|Year : 2015 | Volume
| Issue : 2 | Page : 60-65
The serum high-mobility group box 1 level and RAGE expression in childhood acute lymphoblastic leukemic patients'
Manal H Farahat MD 1, Mohammad A Sharaf2, Tarek A Attia3
1 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Biochemistry, Zagazig University Hospitals, Zagazig University, Zagazig, Egypt
3 Department of Pediatrics, Faculty of Medicine, Zagazig University, Zagazig, Egypt
|Date of Submission||13-Feb-2015|
|Date of Acceptance||18-May-2015|
|Date of Web Publication||22-Jul-2015|
Manal H Farahat
Block 14, Building 14, Jasmine Tower, Apartment 903, Nasr City, 11111, Waha District
Source of Support: None, Conflict of Interest: None
Introduction High-mobility group box 1 (HMGB1) overexpression renders cancer cells resistant to apoptosis, and plays an important role in chemotherapy resistance. The aim of the present study was to measure serum high-mobility group box 1 (sHMGB1) and its receptor expression, advanced glycation end (RAGE) on childhood acute lymphoblastic leukemia (ALL) blast cells, and their correlation with different variables.
Participants and methods Twenty-eight newly diagnosed childhood ALL cases (group I), 24 patients with childhood ALL in complete remission (group II), and 22 apparently normal individuals matched for age and sex as the control group (group III) were included in the study. In addition to routine laboratory investigations, sHMGB1 was measured by enzyme-linked immunosorbent assay, and RAGE expression on mononuclear cells from the bone marrow and/or the peripheral blood was assessed using monoclonal antibodies by flow cytometry for all participants.
Results sHMGB1 and RAGE expression were significantly higher in ALL (group I) when compared with other groups; there was no significant difference between groups II and III with regard to sHMGB1 and RAGE expression. There were significant positive correlations between sHMGB1 and the white blood cell count and the bone marrow blast percentage in group I, and no significant correlations were found between sHMGB1, the RAGE expression, and other variables.
Conclusion The present study showed significant upregulation of sHMGB1 in all cases and RAGE expression in a few cases of childhood ALL. HMGB1 may represent an important potential target for cancer therapeutics. Hence, we recommend the concomitant use of HMGB1-neutralizing antibodies and potential HMGB1 release inhibitors (e.g. quercetin) with the chemotherapeutic agents used in childhood ALL treatment.
Keywords: childhood ALL, HMGB1, RAGE
|How to cite this article:|
Farahat MH, Sharaf MA, Attia TA. The serum high-mobility group box 1 level and RAGE expression in childhood acute lymphoblastic leukemic patients'. Egypt J Haematol 2015;40:60-5
|How to cite this URL:|
Farahat MH, Sharaf MA, Attia TA. The serum high-mobility group box 1 level and RAGE expression in childhood acute lymphoblastic leukemic patients'. Egypt J Haematol [serial online] 2015 [cited 2020 Feb 20];40:60-5. Available from: http://www.ehj.eg.net/text.asp?2015/40/2/60/161290
| Introduction|| |
High-mobility group box 1 (HMGB1) is a nuclear DNA-binding protein that serves as a DNA chaperone to regulate transcription, replication, recombination, repair, and genome stability  ; it interacts with and enhances the activities of a number of transcription factors, including p53  , p73  , and the retinoblastoma protein  . HMGB1 can be expressed under both physiological and pathological conditions. There are two ways for HMGB1 to be released from the cell. One is the active secretion from activated immune cells  , and the other is the passive release under stress conditions such as injury and infection from necrotic and injured cells  . HMGB1 serves as a damage-associated molecular pattern molecule to regulate inflammation and immunity  . Overexpression of HMGB1 by gene transfection renders several cancer cells resistant to apoptosis, whereas its expression suppression by RNA interference increases the sensitivity of cancer cells to anticancer drugs  .
Also, HMGB1 is a novel autophagy regulator through multiple mechanisms  . Studies suggested that autophagy may be important in the regulation of cancer development, progression, and in determining the response of tumor cells to anticancer therapy ,, .
HMGB1 has been reported to transduce cellular signals by interacting with at least three receptors: receptor for advanced glycation end (RAGE) products, toll-like receptor-2, and toll-like receptor-4  . RAGE was the first demonstrated binding partner  . RAGE-mediated signaling plays a role in the pathogenesis of epithelial-derived cancers such as pancreatic cancer by activating key survival pathways such as autophagy in cancer cells, propagating and sustaining protumor host inflammatory responses , , and also in intestinal and colorectal tumorigenesis  . The HMGB1-RAGE signaling axis represents an important potential target for cancer therapeutics  . RAGE promotes pancreatic tumor cell survival after genotoxic or metabolic stress  . Reducible HMGB1 binds to RAGE, induces beclin-1-dependent autophagy, and promotes resistance to chemotherapeutic agents or ionizing radiation  . Moreover, others have demonstrated that the inhibition of HMGB1-RAGE signaling suppresses tumor growth and metastases in C6 gliomas  and skin carcinogenesis  .
Acute lymphoblastic leukemia (ALL) is the most frequent cancer found in children. Karyotyping of leukemic cells identifies recurrent chromosome rearrangements. These are usually translocations that activate genes encoding transcription factors regulating B-cell or T-cell differentiation. This type of cancer usually progresses quickly if it is not treated  . Studies indicate that HMGB1 plays an important role in leukemia pathogenesis and chemotherapy resistance  .
The aim of the present study was to measure the serum levels of HMGB1 and RAGE expression on blast cells of childhood ALL and to study their correlation with different variables.
| Participants and methods|| |
The study protocol was approved by the ethical committee in our institute, and the study included 76 participants categorized into three groups:
Group I: Twenty-eight patients with newly diagnosed childhood ALL, including 16 male and 12 female patients. Their ages ranged from 3 to 12 years.
Group II: Twenty-four patients with childhood ALL in complete remission, including 13 male and 11 female patients. Their ages ranged from 3 to 11 years.
Group III: Twenty-two apparently normal individuals, with matched age and sex, served as a control group. There were 12 male and 10 female patients. Their ages ranged from 2 to 13 years.
Informed consents were obtained from the parents of the participants.
All individuals were subjected to the following:
- Full history taking, clinical examination, and chest radiograph.
- Routine laboratory investigations included the following:
- Complete blood count using KX-21N hematology analyser from Sysmex Corporation, Kobe, Japan, with the examination of Leishman-stained films.
- Liver and Kidney function tests using a dimension RXL MAX autoanalyser (Seimens Medical Solution Diagnostics, UL, USA).
- Bone marrow (BM) aspiration (for patients only) with the examination of Leishman-stained films. Smears were also stained for myeloperoxidase.
- Immunophenotyping of BM or peripheral blood samples was performed by FACScan flowcytometry (Becton Dickinson, San Jose, California, USA) using the following panel of monoclonal antibodies (Moabs): CD45, CD10, CD19, CD20, CD22, CD5, CD7, CD34, HLA-DR, CD13, CD33, CD14, and labeled with FITC/PE.
- Cytogenetic analysis using the G banding technique (cell imaging analyzer) (IMSTAR, Paris, France)  .
- Special investigations:
- Detection of serum levels of HMGB1 by enzyme-linked immunosorbent assay (ELISA) HMGB1 ELISA (IBL International GmbH, Hamburg, Germany):
It is a sandwich-enzyme immunoassay for the quantitative determination of HMGB1 in the serum. Wells of the microtiter strips are coated with purified anti-HMGB1 antibody. HMGB1 in the sample binds specifically to the immobilized antibody and is recognized by a second enzyme-marked antibody. After substrate reaction, the HMGB1 concentration is determined by the color intensity. The intensity of the blue color was proportionate to the amount of HMGB1 bound in the initial step. The color development was stopped, and the intensity of the color was measured and compared with a standard curve. Reading was carried out at 450 nm wavelength.
- Detection of RAGE expression by flow cytometry.
Two milliliters of heparinized BM and/or peripheral blood were collected in sterile tubes, mononuclear cells (MNCs) were separated according to Brach et al.  , by Ficoll-Hypaque density-gradient centrifugation (Histopaque 1077; Sigma, Aldrich, St. Louis, USA) at 2000 rpm for 40 min at 20°C, and the separated cells were suspended in PBS.
Phenotypic analysis was carried out by incubating 100 μl of MNCs with 10 μl of Moab against human RAGE to determine the RAGE expression (Biocompare, USA) for 30 min at 4°C. After washing three times in PBS, cells were incubated for 30 min at 4°C with PE-conjugated (secondary) goat F(ab′) 2 anti-mouse IgG. Cells labeled with irrelevant isotype-matched Moab and incubated with the secondary detection antibody served as negative controls. Data on at least 10 000 cells were collected using FACScan flowcytometry (Becton Dickinson) and analyzed using the CellQuest software (Becton Dickinson).
Data were tabulated statistically and analyzed using SPSS version 20.0 for Windows (SPSS Inc., Chicago, Illinois, USA). Results were expressed as mean ± SD, and analyzed statistically using analysis of variance. The least significant difference was determined to test the difference between the different studied groups. Correlation analysis was performed with the Pearson correlation test. P values below 0.05 were considered to be significant.
| Results|| |
Some laboratory data of ALL groups (group I and II) and the control group (group III) are illustrated in [Table 1].
[Table 2] shows that mean ± SD values of serum HMGB1 were 14 ± 6.22 ng/ml in group I, 1.01 ± 0.54 ng/ml in group II, and 1.0 ± 0.403 ng/ml in group III. There were highly significant differences among the groups (F = 98.73; P < 0.001).
|Table 2 Mean ± SD values, range, and simple ANOVA between the studied groups with regard to sHMGB1 and RAGE % expression |
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[Table 2] also shows that mean ± SD values of the percent of RAGE expression were 6.53 ± 10.0% in group I, 1.6 ± 0.826 in group II, and 1.23 ± 0.52 in group III. There were highly significant differences among the studied groups (F = 5.949; P < 0.01). There were six out of 28 cases (21.4%) in group I that showed increased RAGE expression.
No significant difference was found between the healthy control group and the ALL complete remission group regarding HMGB1 levels and RAGE expression (P > 0.05). [Table 3] and [Figure 1] showed that there was a significant positive correlation between sHMGB1 and the white blood cell count in group I (r = 0.51, P < 0.05).
|Figure 1 A significant positive correlation was found between serum high-mobility group box 1 (sHMGB1) and white blood count (WBC) in group I (r = 0.51, P < 0.05).|
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|Table 3 Correlations between sHMGB1 and some laboratory findings in the studied groups |
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[Table 3] and [Figure 2] show that there was a significant positive correlation between sHMGB1 and the percentage of BM blast cells in group I (r = 0.61, P < 0.01).
|Figure 2 A highly significant positive correlation was found between serum high-mobility group box 1 (sHMGB1) and blast% in group I (r = 0.61, P < 0.001).|
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| Discussion|| |
Many tumor cells upregulate autophagy and downregulate apoptosis to withstand chemotherapeutic treatment. HMGB1 overexpression inhibits apoptosis in leukemia K562 cells by regulating the protein level of Bcl-2 and the activities of caspase-3 and caspase-9  . As a positive regulator of autophagy, intracellular HMGB1 interacts with beclin-1 in leukemia cells, leading to autophagosome formation. In addition, exogenous HMGB1 directly induces autophagy and cell survival in leukemia cells  . Targeting the HMGB1 ligand or its receptor represents an important potential application in cancer therapy  . These findings raise the question as to whether HMGB1 and its receptor RAGE are upregulated in lymphoblastic leukemia. Therefore, this work was planned to study the serum level of HMGB1 and the expression of RAGE on the MNC of patients with childhood ALL and their correlations with some variables.
In this study, HMGB1 protein was constitutively detected by ELISA in the serum of both normal individuals and patients. Serum HMGB1 protein levels were significantly higher in newly diagnosed patients (group I) when compared with other groups. These results are in agreement with Kang et al.  , who reported that there were significant increases in the HMGB1 serum level in pediatric ALL patients with respect to either ALL patients in remission or normal controls. They suggested that HMGB1 stimulates leukemic cells to secrete tumor necrosis factor-a through an MAPK-dependent mechanism, and the measurement of serum HMGB1 is helpful to evaluate the prognosis of childhood ALL. Similarly, Zhao et al.  suggested that HMGB1 is important in the pathogenesis of leukemia. They showed that mRNA levels of HMGB1 are high in leukemia cells, and it is involved in the progression of childhood chronic myeloid leukemia. They argued that HMGB1 overexpression decreases the sensitivity of human myeloid leukemia cells K562 to anticancer drug-induced death by upregulating the autophagy pathway, which is confirmed by the observation of an increase in the formation of autophagosome and autophagolysosome fusion, in mRNA levels of beclin-1, VSP34, and UVRAG, which are key genes involved in mammalian autophagy, and in protein levels of p-Bcl-2 and LC3-II  .
Also, Yang et al.  found that HMGB1 was expressed abundantly in various kinds of both leukemia and nonblood cancer cell-lines, and its expression was positively correlated with the clinical status in childhood leukemia. They found that in leukemia cells, when endogenous HMGB1 increased starvation-induced autophagy, this reaction was inhibited by the suppression of HMGB1. Although the use of the autophagy inhibitor, 3-methyladenine, blocked the autophagic reaction and increased leukemia cell sensitivity to chemotherapy, enhancing HMGB1 expression decreased this sensitivity. In addition, suppressing HMGB1 expression also increased leukemia cell chemosensitivity. Their results suggested that endogenous HMGB1 is an intrinsic regulator of autophagy in leukemia cells  . Kimura and Mori  reported high plasma HMGB1 levels in patients with adult T-cell leukemia (caused by infection with human T-cell lymphotropic virus type I) compared with normal controls. Their results suggested that HMGB1 is a potential biomarker and a therapeutic target for adult T-cell leukemia.
In the present study, the increased levels of serum HMGB1 may be attributed to the release by the leukemic cells. We found a positive relationship between HMGB1 levels and the white blood cell count and the percentages of blast cells in newly diagnosed childhood ALL. Jia et al.  proved that chronic lymphoblastic leukemia (CLL) cells release HMGB1 passively. Also, Liu et al.  showed that HMGB1 was released from leukemia cell lines.
In our study, the assessment of RAGE expression on MNCs revealed very low expression in groups II and III, whereas it was highly increased in a few cases in groups I. Besides the present study, there have been no significant data published concerning RAGE expression in ALL patients. The RAGE expression data determined in our study [6/28 (21.4%) in group I] indicated that HMGB1-induced effects in most of the analyzed samples are not regulated by RAGE as it remains normally expressed in most patients. Results of Chai et al.  showed that the overexpression of the RAGE-1 transcript was found in 28% of the acute myloid leukemia (AML) patients and the level of RAGE-1 transcript decreased significantly in patients who obtained complete remission after treatment. The overall survival of AML patients with RAGE-1 overexpression was similar to that in those without RAGE-1 overexpression. It is concluded that RAGE-1 overexpression is a common event in AML, but has no impact on the prognosis of patients ([Table 4]).
|Table 4 Correlations between RAGE % expression and laboratory findings in the studied groups |
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| Conclusion|| |
The data of the present study showed significant upregulation of HMGB1 in most cases of childhood ALL, but RAGE upregulation was detected in only a few cases in group I. Hence, we support the notion that HMGB1 is a potential drug target for therapeutic interventions in leukemia, representing a therapeutic promise of autophagy inhibitors to render tumor cells more susceptible to conventional therapies. Hence, we may recommend the concomitant use of HMGB1-neutralizing antibodies and potential HMGB1 release inhibitors (e.g. quercetin) with the chemotherapeutic agents that are used in the treatment of childhood ALL.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
| References|| |
Liu Y, Prasad R, Wilson SH. HMGB1: roles in base excision repair and related function. Biochim Biophys Acta
Livesey K, Tang D, Zeh H, Lotze M. Not just nuclear proteins: ′novel′ autophagy cancer treatment targets - p53 and HMGB1. Curr Opin Investig Drugs
Uramoto H, Izumi H, Nagatani G, Ohmori H, Nagasue N, Ise T, et al.
Physical interaction of tumour suppressor p53/p73 with CCAAT-binding transcription factor 2 (CTF2) and differential regulation of human high-mobility group 1 (HMG1) gene expression. Biochem J
2003; 371(Pt 2)
Jiao Y, Wang HC, Fan SJ. Growth suppression and radiosensitivity increase by HMGB1 in breast cancer. Acta Pharmacol Sin
Wang H, Bloom O, Zhang M, Vishnubhakat JM, Ombrellino M, Che J, et al
. HMG-1 as a late mediator of endotoxin lethality in mice. Science
Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature
Andersson U, Tracey KJ. HMGB1 is a therapeutic target for sterile inflammation and infection. Annu Rev Immunol
Guerin R, Arseneault G, Dumont S, Rokeach LA. Calnexin is involved in apoptosis induced by endoplasmic reticulum stress in the fission yeast. Mol Biol Cell
Tang D, Kang R, Livesey KM, Kroemer G, Billiar TR, Van Houten B, et al
. High-mobility group box 1 is essential for mitochondrial quality control. Cell Metab
Amaravadi RK, Lippincott-Schwartz J, Yin XM, Weiss WA, Takebe N, Timmer W, et al
. Principles and current strategies for targeting autophagy for cancer treatment. Clin Cancer Res
Dikic I, Johansen T, Kirkin V. Selective autophagy in cancer development and therapy. Cancer Res
Kimmelman AC. The dynamic nature of autophagy in cancer. Genes Dev
Lotze MT, Tracey KJ. High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol
Hori O, Brett J, Slattery T, Cao R, Zhang J, Chen J, et al.
The receptor for advanced glycation end products (RAGE) is a cellular binding site for amphoterin. Mediation of neurite outgrowth and coexpression of RAGE and amphoterin in the developing nervous system. J Biol Chem
Kang R, Loux T, Tang D, Schapiro NE, Vernon P, Livesey KM, et al
. The expression of the receptor for advanced glycation endproducts (RAGE) is permissive for early pancreatic neoplasia. Proc Natl Acad Sci USA
Weiner L, Lotze M. Tumor-cell death, autophagy, and immunity. N Engl J Med
Heijmans J, Buller NV, Hoff E, Dihal AA, van der Poll T, van Zoelen MA, et al
. Rage signalling promotes intestinal tumourigenesis. Oncogene
Sims GP, Rowe DC, Rietdijk ST, Herbst R, Coyle AJ. HMGB1 and RAGE in inflammation and cancer. Annu Rev Immunol
Kang R, Tang D, Schapiro NE, Livesey KM, Farkas A, Loughran P, et al
. The receptor for advanced glycation end products (RAGE) sustains autophagy and limits apoptosis, promoting pancreatic tumor cell survival. Cell Death Differ
Tang D, Kang R, Cheh CW, Livesey KM, Liang X, Schapiro NE, et al
. HMGB1 release and redox regulates autophagy and apoptosis in cancer cells. Oncogene
Taguchi A, Blood DC, del Toro G, Canet A, Lee DC, Qu W, et al
. Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases. Nature
Gebhardt C, Riehl A, Durchdewald M, Nemeth J, Furstenberger G, Muller-Decker K, et al
. RAGE signaling sustains inflammation and promotes tumor development. J Exp Med
Harrison CJ. The detection and significance of chromosomal abnormalities in childhood acute lymphoblastic leukaemia. Blood Rev
Liu L, Yang M, Kang R, Wang Z, Zhao Y, Yu Y, et al.
HMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells. Leukemia
Seabright M. A rapid banding technique for human chromosomes. Lancet
Brach MA, Buhring HJ, Gruss HJ, Ashman LK, Ludwig WD, Mertelsmann RH, Herrmann F. Functional expression of c-kit by acute myelogenous leukemia blasts is enhanced by tumor necrosis factor-alpha through posttranscriptional mRNA stabilization by a labile protein. Blood
Yu Y, Xie M, He YL, Xu WQ, Zhu S, Cao LZ. Role of high mobility group box 1 in adriamycin-induced apoptosis in leukemia K562 cells. Ai Zheng
. 2008; 27
Yu Y, Xie M, Kang R, Livesey KM, Cao L, Tang D. HMGB1 is a therapeutic target for leukemia. Am J Blood Res
Lotze MT, DeMarco RA. Dealing with death: HMGB1 as a novel target for cancer therapy. Curr Opin Investig Drugs
Kang R, Tang DL, Cao LZ, Yu Y, Zhang GY, Xiao XZ. High mobility group box 1 is increased in children with acute lymphocytic leukemia and stimulates the release of tumor necrosis factor-alpha in leukemic cell. Zhonghua Er Ke Za Zhi
Zhao M, Yang M, Yang L, Yu Y, Xie M, Zhu S, et al.
HMGB1 regulates autophagy through increasing transcriptional activities of JNK and ERK in human myeloid leukemia cells. BMB Rep
Yang L, Yu Y, Kang R, Yang M, Xie M, Wang Z, et al.
Up-regulated autophagy by endogenous high mobility group box-1 promotes chemoresistance in leukemia cells. Leuk Lymphoma
Kimura M, Mori L. Abundant expression of HMGB1 in human Tcell lymphotropic virus type I infected Tcell lines and high plasma levels of HMGB1 in patients with adult Tcell leukemia. Oncol Lett
Jia L, Clear A, Liu FT, Matthews J, Uddin N, McCarthy A, et al
. Extracellular HMGB1 promotes differentiation of nurse-like cells in chronic lymphocytic leukemia. Blood
Chai HY, Qian J, Lin J, Yang J, Li Y, Wang CZ, et al
. Expression of RAGE-1 gene in acute myeloid leukemia. Zhongguo Shi Yan Xue Ye Xue Za Zhi
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]