Identification of potent c-Src inhibitors strongly affecting the proliferation of human neuroblastoma cells

Neuroblastoma (NB) represents the most common extracranial paediatric solid tumor for which no specific FDA-approved treatment is currently available. The tyrosine kinase c-Src has been reported to play an important role in the differentiation, cell-adhesion and survival of NB cells. Starting from dual Src/Abl inhibitors previously found active in NB cell lines (1-3), small modification of the original structures almost abolished the Abl activity with a contemporary improvement of affinity and specificity for c-Src. Among the synthesized compounds, the most potent c-Src inhibitor (10a) showed a very interesting antiproliferative activity in SH-SY5Y cells with an IC(50) of 80 nM and a favourable ADME profile. A 3D SAR analysis was also attempted and may guide the design of more potent c-Src inhibitors as potential agents for NB treatment.     

Inhibition of lysine-specific demethylase 1 by the acyclic diterpenoid geranylgeranoic acid and its derivatives

Lysine-specific demethylase 1 (LSD1) is upregulated in many cancers, especially neuroblastoma. We set out to explore whether geranylgeranoic acid (GGA) inhibits LSD1 activity by using recombinant human LSD1. GGA inhibited LSD1 activity with IC₅₀ similar to that of the clinically used drug tranylcypromine. In human neuroblastoma SH-SY5Y cells, GGA induced NTRK2 gene expression alongside upregulation of histone H3 with dimethylated lysine-4 in the regulatory regions of the NTRK2 gene. Dihydrogenation of GGA reinforced the LSD1-inhibitory effect in a position-dependent manner. The inhibitory effects of dihydro-derivatives of GGA on recombinant LSD1 strongly correlated with the induction of NTRK2 gene expression in SH-SY5Y cells. These data demonstrate for the first time the efficient LSD1-inhibitor activity of GGA and its derivatives, providing a novel prospect of preventing cancer onset by using GGA to regulate epigenetic modification.     

IGF2 derived from SH-SY5Y neuroblastoma cells induces the osteoclastogenesis of human monocytic precursors

The insulin-like growth factors 2 (IGF2) is a peptide hormone that binds to the insulin-like growth factor 1 receptor (IGF1R) and is abundantly stored in bone. IGF1R is deeply involved in the pathogenesis of many cancers that growth within bone and is also involved in osteoclast biology. Among different cell lines representative of osteolytic tumors, we found a very high expression of IGF2 in SH-SY5Y cells derived from neuroblastoma (NB). We previously showed that NB cells induce an osteolytic process through the Osteoprotegerin/RANKL/RANK and the canonical Wnt pathway system. Here, we hypothesized that NB promotes osteoclastogenesis also via IGF2. First, we demonstrated the presence of IGF1R on the osteoclast basolateral membrane, and we observed a cyclic IGF1R activation along with the differentiation process, also when induced by SH-SY5Y. Moreover, we found that IGF2 mRNA expression in SH-SY5Y cells was further increased when co-cultured with mesenchymal stromal cells, suggesting that IGF2 is important for NB interaction with the bone microenvironment. Finally, the treatment of SH-SY5Y cells with an anti-IGF2 siRNA or the addition of anti-IGF1R molecules impaired NB-induced osteoclastogenesis, even though the chemoattraction of monocytes by NB cells was unaffected. Our findings suggest that in IGF2-producing osteolytic tumors IGF1R is a good candidate for targeted therapies in combination with conventional drugs.     

Synthesis and biological evaluation of 3-carbamate smilagenin derivatives as potential neuroprotective agents

Studies indicated that smilagenin, isolated from Anemarrhena asphodeloides Bunge, could improve cognitive impairment and exhibit neuroprotective activity. On the basis of the structure of smilagenin, a series of derivatives were synthesized and evaluated for their neuroprotective effects of H₂O₂-induced, oxygen glucose deprivation-induced neurotoxicity in SH-SY5Y cells and LPS-induced NO production in RAW264.7 cells. Structure activity relationship of derivatives revealed that benzyl-substituted piperazine formate derivatives showed the potent neuroprotective activity such as A12. These findings may provide new insights for the development of neuroprotective agents against Alzheimer’s disease.     

Pyrazolo[3,4-d]pyrimidines-loaded human serum albumin (HSA) nanoparticles: Preparation,characterization and cytotoxicity evaluation against neuroblastoma cell line

Pyrazolo[3,4-d]pyrimidine derivatives 1–5, active as c-Src inhibitors, have been selected to be formulated as drug-loaded human serum albumin (HSA) nanoparticles, with the aim of improving their solubility and pharmacokinetic properties. The present study includes the optimization of a desolvation method-based procedure for preparing HSA nanoparticles. First, characterization by HPLC-MS and Dynamic Light Scattering (DLS) showed a good entrapment efficacy, a controllable particle size (between 100 and 200 nm) and an optimal stability over time, confirmed by an in vitro drug release assay. Then, 1–4 and the corresponding NPs were tested for their antiproliferative activity against neuroblastoma SH-SY5Y cell line. Notably, 3-NPs and 4-NPs were identified as the most promising formulation showing a profitable balance of stability, small size and a similar activity compared to the free drugs in cell-based assays. In addition, albumin formulations increase the solubility of pyrazolo[3,4-d]pyrimidine avoiding the use of DMSO as solubilizing agent.     

Protective Effects of Acetyl-<Emphasis Type="SmallCaps">l</Emphasis>-Carnitine on Cisplatin Cytotoxicity and Oxidative Stress in Neuroblastoma

The most widely used platinum-derived drug is cisplatin in neuroblastoma (NB) chemotherapy, which is severely neurotoxic. Acetyl-l-Carnitine (ALC) is a natural occurring compound with a neuroprotective activity in several experimental paradigms. The aim of this study was to determine the effects of ALC on cisplatin induced cytotoxicity and oxidative stress in NB cells. SH-SY5Y (N-Myc negative) and KELLY (N-Myc positive) human NB cell lines were used. Cisplatin induced apoptosis was assessed by using a Cell Death Detection ELISA^PLUS kit. Lipid peroxidation levels were determined by HPLC analysis. Glutathione levels were determined spectrophotometrically. ALC was used prophylactic or after cisplatin application. The level of cisplatin doses were determined in both type of NB cells at which 50% cell death occurred along with synchronized apoptosis induced. Prophylactic 10 and 50 μmol of ALC concentrations were decreased cisplatin induced lipid peroxidation compared to controls that normally exhibited apoptosis especially in SH-SY5Y cells. Cisplatin caused oxidative stress through decreasing glutathione levels in both cell types. ALC were effectively inhibited the increase in cisplatin induced oxidized glutathione and lipid peroxidation formation in NB cells. We suggested that prophylactic ALC would be a useful agent for cisplatin induced toxicity in NB cells.     

Neuroprotective effects of aucubin on hydrogen peroxide-induced toxicity in human neuroblastoma SH-SY5Y cells via the Nrf2/HO-1 pathway

BackgroundWhen redox balance is lost in the brain, oxidative stress can cause serious damage that leads to neuronal loss, in congruence with neurodegenerative diseases. Aucubin (AU) is an iridoid glycoside and that is one of the active constituents of Eucommia ulmoides, has many pharmacological effects such as anti-inflammation, anti-liver fibrosis, and anti-atherosclerosis.PurposeThe present study aimed to evaluate the inhibitory effects of AU on cell oxidative stress against hydrogen peroxide (H₂O₂)-induced injury in SH-SY5Y cells in vitro.MethodsSH-SY5Y cells were simultaneously treated with AU and H₂O₂ for 24 h. Cell viability was measured by CCK-8. Additionally, mitochondrial membrane depolarization, reactive oxygen species (ROS) generation, and cell apoptosis were measured by flow cytometry.ResultsThe results showed that AU can significantly increase the H₂O₂-induced cell viability and the mitochondrial membrane potential, decrease the ROS generation, malondialdehyde (MDA), and increase glutathione (GSH) contents and the superoxide dismutase (SOD) activity. We also found that H₂O₂ stimulated the production of nitric oxide (NO), which could be reduced by treatment with AU through inhibiting the inducible nitric oxide synthase (iNOS) protein expression. In H₂O₂-induced SH-SY5Y cells, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) content and cell apoptosis were significantly reduced by AU treatment through nuclear factor E2-related factor 2/hemo oxygenase-1 (Nrf2/HO-1) activation, inhibiting the expression of p-NF-κB/NF-κB and down-regulating MAPK and Bcl-2/Bax pathways.ConclusionThese results indicate that AU can reduce inflammation and oxidative stress through the NF-κB, Nrf2/HO-1, and MAPK pathways.     

Angelica polymorpha Maxim Induces Apoptosis of Human SH-SY5Y Neuroblastoma Cells by Regulating an Intrinsic Caspase Pathway

Angelica polymorpha Maxim root extract (APRE) is a popular herbal medicine used for treating stomachache, abdominal pain, stomach ulcers, and rheumatism; however the effect of APRE on cancer cells has not yet been explored. Here, we examined APRE cytotoxicity seen on target neuroblastoma cells (NB) using cell viability assays, DAPI visualization of fragmented DNA, and Western blotting analysis of candidate signaling pathways involved in proliferation and apoptosis. We demonstrated that APRE reduced cell viability in NB to a greater extent than in fibroblast cells. In addition, we found that APRE could inhibit the three classes of MAPK proteins and could also down-regulate the PI3K/AKT/GSK-3β activity all being relevant for proliferation and survival. APRE could also up-regulate Bax expression and down-regulate Bcl-2 and Mcl-1. With APRE treatment, depolarization of mitochondria membrane potential and activation of caspase-3 was demonstrated in the SH-SY5Y cells. We could not found increased activity of death receptor and caspase-8 as markers of the extrinsic apoptosis pathway for the APRE treated cells. In presence of a caspase-3 siRNA and a pan-caspase inhibitor, APRE could not reduce the viability of NB cells to a significant degree. So we predicted that with APRE, the intrinsic pathway was solely responsible for inducing apoptosis as we also showed that the non-caspase autophagy pathway or ER stress-ROS mediated pathways were not involved. These findings demonstrate that an intrinsic mitochondria-mediated apoptosis pathway mediates the apoptotic effects of APRE on SH-SY5Y cells, and that APRE shows promise as a novel agent for neuroblastoma therapy.     

The TrkAIII Oncoprotein Inhibits Mitochondrial Free Radical ROS-Induced Death of SH-SY5Y Neuroblastoma Cells by Augmenting SOD2 Expression and Activity at the Mitochondria,within the Context of a Tumour Stem Cell-like Phenotype

The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H₂O₂, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"GW441756","term_id":"315858226","term_text":"GW441756"}}GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB.     

Identification,characterization and in vitro neuroprotection of N6-(4-hydroxybenzyl) adenine riboside and its metabolites

N⁶-(4-hydroxybenzyl) adenine riboside (NHBA), isolated from Gastrodia elata Blume, has been demonstrated to show great pharmacological effects. The present study aimed to synthesize and identify the metabolites of NHBA, and to determine their neuroprotective potentials in vitro. After incubation with rat liver microsomes in the presence of NADPH, two metabolites were detected, which were further semisynthesized and identified as N⁶-(4-hydroxylbenzyl) purine (NHBP) and N⁶-(3,4-dihydroxylbenzyl) adenine riboside (ONHBA) by UPLC-QTOF-MS, ¹H NMR and ¹³C NMR. Furthermore, the neuroprotective activities of NHBA and two metabolites were evaluated in SH-SY5Y cells. Our results demonstrated that NHBA substantially protected against H₂O₂-induced neuronal death in SH-SY5Y cells. Moreover, both ONHBA and NHBP could significantly prevent Aβ oligomers- and H₂O₂-induced neuronal death in SH-SY5Y cells. These results suggested that NHBA and its metabolites, ONHBA and NHBP, might be suitable for the development of new drugs in the treatment of neurodegenerative diseases, including Alzheimer’s disease in particular.     

Morphological Differentiation Towards Neuronal Phenotype of SH-SY5Y Neuroblastoma Cells by Estradiol,Retinoic Acid and Cholesterol

Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was to examine in the RA and CHOL treated SH-SY5Y cells the additive impacts of estradiol (E₂) and brain-derived neurotrophic factor (BDNF) on cell morphology, cell population growth, synaptic vesicle recycling and presence of neurofilaments. The above features indicate a higher level of neuronal differentiation. Our data show that treatment for 10 days in vitro (DIV) with RA alone or when combined with E₂ (RE) or CHOL (RC), but not when combined with BDNF (RB), significantly (p < 0.01) inhibited the cell population growth. Synaptic vesicle recycling, induced by high-K⁺ depolarization, was significantly increased in all treatments where RA was included (RE, RC, RB, RCB), and when all agents were added together (RCBE). Specifically, our results show for the first time that E₂ treatment can alone increase synaptic vesicle recycling in SH-SY5Y cells. This work contributes to the understanding of the ways to improve suppression of neuroblastoma cells’ population growth by inducing maturation and differentiation.     

Insulin-like growth factor I rescues SH-SY5Y human neuroblastoma cells from hyperosmotic induced programmed cell death

Insulin-like growth factor I (IGF-I) and the type I IGF receptor are widely distributed in developing and adult mammalian nervous systems. In vitro, IGF-I is a mitogen for primary neurons and also for cells from the SH-SY5Y human neuroblastoma cell line, a well-characterized model system of neuronal growth. In the current study, we examined the effects of osmotic stress on SH-SY5Y cell viability and the mechanism by which IGF-I serves as a neuronal osmoprotectant. Within 24 hr, exposure of SH-SY5Y cells to hyperosmotic serum-free media decreased (1) the number of viable cells, (2) the rate of ³H-thymidine incorporation, and (3) cell cycle progression. The inclusion of 10 nM IGF-I with hyperosmotic media prevented the loss of cell viability. The osmoprotective effects of IGF-I were inhibited by α-IR_J, a blocking antibody of the type I IGF receptor. The observed loss of SH-SY5Y cell viability following hyperosmotic shock was due to an induction of programmed cell death as determined by flow cytometry and gel electrophoresis. Our results suggest that IGF-I can protect SH-SY5Y cells from hyperosmotic induced programmed cell death. © 1996 Wiley-Liss, Inc.     

Protective Effect of Paeoniflorin on Aβ25–35-Induced SH-SY5Y Cell Injury by Preventing Mitochondrial Dysfunction

Alzheimer’s disease (AD) is a major neurodegenerative brain disorder affecting about 14 million people worldwide. Aβ-induced cell injury is a crucial cause of neuronal loss in AD, thus the suppression of which might be useful for the treatment of this disease. In this study, we aimed to evaluate the effect of paeoniflorin (PF), a monoterpene glycoside isolated from aqueous extract of Radix Paeoniae Alba, on Aβ_25–35-induced cytotoxicity in SH-SY5Y cells. The results showed PF could attenuate or restore the viability loss, apoptotic increase, and ROS production induced by Aβ_25–35 in SH-SY5Y cells. In addition, PF strikingly inhibited Aβ_25–35-induced mitochondrial dysfunction, which includes decreased mitochondrial membrane potential, increased Bax/Bcl-2 ratio, cytochrome c release and activity of caspase-3 and caspase-9. Therefore, our study provided the first experimental evidence that PF could modulate ROS production and apoptotic mitochondrial pathway in model of neuron injury in vitro and which might provide new insights into its application toward Alzheimer’s disease therapy.     

Peptide gomesin triggers cell death through L-type channel calcium influx,MAPK/ERK,PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurria gomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species.     

Assessment of novel azaanthraquinone derivatives as potent multi-target inhibitors of inflammation and amyloid-β aggregation in Alzheimer’s disease

A series of 6-substituted azaanthraquinone derivatives have been designed, synthesized, and their anti-inflammatory activities, antiaggregation effects on β-amyloid proteins, anticholinesterase and neuroprotective activity were tested. The new derivatives strongly suppressed NO and iNOS production and modulate the production of cytokines by decreasing TNF-a, IL-1β and IL-6 formation in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Meanwhile, the derivatives exhibited a significant in vitro inhibitory activity toward the self-induced Aβ aggregation. While, treatment of SH-SY5Y cells overexpressing the Swedish mutant form of human b-amyloid precursor protein (APPsw) with derivatives was associated with significant reduction of Aβ42 secretion levels. Moreover, the derivatives exhibited moderate inhibitory potency toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Further investigations indicated that compound 7b could attenuate H₂O₂-induced neurotoxicity toward SH-SY5Y neuroblastoma cells and half of the synthetic compounds were predicted to be able to cross the blood–brain barrier (BBB) to reach their targets in the central nervous system (CNS) according to a parallel artificial membrane permeation assay for BBB. Taken together, azaanthraquinone derivatives targeting multiple pathogenetic factors deserves further investigation for prevention and treatment of AD.