosis and inhibit cancer cell proliferation by poorly defined mechanisms. In this report, we identify lysosomes as the essential targets of the anti-cancer activity of ursolic acid. The treatment of MCF7 breast cancer cells with ursolic acid elevates lysosomal pH, alters the cellular lipid profile, and causes lysosomal membrane permeabilization and leakage of lysosomal enzymes into the cytosol. Lysosomal membrane permeabilization precedes the essential hallmarks of apoptosis placing it as an initial event in the cascade of effects induced by ursolic acid. The disruption of the lysosomal function impairs the autophagic pathway and likely partakes in the mechanism by which ursolic acid kills cancer cells. Furthermore, we find that combining treatment with ursolic acid and cationic amphiphilic drugs can significantly enhance the degree of lysosomal membrane permeabilization and cell death in breast cancer cells.

Cholesterol transfer via endoplasmic reticulum contacts mediates lysosome damage repair

Lysosome integrity is essential for cell viability, and lesions in lysosome membranes are repaired by the ESCRT machinery. Here, we describe an additional mechanism for lysosome repair that is activated independently of ESCRT recruitment. Lipidomic analyses showed increases in lysosomal phosphatidylserine and cholesterol after damage. Electron microscopy demonstrated that lysosomal membrane damage is rapidly followed by the formation of contacts with the endoplasmic reticulum (ER), which depends on the ER proteins VAPA/B. The cholesterol‐binding protein ORP1L was recruited to damaged lysosomes, accompanied by cholesterol accumulation by a mechanism that required VAP–ORP1L interactions. The PtdIns 4‐kinase PI4K2A rapidly produced PtdIns4P on lysosomes upon damage, and knockout of PI4K2A inhibited damage‐induced accumulation of ORP1L and cholesterol and led to the failure of lysosomal membrane repair. The cholesterol–PtdIns4P transporter OSBP was also recruited upon damage, and its depletion caused lysosomal accumulation of PtdIns4P and resulted in cell death. We conclude that ER contacts are activated on damaged lysosomes in parallel to ESCRTs to provide lipids for membrane repair, and that PtdIns4P generation and removal are central in this response.

Abstract 11801: Glycoprotein Nonmetastatic Melanoma Proteinbbregulates Lysosomal Integrity and Lifespan of Senescent Endothelial Cells

Introduction: The accumulation of senescent cells during aging relates to the development of various age-related pathologies including atherosclerosis and heart failure, which can be improved by specific elimination of senescent cells, so called “senolysis”. We have identified glycoprotein nonmetastatic melanoma protein B (GPNMB) as an antigen specifically expressed by senescent endothelial cells, so called “seno-antigen”. Also, we previously developed the vaccine against Gpnmb, eliminated senescent cells in mice, leading to an improvement of age-related pathologies. However, detailed function of GPNMB in senescent cells is still uncertain. Purpose: To elucidate the role of GPNMB in senescent cells and cardiovascular-related pathologies. Methods: Overexpression or depletion of GPNMB in endothelial cells (ECs) were generated. Cellular senescence was induced in ECs by doxorubicin. Gpnmb-knockout or overexpressed (Gp-KO or Gp-OE) mice were generated and imposed to hind-limb ischemia treatment or high-fat-diet feeding to evaluate vascular functions. Double transgenic mice (ApoE-KO and Gp-KO or Gp-OE) were also generated and imposed to high-fat diet to develop atherosclerosis. Results: The depletion of GPNMB in ECs was proved to shorten their replicative lifespan, and increase the expression of negative cell cycle regulators including p53, p21 and p16. Conversely, overexpressed GPNMB protected ECs from senescence stress. In vivo studies showed the impairment of vascular function, atherogenesis, and endothelium-dependent vasodilatation were enhanced in Gp-KO mice, but attenuated in Gp-OE mice. Furthermore, GPNMB was found to localize on lysosomal membrane. Cells with depleted GPNMB demonstrated the accumulation of dysfunctional lysosomes, indicated the contribution of GPNMB in maintaining lysosome integrity. Under senescence-associated lysosomal stress, elevated GPNMB expression was detected, contributing to senescent cells survival. Conclusions: GPNMB acts as a protective factor to senescent cells. Seno-antigen targeting GPNMB is possibly considered as an efficient strategy against cardiovascular diseases and other age-associated disorders with higher selectivity and fewer off-target effects.

Antimycotic potential of protein preparation and fixed oil obtained from Moringa oleifera seeds against Trichophyton tonsurans

• This study evaluated the antifungal potential of protein fraction (PF) and fixed oil (FO) obtained from Moringa oleifera seeds. • PF contained lectins and FO had oleic, palmitic, and elaidic acids as major compounds. • PF and FO were fungistatic agents on T. tonsurans but were not fungicidal. • Synergistic effect was detected for PF+itraconazole and FO+itraconazole. • PF and FO cause apoptosis and necrosis of fungal cells. Species of the genus Trichophyton are etiological agents of dermatophytoses. The appearance of fungi resistant to drugs used to treat these diseases has prompted the search for natural compounds with anti- Trichophyton action. This study sought to determine the antifungal potential of protein fraction (PF) and fixed oil (FO) obtained from Moringa oleifera seeds against T. tonsurans . The synergistic potential of the two preparations and their synergy with commercial antifungal drugs were also evaluated. The possible mechanisms of action were assessed using the sorbitol protection assay and flow cytometry using probes to ascertain cell death, lysosomal integrity, and mitochondrial membrane potential. PF contained 72.24 mg/mL of proteins and lectins (specific hemagglutinating activity of 113.24). Further, FO had oleic (66%), palmitic (9.25%), and elaidic (9.06%) acids as major compounds. PF and FO were identified as fungistatic agents (minimum inhibitory concentration of 1024 μg/mL) but were not fungicidal. An additive effect was found for the PF+FO and PF+ketoconazole combinations, while a synergistic effect was detected for PF+itraconazole and FO+itraconazole. By evaluating T. tonsurans growth in the presence of sorbitol, this osmoprotectant was found to prevent the fungistatic action of FO, indicating that the oil acts on cell wall biosynthesis pathways. The number of cells in apoptosis and necrosis increased when the fungi were treated with PF and FO. Further, mitochondrial hyperpolarization increased in cells exposed to the oil. Of note, there were no changes in lysosomal integrity. In conclusion, PF and FO from M. oleifera seeds were identified as fungistatic agents against T. tonsurans, and are thus promising for combating dermatophytosis caused by this fungus.

Stress responses of bivalve mollusc Unio tumidus from two areas to ibuprofen, microplastic and their mixture.

Even though bivalve molluscs are recognized as bioindicators of freshwater quality, their responses to multiple stressors are unpredictable. This study aims to elucidate the inter-population peculiarities of the effect in the sub-chronic environmentally relevant exposure to novel contaminants. The specimens of Unio tumidus from reference (Pr) and contaminated (Ct) areas were treated with ibuprofen (IBU, 0.8 µg L-1), microplastic (MP, 1.0 mg L-1, size 0.1-0.5 mm), or their combination (Mix) for 14 days. Untreated mussels (PrC- and CtC-groups) served as controls. The PrC-group had higher levels of antioxidants Mn-SOD, Cu,Zn-SOD, catalase, and cholinesterase (AChE) as well as lesser levels of oxidative lesions (TBARS and protein carbonyls) in digestive glands, indicating lower environmental impact than in the CtC-group. However, lysosomal stability was similar in both control groups. Among antioxidants, Mn-SOD activity was affected most prominently, increasing in all exposed Ct-groups. TBARS level was increased only in PrMP-group compared to responsive control. IBU and Mix enhanced protein carbonyl concentration in the Pr-groups, and decreased it in the Ct-groups. AChE was induced in the CtIBU- and PrMix-groups, and lysosomal integrity increased in the CtIBU and CtMix-groups. Discriminant analyses indicated lesser differences between Pr-groups, demonstrating lower cumulative stress compared to Ct-groups. Generally, the most remarkable response was revealed in the CtIBU-group, and distortion of individual effects was established in combined exposures. The qualification of stress-neutral and stress-positive populations was proposed for Pr- and Ct-populations correspondingly. Inter-site peculiarities must be taken into consideration when the environmental impact of MP and pharmaceuticals is evaluated.

Lysosomal lipid alterations caused by glucocerebrosidase deficiency promote lysosomal dysfunction, chaperone-mediated-autophagy deficiency, and alpha-synuclein pathology

Mutations in the GBA gene that encodes the lysosomal enzyme β-glucocerebrosidase (GCase) are a major genetic risk factor for Parkinson’s disease (PD). In this study, we generated a set of differentiated and stable human dopaminergic cell lines that express the two most prevalent GBA mutations as well as GBA knockout cell lines as a in vitro disease modeling system to study the relationship between mutant GBA and the abnormal accumulation of α-synuclein. We performed a deep analysis of the consequences triggered by the presence of mutant GBA protein and the loss of GCase activity in different cellular compartments, focusing primarily on the lysosomal compartment, and analyzed in detail the lysosomal activity, composition, and integrity. The loss of GCase activity generates extensive lysosomal dysfunction, promoting the loss of activity of other lysosomal enzymes, affecting lysosomal membrane stability, promoting intralysosomal pH changes, and favoring the intralysosomal accumulation of sphingolipids and cholesterol. These local events, occurring only at a subcellular level, lead to an impairment of autophagy pathways, particularly chaperone-mediated autophagy, the main α-synuclein degradative pathway. The findings of this study highlighted the role of lysosomal function and lipid metabolism in PD and allowed us to describe a molecular mechanism to understand how mutations in GBA can contribute to an abnormal accumulation of different α-synuclein neurotoxic species in PD pathology.

APOL1 variant-expressing endothelial cells exhibit autophagic dysfunction and mitochondrial stress.

Polymorphisms in the Apolipoprotein L1 (APOL1) gene are common in ancestrally African populations, and associate with kidney injury and cardiovascular disease. These risk variants (RV) provide an advantage in resisting Trypanosoma brucei, the causal agent of African trypanosomiasis, and are largely absent from non-African genomes. Clinical associations between the APOL1 high risk genotype (HRG) and disease are stronger in those with comorbid infectious or immune disease. To understand the interaction between cytokine exposure and APOL1 cytotoxicity, we established human umbilical vein endothelial cell (HUVEC) cultures representing each APOL1 genotype. Untreated HUVECs were compared to IFNɣ-exposed; and APOL1 expression, mitochondrial function, lysosome integrity, and autophagic flux were measured. IFNɣ increased median APOL1 expression across all genotypes 22.1 (8.3 to 29.8) fold (p=0.02). Compared to zero risk variant-carrying HUVECs (0RV), HUVECs carrying 2 risk variant copies (2RV) showed both depressed baseline and maximum mitochondrial oxygen consumption (p<0.01), and impaired mitochondrial networking on MitoTracker assays. These cells also demonstrated a contracted lysosomal compartment, and an accumulation of autophagosomes suggesting a defect in autophagic flux. Upon blocking autophagy with non-selective lysosome inhibitor, hydroxychloroquine, autophagosome accumulation between 0RV HUVECs and untreated 2RV HUVECs was similar, implicating lysosomal dysfunction in the HRG-associated autophagy defect. Compared to 0RV and 2RV HUVECs, HUVECs carrying 1 risk variant copy (1RV) demonstrated intermediate mitochondrial respiration and autophagic flux phenotypes, which were exacerbated with IFNɣ exposure. Taken together, our data reveal that IFNɣ induces APOL1 expression, and that each additional RV associates with mitochondrial dysfunction and autophagy inhibition. IFNɣ amplifies this phenotype even in 1RV HUVECs, representing the first description of APOL1 pathobiology in variant heterozygous cell cultures.

Lysosome-targeted cyclometalated iridium(III) complexes: JMJD inhibition, dual induction of apoptosis, and autophagy.

A series of cyclometalated iridium(III) complexes with the formula [Ir(C^N)2L](PF6) (C^N=2-phenylpyridine (ppy, in Ir-1), 2-(2-thienyl)pyridine (thpy, in Ir-2), 2-(2,4-difluorophenyl)pyridine (dfppy, in Ir-3), L=2-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)quinolin-8-ol) were designed and synthesized, which utilize 8-hydroxyquinoline derivative as N^N ligands to chelate the cofactor Fe2+ of the Jumonji domain-containing protein (JMJD) histone demethylase. As expected, the results of UV/Vis titration analysis confirm the chelating capabilities of Ir-1-3 for Fe2+, and molecular docking studies also show that Ir-1-3 can interact with the active pocket of JMJD protein, and treatment of cells with Ir-1-3 results in significant upregulation of trimethylated histone 3 lysine 9 (H3K9Me3), indicating the inhibition of JMJD activity. Meanwhile, Ir-1-3 exhibit much higher cytotoxicity against the tested tumor cell lines compared with the clinical chemotherapeutic agent cisplatin. And Ir-1-3 can block the cell cycle at the G2/M phase and inhibit cell migration and colony formation. Further studies show that Ir-1-3 can specifically accumulate in lysosomes, damage the integrity of lysosomes, and induce apoptosis and autophagy. Reduction of mitochondrial membrane potential and elevation of reactive oxygen species also contribute to the antitumor effects of Ir-1-3. Finally, Ir-1 can inhibit tumor growth effectively in vivo and increase the expression of H3K9Me3 in tumor tissues. Our study demonstrates that these iridium(III) complexes are promising anticancer agents with multiple functions, including the inhibition of JMJD and induction of apoptosis and autophagy.

A phosphoinositide signalling pathway mediates rapid lysosomal repair.

Lysosomal dysfunction has been increasingly linked to disease and normal ageing1,2. Lysosomal membrane permeabilization (LMP), a hallmark of lysosome-related diseases, can be triggered by diverse cellular stressors3. Given the damaging contents of lysosomes, LMP must be rapidly resolved, although the underlying mechanisms are poorly understood. Here, using an unbiased proteomic approach, we show that LMP stimulates a phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway for rapid lysosomal repair. Upon LMP, phosphatidylinositol-4 kinase type 2α (PI4K2A) accumulates rapidly on damaged lysosomes, generating high levels of the lipid messenger phosphatidylinositol-4-phosphate. Lysosomal phosphatidylinositol-4-phosphate in turn recruits multiple oxysterol-binding protein (OSBP)-related protein (ORP) family members, including ORP9, ORP10, ORP11 and OSBP, to orchestrate extensive new membrane contact sites between damaged lysosomes and the endoplasmic reticulum. The ORPs subsequently catalyse robust endoplasmic reticulum-to-lysosome transfer of phosphatidylserine and cholesterol to support rapid lysosomal repair. Finally, the lipid transfer protein ATG2 is also recruited to damaged lysosomes where its activity is potently stimulated by phosphatidylserine. Independent of macroautophagy, ATG2 mediates rapid membrane repair through direct lysosomal lipid transfer. Together, our findings identify that the PITT pathway maintains lysosomal membrane integrity, with important implications for numerous age-related diseases characterized by impaired lysosomal function.

Mannose ameliorates experimental colitis by protecting intestinal barrier integrity

Metabolite alteration has been associated with the pathogenesis of inflammatory bowel disease (IBD), including colitis. Mannose, a natural bioactive monosaccharide that is involved in metabolism and synthesis of glycoproteins, exhibits anti-inflammatory and anti-oxidative activities. We show here that the circulating level of mannose is increased in patients with IBD and mice with experimental colitis. Mannose treatment attenuates intestinal barrier damage in two mouse colitis models, dextran sodium sulfate (DSS)-induced colitis and spontaneous colitis in IL-10-deficient mice. We demonstrate that mannose treatment enhanced lysosomal integrity and limited the release of cathepsin B, preventing mitochondrial dysfunction and myosin light chain kinase (MLCK)-induced tight junction disruption in the context of intestinal epithelial damage. Mannose exerts a synergistic therapeutic effect with mesalamine on mouse colitis. Cumulatively, the results indicate that mannose supplementation may be an optional approach to the treatment of colitis and other diseases associated with intestinal barrier dysfunction.

Induced Zinc Loss Produces Heterogenous Biological Responses in Melanoma Cells.

Zinc levels in serum and/or tissue are reported to be altered in melanoma with unknown effects on melanoma development and biology. The purpose of this study was to examine the effects of acute chelation of free intracellular zinc pools in melanoma cell lines Bowes and A375, as well as selected melanoma tissue explants with high or low intracellular free zinc. Zinc chelating agent TPEN at the concentration of 25 µM was employed during 48 h, which significantly reduced intracellular free zinc while decreasing melanoma cell proliferation, inducing G1/S arrest and cell damage leading to mitochondrial, caspase-dependent apoptosis. Chelation of free zinc was also associated with increased generation of superoxide in cell lines but not marked lysosomal membrane damage. Conversely, melanoma explant cultures mostly displayed time-dependent loss of lysosomal membrane integrity in the presence of slowly growing superoxide levels. Loss of free zinc-dependent p53 activity was similarly disparate in individual melanoma models. Surviving melanoma cells were arrested in the cell cycle, and varying proportions of them exhibited features characteristic of premature senescence, which increased in time despite zinc reloading. The present results show that melanoma cells with varying free zinc levels respond to its acute loss in a number of individual ways, reflecting activated mechanisms including oxidative stress, lysosomal damage, and p53 activity leading to heterogenous outcomes including cell death, transient, and/or permanent cell cycle arrest and premature senescence.

Cationic Carbon Nanoparticles Induce Inflammasome-Dependent Pyroptosis in Macrophages via Lysosomal Dysfunction

Carbon nanomaterials, including carbon dots (CDs), form a growing family of engineered nanoparticles (NPs) with widespread applications. As the rapid expansion of nanotechnologies raises safety concerns, interaction of NPs with the immune system is receiving a lot of attention. Recent studies have reported that engineered NPs may induce macrophage death by pyroptosis. Therefore, this study investigated whether cationic CDs induce pyroptosis in human macrophages and assessed the role of inflammasome and lysosome in this process. Cationic CDs were synthetized by microwave-assisted pyrolysis of citric acid and high molecular weight branched polyethyleneimine. The NPs evoked a dose-dependent viability loss in THP-1-derived macrophages. A cell leakage, an increase in IL-1β secretion and an activation of caspase-1 were also observed in response to the NPs. Inhibition of caspase-1 decreased CD-induced cell leakage and IL-1β secretion, while restoring cell viability. Besides, CDs triggered swelling and loss of integrity of lysosome, and inhibition of the lysosomal enzyme cathepsin B decreased CD-induced IL-1β secretion. Thus, our data provide evidence that cationic CDs induce inflammasome-dependent pyroptosis in macrophages via lysosomal dysfunction.

Iranian Mesobuthus Eupeus Crude Venom Induces Selective Toxicity in Chronic Lymphocytic Leukemia B-Lymphocytes Through Lysosomal/Mitochondrial Dysfunction and Reactive Oxygen Species Formation.

From ancient times to the present-day animal venoms had been used as medicinal and therapeutic agents. Recently it has been reported that the scorpion venom is a potential source of active and therapeutic compounds to design potent drugs against variety of cancerous cells and other diseases. The current study aimed to evaluate the selective toxicity of Iranian Mesobuthus eupeus (IMe) crude venom as a potential source of anticancer compounds on cancerous CLL B-lymphocytes and normal lymphocytes. For this purpose, we isolated cancerous CLL B-lymphocytes and normal lymphocytes from chronic lymphocytic leukemia patients and healthy volunteers. Cancerous CLL B-lymphocytes and normal lymphocytes were treated with different concentration (0, 5, 10, 20, 40 and 80 µg/ml) of IMe crude venom for 12 hours and cytotoxicity, reactive oxygen species (ROS) production, collapse of mitochondrial membrane potential (MMP) and lysosomal membrane integrity were determined. The data demonstrated the significant cytotoxic effect of IMe crude venom on cancerous CLL B-lymphocytes, with a concentration value (IC50) that inhibits 50% of the cell viability of 60 µg/ ml after 12 h of incubation. MTT assay proved that the IMe crude venom is selectively toxic to cancerous CLL B-lymphocytes, and IMe crude venom induced selective cell death via activation of ROS formation and mitochondrial/lysosomal dysfunction. These finding showed that IMe crude venom has a selective mitochondrial/lysosomal-mediated cell death effect on cancerous CLL B-lymphocytes. Therefore, the IMe crude venom and its fractions may be promising in the future anticancer drug development for treatment of CLL and variety of cancers.

Therapeutic advantage of targeting lysosomal membrane integrity supported by lysophagy in malignant glioma.

Lysosomes function as the digestive system of a cell and are involved in macromolecular recycling, vesicle trafficking, metabolic reprogramming, and progrowth signaling. Although quality control of lysosome biogenesis is thought to be a potential target for cancer therapy, practical strategies have not been established. Here, we show that lysosomal membrane integrity supported by lysophagy, a selective autophagy for damaged lysosomes, is a promising therapeutic target for glioblastoma (GBM). In this study, we found that ifenprodil, an FDA‐approved drug with neuromodulatory activities, efficiently inhibited spheroid formation of patient‐derived GBM cells in a combination with autophagy inhibition. Ifenprodil increased intracellular Ca2+ level, resulting in mitochondrial reactive oxygen species–mediated cytotoxicity. The ifenprodil‐induced Ca2+ elevation was due to Ca2+ release from lysosomes, but not endoplasmic reticulum, associated with galectin‐3 punctation as an indicator of lysosomal membrane damage. As the Ca2+ release was enhanced by ATG5 deficiency, autophagy protected against lysosomal membrane damage. By comparative analysis of 765 FDA‐approved compounds, we identified another clinically available drug for central nervous system (CNS) diseases, amoxapine, in addition to ifenprodil. Both compounds promoted degradation of lysosomal membrane proteins, indicating a critical role of lysophagy in quality control of lysosomal membrane integrity. Importantly, a synergistic inhibitory effect of ifenprodil and chloroquine, a clinically available autophagy inhibitor, on spheroid formation was remarkable in GBM cells, but not in nontransformed neural progenitor cells. Finally, chloroquine dramatically enhanced effects of the compounds inducing lysosomal membrane damage in a patient‐derived xenograft model. These data demonstrate a therapeutic advantage of targeting lysosomal membrane integrity in GBM.

Antitumor activity of Tigerinin-1: Necroptosis mediates toxicity in A549 cells

BackgroundTigerinins are antimicrobial peptides (AMPs) derived from the skin secretions of the Indian bullfrog Hoplobatrachus tigerinus. MethodsTigerinin-1 (FCTMIPIPRCY-Am) peptide was synthesized by solid-phase Fmoc chemistry and investigated its antitumor activities. ResultsTigerinin-1 was cytotoxic to human cancer cells. It causes necrosis by damaging the cell membrane and loss of lysosome integrity. Tigerinin-1triggers the expression of necroptosis pathway proteins. It generates reactive oxygen species (ROS) and induces oxidative stress-mediated genotoxicity. Tigerinin-1 inhibits cancer cell proliferation, reduces neovascularization, and down-regulates the vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), and fibroblast growth factor (FGF) genes. ConclusionsTigerinin-1 exhibited its potent antitumor properties in this study. General significanceTigerinin-1 can be beneficial for developing novel therapeutics for cancer.

Cytocompatibility of Pluronics F-127 on adenocarcinomic human alveolar basal epithelial cells (A549 cells).

Pluronics, due to its high water-soluble and thermoreversible ability, attracted much in biomedical applications. They are mainly utilized in drug delivery, gene therapy, and tissue remodeling. The study aims to explore the cytocompatibility of Pluronics F-127, which has gained much popularity due to its various properties. The cells were exposed to varying concentrations of Pluronics F-127 in A549 cells for 24h. According to the MTT and neutral red assay, A549 cells displayed dose-dependent cell viability. The cell's morphology was preserved after treatment, as seen in phase-contrast and Giemsa staining. When exposed to PF-127, lysosomal, cytoskeletal, and nuclear integrity were maintained. The percentage of live cells in all the treated groups was more significant than 90%, according to the live/dead flow cytometric analyses. The study identified the cytocompatibility of Pluronics F-127 required for the breakthrough in biomedical applications.

Hypoxia-Reoxygenation Impairs Autophagy-Lysosomal Machinery in Primary Human Trophoblasts Mimicking Placental Pathology of Early-Onset Preeclampsia.

We have previously described that placental activation of autophagy is a central feature of normal pregnancy, whereas autophagy is impaired in preeclampsia (PE). Here, we show that hypoxia–reoxygenation (H/R) treatment dysregulates key molecules that maintain autophagy–lysosomal flux in primary human trophoblasts (PHTs). Ultrastructural analysis using transmission electron microscopy reveals a significant reduction in autophagosomes and autolysosomes in H/R-exposed PHTs. H/R-induced accumulation of protein aggregates follows a similar pattern that occurs in PHTs treated with a lysosomal disruptor, chloroquine. Importantly, the placenta from early-onset PE deliveries exhibits the same features as seen in H/R-treated PHTs. Taken together, our results indicate that H/R disrupts autophagic machinery in PHTs and that impaired autophagy in the placenta from early-onset PE deliveries mimics the events in H/R-treated PHTs. Notably, assessment of key regulators at each stage of autophagic processes, especially lysosomal integrity, and verification of autophagic ultrastructure are essential for an accurate evaluation of autophagy activity in human trophoblasts and placental tissue from PE deliveries.

Ca2+‐activated Sphingomyelin Scrambling and Turnover Mediate ESCRT‐independent Lysosomal Repair

Lysosomes are vital organelles vulnerable to injuries from diverse materials. Failure to repair or sequester damaged lysosomes poses a threat to cell viability. Here we report that cells exploit a sphingomyelin‐based lysosomal repair pathway that operates independently of ESCRT to reverse potentially lethal membrane damage. Various conditions perturbing organelle integrity trigger a rapid calcium‐activated scrambling and cytosolic exposure of sphingomyelin. Subsequent metabolic conversion of sphingomyelin by neutral sphingomyelinases on the cytosolic surface of injured lysosomes promotes their repair, also when ESCRT function is compromised. Conversely, blocking turnover of cytosolic sphingomyelin renders cells more sensitive to lysosome‐damaging drugs. Our data indicate that calcium‐activated scramblases, sphingomyelin, and neutral sphingomyelinases are core components of a previously unrecognized membrane restoration pathway by which cells preserve the functional integrity of lysosomes.