Normal Neuronal Cells Research Articles

Anti-cancer effects of thymoquinone in mouse neuroblastoma (Neuro-2a) cells through caspase-3 activation with down-regulation of XIAP

Thymoquinone (TQ) is a bioactive component derived from the medicinal plant Nigella sativa. Recent studies reported that TQ exhibited cytotoxic effects in several cancer cell lines. Currently, no information in the literature is found concerning its mechanisms and cytotoxicity on neuroblastoma cells. In this study, the cytotoxicity of TQ in mouse neuroblastoma cells (Neuro-2a) was investigated. Our results showed that TQ significantly reduced viability of Neuro-2a cells than normal neuronal cells. Apoptosis induction by TQ was confirmed by DAPI and AO/PI staining. TQ triggered the apoptotic pathway, which was characterized by increased Bax/Bcl-2 ratio. TQ significantly increased the expression of pro-apoptotic protein Bax, whereas decreased the expression of anti-apoptotic protein Bcl-2, which leads to the release of cytochrome c from mitochondria into the cytoplasm. Moreover, TQ treatment directs the activation of caspase-3 followed by the cleavage of poly(ADP-ribose) polymerase (PARP). Interestingly, we also observed that TQ down-regulated caspase inhibitor X-linked inhibitor of apoptosis protein (XIAP). These results indicate that TQ induces apoptosis via caspase-3 activation with down-regulation of XIAP in Neuro-2a cells.

Regulatory Factor X1-induced Down-regulation of Transforming Growth Factor β2 Transcription in Human Neuroblastoma Cells

Regulatory factor X (RFX) proteins are transcription factors. Seven mammalian RFX proteins have been identified. RFX1 is the prototype RFX. However, its biological functions are not known. Here, RFX1 overexpression reduced fetal bovine serum-stimulated proliferation of SH-SY5Y cells, a human neuroblastoma cell line. This inhibition is associated with decreased transforming growth factor β2 (TGFβ2) and phospho-extracellular signal-regulated kinase (ERK). Exogenous TGFβ2 increased cell proliferation and phospho-ERK in cells overexpressing RFX1. An anti-TGFβ2 antibody and PD98059, an ERK activation inhibitor, inhibited SH-SY5Y cell proliferation. TGFβ2 promoter activity was decreased in cells overexpressing RFX1. Chromosome immunoprecipitation assay showed that RFX1 bound the TGFβ2 promoter. RFX1 down-regulation increased TGFβ2 in SH-SY5Y and HCN-1A cells, a normal human neuronal cell line. More importantly, TGFβ2 concentrations were negatively correlated with RFX1 levels in human medulloblastoma tissues with a R(2) of 0.464. These results suggest that RFX1 reduces cell proliferation through inhibiting the TGFβ2-ERK signaling pathway. RFX1 blocks TGFβ2 expression through its direct action on TGFβ2 transcription. This effect also appears in human brain tumor tissues. Because TGFβ is known to be involved in cancer development, our results provide initial evidence to suggest that RFX1 may play an important role in human tumor biology.

Abstract LB-210: Abrogation of mutant TP53 radiation resistance by lithium induced CTNNB1 (β-catenin) activation in medulloblastoma.

Abstract Introduction: Medulloblastoma (MB) is the most common paediatric malignant brain neoplasm. 30 % of patients will reccur and die of their tumour. TP53 mutations confer radiation resistance; we have recently demonstrated that somatic mutations in TP53 are predictive of poor survival in patients with MB. Patients with both TP53 and WNT activating CTNNB1 (β-catenin) mutations demonstrate improved survival compared to patients with TP53 mutations alone. WNT pathway is abrogated in 10% of MB. Lithium Chloride (LiCl) treatment constitutively activates WNT pathway by inhibition of Gsk3β-mediated degradation of β-catenin. Radiation therapy is key treatment in MB; hence we hypothesized that activation of WNT/β-catenin signalling can rescue radiation resistance of TP53 mutant phenotype, sensitize MB cells to radiation and can be achieved with administration of LiCl. Methods: Human MB, TP53 wild type (wt) and mutant (mut), all CTNNB1 wt cell lines were used to investigate radiation response and effect of LiCl on cell survival by clonogenic assays. Functional roles of TP53 and CTNNB1 mutations in response to LiCl/radiation treatments were assessed by transfection of mutant genes on a wild type background. Double strand (DS) DNA damage-repair was assessed by γH2AX immunofluorescence (IF). Normal neuronal stem cells were used to assess effects of LiCl/radiation on normal brain tissue by clonogenic assay. Results: TP53 mut MB cells were resistant to radiation compared to TP53 wt cells by clonogenic assay (p<0.01), accompanied by more efficient DNA damage response manifested by lower numbers of γH2AX foci. Transfection of R175H dominant-negative TP53 mutation to wt cells resulted in increased clonogenic potential post-radiation (p<0.01). MB cells transfected with CTNNB1 S33Y mutation revealed increased radiosenstivity especially in TP53 mut MB (p<0.01). LiCl treatment resulted in nuclear translocation of β-catenin and signalling activation by luciferase assay, which is consistent with activation of WNT pathway. Treatment with LiCl increased radiation sensitivity of both wt and mut TP53 cells (p<0.01; p<0.05 respectively), accompanied by increased DS DNA damage and delayed repair in both lines by γH2AX IF. LiCl treatment of normal neuronal stem cells did not sensitize them to radiation suggesting that LiCl is a tumour specific treatment. Conclusions: Constitutive activation of β-catenin by LiCl sensitizes TP53 mut MB cells to radiation possibly through activation of β-catenin/TCF mediated transcription and impaired DS DNA repair response. These findings may explain survival advantage in CTNNB1 mut MB patients harbouring TP53 mutation. Knowing safety of LiCl and its neuroprotective capacity in normal neuronal cells, oral administration of LiCl may represent an attractive novel therapy for MB patients. Investigations of the molecular mechanisms governing this phenomenon are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-210. doi:1538-7445.AM2012-LB-210

Abstract 1984: Nature's pharmacy: eburnamonine and its derivative as therapeutics against brain tumors

Abstract Brain tumors are highly aggressive and often afflict younger patients. In 2011 there were 22,340 estimated new cases of brain tumors in the United States with 40% arising from metastatic lesions originating mostly from lung, breast, and melanoma tumors. Treatment options remain limited and include steroids, whole brain radiation, and surgery. Because of difficulty penetrating the blood-brain barrier (BBB), chemotherapy is usually not a viable option. Therefore, any intervention that can block the malignant colonization of the brain and/or eliminate symptomatic and occult brain lesions will significantly improve both quality of life and survival. To target brain tumors, a successful therapy has to meet the following criteria: 1) penetrate the BBB; 2) exhibit specificity for the tumor cells; and 3) have minimal off-target effects. Here we investigate a Periwinkle minor derived natural product, eburnamonine (EBN), and its structural analog, 15-methylene-(-)-eburnamonine (15-M-EBN), as therapeutic agents against brain lesions from glioblastoma multiforme and metastatic breast cancer. The two common subtypes of breast tumors giving rise to brain metastases are triple negative and Her2 positive primary tumors. Potency of EBN and 15-M-EBN was evaluated in cultures of chemosensitive (U87) and chemoresistant (SF767) glioma cell lines and ‘brain-seeking’ metastatic breast cancer cells, MDA-MB-231BR (triple negative) and MDA-MB-231BR-Her2 (Her2 positive). To test whether our compounds can overcome the environment-mediated drug resistance (EM-DR) cells were treated in the context of extracellular matrix (ECM) (hyaluronic acid, collagen IV/laminin, and collagens I and IV/laminin/fibronectin to reconstruct the brain, mammary gland, and bone marrow microenvironments respectively). The off-target toxicity of EBN and 15-M-EBN was tested on normal neuronal cells, bone marrow stromal cells, and non-malignant mammary epithelial cells. To evaluate the capacity of EBN and 15-M-EBN to penetrate the BBB, we utilized a transwell model with hCMEC/D3 cell monolayer mimicking the BBB. We demonstrate that EBN and 15-M-EBN are cytotoxic against glioma and ‘brain-seeking’ metastatic breast cancer cells (LC50=42 micromolar and 14 micromolar respectively) with minimal toxicity against non-malignant cells. Moreover, 15-M-EBN is active in the tumor environment and predicted to cross the BBB and have anti-cancer stem cell activity. Our preclinical data establish that 15-M-EBN meets the major criteria for a successful therapeutic agent to target brain lesions, both primary and metastatic, due to its ability to: 1) exhibit specificity for the tumor cells; 2) have minimal neurotoxicity and off-target effects; and 3) penetrate the BBB. In vivo studies are in progress to establish a pro-drug strategy to increase water solubility and bioavailability of 15-M-EBN for further clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1984. doi:1538-7445.AM2012-1984

Histological analysis of the third ventricle floor in hydrocephalic and nonhydrocephalic brains: application to neuroendocrine complications following third ventriculostomy procedures

Endocrine dysfunction following endoscopic third ventriculostomy (ETV) is rare, but it has been reported. In the present study the authors sought to determine the histological nature of the floor of the third ventricle in hydrocephalic brains to better elucidate this potential association. Five adult cadaveric brains with hydrocephalus were examined. Specifically, the floors of the third ventricle of these specimens were studied histologically. Age-matched controls without hydrocephalus were used for comparison. Although it was thinned in the hydrocephalic brains, the floor of the third ventricle had no significant difference between the numbers of neuronal cell bodies versus nonhydrocephalic brains. Although uncommon following ETV, endocrine dysfunction has been reported. Based on the present study, this is most likely to be due to the injury of normal neuronal cell bodies found in this location, even in very thinned-out tissue.

The Effect of Combined Therapy of Exercise and Nootropic Agent on Cognitive Function in Focal Cerebral Infarction Rat Model

ObjectiveTo investigate the effect of combined therapy of exercise and nootropic agent on cognitive function in a focal cerebral infarction rat model.MethodForty 10-week old male Sprague-Dawley rats were subjected to photothrombotic cerebral infarction of the left parietal lobe. All rats were randomly divided into 4 groups: group A was photothrombotic cerebral infarction rats without any treatment (n=10); group B was photothrombotic cerebral infarction rats with swimming exercise (n=10); group C was photothrombotic cerebral infarction rats with oral administration of acetyl-L-carnitine (n=10); group D was photothrombotic cerebral infarction rats with swimming exercise and oral administration of acetyl-L-carnitine (n=10). Cognitive function was evaluated using the Morris water maze test on the 1st day, and the 1st, 2nd, and 4th week after the induction of cerebral infarction. The activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) in the hippocampus were measured. The neuronal cells of the hippocampus were histopathologically evaluated.ResultsThe escape latency was shorter in groups B, C, and D than in group A. However, the differences were not statistically significant at the 1st, 2nd and 4th week. The activity of SOD was the highest in group D. The level of MDA was the lowest in group D. We observed more normal neuronal cells in groups B, C, and D.ConclusionThe combined therapy of exercise and nootropic agent was helpful in ameliorating oxidative stress in the focal cerebral infarction rat model. However, the effect did not translate into improvement of cognitive function.

Pituitary adenylate cyclase‐activating polypeptide (PACAP) stimulates the expression and the release of tissue plasminogen activator (tPA) in neuronal cells: involvement of tPA in the neuroprotective effect of PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) and tissue plasminogen activator (tPA) play important roles in neuronal migration and survival. However, a direct link between the neurotrophic effects of PACAP and tPA has never been investigated. In this study, we show that, in PC12 cells, PACAP induced a 9.85-fold increase in tPA gene expression through activation of the protein kinase A- and protein kinase C-dependent signaling pathways. In immature cerebellar granule neurons (CGN), PACAP stimulated tPA mRNA expression and release of proteolytically active tPA. Immunocytochemical labeling revealed the presence of tPA in the cytoplasm and processes of cultured CGN. The inhibitory effect of PACAP on CGN motility was not affected by the tPA substrate plasminogen or the tPA inhibitor plasminogen activator inhibitor-1. In contrast, plasminogen activator inhibitor-1 significantly reduced the stimulatory effect of PACAP on CGN survival. Altogether, these data indicate that tPA gene expression is activated by PACAP in both tumoral and normal neuronal cells. The present study also demonstrates that PACAP stimulates the release of tPA which promotes CGN survival by a mechanism dependent of its proteolytic activity.

Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells

Aging is one of the major risk factors of cancer. The onset of cancer can be postponed by pharmacological and dietary anti-aging interventions. We recently found in yeast cellular models of aging that lithocholic acid (LCA) extends longevity. Here we show that, at concentrations that are not cytotoxic to primary cultures of human neurons, LCA kills the neuroblastoma (NB) cell lines BE(2)-m17, SK-n-SH, SK-n-MCIXC and Lan-1. In BE(2)-m17, SK-n-SH and SK-n-MCIXC cells, the LCA anti-tumor effect is due to apoptotic cell death. In contrast, the LCA-triggered death of Lan-1 cells is not caused by apoptosis. While low concentrations of LCA sensitize BE(2)-m17 and SK-n-MCIXC cells to hydrogen peroxide-induced apoptotic cell death controlled by mitochondria, these LCA concentrations make primary cultures of human neurons resistant to such a form of cell death. LCA kills BE(2)-m17 and SK-n-MCIXC cell lines by triggering not only the intrinsic (mitochondrial) apoptotic cell death pathway driven by mitochondrial outer membrane permeabilization and initiator caspase-9 activation, but also the extrinsic (death receptor) pathway of apoptosis involving activation of the initiator caspase-8. Based on these data, we propose a mechanism underlying a potent and selective anti-tumor effect of LCA in cultured human NB cells. Moreover, our finding that LCA kills cultured human breast cancer and rat glioma cells implies that it has a broad anti-tumor effect on cancer cells derived from different tissues and organisms.

Regulation of MET by FOXP2, Genes Implicated in Higher Cognitive Dysfunction and Autism Risk

Autism spectrum disorder (ASD) is a highly heritable, behaviorally defined, heterogeneous disorder of unknown pathogenesis. Several genetic risk genes have been identified, including the gene encoding the receptor tyrosine kinase MET, which regulates neuronal differentiation and growth. An ASD-associated polymorphism disrupts MET gene transcription, and there are reduced levels of MET protein expression in the mature temporal cortex of subjects with ASD. To address the possible neurodevelopmental contribution of MET to ASD pathogenesis, we examined the expression and transcriptional regulation of MET by a transcription factor, FOXP2, which is implicated in regulation of cognition and language, two functions altered in ASD. MET mRNA expression in the midgestation human fetal cerebral cortex is strikingly restricted, localized to portions of the temporal and occipital lobes. Within the cortical plate of the temporal lobe, the pattern of MET expression is highly complementary to the expression pattern of FOXP2, suggesting the latter may play a role in repression of gene expression. Consistent with this, MET and FOXP2 also are reciprocally expressed by differentiating normal human neuronal progenitor cells (NHNPs) in vitro, leading us to assess whether FOXP2 transcriptionally regulates MET. Indeed, FOXP2 binds directly to the 5' regulatory region of MET, and overexpression of FOXP2 results in transcriptional repression of MET. The expression of MET in restricted human neocortical regions, and its regulation in part by FOXP2, is consistent with genetic evidence for MET contributing to ASD risk.

MicroPET/CT Imaging of an Orthotopic Model of Human Glioblastoma Multiforme and Evaluation of Pulsed Low-Dose Irradiation

Glioblastoma multiforme (GBM) is an aggressive tumor that typically causes death due to local progression. To assess a novel low-dose radiotherapy regimen for treating GBM, we developed an orthotopic murine model of human GBM and evaluated in vivo treatment efficacy using micro-positron-emission tomography/computed tomography (microPET/CT) tumor imaging. Orthotopic GBM xenografts were established in nude mice and treated with standard 2-Gy fractionation or 10 0.2-Gy pulses with 3-min interpulse intervals, for 7 consecutive days, for a total dose of 14 Gy. Tumor growth was quantified weekly using the Flex Triumph (GE Healthcare/Gamma Medica-Ideas, Waukesha, WI) combined PET-single-photon emission CT (SPECT)-CT imaging system and necropsy histopathology. Normal tissue damage was assessed by counting dead neural cells in tissue sections from irradiated fields. Tumor engraftment efficiency for U87MG cells was 86%. Implanting 0.5 × 10(6) cells produced a 50- to 70-mm(3) tumor in 10 to 14 days. A significant correlation was seen between CT-derived tumor volume and histopathology-measured volume (p = 0.018). The low-dose 0.2-Gy pulsed regimen produced a significantly longer tumor growth delay than standard 2-Gy fractionation (p = 0.045). Less normal neuronal cell death was observed after the pulsed delivery method (p = 0.004). This study successfully demonstrated the feasibility of in vivo brain tumor imaging and longitudinal assessment of tumor growth and treatment response with microPET/CT. Pulsed radiation treatment was more efficacious than the standard fractionated treatment and was associated with less normal tissue damage.

A novel PKC-ι inhibitor abrogates cell proliferation and induces apoptosis in neuroblastoma

Protein Kinase C-iota (PKC-ι), an atypical protein kinase C isoform manifests its potential as an oncogene by targeting various aspects of cancer cells such as growth, invasion and survival. PKC-ι confers resistance to drug-induced apoptosis in cancer cells. The acquisition of drug resistance is a major obstacle to good prognosis in neuroblastoma. The focus of this research was to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-ι inhibitor in neuroblastoma cell proliferation and apoptosis. ICA-1 specifically inhibits the activity of PKC-ι but not that of PKC-zeta (PKC-ζ), the closely related atypical PKC family member. The IC50 for the kinase activity assay was approximately 0.1μM which is 1000 times less than that of aurothiomalate, a known PKC-ι inhibitor. Cyclin dependent kinase 7 (Cdk7) phosphorylates cyclin dependent kinases (cdks) and promotes cell proliferation. Our data shows that PKC-ι is an in vitro Cdk7 kinase and the phosphorylation of Cdk7 by PKC-ι was potently inhibited by ICA-1. Furthermore, our data shows that neuroblastoma cells proliferate via a PKC-ι/Cdk7/cdk2 cell signaling pathway and ICA-1 mediates its antiproliferative effects by inhibiting this pathway. ICA-1 (0.1μM) inhibited the in vitro proliferation of BE(2)-C neuroblastoma cells by 58% (P=0.01). Additionally, ICA-1 also induced apoptosis in neuroblastoma cells. Interestingly, ICA-1 did not affect the proliferation of normal neuronal cells suggesting its potential as chemotherapeutic with low toxicity. Hence, our results emphasize the potential of ICA-1 as a novel PKC-ι inhibitor and chemotherapeutic agent for neuroblastoma.

Electrophysiological Modeling of Channelrhodophsin-2 in Cardiac Cells

Purpose: With the recent interest in Channelrhodopshin-2 (Chr2) in neurological experiments, researchers have begun to investigate the utility of light-activated ion channels in other electrically active cell types, including human embryonic stem cell-derived cardiomyocytes (Abilez et al. 2010). However, the impact of Chr2 in action potential synchronization in cardiac cells is not yet fully realized, as neuronal and cardiac cells differ in electrical behavior. In the past, baseline electrophysiological models for normal neuronal and cardiac cells have been developed and recent attempts have been made to characterize Chr2 in neuron excitation control. However, these approaches do not capture the resulting ion channel current, nor have they been adapted for cardiac cells. By characterizing Chr2 currents within existing cell models, simulations can be conducted concurrently with experiments for principle validation and experiment optimization.Methods: A kinetic model for Chr2 activation (Nikolic et al. 2006) was extended to an ion current formulation from current-voltage comparisons in the literature. This current was introduced into a ventricular cell model (ten Tusscher et al. 2003) and embedded in an implicit non-linear finite element framework (Wong et al. 2010) to perform simulations at cellular, tissue, and organ levels.Results: To illustrate the features of our novel light-activated cell model, we present selected examples to show the benefits of concurrent modeling. At the cellular level, we explore the impact of photostimulation strength, duration, and frequency in Chr2-manipulated ventricular cells. At the tissue level, we evaluate the feasibility of using such manipulated cells as pacemakers in the heart.Conclusion: By “transducing” cell models with Chr2, we can not only virtually probe characteristics of light-activated functional cells for novel applications of Chr2 in cardiac cells and other electrically active cells, but also optimize experiments by qualitatively predicting experimental results.

Plantaricin A, a peptide pheromone produced by Lactobacillus plantarum, permeabilizes the cell membrane of both normal and cancerous lymphocytes and neuronal cells

Antimicrobial peptides produced by multicellular organisms protect against pathogenic microorganisms, whereas such peptides produced by bacteria provide an ecological advantage over competitors. Certain antimicrobial peptides of metazoan origin are also toxic to eukaryotic cells, with preference for a variety of cancerous cells. Plantaricin A (PlnA) is a peptide pheromone with membrane permeabilizing strain-specific antibacterial activity, produced by Lactobacillus plantarum C11. Recently, we have reported that PlnA also permeabilizes cancerous rat pituitary cells (GH(4) cells), whereas normal rat anterior pituitary cells are resistant. To investigate if preferential effect on cancerous cells is a general feature of PlnA, we have studied effects of the peptide on normal and cancerous lymphocytes and neuronal cells. Normal human B and T cells, Reh cells (from human B cell leukemia), and Jurkat cells (from human T cell leukemia) were studied by flow cytometry to detect morphological changes (scatter) and viability (propidium iodide uptake), and by patch clamp recordings to monitor membrane conductance. Ca(2+) imaging based on a combination of fluo-4 and fura-red was used to monitor PlnA-induced membrane permeabilization in normal rat cortical neurons and glial cells, PC12 cells (from a rat adrenal chromaffin tumor), and murine N2A cells (from a spinal cord tumor). All the tested cell types were affected by 10-100 microM PlnA, whereas concentrations below 10 microM had no significant effect. We conclude that normal and cancerous lymphocytes and neuronal cells show similar sensitivity to PlnA.

Abstract 4276: In-vivo imaging of glioma cancer stem cells

Abstract Recent data support that glioblastoma (GB) is organized hierarchically with a small population of cancer stem cells (CSCs), able to repopulate an entire tumor. The cell of origin in GB is currently unknown, but animal data suggest that CSCs derive from the normal SC population and that the normal stem cell niche in the brain may harbor CSCs in patients with GB. Because therapies targeting CSCs directly may potentially improve patient outcome, novel means are needed to track CSCs and their interaction with the tumor microenvironment to develop and test novel therapeutic approaches. Using limiting dilution assays we studied if glioma CSCs (GCSCs) can be serially transplanted in Rag2−/− γc −/− KO mice. Radiation responses of the population of glioma GCSCs were investigated using FACS. Radiation sensitivity assessed in sphere forming capacity assays. To studying proteasome activity in normal neuronal stem cells a transgenic animal model was generated that reports activities of the proteasome. Finally, the role of the normal tissue SC niche for radiation therapy outcome was investigated in a retrospective analysis of patient data. GCSCs showed increased tumorigenicity over non-tumorigenic cells in the Rag2−/− γc −/− mouse model supporting a hierarchical CSC model in GB. GCSCs could be serially transplanted over at least 5 generations and reassembled the typical histopathology of GB. When unselected glioma cells were subjected to fractionated irradiation, cells were highly enriched for GSCSs. We did not detect differences in H2AX phosphorylation directly after irradiation, which was consistent with previous studies reporting comparable amounts of initial DNA damage in GCSCs and non-tumorigenic cells. Single cell sphere formation after irradiation indicated a 1.3-fold increase in radioresistance for GCSCs (measured at 10% survival) supporting previously reported increased DNA repair in CSCs. Using sections of brains from transgenic animals, which report lack of proteasome function by accumulation of ZsGreen, we identified a rare cell population in the subventricular zone of the brain that lacks proteasome function, consistent with normal stem cell origin of GCSCs. Clinically, radiation dose of 43Gy and higher delivered to the periventricular region of the brain of patients with high-grade glioma was correlated with improved progression-free survival and (n=55, 7.2 vs. 15 months, p=0.028, log-rank) and a decreased risk ratio in a Cox proportional Hazard model (p=0.034). We conclude that lack of proteasome activity is a valuable marker for tracking GCSCs and their treatment responses. Low proteasome activity seems to be shared by malignant and normal stem cells in the brain, supporting the hypothesis that normal tissue stem cells in the brain are the cells of origin in GB. Our clinical data indicate that the normal tissue stem cell niche in the brain may harbor GCSCs and that targeted treatment of this region could improve patient outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4276.

Snake venom toxin inhibits cell growth through induction of apoptosis in neuroblastoma cells

Snake venom toxin from Vipera lebetina turanica can induce apoptosis in many cancer cell lines, but there is no study about the apoptotic effect of snake venom toxin on human neuroblastoma cells. In this study, we investigated the apoptotic effect of snake venom toxin in human neuroblastoma SK-N-MC and SK-N-SH cells. Our result showed that cell detachment and apoptotic cell death were increased by snake venom toxin (1.25-10 microg/mL), but normal neuronal cells were not affected. Consistent with the induction of apoptosis, the level of reactive oxygen species (ROS) was increased, but mitochondrial membrane potential (MMP) was disrupted by treatment with snake venom toxin. However, the glutathione prevented snake venom toxin-induced cell growth inhibition. Snake venom toxin also increased the expression of pro-apoptotic protein Bax, but down-regulated anti-apoptotic protein Bcl-2. Therefore, these results showed that snake venom toxin from Vipera lebetina turanica causes apoptotic cell death of neuroblastoma cells through ROS dependent MMP disruption, and suggested that snake venom toxin may be applicable as an anti-cancer agent for neuroblastoma.

Subcellular localization of APMCF1 and its biological significance of expression pattern in normal and malignant human tissues

BackgroundAPMCF1 is a novel human gene first cloned from apoptotic MCF-7 cells. Our previous study found ectogenic APMCF1 could induce G1 arrest in hepatocarcinoma cell line HHCC. In order to search its broad expression profile for further understanding of its mechanism in tumor, we investigated a subcellular location of APMCF1 and performed an immunohistochemistry study including various tumor and normal tissues. Discovery from the expression characterization of AMPCF1 may have applicability in the analysis of its biological function in tumor.MethodsWe investigated subcellular localization of APMCF1 by transient transfection in green monkey kidney epithelial cells (COS-7) with a fusion protein vector pEGFP-APMCF1 and detected expression profile in a broad range of normal and malignant human tissues via tissue microarray (TMA) by immunohistochemistry with polyclonal antibody first produced in our laboratory.ResultsEGFP-APMCF1 was generally localized in the cytoplasm of COS-7 cell. Positive staining of APMCF1 was found in liver, lung, breast, colon, stomach, esophagus and testis, exhibited a ubiquitous expression pattern while its expression was up-regulated in tumor tissues compared with corresponding normal tissues. Normal brain neuron cells also showed expression of APMCF1, but negative in gliocyte cells and glioma. Both the normal and tumor tissues of ovary were absent of APMCF1 expression. Positive immunostaining for APMCF1 with large samples in liver, colon, esophagus, lung and breast carcinomas were 96% (51/53), 80% (44/55), 57% (30/53), 58% (33/57) and 34% (16/47) respectively.ConclusionThese results revealed a cytoplastic expression pattern of APMCF1 and up-regulated in tumour tissues suggesting APMCF1 may have potential relationship with oncogenesis. The data presented should serve as a useful reference for further studies of APMCF1 functions in tumorigenesis and might provide a potential anti-tumor target.

Markers Distinguishing Cancer Stem Cells from Normal Human Neuronal Stem Cell Populations in Malignant Glioma Patients

Markers Distinguishing Cancer Stem Cells from Normal Human Neuronal Stem Cell Populations in Malignant Glioma Patients

Depletion of Pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatases 1 and 2 by Bcr-Abl Promotes Chronic Myelogenous Leukemia Cell Proliferation through Continuous Phosphorylation of Akt Isoforms

The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway commonly occurs in cancers and is a crucial event in tumorigenesis. Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The PI3K/Akt pathway is activated by Bcr-Abl chimera protein and mediates the leukemogenesis in CML. However, the mechanism by which Bcr-Abl activates the PI3K/Akt pathway is not completely understood. In the present study, we found that pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1 and PHLPP2) were depleted in CML cells. We investigated the interaction between PHLPPs and Bcr-Abl in CML cell lines and Bcr-Abl+ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of PHLPP1 and PHLPP2, which dephosphorylated Ser-473 on Akt1, -2, and -3, resulting in inhibited proliferation of CML cells. The reduction of PHLPP1 and PHLPP2 expression by short interfering RNA in CML cells weakened the Abl kinase inhibitor-mediated inhibition of proliferation. In colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; colony-forming unit-granulocyte, macrophage; and burst-forming unit-erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced PHLPP1 and PHLPP2 expression and inhibited colony formation of Bcr-Abl+ progenitor cells, whereas depletion of PHLPP1 and PHLPP2 weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl+ progenitor cells. Thus, Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and continuously activates Akt1, -2, and -3 via phosphorylation on Ser-473, resulting in the proliferation of CML cells.

Cytopathological diagnosis of adult retinoblastoma in a vitrectomy specimen

Retinoblastoma (RB) is extremely rare in adults. We describe a case of RB diagnosed by cytology in a vitrectomy specimen of a 23-year-old patient who presented with diminished visual acuity and retinal detachment in the absence of a clinically-visible mass. Cytological examination of the vitreous fluid showed clusters of loosely cohesive atypical cells with high nuclear to cytoplasmic ratio and "salt and pepper" chromatin pattern in a background of normal neuronal retinal cells. Nuclear molding was present as well as numerous apoptotic bodies. The cells were focally positive for epithelial markers and showed strong and diffuse positivity for neuroendocrine markers. Ki-67 stained 90% of the "atypical cells" nuclei, in contrast to nonneoplastic retinal neuronal cells, which were negative for the marker. A diagnosis of RB was rendered, and subsequently was confirmed in the enucleation specimen. The cytological differential diagnosis is discussed as well as the role that cytology and immunohistochemistry can play in differentiating neoplastic cells from normal retinal cellular elements in vitreous fluid specimens.

Increased Dosage of Dyrk1A Alters Alternative Splicing Factor (ASF)-regulated Alternative Splicing of Tau in Down Syndrome

Two groups of tau, 3R- and 4R-tau, are generated by alternative splicing of tau exon 10. Normal adult human brain expresses equal levels of them. Disruption of the physiological balance is a common feature of several tauopathies. Very early in their life, individuals with Down syndrome (DS) develop Alzheimer-type tau pathology, the molecular basis for which is not fully understood. Here, we demonstrate that Dyrk1A, a kinase encoded by a gene in the DS critical region, phosphorylates alternative splicing factor (ASF) at Ser-227, Ser-234, and Ser-238, driving it into nuclear speckles and preventing it from facilitating tau exon 10 inclusion. The increased dosage of Dyrk1A in DS brain due to trisomy of chromosome 21 correlates to an increase in 3R-tau level, which on abnormal hyperphosphorylation and aggregation of tau results in neurofibrillary degeneration. Imbalance of 3R- and 4R-tau in DS brain by Dyrk1A-induced dysregulation of alternative splicing factor-mediated alternative splicing of tau exon 10 represents a novel mechanism of neurofibrillary degeneration and may help explain early onset tauopathy in individuals with DS.