Abstract
Background: Recently, it has become obvious that the HMGB1 protein can act as a proinflammatory and proangiogenic mediator when actively secreted by macrophages or passively released from necrotic cells playing an important role in the pathogenesis of several diseases including cancer. Materials and Methods: The absolute and relative amount of HMGB1 was measured with an ELISA in different effusion types. Results: The amount of HMGB1 protein in the samples differed between 0.0004% and 0.0025% of the total sample protein. The mean values of transudates were significantly (p<0.001) lower than the mean values of exudates. Conclusion: HMGB1, a so-called danger signalling protein, was found to be highly expressed in human pleural and peritoneal effusions due to cancer and inflammation. Compared to transudates the average level of HMGB1 was significantly higher in exudates. These results underline the characteristics of HMGB1 as a possible target for treatment in advanced cancer as well.
High mobility group box 1 (HMGB1), also known as amphoterin or HMG1, belongs to a group of chromatin-associated non-histone proteins characterized by low molecular weight, acidic solubility and high content of charged amino acids (1, 2). Proteins of the HMGB family, comprising HMGB1, HMGB2, and HMGB3, are characterized by two DNA-binding domains called HMG boxes (3). Currently the best analyzed member of this group is HMGB1. HMGB1 is an intracellular protein, which can be secreted for example by activated monocytes, macrophages, and astrocytes and can be released by necrotic or damaged cells (4-6). Extracellular HMGB1 can signal through the receptor for advanced glycation end products (RAGE) (7, 8) and the toll-like receptors TLR2 and TLR4 (9-11). Activation of these receptors can enable nuclear factor kappa B (NF-κB) signalling pathways linked to e.g. angiogenesis and/or inflammatory processes (9, 10, 12-15). In cancer, these angiogenic properties of HMGB1 can also lead to a better vascular supply resulting in a faster and greater growth of the tumor (11, 16). Generally, overexpression of HMGB1 has been detected in subsets of carcinomas of the breast (17, 18), tumors of the gastrointestinal tract (19), hepatocellular carcinomas (20), and malignant lymphomas (21).
The aim of this study was to measure the level of HMGB1 in effusions of various origin and to determine if effusions due to cancer or inflammatory disease show elevated levels of HMGB1 protein compared to theses from transudates. Furthermore, it was investigated whether the amount of HMGB1 in effusions due to cancer is consistently increased or if it is not a general phenomenon in effusions of malignant tumors.
Patients and Methods
A total of 36 samples were collected from patients at the General Hospital Bremen Mitte, Bremen, Germany: 30 samples from pleural and 6 from ascitic effusions were obtained (Table I). As a control group patients with transudative effusions were included in this study as well; the most common cause for transudates is congestive heart failure or liver cirrhosis (22, 23), and as a rule no malignant or inflammatory background exists. Samples were subdivided into three groups, namely transudative effusions, infectious or inflammatory exudative effusions, and malignant exudative effusions.
Sample preparation. Samples of 50-100 ml pleural or peritoneal fluid were collected in 50 ml tubes and immediately frozen at −20°C for no more than 15 hours. After thawing, 2 ml of each sample were taken and stored at −80°C until further use. A total of 200 μl of each sample were used for the enzyme-linked immunosorbent assay (ELISA).
Samples from patients with various effusions and their total and relative HMGB1 concentration (T: transudate; E: exudate; P: pleural effusion; A: ascitic effusion).
Protein preparation. Total proteins were isolated with RIPA-buffer (containing: 50 mM Tris-HCl, 150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 1 mM EDTA). Protein concentration was measured with the Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA) following the manufacturer's instructions. All measurements were carried out in triplicate and compared to bovine serum albumin (BSA) and bovine gamma globulin (BGG) standard curves.
ELISA. A 2-step sandwich ELISA was performed using the HMGB1 ELISA Kit (Shino-Test Corporation, Tokyo, Japan) following the manufacturer's instructions.
Analysis of results. All samples were measured at least in duplicate or triplicate, and the results were averaged. Statistical analysis was performed using Student's t-test, with a significance level of p<0.05. In addition the percentage of HMGB1 in comparison to the total protein amount was calculated.
Results
The effusions (Table I) were subdivided into the three groups: transudates (n=11), effusions due to inflammation or infectious diseases (n=12), and malignant effusions (n=13). Whereas the average total protein concentration within the groups showed only slight variation (transudates 6.83 mg/ml; inflammatory effusions 7.83 mg/ml; malignant effusions 8.74 mg/ml), the amount of HMGB1 strongly varied between 2.83 ng/ml (case no. 2, cardiac insufficiency) and 300.00 ng/ml (case no. 14, pneumonia) (Figure 1). The average HMGB1 concentration of the three groups was 36.62 ng/ml for the transudates, 118.00 ng/ml for malignant effusions, and 111.45 ng/ml for inflammatory effusions (Figure 1).
Average protein concentration of HMGB1 in effusion samples quantified with the ELISA.
A highly significant association (t-test, p<0.001) was found between the HMGB1 concentration and the existence of an exudate. Higher HMGB1 protein concentrations did not simply depend on the different cell densities varying in transudates and exudates (Figure 2). With regard to the percentage of HMGB1 present relative to the total protein amount, the increase in exudative effusions was also statistically significant (p<0.001). The total HMGB1 protein level as well as HMGB1 relative to the total protein amount was three-fold higher in exudates than in transudates: 0.0005% in transudative effusions compared to 0.0015% from both groups of exudates.
Eleven patients died from their disease within two months after sampling, among these, one patient with a transudative effusion was affected by cardiac insufficiency, one by an inflammatory effusion and the others died from cancer. The patient dying from cardiac insufficiency showed the highest percentage of HMGB1 by far within this group. Within the exudative samples, no correlation between the HMGB1 concentration and survival of the patients was detected.
Discussion
HMGB1 plays an important role in tumorigenesis and acts as a proinflammatory mediator in the immune response (24, 25). This suggests that HMGB1 can serve as an important target in the diagnosis and management of exudative effusions. In the present study, most of the patients with transudative effusions had cardiac insufficiencies or renal dysfunctions (Table I). The exudative effusions were mainly caused by carcinoma or inflammatory diseases.
Recently several studies were carried out addressing ‘newer’ biomarkers for peritoneal and pleural effusions. A cytological test was performed in which D2-40, X-linked inhibitor of apoptosis (XIAP), MOC-31, and Wilms tumor 1 (WT1) were tested (26), describing the markers MOC-31 and D2-40 as very sensitive and specific markers of epithelial and mesothelial cells, respectively, compared to the established calretinin. In addition to these newer markers, the classification with Light et al.'s criteria is amongst others the most common to differentiate between transudates and exudates (27). In our investigation, only slight differences of total protein amount were detected in the groups of effusions. Nevertheless, the total protein amount was lower for transudates than for exudates (6.83 mg/ml vs. 8.29 mg/ml).
The results of the present study demonstrate that the relative amount of HMGB1 was approximately three-fold higher in exudative effusions than in transudates. This significantly higher protein level was detected with the HMGB1 ELISA alone and also with regard to the total amount of protein (percentage of HMGB1). This definitively proves that the lower HMGB1 protein value is not caused by the lower cell densities in transudate samples.
A high HMGB1 protein level in the effusion may indicate highly active NF-κB or mitogen-activated protein kinase (MAPK) signalling pathways mediated by receptors such as e.g. RAGE or TLR (9, 10, 15). This could serve as an indicator for transformed cells (28). Malignant effusions due to metastatic spread represent a frequent problem in advanced cancer. Herein, it has been shown that malignant effusions are often characterized by high levels of the danger signaling protein HMGB1 that are in the same range as in effusions due to inflammatory processes. Extracellular HMGB1 has been linked to both inflammation and cancer. In the latter group of diseases HMGB1 can promote tumor growth by the stimulation of cell proliferation and by inducing neo-angiogenesis (9). On the other hand, HMGB1 is also able to stimulate dendritic cells and thus may support an immune response against tumor cells (29, 30). Antibodies against HMGB1 are able to reduce inflammation and suppress cell migration, tumor growth and metastasis formation (6, 15). The therapeutic use e.g. of HMGB1 antibodies instilled locally in case of malignant effusions remains to be investigated. While such therapy will reduce the proliferative potential of the cancer cells as well as their further spread, it might also repress a proper response of the immune system against the cancer cells (15, 31).
This is to the best of our knowledge the first report aimed at determining the concentration of HMGB1 in effusions and suggests interesting new approaches for the treatment of patients with malignant effusions.
Concentration of HMGB1 in effusion samples quantified with the ELISA in correlation to total protein amount (light grey: transudates; black: malignant exudates; white: exudates due to inflammation).
Acknowledgements
We thank K. Sobczyk, T. Schwarz and N. Schwochow for excellent technical assistance. Furthermore, the Authors acknowledge the valuable technical support of E. Meyer.
Footnotes
-
↵* Both authors contributed equally to this paper.
- Received July 8, 2009.
- Revision received October 30, 2009.
- Accepted November 2, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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