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Molecules (Basel, Switzerland) Jun 2024This study investigated the mechanism by which fucoxanthin acts as a novel ferroptosis inducer to inhibit tongue cancer. The MTT assay was used to detect the inhibitory...
This study investigated the mechanism by which fucoxanthin acts as a novel ferroptosis inducer to inhibit tongue cancer. The MTT assay was used to detect the inhibitory effects of fucoxanthin on SCC-25 human tongue squamous carcinoma cells. The levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and total iron were measured. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to assess glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor 2 (Nrf2), Keap1, solute carrier family 7 member 11 (SLC7A11), transferrin receptor protein 1 (TFR1), p53, and heme oxygenase 1 (HO-1) expression. Molecular docking was performed to validate interactions. Compared with the control group, the activity of fucoxanthin-treated SCC-25 cells significantly decreased in a dose- and time-dependent manner. The levels of MMP, GSH, and SOD significantly decreased in fucoxanthin-treated SCC-25 cells; the levels of ROS, MDA, and total iron significantly increased. mRNA and protein expression levels of Keap1, GPX4, Nrf2, and HO-1 in fucoxanthin-treated cells were significantly decreased, whereas levels of TFR1 and p53 were significantly increased, in a concentration-dependent manner. Molecular docking analysis revealed that binding free energies of fucoxanthin with p53, SLC7A11, GPX4, Nrf2, Keap1, HO-1, and TFR1 were below -5 kcal/mol, primarily based on active site hydrogen bonding. Our findings suggest that fucoxanthin can induce ferroptosis in SCC-25 cells, highlighting its potential as a treatment for tongue cancer.
Topics: Humans; NF-E2-Related Factor 2; Ferroptosis; Xanthophylls; Heme Oxygenase-1; Cell Line, Tumor; Phospholipid Hydroperoxide Glutathione Peroxidase; Molecular Docking Simulation; Reactive Oxygen Species; Signal Transduction; Tongue Neoplasms; Receptors, Transferrin; Membrane Potential, Mitochondrial; Kelch-Like ECH-Associated Protein 1; Gene Expression Regulation, Neoplastic; Amino Acid Transport System y+; Superoxide Dismutase; Down-Regulation; Antigens, CD
PubMed: 38930897
DOI: 10.3390/molecules29122832 -
Molecules (Basel, Switzerland) Jun 2024Miniaturized weak affinity chromatography is emerging as an interesting alternative to conventional biophysical tools for performing fragment-screening studies in the...
Miniaturized weak affinity chromatography is emerging as an interesting alternative to conventional biophysical tools for performing fragment-screening studies in the context of fragment-based drug discovery. In order to push back the analytical limits, it is necessary not only to control non-specific interactions with chromatographic support, but also to adapt this methodology by comparing the results obtained on an affinity column to a control column. The work presented in this study focused on fragment screening that targets a model membrane protein, the adenosine A2A receptor, embedded in nanodiscs (NDs) as biomimetic membranes. By studying the retention behavior of test fragment mixtures on supports modified with different types of NDs, we were able to determine the contribution of ND-related non-specific interactions, in particular the electrostatic effect of anionic phospholipids and the hydrophobic effect of neutral phospholipids. Different strategies for the preparation of control columns (empty NDs, orthosteric site blocking) were investigated and are presented for the first time. With these two types of control columns, the screening enabled the identification of two new fragments of AAR, which were confirmed by competition experiments and whose K values, estimated directly during the screening or after the competition experiments in frontal mode, were in good agreement.
Topics: Ligands; Chromatography, Affinity; Nanostructures; Receptor, Adenosine A2A; Membrane Proteins; Protein Binding; Humans; Phospholipids; Hydrophobic and Hydrophilic Interactions; Drug Discovery
PubMed: 38930880
DOI: 10.3390/molecules29122814 -
Antioxidants (Basel, Switzerland) Jun 2024(SH) is widely consumed as a healthy seaweed food in the Asia-Pacific region. However, the bioactive components contributing to its biological activity remain unknown....
(SH) is widely consumed as a healthy seaweed food in the Asia-Pacific region. However, the bioactive components contributing to its biological activity remain unknown. Herein, we optimized multifrequency ultrasonic-assisted extraction conditions to achieve higher antioxidant activity using a response surface methodology and an artificial neural network. High-resolution mass spectrometry (HRMS; negative mode) was used to tentatively identify the secondary metabolites in the optimized SH extract, which were further tested against oxidative stress in RAW264.7 cells. Additionally, the identified compounds were analyzed in silico to determine their binding energies with the Keap1 protein (4L7B). We identified 89 compounds using HRMS, among which 19 metabolites (8 polyphenolics, 2 flavonoids, 2 lignans, 2 terpenes, 2 tannins, 2 sulfolipids, and 1 phospholipid) were putatively reported for the first time in SH. The in vitro results revealed that optimized SH extract inhibited oxidative stress via the Nrf2/MAPKs/HO-1 pathway in a dose-dependent manner. This result was validated by performing in silico simulation, indicating that sargaquinoic acid and glycitein-7--glucuronide had the highest binding energies (-9.20 and -9.52 Kcal/mol, respectively) toward Keap1 (4L7B). This study offers a unique approach for the scientific community to identify potential bioactive compounds by optimizing the multivariant extraction processing conditions, which could be used to develop functional and nutraceutical foods.
PubMed: 38929129
DOI: 10.3390/antiox13060690 -
International Journal of Molecular... Jun 2024Our study aimed to investigate the role of ferroptosis in sevoflurane-induced hearing impairment and explore the mechanism of the microRNA-182-5p...
Our study aimed to investigate the role of ferroptosis in sevoflurane-induced hearing impairment and explore the mechanism of the microRNA-182-5p (miR-182-5p)/Glutathione Peroxidase 4 (GPX4) pathway in sevoflurane-induced ototoxicity. Immunofluorescence staining was performed using myosin 7a and CtBP2. Cell viability was assessed using the CCK-8 kit. Fe concentration was measured using FerroOrange and Mi-to-FerroGreen fluorescent probes. The lipid peroxide level was assessed using BODIPY 581/591 C11 and MitoSOX fluorescent probes. The auditory brainstem response (ABR) test was conducted to evaluate the hearing status. Bioinformatics tools and dual luciferase gene reporter analysis were used to confirm the direct targeting of miR-182-5p on GPX4 mRNA. GPX4 and miR-182-5p expression in cells was assessed by qRT-PCR and Western blot. Ferrostatin-1 (Fer-1) pretreatment significantly improved hearing impairment and damage to ribbon synapses in mice caused by sevoflurane exposure. Immunofluorescence staining revealed that Fer-1 pretreatment reduced intracellular and mitochondrial iron overload, as well as lipid peroxide accumulation. Our findings indicated that miR-182-5p was upregulated in sevoflurane-exposed HEI-OC1 cells, and miR-182-5p regulated GPX4 expression by binding to the 3'UTR of GPX4 mRNA. The inhibition of miR-182-5p attenuated sevoflurane-induced iron overload and lipid peroxide accumulation. Our study elucidated that the miR-182-5p/GPX4 pathway was implicated in sevoflurane-induced ototoxicity by promoting ferroptosis.
Topics: Ferroptosis; MicroRNAs; Sevoflurane; Phospholipid Hydroperoxide Glutathione Peroxidase; Animals; Mice; Ototoxicity; Signal Transduction; Cell Line; Male; Hearing Loss; Mice, Inbred C57BL; Phenylenediamines; Cyclohexylamines
PubMed: 38928480
DOI: 10.3390/ijms25126774 -
International Journal of Molecular... Jun 2024Human corneal fibrosis can lead to opacity and ultimately partial or complete vision loss. Currently, corneal transplantation is the only treatment for severe corneal...
Human corneal fibrosis can lead to opacity and ultimately partial or complete vision loss. Currently, corneal transplantation is the only treatment for severe corneal fibrosis and comes with the risk of rejection and donor shortages. Sphingolipids (SPLs) are known to modulate fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to both, transforming growth factor beta (TGF-β) signaling and corneal fibrogenesis. The aim of this study was to investigate the effects of sphingosine-1-phosphate (S1P) and S1P inhibition on specific TGF-β and SPL family members in corneal fibrosis. Healthy human corneal fibroblasts (HCFs) were isolated and cultured in EMEM + FBS + VitC (construct medium) on 3D transwells for 4 weeks. The following treatments were prepared in a construct medium: 0.1 ng/mL TGF-β1 (β1), 1 μM sphingosine-1-phosphate (S1P), and 5 μM Sphingosine kinase inhibitor 2 (I). Five groups were tested: (1) control (no treatment); rescue groups; (2) β1/S1P; (3) β1/I; prevention groups; (4) S1P/β1; and (5) I/β1. Each treatment was administered for 2 weeks with one treatment and switched to another for 2 weeks. Using Western blot analysis, the 3D constructs were examined for the expression of fibrotic markers, SPL, and TGF-β signaling pathway members. Scratch assays from 2D cultures were also utilized to evaluate cell migration We observed reduced fibrotic expression and inactivation of latent TGF-β binding proteins (LTBPs), TGF-β receptors, Suppressor of Mothers Against Decapentaplegic homologs (SMADs), and SPL signaling following treatment with I prevention and rescue compared to S1P prevention and rescue, respectively. Furthermore, we observed increased cell migration following stimulation with I prevention and rescue groups, with decreased cell migration following stimulation with S1P prevention and rescue groups after 12 h and 18 h post-scratch. We have demonstrated that I treatment reduced fibrosis and modulated the inactivation of LTBPs, TGF-β receptors, SPLs, and the canonical downstream SMAD pathway. Further investigations are warranted in order to fully uncover the potential of utilizing SphK I as a novel therapy for corneal fibrosis.
Topics: Humans; Sphingosine; Lysophospholipids; Fibrosis; Cornea; Signal Transduction; Transforming Growth Factor beta; Fibroblasts; Cells, Cultured; Sphingolipids; Phosphotransferases (Alcohol Group Acceptor); Corneal Diseases
PubMed: 38928268
DOI: 10.3390/ijms25126560 -
International Journal of Molecular... Jun 2024LPA receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased...
LPA receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased intracellular calcium and ERK phosphorylation through pertussis toxin-insensitive processes. Phorbol myristate acetate, but not LPA, desensitizes LPA-mediated calcium signaling, the agonists, and the phorbol ester-induced LPA internalization. Pitstop 2 (clathrin heavy chain inhibitor) markedly reduced LPA-induced receptor internalization; in contrast, phorbol ester-induced internalization was only delayed. LPA induced rapid β-arrestin-LPA receptor association. The agonist and the phorbol ester-induced marked LPA receptor phosphorylation, and phosphorylation sites were detected using mass spectrometry. Phosphorylated residues were detected in the intracellular loop 3 (S221, T224, S225, and S229) and in the carboxyl terminus (S321, S325, S331, T333, S335, Y337, and S343). Interestingly, phosphorylation sites are within sequences predicted to constitute β-arrestin binding sites. These data provide insight into LPA receptor signaling and regulation.
Topics: Humans; Receptors, Lysophosphatidic Acid; Phosphorylation; HEK293 Cells; Signal Transduction; Lysophospholipids; beta-Arrestins; Binding Sites; Calcium Signaling
PubMed: 38928196
DOI: 10.3390/ijms25126491 -
Traffic (Copenhagen, Denmark) Jun 2024Processes such as cell migration, phagocytosis, endocytosis, and exocytosis refer to the intense exchange of information between the internal and external environment in... (Review)
Review
Processes such as cell migration, phagocytosis, endocytosis, and exocytosis refer to the intense exchange of information between the internal and external environment in the cells, known as vesicular trafficking. In eukaryotic cells, these essential cellular crosstalks are controlled by Rab GTPases proteins through diverse adaptor proteins like SNAREs complex, coat proteins, phospholipids, kinases, phosphatases, molecular motors, actin, or tubulin cytoskeleton, among others, all necessary for appropriate mobilization of vesicles and distribution of molecules. Considering these molecular events, Rab GTPases are critical components in specific biological processes of immune cells, and many reports refer primarily to macrophages; therefore, in this review, we address specific functions in immune cells, concretely in the mechanism by which the GTPase contributes in dendritic cells (DCs) and, T/B lymphocytes.
Topics: Humans; rab GTP-Binding Proteins; Animals; T-Lymphocytes; Dendritic Cells; Antigen-Presenting Cells
PubMed: 38923715
DOI: 10.1111/tra.12950 -
Biosensors Jun 2024The overall 5-year survival rate of ovarian cancer (OC) is generally low as the disease is often diagnosed at an advanced stage of progression. To save lives, OC must be...
The overall 5-year survival rate of ovarian cancer (OC) is generally low as the disease is often diagnosed at an advanced stage of progression. To save lives, OC must be identified in its early stages when treatment is most effective. Early-stage OC causes the upregulation of lysophosphatidic acid (LPA), making the molecule a promising biomarker for early-stage detection. An LPA assay can additionally stage the disease since LPA levels increase with OC progression. This work presents two methods that demonstrate the prospective application for detecting LPA: the electromagnetic piezoelectric acoustic sensor (EMPAS) and a chemiluminescence-based iron oxide nanoparticle (IONP) approach. Both methods incorporate the protein complex gelsolin-actin, which enables testing for detection of the biomarker as the binding of LPA to the complex results in the separation of gelsolin from actin. The EMPAS was characterized with contact angle goniometry and atomic force microscopy, while gelsolin-actin-functionalized IONPs were characterized with transmission electron microscopy and Fourier transform infrared spectroscopy. In addition to characterization, LPA detection was demonstrated as a proof-of-concept in Milli-Q water, buffer, or human serum, highlighting various LPA assays that can be developed for the early-stage detection of OC.
Topics: Lysophospholipids; Humans; Female; Ovarian Neoplasms; Biomarkers, Tumor; Biosensing Techniques; Gelsolin; Actins; Early Detection of Cancer
PubMed: 38920591
DOI: 10.3390/bios14060287 -
Biosensors May 2024Biosensors play an important role in numerous research fields. Quartz crystal microbalances with dissipation monitoring (QCM-Ds) are sensitive devices, and binding...
Biosensors play an important role in numerous research fields. Quartz crystal microbalances with dissipation monitoring (QCM-Ds) are sensitive devices, and binding events can be observed in real-time. In combination with aptamers, they have great potential for selective and label-free detection of various targets. In this study, an alternative surface functionalization for a QCM-D-based aptasensor was developed, which mimics an artificial cell membrane and thus creates a physiologically close environment for the binding of the target to the sensor. Vesicle spreading was used to form a supported lipid bilayer (SLB) of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphethanolamine-N-(cap biotinyl) (biotin-PE). The SLB was then coated with streptavidin followed by applying a biotinylated aptamer against thrombin. SLB formation was investigated in terms of temperature and composition. Temperatures of 25 °C and below led to incomplete SLB formation, whereas a full bilayer was built at higher temperatures. We observed only a small influence of the content of biotinylated lipids in the mixture on the further binding of streptavidin. The functionalization of the sensor surface with the thrombin aptamer and the subsequent thrombin binding were investigated at different concentrations. The sensor could be reconstituted by incubation with a 5 M urea solution, which resulted in the release of the thrombin from the sensor surface. Thereafter, it was possible to rebind thrombin. Thrombin in spiked samples of human serum was successfully detected. The developed system can be easily applied to other target analytes using the desired aptamers.
Topics: Biosensing Techniques; Thrombin; Lipid Bilayers; Quartz Crystal Microbalance Techniques; Aptamers, Nucleotide; Humans; Phosphatidylcholines
PubMed: 38920574
DOI: 10.3390/bios14060270 -
Bioresources and Bioprocessing Jun 2024Phospholipase A1 (PLA1) is a kind of specific phospholipid hydrolase widely used in food, medical, textile. However, limitations in its expression and enzymatic activity...
Phospholipase A1 (PLA1) is a kind of specific phospholipid hydrolase widely used in food, medical, textile. However, limitations in its expression and enzymatic activity have prompted the investigation of the phospholipase-assisting protein PlaS. In this study, we elucidate the role of PlaS in enhancing the expression and activity of PlaA1 through N-terminal truncation. Our research demonstrates that truncating the N-terminal region of PlaS effectively overcomes its inhibitory effect on host cells, resulting in improved cell growth and increased protein solubility of the protein. The yeast two-hybrid assay confirms the interaction between PlaA1 and N-terminal truncated PlaS (∆N27 PlaS), highlighting their binding capabilities. Furthermore, in vitro studies using Biacore analysis reveal a concentration-dependent and specific binding between PlaA1 and ∆N27 PlaS, exhibiting high affinity. Molecular docking analysis provides insights into the hydrogen bond interactions between ∆N27 PlaS and PlaA1, identifying key amino acid residues crucial for their binding. Finally, the enzyme activity of PLA1 was boost to 8.4 U/mL by orthogonal test. Study significantly contributes to the understanding of the interaction mechanism between PlaS and PlaA1, offering potential strategies for enhancing PlaA1 activity through protein engineering approaches.
PubMed: 38916814
DOI: 10.1186/s40643-024-00777-1