Neuroprotection by Anethum graveolens (Dill) Seeds and Its Phytocompounds in SH-SY5Y Neuroblastoma Cell Lines and Acellular Assays
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PROVIDER: S-EPMC11241255 | biostudies-literature | 2024 Jun
REPOSITORIES: biostudies-literature
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Project description:Alzheimer's disease, a progressive neurodegenerative disease, affects learning and memory resulting from cholinergic dysfunction. Scopolamine has been employed to induce Alzheimer's disease-like pathology in vivo and in vitro through alteration of cholinergic system. N-benzylcinnamide (PT-3), purified from Piper submultinerve, has been shown to exhibit neuroprotective properties against amyloid-β-induced neuronal toxicity in rat cortical primary cell culture and to improve spatial learning and memory of aged rats through alleviating oxidative stress. We proposed a hypothesis that PT3 has a neuroprotective effect against scopolamine-induced cholinergic dysfunction. PT-3 (125-200 nM) pretreatment was performed in human neuroblastoma SH-SY5Y cell line following scopolamine induction. PT-3 (125-200 nM) inhibited scopolamine (2 mM)-induced generation of reactive oxygen species, cellular apoptosis, upregulation of acetylcholinesterase activity, downregulation of choline acetyltransferase level, and activation of p38 and JNK signalling pathways. These findings revealed the underlying mechanisms of scopolamine-induced Alzheimer's disease-like cellular dysfunctions, which provide evidence for developing drugs for the treatment of this debilitating disease.
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Project description:Anethum graveolens cultivar:Gujarat Dill-1 Assembly
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Project description:Anethum graveolens cultivar:Gujarat Dill-1 Assembly
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Project description:Cultured SH-SY5Y human neuroblastoma cells are used in neurotoxicity assays. These cells express markers of the cholinergic and dopaminergic systems. Acetylcholinesterase (AChE) activity has been reported in these cells. Neurotoxic organophosphate compounds that inhibit AChE, also inhibit butyrylcholinesterase (BChE). We confirmed the presence of AChE in the cell lysate by activity assays, Western blot, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of immunopurified AChE. A nondenaturing gel stained for AChE activity identified the catalytically active AChE in SH-SY5Y cells as the unstable monomer. We also identified immature BChE in the cell lysate. The concentration of active BChE protein was similar to that of active AChE protein. The rate of substrate hydrolysis by AChE was 10-fold higher than substrate hydrolysis by BChE. The higher rate was due to the 10-fold higher specific activity of AChE over BChE (5000 units/mg for AChE; 500 units/mg for BChE). Neither cholinesterase was secreted. Tryptic peptides of immunopurified AChE and BChE were identified by LC-MS/MS on an Orbitrap Lumos Fusion mass spectrometer. The unfolded protein chaperone, binding immunoglobulin protein BiP/GRP78, was identified in the mass spectral data from all cholinesterase samples, suggesting that BiP was co-extracted with cholinesterase. This suggests that the cytoplasmic cholinesterases are immature forms of AChE and BChE that bind to BiP. It was concluded that SH-SY5Y cells express active AChE and active BChE, but the proteins do not mature to glycosylated tetramers.
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Project description:Neuroblastoma is a type of developmental childhood cancer that arises from the neural crest. It is the most common pediatric solid tumor in the world. AM580 is a powerful cyto-differentiating molecule on acute promyelocytic leukemia cells and induced pluripotent stem cells, but its effect on neuroblastoma is still unknown. In this study, the neuronal differentiation impact of AM580 was investigated using the human neuroblastoma cell line SH-SY5Y as a model. AM580 successfully stimulated the SH-SY5Y cells to develop into neuron-like cells. Functional enrichment analysis of RNAseq data revealed that differentially expressed genes (DEGs) were substantially enriched for GO keywords and KEGG pathways linked to neuron development. Some potassium ion channel genes associated with neuronal excitation, such as KCNT1, were shown to be upregulated. Through the MEA tests, we found the AM580-induced neurons possessed electrical spikes as mature neurons. AM580 also induced the neuronal marker β-tubulin III and mature neurons marker Neurofilament H. Our study proved that AM580 can promote the differentiation of neurons and has the potential to treat neuroblastoma, neurodevelopmental and neurodegenerative diseases.
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Project description:The increasing appearance of engineered nanomaterials in broad biomedical and industrial sectors poses an escalating health concern from unintended exposure with unknown consequences. Routine in vitro assessments of nanomaterial toxicity are a vital component to addressing these mounting health concerns; however, despite the known role of cell-cell and cell-matrix contacts in governing cell survival, these physical interactions are generally ignored. Herein, we demonstrate that exposure to amorphous silica particles destabilizes mitochondrial membrane potential, stimulates reactive oxygen species (ROS) production and promotes cytotoxicity in SH-SY5Y human neuroblastoma through mechanisms that are potently matrix dependent, with SH-SY5Y cells plated on the softest matrix displaying a near complete recovery in viability compared to dose-matched cells plated on tissue-culture plastic. Cells on the softest matrix (3 kPa) further displayed a 50% reduction in ROS production and preserved mitochondrial membrane potential. The actin cytoskeleton is mechanosensitive and closely related to ROS production. SH-SY5Y cells exposed to a 100 μg/mL dose of 50 nm silica particles displayed distinct cytoskeletal aberrations and a 70% increase in cell stiffness. Overall, this study establishes that the mechanical environment can significantly impact silica nanoparticle toxicity in SH-SY5Y cells. The mechanobiochemical mechanisms behind this regulation, which are initiated at the cell-matrix interface to adjust cytoskeletal structure and intracellular tension, demand specific attention for a comprehensive understanding of nanotoxicity.
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Project description:Manganese (Mn)-associated neurotoxicity has been well recognized. However, Mn is also an essential nutrient to maintain physiological function. Our previous study of human neuroblastoma SH-SY5Y cells showed that Mn treatment comparable to physiological and toxicological concentrations in human brain resulted in different mitochondrial responses, yet cellular metabolic responses associated with such different outcomes remain uncharacterized. Herein, SH-SY5Y cells were examined for metabolic responses discriminated by physiological and toxicological levels of Mn using high-resolution metabolomics (HRM). Before performing HRM, we examined Mn dose (from 0 to100 μM) and time effects on cell death. Although we did not observe any immediate cell death after 5 h exposure to any of the Mn concentrations assessed (0-100 μM), cell loss was present after a 24-h recovery period in cultures treated with Mn ≥ 50 μM. Exposure to Mn for 5 h resulted in a wide range of changes in cellular metabolism including amino acids (AA), neurotransmitters, energy, and fatty acids metabolism. Adaptive responses at 10 μM showed increases in neuroprotective AA metabolites (creatine, phosphocreatine, phosphoserine). A 5-h exposure to 100 µM Mn, a time before any cell death occurred, resulted in decreases in energy and fatty acid metabolites (hexose-1,6 bisphosphate, acyl carnitines). The results show that adjustments in AA metabolism occur in response to Mn that does not cause cell death while disruption in energy and fatty acid metabolism occur in response to Mn that results in subsequent cell death. The present study establishes utility for metabolomics analyses to discriminate adaptive and toxic molecular responses in a human in vitro cellular model that could be exploited in evaluation of Mn toxicity.
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Project description:OBJECTIVE:Isatin has gained attention in recent years owing to its anticancer properties and is thought to offer medical benefits. Isatin is an endogenous oxidized indole with various behavioral and metabolic properties and is commonly found in mammalian tissues and fluids. It has several plausible applications in biomedical research and has also been investigated as a potential anticancer agent. However, its effects on neuroblastoma (NB) cells are unclear. Here, we evaluate the effects of isatin on neuroblastoma cell metastasis and invasion and reveal the underlying mechanism. METHODS:NB cell viability was evaluated with the cell counting kit (CCK)-8 assay. NB cell invasion and migration abilities were tested with transwell and wound healing experiments. The relative mRNA expression of associated molecules was detected with real-time polymerase chain reaction (RT-PCR) and quantitative PCR. The expression level of related proteins was detected with western blotting. RESULTS:Isatin inhibited the proliferation, invasion, and migration of neuroblastoma cells in a dose-dependent manner. Isatin increased the expression level of H3K4m1 and phosphatase and tensin homolog (PTEN) and decreased the phosphorylation level of PTEN downstream proteins phosphoinositide 3-kinase, protein kinase B, mammalian target of rapamycin, focal adhesion kinase, and SHC. Together, these results support the potential anti-metastatic effects of isatin on NB cells.
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