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Serum high mobility group box chromosomal protein 1 is associated with clinicopathologic features in patients with hepatocellular carcinoma
Available online at www.sciencedirect.com
Digestive and Liver Disease 40 (2008) 446–452
Liver, Pancreas and Biliary Tract
Serum high mobility group box chromosomal protein 1 is associated with clinicopathologic features in patients with hepatocellular carcinoma B.-Q. Cheng a,∗ , C.-Q. Jia b , C.-T. Liu a , X.-F. Lu a , N. Zhong a , Z.-L. Zhang c , W. Fan a , Y.-Q. Li a a
Department of Gastroenterology, Qilu Hospital, School of medicine, Shandong University, Jinan 250012, PR China b Department of Epidemiology and Health Statistics, Shandong University, Jinan 250012, PR China c Department of Surgery, Qilu Hospital, School of Medicine, Shandong University, Jinan 250012, PR China Received 18 August 2007; accepted 26 November 2007 Available online 21 February 2008
Abstract Background/aims. The role of high mobility group box chromosomal protein 1 in hepatocellular carcinoma is unknown. The aim of study was to evaluate contributions of high mobility group box chromosomal protein 1 in hepatocellular carcinoma, and analyse the correlation between high mobility group box chromosomal protein 1 and clinicopathologic outcomes. Patients/methods. High mobility group box chromosomal protein 1 levels were analysed by Western blot analysis. Edmondson grade, TNM stage and the Cancer of the Liver Italian Program score were used as analysis variables. Results. The serum high mobility group box chromosomal protein 1 levels in hepatocellular carcinoma (84.2 ± 50.4 ng/ml) was significantly higher than those in chronic hepatitis (39.8 ± 10.5 ng/ml), liver cirrhosis (40.2 ± 11.6 ng/ml) and healthy control (7.0 ± 5.9 ng/ml, p < 0.0001, respectively), and positive correlation were found between high mobility group box chromosomal protein 1 and ␣-fetoprotein (r = 0.952, p < 0.0001), and between high mobility group box chromosomal protein 1 and the size of tumour (r = 0.904, p < 0.0001). High mobility group box chromosomal protein 1 were significant differences among Edmondson grade I, II, III, IV; TNM stage I, II, III, IV and Cancer of the Liver Italian Program score 0–1 points, 2–4 points, >4 points (p < 0.0001, respectively). Conclusions. These results suggest that high mobility group box chromosomal protein 1 may be a useful marker for evaluating the tumour stage and predicting prognosis in hepatocellular carcinoma. Targeting high mobility group box chromosomal protein 1 production or release might have potential approaches for hepatocellular carcinoma treatment. © 2008 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. Keywords: ␣-Fetoprotein; Hepatocellular carcinoma; High mobility group box chromosomal protein 1
1. Introduction High mobility group box chromosomal protein 1 (HMGB1) is a nonhistone, chromatin-binding nuclear protein that functions as a structural co-factor critical for proper transcriptional regulation in somatic cells [1–3]. HMGB1 has a highly conserved sequence among various species, with 98% identity between rodent, bovine and human proteins [4]. Structurally, HMGB1 has 215 residues organized into two DNA-blinding domains (termed A-box and B-box) ∗
Corresponding author. Tel.: +86 531 82169454. E-mail address: [email protected] (B.-Q. Cheng).
and a negatively charged C-terminal tail. HMGB1 knockout mice display a phenotype of normal organ development and cell growth but die within a few hours of birth from hypoglycaemia, demonstrating the crucial role of this protein in cellular function [5]. HMGB1 appears to have two distinct functions in cellular systems. Intranuclearly, it binds without sequence specificity to the minor groove of DNA, which stabilizes nucleoside formation, regulates gene transcription and activates of steroid hormone receptors [6]. Extracellularly, HMGB1 has been demonstrated to be involved in inflammatory processes, the mediation of neurite outgrowth, smooth muscle cell chemotaxia, mesoangioblast migration and prolifera-
1590-8658/$30 © 2008 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. doi:10.1016/j.dld.2007.11.024
B.-Q. Cheng et al. / Digestive and Liver Disease 40 (2008) 446–452
tion, and tumour growth and metastasis [7–9]. Intranuclear HMGB1 reaches the extracellular environment by passive release from necrotic cells and active secretion from cells of innate immune system [10,11]. The preliminary study showed that the tumour cell death induced by a variety of agents such as cancer chemotherapeutic agents, oncolytic viruses, ultraviolet or gamma irradiation, and cytolytic T and nature killer cells indicated classic markers of apoptosis that were associated with significant HMGB1 release [12,13]. Extracellular HMGB1 induces cancer cell growth, mobility, invasion and metastasis via binding to specific membrane receptors including the receptor for advanced glycation end products (RAGE) and multiple toll-like receptors (TLR) with predominant intracellular signalling via p44/p42, p38, Rac1/Cdc42, SAP/JNM mitogen activated protein kinase (MAPK) pathway [14–16]. Blockade of the RAGE–HMGB1 interaction suppresses tumour growth and metastasis [16]. In addition, constant release of HMGB1 as a proinflammatory cytokine from necrotic tumour cells would create a microenvironment similar to chronic inflammations, a condition known to contribute to the development of epithelial malignancies [17]. Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide, with a very poor prognosis [18]. A better understanding of the molecular mechanism involved in the progression of HCC would facilitate future prognosis and treatment strategies. Increased expression of HMGB1 has been reported for several different tumour types, including gastrointestinal stromal tumours [19], breast carcinoma [20], colorectal cancer [21], prostate cancer [22] and pancreatic cancer [23]. Moreover, over expression of HMGB1 has been strongly correlated with tumour genesis and tumour metastasis [24]. However, until now, there is no information about HMGB1 activity and its role in the patients with HCC. The search for a serum biological marker of tumour invasiveness and prognosis in HCC is of clinical importance. In this study, we measured the levels of serum HMGB1 by Western blot analysis and analysed the relationship between the HMGB1 and clinic pathologic parameters in patients with hepatocellular carcinoma.
2. Materials and methods 2.1. Patients All patients diagnosed as having HCC, chronic hepatitis and liver cirrhosis at Department of Gastroenterology and Surgery, Qilu Hospital, Shandong University, were candidates for enrolment. The volunteers and patients who gave written informed consent entered this prospective cohort study, which was approved by the investigation and ethics committee of hospital according to the standards of the Declaration of Helsinki. A total of 161 patients with HCC, 51 patients with chronic hepatitis including 38 hepatitis B, 9 hepatitis C and 4 others, 62 patients with liver cirrhosis including
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49 hepatitis B virus (HBV) cirrhosis, 8 hepatitis C virus (HCV) cirrhosis and 5 others, 56 healthy volunteers were enrolled in this study. The volunteers had no previous liver disease and cancer history. All of patients were diagnosed by histopathological studies and serum viral test. The volunteers and patients were both age- and sex-matched. The procedure workup included ultrasonography (US), colour Doppler US, helical computed tomography (CT), ␣-fetoprotein (AFP), serum viral test and liver function test were performed. Stratification were performed based on the tumour size, levels of AFP, Edmondson grade, TNM stage and the Cancer of the Liver Italian Program (CLIP) score to analyse the correlation between the levels of HMGB1 and these variables. The clinic pathologic characteristics of HCC patients were in Table 1. Table 1 Baseline characteristics of the HCC patients Characteristics
Values
No. of patients
161
Epidemiological Age (years)a Male/femaleb
59.9 (7.1) 117 (73)/44 (27)
Underlying liver disease: HBV/HCV/othersb Tumour description No. of tumours: 1/2/≥3b Size of main tumour (cm)a Ratio of main tumour of <3/3–5/>5 (cm)b ␣-Fetoprotein (ng/ml)b AFP <200 200–400 >400
111 (69)/34 (21)/16 (10)
88 (55)/35 (22)/38 (24) 4.45 (2.05) 39 (24)/52 (32)/70 (43)
97 (60) 10 (6) 54 (34)
Liver function Total bilirubin (mg/dl)a BIL AST (IU/L)a ALT (IU/L)a Albumin (g/dl)a AKP (IU/L)a GGT (IU/L)a BUN (mg/dl)a Platelet count (×1000/mm3 )a Prothrombin activity (%)a PT Presence of ascitesb Child-Pugh class: A/B/Cb
1.60 (1.09) 68.13 (33.61) 88.77 (40.46) 3.65 (0.65) 168.35 (71.38) 128.81 (59.02) 14.36 (4.26) 10.70 (3.65) 74.34 (12.70) 34 (21) 105 (65)/34 (21)/22 (14)
General health Painb Constitutional syndromeb PST (0/1/2)b TNM stage (I/II/III/IV)b
29 (18) 29 (18) 131 (81)/14 (9)/16 (10) 22 (14)/66 (41)/51 (32)/22 (14)
CLIP score (0–1/2–4/>4)b
104 (65)/31 (19)/26 (16)
AST, aspartate aminotransferase; ALT, alanine aminotransferase; AKP, alkaline phosphatase; GGT, gamma-glutamyl transferase; BUN, blood urea nitrogen; PST, performance status test; CLIP, the Cancer of the Liver Italian Program. a Mean (S.D.). b Numbers (%).
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2.2. HMGB1 measurements Whole blood was initially collected in non-heparinized tubes and allowed to clot at room temperature for half an hour, then centrifuged for 15 min, and serum collected for storage at −80 ◦ C in microfuge tubes until assayed. HMGB1 levels were measured by Western blot analysis with reference to standard curves of purified HMGB1 as previously described [25]. In brief, sera samples were thawed on dry ice. Serum samples (100–200 l) were ultrafiltered with Microcon YM-100 filters (Millipore, Billerica, MA, USA). Filtrate was transferred to a new clean Eppendorf tube, 2× Laemmli sample buffer (Bio-Rad, Laemmli Sample Buffer, Hercules, CA, USA) was made with -mercaptoethanol (BioRad) according to manufacture’s instructions, heated at 95 ◦ C for 5 min and spun down at 10,000 rpm for 5 min in tabletop microfuge. Samples were subjected to electrophoresis through 10–20% Tris–HCI acrylamide gels (Bio-Rad) and transferred to pre-activated polyvinyline fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). PVDF membrane was pre-activated with methanol, rinsed and equilibrated in Tris/Glycine/20% methanol transfer buffer (Bio-Rad). After transfer, the membrane was rinsed for 5 min with wash buffer and 0.02% Tween 20 (Bio-Rad). Wash buffer was removed and incubated with 5% blocking buffer (Bio-Rad, Blotting grade blocker nonfat dry milk) for 1 h at room temperature. The membrane was probed overnight at 4 ◦ C with purified IgG from anti-HMGB1 antiserum (2 L/mL in 1% non-fat dry milk in wash buffer). PVDF membrane was washed four times at 15 min intervals in wash buffer and then incubated with peroxides conjugated anti-rabbit secondary antibody (Amersham Biosciences, Piscataway, NJ, USA) at dilution 1:5000 for 1 h. The membrane was washed three times at 15 min internals in wash buffer and 5 min with phosphate-buffered saline. PVDF membrane was
developed using ECL Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ, USA). Equal volumes of ECL A and B were added to the PVDF membrane and incubated for 1 min. PVDF membrane was then exposed to Hyperfilm (Fisher Scientific, Pittsburgh, PA). Autoradiography films were scanned, and densitometry analyses were performed using Quantity One software (Bio-Rad) and Microsoft Excel. The levels of HMGB1 were determined by reference to standard curves generated with purified HMGB1. 2.3. Statistical analysis One-way analysis of variance (ANOVA) with post hoc Bonferroni adjustment for pairwise comparison was used to compare the means of HMGB1 among groups. Pearson correlation analysis was performed to assess the correlations between HMGB1 and the continuous variables. All statistical analyses were carried out with STATA version 9.2 (Stata Corporation, College Station, TX, USA). All reported probabilities (p-value) were two-sided, and less than 0.05 was considered statistically significant.
3. Results 3.1. Serum HMGB1 levels in patients with HCC The mean value of serum HMGB1 levels in 161 patients with HCC was 84.2 ± 50.4 ng/ml and was significantly higher than those in 51 chronic hepatitis (39.8 ± 10.5 ng/ml), 62 liver cirrhosis (40.2 ± 11.6 ng/ml) and 56 healthy control (7.0 ± 5.9 ng/ml, p < 0.0001, respectively, Fig. 1) and no difference was found between cirrhosis and chronic hepatitis (p = 1.00). The levels of HMGB1 were no signif-
Fig. 1. Serum HMGB1 levels in patients with HCC. (*, #, $) Significantly lower vs. HCC (p < 0.0001, respectively).
B.-Q. Cheng et al. / Digestive and Liver Disease 40 (2008) 446–452
icant difference between HBV patients with HCC (n = 111, 84.7 ± 51.6 ng/ml) and HCV patients with HCC (n = 34, 92.1 ± 50.6 ng/ml; p = 0.47); between chronic hepatitis B (n = 38, 40.3 ± 10.8 ng/ml) and chronic hepatitis C (n = 9, 42, 4 ± 7.9 ng/ml; p = 0.59); and between HBV cirrhosis (n = 49, 39.0 ± 12.5 ng/ml) and HCV cirrhosis (n = 8, 44.4 ± 5.2 ng/ml; p = 0.24). 3.2. Relationship between the serum HMGB1 and AFP levels in patients with HCC To assess the correlation between the levels of serum HMGB1 and AFP in patients with HCC, we stratified the value of serum AFP into three levels: <200 ng/ml; 200–400 ng/ml and >400 ng/ml. The mean value of serum HMGB1 levels were 52.8 ± 20.3 ng/ml, 82.6 ± 10.1 ng/ml and 141.1 ± 42.5 ng/ml in correspondence with the serum AFP levels <200 ng/ml, 200–400 ng/ml and >400 ng/ml in the patients with HCC and significant differences were observed among three group (p < 0.0001) and between two groups (AFP of 200–400 ng/ml versus AFP < 200 ng/ml group, p = 0.008; AFP > 400 ng/ml versus AFP of 200–400 ng/ml group, p < 0.001). The significant positive correlation was shown between the levels of serum HMGB1 and AFP (r = 0.952, p < 0.0001). 3.3. Comparison of the serum HMGB1 levels in patients with different size of hepatocellular carcinoma We divided the tumours into three groups by their maximum size: <3 cm; 3–5 cm and >5 cm to compare the serum HMGB1 levels in patients with different size of HCC. For the patients with <3 cm, 3–5 cm and >5 cm HCC
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lesions, the serum HMGB1 levels were 35.2 ± 11.9 ng/ml, 69.3 ± 23.1 ng/ml and 122.7 ± 48.9 ng/ml. There were significant differences among three groups (p < 0.0001) and between two groups (p < 0.001, respectively) and the significant positive correlation between the levels of serum HMGB1 and the size of tumour (r = 0.904, p < 0.0001). 3.4. Correlation between the pathological differentiation of tumour and serum HMGB1 levels in patients with HCC To assess the correlation between the pathological differentiation of tumour and serum HMGB1 levels in patients with HCC, we defined the tumour differentiation with the Edmondson grade. For the HCC patients with tumour differentiation of Edmondson grade I, II, III and IV, the serum HMGB1 levels were 44.0 ± 18 ng/ml, 71.7 ± 10.9 ng/ml, 112.2 ± 14.5 ng/ml and 169.4 ± 48.1 ng/ml. The significant differences were shown among four groups (p < 0.0001) and between two groups (p < 0.001, respectively). 3.5. Comparison of the serum HMGB1 levels in patients with different tumour stage of hepatocellular carcinoma The tumour stage was defined by the AJCC staging (TNM stage) for HCC to evaluate whether HMGB1 was associated with HCC metastases to lymph nodes and distal organs, vascular invasion and tumour characteristics. The serum HMGB1 levels were 30.2 ± 5.9 ng/ml, 58.9 ± 20.0 ng/ml, 100.9 ± 23.4 ng/ml and 175.8 ± 47.6 ng/ml in patients with HCC of TNM stage I, II, III and IV. There were significant differences among four groups (p < 0.0001) and between two groups (p < 0.001, respectively, Fig. 2).
Fig. 2. Serum levels of HMGB1 in patients with tumours of different stages. Value among four groups was significantly different (p < 0.0001); (*) vs. stage I group, p < 0.001; (#) vs. stage II group, p < 0.001; ($) vs. stage III group, p < 0.001.
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Fig. 3. Relationship between serum HMGB1 levels and CLIP score in patients with HCC. Significant difference among three groups, p < 0.0001; (*) vs. 0–1 point group, p < 0.001; (#) vs. 2–4 point group, p < 0.001.
3.6. Relationship between the serum HMGB1 levels and prognostic score for patients with HCC To determine whether the serum HMGB1 was an independent factor that affected the prognosis for the patients with HCC, we used the CLIP score as prognostic system, and stratified this score into three levels: 0–1 points; 2–4 points and >4 points. The serum HMGB1 levels were 54.6 ± 20.8 ng/ml, 113.5 ± 13.0 ng/ml and 167.9 ± 47.7 ng/ml in patients with CLIP score 0–1 points, 2–4 points and >4 points. There were significant differences among three groups (p < 0.0001) and between two groups (p < 0.001, respectively, Fig. 3).
4. Discussion In the present study, we have first demonstrated that serum HMGB1 levels were significantly increased in patients with HCC than those in patients with chronic hepatitis and cirrhosis. No differences were found between chronic hepatitis and cirrhosis and between HBV patients and HCV patients. These results suggest that HMGB1 may play a pivotal role in the development of HCC. Several factors may contribute to elevation in serum HMGB1 levels in the patients with HCC. One is that HCC cells produce and secrete HMGB1. Stimulation of neoplastic cell lines with promoters such as 12-O-tetradecanoyl phorbol acetate (TPA) and the binding of c-Myc to the HMGB1 promoter induces the transcription of HMGB1 [25]. HMGB1 secretion is dependent on active translocation of HMGB1 from the nucleus to the cytoplasm before being released into the extra cellular milieu [26]. On the other hand, it was well known that HMGB1 can be passively released by necrotic and ischeamic cells [10,11].
Tumour cells proliferation may outpace the rate of angiogenesis, resulting in tissue severe ischeamic hypoxia [27]. Considered these results together, HMGB1 release through tumour cell death might have effect on both the local inflammatory responses at the tumour site and the rate of tumour-cell growth. Our results also showed that serum HMGB1 levels were strongly correlated with tumour stage, pathological differentiation grade, known HCC marker (AFP) and CLIP score. Malignancy increases with dedifferentiation, and high serum AFP levels may be an indirect measure of tumour burden [18]. CLIP score accounts for both liver function and tumour characteristics relevant to prognostic assessment for patients with HCC, and has the highest stratification ability regard to prognosis in patients with HCC [28]. These data suggest that HMGB1 may play an important role not only in development of HCC but in metastasis and poor outcome. As an extracellular molecular, HMGB1 induces several intracellular signalling pathways via binding to its receptor RAGE. Malignant cells and immature cells express high levels of HMGB1 and RAGE [3,16,23]. The co-localization of RAGE and HMGB1 indicated their potential contribution to cellular migration and tumour invasion, and the suppression of tumour growth and metastasis by blocking RAGE-HMGB1 complex in mice had been reported [16]. Signalling through RAGE leads to activation of the nuclear factor-kB (NF-kB) pathway, mitogen activated protein kinase (MAPK), type IV collagenase (MMP-2/MMP-9), which are important for cancer cell growth, invasion and metastasis [6,7,16,23]. RAGE and MMP-9 are expressed concordant with the higher metastasis ability of human pancreatic cancer cells [23]. Alternatively, for tumour growth, apoptosis must be blocked in the transformed; HMGB1 could act as an oncoprotein because of its ability to anti-apoptotic prop-
B.-Q. Cheng et al. / Digestive and Liver Disease 40 (2008) 446–452
erties [20]. In addition, overexpression of HMGB1 has been shown to cause modulation of the transcriptional expressing of many groups of genes reported to play key roles in different biological processes of neoplasm progression and metastasis. Enhanced expression of HMGB1 and RAGE has been strongly associated with atypical and increased size of colorectal adenomas, colorectal metastases to lymph nodes and distal organs, and poor prognosis at any colorectal cancer stage [24]. RAGE was found in 19%, 81% and 100% of Dukes’ B, C and D cases, respectively, correlating with invasiveness and poor outcome [29]. Thus, in our study, elevation of HMGB1 in patients with HCC can contribute to HCC genesis and metastasis by altering gene expression with cells. In conclusion, HMGB1 could be a useful and specific marker for evaluating the tumour differentiation grade, tumour stage and predicting prognosis in patients with HCC. This relatively new concept helps us to understand the pathogenesis of HCC and might provide insights that targeting HMGB1 production or release might have substantial potential applications for HCC treatment.
Practice points • The serum HMGB1 levels were significantly increased in patients with HCC than those in chronic hepatitis and liver cirrhosis. No significant differences were found between HBV and HCV infectious patients, and between chronic hepatitis and cirrhosis. • There were positive correlation between HMGB1 and AFP, and between HMGB1 and the size of tumour in patients with HCC. • HMGB1 were significant differences among Edmondson grade I, II, III, IV; TNM stage I, II, III, IV and CLIP score 0–1 points, 2–4 points, >4 points.
Research agenda • Further research will be needed to make an accurate cut-off value of HMGB1 concentrations levels that enable to be used as serological assessment parameters for early HCC and differentiate aggressive and nonaggressive HCCs. • Whether targeting HMGB1 production or release is efficacious method for HCC treatment will be further studied.
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Conflict of interest statement None declared.
References [1] Lotze MT, Tracey KJ. High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol 2005;5:331–42. [2] Parrish W, Ulloa L. High-mobility group box-1 isoforms as potential therapeutic targets in sepsis. Methods Mol Biol 2007;361:145–62. [3] Ulloa L, Messmer D. High-mobility group box 1 (HMGB1) protein: friend and foe. Cytokine Growth Factor Rev 2006;17:189–201. [4] Yotov WV, St-Arnaud R. Nucleotide sequence of a mouse cDNA encoding the nonhistone chromosomal high mobility group protein-1 (HMG1). Nucleic Acids Res 1992;20:3516. [5] Calogero S, Grassi F, Aguzzi A, Voigtlander T, Ferrier P, Ferrari S, et al. The lack of chromosomal protein Hmg1 does not disrupt cell growth but causes lethal hypoglycaemia in newborn mice. Nat Genet 1999;22:276–80. [6] Dumitriu IE, Baruah P, Manfredi AA, Bianchi ME, RovereQuerini P. HMGB1: guiding immunity from within. Trends Immunol 2005;26:381–7. [7] Evans A, Lennard TW, Davies BR. High-mobility group protein 1(Y): metastasis-associated or metastasis-inducing? J Surg Oncol 2004;88:86–99. [8] Palumbo R, Sampaolesi M, De Marchis F, Tonlorenzi R, Colombetti S, Mondino A, et al. Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation. J Cell Biol 2004;164:441–9. [9] Wang H, Yang H, Tracey KJ. Extracellular role of HMGB1 in inflammation and sepsis. J Intern Med 2004;255:320–31. [10] Rovere-Querini P, Capobianco A, Scaffidi P, Valentinis B, Catalanotti F, Giazzon M, et al. HMGB1 is an endogenous immune adjuvant released by necrotic cells. EMBO Rep 2004;5:825–30. [11] Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 2002;418:191–5. [12] Ito N, DeMarco RA, Mailliard RB, Han J, Rabinowich H, Kalinski P, et al. Cytolytic cells induce HMGB1 release from melanoma cell lines. J Leukoc Biol 2007;81:75–83. [13] Zeh III HJ, Lotze MT. Addicted to death: invasive cancer and the immune response to unscheduled cell death. J Immunother 2005;28:1–9. [14] Huttunen HJ, Fages C, Rauvala H. Receptor for advanced glycation end products (RAGE)-mediated neurite outgrowth and activation of NF-kappaB require the cytoplasmic domain of the receptor but different downstream signaling pathways. J Biol Chem 1999;274:19919– 24. [15] Park JS, Gamboni-Robertson F, He Q, Svetkauskaite D, Kim JY, Strassheim D, et al. High mobility group box 1 protein interacts with multiple Toll-like receptors. Am J Physiol Cell Physiol 2006;290:C917–24. [16] 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 2000;405:354–60. [17] Mignogna MD, Fedele S, Lo Russo L, Lo Muzio L, Bucci E. Immune activation and chronic inflammation as the cause of malignancy in oral lichen planus: is there any evidence? Oral Oncol 2004;40:120–30. [18] Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet 2003;362:1907–17. [19] Choi YR, Kim H, Kang HJ, Kim NG, Kim JJ, Park KS, et al. Overexpression of high mobility group box 1 in gastrointestinal stromal tumors with KIT mutation. Cancer Res 2003;63:2188–93. [20] Brezniceanu ML, Volp K, Bosser S, Solbach C, Lichter P, Joos S, et al. HMGB1 inhibits cell death in yeast and mammalian cells
452
[21]
[22]
[23]
[24]
B.-Q. Cheng et al. / Digestive and Liver Disease 40 (2008) 446–452 and is abundantly expressed in human breast carcinoma. FASEB J 2003;17:1295–7. Volp K, Brezniceanu ML, Bosser S, Brabletz T, Kirchner T, Gottel D, et al. Increased expression of high mobility group box 1 (HMGB1) is associated with an elevated level of the antiapoptotic c-IAP2 protein in human colon carcinomas. Gut 2006;55:234–42. Kuniyasu H, Chihara Y, Kondo H, Ohmori H, Ukai R. Amphoterin induction in prostatic stromal cells by androgen deprivation is associated with metastatic prostate cancer. Oncol Rep 2003;10:1863–8. Takada M, Hirata K, Ajiki T, Suzuki Y, Kuroda Y. Expression of receptor for advanced glycation end products (RAGE) and MMP-9 in human pancreatic cancer cells. Hepatogastroenterology 2004;51:928–30. Sasahira T, Akama Y, Fujii K, Kuniyasu H. Expression of receptor for advanced glycation end products and HMGB1/amphoterin in colorectal adenomas. Virchows Arch 2005;446:411–5.
[25] Cmarik JL, Li Y, Ogram SA, Min H, Reeves R, Colburn NH. Tumor promoter induces high mobility group HMG-Y protein expression in transformation-sensitive but not -resistant cells. Oncogene 1998;16:3387–96. [26] Rendon-Mitchell B, Ochani M, Li J, Han J, Wang H, Yang H, et al. IFN-gamma induces high mobility group box 1 protein release partly through a TNF-dependent mechanism. J Immunol 2003;170:3890–7. [27] Carmeliet P. Angiogenesis in life, disease and medicine. Nature 2005;438:932–6. [28] Ueno S, Tanabe G, Sako K, Hiwaki T, Hokotate H, Fukukura Y, et al. Discrimination value of the new western prognostic system (CLIP score) for hepatocellular carcinoma in 662 Japanese patients. Cancer of the Liver Italian Program. Hepatology 2001;34:529–34. [29] Lotze MT, DeMarco RA. Dealing with death: HMGB1 as a novel target for cancer therapy. Curr Opin Invest Drugs 2003;4:1405–9.
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Liver, Pancreas and Biliary Tract
Serum high mobility group box chromosomal protein 1 is associated with clinicopathologic features in patients with hepatocellular carcinoma B.-Q. Cheng a,∗ , C.-Q. Jia b , C.-T. Liu a , X.-F. Lu a , N. Zhong a , Z.-L. Zhang c , W. Fan a , Y.-Q. Li a a
Department of Gastroenterology, Qilu Hospital, School of medicine, Shandong University, Jinan 250012, PR China b Department of Epidemiology and Health Statistics, Shandong University, Jinan 250012, PR China c Department of Surgery, Qilu Hospital, School of Medicine, Shandong University, Jinan 250012, PR China Received 18 August 2007; accepted 26 November 2007 Available online 21 February 2008
Abstract Background/aims. The role of high mobility group box chromosomal protein 1 in hepatocellular carcinoma is unknown. The aim of study was to evaluate contributions of high mobility group box chromosomal protein 1 in hepatocellular carcinoma, and analyse the correlation between high mobility group box chromosomal protein 1 and clinicopathologic outcomes. Patients/methods. High mobility group box chromosomal protein 1 levels were analysed by Western blot analysis. Edmondson grade, TNM stage and the Cancer of the Liver Italian Program score were used as analysis variables. Results. The serum high mobility group box chromosomal protein 1 levels in hepatocellular carcinoma (84.2 ± 50.4 ng/ml) was significantly higher than those in chronic hepatitis (39.8 ± 10.5 ng/ml), liver cirrhosis (40.2 ± 11.6 ng/ml) and healthy control (7.0 ± 5.9 ng/ml, p < 0.0001, respectively), and positive correlation were found between high mobility group box chromosomal protein 1 and ␣-fetoprotein (r = 0.952, p < 0.0001), and between high mobility group box chromosomal protein 1 and the size of tumour (r = 0.904, p < 0.0001). High mobility group box chromosomal protein 1 were significant differences among Edmondson grade I, II, III, IV; TNM stage I, II, III, IV and Cancer of the Liver Italian Program score 0–1 points, 2–4 points, >4 points (p < 0.0001, respectively). Conclusions. These results suggest that high mobility group box chromosomal protein 1 may be a useful marker for evaluating the tumour stage and predicting prognosis in hepatocellular carcinoma. Targeting high mobility group box chromosomal protein 1 production or release might have potential approaches for hepatocellular carcinoma treatment. © 2008 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. Keywords: ␣-Fetoprotein; Hepatocellular carcinoma; High mobility group box chromosomal protein 1
1. Introduction High mobility group box chromosomal protein 1 (HMGB1) is a nonhistone, chromatin-binding nuclear protein that functions as a structural co-factor critical for proper transcriptional regulation in somatic cells [1–3]. HMGB1 has a highly conserved sequence among various species, with 98% identity between rodent, bovine and human proteins [4]. Structurally, HMGB1 has 215 residues organized into two DNA-blinding domains (termed A-box and B-box) ∗
Corresponding author. Tel.: +86 531 82169454. E-mail address: [email protected] (B.-Q. Cheng).
and a negatively charged C-terminal tail. HMGB1 knockout mice display a phenotype of normal organ development and cell growth but die within a few hours of birth from hypoglycaemia, demonstrating the crucial role of this protein in cellular function [5]. HMGB1 appears to have two distinct functions in cellular systems. Intranuclearly, it binds without sequence specificity to the minor groove of DNA, which stabilizes nucleoside formation, regulates gene transcription and activates of steroid hormone receptors [6]. Extracellularly, HMGB1 has been demonstrated to be involved in inflammatory processes, the mediation of neurite outgrowth, smooth muscle cell chemotaxia, mesoangioblast migration and prolifera-
1590-8658/$30 © 2008 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. doi:10.1016/j.dld.2007.11.024
B.-Q. Cheng et al. / Digestive and Liver Disease 40 (2008) 446–452
tion, and tumour growth and metastasis [7–9]. Intranuclear HMGB1 reaches the extracellular environment by passive release from necrotic cells and active secretion from cells of innate immune system [10,11]. The preliminary study showed that the tumour cell death induced by a variety of agents such as cancer chemotherapeutic agents, oncolytic viruses, ultraviolet or gamma irradiation, and cytolytic T and nature killer cells indicated classic markers of apoptosis that were associated with significant HMGB1 release [12,13]. Extracellular HMGB1 induces cancer cell growth, mobility, invasion and metastasis via binding to specific membrane receptors including the receptor for advanced glycation end products (RAGE) and multiple toll-like receptors (TLR) with predominant intracellular signalling via p44/p42, p38, Rac1/Cdc42, SAP/JNM mitogen activated protein kinase (MAPK) pathway [14–16]. Blockade of the RAGE–HMGB1 interaction suppresses tumour growth and metastasis [16]. In addition, constant release of HMGB1 as a proinflammatory cytokine from necrotic tumour cells would create a microenvironment similar to chronic inflammations, a condition known to contribute to the development of epithelial malignancies [17]. Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide, with a very poor prognosis [18]. A better understanding of the molecular mechanism involved in the progression of HCC would facilitate future prognosis and treatment strategies. Increased expression of HMGB1 has been reported for several different tumour types, including gastrointestinal stromal tumours [19], breast carcinoma [20], colorectal cancer [21], prostate cancer [22] and pancreatic cancer [23]. Moreover, over expression of HMGB1 has been strongly correlated with tumour genesis and tumour metastasis [24]. However, until now, there is no information about HMGB1 activity and its role in the patients with HCC. The search for a serum biological marker of tumour invasiveness and prognosis in HCC is of clinical importance. In this study, we measured the levels of serum HMGB1 by Western blot analysis and analysed the relationship between the HMGB1 and clinic pathologic parameters in patients with hepatocellular carcinoma.
2. Materials and methods 2.1. Patients All patients diagnosed as having HCC, chronic hepatitis and liver cirrhosis at Department of Gastroenterology and Surgery, Qilu Hospital, Shandong University, were candidates for enrolment. The volunteers and patients who gave written informed consent entered this prospective cohort study, which was approved by the investigation and ethics committee of hospital according to the standards of the Declaration of Helsinki. A total of 161 patients with HCC, 51 patients with chronic hepatitis including 38 hepatitis B, 9 hepatitis C and 4 others, 62 patients with liver cirrhosis including
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49 hepatitis B virus (HBV) cirrhosis, 8 hepatitis C virus (HCV) cirrhosis and 5 others, 56 healthy volunteers were enrolled in this study. The volunteers had no previous liver disease and cancer history. All of patients were diagnosed by histopathological studies and serum viral test. The volunteers and patients were both age- and sex-matched. The procedure workup included ultrasonography (US), colour Doppler US, helical computed tomography (CT), ␣-fetoprotein (AFP), serum viral test and liver function test were performed. Stratification were performed based on the tumour size, levels of AFP, Edmondson grade, TNM stage and the Cancer of the Liver Italian Program (CLIP) score to analyse the correlation between the levels of HMGB1 and these variables. The clinic pathologic characteristics of HCC patients were in Table 1. Table 1 Baseline characteristics of the HCC patients Characteristics
Values
No. of patients
161
Epidemiological Age (years)a Male/femaleb
59.9 (7.1) 117 (73)/44 (27)
Underlying liver disease: HBV/HCV/othersb Tumour description No. of tumours: 1/2/≥3b Size of main tumour (cm)a Ratio of main tumour of <3/3–5/>5 (cm)b ␣-Fetoprotein (ng/ml)b AFP <200 200–400 >400
111 (69)/34 (21)/16 (10)
88 (55)/35 (22)/38 (24) 4.45 (2.05) 39 (24)/52 (32)/70 (43)
97 (60) 10 (6) 54 (34)
Liver function Total bilirubin (mg/dl)a BIL AST (IU/L)a ALT (IU/L)a Albumin (g/dl)a AKP (IU/L)a GGT (IU/L)a BUN (mg/dl)a Platelet count (×1000/mm3 )a Prothrombin activity (%)a PT Presence of ascitesb Child-Pugh class: A/B/Cb
1.60 (1.09) 68.13 (33.61) 88.77 (40.46) 3.65 (0.65) 168.35 (71.38) 128.81 (59.02) 14.36 (4.26) 10.70 (3.65) 74.34 (12.70) 34 (21) 105 (65)/34 (21)/22 (14)
General health Painb Constitutional syndromeb PST (0/1/2)b TNM stage (I/II/III/IV)b
29 (18) 29 (18) 131 (81)/14 (9)/16 (10) 22 (14)/66 (41)/51 (32)/22 (14)
CLIP score (0–1/2–4/>4)b
104 (65)/31 (19)/26 (16)
AST, aspartate aminotransferase; ALT, alanine aminotransferase; AKP, alkaline phosphatase; GGT, gamma-glutamyl transferase; BUN, blood urea nitrogen; PST, performance status test; CLIP, the Cancer of the Liver Italian Program. a Mean (S.D.). b Numbers (%).
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2.2. HMGB1 measurements Whole blood was initially collected in non-heparinized tubes and allowed to clot at room temperature for half an hour, then centrifuged for 15 min, and serum collected for storage at −80 ◦ C in microfuge tubes until assayed. HMGB1 levels were measured by Western blot analysis with reference to standard curves of purified HMGB1 as previously described [25]. In brief, sera samples were thawed on dry ice. Serum samples (100–200 l) were ultrafiltered with Microcon YM-100 filters (Millipore, Billerica, MA, USA). Filtrate was transferred to a new clean Eppendorf tube, 2× Laemmli sample buffer (Bio-Rad, Laemmli Sample Buffer, Hercules, CA, USA) was made with -mercaptoethanol (BioRad) according to manufacture’s instructions, heated at 95 ◦ C for 5 min and spun down at 10,000 rpm for 5 min in tabletop microfuge. Samples were subjected to electrophoresis through 10–20% Tris–HCI acrylamide gels (Bio-Rad) and transferred to pre-activated polyvinyline fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). PVDF membrane was pre-activated with methanol, rinsed and equilibrated in Tris/Glycine/20% methanol transfer buffer (Bio-Rad). After transfer, the membrane was rinsed for 5 min with wash buffer and 0.02% Tween 20 (Bio-Rad). Wash buffer was removed and incubated with 5% blocking buffer (Bio-Rad, Blotting grade blocker nonfat dry milk) for 1 h at room temperature. The membrane was probed overnight at 4 ◦ C with purified IgG from anti-HMGB1 antiserum (2 L/mL in 1% non-fat dry milk in wash buffer). PVDF membrane was washed four times at 15 min intervals in wash buffer and then incubated with peroxides conjugated anti-rabbit secondary antibody (Amersham Biosciences, Piscataway, NJ, USA) at dilution 1:5000 for 1 h. The membrane was washed three times at 15 min internals in wash buffer and 5 min with phosphate-buffered saline. PVDF membrane was
developed using ECL Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ, USA). Equal volumes of ECL A and B were added to the PVDF membrane and incubated for 1 min. PVDF membrane was then exposed to Hyperfilm (Fisher Scientific, Pittsburgh, PA). Autoradiography films were scanned, and densitometry analyses were performed using Quantity One software (Bio-Rad) and Microsoft Excel. The levels of HMGB1 were determined by reference to standard curves generated with purified HMGB1. 2.3. Statistical analysis One-way analysis of variance (ANOVA) with post hoc Bonferroni adjustment for pairwise comparison was used to compare the means of HMGB1 among groups. Pearson correlation analysis was performed to assess the correlations between HMGB1 and the continuous variables. All statistical analyses were carried out with STATA version 9.2 (Stata Corporation, College Station, TX, USA). All reported probabilities (p-value) were two-sided, and less than 0.05 was considered statistically significant.
3. Results 3.1. Serum HMGB1 levels in patients with HCC The mean value of serum HMGB1 levels in 161 patients with HCC was 84.2 ± 50.4 ng/ml and was significantly higher than those in 51 chronic hepatitis (39.8 ± 10.5 ng/ml), 62 liver cirrhosis (40.2 ± 11.6 ng/ml) and 56 healthy control (7.0 ± 5.9 ng/ml, p < 0.0001, respectively, Fig. 1) and no difference was found between cirrhosis and chronic hepatitis (p = 1.00). The levels of HMGB1 were no signif-
Fig. 1. Serum HMGB1 levels in patients with HCC. (*, #, $) Significantly lower vs. HCC (p < 0.0001, respectively).
B.-Q. Cheng et al. / Digestive and Liver Disease 40 (2008) 446–452
icant difference between HBV patients with HCC (n = 111, 84.7 ± 51.6 ng/ml) and HCV patients with HCC (n = 34, 92.1 ± 50.6 ng/ml; p = 0.47); between chronic hepatitis B (n = 38, 40.3 ± 10.8 ng/ml) and chronic hepatitis C (n = 9, 42, 4 ± 7.9 ng/ml; p = 0.59); and between HBV cirrhosis (n = 49, 39.0 ± 12.5 ng/ml) and HCV cirrhosis (n = 8, 44.4 ± 5.2 ng/ml; p = 0.24). 3.2. Relationship between the serum HMGB1 and AFP levels in patients with HCC To assess the correlation between the levels of serum HMGB1 and AFP in patients with HCC, we stratified the value of serum AFP into three levels: <200 ng/ml; 200–400 ng/ml and >400 ng/ml. The mean value of serum HMGB1 levels were 52.8 ± 20.3 ng/ml, 82.6 ± 10.1 ng/ml and 141.1 ± 42.5 ng/ml in correspondence with the serum AFP levels <200 ng/ml, 200–400 ng/ml and >400 ng/ml in the patients with HCC and significant differences were observed among three group (p < 0.0001) and between two groups (AFP of 200–400 ng/ml versus AFP < 200 ng/ml group, p = 0.008; AFP > 400 ng/ml versus AFP of 200–400 ng/ml group, p < 0.001). The significant positive correlation was shown between the levels of serum HMGB1 and AFP (r = 0.952, p < 0.0001). 3.3. Comparison of the serum HMGB1 levels in patients with different size of hepatocellular carcinoma We divided the tumours into three groups by their maximum size: <3 cm; 3–5 cm and >5 cm to compare the serum HMGB1 levels in patients with different size of HCC. For the patients with <3 cm, 3–5 cm and >5 cm HCC
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lesions, the serum HMGB1 levels were 35.2 ± 11.9 ng/ml, 69.3 ± 23.1 ng/ml and 122.7 ± 48.9 ng/ml. There were significant differences among three groups (p < 0.0001) and between two groups (p < 0.001, respectively) and the significant positive correlation between the levels of serum HMGB1 and the size of tumour (r = 0.904, p < 0.0001). 3.4. Correlation between the pathological differentiation of tumour and serum HMGB1 levels in patients with HCC To assess the correlation between the pathological differentiation of tumour and serum HMGB1 levels in patients with HCC, we defined the tumour differentiation with the Edmondson grade. For the HCC patients with tumour differentiation of Edmondson grade I, II, III and IV, the serum HMGB1 levels were 44.0 ± 18 ng/ml, 71.7 ± 10.9 ng/ml, 112.2 ± 14.5 ng/ml and 169.4 ± 48.1 ng/ml. The significant differences were shown among four groups (p < 0.0001) and between two groups (p < 0.001, respectively). 3.5. Comparison of the serum HMGB1 levels in patients with different tumour stage of hepatocellular carcinoma The tumour stage was defined by the AJCC staging (TNM stage) for HCC to evaluate whether HMGB1 was associated with HCC metastases to lymph nodes and distal organs, vascular invasion and tumour characteristics. The serum HMGB1 levels were 30.2 ± 5.9 ng/ml, 58.9 ± 20.0 ng/ml, 100.9 ± 23.4 ng/ml and 175.8 ± 47.6 ng/ml in patients with HCC of TNM stage I, II, III and IV. There were significant differences among four groups (p < 0.0001) and between two groups (p < 0.001, respectively, Fig. 2).
Fig. 2. Serum levels of HMGB1 in patients with tumours of different stages. Value among four groups was significantly different (p < 0.0001); (*) vs. stage I group, p < 0.001; (#) vs. stage II group, p < 0.001; ($) vs. stage III group, p < 0.001.
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Fig. 3. Relationship between serum HMGB1 levels and CLIP score in patients with HCC. Significant difference among three groups, p < 0.0001; (*) vs. 0–1 point group, p < 0.001; (#) vs. 2–4 point group, p < 0.001.
3.6. Relationship between the serum HMGB1 levels and prognostic score for patients with HCC To determine whether the serum HMGB1 was an independent factor that affected the prognosis for the patients with HCC, we used the CLIP score as prognostic system, and stratified this score into three levels: 0–1 points; 2–4 points and >4 points. The serum HMGB1 levels were 54.6 ± 20.8 ng/ml, 113.5 ± 13.0 ng/ml and 167.9 ± 47.7 ng/ml in patients with CLIP score 0–1 points, 2–4 points and >4 points. There were significant differences among three groups (p < 0.0001) and between two groups (p < 0.001, respectively, Fig. 3).
4. Discussion In the present study, we have first demonstrated that serum HMGB1 levels were significantly increased in patients with HCC than those in patients with chronic hepatitis and cirrhosis. No differences were found between chronic hepatitis and cirrhosis and between HBV patients and HCV patients. These results suggest that HMGB1 may play a pivotal role in the development of HCC. Several factors may contribute to elevation in serum HMGB1 levels in the patients with HCC. One is that HCC cells produce and secrete HMGB1. Stimulation of neoplastic cell lines with promoters such as 12-O-tetradecanoyl phorbol acetate (TPA) and the binding of c-Myc to the HMGB1 promoter induces the transcription of HMGB1 [25]. HMGB1 secretion is dependent on active translocation of HMGB1 from the nucleus to the cytoplasm before being released into the extra cellular milieu [26]. On the other hand, it was well known that HMGB1 can be passively released by necrotic and ischeamic cells [10,11].
Tumour cells proliferation may outpace the rate of angiogenesis, resulting in tissue severe ischeamic hypoxia [27]. Considered these results together, HMGB1 release through tumour cell death might have effect on both the local inflammatory responses at the tumour site and the rate of tumour-cell growth. Our results also showed that serum HMGB1 levels were strongly correlated with tumour stage, pathological differentiation grade, known HCC marker (AFP) and CLIP score. Malignancy increases with dedifferentiation, and high serum AFP levels may be an indirect measure of tumour burden [18]. CLIP score accounts for both liver function and tumour characteristics relevant to prognostic assessment for patients with HCC, and has the highest stratification ability regard to prognosis in patients with HCC [28]. These data suggest that HMGB1 may play an important role not only in development of HCC but in metastasis and poor outcome. As an extracellular molecular, HMGB1 induces several intracellular signalling pathways via binding to its receptor RAGE. Malignant cells and immature cells express high levels of HMGB1 and RAGE [3,16,23]. The co-localization of RAGE and HMGB1 indicated their potential contribution to cellular migration and tumour invasion, and the suppression of tumour growth and metastasis by blocking RAGE-HMGB1 complex in mice had been reported [16]. Signalling through RAGE leads to activation of the nuclear factor-kB (NF-kB) pathway, mitogen activated protein kinase (MAPK), type IV collagenase (MMP-2/MMP-9), which are important for cancer cell growth, invasion and metastasis [6,7,16,23]. RAGE and MMP-9 are expressed concordant with the higher metastasis ability of human pancreatic cancer cells [23]. Alternatively, for tumour growth, apoptosis must be blocked in the transformed; HMGB1 could act as an oncoprotein because of its ability to anti-apoptotic prop-
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erties [20]. In addition, overexpression of HMGB1 has been shown to cause modulation of the transcriptional expressing of many groups of genes reported to play key roles in different biological processes of neoplasm progression and metastasis. Enhanced expression of HMGB1 and RAGE has been strongly associated with atypical and increased size of colorectal adenomas, colorectal metastases to lymph nodes and distal organs, and poor prognosis at any colorectal cancer stage [24]. RAGE was found in 19%, 81% and 100% of Dukes’ B, C and D cases, respectively, correlating with invasiveness and poor outcome [29]. Thus, in our study, elevation of HMGB1 in patients with HCC can contribute to HCC genesis and metastasis by altering gene expression with cells. In conclusion, HMGB1 could be a useful and specific marker for evaluating the tumour differentiation grade, tumour stage and predicting prognosis in patients with HCC. This relatively new concept helps us to understand the pathogenesis of HCC and might provide insights that targeting HMGB1 production or release might have substantial potential applications for HCC treatment.
Practice points • The serum HMGB1 levels were significantly increased in patients with HCC than those in chronic hepatitis and liver cirrhosis. No significant differences were found between HBV and HCV infectious patients, and between chronic hepatitis and cirrhosis. • There were positive correlation between HMGB1 and AFP, and between HMGB1 and the size of tumour in patients with HCC. • HMGB1 were significant differences among Edmondson grade I, II, III, IV; TNM stage I, II, III, IV and CLIP score 0–1 points, 2–4 points, >4 points.
Research agenda • Further research will be needed to make an accurate cut-off value of HMGB1 concentrations levels that enable to be used as serological assessment parameters for early HCC and differentiate aggressive and nonaggressive HCCs. • Whether targeting HMGB1 production or release is efficacious method for HCC treatment will be further studied.
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Conflict of interest statement None declared.
References [1] Lotze MT, Tracey KJ. High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol 2005;5:331–42. [2] Parrish W, Ulloa L. High-mobility group box-1 isoforms as potential therapeutic targets in sepsis. Methods Mol Biol 2007;361:145–62. [3] Ulloa L, Messmer D. High-mobility group box 1 (HMGB1) protein: friend and foe. Cytokine Growth Factor Rev 2006;17:189–201. [4] Yotov WV, St-Arnaud R. Nucleotide sequence of a mouse cDNA encoding the nonhistone chromosomal high mobility group protein-1 (HMG1). Nucleic Acids Res 1992;20:3516. [5] Calogero S, Grassi F, Aguzzi A, Voigtlander T, Ferrier P, Ferrari S, et al. The lack of chromosomal protein Hmg1 does not disrupt cell growth but causes lethal hypoglycaemia in newborn mice. Nat Genet 1999;22:276–80. [6] Dumitriu IE, Baruah P, Manfredi AA, Bianchi ME, RovereQuerini P. HMGB1: guiding immunity from within. Trends Immunol 2005;26:381–7. [7] Evans A, Lennard TW, Davies BR. High-mobility group protein 1(Y): metastasis-associated or metastasis-inducing? J Surg Oncol 2004;88:86–99. [8] Palumbo R, Sampaolesi M, De Marchis F, Tonlorenzi R, Colombetti S, Mondino A, et al. Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation. J Cell Biol 2004;164:441–9. [9] Wang H, Yang H, Tracey KJ. Extracellular role of HMGB1 in inflammation and sepsis. J Intern Med 2004;255:320–31. [10] Rovere-Querini P, Capobianco A, Scaffidi P, Valentinis B, Catalanotti F, Giazzon M, et al. HMGB1 is an endogenous immune adjuvant released by necrotic cells. EMBO Rep 2004;5:825–30. [11] Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 2002;418:191–5. [12] Ito N, DeMarco RA, Mailliard RB, Han J, Rabinowich H, Kalinski P, et al. Cytolytic cells induce HMGB1 release from melanoma cell lines. J Leukoc Biol 2007;81:75–83. [13] Zeh III HJ, Lotze MT. Addicted to death: invasive cancer and the immune response to unscheduled cell death. J Immunother 2005;28:1–9. [14] Huttunen HJ, Fages C, Rauvala H. Receptor for advanced glycation end products (RAGE)-mediated neurite outgrowth and activation of NF-kappaB require the cytoplasmic domain of the receptor but different downstream signaling pathways. J Biol Chem 1999;274:19919– 24. [15] Park JS, Gamboni-Robertson F, He Q, Svetkauskaite D, Kim JY, Strassheim D, et al. High mobility group box 1 protein interacts with multiple Toll-like receptors. Am J Physiol Cell Physiol 2006;290:C917–24. [16] 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 2000;405:354–60. [17] Mignogna MD, Fedele S, Lo Russo L, Lo Muzio L, Bucci E. Immune activation and chronic inflammation as the cause of malignancy in oral lichen planus: is there any evidence? Oral Oncol 2004;40:120–30. [18] Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet 2003;362:1907–17. [19] Choi YR, Kim H, Kang HJ, Kim NG, Kim JJ, Park KS, et al. Overexpression of high mobility group box 1 in gastrointestinal stromal tumors with KIT mutation. Cancer Res 2003;63:2188–93. [20] Brezniceanu ML, Volp K, Bosser S, Solbach C, Lichter P, Joos S, et al. HMGB1 inhibits cell death in yeast and mammalian cells
452
[21]
[22]
[23]
[24]
B.-Q. Cheng et al. / Digestive and Liver Disease 40 (2008) 446–452 and is abundantly expressed in human breast carcinoma. FASEB J 2003;17:1295–7. Volp K, Brezniceanu ML, Bosser S, Brabletz T, Kirchner T, Gottel D, et al. Increased expression of high mobility group box 1 (HMGB1) is associated with an elevated level of the antiapoptotic c-IAP2 protein in human colon carcinomas. Gut 2006;55:234–42. Kuniyasu H, Chihara Y, Kondo H, Ohmori H, Ukai R. Amphoterin induction in prostatic stromal cells by androgen deprivation is associated with metastatic prostate cancer. Oncol Rep 2003;10:1863–8. Takada M, Hirata K, Ajiki T, Suzuki Y, Kuroda Y. Expression of receptor for advanced glycation end products (RAGE) and MMP-9 in human pancreatic cancer cells. Hepatogastroenterology 2004;51:928–30. Sasahira T, Akama Y, Fujii K, Kuniyasu H. Expression of receptor for advanced glycation end products and HMGB1/amphoterin in colorectal adenomas. Virchows Arch 2005;446:411–5.
[25] Cmarik JL, Li Y, Ogram SA, Min H, Reeves R, Colburn NH. Tumor promoter induces high mobility group HMG-Y protein expression in transformation-sensitive but not -resistant cells. Oncogene 1998;16:3387–96. [26] Rendon-Mitchell B, Ochani M, Li J, Han J, Wang H, Yang H, et al. IFN-gamma induces high mobility group box 1 protein release partly through a TNF-dependent mechanism. J Immunol 2003;170:3890–7. [27] Carmeliet P. Angiogenesis in life, disease and medicine. Nature 2005;438:932–6. [28] Ueno S, Tanabe G, Sako K, Hiwaki T, Hokotate H, Fukukura Y, et al. Discrimination value of the new western prognostic system (CLIP score) for hepatocellular carcinoma in 662 Japanese patients. Cancer of the Liver Italian Program. Hepatology 2001;34:529–34. [29] Lotze MT, DeMarco RA. Dealing with death: HMGB1 as a novel target for cancer therapy. Curr Opin Invest Drugs 2003;4:1405–9.