Japanese Journal of Clinical Oncology 31:584-588 (2001)
© 2001 Foundation for Promotion of Cancer Research
Levels of Soluble Intercellular Adhesion Molecule-1 and Total Sialic Acid in Serum of Patients with Laryngeal Cancer
1Department of Biochemistry, School of Medicine, 2Department of Otorhinolaryngology–Head and Neck Surgery and 3Department of Physiology, Atatürk University, Erzurum, Turkey
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Background: Adhesion molecules have been implicated in tumor progression. In this study, we aimed to investigate serum soluble intercellular adhesion molecule-1 (sICAM-1) and total sialic acid (TSA) levels in laryngeal carcinoma and correlate their levels with the cancer stage.
Method: The sera from 35 patients with laryngeal cancer (10 at stage II, 12 at stage III and 13 at stage IV) were extracted before treatment. The concentrations of sICAM-1 and TSA were measured by enzyme-linked immunoassay and the thiobarbituric acid method, respectively and compared with those from a healthy control group (n = 34).
Results: Mean serum sICAM-1 and TSA levels were found to be higher in the total patient group (the lowest level belonging to stage II) than in the control group (p < 0.001, control versus total patient group). As the stage of the disease increased, higher levels of sICAM-1 and TSA were determined. The correlations between TSA and sICAM-1 became more significant as the stage of the disease increased (r = 0.67, p < 0.05 in stage II, r = 0.86, p < 0.001 in stage III and r = 0.90, p < 0.001 in stage IV).
Conclusion: These data reveal that the significant correlations between sICAM-1 and TSA in laryngeal cancer, more prominent at advanced stage, might reflect the similar nature of these molecules, which function as adhesion molecules.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the most important problems in the treatment of head and neck cancer is the management of metastatic spread to locoregional lymph nodes. The major determinant of survival following laryngeal cancer appears to be the development of metastases. Most cases are diagnosed at an advanced stage and the 5-year survival rate at advanced stage is ~40% (1). Chemotherapy in the advanced stage is of limited use. Therefore, identification of biomolecules involved in the progression and dissemination of laryngeal cancer has gained considerable interest in the recent years. If a tumor marker is able to reflect overall survival, the data should be potentially useful in guiding additional treatment for these types of cancer (2,3).
The surface of cancer cells differs in many respects from that of normal cells. Neoplastic transformations of a variety of cell types are associated with changes in the composition of membrane glycoproteins (4). Cell surface adhesive glycoproteins are involved in cell adhesion and migration in inflammatory diseases and malignant disorders and identified together with specific aberrations in malignant diseases (5,6). Although not much investigated so far, soluble forms of cell adhesion molecules have been linked to the clinical behavior of tumors. Positive and negative regulation of cell adhesion will influence the process as metastatic cells break away from the primary tumor, enter the circulation and then adhere to cellular and extracellular matrix elements in particular secondary sites. It has been suggested that several cell adhesion molecules may play a role in infiltrative growth and metastatic processes (6).
Intercellular adhesion molecule-1 (ICAM-1) is a monomeric, transmembrane molecule of the immunoglobulin superfamily with a molecular weight of 95–110 kDa. Two ligands, the lymphocyte function-associated antigen-1 and the membrane adhesion complex-1, mediate adhesion and transvascular migration (5). Although the source of sICAM-1 has not been fully elucidated, it can be released by cancer cells and also by mononuclear blood, endothelial and fibroblastic cells (7). In one study, it was reported that increased expression of sICAM-1 was found in small vessels around colon neoplasms. Recently, circulating forms of several adhesion molecules, including E-cadherin, E-selectin and ICAM-1, have been described with their concentrations being increased in inflammatory and malignant diseases such as malignant melanoma, gastrointestinal cancer, lymphoma and hepatocellular cancer (6). The significance of adhesion molecule shedding is not clear but it may have profound implications for tumor metastasis. Shedding of ICAM-1 by circulating tumor cells may allow their escape from surveillance by cytotoxic T cell and natural killer cells and thus promote metastasis (8).
Sialic acid (SA), a family of acylated derivatives of neuraminic acid, is widely distributed in mammals, usually occurring as a terminal component at the non-reducing end of carbohydrate chains of glycoproteins and glycolipids. Studies of malignant cells have revealed alterations in cell surfaces and membranes in terms of sialic acid content of glycoproteins and glycolipids. Serum/plasma total or bound sialic acid content has been studied in patients with various types of malignancy such as malignant melanoma, lung, prostate, breast, ovary and colon carcinoma. Marked elevation of serum sialic acid concentrations that correlates with the clinical activity of a disease has been documented in such malignancies (9).
To our knowledge, there has been no study measuring serum levels of both total sialic acid (TSA) and sICAM (both are the important components in cell to cell interaction and cell adhesion) in any type of cancer. Therefore, in the present study, we aimed to investigate serum TSA and sICAM-1 levels in laryngeal carcinoma and correlate their levels with the cancer stage.
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PATIENTS AND METHODS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thirty-five cases (35 males) with laryngeal cancer comprised the patient group and 34 healthy male subjects the control group. The age range was 37–63 years (mean 54.2 ± 8.1 years) for the laryngeal cancer group and 35–58 years (mean 50.3 ± 6.3 years) for the control group. The control and laryngeal cancer subjects were smokers but not drinkers. All of the patients and control subjects were men. Tumor stage was determined according to the criteria of the American Joint Committee on Cancer (AJCC), 10 cases at stage II, 12 cases at stage III and 13 cases at stage IV (10). Two patients were T2, 12 were T3 and 11 were T4 according to the classification of the AJCC (1992) (11). No lymph node metastasis was present in 12 patients. Ten patients were N1, two were N2 and one was N3. None of our patients had distant metastasis. Laryngeal cancer patients and healthy control subjects were recruited into the study after obtaining their informed consent. Samples of 10 ml of venous blood were taken from controls and patients. The blood samples were centrifuged and the serum samples obtained were stored at –80°C until the analysis date.
Total sialic acid levels were determined according to the thiobarbituric acid method described by Aminoff (12) after hydrolysis of the samples in 5 volumes of 0.1 N H2SO4 at 80°C for 1 h. Briefly, 5 ml serum are treated with 0.25 ml of 25 mM periodic acid in 0.125 N H2SO4 (pH 1.2) for 30 min in a water-bath at 37°C. The excess of periodate is then reduced with 0.2 ml of 2% sodium arsenite in 0.5 N HCl. As soon as the yellow color of the liberated iodine has disappeared (1–2 min), 2 ml of 0.1 M thiobarbituric acid in water are added and the test sample is covered and heated in a boiling water-bath for 7.5 min. The colored solutions are cooled in ice-water and shaken with 5 ml of acidic butanol (1-butanol containing 5% v/v of 12 N HCl). The separation of the phases is facilitated by a short, rapid centrifugation and the intensities of the colors in the butanol layer are measured at 549 nm using a spectrophotometer (Cecil CE 3041). A calibration curve is obtained by plotting absorbance versus concentration. The TSA concentration of unknown samples is then calculated from this calibration curve.
Levels of s-ICAM-1 were measured with a commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit using two monoclonal antibodies, directed against different epitopes of sICAM-1 (Cat. No. 1 573 659, Roche). During the first incubation step, sICAM-1 in standards/samples is simultaneously bound by the biotin-labeled antibody and the peroxidase-conjugated detection antibody, forming a complex which binds via the biotin-labeled antibody to the streptavidin-coated surface of a microtiter plate. Subsequent to the washing step, the peroxidase bound in the complex is developed with tetramethylbenzidine as a substrate and determined spectrophotometrically. The developed color is proportional to the concentration of sICAM-1. A calibration curve is obtained by plotting absorbance versus concentration. The sICAM-1 concentration of unknown samples is then calculated from this calibration curve.
Statistical and correlation analyses were undertaken using the Mann–Whitney U-test and Pearson’s rank correlation test, respectively. A p value of <0.05 was accepted as statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results are given in Tables 1 and 2.
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Mean serum sICAM-1 levels were found to be higher in the total patient group (the lowest level belonging to stage II) than in the control group (p < 0.001, control versus total patient group). Among patient groups, while there was no significant difference between stage II and III patients, a significant difference was found between stages II and IV. As can be seen from Table 2, as the stage of the disease increased, higher levels of sICAM-1 were determined.
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The mean serum TSA level was found to be lowest in the control group and highest in the stage IV patients. The difference between the control group with stage II group was at the p < 0.01 level and with stage III and IV groups was at the p < 0.001 level. Additionally, both stage III and IV patients had higher mean serum TSA levels than the stage II patients (p < 0.05 and p < 0.001, respectively).
In correlation analysis (Figs 1 and 2), there were significant positive correlations between TSA and SICAM-1 in stage II (r = 0.67, p < 0.05), stage III (r = 0.86, p < 0.001) and stage IV (r = 0.90, p < 0.001). The correlations became more significant as the stage of the disease increased.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The adhesion molecules mediating adhesive interactions have recently drawn considerable attention. Neoplastic transformation and the evolution to metastatic disease are characterized by a dramatic aberration in cellular cohesive interactions. The adhesion molecules have also been shown to facilitate tumor cell motility, adhesion of tumor cells to endothelium, neovascularization at the metastatic sites and host inflammatory response to cancer (3,6).
The present study revealed that serum sICAM-1 and TSA levels were elevated in patients with laryngeal cancer. These parameters were elevated in both local and metastatic disease and significant correlations between these parameters and stage of disease were seen.
sICAM-1 levels have been found to be markedly increased in several types of carcinoma and associated with disease stage and prognosis (13). Wollenberg et al. (5) reported that sICAM-1 levels in serum are high in patients with head and neck squamous cell carcinoma, particularly in the advanced tumor stages. Our data confirm this condition in laryngeal cancer patients for the first time. We have also demonstrated that concentrations of sICAM-1 are increased in laryngeal cancer, particularly in patients with distant metastasis.
Some mechanisms have been proposed concerning the elevation of sICAM-1 in serum, such as enzymatic cleavage of cell surface adhesion molecules or secretion of alternatively spliced forms lacking the transmembrane domain. Additionally, that sICAM-1 has been described on malignant epithelial tissue may be the source of at least some of the sICAM-1 present in sera of cancer patients (6).
Contradictory results have also been reported in the literature. Ura et al. (14), in contrast to our study, demonstrated that the expression of sICAM-1 was inversely correlated with liver metastasis. Yasoshima et al. (15) also showed a reduced expression of sICAM-1 and of lymphocyte function-associated antigen-1 and increased surface expression of ß1 integrins in an experimental animal model of high metastatic gastric cancer cell line.
Liu et al. (16) measured the circulating ICAM-1 in the sera of nasopharyngeal, oral and laryngeal cancer cases and indicated that the circulating ICAM-1 was not elevated in the sera of oral and laryngeal cancer patients, but increased in nasopharyngeal cancer patients. They speculated that the discrepancy in the level of ICAM-1 among these three groups of patients with head and neck carcinoma might be attributed to either the different immunological reaction profiles or a cell-specific response.
The cellular source and the mechanisms for releasing the soluble components of these endothelial adhesion molecules, although not well known, could involve either shedding or enzymatic cleavage from endothelial cells, leucocyte surfaces or tumor cells (17). sICAM-1 was first described as an adhesion molecule regulating adherence of several cell types via lymphocyte function-associated antigen-1 and membrane adhesion complex-1. There is evidence that the sICAM-1 is shed by tumors, which could be demonstrated in a nude mouse model (4). It has been claimed that various tumor cells activate T cells to produce cytokines, resulting in strong expression of the ICAM on surrounding fibroblasts, and also on tumor cells (13).
Surface glycoproteins and glycolipids of tumor cells have altered carbohydrate composition, that may contribute to aberrant cell–cell recognition, cell adhesion, antigenicity and invasiveness demonstrated by malignant cells. Since sialic acids are major constituents of glycoproteins and glycolipids, recent studies showed that the values of total bound sialic acid or protein-bound sialic acid were higher in patients with cancer than normal subjects. Lipid-bound sialic acid levels are correlated with stage of the disease, degree of metastatic involvement and recurrence of disease (9).
Serum TSA has been found to be increased in malignant melanoma (17), colorectal cancer (18), gastric cancer (19), head and neck cancer (20) and laryngeal cancer (21) and higher TSA levels were observed in cases of more developed malignancies (22). Additionally, in a study by Romppanen et al. (23), it was reported that patients with malignant breast disease had the highest TSA, those with benign breast disease had intermediate TSA and healthy control women had the lowest TSA values.
The data of the present study are in agreement with those previously reported which described an increase in the TSA content of serum in several types of carcinoma.
The significant correlations found between sICAM-1 and TSA probably reflect the similar nature of these molecules, which function as adhesion molecules. These findings indicate a relationship between serum TSA and sICAM-1 levels and the stage of the disease in laryngeal cancer patients.
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FOOTNOTES |
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+ For reprints and all correspondence: Fatih Akcay, Department of Biochemistry, School of Medicine, Atatürk University, 25240 Erzurum, Turkey. E-mail: fakcay32@hotmail.com
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Received June 4, 2001; accepted August 27, 2001.
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