SH-SY5Y Cell Line Research Articles

Effect of napabucasin and doxorubicin via the Jak2/Stat3 signaling pathway in suppressing the proliferation of neuroblastoma cells.

Napabucasin (NP) is a natural compound that can suppress cancer cell proliferation and cell cycle by inhibition of the signal transducer and activator of transcription 3 (STAT3) gene. We examined the effects of NP on the proliferation and invasion of neuroblastoma cells (SH-SY5Y). Human neuroblastoma SH-SY5Y cell line was used in this study. NP was administered to groups at the doses of 0.3-1 µM. Cell viability was analyzed by MTT assay. Real-time quantitative reverse transcription polymerase chain reaction and western blot analysis assessed the expressions of interleukin (IL)-6 dependent Jak2/Stat3 signaling pathway. The MTT cell viability method was applied to determine the antagonistic-synergistic effects and inhibitory concentration (IC50) doses of doxorubicin (DX) and NP. It was determined that 0.3-1 µM doses of NP killed the cells almost completely after 48 hours, and also that Jak2/Stat3 expressions decreased dose-dependently via IL-6. At the protein level, NP and DX were found to reduce Jak2 and Stat3 levels. NP showed that it suppresses the proliferation of neuroblastoma cells. Due to its inhibitory effect on Jak2 and Stat3, it can be used to prevent invasion of SH-SY5Y cells. NP, which can inactivate Jak2/Stat3, can be used as a treatment agent by combining with DX in proliferation pathway in neuroblastoma.

Targeted Modification of Epigenetic Marks Using CRISPR/dCas9-SunTag-Based Modular Epigenetic Toolkit.

The epigenome, consisting of chemical modifications to DNA and histone proteins, can alter gene expression. Clustered regularly interspaced short palindromic repeats/dead CRISPR-associated protein 9 (CRISPR/dCas9) systems enable precise target gene-specific gene modulation by attaching different "effector" domains to the dCas9 protein to activate or repress specific genes. CRISPR/dCas9-SunTag is an improved system version, allowing more efficient and precise gene activation or repression by recruiting multiple copies of the protein of interest. A CRISPR/dCas9-SunTag-based modular epigenetic toolkit was developed, enabling gene-specific epigenetic architecture modulation. This protocol generated a stable SH-SY5Y cell line expressing the CRISPR/dCas9-SunTag-JARID1A system to study H3K4Me3-mediated promoter regulation at a 200-400bp of fine resolution. The procedure involved designing sgRNAs, subcloning dCas9-5XGCN4 into pLvx-DsRed, validating epigenetic mark changes with ChIP, and validating gene expression changes with RT-qPCR. This epigenetic toolkit is valuable for researchers to understand the relationship between gene-specific epigenetic modifications and gene expression.

A mitochondria targeting, de novo designed, aggregation-induced emission probe for selective detection of neurotoxic amyloid-β aggregates.

A striking issue is the scarcity of imaging probes for the early diagnosis of Alzheimer's disease. For the development of Aβ biomarkers, a mitochondria targeting, de novo designed, aggregation-induced emission (AIE) probe Cou-AIE-TPP+ is constructed by engineering the aromatic coumarin framework into the bridge of electron donor-acceptor-donor tethered with a lipophilic cationic triphenylphosphonium (TPP+) group. The synthesized Cou-AIE-TPP+ probe exhibits biocompatibility, noncytotoxicity, and a huge Stokes shift (124 nm in PBS). Cou-AIE-TPP+ has respectable fluorescence augmentation inside the aggregated Aβ40 in comparison with monomeric Aβ40 with a high binding affinity (Kd = 83 nM) to Aβ40 aggregates, is capable of detecting the kinetics of amyloid aggregation, and is superior to the gold standard probe thioflavin T. Fluorescence lifetime and brightness are also augmented when the probe Cou-AIE-TPP+ binds with Aβ aggregates in PBS. Cou-AIE-TPP+ (λem 604 nm) selectively targets and images neuronal cell mitochondria, is useful to monitor mitochondrial morphology alteration and damage during Aβ40-induced neurotoxicity, recognizes neurotoxic Aβ fibrils, and is highly colocalized with thioflavin T, showing a decent Pearson correlation coefficient of 0.91 in the human neuroblastoma SH-SY5Y cell line. These findings indicate that the mitochondria targeting, de novo designed, functional AIE-based solvatofluorochromic Cou-AIE-TPP+ probe is a promising switch on biomarkers for fluorescence imaging of Aβ aggregates and to monitor mitochondrial morphology change and dysfunction during Aβ-induced neurotoxicity, which may offer imperative direction for the advancement of compelling AIE biomarkers for targeted early stage Aβ diagnosis in the future.

Differentiating SH-SY5Y Cells into Polarized Human Neurons for Studying Endogenous and Exogenous Tau Trafficking: Four Protocols to Obtain Neurons with Noradrenergic, Dopaminergic, and Cholinergic Properties.

Pathological alterations of the neuronal Tau protein are characteristic for many neurodegenerative diseases, called tauopathies. To investigate the underlying mechanisms of tauopathies, human neuronal cell models are required to study Tau physiology and pathology in vitro. Primary rodent neurons are an often used model for studying Tau, but rodent Tau differs in sequence, splicing, and aggregation propensity, and rodent neuronal physiology cannot be compared to humans. Human-induced pluripotent stem cell (hiPSC)-derived neurons are expensive and time-consuming. Therefore, the human neuroblastoma SH-SY5Y cell line is a commonly used cell model in neuroscience as it combines convenient handling and low costs with the advantages of human-derived cells. Since naïve SH-SY5Y cells show little similarity to human neurons and almost no Tau expression, differentiation is necessary to obtain human-like neurons for studying Tau protein-related aspects of health and disease. As they express in principle all six Tau isoforms seen in the human brain, differentiated SH-SY5Y-derived neurons are suitable for investigating the human microtubule-associated protein Tau and, for example, its sorting and trafficking. Here, we describe and discuss a general cultivation procedure as well as four differentiation methods to obtain SH-SY5Y-derived neurons resembling noradrenergic, dopaminergic, and cholinergic properties, based on the treatment with retinoic acid (RA), brain-derived neurotrophic factor (BDNF), and 12-O-tetrade canoylphorbol-13-acetate (TPA). TPA and RA-/TPA-based protocols achieve differentiation efficiencies of 40-50% after 9 days of treatment. The highest differentiation efficiency (~75%) is accomplished by a combination of RA and BDNF; treatment only with RA is the most time-efficient method as ~50% differentiated cells can be obtained already after 7 days.

Drug Synergism of Anticancer Action in Combination with Favipiravir and Paclitaxel on Neuroblastoma Cells.

Background and Objectives: Favipiravir (FPV) is an antiviral medication and has an inhibitory effect on Cytochrome P450 (CYP2C8) protein, which is mainly involved in drug metabolism in the liver, and the expression of this gene is known to be enhanced in neuronal cells. The metabolization of Paclitaxel (PTX), a chemotherapeutic drug used in cancer patients, was analyzed for the first time in the human SH-SY5Y neuroblastoma cell line for monitoring possible synergistic effects when administered with FPV. Materials and Methods: Further, in vitro cytotoxic and genotoxic evaluations of FPV and PTX were also performed using wide concentration ranges in a human fibroblast cell culture (HDFa). Nuclear abnormalities were examined under a fluorescent microscope using the Hoechst 33258 fluorescent staining technique. In addition, the synergistic effects of these two drugs on cultured SH-SY5Y cells were determined by MTT cell viability assay. In addition, the death mechanisms that can occur in SHSY-5Y were revealed by using the flow cytometry technique. Results: Cell viability analyses on the HDFa healthy cell culture showed that both FPV and PTX have inhibitory effects at higher concentrations. On the other hand, there were no significant differences in nuclear abnormality numbers when both of the compounds were applied together. Cell viability analyses showed that FPV and PTX applications have higher cytotoxicity, which indicated synergistic toxicity against the SHSY-5Y cell line. Also, PTX exhibited higher anticancer properties against the neuroblastoma cell line when applied with FPV, as shown in both cytotoxicity and flow cytometry analyses. Conclusions: In light of our findings, the anticancer properties of PTX can be enhanced when the drug application is coupled with FPV exposure. Moreover, these results put forth that the anticancer drug dosage should be evaluated carefully in cancer patients who take COVID-19 treatment with FPV.

Investigation of the Protective Effects of Chrysin Against Paclitaxel-Induced Oxidative Stress and Apoptosis in Human Neuronal SH-SY5Y Cells

In this study, the potential protective effects of chrysin, an important flavonoid, against paclitaxel-induced cell toxicity in the SH-SY5Y nerve cell line as an in vitro model, were investigated by cell viability analysis, lipid peroxidation analysis and quantitative simultaneous PCR methods. In the study, firstly, paclitaxel and chrysin were applied to the SH-SY5Y cell line at different concentrations in the range of 0-30 µM, and the results showed that 15 and 30 µM paclitaxel reduced cell viability, and 500 and 1000 µM chrysin application reduced these effects. In addition, chrysin application has been shown to significantly reduce malondialdehyde levels in paclitaxel-induced cells. The study also examined the effects of paclitaxel and chrysin application on apoptotic and antiapoptotic genes, mostly located in the intrinsic pathway, and showed that chrysin significantly reduced the levels of caspase 10, caspase 8, caspase 6, p53 and NFKB, and increased the Bcl-2 level compared to the paclitaxel-treated group. The results of this study suggest that chrysin's suppression of oxidative stress and apoptotic cell death may be an effective strategy for the treatment of paclitaxel-induced SH-SY5Y cytotoxicity.

Modulation of TRPV1 and TRPA1 Channels Function by Sea Anemones' Peptides Enhances the Viability of SH-SY5Y Cell Model of Parkinson's Disease.

Cellular dysfunction during Parkinson's disease leads to neuroinflammation in various brain regions, inducing neuronal death and contributing to the progression of the disease. Different ion channels may influence the process of neurodegeneration. The peptides Ms 9a-1 and APHC3 can modulate the function of TRPA1 and TRPV1 channels, and we evaluated their cytoprotective effects in differentiated to dopaminergic neuron-like SH-SY5Y cells. We used the stable neuroblastoma cell lines SH-SY5Y, producing wild-type alpha-synuclein and its mutant A53T, which are prone to accumulation of thioflavin-S-positive aggregates. We analyzed the viability of cells, as well as the mRNA expression levels of TRPA1, TRPV1, ASIC1a channels, alpha-synuclein, and tyrosine hydroxylase after differentiation of these cell lines using RT-PCR. Overexpression of alpha-synuclein showed a neuroprotective effect and was accompanied by a reduction of tyrosine hydroxylase expression. A mutant alpha-synuclein A53T significantly increased the expression of the pro-apoptotic protein BAX and made cells more susceptible to apoptosis. Generally, overexpression of alpha-synuclein could be a model for the early stages of PD, while expression of mutant alpha-synuclein A53T mimics a genetic variant of PD. The peptides Ms 9a-1 and APHC3 significantly reduced the susceptibility to apoptosis of all cell lines but differentially influenced the expression of the genes of interest. Therefore, these modulators of TRPA1 and TRPV1 have the potential for the development of new therapeutic agents for neurodegenerative disease treatment.

New metal-organic framework coated sodium alginate for the delivery of curcumin as a sustainable drug delivery and cancer therapy system

The utilization of biocompatible drug delivery systems with extended drug release capabilities is highly advantageous in cancer therapy, as they can mitigate adverse effects. To establish such a biocompatible system with prolonged drug release behavior, researchers developed an innovative drug carrier. In this study, a sustainable approach was employed to synthesize a new zinc-based metal-organic framework (Zn-MOF) through the reaction between synthesized Schiff base ligands and zinc ions. Comprehensive analyses, including FT-IR, XRD, SEM, BET surface area, and TGA techniques, were employed to thoroughly characterize the frameworks. Following comprehensive characterization, curcumin (CUR) was loaded onto the Zn-MOF, resulting in CUR entrapment efficiency and loading capacity of 79.23 % and 26.11 %, respectively. In vitro evaluations of CUR release from CUR@MOF exhibited controlled release patterns, releasing 78.9 % and 50.0 % of CUR at pH 5.0 and pH 7.4, respectively. To mitigate initial burst release, a coating of the biopolymer sodium alginate (SA) was applied to CUR@Zn-MOF. In vitro CUR release tests indicated that SA/CUR@Zn-MOF outperformed pristine CUR@Zn-MOF. The release of CUR conformed to the Korsmeyer-Peppas model, displaying non-Fickian diffusion. Furthermore, an in vitro cytotoxicity study clearly demonstrated the potent anti-tumor activity of the synthesized CUR@Zn-MOF attributed to its controlled release of CUR. This led to the induction of apoptotic effects and cell death across HeLa, HEK293, and SH-SY5Y cell lines. These findings strongly suggest that the developed pH-sensitive carriers hold remarkable potential as targeted vehicles for drug delivery in cancer therapy.

Zika Virus Infection Alters the Circadian Clock Expression in Human Neuronal Monolayer and Neurosphere Cultures.

Rhythmic regulations are virtually described in all physiological processes, including central nervous system development and immunologic responses. Zika virus (ZIKV), a neurotropic arbovirus, has been recently linked to a series of birth defects and neurodevelopmental disorders. Given the well-characterized role of the intrinsic cellular circadian clock within neurogenesis, cellular metabolism, migration, and differentiation among other processes, this study aimed to characterize the influence of ZIKV infection in the circadian clock expression in human neuronal cells. For this, in vitro models of human-induced neuroprogenitor cells (hiNPCs) and neuroblastoma cell line SH-SY5Y, cultured as monolayer and neurospheres, were infected by ZIKV, followed by RNA-Seq and RT-qPCR investigation, respectively. Targeted circadian clock components presented mRNA oscillations only after exogenous synchronizing stimuli (Forskolin) in SH-SY5Y monolayer culture. Interestingly, when these cells were grown as 3D-arranged neurospheres, an intrinsic oscillatory expression pattern was observed for some core clock components without any exogenous stimulation. The ZIKV infection significantly disturbed the mRNA expression pattern of core clock components in both neuroblastoma cell culture models, which was also observed in hiNPCs infected with different strains of ZIKV. The ZIKV-mediated desynchronization of the circadian clock expression in human cells might further contribute to the virus impairment of neuronal metabolism and function observed in adults and ZIKV-induced congenital syndrome. In vitro models of Zika virus (ZIKV) neuronal infection. Human neuroprogenitor cells were cultured as monolayer and neurospheres and infected by ZIKV. Monolayer-cultured cells received forskolin (FSK) as a coupling factor for the circadian clock rhythmicity, while 3D-arranged neurospheres showed an intrinsic oscillatory pattern in the circadian clock expression. The ZIKV infection affected the mRNA expression pattern of core clock components in both cell culture models. The ZIKV-mediated desynchronization of the circadian clock machinery might contribute to the impairment of neuronal metabolism and function observed in both adults (e.g., Guillain-Barré syndrome) and ZIKV-induced congenital syndrome (microcephaly). The graphical abstract has beencreated with Canva at the canva.com website.

The HSP40 family chaperone isoform DNAJB6b prevents neuronal cells from tau aggregation

BackgroundAlzheimer’s disease (AD) is the most common neurodegenerative disorder with clinical presentations of progressive cognitive and memory deterioration. The pathologic hallmarks of AD include tau neurofibrillary tangles and amyloid plaque depositions in the hippocampus and associated neocortex. The neuronal aggregated tau observed in AD cells suggests that the protein folding problem is a major cause of AD. J-domain-containing proteins (JDPs) are the largest family of cochaperones, which play a vital role in specifying and directing HSP70 chaperone functions. JDPs bind substrates and deliver them to HSP70. The association of JDP and HSP70 opens the substrate-binding domain of HSP70 to help the loading of the clients. However, in the initial HSP70 cycle, which JDP delivers tau to the HSP70 system in neuronal cells remains unclear.ResultsWe screened the requirement of a diverse panel of JDPs for preventing tau aggregation in the human neuroblastoma cell line SH-SY5Y by a filter retardation method. Interestingly, knockdown of DNAJB6, one of the JDPs, displayed tau aggregation and overexpression of DNAJB6b, one of the isoforms generated from the DNAJB6 gene by alternative splicing, reduced tau aggregation. Further, the tau bimolecular fluorescence complementation assay confirmed the DNAJB6b-dependent tau clearance. The co-immunoprecipitation and the proximity ligation assay demonstrated the protein–protein interaction between tau and the chaperone–cochaperone complex. The J-domain of DNAJB6b was critical for preventing tau aggregation. Moreover, reduced DNAJB6 expression and increased tau aggregation were detected in an age-dependent manner in immunohistochemical analysis of the hippocampus tissues of a mouse model of tau pathology.ConclusionsIn summary, downregulation of DNAJB6b increases the insoluble form of tau, while overexpression of DNAJB6b reduces tau aggregation. Moreover, DNAJB6b associates with tau. Therefore, this study reveals that DNAJB6b is a direct sensor for its client tau in the HSP70 folding system in neuronal cells, thus helping to prevent AD.

REC8 regulates neuroblastoma cell proliferation, migration, invasion, and angiogenesis via STAT3/VEGF signaling

BackgroundNeuroblastoma, one of the most prevalent childhood cancers, is often treated with surgery, radiation, and chemotherapy. However, prognosis and survival are still dismal for children with neuroblastoma at high risk. Consequently, it is vital to identify new and effective treatment targets. As a component of the meiotic cohesion complex, REC8 is involved in a wide range of malignancies. The current work assessed the impact of REC8 knockdown on SH-SY5Y and SK-N-AS neuroblastoma cells and delved into the molecular mechanism behind this effect.MethodsKnockdown of REC8 using the small interfering (si) RNA technology, and the results were verified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot. The Cell Counting Kit-8 (CCK-8) was used to examine cell proliferation, while flow cytometry was used to examine cell cycle progression and apoptosis. Analyses of angiogenesis included tube formation experiments. Transwell tests were used to examine cell migration and invasion.ResultsThe data showed that downregulation of the REC8 led to a substantial decrease in cell proliferation by stopping the cell cycle in the G1 phase. REC8 knockdown significantly reduced neuroblastoma cell proliferation, migration, invasion, angiogenesis, induced cell cycle arrest, and enhanced apoptosis. We also discovered that repressing REC8 expression in neuroblastoma cell lines SH-SY5Y and SK-N-AS reduced their ability to activate the STAT3/VEGF signaling pathway.ConclusionsNeuroblastoma therapy may benefit from targeting REC8 and its downstream targets.

Effects of cylindrospermopsin, chlorpyrifos and their combination in a SH-SY5Y cell model concerning developmental neurotoxicity

The cyanotoxin cylindrospermopsin (CYN) has been postulated to cause neurotoxicity, although the studies in this concern are very few. In addition, some studies in vitro indicate its possible effects on development. Furthermore, pesticides can be present in the same environmental samples as cyanotoxins. Therefore, chlorpyrifos (CPF) has been one of the most common pesticides used worldwide. The aim of this report was to study the effects of CYN, isolated and in combination with CPF, in a developmental neurotoxicity in vitro model. The human neuroblastoma SH-SY5Y cell line was exposed during 6 days of differentiation to both toxics to study their effects on cell viability and neurite outgrowth. To further evaluate effects of both toxicants on cholinergic signaling, their agonistic and antagonistic activities on the α7 homomeric nicotinic acetylcholine receptor (nAChR) were studied upon acute exposure. Moreover, a transcriptomic analysis by qPCR was performed after 6 days of CYN-exposure during differentiation. The results showed a concentration-dependent decrease on both cell viability and neurite outgrowth for both toxics isolated, leading to effective concentration 20 (EC20) values of 0.35 µM and 0.097 µM for CYN on cell viability and neurite outgrowth, respectively, and 100 µM and 58 µM for CPF, while the combination demonstrated no significant variations. In addition, 95 µM and 285 µM CPF demonstrated to act as an antagonist to nicotine on the nAChR, although CYN up to 2.4 µM had no effect on the efficacy of these receptors. Additionally, the EC20 for CYN (0.097 µM) on neurite outgrowth downregulated expression of the 5 genes NTNG2 (netrin G2), KCNJ11 (potassium channel), SLC18A3 (vesicular acetylcholine transporter), APOE (apolipoprotein E), and SEMA6B (semaphorin 6B), that are all important for neuronal development. Thus, this study points out the importance of studying the effects of CYN in terms of neurotoxicity and developmental neurotoxicity.

Three new schinortriterpenoids from the leaves of Schisandra chinensis (Turcz.) Baill

Three undescribed schinortriterpenoids, schinensilactones D-F (1–3), together with five known ones, namely, wuweizidilactone A (4), wuweizidilactone C (5), wuweizidilactone F (6), wuweizidilactone J (7) and wuweizidilactone N (8), were isolated from the leaves of Schisandra chinensis (Turcz.) Baill. The structures of new compounds were established by analysis of their spectroscopic data including MS, IR, 1D- and 2D-NMR spectra. The absolute configuration of 1 was confirmed by single-crystal X-ray diffraction and calculated electronic circular dichroism (ECD) spectra. All compounds were evaluated for their neuroprotective effects against H2O2-induced injury in human neuroblastoma SH-SY5Y cell lines. Cell viability was remarkably reduced to 52.33% in H2O2-treated cells. Compounds 5–7 exhibited moderate neuroprotective activities at 50 μM, with cell viability of 64.84%, 67.34% and 63.73%, respectively.

Histone H3 Acetylation Is Involved in Retinoid Acid-Induced Neural Differentiation through Increasing Mitochondrial Function.

Histone acetylation and mitochondrial function contribute importantly to neural differentiation, which is critically associated with neurodevelopmental disorders such as Down Syndrome (DS). However, whether and how histone acetylation regulates mitochondrial function and further affects neural differentiation has not been well described. In this study, when treated with retinoid acid (RA), the human neuroblastoma SH-SY5Y cell line was used as a neural differentiation model. We found that the acetylation of histone H3, especially H3 lysine 14 acetylation (H3K14ac), and mitochondrial function, including biogenesis and electron transport chain, were enhanced during neural differentiation. Specific inhibition of histone acetyltransferases (HATs) induced neural differentiation deficits, accompanied by downregulation of mitochondrial function. Furthermore, RA receptors (RARs) interacting with HATs were involved in the increased H3K14ac and the enhanced mitochondrial function during the neural differentiation process. Finally, receptor-interacting protein 140 (RIP140), a co-repressor of RARs, was also involved in regulating histone acetylation. RIP140 overexpression inhibited histone acetylation and mediated negative feedback on target genes which are involved in RA signaling. These findings evidenced that when interacting with RARs which had been negatively regulated by RIP140, RA promoted neural differentiation by promoting H3K14ac and enhanced mitochondrial function. This provides a molecular foundation for further investigations into abnormal neural development.

Novel tetrahydronaphthalen-1-yl-phenethyl ureas: synthesis and dual antibacterial-anticancer activities

Cancer and antibiotic-resistant bacterial infections are significant global health challenges. The resistance developed in cancer treatments intensifies therapeutic difficulties. In addressing these challenges, this study synthesised a series of N,N′-dialkyl urea derivatives containing methoxy substituents on phenethylamines. Using isocyanate for the efficient synthesis yielded target products 14–18 in 73–76% returns. Subsequently, their antibacterial and anticancer potentials were assessed. Cytotoxicity tests on cancer cell lines, bacterial strains, and a healthy fibroblast line revealed promising outcomes. All derivatives demonstrated robust antibacterial activity, with MIC values ranging from 0.97 to 15.82 µM. Notably, compounds 14 and 16 were particularly effective against the HeLa cell line, while compounds 14, 15, and 17 showed significant activity against the SH-SY5Y cell line. Importantly, these compounds had reduced toxicity to healthy fibroblast cells than to cancer cells, suggesting their potential as dual-functioning agents targeting both cancer and bacterial infections.

Chimeric single-chain variable fragment-human immunoglobulin G crystallizable fragment antibody against GD2 for neuroblastoma targeted immunotherapy

Aim: The present study aims to generate chimeric mouse single-chain variable fragment (scFv) and immunoglobulin G1 (IgG1) crystallizable fragment (Fc) antibody against disialoganglioside (GD2) for the treatment of neuroblastoma (NB). The generated scFv-IgG Fc antibody, lacking first constant domain of heavy chain (CH1), is of a smaller size than the natural antibody and has anti-tumor activity. Methods: Vector for scFv-IgG Fc antibody was constructed and scFv-IgG Fc antibody was expressed in human embryonic kidney 293T (HEK293T) cell line. Purification of scFv-IgG Fc antibody from the culture supernatant of transfected HEK293T cells was performed by Protein G affinity chromatography. The structure and binding activity of scFv-IgG Fc antibody were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting (WB), and immunofluorescence techniques. Anti-tumor activities by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) were determined. Results: Using plasmid fusion-human IgG1-Fc2 tag vector (pFUSE-hIgG1-Fc2), a plasmid vector encoding chimeric mouse scFv and hIgG1 Fc antibody against GD2 was successfully constructed. This vector was transfected into human HEK293T cells to produce scFv-IgG Fc antibody. The transfected HEK293T cells could produce chimeric scFv-IgG Fc antibody against GD2, which lacks the IgG heavy chain CH1 domain but carries CH2 and CH3 domains. The chimeric antibodies could be purified from the culture supernatant of the transfected HEK293T culture in the presence of zeocin drug. The produced GD2 scFv-IgG Fc antibodies, which are smaller in size than the intact antibody, could trigger the killing of GD2 expressed NB cell line SH-SY5Y by ADCC and ADCP mechanisms. Conclusions: The results indicate that chimeric scFv-hIgG Fc antibody, lacking heavy chain CH1 domain, could mediate antibody induced anti-tumor activities. The small size of this type of chimeric antibody may be employed as anti-GD2 antibody for NB therapy.

Design, Synthesis, and Pharmacological Evaluation of Novel Tacrine Derivatives as Multi-target ANTI-Alzheimer's Agents In Rat Models.

Alzheimer's disease is a progressive neurodegenerative disorder for which no curative drugs are available and treatment available is just palliative. Current research focused on design of Tacrine-Flavone hybrids as multitargeted cholinesterase and monoamine oxidase B inhibitors. A total of 10 Tacrine- Flavone hybrids were designed, synthesized and characterized. The in vitro neurotoxicity and hepatotoxicity of the synthesized compounds determined using SHSY5Y cell line and HEPG2 cell line. One most active compound (AF1) with least toxicity in in vitro studies was chosen for in vivo studies. Acute and subacute toxicity of the novel compound AF1 conducted on Wistar rats according to OECD guideline 423 and 407. The LD50 value of the novel compound calculated according to Finney's method using Probit analysis. Anti-Alzheimer's activity studies conducted on male Wistar rats. Behavioral studies conducted and AChE and MAO-B activity determined in rat brain. All the compounds exhibited good inhibitory effect on MAO B and AChE. The neurotoxicity studies of the active compound AF1 did not show toxicity up to 100μg. The hepatotoxicity study of the most active compound AF1, showed the compound to be safe up to 200μg. The LD 50 value of the novel compound after a single oral administration was found to be 64 mg/kg bodyweight in rats. Subacute toxicity studies did not show any remarkable toxicity in the vital organs up to 40 mg/kg. Activity studies showed comparable results with standard at 20 mg/kg. The results showed that the novel Tacrine-Flavone hybrids are multitarget-directed ligands, which are safe and active compared to tacrine and can be a promising lead molecule for further study.

Assessment of poly(hexamethylenebicyanoguanide-hexamethylenediamine) hydrochloride-induced developmental neurotoxicity via oxidative stress mechanism: Integrative approaches with neuronal cells and zebrafish

Poly(hexamethylenebicyanoguanide-hexamethylenediamine) hydrochloride (PHMB) is a biocide with a broad spectrum of antibacterial activity. Its use as a disinfectant and preservative in consumer products results in human exposure to PHMB. Toxicity studies on PHMB mainly focus on systemic toxicity or skin irritation; however, its effects on developmental neurotoxicity (DNT) and the underlying mechanisms are poorly understood. In this study, the DNT effects of PHMB were evaluated using IMR-32 and SH-SY5Y cell lines and zebrafish. In both cell lines, PHMB concentrations ≥ 10 µM reduced neurite outgrowth, and cytotoxicity was observed at concentrations up to 40 µM. PHMB regulated expression of neurodevelopmental genes and induced reactive oxygen species (ROS) production and mitochondrial dysfunction. Treatment with N-acetylcysteine reversed the toxic effects of PHMB. Toxicity tests on zebrafish embryos showed that PHMB reduced viability and heart rate and caused irregular hatching. PHMB concentrations of 1–4 µM reduced the width of the brain and spinal cord of transgenic zebrafish and attenuated myelination processes. Furthermore, PHMB modulated expression of neurodevelopmental genes in zebrafish and induced ROS accumulation. These results suggested that PHMB exerted DNT effects in vitro and in vivo through a ROS-dependent mechanism, highlighting the risk of PHMB exposure.

Evaluation of the SH-SY5Y cell line as an in vitro model for potency testing of a neuropeptide-expressing AAV vector

Viral vectors have become important tools for basic research and clinical gene therapy over the past years. However, in vitro testing of vector-derived transgene function can be challenging when specific post-translational modifications are needed for biological activity. Similarly, neuropeptide precursors need to be processed to yield mature neuropeptides. SH-SY5Y is a human neuroblastoma cell line commonly used due to its ability to differentiate into specific neuronal subtypes. In this study, we evaluate the suitability of SH-SY5Y cells in a potency assay for neuropeptide-expressing adeno-associated virus (AAV) vectors. We looked at the impact of neuronal differentiation and compared single-stranded (ss) AAV and self-complementary (sc) AAV transduction at increasing MOIs, RNA transcription kinetics, as well as protein expression and mature neuropeptide production. SH-SY5Y cells proved highly transducible with AAV1 already at low MOIs in the undifferentiated state and even better after neuronal differentiation. Readouts were GFP or neuropeptide mRNA expression. Production of mature neuropeptides was poor in undifferentiated cells. By contrast, differentiated cells produced and sequestered mature neuropeptides into the medium in a MOI-dependent manner.

Bioactive And Cytotoxic Potentials of the Extract Fractions of Strophanthus Hispidus on Neuroblastoma (SH-SY5Y) Cell Line Model of Alzheimer’s Disease

Alzheimer disease (AD) is a neurodegenerative disorder that affect memory and cognitive function. Currently, the available agents used in managing this condition are associated with severe side effects and have limited efficacy in reducing or modulating events associated with AD. Hence, there is a need for an alternative therapy with fewer side effects and better efficacy. Strophantus hispidus (SH) is a plant product widely used in Africa for its therapeutic potential. This study was conducted to assess the bioactive constituent of the SH extract fractions, as well as the cytotoxic potential of these fractions. The root of SH was pulverized and extracted with 80% methanol. The crude extract was then fractionated with Chloroform, Hexane, and Ethylacetate. The extract and fractions were then subjected to Gas Chromatography-Mass Spectrometry (GC-MS). SH-SY5Y cell line model of AD was treated with graded concentrations (9.77 – 5000 µg/mL) of the SH extract and fractions for 24 hours. Cell viability assay was performed using WST-8 proliferation assay. The EC50 was determined to be 48.4 μg/mL, 15.16 μg/mL, and 151 μg/mL for cells treated with Chloroform, Ethylacetate, and Hexane fractions of Strophantus hispidus respectively. The selectivity index (SI) greater than 3 of the hexane and ethylacetate fractions were determined to be safe, less toxic, and effective in improving cellular growth.