Human Neuroblastoma Cell Line SH-SY5Y Research Articles

The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines.

Sequence variants or mutations in the GBA gene are numerically the most important risk factor for Parkinson disease (PD). The GBA gene encodes for the lysosomal hydrolase enzyme, glucocerebrosidase (GCase). GBA mutations often reduce GCase activity and lead to the impairment of the autophagy-lysosomal pathway, which is important in the turnover of alpha-synuclein, accumulation of which is a key pathological hallmark of PD. Although the E326K variant is one of the most common GBA variants associated with PD, there is limited understanding of its biochemical effects. We have characterized homozygous and heterozygous E326K variants in human fibroblasts. We found that E326K variants did not cause a significant loss of GCase protein or activity, endoplasmic reticulum (ER) retention or ER stress, in contrast to the L444P GBA mutation. This was confirmed in human dopaminergic SH-SY5Y neuroblastoma cell lines overexpressing GCase with either E326K or L444P protein. Despite no loss of the GCase activity, a significant increase in insoluble alpha-synuclein aggregates in E326K and L444P mutants was observed. Notably, SH-SY5Y overexpressing E326K demonstrated a significant increase in the lipid droplet number under basal conditions, which was exacerbated following treatment with the fatty acid oleic acid. Similarly, a significant increase in lipid droplet formation following lipid loading was observed in heterozygous and homozygous E326K fibroblasts. In conclusion, the work presented here demonstrates that the E326K mutation behaves differently to the common loss of function GBA mutations; however, lipid dyshomeostasis and alpha-synuclein pathology are still evident.

Neuroblastoma SH-SY5Y Cell Differentiation to Mature Neuron by AM580 Treatment

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.

Mechanisms underlying citrinin-induced toxicity via oxidative stress and apoptosis-mediated by mitochondrial-dependent pathway in SH-SY5Y cells

Citrinin (CIT) is a mycotoxin produced as a secondary product by the genera Aspergillus, Penicillium, Monascus, and other strains. CIT has the potential for contaminating animal feed and human food such as maize, wheat, rye, barley, oats, rice, cheese, and sake. Although CIT is primarily known as a nephrotoxic mycotoxin, it also affects other organs, including the liver and bone marrow, and its mechanisms of toxicity have not been clearly elucidated. There is a further lack of studies investigating the potential for CIT-induced neurotoxicity and its mechanisms. In the current study, SH-SY5Y human neuroblastoma cell line was treated with CIT for 24 h to evaluate various toxicological endpoints, such as reactive oxygen species (ROS) production and apoptosis induction. Results indicate that CIT has an IC50 value of 250.90 μM and cell proliferation decreased significantly at 50 and 100 μM CIT concentrations. These same concentrations also caused elevated ROS production (≥34.76%), apoptosis (≥9.43-fold) and calcium ion mobilization (≥36.52%) in the cells. Results show a significant decrease in the mitochondrial membrane potential (≥86.8%). We also found that CIT significantly upregulated the expression of some genes related to oxidative stress and apoptosis, while downregulating others. These results suggest that apoptosis and oxidative stress may be involved in the mechanisms underlying CIT-induced neurotoxicity.

P16-09 Synthetic opioids induce oxidative stress and DNA damage in human neuroblastoma SH-SY5Y cell line

P16-09 Synthetic opioids induce oxidative stress and DNA damage in human neuroblastoma SH-SY5Y cell line

Bioactivity of Ionic Liquids Based on Valproate in SH-SY5Y Human Neuroblastoma Cell Line

The search for alternative and effective therapies to fight cancer is one of the main goals of the pharmaceutical industry. Recently, ionic liquids (ILs) have emerged as potential therapeutic agents with antitumor properties. The goal of this study was to synthesize and evaluate the bioactivity of different ILs coupled with the active pharmaceutical ingredient (API) valproate (VPA) as an antitumor agent. The toxicity of the prepared ionic liquids was evaluated by the MTT cell metabolic assay in human neuroblastoma SH-SY5Y and human primary Gingival Fibroblast (GF) cell lines, in which they showed inhibitory effects during the study period. In addition, low cytotoxicity against GF cell lines was observed, suggesting that these compounds are not toxic to human cell lines. [C2OHDMiM][VPA] demonstrated an outstanding antitumor activity against SH-SY5Y and lower activity against the non-neoplastic GF line. The herein assessed compounds played an important role in the modulation of the signaling pathways involved in the cellular behavior. This work also highlights the potential of these ILs-API as possible antitumor agents.

Antagonism of the Muscarinic Acetylcholine Type 1 Receptor Enhances Mitochondrial Membrane Potential and Expression of Respiratory Chain Components via AMPK in Human Neuroblastoma SH-SY5Y Cells and Primary Neurons

Impairments in mitochondrial physiology play a role in the progression of multiple neurodegenerative conditions, including peripheral neuropathy in diabetes. Blockade of muscarinic acetylcholine type 1 receptor (M1R) with specific/selective antagonists prevented mitochondrial dysfunction and reversed nerve degeneration in in vitro and in vivo models of peripheral neuropathy. Specifically, in type 1 and type 2 models of diabetes, inhibition of M1R using pirenzepine or muscarinic toxin 7 (MT7) induced AMP-activated protein kinase (AMPK) activity in dorsal root ganglia (DRG) and prevented sensory abnormalities and distal nerve fiber loss. The human neuroblastoma SH-SY5Y cell line has been extensively used as an in vitro model system to study mechanisms of neurodegeneration in DRG neurons and other neuronal sub-types. Here, we tested the hypothesis that pirenzepine or MT7 enhance AMPK activity and via this pathway augment mitochondrial function in SH-SY5Y cells. M1R expression was confirmed by utilizing a fluorescent dye, ATTO590-labeled MT7, that exhibits great specificity for this receptor. M1R antagonist treatment in SH-SY5Y culture increased AMPK phosphorylation and mitochondrial protein expression (OXPHOS). Mitochondrial membrane potential (MMP) was augmented in pirenzepine and MT7 treated cultured SH-SY5Y cells and DRG neurons. Compound C or AMPK-specific siRNA suppressed pirenzepine or MT7-induced elevation of OXPHOS expression and MMP. Moreover, muscarinic antagonists induced hyperpolarization by activating the M-current and, thus, suppressed neuronal excitability. These results reveal that negative regulation of this M1R-dependent pathway could represent a potential therapeutic target to elevate AMPK activity, enhance mitochondrial function, suppress neuropathic pain, and enhance nerve repair in peripheral neuropathy.

In Vitro Effects of Acitretin on Human Neuronal SH-SY5Y Cells

Acitretin is an oral drug approved by the Food and Drug Administration that is commonly used to treat psoriasis. In recent years, acitretin has been identified as a candidate drug for the treatment of Alzheimer’s disease, but its role in neuronal development is still unclear. In this study, the human neuroblastoma cell line SH-SY5Y was used as a model to study neuronal differentiation. We found that acitretin effectively promoted the differentiation of SH-SY5Y cells into neuronal cells and upregulated the expression of the neuronal marker β-III tubulin and the mature neuronal marker NFH. Differentially expressed genes were identified by RNA sequencing and analyzed by bioinformatics approaches. The results showed that genes associated with neuron development-related pathways, such as SSPO and KCNT1, had significant changes in expression. Analysis showed that PRKCA and CAMK2B may play important roles in the process by which acitretin promotes neurodevelopment. Through whole-cell patch clamping and a microelectrode array assay, we found that acitretin-treated neurons generated electrical spikes similar to those generated by mature neurons. This study provided evidence to support an accessible and safe model of neuron-like cells and verified that acitretin can promote the differentiation of neurons and has the potential to treat brain tumors and neurodevelopmental and neurodegenerative diseases.

Syzysamalactone, an Unusual 11-Carbon δ-Lactone Derivative from the Fresh Ripe Fruits of Syzygium samarangense (Wax Apple).

To study the chemical constituents from the ripe fresh fruits of Syzygium samarangense (wax apple) and their potential health effects, a phytochemical investigation was undertaken. A new δ-lactone derivative, syzysamalactone (1), along with a known biogenetically related δ-lactone derivative, 6-pentyl-α-pyrone (2), were isolated from the fresh ripe fruits of S. samarangense. Syzysamalactone (1) is an unusual 11-carbon δ-lactone derivative, and its chemical structure and absolute configuration were elucidated by spectroscopic data analysis. A plausible biogenetic pathway for 1 was also proposed. Furthermore, the potential neuroprotective effects of compounds 1 and 2 were assessed. As a result, compounds 1 and 2 displayed notable neuroprotective effects with EC50 values of 0.29 ± 0.03 and 1.28 ± 0.06 μM, respectively, using the SH-SY5Y human neuroblastoma cell line. This is the first report of δ-lactone derivatives showing significant neuroprotective activities.

Nicotinic receptor components of amyloid beta 42 proteome regulation in human neural cells

Alzheimer’s disease (AD) is associated with chronic neurodegeneration often accompanied by elevated levels of the neurotoxic peptide amyloid-beta 1–42 (Aβ42) in the brain. Studies show that extracellular Aβ42 binds to various cell surface receptors including the human α7 nicotinic acetylcholine receptor (nAChR) and activates pathways of neurotoxicity leading to cell death. The α7 nAChR is thus considered a promising drug target for therapy against neurodegenerative disease such as AD. In this study, we use mass spectrometry-based label-free precursor ion quantification to identify proteins and pathways that are changed by a 72-hour treatment with Aβ42 or Aβ42 in the presence of the α7 nAChR blocker, α-bungarotoxin (Bgtx) in the human neuroblastoma SH-SY5Y cell line. Bioinformatic gene ontology enrichment analysis was used to identify and characterize proteins and pathways altered by Aβ42 presentation. The results support evidence on the involvement of mitochondrial proteins in Aβ42 responses and define potential mechanisms of α7 nAChR mediated amyloid toxicity. These findings can inform pharmacological strategies for drug design and treatment against amyloid disease.

Alpha-Amylase Inhibits Cell Proliferation and Glucose Uptake in Human Neuroblastoma Cell Lines

The present article describes a study of the effects of alpha-amylase (α-amylase) on the human neuroblastoma (NB) cell lines SH-SY5Y, IMR-32, and LA-N-1. NB is the most common malignancy diagnosed in infants younger than 12 months. Some clinical observations revealed an inverse association between the risk of NB development and breastfeeding. α-Amylase which is present in breast milk was shown to have anticancer properties already in the beginning of the 20th century. Data presented here show that pancreatic α-amylase inhibits cell proliferation and has a direct impact on glucose uptake in the human NB cell lines. Our results point out the importance of further research which could elucidate the α-amylase mode of action and justify the presence of this enzyme in breast milk as a possible inhibitor of NB development. α-Amylase can be thus recognized as a potential safe and natural mild/host anticancer agent minimizing chemotherapy-related toxicity in the treatment of NB.

Semaglutide Protects against 6-OHDA Toxicity by Enhancing Autophagy and Inhibiting Oxidative Stress.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder for which no effective treatment is available. Studies have demonstrated that improving insulin resistance in type 2 diabetes mellitus (T2DM) can benefit patients with PD. In addition, a neuroprotective effect of glucagon-like peptide-1 (GLP-1) receptor agonists was demonstrated in experimental models of PD. In addition, there are some clinical trials to study the neuroprotective effect of GLP-1 analog on PD patients. Semaglutide is a long-acting, once-a-week injection treatment and the only available oral form of GLP-1 analog. In the present study, we treated the human neuroblastoma SH-SY5Y cell line with 6-hydroxydopamine (6-OHDA) as a PD in vitro model to explore the neuroprotective effects and potential mechanisms of semaglutide to protect against PD. Moreover, we compared the effect of semaglutide with liraglutide given at the same dose. We demonstrated that both semaglutide and liraglutide protect against 6-OHDA cytotoxicity by increasing autophagy flux and decreasing oxidative stress as well as mitochondrial dysfunction in SH-SY5Y cells. Moreover, by comparing the neuroprotective effects of semaglutide and liraglutide on PD cell models at the same dose, we found that semaglutide was superior to liraglutide for most parameters measured. Our results indicate that semaglutide, the new long-acting and only oral GLP-1 analog, may be represent a promising treatment for PD.

Antioxidant and Neuroprotective Activity of Vitamin E Homologues: In Vitro Study.

Here we present comparative data on the inhibition of lipid peroxidation by a variety of tocochromanols in liposomes. We also show for the first time the potential neuroprotective role of all the vitamin E homologues investigated on the neuronally differentiated human neuroblastoma SH-SY5Y cell line. α-Tocopherol had nearly no effect in the inhibition of lipid peroxidation, while β-, γ-, and δ-tocopherols inhibited the reaction completely when it was initiated in a lipid phase. Similar effects were observed for tocotrienol homologues. Moreover, in this respect plastochromanol-8 was as effective as β-, γ-, and δ-tocochromanols. When the prenyllipids were investigated in a 1,1-diphenyl-2-picrylhydrazyl (DPPH) test and incorporated into different lipid carriers, the radical oxidation was most pronounced in liposomes, followed by mixed micelles and the micellar system. When the reaction of tocochromanols was examined in niosomes, the oxidation was most pronounced for α-tocopherol and plastochromanol-8, followed by α-tocotrienol. Next, using retinoic acid-differentiated SH-SY5Y cells, we tested the protective effects of the compounds investigated on hydrogen peroxide (H2O2)-induced cell damage. We showed that tocotrienols were more active than tocopherols in the oxidative stress model. Plastochromanol-8 had a strong inhibitory effect on H2O2-induced lactate dehydrogenase (LDH) release and H2O2-induced decrease in cell viability. The water-soluble α-tocopherol phosphate had neuroprotective effects at all the concentrations analyzed. The results clearly indicate that structural differences between vitamin E homologues reflect their different biological activity and indicate their potential application in pharmacological treatments for neurodegenerative diseases. In this respect, the application of optimal tocochromanol-carrying structures might be critical.

Rosmarinic Acid-Loaded Polymeric Nanoparticles Prepared by Low-Energy Nano-Emulsion Templating: Formulation, Biophysical Characterization, and In Vitro Studies.

Rosmarinic acid (RA), a caffeic acid derivative, has been loaded in polymeric nanoparticles made up of poly(lactic-co-glycolic acid) (PLGA) through a nano-emulsion templating process using the phase-inversion composition (PIC) method at room temperature. The obtained RA-loaded nanoparticles (NPs) were colloidally stable exhibiting average diameters in the range of 70–100 nm. RA was entrapped within the PLGA polymeric network with high encapsulation efficiencies and nanoparticles were able to release RA in a rate-controlled manner. A first-order equation model fitted our experimental data and confirmed the prevalence of diffusion mechanisms. Protein corona formation on the surface of NPs was assessed upon incubation with serum proteins. Protein adsorption induced an increase in the hydrodynamic diameter and a slight shift towards more negative surface charges of the NPs. The radical scavenging activity of RA-loaded NPs was also studied using the DPPH·assay and showed a dose–response relationship between the NPs concentration and DPPH inhibition. Finally, RA-loaded NPs did not affect the cellular proliferation of the human neuroblastoma SH-SY5Y cell line and promoted efficient cellular uptake. These results are promising for expanding the use of O/W nano-emulsions in biomedical applications.

A Class I HDAC Inhibitor Rescues Synaptic Damage and Neuron Loss in APP-Transfected Cells and APP/PS1 Mice through the GRIP1/AMPA Pathway.

As a neurodegenerative disease, Alzheimer’s disease (AD) seriously affects the health of older people. Changes in synapses occur first over the course of the disease, perhaps even before the formation of Aβ plaques. Histone deacetylase (HDAC) mediates the damage of Aβ oligomers to dendritic spines. Therefore, we examined the relationship between HDAC activity and synaptic defects using an HDAC inhibitor (HDACI), BG45, in the human neuroblastoma SH-SY5Y cell line with stable overexpression of Swedish mutant APP (APPsw) and in APP/PS1 transgenic mice during this study. The cells were treated with 15 μM BG45 and the APP/PS1 mice were treated with 30 mg/kg BG45. We detected the levels of synapse-related proteins, HDACs, tau phosphorylation, and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors using Western blotting and immunohistochemistry. We also measured the expression of cytoskeletal proteins in the cell model. The mRNA levels of the glutamate ion receptor alginate subunit 2 (GRIK2), sodium voltage-gated channel beta subunit (SCN3B), synaptophysin (SYP), Grm2 (the gene encoding glutamate receptor subunit 2 (GluR2)), Grid2IP, glutamate receptor interacting protein 1 (GRIP1), and GRIP2 were detected to explore the effects of the HDACI on regulating the expression of synaptic proteins and AMPA receptors. According to our studies, the expressions of HDAC1, HDAC2, and HDAC3 were increased, which were accompanied by the downregulation of the synapse-related proteins SYP, postsynaptic dendritic protein (PSD-95), and spinophilin as early as 24 h after transfection with the APPsw gene. BG45 upregulated the expression of synapse-related proteins and repaired cytoskeletal damage. In vivo, BG45 alleviated the apoptosis-mediated loss of hippocampal neurons, upregulated synapse-related proteins, reduced Aβ deposition and phosphorylation of tau, and increased the levels of the synapse-related genes GRIK2, SCN3B, SYP, Grm2, and Grid2IP. BG45 increased the expression of the AMPA receptor subunits GluA1, GluA2, and GluA3 on APPsw-transfected cells and increased GRIP1 and GRIP2 expression and AMPA receptor phosphorylation in vivo. Based on these results, HDACs are involved in the early process of synaptic defects in AD models, and BG45 may rescue synaptic damage and the loss of hippocampal neurons by specifically inhibiting HDAC1, HDAC2, and HDAC3, thereby modulating AMPA receptor transduction, increasing synapse-related gene expression, and finally enhancing the function of excitatory synapses. BG45 may be considered a potential drug for the treatment of early AD in further studies.

Artemisia Leaf Extract protects against neuron toxicity by TRPML1 activation and promoting autophagy/mitophagy clearance in both in vitro and in vivo models of MPP+/MPTP-induced Parkinson's disease

BackgroundParkinson's disease (PD) is a neurodegenerative disorder involving the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Cellular clearance mechanisms, including the autophagy-lysosome pathway, are commonly affected in the pathogenesis of PD. The lysosomal Ca2+ channel mucolipin TRP channel 1 (TRPML1) is one of the most important proteins involved in the regulation of autophagy. Artemisia argyi Lev. et Vant., is a traditional Chinese herb, that has diverse therapeutic properties and is used to treat patients with skin diseases and oral ulcers. However, the neuroprotective effects of A. argyi are not explored yet. HypothesisThis study aims is to investigate the neuroprotective effects of A. argyi in promoting the TRPML1-mediated autophagy/mitophagy-enhancing effect MethodsIn this study, we used 1-methyl-4-phenyl-pyridinium (MPP+)-induced PD model established in an SH-SY5Y human neuroblastoma cell line as well as in a 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-induced PD model in C57BL/6 J mice. MTT assay was conducted to measure the cell viability and further MitoSoX and DCFDA assay were used to measure the ROS. Western blot analysis was used to access levels of TRPML1, p-DRP1 (ser616), p-AKT, PI3K, and β-catenin, Additionally, IF and IHC analysis to investigate the expression of TRPML1, LC3B, β-catenin, TH+, α-synuclein. Mitotracker stain was used to check mitophagy levels and a lysosomal intracellular activity kit was used to measure the lysosomal dysfunction. Behavioral studies were conducted by rotarod and grip strength experiments to check motor functions. ResultsIn our in vitro study, A. argyi rescued the MPP+-induced loss of cell viability and reduced the accumulation of mitochondrial and total reactive oxygen species (ROS). Subsequently, it increased the expression of TRPML1 protein, thereby inducing autophagy, which facilitated the clearance of toxic accumulation of α-synuclein. Furthermore, A. argyi played a neuroprotective role by activating the PI3K/AKT/β-catenin cell survival pathway. MPP+-mediated mitochondrial damage was overcome by upregulation of mitophagy and downregulation of the mitochondrial fission regulator p-DRP1 (ser616) in SH-SY5Y cells. In the in vivo study, A. argyi ameliorated impaired motor function and rescued TH+ neurons in the SNpc region. Similar to the results of the in vitro study, TRPML1, LC3B, and β-catenin expression was enhanced in the SNpc region in the A. argyi-treated mice brain. ConclusionThus, our results first demonstrate that A. argyi can exert neuroprotective effects by stimulating TRPML1 and rescuing neuronal cells by boosting autophagy/mitophagy and upregulating a survival pathway, suggesting that A. argyi can further be exploited to slow the progression of PD.

Abstract 5069: Delivering docetaxel and curcumin via a nano-combination-therapy for modulating the progression of neuroblastoma

Abstract Introduction: Neuroblastoma (NB) is a form of extracranial tumor derived from the sympathetic nervous system that affects most often infants and young children. It is a very heterogeneous tumor with different levels of aggressiveness. Despite the multiple therapeutic strategies (i.e. aggressive chemotherapy, surgery, radiotherapy, immunotherapy), the outcome in advanced stages or recurrent diseases is negative. New strategies are needed to improve the therapeutic efficacy of existing drugs and reduce their toxicity. Nanotechnology represents a good tool for reaching this goal. Taken this in mind, the focus of this experimental work was to engineer polymeric biodegradable nanomedicines for co-delivering anti-inflammatory and chemotherapeutic molecules to NB malignant masses. More specifically, the work focused on the synthesis, physico-chemical and biopharmaceutical characterization, in vitro testing and in vivo validation of nanomedicines loaded with the cytotoxic drug Docetaxel (DTXL) and the natural anti-inflammatory compound, Curcumin (CURC). Methods: Four configurations of Spherical Polymeric Nanoparticles (SPNs) - loaded with CURC (CURC-SPNs), loaded with DTXL (DTXL-SPNs), loaded with the combination thereof (CURC/DTXL-SPNs), and empty (SPNs) - were synthesized using an oil-in water emulsion/solvent evaporation technique. SPNs size, zeta potential, and polydispersity index (PDI) were measured by dynamic light scattering. The toxicity of SPNs was determined by an MTT assay on the human NB cell line SH-SY5Y. For in vivo efficacy and biodistribution experiments, homozygous CD1 nu/nu athymic female mice (4 to 6-weeks old) were orthotopically injected with SH-SY5Y cells in the left adrenal gland. Results: Empty, DTXL-SPNs, CURC-SPNs, and CURC/DTXL-SPNs were characterized by a narrow size distribution (PdI < 0.15) with an average hydrodynamic diameter of about 185 nm. All the formulations showed a negative surface ζ-potential, associated with the carboxylate groups in the DSPE-PEG coating. A biphasic release profile was observed for all the 3 formulations, with almost 90% of the total drug mass released within the first 24 hours. In vivo results indicated that mice treated with CURC/DTXL -SPNs had a significant increase in life span as compared to untreated mice (control) (p=0.0002), mice treated with CURC-SPNs (p=0.0205), DTXL-SPNs (p=0.0391), and free DTXL (p=0.0054). Biodistribution experiments showed a 2% ID/g accumulation of the injected dose per tumor mass, regardless of the tumor development stage. This behavior is in agreement with results from a longitudinal Magnetic Resonance Imaging analysis of the malignant masses. Conclusion: The obtained results would suggest that nanomedicines could effectively delivery two therapeutic molecules within the malignant mass and modulate its progression leading to a significant increase in overall survival. Citation Format: Agnese Fragassi, Martina Di Francesco, Fabio Pastorino, Miguel Ferreira, Valentina Di Francesco, Anna Lisa Palange, Christian Celia, Luisa Di Marzio, Michele Cilli, Veronica Bensa, Mirco Ponzoni, Paolo Decuzzi. Delivering docetaxel and curcumin via a nano-combination-therapy for modulating the progression of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5069.

The efficacy and neuroprotective effects of edaravone-loaded mPEG-b-PLGA polymeric nanoparticles on human neuroblastoma SH-SY5Y cell line as in vitro model of ischemia

Stroke is the second leading cause of death and disability worldwide. The present study was designed to investigate the neuroprotective effects of polymeric micelles loaded with Edaravone on ischemia-induced in human neuroblastoma cell line (SH-SY5Y). Polymethoxy ethylene glycol-polylactic-co-glycolic acid (mPEG-b-PLGA) was first synthesized, and its physicochemical properties were determined. Micelles were prepared by the nanoprecipitation method. The size of micelles was 155 ± 2.5 nm with a surface charge of −15 ± 0.94 and polydispersity index (PDI) of 0.11. The examination of the morphology of micelles performed by transmission electron microscopy (TEM) and atomic force microscopy (AFM) showed that the size of particles was less than 155 ± 2.5 nm, and the morphology was spherical. The drug loading (DL) was determined about 13 ± 1.4% and encapsulation efficiency was 65 ± 0.79%. Drug delivery was analyzed using UV-VIS spectroscopy. The impact of different concentrations of free and micelle-containing Edaravone was performed on SH-SY5Y cells under normal and oxygen and glucose deprivation (OGD) conditions. Lactate dehydrogenase (LDH), nitric oxide (NO) activity, Intracellular reactive oxygen species (ROS) and apoptosis rate were significantly reduced in cells treated with edaravone loaded-micelle in addition to a significant reduction in the expression of pro-apoptotic gene (Bax) and increase in the expression of anti-apoptotic genes (HSP70 and Bcl-2). Consequently, according to the result of the study Edaravone-loaded micelles had a significant rate of neuroprotective effects than free Edaravone against ischemia-induced oxidative damage.

NMR-Driven Identification of Cinnamon Bud and Bark Components With Anti-Aβ Activity.

The anti-Alzheimer disease (AD) activity reported for an aqueous cinnamon bark extract prompted us to investigate and compare the anti-amyloidogenic properties of cinnamon extracts obtained from both bark and bud, the latter being a very little explored matrix. We prepared the extracts with different procedures (alcoholic, hydroalcoholic, or aqueous extractions). An efficient protocol for the rapid analysis of NMR spectra of cinnamon bud and bark extracts was set up, enabling the automatic identification and quantification of metabolites. Moreover, we exploited preparative reverse-phase (RP) chromatography to prepare fractions enriched in polyphenols, further characterized by UPLC-HR-MS. Then, we combined NMR-based molecular recognition studies, atomic force microscopy, and in vitro biochemical and cellular assays to investigate the anti-amyloidogenic activity of our extracts. Both bud and bark extracts showed a potent anti-amyloidogenic activity. Flavanols, particularly procyanidins, and cinnamaldehydes, are the chemical components of cinnamon hindering Aβ peptide on-pathway aggregation and toxicity in a human neuroblastoma SH-SY5Y cell line. Together with the previously reported ability to hinder tau aggregation and filament formation, these data indicate cinnamon polyphenols as natural products possessing multitarget anti-AD activity. Since cinnamon is a spice increasingly present in the human diet, our results support its use to prepare nutraceuticals useful in preventing AD through an active contrast to the biochemical processes that underlie the onset of this disease. Moreover, the structures of cinnamon components responsible for cinnamon anti-AD activities represent molecular templates for designing and synthesizing new anti-amyloidogenic drugs.

DNA-hydrolyzing catalytic IgGs from schizophrenia patients do not affect cell viability of the SH-SY5Y human neuroblastoma cell line

IntroductionDNA-hydrolyzing catalytic IgGs have caspase-dependent cytotoxic effects in autoimmune diseases. Recently, DNA-hydrolyzing IgGs have been discovered in schizophrenia. However, their cytotoxic properties have not been studied.ObjectivesTo assess the effect of serum IgGs with DNA-hydrolyzing activity of schizophrenia patients on the cell viability of the SH-SY5Y human neuroblastoma cell line.MethodsSerum of 8 patients with paranoid schizophrenia in the acute phase and 7 mentally and somatically healthy persons were used. IgG was purified from serum by affinity chromatography on Protein-G-Sepharose columns. The DNA hydrolyzing activity of IgG was assessed by the degree of hydrolysis of the pBluescript plasmid. The cell viability of the SH-SY5Y human neuroblastoma cell line after exposure to purified IgG preparations was assessed by high-throughput screening on the CellInsight CX7 platform (Thermo Scientific, USA) using the fluorescent dyes propidium iodide and Hoechst.ResultsOf the 8 IgG preparation obtained, 4 drugs had high DNA-hydrolyzing activity. All tested IgG preparations from healthy donors were inactive. One-way ANOVA analysis of the proportion of dead cells of the SH-SY5Y line after exposure to antibodies (0.1 mg/ml) showed no significant differences in the proportion of dead cells (p=0.688 after 24 hours; p=0.831 after 48 hours). Similar results were obtained at a higher concentration of antibodies - 0.2 mg/ml.ConclusionsThus, it has been shown in vitro that IgGs isolated from the serum of schizophrenia patients with or without DNA-hydrolyzing activity does not exhibit cytotoxic properties against the SH-SY5Y human neuroblastoma cell line. Support by Grant of RSF № 18-15-00053P.DisclosureNo significant relationships.

LncRNA TUG1 compromised neuronal mitophagy in cerebral ischemia/reperfusion injury by targeting sirtuin 1.

Mitophagy protects against cerebral ischemia/reperfusion (CI/R)-induced neuronal apoptosis via mitochondrial clearance. Although taurine-upregulated gene 1 (lncRNA TUG1) has been proposed to be involved in the neuronal apoptosis evoked by CI/R, its specific role in mitophagy during the progression of CI/R injury remains unknown. The CI/R rat model was established using middle cerebral artery occlusion/reperfusion (MCAO/R). Human neuroblastoma cell line SH-SY5Y was subjected to oxygen-glucose deprivation and reoxygenation (OGD/R). Ubiquitination assay, co-immunoprecipitation assay, RNA pull-down, and RNA immunoprecipitation were used to determine the interplay among TUG1, sirtuin 1 (SIRT1), and F-box and WD repeat domain-containing 7 (FBXW7). The upregulation of the TUG1 level and downregulation of the mitophagy were observed in both MCAO/R-treated rats and OGD/R-treated cells. The administration of si-TUG1 (a siRNA directed against TUG1) potentiated mitophagy and suppressed neuronal apoptosis in OGD/R-treated cells. However, the neuroprotective effect of si-TUG1 was reversed by mitophagy inhibitor or SIRT1 knockdown in vitro. Functionally, TUG1 enhanced FBXW7-mediated SIRT1 ubiquitination by upregulating FBXW7 expression. The overexpression of FBXW7 abrogated the si-TUG1-reinforced mitophagy by decreasing SIRT1 expression, thus aggravating neuronal apoptosis in the OGD/R+si-TUG1-treated cells. In rats with MCAO/R, the interference of TUG1 clearly decreased neuronal apoptosis, lessened the infarct volume, and relieved the neurological deficits. TUG1 knockdown promotes SIRT1-induced mitophagy by suppressing FBXW7-mediated SIRT1 degradation, thus relieving the neuronal apoptosis induced by CI/R injury. LncRNA TUG1 promotes neuronal apoptosis through inhibition of mitophagy. TUG1 decreased SIRT1 expression by promoting FBXW7-mediated SIRT1 ubiquitination. FBXW7/SIRT1 axis mediated the effect of TUG1 on OGD/R-induced neuronal apoptosis by regulating mitophagy.