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Cell Death & Disease Jun 2024The repurposing of medications developed for central nervous system (CNS) disorders, possessing favorable safety profiles and blood-brain barrier permeability,...
The repurposing of medications developed for central nervous system (CNS) disorders, possessing favorable safety profiles and blood-brain barrier permeability, represents a promising strategy for identifying new therapies to combat glioblastoma (GBM). In this study, we investigated the anti-GBM activity of specific antipsychotics and antidepressants in vitro and in vivo. Our results demonstrate that these compounds share a common mechanism of action in GBM, disrupting lysosomal function and subsequently inducing lysosomal membrane rupture and cell death. Notably, PTEN intact GBMs possess an increased sensitivity to these compounds. The inhibition of lysosomal function synergized with inhibitors targeting the EGFR-PI3K-Akt pathway, leading to an energetic and antioxidant collapse. These findings provide a foundation for the potential clinical application of CNS drugs in GBM treatment. Additionally, this work offers critical insights into the mechanisms and determinants of cytotoxicity for drugs currently undergoing clinical trials as repurposing agents for various cancers, including Fluoxetine, Sertraline, Thioridazine, Chlorpromazine, and Fluphenazine.
Topics: Humans; Glioblastoma; PTEN Phosphohydrolase; Lysosomes; Signal Transduction; Antipsychotic Agents; Animals; Cell Line, Tumor; Mice; Brain Neoplasms; Proto-Oncogene Proteins c-akt; Mice, Nude; Drug Repositioning; Phosphatidylinositol 3-Kinases; ErbB Receptors; Chlorpromazine
PubMed: 38871731
DOI: 10.1038/s41419-024-06779-3 -
Cancer Letters Jun 2024Lysosomes are single membrane bounded group of acidic organelles that can be involved in a process called lysosomal exocytosis which leads to the extracellular release... (Review)
Review
Lysosomes are single membrane bounded group of acidic organelles that can be involved in a process called lysosomal exocytosis which leads to the extracellular release of their content. Lysosomal exocytosis is required for plasma membrane repair or remodeling events such as bone resorption, antigen presentation or mitosis, and for protection against toxic agents such as heavy metals. Recently, it has been showed that to fulfill this protective role, lysosomal exocytosis needs some autophagic proteins, in an autophagy-independent manner. In addition to these crucial physiological roles, lysosomal exocytosis plays a major protumoral role in various cancers. This effect is exerted through tumor microenvironment modifications, including extracellular matrix remodeling, acidosis, oncogenic and profibrogenic signals. This review provides a comprehensive overview of the different elements released in the microenvironment during lysosomal exocytosis, i.e. proteases, exosomes, and protons, and their effects in the context of tumor development and treatment.
PubMed: 38871244
DOI: 10.1016/j.canlet.2024.217024 -
International Journal of Nanomedicine 2024Acute myeloid leukemia (AML) remains difficult to treat due to its heterogeneity in molecular landscape, epigenetics and cell signaling alterations. Precision medicine...
INTRODUCTION
Acute myeloid leukemia (AML) remains difficult to treat due to its heterogeneity in molecular landscape, epigenetics and cell signaling alterations. Precision medicine is a major goal in AML therapy towards developing agents that can be used to treat patients with different 'subtypes' in combination with current chemotherapies. We have previously developed dextrin-colistin conjugates to combat the rise in multi-drug resistant bacterial infections and overcome dose-limiting nephrotoxicity. Recent evidence of colistin's anticancer activity, mediated through inhibition of intracellular lysine-specific histone demethylase 1 (LSD1/KDM1A), suggests that dextrin-colistin conjugates could be used to treat cancer cells, including AML. This study aimed to evaluate whether dextrin conjugation (which reduces in vivo toxicity and prolongs plasma half-life) could enhance colistin's cytotoxic effects in myeloid leukemia cell lines and compare the intracellular uptake and localization of the free and conjugated antibiotic.
RESULTS
Our results identified a conjugate (containing 8000 g/mol dextrin with 1 mol% succinoylation) that caused significantly increased toxicity in myeloid leukemia cells, compared to free colistin. Dextrin conjugation altered the mechanism of cell death by colistin, from necrosis to caspase 3/7-dependent apoptosis. In contrast, conjugation via a reversible ester linker, instead of an amide, had no effect on the mechanism of the colistin-induced cell death. Live cell confocal microscopy of fluorescently labelled compounds showed both free and dextrin-conjugated colistins were endocytosed and co-localized in lysosomes, and increasing the degree of modification by succinoylation of dextrin significantly reduced colistin internalization.
DISCUSSION
Whilst clinical translation of dextrin-colistin conjugates for the treatment of AML is unlikely due to the potential to promote antimicrobial resistance (AMR) and the relatively high colistin concentrations required for anticancer activity, the ability to potentiate the effectiveness of an anticancer drug by polymer conjugation, while reducing side effects and improving biodistribution of the drug, is very attractive, and this approach warrants further investigation.
Topics: Colistin; Dextrins; Humans; Apoptosis; Cell Line, Tumor; Anti-Bacterial Agents; Leukemia, Myeloid, Acute; Cell Survival
PubMed: 38868592
DOI: 10.2147/IJN.S449185 -
BMC Veterinary Research Jun 2024Antimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture,...
BACKGROUND
Antimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture, are promoting AMR while also encouraging the research and employment of alternative drugs. The addition of antibiotics to the cell media is strongly recommended in sperm preservation, being gentamicin the most used for boar semen. Because of its continued use, several bacterial strains present in boar semen have developed resistance to this antibiotic. Antimicrobial peptides and proteins (AMPPs) are promising candidates as alternative antibiotics because their mechanism of action is less likely to promote AMR. In the present study, we tested two AMPPs (lysozyme and nisin; 50 and 500 µg/mL) as possible substitutes of gentamicin for boar semen preservation up to 48 h of storage.
RESULTS
We found that both AMPPs improved sperm plasma membrane and acrosome integrity during semen storage. The highest concentration tested for lysozyme also kept the remaining sperm parameters unaltered, at 48 h of semen storage, and reduced the bacterial load at comparable levels of the samples supplemented with gentamicin (p > 0.05). On the other hand, while nisin (500 µg/mL) reduced the total Enterobacteriaceae counts, it also decreased the rapid and progressive sperm population and the seminal oxidation-reduction potential (p < 0.05).
CONCLUSIONS
The protective effect of lysozyme on sperm function together with its antimicrobial activity and inborn presence in body fluids, including semen and cervical mucus, makes this enzyme a promising antimicrobial agent for boar semen preservation.
Topics: Animals; Semen Preservation; Male; Anti-Bacterial Agents; Swine; Muramidase; Nisin; Semen; Spermatozoa; Antimicrobial Peptides; Cell Membrane; Gentamicins; Acrosome
PubMed: 38867200
DOI: 10.1186/s12917-024-04105-9 -
Human Genomics Jun 2024Diabetic foot ulcers (DFU) is the most serious complication of diabetes mellitus, which has become a global health problem due to its high morbidity and disability rates...
BACKGROUND
Diabetic foot ulcers (DFU) is the most serious complication of diabetes mellitus, which has become a global health problem due to its high morbidity and disability rates and the poor efficacy of conventional treatments. Thus, it is urgent to identify novel molecular targets to improve the prognosis and reduce disability rate in DFU patients.
RESULTS
In the present study, bulk RNA-seq and scRNA-seq associated with DFU were downloaded from the GEO database. We identified 1393 DFU-related DEGs by differential analysis and WGCNA analysis together, and GO/KEGG analysis showed that these genes were associated with lysosomal and immune/inflammatory responses. Immediately thereafter, we identified CLU, RABGEF1 and ENPEP as DLGs for DFU using three machine learning algorithms (Randomforest, SVM-RFE and LASSO) and validated their diagnostic performance in a validation cohort independent of this study. Subsequently, we constructed a novel artificial neural network model for molecular diagnosis of DFU based on DLGs, and the diagnostic performance in the training and validation cohorts was sound. In single-cell sequencing, the heterogeneous expression of DLGs also provided favorable evidence for them to be potential diagnostic targets. In addition, the results of immune infiltration analysis showed that the abundance of mainstream immune cells, including B/T cells, was down-regulated in DFUs and significantly correlated with the expression of DLGs. Finally, we found latamoxef, parthenolide, meclofenoxate, and lomustine to be promising anti-DFU drugs by targeting DLGs.
CONCLUSIONS
CLU, RABGEF1 and ENPEP can be used as novel lysosomal molecular signatures of DFU, and by targeting them, latamoxef, parthenolide, meclofenoxate and lomustine were identified as promising anti-DFU drugs. The present study provides new perspectives for the diagnosis and treatment of DFU and for improving the prognosis of DFU patients.
Topics: Humans; Lysosomes; Diabetic Foot; RNA-Seq; Single-Cell Analysis; Gene Expression Profiling; Prognosis; Male; Female; Machine Learning; Single-Cell Gene Expression Analysis
PubMed: 38862997
DOI: 10.1186/s40246-024-00629-1 -
Scientific Reports Jun 2024The primary hurdles for small interference RNA (siRNA) in clinical use are targeted and cytosolic delivery. To overcome both challenges, we have established a novel...
The primary hurdles for small interference RNA (siRNA) in clinical use are targeted and cytosolic delivery. To overcome both challenges, we have established a novel platform based on phage display, called NNJA. In this approach, a lysosomal cathepsin substrate is engineered within the flexible loops of PIII, that is displaying a unique random sequence at its N-terminus. NNJA library selection targeting cell-expressed targets should yield specific peptides localized in the cytoplasm. That is because phage internalization and subsequent localization to lysosome, upon peptide binding to the cell expressed target, will result in cleavage of PIII, rendering phage non-infective. Such phage will be eliminated from the selected pool and only peptide-phage that escapes lysosomes will advance to the next round. Proof of concept studies with the NNJA library demonstrated cytosolic localization of selected peptide-phage and peptide-siRNA, confirmed through confocal microscopy. More importantly, conjugation of siHPRT to monomeric or multimeric NNJA peptides resulted in significant reduction in HPRT mRNA in various cell types without significant cytotoxicity. Sequence similarity and clustering analysis from NGS dataset provide insights into sequence composition facilitating cell penetration. NNJA platform offers a highly efficient peptide discovery engine for targeted delivery of oligonucleotides to cytosol.
Topics: Cell-Penetrating Peptides; Humans; RNA, Small Interfering; Peptide Library; Lysosomes; Cell Surface Display Techniques; Cytosol
PubMed: 38862601
DOI: 10.1038/s41598-024-64405-w -
Frontiers in Molecular Biosciences 2024Autophagy mediates the degradation of intracellular macromolecules and organelles within lysosomes. There are three types of autophagy: macroautophagy, microautophagy,... (Review)
Review
Autophagy mediates the degradation of intracellular macromolecules and organelles within lysosomes. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Heat shock protein 70.1 (Hsp70.1) exhibits dual functions as a chaperone protein and a lysosomal membrane stabilizer. Since chaperone-mediated autophagy participates in the recycling of ∼30% cytosolic proteins, its disorder causes cell susceptibility to stress conditions. Cargo proteins destined for degradation such as amyloid precursor protein and tau protein are trafficked by Hsp70.1 from the cytosol into lysosomes. Hsp70.1 is composed of an N-terminal nucleotide-binding domain (NBD) and a C-terminal domain that binds to cargo proteins, termed the substrate-binding domain (SBD). The NBD and SBD are connected by the interdomain linker L, which modulates the allosteric structure of Hsp70.1 in response to ADP/ATP binding. After the passage of the Hsp70.1-cargo complex through the lysosomal limiting membrane, high-affinity binding of the positive-charged SBD with negative-charged bis(monoacylglycero)phosphate (BMP) at the internal vesicular membranes activates acid sphingomyelinase to generate ceramide for stabilizing lysosomal membranes. As the integrity of the lysosomal limiting membrane is critical to ensure cargo protein degradation within the acidic lumen, the disintegration of the lysosomal limiting membrane is lethal to cells. After the intake of high-fat diets, however, β-oxidation of fatty acids in the mitochondria generates reactive oxygen species, which enhance the oxidation of membrane linoleic acids to produce 4-hydroxy-2-nonenal (4-HNE). In addition, 4-HNE is produced during the heating of linoleic acid-rich vegetable oils and incorporated into the body via deep-fried foods. This endogenous and exogenous 4-HNE synergically causes an increase in its serum and organ levels to induce carbonylation of Hsp70.1 at Arg469, which facilitates its conformational change and access of activated μ-calpain to L. Therefore, the cleavage of Hsp70.1 occurs prior to its influx into the lysosomal lumen, which leads to lysosomal membrane permeabilization/rupture. The resultant leakage of cathepsins is responsible for lysosomal cell death, which would be one of the causative factors of lifestyle-related diseases.
PubMed: 38859931
DOI: 10.3389/fmolb.2024.1378656 -
Scientific Reports Jun 2024Lung cancer is the most common oncological disease worldwide, with non-small cell lung cancer accounting for approximately 85% of lung cancer cases. α-Hederin is a...
Lung cancer is the most common oncological disease worldwide, with non-small cell lung cancer accounting for approximately 85% of lung cancer cases. α-Hederin is a monodesmosidic triterpenoid saponin isolated from the leaves of Hedera helix L. or Nigella sativa and has been extensively studied for its antitumor activity against a variety of tumor cells. It has been suggested that α-Hederin is a potential regulator of autophagy and has high promise for application. However, the specific mechanism and characteristics of α-Hederin in regulating autophagy are not well understood. In this study, we confirmed the potential of α-Hederin application in lung cancer treatment and comprehensively explored the mechanism and characteristics of α-Hederin in regulating autophagy in lung cancer cells. Our results suggest that α-Hederin is an incomplete autophagy inducer that targets mTOR to activate the classical autophagic pathway, inhibits lysosomal acidification without significantly affecting the processes of autophagosome transport, lysosome biogenesis, autophagosome and lysosome fusion, and finally leads to impaired autophagic flux and triggers autophagic damage in NSCLC.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Lysosomes; Autophagy; Lung Neoplasms; Oleanolic Acid; Saponins; Cell Line, Tumor; TOR Serine-Threonine Kinases; Autophagosomes; A549 Cells
PubMed: 38858422
DOI: 10.1038/s41598-024-63348-6 -
Journal of Lipid Research Jun 2024Bis(monoacylglycerol)phosphate (BMP) is an acidic glycerophospholipid localized to late endosomes and lysosomes. However, the metabolism of BMP is poorly understood....
Bis(monoacylglycerol)phosphate (BMP) is an acidic glycerophospholipid localized to late endosomes and lysosomes. However, the metabolism of BMP is poorly understood. Because many drugs that cause phospholipidosis inhibit lysosomal phospholipase A2 (LPLA2, PLA2G15, LYPLA3) activity, we investigated whether this enzyme has a role in BMP catabolism. The incubation of recombinant human LPLA2 (hLPLA2) and liposomes containing the naturally occurring BMP (sn-(2-oleoyl-3-hydroxy)-glycerol-1-phospho-sn-1'-(2'-oleoyl-3'-hydroxy)-glycerol (S,S-(2,2',C)-BMP) resulted in the deacylation of this BMP isomer. The deacylation rate was 70 times lower than that of dioleoyl phosphatidylglycerol (DOPG), an isomer and precursor of BMP. The release rates of oleic acid from DOPG and four BMP stereoisomers by LPLA2 differed. The rank order of the rates of hydrolysis were DOPG>S,S-(3,3',C)-BMP>R,S-(3,1',C)-BMP>R,R-(1,1',C)>S,S-(2,2')-BMP. The cationic amphiphilic drug amiodarone (AMD) inhibited the deacylation of DOPG and BMP isomers by hLPLA2 in a concentration dependent manner. Under these experimental conditions, the ICs of amiodarone-induced inhibition of the four BMP isomers and DOPG were less than 20 μM and approximately 30 μM, respectively. BMP accumulation was observed in AMD-treated RAW 264.7 cells. The accumulated BMP was significantly reduced by exogenous treatment of cells with active recombinant hLPLA2 but not with diisopropylfluorophosphate-inactivated recombinant hLPLA2. Finally, a series of cationic amphiphilic drugs known to cause phospholipidosis were screened for inhibition of LPLA2 activity as measured by either the transacylation or fatty acid hydrolysis of BMP or phosphatidylcholine as substrates. Fifteen compounds demonstrated significant inhibition with ICs ranging from 6.8 to 63.3 μM. These results indicate that LPLA2 degrades BMP isomers with different substrate specificities under acidic conditions and may be the key enzyme associated with BMP accumulation in drug-induced phospholipidosis.
PubMed: 38857781
DOI: 10.1016/j.jlr.2024.100574 -
BioRxiv : the Preprint Server For... May 2024Accumulated levels of mutant huntingtin protein (mHTT) and its fragments are considered contributors to the pathogenesis of Huntington's disease (HD). Although lowering...
Accumulated levels of mutant huntingtin protein (mHTT) and its fragments are considered contributors to the pathogenesis of Huntington's disease (HD). Although lowering mHTT by stimulating autophagy has been considered a possible therapeutic strategy, the role and competence of autophagy-lysosomal pathway (ALP) during HD progression in the human disease remains largely unknown. Here, we used multiplex confocal and ultrastructural immunocytochemical analyses of ALP functional markers in relation to mHTT aggresome pathology in striatum and the less affected cortex of HD brains staged from HD2 to HD4 by Vonsattel neuropathological criteria compared to controls. Immunolabeling revealed the localization of HTT/mHTT in ALP vesicular compartments labeled by autophagy-related adaptor proteins p62/SQSTM1 and ubiquitin, and cathepsin D (CTSD) as well as HTT-positive inclusions. Although comparatively normal at HD2, neurons at later HD stages exhibited progressive enlargement and clustering of CTSD-immunoreactive autolysosomes/lysosomes and, ultrastructurally, autophagic vacuole/lipofuscin granules accumulated progressively, more prominently in striatum than cortex. These changes were accompanied by rises in levels of HTT/mHTT and p62/SQSTM1, particularly their fragments, in striatum but not in the cortex, and by increases of LAMP1 and LAMP2 RNA and LAMP1 protein. Importantly, no blockage in autophagosome formation and autophagosome-lysosome fusion was detected, thus pinpointing autophagy substrate clearance deficits as a basis for autophagic flux declines. The findings collectively suggest that upregulated lysosomal biogenesis and preserved proteolysis maintain autophagic clearance in early-stage HD, but failure at advanced stages contributes to progressive HTT build-up and potential neurotoxicity. These findings support the prospect that ALP stimulation applied at early disease stages, when clearance machinery is fully competent, may have therapeutic benefits in HD patients.
PubMed: 38854113
DOI: 10.1101/2024.05.29.596470