Casein kinase 1 (CK1) promotes the proliferation and metastasis of glioma cells via the phosphatidylinositol 3 kinase-matrix metalloproteinase 2 (AKT-MMP2) pathway.

BACKGROUND: Glioma is a type of tumor that usually occurs in the adult central nervous system. Protein kinases have become important targets for oncotherapy since they are closely correlated with signal transduction. The role of the casein kinase 1 (CK1) gene in glioma remains to be fully elucidated. METHODS: The mRNA and protein expression of CK1 were analyzed by Realtime PCR, Western blot and immunohistochemistry. The cell behavior was assayed by MTT, Transwell and cell scratch methods. Cell cycle and cell apoptosis were performed by flow cytometer. Construction of stable cell line was completed by lentivirus infection. The nude mouse model was used for in vivo analysis on the role of CK1 by injecting the cells into subcutaneous tissue, tail vein and cerebral cortex. The prognostic role of CK1 in glioma was evaluated using Kaplan-Meier and Cox regression analyses. RESULTS: immunohistochemical staining demonstrated that the expression of CK1 in glioma samples was correlated with the grade of glioma. Survival analysis using Kaplan-Meier and multivariate analysis by Cox regression indicated that CK1 could be used as an independent prognostic marker for glioma. The methyl thiazolyl tetrazolium (MTT), transwell, and cell scratch assays demonstrated that the CK1 gene promoted cell proliferation and invasion through the phosphatidylinositol 3 kinase/matrix metalloproteinase 2 (AKT-MMP2) signaling pathway. In vivo experiments in mice also confirmed the ability of CK1 to enhance tumor proliferation and metastasis, with the metastatic site being the small intestine. CONCLUSIONS: the expression of CK1 was correlated with glioma grade and patient survival and it may enhance glioma proliferation and metastasis via AKT-MMP2 pathway.

Assessment spermatogenic cell apoptosis and the transcript levels of metallothionein and p53 in Meretrix meretrix induced by cadmium.

Cadmium (Cd) has been widely used in industry and can accumulate in the water, soil, and food. Meretrix meretrix is one of the marine shellfishes cultivated for economic purpose in China. The increasing Cd levels in coastal marine water could adversely affect the economic benefits of shellfish cultivation. In the present study, M. meretrix were exposed to different Cd2+ concentrations (0, 1.5, 3, 6, and 12 mg L-1) for 5 d to evaluate the effects of Cd on spermatogenic cell. The Cd accumulation, survival rate and the indices of oxidative stress and apoptosis were determined in the spermatogenic cells of M. meretrix. The expression levels of p53 and metallothionein (MT) mRNA were also measured in the spermatogenic cells. Cd accumulation and the mortality rate of spermatogenic cells were found to increase in a dose-response manner with Cd2+ concentrations. Histopathology changes, especially the damage of membranous structure, were more severe as the Cd2+ levels in the testis became higher. The indexes of oxidative stress, including reactive oxygen species, malondialdehyde, protein carbonyl derivates and DNA-protein crosslinks all increased after exposure to Cd2+. However, the total antioxidant capacity gradually decreased with the increasing Cd2+ concentration. In addition, exposure to Cd2+ increased the apoptotic rate and caspase-3 and 9 activities but decreased the level of mitochondrial membrane potential and cytochrome C oxidase in the spermatogenic cells. MT mRNA expression increased in lower Cd2+ concentration treated groups whereas decreased in higher groups, while the p53 mRNA expression increased in a dose-response manner with Cd2+ and was positively correlated with the oxidative damage indices. These results indicated that Cd2+ caused oxidative stress and p53 induced apoptosis in the spermatogenic cells, and thus decreased the survival rate of sperm cells. This finding highlights that Cd can reduce the reproductive capacity of M. meretrix, thus threatening to wild shellfish populations and reducing the efficiency of shellfish farming.

Umbilical cord-derived mesenchymal stromal/stem cells expressing IL-24 induce apoptosis in gliomas.

Although much progress has been made in the treatment of gliomas, the prognosis for patients with gliomas is still very poor. Stem cell-based therapies may be promising options for glioma treatment. Recently, many studies have reported that umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) are ideal gene vehicles for tumor gene therapy. Interleukin 24 (IL-24) is a pleiotropic immunoregulatory cytokine that has an apoptotic effect on many kinds of tumor cells and can inhibit the growth of tumors specifically without damaging normal cells. In this study, we investigated UC-MSCs as a vehicle for the targeted delivery of IL-24 to tumor sites. UC-MSCs were transduced with lentiviral vectors carrying green fluorescent protein (GFP) or IL-24 complementary DNA. The results indicated that UC-MSCs could selectively migrate to glioma cells in vitro and in vivo. Injection of IL-24-UC-MSCs significantly suppressed tumor growth of glioma xenografts. The restrictive efficacy of IL-24-UC-MSCs was associated with the inhibition of proliferation as well as the induction of apoptosis in tumor cells. These findings indicate that UC-MSC-based IL-24 gene therapy may be able to suppress the growth of glioma xenografts, thereby suggesting possible future therapeutic use in the treatment of gliomas.

The burden of stroke in China: Results from a nationwide population-based epidemiological survey.

Stroke is a serious threat to human health that often leads to severe complications, and currently ranks first as leading cause of death in China. However, reliable data on stroke burden in China in the 21st century are lacking. We used the data from NESS-China (National Epidemiological Survey of Stroke in China) for assessing the adverse health effects of stroke in Chinese population. We carried out inter-regional comparative study in order to obtain regular burden related characteristics of stroke in China, as measured by YLLs (years of life lost due to premature mortality), YLDs (years lived with disability) and DALYs (disability adjusted life years). Amongst the nationwide population of 596,536 individuals of all ages in 2013, the YLLs for stroke was 1748, the YLDs was 262, and the DALYs was 2010(per 100,000). The gender subtype analysis of DALYs was 2171(male) and 1848(female). The YLLs, YLDs and DALYs in rural areas were higher compared to urban areas. Among the 18 age groups, the highest YLLs was observed in ≥ 80 years old group. The impact of stroke on Chinese population is more severe compared to the global average levels. Stroke results as the main cause of YLLs in China, while there is no significant difference for the YLDs. Nevertheless, DALYs caused by stroke rank 3th in global epidemiologic study territories, 1st in China.

Knockdown of DSPP inhibits the migration and invasion of glioma cells.

Dentin sialophosphoprotein (DSPP) is a member of the SIBLING (Small integrin-binding ligand N-linked glycoproteins) family of phosphoglycoproteins and has been proved to contribute to the migration of a variety of solid tumor cells. However, whether DSPP participates in the pathogenic process of glioma remains unknown. In this study, we aimed to investigate the expression and biological function of DSPP in human glioma cells. We demonstrated through Western blot that DSPP is overexpressed in glioma tissues comparing to normal brain tissues. To investigate the role of DSPP in glioma carcinogenesis, we reduced the DSPP expression by small interfering RNA (siRNA) and found that DSPP silencing significantly inhibited the migration and invasion of glioma cells, the critical characteristics of glioma. Furthermore, we showed that DSPP down-regulation significantly decreased the activation of the AKT/mTOR/p70S6K pathway in glioma cells. Taken together, these findings indicate that knockdown of DSPP inhibits glioma cells migration and invasion, suggesting that targeting DSPP might be a potentially effective therapeutic strategy for treating glioma.

The Role of Smurf1 in Neuronal Necroptosis after Lipopolysaccharide-Induced Neuroinflammation.

The role of inflammation in neurological disorders such as Alzheimer's disease and Parkinson's disease is gradually recognized and leads to an urgent challenge. Smad ubiquitination regulatory factor 1 (Smurf1), one member of the HECT family, is up-regulated by proinflammatory cytokines and associated with apoptosis in acute spinal cord injury. However, the function of Smurf1 through promoting neuronal necroptosis is still limited in the central nervous system (CNS). Hence, we developed a neuroinflammatory model in adult rats following lipopolysaccharide (LPS) lateral ventral injection to elaborate whether Smurf1 is involved in necroptosis in CNS injury. The up-regulation of Smurf1 detected in the rat brain cortex was similar to the necroptotic marker RIP1 expression in a time-dependent manner after LPS-induced neuroinflammation. Meanwhile, Smurf1 knockdown with siRNA inhibited neuronal necroptosis following LPS-stimulated rat pheochromocytomal PC12 cells. Thus, it was indicated that LPS-induced necroptosis could be promoted by Smurf1. In short, these studies suggest that Smurf1 might promote neuronal necroptosis after LPS-induced neuroinflammation, which might act as a novel and potential molecular target for the treatment of neuroinflammation associated diseases.

The Contribution of Necroptosis in Neurodegenerative Diseases.

Over the past decades, cell apoptosis has been significantly reputed as an accidental, redundant and alternative manner of cell demise which partakes in homeostasis in the development of extensive diseases. Nevertheless, necroptosis, another novel manner of cell death through a caspase-independent way, especially in neurodegenerative diseases remains ambiguous. The cognition of this form of cell demise is helpful to understand other forms of morphological resemblance of necrosis. Additionally, the concrete signal mechanism in the regulation of necroptosis is beneficial to the diagnosis and treatment of neurodegenerative diseases. Recent studies have demonstrated that necroptotic inhibitor, 24(S)-Hydroxycholesterol and partial specific histone deacetylase inhibitors could alleviate pathogenetic conditions of neurodegenerative diseases via necroptosis pathway. In this review, we summarize recent researches about mechanisms and modulation of necroptotic signaling pathways and probe into the role of programmed necroptotic cell demise in neurodegenerative diseases such as Parkinson's disease, Multiple sclerosis, Amyotrophic lateral sclerosis.

O-GlcNAc Glycosylation of nNOS Promotes Neuronal Apoptosis Following Glutamate Excitotoxicity.

Ischemic stroke is a dominant health problem with extremely high rates of mortality and disability. The main mechanism of neuronal injury after stroke is excitotoxicity, during which the activation of neuronal nitric oxide synthase (nNOS) exerts a vital role. However, directly blocking N-methyl-D-aspartate receptors or nNOS can lead to severe undesirable effects since they have crucial physiological functions in the central nervous system. Here, we report that nNOS undergoes O-linked-ß-N-acetylglucosamine (O-GlcNAc) modification via interacting with O-GlcNAc transferase, and the O-GlcNAcylation of nNOS remarkably increases during glutamate-induced excitotoxicity. In addition, eliminating the O-GlcNAcylation of nNOS protects neurons from apoptosis during glutamate stimulation by decreasing the formation of nNOS-postsynaptic density protein 95 complexes. Taken together, our data suggest a novel function of the O-GlcNAcylation of nNOS in neuronal apoptosis during glutamate excitotoxicity, suggesting a novel therapy strategy for ischemic stroke.

Hck Promotes Neuronal Apoptosis Following Intracerebral Hemorrhage.

The hematopoietic cell kinase (Hck) is a member of the Src family protein kinases which regulates many signal transduction pathways including cell growth, proliferation, differentiation, migration, and apoptosis. However, the expression and function of Hck after intracerebral hemorrhage (ICH) are unknown. Western blot, immunohistochemistry, and immunofluorescence showed that Hck was obviously up-regulation in neurons adjacent to the hematoma after ICH. In addition, the temporary raise of Hck expression was paralleled with the expression of p53, Bax, and active caspase-3, suggesting that Hck was involved in neuronal apoptosis. Hck siRNA dramatically decrease hemin-induced expression of p53, Bax, and active caspase-3 as well as the amount of apoptotic SH-SY5Y cells in vitro. Furthermore, Hck interacted with p53. Hence, Hck might promote neuronal apoptosis via p53 signaling pathway after ICH.

Up-regulation of MCM3 Relates to Neuronal Apoptosis After Traumatic Brain Injury in Adult Rats.

Minichromosome maintenance complex component 3, one of the minichromosome maintenance proteins, functions as a part of pre-replication complex to initiate DNA replication in eukaryotes. Minichromosome maintenance complex component 3 (MCM3) was mainly implied in cell proliferation and tumorigenesis. In addition, MCM3 might play an important role in neuronal apoptosis. However, the functions of MCM3 in central nervous system are still with limited acquaintance. In this study, we performed a traumatic brain injury (TBI) model in adult rats. Western blot and immunohistochemistry staining showed up-regulation of MCM3 in the peritrauma brain cortex. The expression patterns of active caspase-3 and Bax, Bcl-2 were parallel with that of MCM3. Immunofluorescent staining and terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling suggested that MCM3 was involved in neuronal apoptosis. In conclusion, our data indicated that MCM3 might play an important role in neuronal apoptosis following TBI. Further understanding of these insights could serve as the basis for broadening the therapeutic scope against TBI.

Spatiotemporal Patterns of RING1 Expression after Rat Spinal Cord Injury.

Ring finger protein 1 (RING1) is a RING domain characterized protein belonging to the RING finger family. It is an E3 ubiquitin-protein ligase that mediated monoubiquitination of histone H2A and the core component of PRC1 complex, which is the repressive multiprotein complex of Polycomb group (PcG). Previous studies showed the important tumorigenic role of RING1 via promoting cell proliferation and the crucial function in maintaining transcriptional program stability during development. However, its mechanism for spinal cord injury (SCI) is still unknown. In our research, we established an acute SCI model in adult rats and studied the expression and function profiles of RING1. RING1 protein level detected by western blot peaked at day 3 after trauma and then decreased gradually. Immunohistochemistry showed the increase of RING1 expression displayed in the white matter more obviously than in the gray matter. Furthermore, increased co-expression of RING1 and GFAP confirmed activated astrocytes in injured spinal cord via double immunofluorescence staining. Meanwhile, we also found the co-localization of PCNA, a famous marker of proliferative cells, with RING1 and GFAP, which indicated RING1 might play a role in astrocyte proliferation after SCI. In vitro studies, RING1 protein level in C6 cells increased after LPS challenge and RING1 was required for astrocyte proliferation and activation induced by LPS. In summary, we took a new insight into the function of RING1 in the cellular and molecular mechanism underlying the pathophysiology of SCI.

GFAP expression is regulated by Pax3 in brain glioma stem cells.

Glioblastomas are understood to evolve from brain glioma stem cells (BGSCs), and yet the biology underlying this model of tumorigenesis is largely unknown. Paired box 3 protein (Pax3) is a member of the paired box (Pax) family of transcription factors that is normally expressed during embryonic development, but has recently been implicated in tumorigenesis. The present study demonstrated that Pax3 is differentially expressed in U87MG human glioma cell, BGSC and normal 1800 human astrocyte lines. Herein, we identified that the glial fibrillary acidic protein (GFAP), a major intermediate filament protein of mature astrocytes, is directly downregulated during the differentiation of BGSCs via the binding of Pax3 to the promoter region of GFAP. Moreover, siRNA silencing of Pax3 arrested BGSC differentiation, while overexpression of Pax3 promoted the differentiation in BGSCs. Furthermore, we studied the cell proliferation, invasion, apoptosis, differentiation and expression of Pax3 and GFAP in Pax3 siRNA-knockdown and Pax3-overexpressing BGSC models by CCK-8, Transwell migration, flow cytometry and western blot assays. The results indicate that Pax3 regulates GFAP expression, and that Pax3 may contribute to the evolution of BGSCs towards malignancy.

Knockdown of PFTK1 Inhibits the Migration of Glioma Cells.

The prognosis of glioma patients is generally poor, so it is urgent to find out the underlying molecular mechanisms. PFTK1 is a member of cyclin-dependent kinases (Cdks) family and has been reported to contribute to tumor migration and invasion. In this study, we aimed to explore the expression and function in human glioma. Western blot and immunohistochemistry were used to evaluate the expression of PFTK1. PFTK1 expression was higher in glioma tissues compared with normal brain tissues, and its level was associated with the WHO grade in Western blot analysis. The suppression of PFTK1 expression by RNA interference was shown to inhibit the migration of glioma cells. Knockdown of PFTK1 increases E-cadherin expression and decreases vimentin expression. These data show that PFTK1 may participate in the pathogenic process of glioma, suggesting that PFTK1 can become a potential therapeutic strategy for gastric cancer.

Astrocyte-conditioned medium attenuates glutamate-induced apoptotic cell death in primary cultured spinal cord neurons of rats.

OBJECTIVES: To better understand the neuroprotective role of astrocytes in spinal cord injury (SCI), we investigated whether astrocyte-conditioned medium (ACM) can attenuate glutamate-induced apoptotic cell death in primary cultured spinal cord neurons. METHODS: Spinal cord neurons were pretreated with ACM for 24 hours. Subsequently, they were exposed to glutamate (125 µM) for 1 hour. The neurons were then incubated for 24 hours. Following that, measurements assessing cell viability and lactate dehydrogenase (LDH) release were performed. Apoptosis was confirmed through cell morphology using Hoechst 33342 staining and terminal deoxynucleotidyl transferase dUTP-mediated nicked end labeling (TUNEL) assay. Assessment for expression of apoptotic enzymes, including Caspase-3, Bcl-2 and Bax, was performed using Western Blot Analysis. RESULTS: Astrocyte-conditioned medium pretreatment of neurons showed both an increase in spinal cord neuron viability and a decrease in LDH release in a dose-dependent pattern. Moreover, pretreatment seems to attenuate glutamate-induced apoptotic cell death, antagonise glutamate-induced up-regulation of Caspase-3 expression and downregulate Bcl-2/Bax protein expression ratio. CONCLUSIONS: By attenuating glutamate-induced apoptotic cell death in primary cultured spinal cord neurons of rats, ACM seems to provide a neuroprotective effect by regulating apoptosis-related protein expression. Our results provide an experimental basis for clinical applications and potential therapeutic use of ACM in SCI.

Nrdp1 is Associated with Neuronal Apoptosis in Lipopolysaccharide-Induced Neuroinflammation.

Neuregulin receptor degradation protein-1 (Nrdp1), a kind of ring finger E3 ubiquitin ligase, is expressed in several adult tissues, including the heart, testis, prostate and brain. Studies of this molecule have demonstrated its great importance in regulating cell growth, apoptosis and oxidative stress in various cell types. However, information regarding its expression and possible function in the central nervous system is still limited. In this study, we performed a neuroinflammation model by lipopolysaccharide (LPS) lateral ventral injection in adult rats. It was found that the expression of Nrdp1 was significantly increased in cerebral cortex after LPS injection. Immunofluorescence indicated that Nrdp1 was located in the neurons, but not astrocytes or microglia. Furthermore, there was a concomitant up-regulation of active caspase-3 and decreased expression of BRUCE (an inhibitor of apoptosis protein). In addition, decreasing Nrdp1 levels by RNA interference in cortical primary neurons reduced active caspase-3 expression but induced up-regulation of BRUCE. Collectively, all these results suggested that Nrdp1 might play a role in neuronal apoptosis by reducing the expression of BRUCE in neuroinflammation after LPS injection.

microRNA response element­regulated TIKI2 expression suppresses the tumorigencity of malignant gliomas.

Glioma is the most common brain malignancy and has a very poor prognosis. The current treatment options have a minimal benefit on prolonging patient survival time. Accumulating data have shown that the WNT signaling pathway has a critical function in the progression and invasion of glioma. Thus, targeting WNT signaling appears to be an effective anti­glioma strategy. TIKI2 was recently found to suppress the activation of the WNT signaling pathway by post­translationally modifying secreted WNT proteins. The implication of TIKI2 aberrance in cancers and its potential therapeutic effect, however, has not been studied. In the present study, a glioma­specific adenoviral vector was constructed, which was regulated by response elements of miR­124, to express TIKI2 in glioma cells (Ad­TIKI2­124). Ad­TIKI2­124 was found to potently suppress the activation of WNT signaling in glioma cells. This inhibitory effect on the WNT signaling pathway lead to the reduction in proliferation, colony formation ability and invasion of glioma cell lines. In addition, animal experiments confirmed that the expression of the Ad­TIKI2­124 construct could compromise the tumorigenicity of glioma cells in vivo. Furthermore, this glioma­selective TIKI2 expression protected normal cells from toxicity. In conclusion, the present study demonstrated that adenovirus­mediated TIKI2 therapy may be used for glioma treatment and therefore warrants further clinical studies.

Up-regulated expression of Bnip3L after intracerebral hemorrhage in adult rats.

Bnip3L, also known as NIX, is a homolog of the E1B 19K/Bcl-2 binding and pro-apoptotic protein Bnip3 which can bind to Bcl-2 to elaborate that effect. In tumor cells, Bnip3L played a role in tumor growth inhibition, but some studies argued hypoxia-induced autophagy via Bnip3L was a survival mechanism that promoted tumor progression. In heart muscle, it related to decreased myocardial function. However, its function in intracerebral hemorrhage (ICH) is still not clear. In this frame, we found the Bnip3L expression increased in the perihematomal region in adult rats after performed ICH. Double immunofluorenscence staining manifested that Bnip3L co-located with neurons, not astrocytes or oligodendrocytes. Furthermore, we detected that neuronal apoptosis marker active caspase-3 had colocalizations with Bnip3L. In addition, colocalizations and co-immunoprecipitation between Bnip3L and Bcl-2, consistent with previous study, were also found. All our findings suggested that Bnip3L might be involved in the pathophysiology of ICH.

Upregulation of Ras homolog enriched in the brain (Rheb) in lipopolysaccharide-induced neuroinflammation.

Ras homolog enriched in the brain (Rheb) is a homolog of Ras GTPase that regulates cell growth, proliferation, and cell cycle via mammalian target of rapamycin (mTOR). Recently, it has been confirmed that Rheb activation not only promotes cellular proliferation and differentiation but also enhances cellular apoptosis in response to diverse toxic stimuli. However, the function of Rheb in the central nervous system (CNS) is still with limited understanding. To elaborate whether Rheb was involved in CNS injury, we performed a neuroinflammatory model by lipopolysaccharide (LPS) lateral ventral injection in adult rats. Upregulation of Rheb was observed in the brain cortex by performing western blotting and immunohistochemistry. Double immunofluorescent staining demonstrated that Rheb was mainly in active astrocytes and neurons. PCNA and active caspase-3 were upregulated, and co-labeling with Rheb, which indicated that Rheb might be relevant to astrocytic proliferation and neuronal apoptosis following the inflammatory response by LPS-induced. Furthermore, we also found that the expression profiles of cyclinD1 and CDK4 were parallel with that of Rheb in a time-space dependent manner. Finally, knocking down Rheb by siRNA and treatment with rapamycin or lovastatin showed that not only astrocytic proliferation decreased but also neuronal protection. Based on our data, we suggested that Rheb might play an important role in physiological and pathological functions following neuroinflammation caused by LPS, which might provide a potential target to the treatment of neuroinflammation.

Expression of G-protein-coupled receptor kinase 6 (GRK6) after acute spinal cord injury in adult rat.

Spinal cord injury frequently results in permanent loss of neurological function. It includes many complex molecular and biochemical mechanisms. G-protein-coupled receptor kinase 6 (GRK6) is an intracellular kinase that regulates the sensitivity of certain G-protein-coupled receptors. Some studies reported GRK2 and GRK5 modulate the NFκB pathway in macrophages. Additionally, GRK2 is referred to as regulating activation of spinal cord microglia and GRK6 expression is significantly elevated in most brain regions in the MPTP-lesioned parkinsonian monkeys. However, the expression and function of GRK6 in nervous system lesion and repair are not well understood. In this study, we performed an acute spinal cord injury (SCI) model in adult rats. Western blot analysis showed the expression of GRK6 was upregulated significantly at protein level in spinal cord after SCI. Immunohistochemistry and immunofluorescence revealed wide expression of GRK6 in the normal spinal cord. After injury, GRK6 expression was increased predominantly in microglia, which expressed F4/80 (marker of macrophages and activated microglia) strongly. To understand whether GRK6 played a role in microglia activation, we applied lipopolysaccharide (LPS) to induce microglia activation in vitro. Western blot analysis demonstrated up-regulation in GRK6 protein expression after LPS stimulation was time- and dose-dependent and that up-regulation in F4/80 expression was concomitant with GRK6. These data suggested that GRK6 might be involved in the pathophysiology of SCI.