PXR激活在拮抗SH-SY5Y细胞凋亡中的作用

人神经母细胞瘤细胞SH-SY5Y细胞可以表达神经元特异性的酪氨酸羟化酶、多巴胺-β-羟化酶以及多巴胺转运体等,因此可用于建立帕金森病的体外模型。虽然帕金森综合症发病的确切机制至今尚不清楚,但众多的病理学资料证实该病患者存在中脑黑质多巴胺能神经元的凋亡。自由基、兴奋性     

Garlic compounds induced calpain and intrinsic caspase cascade for apoptosis in human malignant neuroblastoma SH-SY5Y cells

Malignant (N-type) neuroblastoma continues to defy current chemotherapeutic regimens. We tested the garlic compounds diallyl sulfide (DAS) and diallyl disulfide (DADS) for induction of apoptosis in human malignant neuroblastoma SH-SY5Y cells. Viability of human primary neurons was unaffected after 24 h treatment with 50 and 100 μM DAS and 50 μM DADS but slightly affected with 100 μM DADS. Treatment with 50 and 100 μM DAS or DADS significantly decreased viability in SH-SY5Y cells. Wright staining showed morphological features of apoptosis in SH-SY5Y cells treated with 50 and 100 μM DAS or DADS for 24 h. ApopTag assay demonstrated DNA fragmentation in apoptotic cells. Apoptosis was associated with an increase in [Ca²⁺]_i, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, increase in cytosolic Smac/Diablo, and down regulation of inhibitor-of-apoptosis proteins and nuclear factor kappa B (NFκB). Activation of caspase-9 and caspase-3 indicated involvement of intrinsic pathway of apoptosis. Calpain and caspase-3 activities produced 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Also, caspase-3 activity cleaved inhibitor of caspase-activated DNase (ICAD). Results strongly suggested that the garlic compounds DAS and DADS suppressed anti-apoptotic factors and activated calpain and intrinsic caspase cascade for apoptosis in SH-SY5Y cells.     

p38 MAPK and ERK1/2 pathways are involved in the pro-apoptotic effect of notoginsenoside Ft1 on human neuroblastoma SH-SY5Y cells

Aims

This study aims to investigate the effect and the mechanisms of notoginsenoside Ft1, a natural compound exclusively found in P. notoginseng, on the proliferation and apoptosis of human neuroblastoma SH-SY5Y cells.

Main methods

CCK-8 assay was used to assess the cell proliferation. Flow cytometry was performed to measure the cell cycle distribution and cell apoptosis. Hoechst 33258 staining was conducted to confirm the morphological changes of apoptotic cells. Protein expression was detected by western blot analysis and caspase 3 activity was measured by colorimetric assay kit.

Key findings

Among the saponins examined, Ft1 showed the best inhibitory effect on cell proliferation of SH-SY5Y cells with IC₅₀ of 45 μM. Ft1 not only arrested the cell cycle at S, G₂/M stages, but also promoted cell apoptosis, which was confirmed by Hoechst 33258 staining. Further studies demonstrated that Ft1 up-regulated the protein expressions of cleaved caspase 3, phospho-p53, p21, and cyclin B1, but down-regulated that of Bcl-2. Moreover, Ft1 enhanced the phosphorylation of ERK1/2, JNK and p38 MAPK. However, the phosphorylation of Jak2 and p85 PI3K was reduced by Ft1. Inhibitors of p38 MAPK and ERK1/2 but not JNK abrogated the up-regulated protein expressions of cleaved caspase 3, p21 and down-regulated protein expression of Bcl-2 as well as elevated caspase 3 activity induced by Ft1.

Significance

Ft1 arrested the proliferation and elicited the apoptosis of SH-SY5Y cells possibly via p38 MAPK and ERK1/2 pathways, which indicates the potential therapeutic effect of it on human neuroblastoma.     

Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin

We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH₂-RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinson’s disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27^Kip1 protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27^Kip1 significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27^Kip1 degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin.     

Oral Metronomic Topotecan Sensitizes Crizotinib Antitumor Activity in ALKF1174L Drug-Resistant Neuroblastoma Preclinical Models

BACKGROUND: Anaplastic lymphoma kinase (ALK) inhibitor crizotinib has proven to be effective in the treatment of ALK-mutated neuroblastoma, but crizotinib resistance was commonly observed in patients. We aimed to overcome crizotinib resistance by combining with the MEK inhibitor trametinib or low-dose metronomic (LDM) topotecan in preclinical neuroblastoma models. METHODS: We selected a panel of neuroblastoma cell lines carrying various ALK genetic aberrations to assess the therapeutic efficacy on cell proliferation in vitro. Downstream signals of ALK activation, including phosphorylation of ERK1/2, Akt as well as HIF-1α expression were evaluated under normoxic and hypoxic conditions. Tumor growth inhibition was further assessed in NOD/SCID xenograft mouse models. RESULTS: All NBL cell lines responded to crizotinib treatment but at variable ED50 levels, ranging from 0.25 to 5.58 μM. ALK-mutated cell lines SH-SY5Y, KELLY, LAN-5, and CHLA-20 are more sensitive than ALK wild-type cell lines. In addition, we demonstrated that under hypoxic conditions, all NBL cell lines showed marked decrease of ED50s when compared to normoxia except for KELLY cells. Taking into consideration the hypoxia sensitivity to crizotinib, combined treatment with crizotinib and LDM topotecan demonstrated a synergistic effect in ALK^F1174L-mutated SH-SY5Y cells. In vivo, single-agent crizotinib showed limited antitumor activity in ALK^F1174L-mutated SH-SY5Y and KELLY xenograft models; however, when combined with topotecan, significantly delayed tumor development was achieved in both SH-SY5Y and KELLY tumor models. CONCLUSIONS: Oral metronomic topotecan reversed crizotinib drug resistance in the ALK^F1174L-mutated neuroblastoma preclinical model.     

Growth Inhibition and Apoptosis of Neuroblastoma Cells Through ROS-Independent MEK/ERK Activation by Sulforaphane

Deregulation of apoptosis alters the balance of cell proliferation and cell death, resulting in a variety of diseases, including cancer. In recent studies, sulforaphane (SFN) has demonstrated potent anti-tumor and chemopreventive activities. A possible signal transduction pathway has also been elucidated for SFN-induced apoptosis in human neuroblastoma SH-SY5Y cells. The present study further investigates the anti-proliferation activities of SFN through induced apoptosis in SH-SY5Y cells. We found that treating SH-SY5Y cells with SFN resulted in the depletion of mitochondrial membrane potential (Δψ), which in turn increased caspase 9, caspase 3, and the up-regulation of phosphorylated MEK/ERK without generating reactive oxygen species. Results were confirmed by MTT assay, which demonstrated the cytotoxic activity of SFN against SH-SY5Y cells (IC₅₀ values of 20 μM).     

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.     

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.     

八氯腺苷诱导SH-SY5Y和SK-N-SH细胞G2/M期阻滞的分子机制不同

为探索八氯腺苷的抗肿瘤作用机制,以神经母细胞瘤SH-SY5Y和SK-N-SH细胞为对象,采用四唑盐比色实验（MTT法）证明,八氯腺苷具有明显的抑制肿瘤细胞增殖的作用,这种抑制作用呈剂量-时间依赖性.流式细胞分析显示,10 μmol/L八氯腺苷作用48 h后可导致靶细胞生长停滞于G ^₂/M期;SH-SY5Y细胞发生明显细胞凋亡,但SK-N-SH细胞却未见凋亡.Hoechst 33342染色显示,SK-N-SH细胞发生了核分裂异常.蛋白质免疫印迹分析证明,10 μmol/L 八氯腺苷处理SH SY5Y 48～72 h后,G^₂检验点调节蛋白ATM、Chk1、Cdc25C和Cdc2磷酸化形式明显上调,同时伴有caspase-3的激活,提示SH-SY5Y细胞发生了G^₂检验点通路和细胞凋亡途径的激活.与SH-SY5Y细胞不同,在SK-N-SH细胞中,八氯腺苷处理24～96 h时,磷酸化ATM、磷酸化Chk1/Chk2、磷酸化Cdc25C以及磷酸化Cdc2的水平呈现逐渐降低的趋势.结果提示,SK-N-SH细胞在八氯腺苷处理后发生了G^₂检验点失败.蛋白质免疫印迹分析还显示,八氯腺苷可诱导p53在SH-SY5Y细胞的表达,但却不能影响SK—N-SH细胞的p53组成性表达水平.p21在SK-N-SH的组成性表达随八氯腺苷处理时间延长而逐渐减少,但在处理前后的SH-SY5Y细胞均未检测到p21蛋白的表达.上述实验结果提示,八氯腺苷抑制两种细胞增殖的机制不同：在SH-SY5Y细胞,八氯腺苷可激活ATM-Chk-Cdc25C-Cdc2/cyclin途径和凋亡通路,使细胞发生G_²/M期阻滞和细胞凋亡;在SK-N-SH细胞,八氯腺苷诱导G^₂检验点失败,导致细胞阻滞在有丝分裂期,并发生有丝分裂异常.2种不同的细胞命运可能还与p53和p21表达不同有关.     

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.     

ASK1 resistant neuroblastoma is deficient in activation of p38 kinase

Apoptosis Signal-regulating Kinase 1 (ASK1) is known to either induce apoptosis or differentiation in various cell lines of neuronal origin. We analyzed the effect of the constitutively active mutant of ASK1 (ASK1-Delta N) in an adenoviral vector in four neuroblastoma cell lines, two murine, C1300 and NXS2, and two human, SH-SY5Y and IMR-32. Already after 24 h upon infection, C1300 and SH-SY5Y cells arrested in growth when judged by [(3)H]thymidine incorporation, and the majority of the cells demonstrated apoptotic appearance, which was confirmed by DNA-laddering in gel electrophoresis. In contrast, NXS2 and IMR-32 cell lines remained unaffected. Immunoblotting revealed strongly phosphorylated p38 MAPK accompanied by weakly phosphorylated JNK in C1300 and SH-SY5Y, whereas none of these kinases were activated by adenoviruses expressing the kinase negative ASK1 mutant or beta-galactosidase. There was no expression of phosphorylated kinases in IMR-32 cells, but NXS2 showed a faint band of phosphorylated p38 MAPK. Addition of the p38 MAPK specific inhibitor, SB203580, protected C1300 and SH-SY5Y cells from apoptosis induced by ASK1-Delta N. The anti-neoplastic agent, paclitaxel, activates ASK1 and JNK, and promotes the in vitro assembly of stable microtubules. Addition of 10 nM paclitaxel sensitised the NXS2 cell line to ASK1-induced cell death. Our results indicate that ASK1 induces apoptosis in neuroblastoma cells mainly via the p38 MAPK pathway, and resistant neuroblastoma cells can be sensitised to ASK1 by paclitaxel.     

Activation of AMP-Activated Protein Kinase Alleviates Homocysteine-Mediated Neurotoxicity in SH-SY5Y Cells

Mammalian AMP-activated protein kinase (AMPK) acts as a metabolite-sensing protein kinase in multiple tissues. Recent studies have shown that AMPK activation also regulates intracellular signaling pathways involved in cellular survival and apoptosis. Previously, we have reported that AMPK activation alleviates the endoplasmic reticulum (ER) stress-mediated neurotoxicity and tau hyperphosphorylation caused by palmitate. Therefore, we investigated whether AMPK activation alleviates ER stress-mediated neurotoxicity in SH-SY5Y human neuroblastoma cells incubated with homocysteine. Regulation of AMPK activity by isoflavone was also determined to investigate the underlying mechanism of its neuroprotective effect. Treatment of SH-SY5Y human neuroblastoma cells with N ¹-(β-D-ribofuranosyl)-5-aminoimidazole-4-carboxamide (AICAR), a pharmacological activator of AMPK, significantly protected cells against cytotoxicity imposed by tunicamycin and homocysteine. Homocysteine significantly suppressed AMPK activation, which was alleviated by AICAR. We observed a significant inhibition of the unfolded protein response by AICAR in cells incubated with homocysteine, suggesting a protective role of AMPK activation against ER stress-mediated neurotoxicity. AICAR also significantly reduced tau hyperphosphorylation by inactivating glycogen synthase kinase-3β and c-Jun N-terminal kinase in cells incubated with homocysteine. Furthermore, treatment of cells with soy isoflavone, genistein and daidzein significantly activated AMPK, which was repressed by tunicamycin and homocysteine. Therefore, our results suggest that AMPK activation by isoflavone as well as AICAR alleviates homocysteine-mediated neurotoxicity in SH-SY5Y cells.     

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.     

Neural stem and progenitor cell fate transition requires regulation of Musashi1 function

Background

There is increasing evidence of a pivotal role for regulated mRNA translation in control of developmental cell fate transitions. Physiological and pathological stem and progenitor cell self-renewal is maintained by the mRNA-binding protein, Musashi1 through repression of translation of key mRNAs encoding cell cycle inhibitory proteins. The mechanism by which Musashi1 function is modified to allow translation of these target mRNAs under conditions that require inhibition of cell cycle progression, is unknown.

Results

In this study, we demonstrate that differentiation of primary embryonic rat neural stem/progenitor cells (NSPCs) or human neuroblastoma SH-SY5Y cells results in the rapid phosphorylation of Musashi1 on the evolutionarily conserved site serine 337 (S337). Phosphorylation of this site has been shown to be required for cell cycle control during the maturation of Xenopus oocytes. S337 phosphorylation in mammalian NSPCs and human SH-SY5Y cells correlates with the de-repression and translation of a Musashi reporter mRNA and with accumulation of protein from the endogenous Musashi target mRNA, p21^WAF1/CIP1. Inhibition of Musashi regulatory phosphorylation, through expression of a phospho-inhibitory mutant Musashi1 S337A or over-expression of the wild-type Musashi, blocked differentiation of both NSPCs and SH-SY5Y cells. Musashi1 was similarly phosphorylated in NSPCs and SH-SY5Y cells under conditions of nutrient deprivation-induced cell cycle arrest. Expression of the Musashi1 S337A mutant protein attenuated nutrient deprivation-induced NSPC and SH-SY5Y cell death.

Conclusions

Our data suggest that in response to environmental cues that oppose cell cycle progression, regulation of Musashi function is required to promote target mRNA translation and cell fate transition. Forced modulation of Musashi1 function may present a novel therapeutic strategy to oppose pathological stem cell self-renewal.