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ORIGINAL ARTICLE
Year : 2014  |  Volume : 10  |  Issue : 1  |  Page : 107-111

The investigation of changes in proteins expression (Apolipoprotein A1 and albumin) in malignant astrocytoma brain tumor


1 Department of Genetics, Islamic Azad University, Tehran Medical Sciences Branch, Tehran, Iran
2 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

Date of Web Publication23-Apr-2014

Correspondence Address:
Mehrdad Hashemi
Department of Genetics, Islamic Azad University, Tehran Medical Sciences Branch, Tehran - 19395/1495
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.131413

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 > Abstract 

Objective: Angiogenesis performs a critical role in the development of cancer. Angiogenesis research is a cutting-edge field in cancer research. Proteomics is a powerful tool in identifying multiple proteins that are altered following a neuropharacological intervention in a disease of the central nervous system. Diagnostic oncoproteomics is the application of proteomic techniques for the diagnosis of malignancies.
Materials and Methods: We extracted proteins of tumor and normal brain tissues and then evaluated the protein purity by Bradford test and spectrophotometery method. In this study, we separated proteins by two-dimensional (2D) gel electrophoresis method and the spots were then analyzed and compared using statistical data and specific software, after providing three-dimensional images of spots alteration. Spots were identified by pH isoelectric, molecular weights, and data banks.
Results: Simple statistical test were used to establish a putative hierarchy in which the change in protein level were ranked according a cutoff point with P < 0.05. Apolipoprotein A1 (apo A1) protein and albumin were consistently upregulated in astrocytoma brain tumors.
Conclusion: The vascular microenvironment of glioma play a major role in determining the pathophysiological character is tics of the tumor. apo A1 and albumin are very significant due to their functional consequences in glioma tumor growth, migration and angiogenesis.

 > Abstract in Chinese 

恶性星形细胞脑瘤中蛋白质表达(阿朴脂蛋白和白蛋白)改变的研究
目的:血管生成在肿瘤发展中起到关键性作用。血管生成的研究是肿瘤研究中的前沿领域。在中枢神经系统疾病中,蛋白质组学在确认复杂的被神经干预而改变的蛋白质是强有力的工具。诊断癌蛋白质组学是癌症诊断中蛋白质组学技术的应用。
材料和方法:我们提取了肿瘤和正常脑组织中的蛋白质,然后用布拉德福试验和分光光度计评价了它们的纯度。此研究中,我们通过二维凝胶电泳法分离蛋白质,通过三维影像技术转化,再用统计学方法和特定软件对蛋白谱分析比较。蛋白谱通过等电位pH,分子量和数据库辩认。
结果:简单的统计检验用来建立假设的蛋白质改变层次水平,去掉不可信区间P<0.05。阿朴脂蛋白A1(apo A1)和白蛋白在脑星形细胞瘤中总是呈正调节。
结论:神经胶质瘤的血管微环境在决定肿瘤的病理生理特性中起主要作用。阿朴脂蛋白A1和白蛋白在神经胶质瘤生长、转移和血管生成方面需要引起注意,它们在上述方面有较强功能。
关键词:白蛋白二维凝胶电泳,星形细胞瘤,蛋白质组学,阿朴脂蛋白A1


Keywords: Albumin and 2G gel electrophoresis, astrocytoma, proteomics apolipoprotein A1


How to cite this article:
Hashemi M, Pooladi M, Razi Abad SK. The investigation of changes in proteins expression (Apolipoprotein A1 and albumin) in malignant astrocytoma brain tumor. J Can Res Ther 2014;10:107-11

How to cite this URL:
Hashemi M, Pooladi M, Razi Abad SK. The investigation of changes in proteins expression (Apolipoprotein A1 and albumin) in malignant astrocytoma brain tumor. J Can Res Ther [serial online] 2014 [cited 2019 Dec 9];10:107-11. Available from: http://www.cancerjournal.net/text.asp?2014/10/1/107/131413


 > Introduction Top


Angiogenesis was described in glioblastoma multiforme (GBM) as early as 1976, when Brem observed tense neovascularization in rabbit corneas transplanted with GBM. [1] GBM or grade IV astrocytoma is the most common and lethal adult malignant glioma tumor. [2] Glioma accounts for the majority of primary brain tumors. [3],[4] The glioblastomas are exceptionally aggressive malignancies with increased mitotic active pronounced angiogenesis (microvascular proliferation), necrosis, and proliferative rates 3-5 times higher than grade III astrocytoma tumors. [5] World Health Organization (WHO) grade III and IV tumors, comprising anaplastic glioma and glioblastoma, are highly malignant with median survival time for glioblastoma of around 14 months in the latest randomized controlled trials. [6] At least five distinct mechanisms of neovascularization and malignant in glioblastoma have been identified: I) Vascular cooption, II) angiogenesis, III) vasculogenesis, IV) vascular mimicry, and V) glioblastoma-endothelial cell transdifferentiation. [1]

Malignant gliomas exhibit a high degree of vascular proliferation histologically, and angiogenesis is a crucial, defining process in the progression of the disease. As such, angiogenesis is one on of the major there poetic targets for the development of never therapies. [7] A number of drugs that block angiogansis are in clinical development. Early clinical studies have employed antibodies or small molecule tyrosine kinase inhibitors. [8],[9]

Conversely, antibodies are costly to produce, must be administered by intravenous infusion, and are large molecular weight protein molecules with limited penetration of the intact blood-brain barrier (BBB). [10] Antibodies normally do not cress the BBB and cannot bind and intercellular cerebral antigen. [11] GBMs in particular have immature vasculature, with excessive leakiness, that con contributes to the breakdown of the BBB. [1]

Proteomics analysis is now applied widely in every area of neuroscience research in clouding brain cancer. [12],[13] Proteomics is a powerful tool in identifying multiple proteins that are altered following a neuropharacological intervention in a disease of the central nervous system (CNS). [14],[15] Diagnostic oncoproteomics is the application of proteomic techniques for the diagnosis of malignancies. [16]

Evaluation of protein structure, function, and regulation has evolved rapidly over the past several decades. Large amounts of information about a particular protein's activation status and interactions can now be obtained in a matter of minutes with new high-throughput approaches. [17] Well-designed studies are now required to define quantitative and qualitative differences between glioma and control brain proteomics. Clinical correlates of proteomics differences can be explored with respect to their role in tumors an etiology, implications for treatment and prognosis, and relationship to neuropathology. [18] In the present study, we investigate the apolipoprotein A1 (apo A1) and albumin proteins expression change in human brain astrocytoma tumor. To get an understanding of data and specific software molecular diagnosis of astrocytomas, we extracted proteins of tumor and normal brain tissues and evaluated the protein purity. After providing three-dimensional (3D) images of spots, we identified spot alteration using statistical data and specific software. Using different proteomics approaches, we identified multiple differentially expressed astrocytoma proteins, few of which could further be investigated as potential surrogate marker for astrocytoma.


 > Matherial and Methods Top


Patient samples

Tissues ware obtained, with informed consent and institutional review board approval, from patients undergoing tumor resection. For this study, all individuals filled a written informed consent form. Astrocytoma tumors were surgically removed at hospitals in Tehran. The tumors were classified by a team of neurophathologists according to the guidelines of the WHO classification of tumors of the CNS. Nontumoros brain tissues were obtained from normal areas (either grey or white matter) of brain tissues removed from patient undergoing nontumor epileptic surgery.

Tissue and samples preparation

Tissue samples of both tumor and normal brain tissue were snap-frozen immediately after operation in liquid nitrogen and stored at -80° until used for proteomic analysis. To obtain tissue extracts, the samples were broken into suitable pieces and were homogenized in lysis buffer II consisting of lysis buffer I {7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 0.2% 100 × Bio-Lyte 3/10}, dithiothreitol (DTT), and 1 mM ampholyte and protease inhibitor on ice. Cell lysis was completed by subsequent sonication (4 × 30 pulses). The samples were then centrifuged 20,000 g at 4°C for 30 min to remove insoluble debris. The supernatants were combined with acetone 100% and centrifuged at 15,000 g, and then the supernatants were decanted and removed (3 times). Acetone 100% was added to the protein precipitant and kept at - 20° overnight. The samples were then centrifuged again at 15,000 g and the precipitant incubated 1 h at room temperature. The protein samples were dissolved in rehydration buffer [8 M urea, 1% CHAPS, DTT, ampholyte pH (4) and protease inhibitor]. Protein concentrations were determined using the Bradford test and spectrophotometry method, and the protein extracts were then separated and used for two-dimensional (2D) gel electrophoresis.

2D gel electrophoresis

The isoelectric focusing for first-dimensional electrophoresis was performed using 18 cm, pH 3-10 immobilized pH gradient (IPG) strips. The samples were diluted in a solution containing rehydration buffer, IPG buffer, and DTT to reach a final protein amount of 500 μg per strip. The strips were subsequently subjected to voltage gradient as described in the instructions of the manufacturer. Once focused, the IPG strips were equilibrated twice for 15 min in equilibration buffer I [50 mM tris-Hcl (pH: 8.8), 6 M urea, 30% glycerol, 2% sodium dodecyl sulfate (SDS), and DTT] and equilibration buffer II. The second-dimensional SDS-PAGE was carried out using 12% PAGEs. Following SDS-PAGE, the gels were stained using the Coomassie Blue method.

Image analysis

The gel images were analyzed by Progenesis SameSpots software to identify spots differentially expressed between tumor and control samples based on their volume and density. The spots were carefully matched individually and only spots that showed a definite difference were defined as altered.


 > Results Top


Using 2D-PAGE proteomic analysis, we compared protein expression patterns between asterocytoma samples relative to control tissue. The 2D- difference gel electrophoresis revealed consistent protein profiles for each group. Simple statistical test were used to establish a putative hierarchy in which the change in protein level were ranked according a cutoff point with P < 0.05. The 2D gel showed totally 800 spots. A total of 343 spots showed statistically significant differences (Student's t-test; P < 0.05) in gel, of which 164 spots exhibited up regulation in expression level, whereas the remaining 179 spots were decreased in astrocytoma tumor relative to normal tissue. Among them, the statistically significant protein spots (P < 0.05) apo A1 protein was definitely with pI 5.11 and MW 28 kDa detected which has an upregulation about 3.7 (fold = 3.7) in astrocytoma brain tumors than normal brain tissue [Figure 1] and [Figure 2]. After providing 3D images of apo A1 protein spot alteration [Figure 3].
Figure 1: Images of apo A1 protein in (a) normal brain tissue (b) astrocytoma tumor tissue

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Figure 2: Apo A1 protein has an up-regulation about 3.7 (fold = 3.7) in astrocytoma brain tumor than normal brain tissue

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Figure 3: Three-dimensional images of apo A1 protein in normal brain tissue and astrocytoma tumor tissue

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Among them the statistically significant protein spots (P < 0.05) albumin protein was definitely with pI 6.09 and MW 69 kDa detected which has an upregulation about 1.8 (fold = 1.8) in asterocytoma brain tumors than normal brain tissue [Figure 4] and [Figure 5]. After providing 3D images of albumin protein spot alteration [Figure 6].
Figure 4: Images of albumin protein in A) normal brain tissue B) astrocytoma tumor tissue

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Figure 5: Albumin protein has a upregulation about 1.8 (fold = 1.8) in astrocytoma brain tumor than normal brain tissue

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Figure 6: Three-dimensional images of albumin protein in normal brain tissue and astrocytoma tumor tissue

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 > Discussion Top


Proteomics profiling in glioma research is described in a number of papers in human tissue. [19 - 22] The aims, experimental design, methodology and analysis, classification of control brain and size of these studies has been different and there appear inconsistency in some findings. [23]

The vascular microenvironment of glioma play a major role in determining the pathophysiological character is tics of the tumor. [24] Growing tumors reach a point at which the existing blood supply can no longer support the needs of the tumor leading to areas of hypoxia. In response to this hypoxia, GBMs undergo one angiogenic switch and increase section of various growth factors to promote new blood vessel formation. [10],[25] A vast number of growth factors have been implicated in glioma angiogenesis with vascular endothelial growth factor (VEGF) being attributed as the principal proangiopgenic factor. [25 - 27] VEGF is one of the mast critical growth factors and plays a central role in GBM angiogenesis by interacting with a number of pathways to promote GBM growth. [28],[29] Using fresh-frozen human tissue showed in several tumor types that VEGF-A was upregulated in the absence of markers of hypoxia. [1],[26] According to this hypothesis, a novel strategy can be suggested for human brain tumor management that enhances the respiratory potential of normal brain cells while metabolically targeting the tumor cells. The approach would involve a sequential series of therapeutic steps and should be effective against any primary or secondary brain tumor regardless of cell of origin, anatomical location, or histological grade. [6],[30],[31]

High-grade gliomas are among the most vascular of all solid tumors and vascular proliferation is a pathological hallmark of GBMs. [1],[2],[32] The comparison of GBM and healthy subjects revealed differentially expressed and statically significant (P < 0/05) serum proteins in brain tumor [Figure 7]. Among the identified proteins, apo A1 and albumin are very significant due to their functional consequences in glioma tumor growth, migration, and angiogenesis. Apo A1 and albumin are overexpressed in malignant tumor [Figure 8].
Figure 7: Apo A1 and albumin proteins position alteration in normal brain tissue and astrocytoma tumor tissue

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Figure 8: Proteins expression chart: Showing differential expression between the control (blue) and tumor (red)

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Apo A1 gene is adjacent to the genes encoding apo c3 and A4 on chromosome 11q23. Mature apo A1 is a 243 - amino acid. Single polypeptide chain synthesized by the liver and intestine. Apo A1 gene mRNA translates the 267 amino acid prepropeptide, which undergoes intracellular cleavage leading to a 249 - amino acid propertied. The propeptide is secreted and undergoes extracellular proteolysis giving rise to mature apo A1. [33],[34]

Apo A1 is known to play a central role in regulation of the efflux and transport of cholesterol from peripheral tissues to the liver, an as a cofactor for lecithin. Cholesterol acyltransferase is responsible for the formation of most cholesterol esters in plasma. [35] Apo A1 the major protein component of high-density lipoprotein (HDL). [36] Indeed, binding of HDL on hepatocyte membranes displayed two-binding sites with high and low affinities. [37] A high-affinity receptor for lipid-free Apo A1 was purified by Biacore and identified as the β-chain of adenosine triphosphate synthase. [36] Of screening of human serum or plasma by 2-DE is to become commonplace, a rapid, inexpensive and simple method is required to remove high-abundance proteins such as albumin. [38],[39],[40],[41] Glial albumin uptake was restricted to long-term epilepsy associated, vascularized-containing tumors. Intratumoral BBB dysfunction in concert with subsequent accumulation of albumin by neoplastic glial cell elements represent a new putatively epileptogenic mechanism for long-term epilepsy-associated tumors. [42]

Logically, one would expect the protein content of the filtrate (<30 KDa proteins) to be considerably less than the protein content of the retentive (>30 KDa proteins) which should contain proteins such as albumin. [43] must be administered by intravenous infusion and are large molecular weight protein molecules with limited penetration of the intact BBB. [10],[25]

The upregulation of serum albumin and apo A1 in malignant gliomas is thought to reflect the ability of both these proteins to pass into the interstitium of malignant gliomas because either the BBB has broken down and/or the tumor capillaries have no BBB. [19] The resulting neovasculature in glioma is structurally and functionally abnormal. [24] And, irregularities of the endothelial lining result in increased BBB permeability. [44],[45]


 > Acknowledgment Top


I would like to thank the department of genetics of Tehran medical branch, Islamic Azad University, Tehran, Iran.

 
 > References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]



 

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