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Serum TIMP1 and TIMP2 concentration in patients with different grades of meningioma
Clinical Neurology and Neurosurgery 170 (2018) 84–87
Contents lists available at ScienceDirect
Clinical Neurology and Neurosurgery journal homepage: www.elsevier.com/locate/clineuro
Serum TIMP1 and TIMP2 concentration in patients with different grades of meningioma
T
⁎
Farhad Mashayekhia, , Alia Saberib, Sohail Mashayekhib a b
Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran Neuroscience Research Center, Poursina Hospital, Guilan University of Medical Sciences, Rasht, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Meningioma Serum Concentration TIMP-1 TIMP-2
Objective: Meningiomas are common primary brain tumors that constitute about 13% of all intracranial tumors. Matrix metalloproteinase-9 (MMP-9) is able to degrade the extracellular matrix and basement membrane leading to cancer cell invasion and metastasis. MMPs are specifically inhibited by a family of small extracellular proteins known as the tissue inhibitors of metalloproteinases (TIMPs). The objective of this project was to evaluate serum concentration of TIMP-1 and TIMP-2 in patients with different grades of meningioma. Patients and Methods: Ninety samples from different stages of patients with meningitis (42 cases of grade I, 28 grade II, 20 grade III) and 51 samples from normal healthy were included in this study. Total protein concentration (TPC) and the level TIMP-1 and TIMP-2 serum were determined by Bio-Rad protein assay based on the Bradford dye procedure and enzyme-linked immunosorbent assay (ELISA), respectively. Results: No significant change in the TPC was seen in the serum of patients with meningioma when compared with normal controls. Results obtained demonstrated that all serum samples presented TIMP-1 and TIMP-2 expression, whereas, starting from grade I to III meningiomas, a significant decrease of TIMP-1 and TIMP-2 expression was observed as compared to controls. Conclusion: The results of this study show that a low expression of TIMP1 and TIMP2 is correlated with advanced stages of meningioma. It is also concluded that the detection of serum TIMP1 and TIMP2 may be useful in classifying different grades of meningioma.
1. Introduction Meningioma is the most common primary intracranial tumor which originates from the meninges that can be classified histologically into 15 subtypes and three grades (I–III). The frequency of grade I, II and III meningioma are 90%, 5–7% and 3% of all meningiomas, respectively [1]. The extracellular matrix (ECM) plays a crucial role in many physiological and pathological processes. Matrix metalloproteinases (MMPs), which are important in the process of ECM degradation, play key role in tumor progression [2]. MMPs are the family of endopeptidases mainly involved in tissue remodeling by selective proteolytic activity. MMPs activity is regulated by binding of endogenous tissue inhibitor of metalloproteinases (TIMPs) and hence play key role in the regulation of ECM degradation [3]. The association between higher expression of MMP and/or low TIMP expression during tumor progression has been demonstrated [4]. It has been shown that MMPs and TIMPs play important role in cancer progression and can be regarded as prognostic factor in cancer development [5]. MMPs, classified as the matrixin subfamily of zinc metalloprotease ⁎
Corresponding author. E-mail address: [email protected] (F. Mashayekhi).
https://doi.org/10.1016/j.clineuro.2018.05.001 Received 30 January 2018; Received in revised form 22 April 2018; Accepted 1 May 2018 Available online 02 May 2018 0303-8467/ © 2018 Elsevier B.V. All rights reserved.
family, are the main proteinases involved in ECM degradation. More than 50 similar MMPs including 23 MMPs, 13 ADAMs and 19 ADAMTSs were found in human. TIMPs were shown to function as the endogenous inhibitors of MMPs. The ECM proteolysis was shown to be controlled by the balance between MMPs and TIMPs. TIMPs are also able to control ECM degradation directly by control of the release of TGFβ1 activation [6].The MMPs were shown to play key role in different physiological and pathological processes [7–10]. TIMP-1 was shown to play important role in cell cycle regulation and cancer progression [11]. TIMPs play key role in MMPs function and the amount of ECM degradation is determined by the ratio of MMP/TIMP. The balance between MMPs/TIMPs regulates the ECM turnover and remodeling during normal development and pathogenesis. An imbalance of MMP/ TIMP regulation has been implicated in several inflammatory diseases of the central nervous system (CNS) [12]. Biochemical brain modifications was shown to be reflected in the biological fluids including serum, hence, measurement of peptides in the serum might identify biomarkers of meningioma and tumor
Clinical Neurology and Neurosurgery 170 (2018) 84–87
F. Mashayekhi et al.
progression. In this project we aimed to analyze the levels of TIMP-1 and TIMP-2 in the serum of patients with different grades of meningioma. 2. Material and methods 2.1. Patient samples Ninety samples from different stages of patients with meningitis (42 cases of grade I, 28 grade II, 20 grade III) and 51 samples from normal healthy were included in this study. The samples were collected between March 2013 and January 2018 at Poursina Hospital, Rasht, Iran. Meningioma cases were confirmed by magnetic resonance imaging (MRI) and histopathology according to the World Health Organization (WHO) classification [1]. Patients who had any history of cancer/intracranial surgery, and received either radiotherapy or chemotherapy before surgery were excluded in this study. The physical examination and imaging results of the patients at presentation and follow-up were recorded. Clinical information was collected and regularly updated for the patients through follow-up and questionnaires. One hundred and thirty two healthy volunteers were randomly selected from annual check-up visitors at the same hospital during the similar time period. The healthy controls with a self-reported history of CNS-related diseases or cancer and previously receiving radiotherapy/chemotherapy were excluded in this study. The research protocol was performed in accordance with the Declaration of Helsinki. Informed consent was obtained from all study subjects after explanation of the nature and possible consequences of the study. Then, the serum samples from control subjects and patients with different stages of meningioma were collected and stored at −70 °C until used.
Fig. 1. Total protein concentration in the serum of normal subjects and patients with various grades of meningioma (g/L). No significant difference has been seen in total protein concentration between the groups (P > 0.5).
Table 1 Total protein concentration in serum samples from patients with different grades of meningioma and controls (g/l).
Grade I Grade II Grade III Controls
Serum total protein concentration (Mean ± SD) g/l
P Compared to control group
0.37 0.37 0.37 0.37
0.939 0.970 0.933 –
± ± ± ±
0.04 0.06 0.05 0.05
different grades of meningioma and controls was measured by the BioRad protein assay. The results showed that no significant alterations in all serum samples, starting from grades I to III meningioma, was seen (0.37 ± 0.04, 0.37 ± 0.06, 0.37 ± 0.05 g/l as compared to control group (0.37 ± 0.05 g/l) (Fig. 1, Table 1) (P > 0.05).
2.2. Protein analysis 2.2.1. Total concentration of protein The total concentration of proteins in serum and serum was determined by the Bio-Rad protein assay based on the Bradford dye procedure.
3.2. Examination of TIMP-1 and TIMP-2 serum levels by ELISA The level of TIMP-1 and TIMP-2 was measured by ELISA. We showed that the serum concentrations of TIMP-1 and TIMP-2 in the patients with meningioma were lower than in normal subjects. TIMP1 and TIMP-2 were detected in all samples and starting from grades I to III meningioma, a significant decrease of TIMP-1 and TIMP-2 concentration was seen (TIMP-1 serum concentrations of 323.72 ± 45.51, 363.18 ± 21.50, 425.18 ± 4846 ng/ml) as compared to healthy subjects which was 276.27 ± 32.77 ng/ml (P < 0.001) (Fig. 2 and Table 2) and TIMP-2 serum levels of 139.09 ± 36.13, 189.81 ± 39.79, 254.54 ± 55.58 ng/ml as compared to controls (90.18 ± 41.13 ng/ml) (P < 0.01) (Fig. 3 and Table 3). A low level of TIMP-1 and TIMP-2 was demonstrated to be associated with advanced grades of meningioma.
2.2.2. TIMP-1 and TIMP-2 levels by ELISA TIMP-1 and TIMP-2 in serum was measured using the sensitive two sided ELISA and antiserum against Human TIMP-1 ELISA Kit (ab187394), Human TIMP-2 ELISA Kit (ab100653) (Cambridge, UK) were first coated with 80 ng primary antibody per well in 0.1 M Tris buffer. After overnight incubation, the plates were blocked with EIA buffer (50 mM Tris, pH 7.5, 0.3 M NaCl, 0.1% Triton X-100, 1% BSA and 1% Gelatine). The samples and standards were placed in triplicate wells and incubated overnight at room temperature. After washing a biotinylated secondary antibody (8 ng/ml) was added per well and the incubation was carried out for 24 h at room temperature. bGalactosidase coupled to Avidin was then added and after 2 h was followed by washing. Finally 200 l M 4-methylumbelliferyl-b-galactoside (Sigma, Poole, UK) in 50 mM sodium phosphate and 10 mM MgCl2 buffer were added and the amount of fluorescence was measured after 40 min incubation at 37 °C using a fluorimeter (Dynatech). 2.3. Statistical analysis All values were expressed as mean ± standard error of the mean (SEM). Statistical analysis was done using one-way ANOVA and only values with P ≤ 0.05 were considered as significant. 3. Results
Fig. 2. TIMP-1 concentration in the serum samples from controls and patients with different grades of meningioma (ng/ml). Significant increase in serum TIMP-1 level has been seen in the patients' samples when compared with normal controls (P < 0.05).
3.1. Total protein levels The total level of proteins in serum samples from patients with 85
Clinical Neurology and Neurosurgery 170 (2018) 84–87
F. Mashayekhi et al.
meningioma migration and invasion [23]. MMPs and their TIMPs were shown to be important in the pathogenesis of meningioma [24]. Increased levels of MMP-2 and MMP-9 in meningioma patients were shown as prognostic or predictive factors of recurrence [25]. Serum MMP-2 and -9 concentrations were shown to be significantly increased in meningioma patients as compared to normal subjects [26]. It was shown that TIMP-1 concentration increases in the serum of patients with hydrocephalus [27]. It was found that a CSF MMP-9 level in the patients with Seizure is higher than normal controls [28]. It has been suggested that MMPs and TIMPs play key role in cell proliferation, invasion and migration in different cancers including laryngeal carcinoma cells and ovarian cancer [29,30]. It was suggested that a balance between MMPs and TIMPs plays key role in human brain tumors and their expression may be valuable markers for tumor malignancy [31] and the imbalance between MMP-2 and TIMP-2 may have a key role in glioblastoma invasion by degrading the extracellular matrix [32]. The results of this study showed that the serum TIMP-1 and TIMP-2 concentrations were significantly decreased in patients with meningioma. Furthermore, among patients with meningiomas, the concentration of TIMP-1 and TIMP-2 were significantly lower in patients with advanced grade of the disease. The result of this study suggests that TIMP-1 and TIMP-2 serum concentrations may provide a reliable and practical indicator of malignant potential and tumor progression. It is also concluded that TIMP1 and TIMP2 might be involved in the pathophysiology of meningioma and the detection of serum TIMP1 and TIMP2 may be useful in classifying meningioma. Taken together, these data suggest that TIMP-1 and -2 expressions in serum may be valuable markers for tumor malignancy.
Table 2 TIMP-1 concentration in serum samples from patients with different grades of meningioma and controls (ng/ml).
Grade I Grade II Grade III Controls
Serum TIMP-1 concentration (Mean ± SD) ng/ml
P Compared to control group
323.72 363.18 425.18 276.27
0.01 < 0.0001 < 0.0001 –
± ± ± ±
45.51 21.50 4846 32.77
Fig. 3. TIMP-2 concentration in the serum samples from controls and patients with different grades of meningioma (ng/ml). Significant increase in serum TIMP-2 level has been seen in the patients' samples when compared with normal controls (P < 0.05).
Table 3 TIMP-2 concentration in serum samples from patients with different grades of meningioma and controls (ng/ml). Serum TIMP-2 concentration (Mean ± SD) ng/ml
Grade I Grade II Grade III Controls
139.09 ± 36.13 189.81 ± 39.79 254.54 ± 55.58 90.18 ± 41.13
Conflict of interest
P Compared to control group
None
0.007 < 0.00001 < 0.00001 –
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
4. Discussion
Acknowledgements
Meningiomas, the most common benign intracranial tumor, arise from the coverings of the brain and spinal cord and with the annual frequency of around 6 per 100.000 individuals. Ionizing radiation is an important environmental risk factor for meningioma. Several protooncogenes and tumor suppressor genes were shown to be implicated in meningioma development [13,14]. Meningiomas originate from the arachnoid layer of the meninges. The etiology of meningiomas is still unclear; specific genetic profile is the known risk factors [15]. Cytogenetic alterations of chromosome 22 and NF2 gene mutations are characteristic genetic alterations in early tumorigenesis [16]. Mutations in chromosome 10 (PTEN), CDKN2A (p14ARF), and CDKN2B and were shown to be involved in meningioma initiation and progression [14]. Genetic variation in many genes was shown to be involved in the susceptibility to meningioma [17]. The expression of many genes including Multidrug resistance-associated proteins (MRPs), DAL-1 (differentially expressed in adenocarcinoma of the lung), insulin like growth factor-2 (IGF2) and IGF-binding protein were shown to be altered in meningiomas [18–20]. Tissue inhibitors of matrix metalloproteinases (TIMPs) genes have been implicated in the aggressive behavior and invasion of meningiomas. Invasive meningiomas produce significantly lower levels of TIMP-1 compared with noninvasive meningiomas [21]. TIMP3 inactivation via methylation was associated with a more aggressive and higher-grade meningioma phenotype [22]. Moreover, suppression of MMP-9 expression decreases
This study was supported partly by the University of Guilan and GUMS, Rasht, Iran. The authors thank all people in the Genetics and Developmental Biology laboratories, Department of Biology, Faculty of Sciences, University of Guilan, for their technical assistance. References [1] D.N. Louis, A. Perry, G. Reifenberger, A. von Deimling, D. Figarella-Branger, W.K. Cavenee, H. Ohgaki, O.D. Wiestler, P. Kleihues, D.W. Ellison, The 2016 World Health Organization classification of tumors of the Central nervous system: a summary, Acta Neuropathol. 131 (2016) 803–820. [2] A.J. von Randow, S. Schindler, D.S. Tews, Expression of extracellular matrix-degrading proteins in classic, atypical, and anaplastic meningiomas, Pathol. Res. Pract. 202 (2006) 365–372. [3] D. Bourboulia, W.G. Stetler-Stevenson, Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs): positive and negative regulators in tumor cell adhesion, Semin. Cancer Biol. 20 (2010) 161–168. [4] A.E. Stanciu, A. Zamfir-Chiru-Anton, M.M. Stanciu, D.C. Gheorghe, Imbalance between matrix metalloproteinases and tissue inhibitors of metalloproteinases promotes invasion and metastasis of head and neck squamous cell carcinoma, Clin. Lab. 63 (2017) 1613–1620. [5] X. Lu, L. Duan, H. Xie, X. Lu, D. Lu, D. Lu, N. Jiang, Y. Chen, Evaluation of MMP-9 and MMP-2 and their suppressor TIMP-1 and TIMP-2 in adenocarcinoma of esophagogastric junction, Onco Targets Ther. 9 (2016) 4343–4349. [6] X. Wang, R.A. Khalil, Matrix Metalloproteinases, Vascular remodeling, and vascular disease, Adv. Pharmacol. 81 (2018) 241–330. [7] F. Toroghi, F. Mashayekhi, V. Montazeri, H. Saeedi Saedi, Z. Salehi, Association of MMP-9 promoter polymorphism and breast cancer among Iranian patients, Eur. J. Oncol. 22 (2017) 38–42.
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[8] A. Saberi, Z. Salehi, B. Naderinabi, S. Hojaat Ansari, S. Mashayekhi, Genetic dimension of intervertebral disc degeneration: polymorphism of matrix metalloproteinase 1 and 3 in the North Iranian population, Turk. Neurosurg. 4 (April) (2017), http://dx.doi.org/10.5137/1019-5149.JTN.19978-17.0. [9] F. Toroghi, F. Mashayekhi, V. Montazeri, H. Saeedi Saedi, Association between MMP9 promoter polymorphism and breast cancer progression in Northwest of Iran, Amuj 19 (2016) 46–53. [10] S. Shabanipour, F. Mashayekhi, M.H. Bahadori, Z.Z. Soruri, The relationship between MMP-9 promoterpolymorphism and IVF outcome, Cell. Mol. Biol. (Noisy-legrand) 61 (2015) 64–67. [11] Y.S. Kim, S.H. Kim, J.G. Kang, J.H. KO, Expression level and glycan dynamics determine the net effects of TIMP-1 on cancer progression, BMB Rep. 45 (2012) 623–628. [12] J. Gardner, A. Ghorpade, Tissue inhibitor of metalloproteinase (TIMP)-1: the TIMPed balance of matrix metalloproteinases in the central nervous system, J. Neurosci. Res. 74 (2003) 801–806. [13] T. Hortobágyi, J. Bencze, G. Varkoly, M.C. Kouhsari, Á Klekner, Meningioma recurrence, Open Med. (Wars) 11 (2016) 168–173. [14] K. Lamszus, Meningioma pathology, genetics, and biology, J. Neuropathol. Exp. Neurol. 63 (2004) 275–286. [15] V. Galani, E. Lampri, A. Varouktsi, G. Alexiou, A. Mitselou, A.P. Kyritsis, Genetic and epigenetic alterations in meningiomas, Clin. Neurol. Neurosurg. 157 (2017) 119–125. [16] S. Yuzawa, H. Nishihara, S. Tanaka, Genetic landscape of meningioma, Brain Tumor Pathol. 33 (2016) 237–247. [17] S. Mashayekhi, Z. Salehi, A. Saberi, M. Shakiba, F. Mashayekhi, S. YousefzadehChabok, Functional variants of p21 gene alter susceptibility to meningioma, Br. J. Biomed. Sci. 75 (2018) 92–94. [18] G.A. Alexiou, A. Goussia, A. Ntoulia, P. Zagorianakou, V. Malamou-Mitsi, S. Voulgaris, A.P. Kyritsis, Immunohistochemical study of MRP5 expression in meningiomas, Cancer Chemother. Pharmacol. 71 (2013) 825–828. [19] F. Nunes, Y. Shen, Y. Niida, R. Beauchamp, A.O. Stemmer-Rachamimov, V. Ramesh, J. Gusella, M. MacCollin, Inactivation patterns of NF2 and DAL-1/4.1B (EPB41L3) in sporadic meningioma, Cancer Genet. Cytogenet. 162 (2005) 135–139. [20] G. Wrobel, P. Roerig, F. Kokocinski, K. Neben, M. Hahn, G. Reifenberger, P. Lichter, Microarray-based gene expression profiling of benign, atypical and anaplastic meningiomas identifies novel genes associated with meningioma progression, Int. J. Cancer 114 (2005) 249–256. [21] H.K. Rooprai, A.J. Martin, A. King, U.D. Appadu, H. Jones, R.P. Selway, R.W. Gullan, G.J. Pilkington, Comparative gene expression profiling of ADAMs, MMPs, TIMPs, EMMPRIN, EGF-R and VEGFA in low grade meningioma, Int. J.
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Contents lists available at ScienceDirect
Clinical Neurology and Neurosurgery journal homepage: www.elsevier.com/locate/clineuro
Serum TIMP1 and TIMP2 concentration in patients with different grades of meningioma
T
⁎
Farhad Mashayekhia, , Alia Saberib, Sohail Mashayekhib a b
Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran Neuroscience Research Center, Poursina Hospital, Guilan University of Medical Sciences, Rasht, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Meningioma Serum Concentration TIMP-1 TIMP-2
Objective: Meningiomas are common primary brain tumors that constitute about 13% of all intracranial tumors. Matrix metalloproteinase-9 (MMP-9) is able to degrade the extracellular matrix and basement membrane leading to cancer cell invasion and metastasis. MMPs are specifically inhibited by a family of small extracellular proteins known as the tissue inhibitors of metalloproteinases (TIMPs). The objective of this project was to evaluate serum concentration of TIMP-1 and TIMP-2 in patients with different grades of meningioma. Patients and Methods: Ninety samples from different stages of patients with meningitis (42 cases of grade I, 28 grade II, 20 grade III) and 51 samples from normal healthy were included in this study. Total protein concentration (TPC) and the level TIMP-1 and TIMP-2 serum were determined by Bio-Rad protein assay based on the Bradford dye procedure and enzyme-linked immunosorbent assay (ELISA), respectively. Results: No significant change in the TPC was seen in the serum of patients with meningioma when compared with normal controls. Results obtained demonstrated that all serum samples presented TIMP-1 and TIMP-2 expression, whereas, starting from grade I to III meningiomas, a significant decrease of TIMP-1 and TIMP-2 expression was observed as compared to controls. Conclusion: The results of this study show that a low expression of TIMP1 and TIMP2 is correlated with advanced stages of meningioma. It is also concluded that the detection of serum TIMP1 and TIMP2 may be useful in classifying different grades of meningioma.
1. Introduction Meningioma is the most common primary intracranial tumor which originates from the meninges that can be classified histologically into 15 subtypes and three grades (I–III). The frequency of grade I, II and III meningioma are 90%, 5–7% and 3% of all meningiomas, respectively [1]. The extracellular matrix (ECM) plays a crucial role in many physiological and pathological processes. Matrix metalloproteinases (MMPs), which are important in the process of ECM degradation, play key role in tumor progression [2]. MMPs are the family of endopeptidases mainly involved in tissue remodeling by selective proteolytic activity. MMPs activity is regulated by binding of endogenous tissue inhibitor of metalloproteinases (TIMPs) and hence play key role in the regulation of ECM degradation [3]. The association between higher expression of MMP and/or low TIMP expression during tumor progression has been demonstrated [4]. It has been shown that MMPs and TIMPs play important role in cancer progression and can be regarded as prognostic factor in cancer development [5]. MMPs, classified as the matrixin subfamily of zinc metalloprotease ⁎
Corresponding author. E-mail address: [email protected] (F. Mashayekhi).
https://doi.org/10.1016/j.clineuro.2018.05.001 Received 30 January 2018; Received in revised form 22 April 2018; Accepted 1 May 2018 Available online 02 May 2018 0303-8467/ © 2018 Elsevier B.V. All rights reserved.
family, are the main proteinases involved in ECM degradation. More than 50 similar MMPs including 23 MMPs, 13 ADAMs and 19 ADAMTSs were found in human. TIMPs were shown to function as the endogenous inhibitors of MMPs. The ECM proteolysis was shown to be controlled by the balance between MMPs and TIMPs. TIMPs are also able to control ECM degradation directly by control of the release of TGFβ1 activation [6].The MMPs were shown to play key role in different physiological and pathological processes [7–10]. TIMP-1 was shown to play important role in cell cycle regulation and cancer progression [11]. TIMPs play key role in MMPs function and the amount of ECM degradation is determined by the ratio of MMP/TIMP. The balance between MMPs/TIMPs regulates the ECM turnover and remodeling during normal development and pathogenesis. An imbalance of MMP/ TIMP regulation has been implicated in several inflammatory diseases of the central nervous system (CNS) [12]. Biochemical brain modifications was shown to be reflected in the biological fluids including serum, hence, measurement of peptides in the serum might identify biomarkers of meningioma and tumor
Clinical Neurology and Neurosurgery 170 (2018) 84–87
F. Mashayekhi et al.
progression. In this project we aimed to analyze the levels of TIMP-1 and TIMP-2 in the serum of patients with different grades of meningioma. 2. Material and methods 2.1. Patient samples Ninety samples from different stages of patients with meningitis (42 cases of grade I, 28 grade II, 20 grade III) and 51 samples from normal healthy were included in this study. The samples were collected between March 2013 and January 2018 at Poursina Hospital, Rasht, Iran. Meningioma cases were confirmed by magnetic resonance imaging (MRI) and histopathology according to the World Health Organization (WHO) classification [1]. Patients who had any history of cancer/intracranial surgery, and received either radiotherapy or chemotherapy before surgery were excluded in this study. The physical examination and imaging results of the patients at presentation and follow-up were recorded. Clinical information was collected and regularly updated for the patients through follow-up and questionnaires. One hundred and thirty two healthy volunteers were randomly selected from annual check-up visitors at the same hospital during the similar time period. The healthy controls with a self-reported history of CNS-related diseases or cancer and previously receiving radiotherapy/chemotherapy were excluded in this study. The research protocol was performed in accordance with the Declaration of Helsinki. Informed consent was obtained from all study subjects after explanation of the nature and possible consequences of the study. Then, the serum samples from control subjects and patients with different stages of meningioma were collected and stored at −70 °C until used.
Fig. 1. Total protein concentration in the serum of normal subjects and patients with various grades of meningioma (g/L). No significant difference has been seen in total protein concentration between the groups (P > 0.5).
Table 1 Total protein concentration in serum samples from patients with different grades of meningioma and controls (g/l).
Grade I Grade II Grade III Controls
Serum total protein concentration (Mean ± SD) g/l
P Compared to control group
0.37 0.37 0.37 0.37
0.939 0.970 0.933 –
± ± ± ±
0.04 0.06 0.05 0.05
different grades of meningioma and controls was measured by the BioRad protein assay. The results showed that no significant alterations in all serum samples, starting from grades I to III meningioma, was seen (0.37 ± 0.04, 0.37 ± 0.06, 0.37 ± 0.05 g/l as compared to control group (0.37 ± 0.05 g/l) (Fig. 1, Table 1) (P > 0.05).
2.2. Protein analysis 2.2.1. Total concentration of protein The total concentration of proteins in serum and serum was determined by the Bio-Rad protein assay based on the Bradford dye procedure.
3.2. Examination of TIMP-1 and TIMP-2 serum levels by ELISA The level of TIMP-1 and TIMP-2 was measured by ELISA. We showed that the serum concentrations of TIMP-1 and TIMP-2 in the patients with meningioma were lower than in normal subjects. TIMP1 and TIMP-2 were detected in all samples and starting from grades I to III meningioma, a significant decrease of TIMP-1 and TIMP-2 concentration was seen (TIMP-1 serum concentrations of 323.72 ± 45.51, 363.18 ± 21.50, 425.18 ± 4846 ng/ml) as compared to healthy subjects which was 276.27 ± 32.77 ng/ml (P < 0.001) (Fig. 2 and Table 2) and TIMP-2 serum levels of 139.09 ± 36.13, 189.81 ± 39.79, 254.54 ± 55.58 ng/ml as compared to controls (90.18 ± 41.13 ng/ml) (P < 0.01) (Fig. 3 and Table 3). A low level of TIMP-1 and TIMP-2 was demonstrated to be associated with advanced grades of meningioma.
2.2.2. TIMP-1 and TIMP-2 levels by ELISA TIMP-1 and TIMP-2 in serum was measured using the sensitive two sided ELISA and antiserum against Human TIMP-1 ELISA Kit (ab187394), Human TIMP-2 ELISA Kit (ab100653) (Cambridge, UK) were first coated with 80 ng primary antibody per well in 0.1 M Tris buffer. After overnight incubation, the plates were blocked with EIA buffer (50 mM Tris, pH 7.5, 0.3 M NaCl, 0.1% Triton X-100, 1% BSA and 1% Gelatine). The samples and standards were placed in triplicate wells and incubated overnight at room temperature. After washing a biotinylated secondary antibody (8 ng/ml) was added per well and the incubation was carried out for 24 h at room temperature. bGalactosidase coupled to Avidin was then added and after 2 h was followed by washing. Finally 200 l M 4-methylumbelliferyl-b-galactoside (Sigma, Poole, UK) in 50 mM sodium phosphate and 10 mM MgCl2 buffer were added and the amount of fluorescence was measured after 40 min incubation at 37 °C using a fluorimeter (Dynatech). 2.3. Statistical analysis All values were expressed as mean ± standard error of the mean (SEM). Statistical analysis was done using one-way ANOVA and only values with P ≤ 0.05 were considered as significant. 3. Results
Fig. 2. TIMP-1 concentration in the serum samples from controls and patients with different grades of meningioma (ng/ml). Significant increase in serum TIMP-1 level has been seen in the patients' samples when compared with normal controls (P < 0.05).
3.1. Total protein levels The total level of proteins in serum samples from patients with 85
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meningioma migration and invasion [23]. MMPs and their TIMPs were shown to be important in the pathogenesis of meningioma [24]. Increased levels of MMP-2 and MMP-9 in meningioma patients were shown as prognostic or predictive factors of recurrence [25]. Serum MMP-2 and -9 concentrations were shown to be significantly increased in meningioma patients as compared to normal subjects [26]. It was shown that TIMP-1 concentration increases in the serum of patients with hydrocephalus [27]. It was found that a CSF MMP-9 level in the patients with Seizure is higher than normal controls [28]. It has been suggested that MMPs and TIMPs play key role in cell proliferation, invasion and migration in different cancers including laryngeal carcinoma cells and ovarian cancer [29,30]. It was suggested that a balance between MMPs and TIMPs plays key role in human brain tumors and their expression may be valuable markers for tumor malignancy [31] and the imbalance between MMP-2 and TIMP-2 may have a key role in glioblastoma invasion by degrading the extracellular matrix [32]. The results of this study showed that the serum TIMP-1 and TIMP-2 concentrations were significantly decreased in patients with meningioma. Furthermore, among patients with meningiomas, the concentration of TIMP-1 and TIMP-2 were significantly lower in patients with advanced grade of the disease. The result of this study suggests that TIMP-1 and TIMP-2 serum concentrations may provide a reliable and practical indicator of malignant potential and tumor progression. It is also concluded that TIMP1 and TIMP2 might be involved in the pathophysiology of meningioma and the detection of serum TIMP1 and TIMP2 may be useful in classifying meningioma. Taken together, these data suggest that TIMP-1 and -2 expressions in serum may be valuable markers for tumor malignancy.
Table 2 TIMP-1 concentration in serum samples from patients with different grades of meningioma and controls (ng/ml).
Grade I Grade II Grade III Controls
Serum TIMP-1 concentration (Mean ± SD) ng/ml
P Compared to control group
323.72 363.18 425.18 276.27
0.01 < 0.0001 < 0.0001 –
± ± ± ±
45.51 21.50 4846 32.77
Fig. 3. TIMP-2 concentration in the serum samples from controls and patients with different grades of meningioma (ng/ml). Significant increase in serum TIMP-2 level has been seen in the patients' samples when compared with normal controls (P < 0.05).
Table 3 TIMP-2 concentration in serum samples from patients with different grades of meningioma and controls (ng/ml). Serum TIMP-2 concentration (Mean ± SD) ng/ml
Grade I Grade II Grade III Controls
139.09 ± 36.13 189.81 ± 39.79 254.54 ± 55.58 90.18 ± 41.13
Conflict of interest
P Compared to control group
None
0.007 < 0.00001 < 0.00001 –
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
4. Discussion
Acknowledgements
Meningiomas, the most common benign intracranial tumor, arise from the coverings of the brain and spinal cord and with the annual frequency of around 6 per 100.000 individuals. Ionizing radiation is an important environmental risk factor for meningioma. Several protooncogenes and tumor suppressor genes were shown to be implicated in meningioma development [13,14]. Meningiomas originate from the arachnoid layer of the meninges. The etiology of meningiomas is still unclear; specific genetic profile is the known risk factors [15]. Cytogenetic alterations of chromosome 22 and NF2 gene mutations are characteristic genetic alterations in early tumorigenesis [16]. Mutations in chromosome 10 (PTEN), CDKN2A (p14ARF), and CDKN2B and were shown to be involved in meningioma initiation and progression [14]. Genetic variation in many genes was shown to be involved in the susceptibility to meningioma [17]. The expression of many genes including Multidrug resistance-associated proteins (MRPs), DAL-1 (differentially expressed in adenocarcinoma of the lung), insulin like growth factor-2 (IGF2) and IGF-binding protein were shown to be altered in meningiomas [18–20]. Tissue inhibitors of matrix metalloproteinases (TIMPs) genes have been implicated in the aggressive behavior and invasion of meningiomas. Invasive meningiomas produce significantly lower levels of TIMP-1 compared with noninvasive meningiomas [21]. TIMP3 inactivation via methylation was associated with a more aggressive and higher-grade meningioma phenotype [22]. Moreover, suppression of MMP-9 expression decreases
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