Interdomain Ca effects in Escherichia coli α-haemolysin: Ca binding to the C-terminal domain stabilizes both C- and N-terminal domains

α-Haemolysin (HlyA) is a toxin secreted by pathogenic Escherichia coli, whose lytic activity requires submillimolar Ca²⁺ concentrations. Previous studies have shown that Ca²⁺ binds within the Asp and Gly rich C-terminal nonapeptide repeat domain (NRD) in HlyA. The presence of the NRD puts HlyA in the RTX (Repeats in Toxin) family of proteins. We tested the stability of the whole protein, the amphipathic helix domain and the NRD, in both the presence and absence of Ca²⁺ using native HlyA, a truncated form of HlyAΔN601 representing the C-terminal domain, and a novel mutant HlyA W914A whose intrinsic fluorescence indicates changes in the N-terminal domain. Fluorescence and infrared spectroscopy, tryptic digestion, and urea denaturation techniques concur in showing that calcium binding to the repeat domain of α-haemolysin stabilizes and compacts both the NRD and the N-terminal domains of HlyA. The stabilization of the N-terminus through Ca²⁺ binding to the C-terminus reveals long-range inter-domain structural effects. Considering that RTX proteins consist, in general, of a Ca²⁺-binding NRD and separate function-specific domains, the long-range stabilizing effects of Ca²⁺ in HlyA may well be common to other members of this family.     

The Role of Mitochondria in Glioma Pathophysiology

It has long been recognised that malignant tumours favour aerobic glycolysis to generate ATP and contain abnormalities of the intrinsic, mitochondria-dependent, apoptotic pathway, suggesting the involvement of dysfunctional mitochondria in tumour pathophysiology. However, the mechanisms underlying such processes in gliomas are poorly understood. Few recent studies have evaluated mitochondrial ultrastructure and proteomics in the pathophysiology of malignant gliomas. However, aberrant energy metabolism has been reported in gliomas and mitochondrial dysfunction links to glioma apoptotic signalling have been observed. Mitochondrial structural abnormalities and dysfunction in malignant gliomas is a neglected area of research. Definition of abnormalities in mitochondrial proteomics, membrane potential regulation, energy metabolism and intrinsic apoptotic pathway signalling in gliomas may open novel therapeutic opportunities.     

Lipoxygenbase-derived products of arachidonic acid mediate stimulation of hexose uptake in human polymorphonuclear leukocytes

The titration of metal-freed bovine α-lactalbumin with Mg²⁺ ions causes a two-stepped decrease in the tryptophan fluorescence quantum yield and a pronounced spectral shift towards shorter wavelengths, which seems to be a result of the binding of two magnesium ions to the protein molecule. The magnesium binding constants evaluated from the fluorimetric Mg²⁺-titration are 2·10³ and 2·10² M^?1. Mg²⁺ ions in millimolar concentrations almost do not influence the binding of Ca²⁺ ions to the protein.     

YKL-40在脑胶质母细胞瘤微环境中的作用

胶质母细胞瘤是大脑及其他中枢神经系统最常见的恶性肿瘤,其复杂的肿瘤微环境是胶质母细胞瘤临床治疗的主要挑战,也是胶质母细胞瘤患者复发率高、生存率低的主要原因。YKL-40,这一分泌性蛋白质与多种类型的癌症预后不良相关,且在高级别胶质瘤尤其是胶质母细胞瘤患者中血清水平与肿瘤组织表达水平显著升高,而在低级别胶质瘤中并未发现这一特征。这提示,YKL-40与胶质瘤分级及胶质母细胞瘤恶性发展过程密切相关。针对YKL-40的抗体治疗也被证明能够与电离辐射协同抑制胶质母细胞瘤血管生成及恶性发展。基于YKL-40的临床价值,本文将从肿瘤微环境的角度,归纳总结YKL-40在恶性肿瘤中的相关研究成果,并讨论其在胶质母细胞瘤发生发展中的相关作用及临床应用前景。     

From bench to the clinic: gamma-linolenic acid therapy of human gliomas

Malignant gliomas are among the most devastating of cancers and are a major cause of mortality in a young population with a median survival time of 9 months following cytoreductive surgery, radiotherapy and chemotherapy. Recent studies showed that polyunsaturated fatty acids especially gamma-linolenic acid (GLA) have selective tumoricidal action especially against malignant glioma cells both in vitro and in vivo. Limited open label clinical studies showed that intratumoral injection/infusion of GLA is safe and effective against malignant gliomas. In view of this, large-scale, double blind studies are needed to establish the usefulness of GLA in the treatment of malignant brain tumors.     

Proteomics for the discovery of biomarkers and diagnosis of periodontitis: a critical review

Periodontitis is a common chronic and destructive disease whose pathogenetic mechanisms remain unclear. Due to their sensitivity and global scale, proteomics studies offer the opportunity to uncover critical host and pathogen activity indicators and can elucidate clinically applicable biomarkers for improved diagnosis and treatment of the disease. This review summarizes the literature of proteomics studies on periodontitis and comprehensively discusses commonly found candidate biomarkers. Key considerations in the design of an experimental proteomics platform are also outlined. The applicability of protein biomarkers across the progression of periodontitis and unexplored areas of research are highlighted.     

Gene Fusion in Malignant Glioma: An Emerging Target for Next-Generation Personalized Treatment

Malignant gliomas are heterogeneous diseases in genetic basis. The development of sequencing techniques has identified many gene rearrangements encoding novel oncogenic fusions in malignant glioma to date. Understanding the gene fusions and how they regulate cellular processes in different subtypes of glioma will shed light on genomic diagnostic approaches for personalized treatment. By now, studies of gene fusions in glioma remain limited, and no medication has been approved for treating the malignancy harboring gene fusions. This review will discuss the current characterization of gene fusions occurring in both adult and pediatric malignant gliomas, their roles in oncogenesis, and the potential clinical implication as therapeutic targets.     

Glutamate as chemotactic fuel for diffuse glioma cells: Are they glutamate suckers?

Diffuse gliomas comprise a group of primary brain tumors that originate from glial (precursor) cells and present as a variety of malignancy grades which have in common that they grow by diffuse infiltration. This phenotype complicates treatment enormously as it precludes curative surgery and radiotherapy. Furthermore, diffusely infiltrating glioma cells often hide behind a functional blood–brain barrier, hampering delivery of systemically administered therapeutic and diagnostic compounds to the tumor cells. The present review addresses the biological mechanisms that underlie the diffuse infiltrative phenotype, knowledge of which may improve treatment strategies for this disastrous tumor type. The invasive phenotype is specific for glioma: most other brain tumor types, both primary and metastatic, grow as delineated lesions. Differences between the genetic make-up of glioma and that of other tumor types may therefore help to unravel molecular pathways, involved in diffuse infiltrative growth. One such difference concerns mutations in the NADP⁺-dependent isocitrate dehydrogenase (IDH1 and IDH2) genes, which occur in > 80% of cases of low grade glioma and secondary glioblastoma. In this review we present a novel hypothesis which links IDH1 and IDH2 mutations to glutamate metabolism, possibly explaining the specific biological behavior of diffuse glioma.     

CD147 confers temozolomide resistance of glioma cells via the regulation of β-TrCP/Nrf2 pathway

Background: Drug resistance is one of the biggest challenges in cancer therapy. temozolomide (TMZ) represents the most important chemotherapeutic option for glioma treatment. However, the therapeutic efficacy of TMZ remains very limited due to its frequent resistance in glioma, and the underlying mechanisms were not fully addressed. Herein, we demonstrate that the elevated expression of CD147 contributes to TMZ resistance in glioma cells, potentially through the post-translational regulation of Nrf2 expression.Methods: Cell-based assays of CD147 triggered drug resistance were performed through Edu-incorporation assay, CCK8 assay, TUNEL staining assay and flow cytometric assay. Luciferase reporter assay, protein stability related assays, co-immunoprecipitation assay were used to determine CD147 induction of Nrf2 expression through β-TrCP dependent ubiquitin system. Finally, the effect of the CD147/Nrf2 signaling on glioma progression and TMZ resistance were evaluated by functional experiments and clinical samples.Results: Based on the analysis of clinical glioma tissues, CD147 is highly expressed in glioma tissues and positively associated with tumor malignancy. Suppression of CD147 expression increased the inhibitory effect of TMZ on cell survival in both U251 and T98G cells, whereas the gain of CD147 function blocked TMZ-induced ROS production and cell death. Mechanistic study indicates that CD147 inhibited GSK3β/β-TrCP-dependent Nrf2 degradation by promoting Akt activation, and subsequently increased Nrf2-mediated anti-oxidant gene expressions. Supporting the biological significance, the reciprocal relationship between CD147 and Nrf2 was observed in glioma tissues, and associated with patient outcome.Conclusions: Our data provide the first evidence that glioma resistance to TMZ is potentially due to the activation of CD147/Nrf2 axis. CD147 promotes Nrf2 stability through the suppression of GSK3β/β-TrCP dependent Nrf2 protein degradation, which results in the ablation of TMZ induced ROS production. As such, we point out that targeting CD147/Nrf2 axis may provide a new strategy for the treatment of TMZ resistant gliomas.     

Retracted: Superoxide‐dependent uptake of vitamin C in human glioma cells

Glioblastomas are lethal brain tumors that resist current cytostatic therapies. Vitamin C may antagonize the effects of reactive oxygen species (ROS) generating therapies; however, it is often used to reduce therapy‐related side effects despite its effects on therapy or tumor growth. Because the mechanisms of vitamin C uptake in gliomas are currently unknown, we evaluated the expression of the sodium‐vitamin C cotransporter (SVCT) and facilitative hexose transporter (GLUT) families in human glioma cells. In addition, as microglial cells can greatly infiltrate high‐grade gliomas (constituting up to 45% of cells in glioblastomas), the effect of TC620 glioma cell interactions with microglial‐like HL60 cells on vitamin C uptake (Bystander effect) was determined. Although glioma cells expressed high levels of the SVCT isoform‐2 (SVCT2), low functional activity, intracellular localization and the expression of the dominant‐negative isoform (dnSVCT2) were observed. The increased glucose metabolic activity of glioma cells was evident by the high 2‐Deoxy‐d ‐glucose and dehydroascorbic acid (DHA) uptake rates through the GLUT isoform‐1 (GLUT1), the main DHA transporter in glioblastoma. Co‐culture of glioma cells and activated microglial‐like HL60 cells resulted in extracellular ascorbic acid oxidation and high DHA uptake by glioma cells. This Bystander effect may explain the high antioxidative potential observed in high‐grade gliomas.

     

Cyclosporine a induces growth arrest or programmed cell death of human glioma cells

Human malignant gliomas are highly resistant to current therapeutic approaches. We previously demonstrated that cyclosporine A (CsA) induces an apoptotic cell death in rat C6 glioma cells. In the present study, we found the induction of growth arrest or cell death of human malignant glioma cells exposed to CsA. In studied glioma cells, an accumulation of p21Cip1/Waf1 protein, a cell cycle inhibitor, was observed following CsA treatment, even in the absence of functional p53 tumour suppressor. CsA induced a senescence-associated growth arrest, in U87-MG glioma cells with functional p53, while in U373 and T98G glioma cells with mutated p53, CsA treatment triggered cell death associated with alterations of cell morphology, cytoplasm vacuolation, and condensation of chromatin. In T98G cells this effect was completely abolished by simultaneous treatment with an inhibitor of protein synthesis, cycloheximide (CHX). Moreover, CsA-induced cell death was accompanied by activation of executory caspases followed by PARP cleavage. CsA treatment did not elevate fasL expression and had no effect on mitochondrial membrane potential. We conclude that CsA triggers either growth arrest or non-apoptotic, programmed cell death in human malignant glioma cells. Moreover, CsA employs mechanisms different to those in the action of radio- and chemotherapeutics, and operating even in cells resistant to conventional treatments. Thus, CsA or related drugs may be an effective novel strategy to treat drug-resistant gliomas or complement apoptosis-based therapies.     

TRAIL-induced apoptosis in gliomas is enhanced by Akt-inhibition and is independent of JNK activation

Patients with malignant gliomas have a poor prognosis and new treatment paradigms are needed against this disease. TRAIL/Apo2L selectively induces apoptosis in malignant cells sparing normal cells and is hence of interest as a potential therapeutic agent against gliomas. To determine the factors that modulate sensitivity to TRAIL, we examined the differences in TRAIL-activated signaling pathways in glioma cells with variable sensitivities to the agent. Apoptosis in response to TRAIL was unrelated to DR5 expression or endogenous p53 status in a panel of 8 glioma cell lines. TRAIL activated the extrinsic (cleavage of caspase-8, caspase-3 and PARP) and mitochondrial apoptotic pathways and reduced FLIP levels. It also induced caspase-dependent JNK activation, which did not influence TRAIL-induced apoptosis. Because the pro-survival PI3K/Akt pathway is highly relevant to gliomas, we assessed whether Akt could protect against TRAIL-induced apoptosis. Pretreatment with SH-6, a novel Akt inhibitor, enhanced TRAIL-induced apoptosis, suggesting a protective role for Akt. Conversely, TRAIL induced caspase-dependent cleavage of Akt neutralizing its anti-apoptotic effects. These results demonstrate that TRAIL-induced apoptosis in gliomas involves both activation of death pathways and downregulation of survival pathways. Additional studies are warranted to determine the therapeutic potential of TRAIL against gliomas.Supported in part by the NIH grant PO1 CA55261     

Inhibition of TGF-beta2 with AP 12009 in recurrent malignant gliomas: from preclinical to phase I/II studies

Transforming growth factor-beta2 (TGF-beta2) is known to suppress the immune response to cancer cells and plays a pivotal role in tumor progression by regulating key mechanisms including proliferation, metastasis, and angiogenesis. For targeted protein suppression the TGF-beta2-specific antisense oligodeoxynucleotide AP 12009 was developed. In vitro experiments have been performed to prove specificity and efficacy of the TGF-beta2 inhibitor AP 12009 employing patient-derived malignant glioma cells as well as peripheral blood mononuclear cells (PBMCs) from patients. Clinically, the antisense compound AP 12009 was assessed in three Phase I/II-studies for the treatment of patients with recurrent or refractory malignant (high-grade) glioma WHO grade III or IV. Although the study was not primarily designed as an efficacy evaluation, prolonged survival compared to literature data and response data were observed, which are very rarely seen in this tumor indication. Two patients experienced long-lasting complete tumor remissions. These results implicate targeted TGF-beta2-suppression using AP 12009 as a promising novel approach for malignant gliomas and other highly aggressive, TGF-beta-2-overexpressing tumors.     

cDNA clone,prokaryotic expression and purification of human interleukin-13 receptor {alpha}2 chain

Despite advances in surgical technology and radiation therapy, the prognosis in the patients with malignant glioma remains poor. Recent studies show that interleukin-13 receptor {alpha}2 chain (IL-13Ra2), a brain tumor-associated receptor for IL-13, may play a role in immunotherapy for glioblastoma. We thus amplified human IL-13Ra2 gene from the human glioblastoma cell line using RT-PCR and cloned the target gene into the pET-28a, a prokaryotic expressing plasmid. After transformation, the recombinant plasmid expressed a soluble protein induced by IPTG. The purified recombinant protein was shown to be a single band on the SDS-PAGE with a predicated molecular weight of human IL-13Ra2 gene, suggesting that the recombinant protein of human IL-13Ra2 was successfully expressed. Recombinant IL-13Ra2 protein can be used as an anti-tumor vaccine, which may provide a promising new strategy for the treatment of brain malignant gliomas.     

Inhibition of matrix metalloproteinase-9 gene expression by an isoflavone metabolite, irisolidone in U87MG human astroglioma cells

Matrix metalloproteinase-9 (MMP-9) plays an important role in mediating the invasion and angiogenic process of malignant gliomas. This study was undertaken to determine if an isoflavone metabolite, irisolidone, inhibits MMP-9 expression in human astroglioma cells. Irisolidone was found to inhibit the secretion and protein expression of MMP-9 induced by PMA in U87 MG glioma cells, accompanied by the inhibition of MMP-9 mRNA expression and promoter activity. Further mechanistic studies revealed that irisolidone inhibits the binding of NF-κB and AP-1 to the MMP-9 promoter and suppresses the PMA-induced phosphorylation of ERK and JNK, which are upstream signaling molecules in MMP-9 expression. The Matrigel-invasion assay showed that irisolidone suppresses the in vitro invasiveness of glioma cells. Therefore, the strong inhibition of MMP-9 expression by irisolidone might be a potential therapeutic modality for controlling the growth and invasiveness of gliomas.     

Histidine-Rich Glycoprotein Can Prevent Development of Mouse Experimental Glioblastoma

Extensive angiogenesis, formation of new capillaries from pre-existing blood vessels, is an important feature of malignant glioma. Several antiangiogenic drugs targeting vascular endothelial growth factor (VEGF) or its receptors are currently in clinical trials as therapy for high-grade glioma and bevacizumab was recently approved by the FDA for treatment of recurrent glioblastoma. However, the modest efficacy of these drugs and emerging problems with anti-VEGF treatment resistance welcome the development of alternative antiangiogenic therapies. One potential candidate is histidine-rich glycoprotein (HRG), a plasma protein with antiangiogenic properties that can inhibit endothelial cell adhesion and migration. We have used the RCAS/TV-A mouse model for gliomas to investigate the effect of HRG on brain tumor development. Tumors were induced with platelet-derived growth factor-B (PDGF-B), in the presence or absence of HRG. We found that HRG had little effect on tumor incidence but could significantly inhibit the development of malignant glioma and completely prevent the occurrence of grade IV tumors (glioblastoma).     

Targeted nitric oxide delivery preferentially induces glioma cell chemosensitivity via altered p53 and O6‐Methylguanine‐DNA Methyltransferase activity

Glioblastoma multiforme is the most common malignant central nervous system tumor, and also among the most difficult to treat due to a lack of response to chemotherapeutics. New methods of countering the mechanisms that confer chemoresistance to malignant gliomas could lead to significant advances in the quest to identify novel drug combinations or targeted drug delivery systems for cancer therapy. In this study, we investigate the use of a targeted nitric oxide (NO) donor as a pretreatment to sensitize glioma cells to chemotherapy. The protein chlorotoxin (CTX) has been shown to preferentially target glioma cells, and we have developed CTX–NO, a glioma‐specific, NO‐donating CTX derivative. Pretreatment of cells with CTX–NO followed by 48‐h exposure to either carmustine (BCNU) or temozolomide (TMZ), both common chemotherapeutics used in glioma treatment, resulted in increased efficacy of both therapeutics. After CTX–NO exposure, both T98G and U‐87MG human malignant glioma cells show increased sensitivity to BCNU and TMZ. Further investigation revealed that the consequences of this combination therapy was a reduction in active levels of the cytoprotective enzyme MGMT and altered p53 activity, both of which are essential in DNA repair and tumor cell resistance to chemotherapy. The combination of CTX–NO and chemotherapeutics also led to decreased cell invasion. These studies indicate that this targeted NO donor could be an invaluable tool in the development of novel approaches to treat cancer. Biotechnol. Bioeng. 2013; 110: 1211–1220. © 2012 Wiley Periodicals, Inc.     

Type 1 diabetes: entering the proteomic era

During the last decade, a major breakthrough in the field of proteomics has been achieved. This review describes available techniques for proteomic analyses, both gel and non-gel based, particularly concentrating on relative quantification techniques. The principle of the different techniques is discussed, highlighting the advantages and drawbacks of recently available visualization methods in gel-based assays. In addition, recent developments for quantitative analysis in non-gel-based approaches are summarized. This review focuses on applications in Type 1 diabetes. These mainly include proteomic studies on pancreatic islets in animal models and in the human situation. Also discussed are mass spectrometry-based studies on T-cells, and studies on the development of diagnostic markers for diabetic nephropathology by capillary electrophoresis coupled to mass spectrometry.