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1.  Exogenous thioredoxin prevents ethanol-induced oxidative damage and apoptosis in mouse liver 
Hepatology (Baltimore, Md.)  2009;49(5):1709-1717.
Ethanol-induced liver injury is characterized by increased formation of reactive oxygen species (ROS) and inflammatory cytokines, resulting in the development of hepatic steatosis, injury and cell death by necrosis and apoptosis. Thioredoxin (Trx), a potent antioxidant and anti-inflammatory molecule with anti-apoptotic properties, protects animals from a number of inflammatory diseases. However, the effects of ethanol on Trx or its role in ethanol-induced liver injury are not known. Female C57BL/6 mice were allowed ad libitum access to a Lieber-deCarli ethanol diet with 5.4% of calories as ethanol for 2 days to acclimate them to the diet, followed by 2 days 32.4% of calories as ethanol or pair-fed control diet. Hepatic Trx-1 was decreased by ethanol feeding; daily supplementation with recombinant human Trx (rhTrx) prevented this ethanol-induced decrease. Therefore, we tested the hypothesis that administration of rhTrx during ethanol exposure would attenuate ethanol-induced oxidative stress, inflammatory cytokine production and apoptosis. Mice were treated with a daily intraperitoneal injection of either 5 g/kg of rhTrx or phosphate buffered saline (PBS).
Ethanol feeding increased accumulation of hepatic 4-hydroxynonenal (4-HNE) protein adducts, expression of hepatic tumor necrosis factor α (TNFα) and resulted in hepatic steatosis and increased plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In ethanol-fed mice, treatment with rhTrx reduced 4-HNE adduct accumulation, inflammatory cytokine expression, decreased hepatic triglyceride and improved liver enzyme profiles. Ethanol feeding also increased TUNEL positive cells, caspase-3 activity, and cytokeratin-18 staining in the liver. rhTrx treatment prevented these increases. In summary, rhTrx attenuated ethanol-induced increases in markers of oxidative stress, inflammatory cytokine expression, and apoptosis.
PMCID: PMC2895317  PMID: 19205032
alcohol; antioxidant; cytokines; TUNEL; inflammation
2.  Glioma cell integrin expression and their interactions with integrin antagonists 
Cancer therapy  2005;3A:325-340.
A panel of human glioma cell explants was screened for integrin expression by flow cytometry using ανβ-specific antibodies. A lower percentage of the glioma cells were positive for the ανβ3 (mean % positive = 20.8%) integrin, whereas higher percentages were positive for the ανβ5 (mean % positive = 72.7%), VLA5α (mean % positive = 87%) and VLAβ1 (mean % positive = 41.7%) integrins. A series of RGD peptides was designed, synthesized and tested for binding to integrin receptors. Based on the results of the binding to the isolated integrin receptors and the expression of integrins on glioma cell lines, a peptide that binds potently to the ανβ3, ανβ5 and α5β1 was selected for further investigations with regards to its effect on glioma cells. The peptide, Ac-c[(Pen)-Tyr(Me)-Ala-Arg-Gly-Asp-Asn-Tic-Cys]NH2 (RGD peptide), exhibited high potential for use in clinical intracranial administration since it had good stability in rat brain cell homogenates placed into artificial cerebrospinal fluid. Using an HPLC method for quantification of peptides in rat brain cell homogenates, we could demonstrate the half-life of the RGD peptide approximated 20 hr. Relative to a scrambled peptide control (non-RGD sequence, same amino acids), the experimental RGD peptide significantly decreased glioma cell proliferation of the entire panel of rat and human glioma cells tested. Adhesion of recently passaged glioma cells to glioma-derived extracellular matrix protein-coated plates was inhibited significantly by the RGD peptide. The peptide also reversed attachment of plated glioma cells. The RGD peptide caused some, but not substantial, glioma cell injury, as evidenced by a quantitative in vitro nuclear DNA morphologic assay and by a flow cytometric assay employing 7-amino actinomycin D (7AAD). We histologically monitored for toxicity caused by various doses of the RGD peptide infused repeatedly into normal cannulated rat brain. At safe doses, the experimental RGD peptide-treated brains did not show significant differences from those infused with scrambled peptide or buffer-treated controls. In tumor-bearing brains, slightly smaller tumor areas were measured with a higher necrotic-to-tumor index in the RGD peptide treated relative to the scrambled peptide-treated controls. This was obtained with intracranial peptide administrations or combined intracranial and intraperitoneal injections. From this in vitro work, we conclude that the anti-glioma effects of the RGD peptide tested resulted from lowered glioma proliferation and adhesion/mobility, rather than from significant glioma cell injury in the timeframe analyzed. Although other mechanisms not discerned from our limited histopathological observations may be operational, from our in vivo work, we conclude that repeated administration of RGD peptide into brain is safe but that better delivery of the peptides to infiltrating tumor cells is necessary.
PMCID: PMC1351132  PMID: 16467916
RGD peptides; brain tumors; apoptosis; adhesion; motility; proliferation
3.  Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy 
BMC Cancer  2011;11:315.
Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells.
To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model.
The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1α (HIF-1α) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival.
In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways.
PMCID: PMC3199865  PMID: 21791085
4.  Regression of glioma tumor growth in F98 and U87 rat glioma models by the Nitrone OKN-007 
Neuro-Oncology  2013;15(3):330-340.
Glioblastoma multiforme, a World Health Organization grade IV glioma, has a poor prognosis in humans despite current treatment options. Here, we present magnetic resonance imaging (MRI) data regarding the regression of aggressive rat F98 gliomas and human U87 glioma xenografts after treatment with the nitrone compound OKN-007, a disulfonyl derivative of α-phenyl-tert-butyl nitrone.
MRI was used to assess tumor volumes in F98 and U87 gliomas, and bioluminescence imaging was used to measure tumor volumes in F98 gliomas encoded with the luciferase gene (F98luc). Immunohistochemistry was used to assess angiogenesis (vascular endothelial growth factor [VEGF] and microvessel density [MVD]), cell differentiation (carbonic anhydrase IX [CA-IX]), hypoxia (hypoxia-inducible factor-1α [HIF-1α]), cell proliferation (glucose transporter 1 [Glut-1] and MIB-1), proliferation index, and apoptosis (cleaved caspase 3) markers in F98 gliomas. VEGF, CA-IX, Glut-1, HIF-1α, and cleaved caspase 3 were assessed in U87 gliomas.
Animal survival was found to be significantly increased (P < .001 for F98, P < .01 for U87) in the group that received OKN-007 treatment compared with the untreated groups. After MRI detection of F98 gliomas, OKN-007, administered orally, was found to decrease tumor growth (P < .05). U87 glioma volumes were found to significantly decrease (P < .05) after OKN-007 treatment, compared with untreated animals. OKN-007 administration resulted in significant decreases in tumor hypoxia (HIF-1α [P < .05] in both F98 and U87), angiogenesis (MVD [P < .05], but not VEGF, in F98 or U87), and cell proliferation (Glut-1 [P < .05 in F98, P < .01 in U87] and MIB-1 [P < .01] in F98) and caused a significant increase in apoptosis (cleaved caspase 3 [P < .001 in F98, P < .05 in U87]), compared with untreated animals.
OKN-007 may be considered as a promising therapeutic addition or alternative for the treatment of aggressive human gliomas.
PMCID: PMC3578492  PMID: 23328810
anti-glioma therapy; bioluminescence imaging; F98 rat glioma model; in vivo; magnetic resonance imaging; nitrone OKN-007; U87 xenografts
5.  WNK1-OSR1 kinase-mediated phospho-activation of Na+-K+-2Cl- cotransporter facilitates glioma migration 
Molecular Cancer  2014;13:31.
The bumetanide (BMT)-sensitive Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) maintains cell volume homeostasis by increasing intracellular K+ and Cl- content via regulatory volume increase (RVI). Expression levels of NKCC1 positively correlate with the histological grade and severity of gliomas, the most common primary adult brain tumors, and up-regulated NKCC1 activity facilitates glioma cell migration and apoptotic resistance to the chemotherapeutic drug temozolomide (TMZ). However, the cellular mechanisms underlying NKCC1 functional up-regulation in glioma and in response to TMZ administration remain unknown.
Expression of NKCC1 and its upstream kinases With-No-K (Lysine) kinase 1 (WNK1) and oxidative stress-responsive kinase-1 (OSR1) in different human glioma cell lines and glioma specimens were detected by western blotting and immunostaining. Live cell imaging and microchemotaxis assay were applied to record glioma cell movements under different treatment conditions. Fluorescence indicators were utilized to measure cell volume, intracellular K+ and Cl- content to reflect the activity of NKCC1 on ion transportation. Small interfering RNA (siRNA)-mediated knockdown of WNK1 or OSR1 was used to explore their roles in regulation of NKCC1 activity in glioma cells. Results of different treatment groups were compared by one-way ANOVA using the Bonferroni post-hoc test in the case of multiple comparisons.
We show that compared to human neural stem cells and astrocytes, human glioma cells exhibit robust increases in the activation and phosphorylation of NKCC1 and its two upstream regulatory kinases, WNK1 and OSR1. siRNA-mediated knockdown of WNK1 or OSR1 reduces intracellular K+ and Cl- content and RVI in glioma cells by abolishing NKCC1 regulatory phospho-activation. Unexpectedly, TMZ activates the WNK1/OSR1/NKCC1 signaling pathway and enhances glioma migration. Pharmacological inhibition of NKCC1 with its potent inhibitor BMT or siRNA knockdown of WNK1 or OSR1 significantly decreases glioma cell migration after TMZ treatment.
Together, our data show a novel role for the WNK1/OSR1/NKCC1 pathway in basal and TMZ-induced glioma migration, and suggest that glioma treatment with TMZ might be improved by drugs that inhibit elements of the WNK1/OSR1/NKCC1 signaling pathway.
PMCID: PMC3936893  PMID: 24555568
Bumetanide; Cell volume; Ezrin; Ion cotransporter; Temozolomide
6.  Thioredoxin Reductase Was Nitrated in the Aging Heart After Myocardial Ischemia/Reperfusion 
Rejuvenation Research  2013;16(5):377-385.
The age-related loss of anti-oxidant defense reduces recovery from myocardial ischemia/reperfusion injury (MI/R) in aged people. Our previous data showed that inactivation of thioredoxin (Trx) was involved in enhanced aging MI/R injury. Thioredoxin reductase (TrxR), the enzyme known to regulate Trx, is less efficient with age. The aim of the current study was to determine why TrxR activity was reduced and whether reduced TrxR activity contributed to enhanced aging MI/R injury. Both Trx and TrxR activity were decreased in the aging heart, and this difference was further amplified after MI/R. However, MI/R injury did not change TrxR expression between young and aging rats. Increased nitrogen oxide (NOx) but decreased nitric oxide (NO) bioavailability (decreased phosphorylated vasodilator-stimulated phosphoprotein) was observed in aging hearts. Peroxynitrite (ONOO−) was increased in aging hearts and was further amplified after MI/R. TrxR nitration in young and aging hearts was detected by immunoprecipitation (anti-nitrotyrosine) followed by immunoblotting (anti-TrxR). Compared with young hearts, TrxR nitration was increased in the aging hearts, and this was further intensified after MI/R. The ONOO− decomposition catalyst (FeTMPyp) reduced TrxR nitration and increased TrxR and Trx activity. More importantly, FeTMPyp attenuated the MI/R injury in aging hearts as evidenced by decreased caspase-3 and malondialdehyde (MDA) concentration and increased cardiac function. Increased ONOO− nitrated TrxR in the aging heart as a post-translational modification, which may be related to the enhanced MI/R injury of aging rats. Interventions that inhibit nitration and restore TrxR activity might be a therapy for attenuating enhanced MI/R injury in aging heart.
PMCID: PMC3804317  PMID: 23802942
7.  Annexin A2 Promotes Glioma Cell Invasion and Tumor Progression 
Gliomas are highly invasive, lethal brain tumors. Tumor-associated proteases play an important role in glioma progression. Annexin A2 is overexpressed in many cancers and correlates with increased plasmin activity on the tumor cell surface, which mediates degradation of extracellular matrix and promotes neoangiogenesis to facilitate tumor growth. In this study, we used two glioma cell lines, mouse GL261-EGFP and rat C6/lacZ, as well as stable clones transfected with an annexin A2 knockdown construct. We find that the annexin A2 knockdown decreased glioma cell migration in vitro and decreased membrane-bound plasmin activity. In vivo we injected the glioma cells into the rodent brain and followed glioma progression. Knockdown of annexin A2 in glioma cells decreased tumor size and slowed tumor progression, as evidenced by decreased invasion, angiogenesis and proliferation, as well as increased apoptosis in the tumor tissue of the annexin A2 knockdown group. Moreover, we report that the levels of expression of annexin A2 in human glioma samples correlate with their degree of malignancy. Taken together, our findings demonstrate that inhibition of annexin A2 expression in glioma cells could become a new target for glioma therapy.
PMCID: PMC3201988  PMID: 21976520
Glioma; annexin A2; plasmin; tPA; mouse
8.  In vivo glioblastoma growth is reduced by apyrase activity in a rat glioma model 
BMC Cancer  2006;6:226.
ATP is an important signalling molecule in the peripheral and central nervous system. Both glioma growth and tumor resection induces cell death, thus liberating nucleotides to the extracellular medium. Nucleotides are hydrolyzed very slowly by gliomas when compared with astrocytes and induce neuronal cell death and glioma proliferation. The objective of the present study was to test the involvement of extracellular ATP in glioblastoma growth in a rat glioma model.
To deplete the extracellular ATP, the enzyme apyrase was tested on the treatment of gliomas implanted in the rats CNS. One million glioma C6 cells in 3 microliters of DMEM/FCS were injected in the right striata of male Wistar rats, 250–270 g. After 20 days, the rats were decapitated and the brain sectioning and stained with hematoxylin and eosine. We performed immunohistochemical experiments with Ki67, CD31 and VEGF. Total RNA was isolated from cultured glioma C6 cells and the cDNA was analyzed by Real Time-PCR with primers for the NTPDase family.
C6 glioma cells effectively have a low expression of all NTPDases investigated, in comparison with normal astrocytes. The implanted glioma co-injected with apyrase had a significant reduction in the tumor size (p < 0.05) when compared with the rats injected only with gliomas or with gliomas plus inactivated apyrase. According to the pathological analysis, the malignant gliomas induced by C6 injection and co-injected with apyrase presented a significant reduction in the mitotic index and other histological characteristics that indicate a less invasive/proliferative tumor. Reduction of proliferation induced by apyrase co-injection was confirmed by counting the percentage of Ki67 positive glioma cell nuclei. According to counts with CD31, vessel density and neoformation was higher in the C6 group 20 days after implantation. Confirming this observation, rats treated with apyrase presented less VEGF staining in comparison to the control group.
These results indicate that the participation of extracellular ATP and the ecto-nucleotidases may be associated with the development of this type of brain tumor in an in vivo glioma model.
PMCID: PMC1592110  PMID: 16995949
9.  Single doublecortin gene therapy significantly reduces glioma tumor volume 
Journal of neuroscience research  2010;88(2):304-314.
We employed lentivirus based doublecortin (DCX), as a glioma suppressor gene therapy in an intracranial glioma tumor xenograft model in nude rats. Single DCX expressing lentivirus was directly administered into the tumor on day 8 after U87 tumor cell implantation. DCX treatment significantly reduced U87 glioma tumor volume (~60%) on day 14 after DCX lentivirus injection and significantly improved median survival of tumor bearing nude rats. DCX synthesis induced neuronal markers MAP2, TUJ1 and PSA-NCAM, and the glial marker, glial fibrillary acidic protein (GFAP) in the implanted U87 glioma tumors. DCX synthesis induced GFAP that co-localized with tubulin in the mitotic stage, inhibited cleavage furrow during cytokinesis and blocked mitosis in glioma cells. DCX lentivirus infection did not induce apoptosis, but significantly inhibited expression of the proliferation marker Ki-67 and the blood vessel marker von-Willebrand Factor (vWF). U87 and other glioma cells except brain tumor stem cells (BTSCs) do not express neuronal markers or both neuronal and glial markers. DCX synthesizing glioma cells express a phenotype of anti-angiogenic BTSC-like cells with terminal differentiation that causes remission of glioma cells by blocking mitosis via a novel DCX/GFAP pathway. Direct local delivery of lentivirus based DCX gene therapy is a potential differentiation based therapeutic approach for the treatment of glioma.
PMCID: PMC2795007  PMID: 19681167
Doublecortin; glioma suppression; neuronal and glial phenotype; brain tumor stem cells; differentiation therapy
10.  Progression of Brainstem Glioma in Young and Adult Rats 
Journal of neurosurgery  2008;109(5):849-855.
Brainstem gliomas are common in children and have the worst prognosis of any brain tumor in children. On the other hand, brainstem gliomas are rare in adult, and clinical studies have suggested different biological behavior between young and adult. The present study was designed to develop an orthotropic C6 brainstem glioma model in young and adult rats, and to investigate the tumor biological behavior in the two age groups.
C6 glioma cells were stereotactically implanted into the pons of young or adult male rats. Neurological presentation and survival time were recorded. Tumor proliferation and apoptosis in brainstem gliomas of young and adult rats were determined by immunohistochemical staining of Ki-67 and TUNEL assay, respectively.
Striking difference were found between young and adult brainstem glioma in the onset of neurological signs, duration of symptoms, survival time, tumor growth pattern, as well as tumor proliferation and apoptosis. Relatively focal tumors were observed in adult rats harboring brainstem glioma, while diffusive tumors were found in young rats. Furthermore, brainstem gliomas in adult rats were less proliferative and had more apoptosis than those in young rats.
The present study demonstrated that C6 brainstem glioma model in young and adult rats closely imitates human brainstem glioma in neurological findings and histopathology. Our findings suggest that the different growth pattern and invasiveness of brainstem glioma between children and adult could be due to the different host cellular environments between the two age groups, hence, warrant further investigation of the different host-response between childhood and adult brainstem glioma in human.
PMCID: PMC2693119  PMID: 18976074
Brainstem; glioma; progression; animal model
11.  Cellular Host Responses to Gliomas 
PLoS ONE  2012;7(4):e35150.
Glioblastoma multiforme (GBM) is the most aggressive type of malignant primary brain tumors in adults. Molecular and genetic analysis has advanced our understanding of glioma biology, however mapping the cellular composition of the tumor microenvironment is crucial for understanding the pathology of this dreaded brain cancer. In this study we identified major cell populations attracted by glioma using orthotopic rodent models of human glioma xenografts. Marker-specific, anatomical and morphological analyses revealed a robust influx of host cells into the main tumor bed and tumor satellites.
Methodology/Principal Findings
Human glioma cell lines and glioma spheroid orthotopic implants were used in rodents. In both models, the xenografts recruited large numbers of host nestin-expressing cells, which formed a ‘network’ with glioma. The host nestin-expressing cells appeared to originate in the subventricular zone ipsilateral to the tumor, and were clearly distinguishable from pericytes that expressed smooth muscle actin. These distinct cell populations established close physical contact in a ‘pair-wise’ manner and migrated together to the deeper layers of tumor satellites and gave rise to tumor vasculature. The GBM biopsy xenografts displayed two different phenotypes: (a) low-generation tumors (first in vivo passage in rats) were highly invasive and non-angiogenic, and host nestin-positive cells that infiltrated into these tumors displayed astrocytic or elongated bipolar morphology; (b) high-generation xenografts (fifth passage) had pronounced cellularity, were angiogenic with ‘glomerulus-like’ microvascular proliferations that contained host nestin-positive cells. Stromal cell-derived factor-1 and its receptor CXCR4 were highly expressed in and around glioma xenografts, suggesting their role in glioma progression and invasion.
Our data demonstrate a robust migration of nestin-expressing host cells to glioma, which together with pericytes give rise to tumor vasculature. Mapping the cellular composition of glioma microenvironment and deciphering the complex ‘crosstalk’ between tumor and host may ultimately aid the development of novel anti-glioma therapies.
PMCID: PMC3335155  PMID: 22539956
12.  Glutaredoxin 2 Reduces Both Thioredoxin 2 and Thioredoxin 1 and Protects Cells from Apoptosis Induced by Auranofin and 4-Hydroxynonenal 
Antioxidants & Redox Signaling  2014;21(5):669-681.
Aims: Mitochondrial thioredoxin (Trx) is critical for defense against oxidative stress-induced cell apoptosis. To date, mitochondrial thioredoxin reductase (TrxR) is the only known enzyme catalyzing Trx2 reduction in mitochondria. However, TrxR is sensitive to inactivation by exo/endogenous electrophiles, for example, 4-hydroxynonenal (HNE). In this study, we characterized the mitochondrial glutaredoxin 2 (Grx2) system as a backup for the mitochondrial TrxR. Meanwhile, as Grx2 is also present in the cytosol/nucleus of certain cancer cell lines, the reducing activity of Grx2 on Trx1 was also tested. Results: Glutathione alone could reduce oxidized Trx2, and the presence of physiological concentrations of Grx2 markedly increased the reaction rate. HeLa cells with Grx2 overexpression (particularly in the mitochondria) exhibited higher viabilities than the wild-type cells after treatment with TrxR inhibitors (Auranofin or HNE), whereas knockdown of Grx2 sensitized the cells to TrxR inhibitors. Accordingly, Grx2 overexpression in the mitochondria had protected Trx2 from oxidation by HNE treatment, whereas Grx2 knockdown had sensitized Trx2 to oxidation. On the other hand, Grx2 reduced Trx1 with similar activities as that of Trx2. Overexpression of Grx2 in the cytosol had protected Trx1 from oxidation, indicating a supportive role of Grx2 in the cytosolic redox balance of cancer cells. Innovation: This work explores the reductase activity of Grx2 on Trx2/1, and demonstrates the physiological importance of the activity by using in vivo redox western blot assays. Conclusion: Grx2 system could help to keep Trx2/1 reduced during an oxidative stress, thereby contributing to the anti-apoptotic signaling. Antioxid. Redox Signal. 21, 669–681.
PMCID: PMC4098818  PMID: 24295294
13.  MicroRNA-195 Inhibits the Proliferation of Human Glioma Cells by Directly Targeting Cyclin D1 and Cyclin E1 
PLoS ONE  2013;8(1):e54932.
Glioma proliferation is a multistep process during which a sequence of genetic and epigenetic alterations randomly occur to affect the genes controlling cell proliferation, cell death and genetic stability. microRNAs are emerging as important epigenetic modulators of multiple target genes, leading to abnormal cellular signaling involving cellular proliferation in cancers.In the present study, we found that expression of miR-195 was markedly downregulated in glioma cell lines and human primary glioma tissues, compared to normal human astrocytes and matched non-tumor associated tissues. Upregulation of miR-195 dramatically reduced the proliferation of glioma cells. Flow cytometry analysis showed that ectopic expression of miR-195 significantly decreased the percentage of S phase cells and increased the percentage of G1/G0 phase cells. Overexpression of miR-195 dramatically reduced the anchorage-independent growth ability of glioma cells. Furthermore, overexpression of miR-195 downregulated the levels of phosphorylated retinoblastoma (pRb) and proliferating cell nuclear antigen (PCNA) in glioma cells. Conversely, inhibition of miR-195 promoted cell proliferation, increased the percentage of S phase cells, reduced the percentage of G1/G0 phase cells, enhanced anchorage-independent growth ability, upregulated the phosphorylation of pRb and PCNA in glioma cells. Moreover, we show that miR-195 inhibited glioma cell proliferation by downregulating expression of cyclin D1 and cyclin E1, via directly targeting the 3′-untranslated regions (3′-UTR) of cyclin D1 and cyclin E1 mRNA. Taken together, our results suggest that miR-195 plays an important role to inhibit the proliferation of glioma cells, and present a novel mechanism for direct miRNA-mediated suppression of cyclin D1 and cyclin E1 in glioma.
PMCID: PMC3557299  PMID: 23383003
14.  Monocarboxylate transporters (MCTs) in gliomas: expression and exploitation as therapeutic targets 
Neuro-Oncology  2012;15(2):172-188.
Gliomas exhibit high glycolytic rates, and monocarboxylate transporters (MCTs) play a major role in the maintenance of the glycolytic metabolism through the proton-linked transmembrane transport of lactate. However, their role in gliomas is poorly studied. Thus, we aimed to characterize the expression of MCT1, MCT4, and their chaperone CD147 and to assess the therapeutic impact of MCT inhibition in gliomas.
MCTs and CD147 expressions were characterized by immunohistochemistry in nonneoplastic brain and glioma samples. The effect of CHC (MCT inhibitor) and MCT1 silencing was assessed in in vitro and in vivo glioblastoma models.
MCT1, MCT4, and CD147 were overexpressed in the plasma membrane of glioblastomas, compared with diffuse astrocytomas and nonneoplastic brain. CHC decreased glycolytic metabolism, migration, and invasion and induced cell death in U251 cells (more glycolytic) but only affected proliferation in SW1088 (more oxidative). The effectiveness of CHC in glioma cells appears to be dependent on MCT membrane expression. MCT1 downregulation showed similar effects on different glioma cells, supporting CHC as an MCT1 inhibitor. There was a synergistic effect when combining CHC with temozolomide treatment in U251 cells. In the CAM in vivo model, CHC decreased the size of tumors and the number of blood vessels formed.
This is the most comprehensive study reporting the expression of MCTs and CD147 in gliomas. The MCT1 inhibitor CHC exhibited anti-tumoral and anti-angiogenic activity in gliomas and, of importance, enhanced the effect of temozolomide. Thus, our results suggest that development of therapeutic approaches targeting MCT1 may be a promising strategy in glioblastoma treatment.
PMCID: PMC3548586  PMID: 23258846
CD147; CHC; glioblastomas; gliomas; glycolytic metabolism; lactate; monocarboxylate transporters
15.  Therapeutic Impact of Human Serum Albumin–Thioredoxin Fusion Protein on Influenza Virus-Induced Lung Injury Mice 
Reactive oxygen species (ROS) are the primary pathogenic molecules produced in viral lung infections. We previously reported on the use of a recombinant human serum albumin (HSA)–thioredoxin 1 (Trx) fusion protein (HSA–Trx) for extending the half-life Trx, an endogenous protein with anti-oxidant properties. As a result, it was possible to overcome the unfavorable pharmacokinetic and short pharmacological properties of Trx. We hypothesized that HSA–Trx would attenuate the enhanced ROS production of species such as hydroxyl radicals by neutrophils during an influenza viral infection. The levels of 8-hydroxy-2′-deoxyguanosine and 3-nitrotyrosine were used as indices of the anti-oxidant activity of HSA–Trx. In addition, the cytoprotective effects of HSA–Trx were examined in PR8 (H1N1) influenza virus-induced lung injured mice. The findings show that HSA–Trx reduced the number of total cells, neutrophils, and total protein in BALF of influenza virus-induced lung injured mice. The HSA–Trx treatment significantly decreased the level of 8-hydroxy-2′-deoxyguanosine and 3-nitrotyrosine, but failed to inhibit inducible nitric oxide synthase expression, in the lungs of the virus-infected mice. On the other hand, Tamiflu® treatment also significantly suppressed the production of inflammatory cells and neutrophil infiltration, as well as the protein level in BALF and lung histopathological alterations caused by the influenza virus. The suppressive effect of Tamiflu® was slightly stronger than that of HSA–Trx. Interestingly, Tamiflu® significantly decreased virus proliferation, while HSA–Trx had no effect. These results indicate that HSA–Trx may be of therapeutic value for the treatment of various acute inflammatory disorders such as influenza-virus-induced pneumonia, by inhibiting inflammatory-cell responses and suppressing the overproduction of NO in the lung.
PMCID: PMC4220708  PMID: 25414704
albumin; thioredoxin; fusion; anti-oxidization; influenza virus
16.  Angiogenic Signalling Pathways Altered in Gliomas: Selection Mechanisms for More Aggressive Neoplastic Subpopulations with Invasive Phenotype 
Journal of Signal Transduction  2012;2012:597915.
The angiogenesis process is a key event for glioma survival, malignancy and growth. The start of angiogenesis is mediated by a cascade of intratumoural events: alteration of the microvasculature network; a hypoxic microenvironment; adaptation of neoplastic cells and synthesis of pro-angiogenic factors. Due to a chaotic blood flow, a consequence of an aberrant microvasculature, tissue hypoxia phenomena are induced. Hypoxia inducible factor 1 is a major regulator in glioma invasiveness and angiogenesis. Clones of neoplastic cells with stem cell characteristics are selected by HIF-1. These cells, called “glioma stem cells” induce the synthesis of vascular endothelial growth factor. This factor is a pivotal mediator of angiogenesis. To elucidate the role of these angiogenic mediators during glioma growth, we have used a rat endogenous glioma model. Gliomas induced by prenatal ENU administration allowed us to study angiogenic events from early to advanced tumour stages. Events such as microvascular aberrations, hypoxia, GSC selection and VEGF synthesis may be studied in depth. Our data showed that for the treatment of gliomas, developing anti-angiogenic therapies could be aimed at GSCs, HIF-1 or VEGF. The ENU-glioma model can be considered to be a useful option to check novel designs of these treatment strategies.
PMCID: PMC3407647  PMID: 22852079
17.  Glioma morphology and tumor-induced vascular alterations revealed in 7 rodent glioma models by in vivo magnetic resonance imaging and angiography 
To evaluate the added value of non-contrast-enhanced magnetic resonance angiography (MRA) to conventional MR imaging for a detailed characterization of different rodent glioma models.
Materials and Methods
Intracerebral tumor cell implantation and chemical induction methods were implemented to obtain rat C6, 9L/LacZ, F98, RG2 and ENU-induced glioma models, a human U87 MG tumor model as well as a mouse GL261 glioma model. MR assessments were regularly conducted on a 7 Tesla Bruker BioSpin system. The tumor border sharpness and growth characteristics of each glioma model were assessed from T2-weighted images. Neovascularization and vascular alterations inherent to each model were characterized by assessing absolute blood volumes, vessel density, length and diameter using Mathematica and Amira software.
9L/LacZ and ENU gliomas both presented flaws that hinder their use as reliable brain tumor models. C6 gliomas were slightly invasive and induced moderate vascular alterations, whereas GL261 tumors dramatically altered the brain vessels in the glioma region. F98, RG2 and U87 are infiltrative models which produced dramatic vascular alterations.
MRI and MRA provided crucial in vivo information to identify a distinctive “fingerprint” for each of our 7 rodent glioma models.
PMCID: PMC2915452  PMID: 20677250
Glioma; Magnetic resonance Angiography; Peritumoral region; Neovascularization
18.  Tetramethylpyrazine inhibits activities of glioma cells and glutamate neuro-excitotoxicity: Potential therapeutic application for treatment of gliomas 
Neuro-Oncology  2008;10(2):139-152.
We tested the herbal extract 2,3,5,6-tetramethylpyrazine (TMP) for possible therapeutic efficacy against a glioma cell line and against gliomas transplanted into rat brains. In the cultured glioma cells, 50 μM TMP significantly inhibited glutamate-induced increase in intracellular calcium. Significant cell damage (30%) and proliferation suppression (10%), however, occurred only at higher concentrations (200–400 μM). Glioma- neuronal co-culturing resulted in significant neuronal damage and higher proliferation of the glioma cells (140%) compared with single cultures. Low concentrations of TMP (⩽200 μM) attenuated the neuronal damage, suppressed glioma migration, and decreased glioma proliferation in the neuronal-glioma co-culture. Gliomas transplanted into the frontal cortical area exhibited high proliferation, with untreated rats dying 10–23 days later. TMP treatment inhibited tumor growth and significantly extended survival time. The results indicate that TMP can suppress glioma activity, including growth, and protect neurons against glioma-induced excitotoxicity, suggesting that TMP may have therapeutic potential in the treatment of malignant gliomas.
PMCID: PMC2613816  PMID: 18314418
calcium; excitotoxicity; glioblastoma multiforme; tetramethylpyrazine
19.  Overexpression of DcR3 and Its Significance on Tumor Cell Differentiation and Proliferation in Glioma 
The Scientific World Journal  2014;2014:605236.
Background. Overexpression of decoy receptor 3 (DcR3) have been reported in various classes of malignancies. However, its expression and clinicopathological contribution in gliomas has not been fully elucidated. Objective. To explore the expression and clinical significance of DcR3 protein in relation to tumor cell differentiation and proliferation in glioma cell lines and tissues. Methods. One hundred and twenty-five samples of glioma patients and 18 cases of normal brain tissues were recruited. The expression of DcR3 protein was detected using immunohistochemistry. Tumor differentiation was assessed by histologic characters and the status of glial fibrillary acidic protein (GFAP). Tumor cell labeling indexes (LIs) of Ki-67 and PCNA were also obtained. The relationship between the DcR3 level and clinicopathological features was investigated, including tumor differentiation, LIs, and survival. Meanwhile, the expression of DcR3 protein was also measured in the supernatants of 8 glioma cell lines and glioma cells freshly prepared from 8 human glioblastoma specimens by using western blot. Results. The level of DcR3 protein in gliomas was significantly higher than that in normal brain tissues (P < 0.01). DcR3 expression showed positive correlations with tumor pathological grade (r = 0.621, P < 0.01) and negative with GFAP expression (r = −0.489, P < 0.01). Furthermore, there were positive correlations between DcR3 expression and Ki-67, PCNA LIs (r = 0.529, P < 0.01; r = 0.556, P < 0.01). The survival in the DcR3 negative group was 50 ± 1.79 months, longer than that of the DcR3 positive group (48.36 ± 2.90), however, without significance (P = 0.149). Different levels of DcR3 could also be detected in the culturing supernatants of all the 8 glioma cell lines and glioma cells freshly obtained from 8 human glioblastoma specimens. Conclusions. The overexpression of DcR3 might play a crucial role in the tumorigenesis, differentiation, and proliferation of glioma.
PMCID: PMC3972858  PMID: 24741354
20.  Proinflammatory-activated glioma cells induce a switch in microglial polarization and activation status, from a predominant M2b phenotype to a mixture of M1 and M2a/B polarized cells 
ASN NEURO  2014;6(3):e00144.
Malignant gliomas are primary brain tumors characterized by morphological and genetic complexities, as well as diffuse infiltration into normal brain parenchyma. Within gliomas, microglia/macrophages represent the largest tumor-infiltrating cell population, contributing by at least one-third to the total tumor mass. Bi-directional interactions between glioma cells and microglia may therefore play an important role on tumor growth and biology. In the present study, we have characterized the influence of glioma-soluble factors on microglial function, comparing the effects of media harvested under basal conditions with those of media obtained after inducing a pro-inflammatory activation state in glioma cells. We found that microglial cells undergo a different pattern of activation depending on the stimulus; in the presence of activated glioma-derived factors, i.e. a condition mimicking the late stage of pathology, microglia presents as a mixture of polarization phenotypes (M1 and M2a/b), with up-regulation of iNOS (inducible nitric oxide synthase), ARG (arginase) and IL (interleukine)-10. At variance, microglia exposed to basal glioma-derived factors, i.e. a condition resembling the early stage of pathology, shows a more specific pattern of activation, with increased M2b polarization status and up-regulation of IL-10 only. As far as viability and cell proliferation are concerned, both LI-CM [LPS (lipopolysaccharide)–IFNγ (interferon γ) conditioned media] and C-CM (control-conditioned media) induce similar effects on microglial morphology. Finally, in human glioma tissue obtained from surgical resection of patients with IV grade glioblastoma, we detected a significant amount of CD68 positive cells, which is a marker of macrophage/microglial phagocytic activity, suggesting that in vitro findings presented here might have a relevance in the human pathology as well.
We have characterized the influence of glioma-soluble factors on microglial, comparing the effects of media harvested under-basal conditions to those of media obtained after inducing a pro-inflammatory activation in glioma cells. Our data suggest that microglia might exert different effects on glioma depending on the stage of disease.
PMCID: PMC4013688  PMID: 24689533
arginase; glioma; IL-10; microglia; microglial polarization; mTOR; NOS; ARG, arginase; BBB, blood–brain barrier; BrdU, bromodeoxyuridine or 5-bromo-2-deoxyuridine; CM, conditioned media; C-CM, control-conditioned media; CNS, central nervous system; COX, cyclooxygenase; DMEM, Dulbecco’s modified Eagle’s medium; GBM, glioblastoma multiforme; H2DCF-DA, 2′,7-dichlorodihydrofluorescein diacetate; IFN, interferon; IL, interleukine; iNOS, inducible nitric oxide synthase; LI-CM, LPS–IFNγ-conditioned media; LPS, lipopolysaccharide; mTOR, mammalian target of rapamycin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; TGF, transforming growth factor; TNF, tumor necrosis factor
21.  Rat brain tumor models in experimental neuro-oncology: the C6, 9L, T9, RG2, F98, BT4C, RT-2 and CNS-1 gliomas 
Journal of neuro-oncology  2009;94(3):299-312.
In this review we will describe eight commonly used rat brain tumor models and their application for the development of novel therapeutic and diagnostic modalities. The C6, 9L and T9 gliomas were induced by repeated injections of methylnitrosourea (MNU) to adult rats. The C6 glioma has been used extensively for a variety of studies, but since it arose in an outbred Wistar rat, it is not syngeneic to any inbred strain, and its potential to evoke an alloimmune response is a serious limitation. The 9L gliosarcoma has been used widely and has provided important information relating to brain tumor biology and therapy. The T9 glioma, although not generally recognized, was and probably still is the same as the 9L. Both of these tumors arose in Fischer rats and can be immunogenic in syngeneic hosts, a fact that must be taken into consideration when used in therapy studies, especially if survival is the endpoint. The RG2 and F98 gliomas were both chemically induced by administering ethylnitrosourea (ENU) to pregnant rats, the progeny of which developed brain tumors that subsequently were propagated in vitro and cloned. They are either weakly or non-immunogenic and have an invasive pattern of growth and uniform lethality, which make them particularly attractive models to test new therapeutic modalities. The CNS-1 glioma was induced by administering MNU to a Lewis rat. It has an infiltrative pattern of growth and is weakly immunogenic, which should make it useful in experimental neuro-oncology. Finally, the BT4C glioma was induced by administering ENU to a BD IX rat, following which brain cells were propagated in vitro until a tumorigenic clone was isolated. This tumor has been used for a variety of studies to evaluate new therapeutic modalities. The Avian Sarcoma Virus (ASV) induced tumors, and a continuous cell line derived from one of them designated RT-2, have been useful for studies in which de novo tumor induction is an important requirement. These tumors also are immunogenic and this limits their usefulness for therapy studies. It is essential to recognize the limitations of each of the models that have been described, and depending upon the nature of the study to be conducted, it is important that the appropriate model be selected.
PMCID: PMC2730996  PMID: 19381449
C6; 9L; T9; RG2; F98; BT4C; RT-2; CNS-1; Rat brain tumor models
22.  Promoter methylation and downregulation of SLC22A18 are associated with the development and progression of human glioma 
Downregulation of the putative tumor suppressor gene SLC22A18 has been reported in a number of human cancers. The aim of this study was to investigate the relationship between SLC22A18 downregulation, promoter methylation and the development and progression of human glioma.
SLC22A18 expression and promoter methylation was examined in human gliomas and the adjacent normal tissues. U251 glioma cells stably overexpressing SLC22A18 were generated to investigate the effect of SLC22A18 on cell growth and adherence in vitro using the methyl thiazole tetrazolium assay. Apoptosis was quantified using flow cytometry and the growth of SLC22A18 overexpressing U251 cells was measured in an in vivo xenograft model.
SLC22A18 protein expression is significantly decreased in human gliomas compared to the adjacent normal brain tissues. SLC22A18 protein expression is significantly lower in gliomas which recurred within six months after surgery than gliomas which did not recur within six months. SLC22A18 promoter methylation was detected in 50% of the gliomas, but not in the adjacent normal tissues of any patient. SLC22A18 expression was significantly decreased in gliomas with SLC22A18 promoter methylation, compared to gliomas without methylation. The SLC22A18 promoter is methylated in U251 cells and treatment with the demethylating agent 5-aza-2-deoxycytidine increased SLC22A18 expression and reduced cell proliferation. Stable overexpression of SLC22A18 inhibited growth and adherence, induced apoptosis in vitro and reduced in vivo tumor growth of U251 cells.
SLC22A18 downregulation via promoter methylation is associated with the development and progression of glioma, suggesting that SLC22A18 is an important tumor suppressor in glioma.
PMCID: PMC3184631  PMID: 21936894
23.  Targeting A20 Decreases Glioma Stem Cell Survival and Tumor Growth 
PLoS Biology  2010;8(2):e1000319.
The A20 protein is a known inhibitor of apoptosis that here is shown to be a novel cancer stem cell-promoting factor associated with poor glioma patient survival.
Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioblastoma stem cells (GSCs). GSCs are regulated by molecular pathways distinct from the bulk tumor that may be useful therapeutic targets. We determined that A20 (TNFAIP3), a regulator of cell survival and the NF-κB pathway, is overexpressed in GSCs relative to non-stem glioblastoma cells at both the mRNA and protein levels. To determine the functional significance of A20 in GSCs, we targeted A20 expression with lentiviral-mediated delivery of short hairpin RNA (shRNA). Inhibiting A20 expression decreased GSC growth and survival through mechanisms associated with decreased cell-cycle progression and decreased phosphorylation of p65/RelA. Elevated levels of A20 in GSCs contributed to apoptotic resistance: GSCs were less susceptible to TNFα-induced cell death than matched non-stem glioma cells, but A20 knockdown sensitized GSCs to TNFα-mediated apoptosis. The decreased survival of GSCs upon A20 knockdown contributed to the reduced ability of these cells to self-renew in primary and secondary neurosphere formation assays. The tumorigenic potential of GSCs was decreased with A20 targeting, resulting in increased survival of mice bearing human glioma xenografts. In silico analysis of a glioma patient genomic database indicates that A20 overexpression and amplification is inversely correlated with survival. Together these data indicate that A20 contributes to glioma maintenance through effects on the glioma stem cell subpopulation. Although inactivating mutations in A20 in lymphoma suggest A20 can act as a tumor suppressor, similar point mutations have not been identified through glioma genomic sequencing: in fact, our data suggest A20 may function as a tumor enhancer in glioma through promotion of GSC survival. A20 anticancer therapies should therefore be viewed with caution as effects will likely differ depending on the tumor type.
Author Summary
Glioblastomas are the most common and aggressive primary brain tumors in adults, with a median survival of only 12–15 months. Glioblastomas display a cellular hierarchy with a subset of cells having stem cell–like properties, including the capacity to self-renew and propagate tumors. Specific ablation of cancer stem cells is widely thought to be critical for effective and long-lasting treatment of cancers. We report the identification of the antiapoptotic protein A20 (which is also known as TNFAIP3) as a novel regulator of glioma stem cell survival. Glioma stem cells overexpress A20 relative to non-stem glioma cells, and this protects them from cell death, whereas depletion of A20 attenuates glioma stem cell survival and tumor growth. Interrogation of a molecular glioma database reveals that A20 levels correlate with decreased survival in patients. These data indicate that A20 is a tumor enhancer in the context of glioma, which importantly contrasts with its known function as a tumor suppressor in the context of lymphoma. Therefore, A20 may be a context-specific regulator of cancer stem cell survival and growth.
PMCID: PMC2826371  PMID: 20186265
24.  c-Myc Is Required for Maintenance of Glioma Cancer Stem Cells 
PLoS ONE  2008;3(11):e3769.
Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells.
Methodology/Principal Findings
Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G0/G1 phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice.
These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.
PMCID: PMC2582454  PMID: 19020659
25.  Molecular MRI differentiation of VEGF receptor-2 levels in C6 and RG2 glioma models 
Vascular endothelial growth factor receptor 2 (VEGFR2) is an important angiogenic marker over-expressed in gliomas. With the use of molecular magnetic resonance imaging (mMRI) differing levels of VEGFR2 can be characterized in vivo with in rodent gliomas varying in angiogenesis. VEGFR2 levels were assessed by intravenous administration of an anti-VEGFR2 probe (anti-VEGFR2-albumin-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin) into C6 or RG2 glioma-bearing rats, and visualized with mMRI. A non-specific IgG was coupled to the albumin-Gd-DTPA-biotin construct as a contrast agent molecular weight control. VEGFR2 levels are heterogeneous in different regions of C6 gliomas, whereas VEGFR2 was more homogenous or evenly distributed in RG2 gliomas. RG2 gliomas have less VEGFR2 within tumor periphery and peri-necrotic (p<0.05) regions, but more VEGFR2 within tumor interior regions (p<0.01), compared to C6 gliomas. mMRI results were confirmed with fluorescence staining and mean fluorescence intensity (MFI) quantification of the anti-VEGFR2 probe in excised glioma and brain tissues, as well as detection of VEGFR2 in C6 and RG2 gliomas and corresponding contalateral brain tissues. Ex vivo VEGFR2 levels were found to be significantly higher in C6 gliomas compared to RG2 tumors (p<0.001), which corresponded with in vivo detection using the VEGFR2 probe. Immunohistochemistry staining for HIF-1α (hypoxia inducible factor 1α), which is associated with angiogenesis, indicated higher levels in RG2 (p<0.01) compared to C6 gliomas. The data suggests that C6 gliomas have angiogenesis which is associated more with large blood vessels in tumor periphery and peri-necrotic regions, and less microvascular angiogenesis within the tumor interior, compared to RG2 gliomas.
PMCID: PMC3715774  PMID: 23901356
Molecular magnetic resonance imaging (mMRI); vascular endothelial growth factor receptor 2 (VEGFR2); C6 and RG2 rat gliomas; in vivo; fluorescence imaging

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