Apoptosis In Neuroblastoma SH-SY5Y Cells Research Articles

Human Bone Marrow Mesenchymal Stem Cells-Derived Exosomal miRNA-21-5p Inhibits Lidocaine-Induced Apoptosis in SH-SY5Y Neuroblastoma Cells.

Local anesthetic lidocaine is one of the most common pain therapies, but high concentration of lidocaine induced neurotoxicity and its mechanism is unclear. Exosomal microRNAs (miRNAs) is implicated in neuronal diseases, but its role in lidocaine induced neurotoxicity remains to be elucidated. All the experiments were performed at Huzhou Key Laboratory of Molecular Medicine, Huzhou City, Jiangsu Province, China in 2022. Lidocaine was used to induce apoptosis of SH-SY5Y cells. Exosomes isolated from bone marrow mesenchymal stem cells (BMSC-exos) were used to co-treat SH-SY5Y cells with lidocaine. Cell apoptosis was measured using a flow cytometer. PKH-67 Dye was used for exosome uptake assay. miR-21-5p mimics/inhibitors, or negative controls were transfected with Lipo2000 to study its effect on lid-induced injury. Interactions between miR-21-5p and PDCD4 was analyzed by luciferase reporter assay. Administration of BMSC-exo protected SH-SY5Y cells against lidocaine induced apoptosis. Suppressing miR-21-5p dramatically enhanced PDCD4, but miR-21-5p overexpression sharply down-regulated PDCD4. Mechanism study showed that miR-21-5p bound to 3'-UTR of PDCD4 to inhibit it. Suppressing miR-21-5p reversed the effect of BMSC-exo on Lid-induced injury. Results also indicate that miR-21-5p regulated lidocaine-induced injury through targeting PDCD4. BMSC-exos protected SH-SY5Y cells against lidocaine induced apoptosis through miR-21-5p by targeting PDCD4, which may develop new strategy in the management of lidocaine-induced neurotoxicity.

Corin is regulated by circ-0012397/miR-200a-3p and inhibits the oxygen-glucose deprivation-induced apoptosis of SHSY5Y neuroblastoma cells

As a type II transmembrane serine protease, corin plays a role in several important physiological and pathological processes. We conducted a bioinformatics analysis to explore the roles of both corin and circ-0012397/miR-200a-3p in ischemic stroke. We established an in vitro model using oxygen-glucose deprivation (OGD)-induced SHSY5Y cells. The proliferation and apoptosis of SHSY5Y cells was determined using Cell Counting Kit-8 (CCK-8) and flow cytometry/Hoechst 33258 staining, respectively. The RNA and protein level was tested using Real Time Quantitative Polymerase Chain Reaction (RT-qPCR) and western blot, respectively. The regulatory relationship of corin and circ-0012397/miR-200a-3p were detected by dual-luciferase reporter assays. We found that OGD downregulated the expression of corin in a time-dependent manner; this change was inversely proportional to the rate of apoptosis of the SHSY5Y cells. Further, high expression levels of corin enhanced the proliferation of SHSY5Y cells and inhibited the apoptosis of SHSY5Y cells by downregulating the expression of cleaved caspase-3, B-cell lymphoma 2 (BCL-2)-associated death promoter, extracellular-regulated protein kinase (ERK), and protein 38 (p38), and upregulated the expression of Bcl-2. Further, the dual-luciferase reporter assays and RT-qPCR showed that corin expression was regulated by circ-0012397/miR-200a-3p. Corin expression was affected by changes in circ-0012397 and miR-200a-3p expression, which were overexpressed or inhibited. Further, corin exerted different regulatory effects on apoptosis signaling-related proteins, including AD Bcl-2, cleaved caspase-3, ERK, and p38, under different expression levels of circ-0012397 and miR-200a-3p. Corin promotes the cell proliferation and inhibits OGD-induced apoptosis of SHSY5Y cells, and that its expression is regulated by circ-0012397/miR-200a-3p. Thus, corin may be a potential target for ischemic stroke patients.

Cryptotanshinone ameliorates MPP+-induced oxidative stress and apoptosis of SH-SY5Y neuroblastoma cells: the role of STAT3 in Parkinson's disease.

Cryptotanshinone (CTN) has shown its neuroprotective and anti-inflammatory qualities in non-genetic mouse model of Alzheimer's disease. According to bioinformatics analysis, CTN and Signal Transducer and Activator of Transcription 3 (STAT3) may interact to form a drug-target network. This study was conducted to identify the role of CTN-STAT3 interaction in Parkinson's disease (PD). PD model was established with MMP+-stimulated SH-SY5Y cells. After pre-treatment with CTN or co-treatment with CTN and STAT3 agonist, MTT assay was performed to observe cell viability; ELISA kit was used to measure the expression level of pro-inflammatory cytokines; DCFH-DA and corresponding assay kits were employed to determine the production of ROS, SOD, CAT and GSH-px; TUNEL assay and western blot were performed to detect cell apoptosis. STAT3 activity was also detected by western blot. Treatment with CTN alone had no impact on SH-SY5Y cell viability, but CTN pre-treatment effectively improved MPP+-induced loss of viability in SH-SY5Y cells. Moreover, pre-treatment with CTN inhibited MPP+-induced oxidative stress, apoptosis and STAT3 activity in SH-SY5Y cells, whereas this inhibitory effect was diminished after additional treatment with STAT3 agonist. CTN ameliorates MPP+-induced oxidative stress and apoptosis of SH-SY5Y neuroblastoma cells by inhibiting the expression of STAT3. Therefore, CTN could be a promising therapeutic agent, and STAT3 could be a potential target for PD treatment.

Reactive Oxygen Species (ROS) Are Not a Key Determinant for Zika Virus-Induced Apoptosis in SH-SY5Y Neuroblastoma Cells.

Introduction: ZIKV is a highly neurotropic virus that can cause the death of infected neuroprogenitor cells through mitochondrial damage and intrinsic apoptotic signaling. In this context, the role of reactive oxygen species (ROS) in neuronal cell death caused by ZIKV still remains elusive. Objective: We aimed at evaluating the role of these cellular components in the death of human undifferentiated neuroblastoma cell line infected with ZIKV. Results: ZIKV infection resulted in the extensive death of SH-SY5Y cells with the upregulation of several genes involved in survival and apoptotic responses as well as the colocalization of mitochondrial staining with ZIKV Envelope (E) protein. Notably, levels of intracellular reactive oxygen species (ROS) were not altered during ZIKV infection in undifferentiated SH-SY5Y cells, and consistent with these results, the treatment of infected cells with the widely studied ROS scavenger N-acetylcysteine (NAC) did not prevent cell death in these cells. Conclusion: Altogether, our results suggest that excessive ROS production is not the main trigger of SH-SY5Y cells death in ZIKV infection.

MicroRNA-216a inhibits neuronal apoptosis in a cellular Parkinson's disease model by targeting Bax.

The study found that microRNAs play an important role in Parkinson's disease (PD). However, the function of MicroRNA-216a (miR-216a) in PD is unclear. Therefore, this experiment aimed to investigate the pathogenesis of miR-216a in PD. Using the toxicity of MPP+ to polyhexamine neurons, apoptosis of SH-SY5Y neuroblastoma cells was induced at different time by MPP+ to construct a stable acute PD cell model. The effects of DNA breakage, mitochondrial membrane potential (A ^ m), caspase-3 activity and nucleosome enrichment on cell apoptosis were detected by flow cytometry, TUNEL. MPP+ increased the toxic effects of dopaminergic neurons in a PD model. The introduction of miR-216a inhibited MPP + -induced neuronal apoptosis. The main manifestations were the decreased levels of positive rate of Tunel cells, caspase 3 activity and nucleosome enrichment factor. Bax was a direct target of miR-216a. In addition, Bax overexpression reversed the effects of miR-216a on neural cells. Bax downstream factors were also involved in miR-216a regulation of MPP + -triggered neuronal apoptosis. miR-216a regulated the progression of PD by regulating Bax, and miR-216a may be a potential target for PD.

FTY720 Inhibits MPP+-Induced Microglial Activation by Affecting NLRP3 Inflammasome Activation.

Parkinson's disease (PD) is characterized by the degeneration of dopaminergic neurons and excessive microglial activation in the substantia nigra pars compacta (SNpc). In the present study, we aimed to demonstrate the therapeutic effectiveness of the potent sphingosine-1-phosphate receptor antagonist fingolimod (FTY720) in an animal model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and to identify the potential mechanisms underlying these therapeutic effects. C57BL/6J mice were orally administered FTY720 before subcutaneous injection of MPTP. Open-field and rotarod tests were performed to determine the therapeutic effect of FTY720. The damage to dopaminergic neurons and the production of monoamine neurotransmitters were assessed using immunohistochemistry, high-performance liquid chromatography, and flow cytometry. Immunofluorescence (CD68- positive) and enzyme-linked immunosorbent assay were used to analyze the activation of microglia, and the levels of activated signaling molecules were measured using Western blotting. Our findings indicated that FTY720 significantly attenuated MPTP-induced behavioral deficits, reduced the loss of dopaminergic neurons, and increased dopamine release. FTY720 directly inhibited MPTP-induced microglial activation in the SNpc, suppressed the production of interleukin (IL)-6, IL-1β, and tumor necrosis factor-α in BV-2 microglial cells treated with 1-methyl-4-phenylpyridinium (MPP+), and subsequently decreased apoptosis in SH-SY5Y neuroblastoma cells. Moreover, in MPP+-treated BV-2 cells and primary microglia, FTY720 treatment significantly attenuated the increases in the phosphorylation of PI3K/AKT/GSK-3β, reduced ROS generation and p65 activation, and also inhibited the activation of NLRP3 inflammasome and caspase-1. In conclusion, FTY720 may reduce PD progression by inhibiting NLRP3 inflammasome activation via its effects on ROS generation and p65 activation in microglia. These findings provide novel insights into the mechanisms underlying the therapeutic effects of FTY720, suggesting its potential as a novel therapeutic strategy against PD. Graphical Abstract FTY720 may reduce ROS production by inhibiting the PI3K/AKT/GSK-3β signaling pathway, while at the same time reducing p65 phosphorylation, thus decreasing NLRP3 inflammasome activation through these two pathways, ultimately reducing microglia activation-induced neuronal damage.

Dopaminergic neurotoxins induce cell death by attenuating NF-κB-mediated regulation of TRPC1 expression and autophagy.

Alterations in Ca2+ homeostasis affect neuronal survival. However, the identity of Ca2+ channels and the mechanisms underlying neurotoxin-induced neuronal degeneration are not well understood. In this study, the dopaminergic neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridium ions (MPP+)/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which mimic Parkinson's disease (PD), induced neuronal degeneration by decreasing store-mediated Ca2+ entry. The function of the transient receptor potential canonical (TRPC)-1 channel was decreased upon exposure to the neurotoxins, followed by a decrease in TRPC1 expression. Similar to neurotoxins, samples from patients with PD exhibited attenuated TRPC1 expression, which was accompanied by a decrease in autophagic markers and a subsequent increase in apoptosis markers. Furthermore, exposure to neurotoxins attenuated PKC phosphorylation, decreased expression of autophagic markers, and increased apoptosis in SHSY-5Y neuroblastoma cells, which was again dependent on TRPC1. Prolonged neurotoxin treatment attenuated the binding of NF-κB to the TRPC1 promoter, which resulted in a decrease in TRPC1 expression, thereby attenuating autophagy and activating cell death. Restoration of TRPC1 expression rescued the effects of the dopaminergic neurotoxins in neuroblastoma cells by increasing Ca2+ entry, restoring NF-κB activity, and promoting autophagy. Overall, these results suggest that dopaminergic neurotoxins initially decreased Ca2+ entry, which inhibited the binding of NF-κB to the TRPC1 promoter, thereby inhibiting TRPC1 expression and resulting in cell death by preventing autophagy.-Sukumaran, P., Sun, Y., Antonson, N., Singh, B. B. Dopaminergic neurotoxins induce cell death by attenuating NF-κB-mediated regulation of TRPC1 expression and autophagy.

ASPP2 Plays a Dual Role in gp120-Induced Autophagy and Apoptosis of Neuroblastoma Cells.

HIV invasion of the central nervous system (CNS) in the majority of patients infected with HIV-1, leads to dysfunction and injury within the CNS, showing a variety of neurological symptoms which was broadly termed HIV-associated neurocognitive disorder (HAND). But the molecular mechanisms are not completely understood. It has been suggested that apoptosis and autophagic dysfunction in neurons may play an important role in the development of HAND. Previous studies have indicated that p53 may be involved in the onset of neurological disorder in AIDS. Apoptosis-stimulating protein of p53-2 (ASPP2), a p53-binding protein with specific function of inducing p53, has been reported to modulate autophagy. In the present study, we observed that gp120 induces autophagy and apoptosis in SH-SY5Y neuroblastoma cells. Adenovirus-mediated overexpression of ASPP2 significantly inhibited autophagy and apoptosis induced by low dose of gp120 protein (50 ng/mL), but induced autophagy and apoptosis when treated by high dose of gp120 protein (200 ng/mL). Further, ASPP2 knockdown attenuated autophagy and apoptosis induced by gp120.Conclusion: ASPP2 had different effects on the autophagy and apoptosis of neurons induced by different concentration of gp120 protein. It may be a potential therapeutic agent for HAND through modulating autophagy and apoptosis in CNS.

Cearoin Induces Autophagy, ERK Activation and Apoptosis via ROS Generation in SH-SY5Y Neuroblastoma Cells.

Neuroblastomas are the most common solid extracranial tumors in childhood. We investigated the anticancer effect of cearoin isolated from Dalbergia odorifera in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were treated with various doses of cearoin. The viability was measured by MTT assay. DCFDA fluorescence assay and Griess assay were used for the measurement of intracellular reactive oxygen species (ROS) and nitric oxide (NO), respectively. Western blot analysis was performed to clarify the molecular pathway involved. Cearoin induced cell death in a dose-dependent manner. Cearoin increased the phosporylation of ERK, the conversion of LC3B-I to LC3B-II, decrease in Bcl2 expression, the activation of caspase-3, and the cleavage of PARP, indicating the induction of autophagy and apoptosis. Furthermore, cearoin treatment increased the production of ROS and NO. Co-treatment with the antioxidant N-acetylcysteine completely abolished cearoin-mediated autophagy, ERK activation and apoptosis, suggesting the critical role of ROS in cearoin-induced anticancer effects. Moreover, co-treatment with ERK inhibitor PD98059 partially reversed cearoin-induced cell death, indicating the involvement of ERK in cearoin anticancer effects. These data reveal that cearoin induces autophagy, ERK activation and apoptosis in neuroblastoma SH-SY5Y cells, which is mediated primarily by ROS generation, suggesting its therapeutic application for the treatment of neuroblastomas.

Silica nanoparticles induced intrinsic apoptosis in neuroblastoma SH-SY5Y cells via CytC/Apaf-1 pathway.

The present study was to investigate effects of Silica nanoparticles (SiNPs) on nervous system and explore potential mechanisms in human neuroblastoma cells (SH-SY5Y). Cytotoxicity was detected by cell viability and Lactate dehydrogenase (LDH) release. Flow cytometry analysis was applied to assess mitochondrial membrane potential (MMP) loss, intracellular Ca2+ and apoptosis. To clarify the mechanism of SiNPs-induced apoptosis, intrinsic apoptosis-related proteins were detected. Our results showed that SiNPs caused cytotoxicity, cell membrane damage and Ca2+ increase in a dose-dependent manner in SH-SY5Y cells. Both the mitochondrial membrane potential (MMP) loss and potential mitochondria damage resulted in Cyt C release to the cytoplasm. The elevated Cyt C and Apaf1 further triggered intrinsic apoptosis via executive molecular caspase-9 and caspase-3. The present study confirmed that SiNPs induced intrinsic apoptosis in neuroblastoma SH-SY5Y cells via CytC/Apaf-1 pathway and provided a better understanding of the potential toxicity induced by SiNPs on human neurocyte.

Cx43 Mediates Resistance against MPP⁺-Induced Apoptosis in SH-SY5Y Neuroblastoma Cells via Modulating the Mitochondrial Apoptosis Pathway.

Neuronal apoptosis in the substantia nigra par compacta (SNpc) appears to play an essential role in the pathogenesis of Parkinson’s disease. However, the mechanisms responsible for the death of dopaminergic neurons are not fully understood yet. To explore the apoptotic mechanisms, we used a well-known parkinsonian toxin, 1-methyl-4-phenylpyridine (MPP+), to induce neuronal apoptosis in the human dopaminergic SH-SY5Y cell line. The most common method of interaction between cells is gap junctional intercellular communication (GJIC) mediated by gap junctions (GJs) formed by transmembrane proteins called connexins (Cx). Modulation of GJIC affects cell viability or growth, implying that GJIC may have an important role in maintaining homeostasis in various organs. Here, we hypothesized that increasing the level of the gap junction protein Cx43 in SH-SY5Y neuroblastoma cells could provide neuroprotection. First, our experiments demonstrated that knocking down Cx43 protein by using Cx43-specific shRNA in SH-SY5Y neuroblastoma cells potentiated MPP+-induced neuronal apoptosis evident from decreased cell viability. In another experiment, we demonstrated that over-expression of Cx43 in the SH-SY5Y cell system decreased MPP+-induced apoptosis based on the MTT assay and reduced the Bax/Bcl-2 ratio and the release of cytochrome C based on Western blot analysis. Taken together, our results suggest that Cx43 could mediate resistance against MPP+-induced apoptosis in SH-SY5Y neuroblastoma cells via modulating the mitochondrial apoptosis pathway.

The inhibitory effect of dihydroartemisinin on the growth of neuroblastoma cells

ObjectiveTo evaluate the inhibitory effect of dihydroartemisinin on neuroblastoma cell line SH-SY5Y, explore the possible mechanism of dihydroartemisinin against neuroblastoma cells. MethodsThe cell viability of dihydroartemisinin treated SH-SY5Y cells was examined by MTT assay and morphology of cells was observed by using inverted microscope. Cell cycle was examined with flowcytometry assay, then cyclin D1 and caspase-3 proteins expression was detected by ELISA and western blotting assay. ResultsMTT analysis results showed that cell viability significantly decreased after exposure to 0.05, 0.50, 5.00 and 50.00 μmol/L dihydroartemisinin in a dose-dependent manner, and the lower density of cells was observed in treated groups. The number of cells in sub-G1 phase was increased after treatment with different doses of dihydroartemisinin compared with the control group. The expression of cyclin D1 protein was decreased, while the expression of caspase-3 protein was increased in treated group. ConclusionsDihydroartemisinin could inhibit the proliferation through stopping the cell cycle and inducing the apoptosis in neuroblastoma SH-SY5Y cells.

Gamma-tocotrienol acts as a BH3 mimetic to induce apoptosis in neuroblastoma SH-SY5Y cells

Bcl-2 family proteins are crucial regulators of apoptosis. Both pro- and antiapoptotic members exist, and overexpression of the latter facilitates evasion of apoptosis in many cancer types. Bcl-2 homology domain 3 (BH3) mimetics are small molecule inhibitors of antiapoptotic Bcl-2 family members, and these inhibitors are promising anticancer agents. In this study, we report that gamma-tocotrienol (γT3), an isomer of vitamin E, can inhibit Bcl-2 to induce apoptosis. We demonstrate that γT3 induces cell death in human neuroblastoma SH-SY5Y cells by depolarising the mitochondrial membrane potential, enabling release of cytochrome c to the cytosol and increasing the activities of caspases-9 and -3. Treatment of cells with inhibitors of Bax or caspase-9 attenuated the cell death induced by γT3. Simulated docking analysis suggested that γT3 binds at the hydrophobic groove of Bcl-2, while a binding assay showed that γT3 competed with a fluorescent probe to bind at the hydrophobic groove. Our data suggest that γT3 mimics the action of BH3-only protein by binding to the hydrophobic groove of Bcl-2 and inducing apoptosis via the intrinsic pathway in a Bax- and caspase-9-dependent manner.

The protein l-isoaspartyl (d-aspartyl) methyltransferase protects against dopamine-induced apoptosis in neuroblastoma SH-SY5Y cells

Parkinson’s disease (PD) is the most common neurodegenerative motor disorder in the world. The main causes of neurodegeneration in PD are mitochondrial impairment and oxidative stress promoted by dopamine (DA) metabolism in the cytosol. Protein l-isoaspartyl (d-aspartyl) methyltransferase (PIMT) is a protein repair enzyme with anti-apoptotic properties. We previously reported that PIMT was downregulated at both gene and protein levels by DA-induced oxidative stresses in SH-SY5Y neuroblastoma cells. The purpose of the current study was to investigate the anti-apoptotic function of PIMT toward DA-induced cell death to better understand its specific neuroprotective role. Overexpression of wild-type PIMT, in contrast to its inactive mutant, protected SH-SY5Y cells from cell death and caspase 3 activation upon DA treatments. The intrinsic pathway of apoptosis as measured by caspase 9 activity was triggered by reactive oxygen species produced from DA metabolism, and overexpression of wild-type PIMT completely prevented caspase 9 activity stimulated by DA. In addition, cells overexpressing wild-type PIMT produced significantly less reactive oxygen species despite DA treatment compared to cells that do not overexpress PIMT. Together, these data indicate that DA-associated PIMT downregulation is an important event contributing to neuronal cell death. More importantly, the PIMT anti-apoptotic capacity seems to be dependent on its involvement in the cellular antioxidant machinery.

Myristoylated Alanine-Rich C Kinase Substrate Accelerates TNF-<i>α</i>-Induced Apoptosis in SH-SY5Y Cells in a Caspases-6 and/or -7-Dependent Manner

Cell proliferation, differentiation, and the elimination of unnecessary cells by apoptosis occur in the development of the nervous system. It is reported that brain dysplasia appears as the results of myristoylated alanine-rich C kinase substrate (MARCKS) knockout or the mutant mouse. We therefore expect that MARCKS participates in the development of the nervous system. However, the mechanism underlying such participation has not been identified. In this study, we observed the effects of the overexpression of MARCKS or unphosphorylatable MARCKS on cell proliferation and TNF-α-induced apoptosis in neuroblastoma SH-SY5Y cells. Furthermore, we restrained MARCKS expression by the RNAi method. In the results, MARCKS-overexpressing cells and not unphosphorylatable MARCKS-overexpressing cells showed increased cell proliferation rates. On the other hand, the RNAi decreased the proliferation of MARCKS-knocked down SH-SY5Y cells. These results indicated that MARCKS-overexpressing cells were more sensitive to TNF-α than normal SH-SY5Y cells. Moreover, in MARCKS-overexpressing cells TNF-α-induced apoptosis was inhibited by caspase-6 and -7 inhibitors but not by caspase-3 inhibitor. These results suggested that MARCKS participated in TNF-α-induced apoptosis in a caspase-6 and/or -7-dependent manner.

Endoplasmic reticulum stress is involved in the lidocaine-induced apoptosis in SH-SY5Y neuroblastoma cells.

Lidocaine has been indicated to promote apoptosis and to promote endoplasmic reticulum (ER) stress. However, the mechanism underlining ER stress-mediated apoptosis is unclear. In the present study, we investigated the promotion to ER stress in the lidocaine-induced apoptosis in human neuroblastoma SH-SY5Y cells. Firstly, we confirmed that lidocaine treatment induced apoptosis in SH-SY5Y cells, time-dependently and dose-dependently, via MTT cell viability assay and annexin V/FITC apoptosis detection with a FACScan flow cytometer. And the anti-apoptosis Bcl-2 and Bcl-xL were downregulated, whereas the apoptosis-executive caspase 3 was promoted through Western blot assay and caspase 3 activity assay. Moreover, the ER stress-associated binding immunoglobulin protein (BiP), PKR-like ER kinase (PERK), activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP) were also upregulated at both mRNA and protein levels by lidocaine treatment. On the other hand, downregulation of the ER stress-associated BiP by RNAi method not only blocked the lidocaine-promoted ER stress but also attenuated the lidocaine-induced SH-SY5Y cell apoptosis. In conclusion, the present study confirmed the involvement of ER stress in the lidocaine-induced apoptosis in human neuroblastoma SH-SY5Y cells. Our study provides a better understanding on the mechanism of lidocaine's neurovirulence.

Downregulation of survivin by siRNA inhibits invasion and promotes apoptosis in neuroblastoma SH-SY5Y cells.

Neuroblastoma is a solid tumor that occurs mainly in children. Malignant neuroblastomas have a poor prognosis because conventional chemotherapeutic agents are not very effective. Survivin, a member of the inhibitor of the apoptosis protein family, plays a significant role in cell division, inhibition of apoptosis, and promotion of cell proliferation and invasion. Previous studies found that survivin is highly expressed in some malignant neuroblastomas and is correlated with poor prognosis. The aim of this study was to investigate whether survivin could serve as a potential therapeutic target of human neuroblastoma. We employed RNA interference to reduce survivin expression in the human neuroblastoma SH-SY5Y cell line and analyzed the effect of RNA interference on cell proliferation and invasion in vitro and in vivo. RNA interference of survivin led to a significant decrease in invasiveness and proliferation and increased apoptosis in SH-SY5Y cells in vitro. RNA interference of survivin inhibited tumor growth in vivo by 68±13% (P=0.002) and increased the number of apoptotic cells by 9.8±1.2% (P=0.001) compared with negative small interfering RNA (siRNA) treatment controls. Moreover, RNA interference of survivin inhibited the formation of lung metastases by 92% (P=0.002) and reduced microvascular density by 60% (P=0.0003). Survivin siRNA resulted in significant downregulation of survivin mRNA and protein expression both in vitro and in vivo compared with negative siRNA treatment controls. RNA interference of survivin was found to be a potent inhibitor of SH-SY5Y tumor growth and metastasis formation. These results support further clinical development of RNA interference of survivin as a treatment of neuroblastoma and other cancer types.

Overexpression of the dependence receptor UNC5H4 inhibits cell migration and invasion, and triggers apoptosis in neuroblastoma cell

UNC5H4 is a newly identified member of the UNC5H receptor family. Previously, we have demonstrated that UNC5H4 expression is significantly higher in favorable neuroblastomas than in unfavorable ones, and higher UNC5H4 level is correlated with longer survival time. However, the function of UNC5H4 in the tumorigenesis of neuroblastoma still remains elusive. In the present study, the effects of UNC5H4 overexpression on neuroblastoma SH-SY5Y cells were investigated. We showed that enforced expression of UNC5H4 receptor significantly inhibited anchorage-dependent and anchorage-independent growth of SH-SY5Y cells. Cell migration and invasion of SH-SY5Y cells transfected with UNC5H4-expressing plasmid were obviously suppressed as compared to those transfected with emptor vector or non-transfected cells. Moreover, overexpression of UNC5H4 resulted in apoptosis in SH-SY5Y cells. The induction of apoptosis by UNC5H4 was completely abolished in the presence of its ligand, netrin-1. Finally, caspase cleavage and the presence of death domain were required for UNC5H4 to induce apoptosis in neuroblastoma SH-SY5Y cells. These data suggest that the dependence receptor UNC5H4 may act as a putative tumor suppressor in neuroblastoma.

Neuroprotective effects of propofol on ER stress-mediated apoptosis in neuroblastoma SH-SY5Y cells

Anesthetic treatment has been associated with widespread apoptotic neurodegeneration in the neonatal rodent brain. It has recently been suggested that propofol, a short-acting intravenous anesthetic agent, may have a potential as a neuroprotective agent. An apoptotic pathway mediated through endoplasmic reticulum (ER) stress has been attracting attention. ER stress is associated with accumulation of unfolded or misfolded proteins in ER, and ER stress-induced apoptosis is implicated in a wide range of diseases, including ischemia/reperfusion injury, neurodegeneration, and diabetes. We investigated whether thapsigargin-induced ER stress is prevented by propofol in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were pretreated with various concentrations of propofol (1–10μM) for 3h before co-treatment with 0.5μM thapsigargin and propofol for 20h. Levels of ssDNA, specific evidence of apoptosis, and biomarkers of ER stress (mRNA expression of Chop and sXbp-1) were determined. We also assayed calpain and caspase-4 activities and intracellular Ca2+ ([Ca2+]i) levels. Thapsigargin-induced increases in ssDNA levels, expressions of ER stress biomarkers, activities of caspase-4 and calpain, and level of [Ca2+]i were suppressed by co-incubation with propofol. Our data indicate the possibility that propofol inhibits the Ca2+ release from ER at clinically employed dose levels. These results demonstrate that propofol suppresses the ER stress-induced apoptosis in this cell system, and may have the neuroprotective potency. It may also be a promising agent for preventing damage from cerebral ischemia or edema.

RNF13 protein regulates endoplasmic reticulum stress induced apoptosis in dopaminergic SH-SY5Y cells by enhancing IRE1α stability

Any interruption in the folding capacity of endoplasmic reticulum (ER) may result in inducing ER stress which would initiate an adaptive signaling mechanism called Unfolded Protein Response (UPR) in order to restore homeostasis, failing to which would initiate signaling pathway leading to death of the cell. Finding new mediators could help better understand the molecular mechanisms of ER stress-induced apoptosis. Our lab initiated a genetic screen method using retroviral insertion mutation system to look for genes whose inactivation would confer resistance to apoptosis. In our previous findings, Ring finger protein 13 (RNF13) was identified whose down-regulation conferred survival against ER stress-induced apoptosis. Our previous results also showed important role of RNF13 in apoptotic signaling in 293T cells as a result of strong RNF13-IRE1α interaction. In current study, using SH-SY5Y cells, overexpression of RNF13 in apoptosis assays and RT-PCR analysis has shown to induce apoptosis as well as splicing of X-box binding Protein 1 (XBP1) confirming its role in ER stress mediated cell death in this cell line as well. Western blot analysis has revealed that overexpression of both N-terminal as well as C-terminal tagged RNF13 resulted in activation of c-Jun N-terminal kinase (JNK) in SH-SY5Y cells. Our co-immunoprecipitation assays in SH-SY5Y cells also showed a strong interaction of RNF13 with IRE1α. Finally, Cycloheximide chase experiment exhibited that RNF13-IRE1α interaction increased the stability of IRE1α. Altogether, our data suggest that RNF13 may act as an important regulator of IRE1α, mediating ER stress-mediated apoptosis in neuroblastoma SH-SY5Y cells.