Abstract
Background: Hepatocellular carcinoma (HCC) is difficult to treat with anticancer drugs. Therefore, development of new drugs for HCC is required. Materials and Methods: The livers of 14 hepatoma patients accompanied by hepatitis B (2 cases) and hepatitis C (12 cases) were used. The biotinidase kinetics of HCC tissues were compared to those of the adjacent liver tissues of 13 liver cirrhosis (LC) and 1 chronic active hepatitis (CAH). Results: The Kip (the inhibition constant by biotin) of HCC tissues were consistently higher than those of LC (plus CAH) tissues: the Kip was 450±231 μmol/l in HCC tissues and 240±111 μmol/l in LC (plus CAH) tissues, p<0.001. This increase of Kip is considered to be due to an increase of biotin repulsion by biotinidase in the HCC tissues. Fucoidan, a sulfated poly-fucose, was found to decrease the Kip of biotinidase in HCC tissues, and conversely to increase it in LC tissues. Fucoidan was also found to decrease the Kip of the hepatoma HuH-6 cells. Conclusion: These findings suggest that fucoidan has a potential therapeutic effect on HCC.
Abbreviations: HCC, hepatocellular carcinoma; LC, liver cirrhosis; Kip, inhibition constant by product of biotin; Amo, affinity for substrate; Rep, repulsion to product; Cap, enzymatic capacity; HPAC, high-performance affinity chromatography; DEN, diethylnitrosamine; BAQ, biotinyl-6-aminoquinoline.
Biotinidase (EC 3.5.1.12) (1, 2), ubiquitous in mammalian cells (3, 4), hydrolyzes biocytin to biotin and lysine. Human serum and urine biotinidase is a glycoprotein enzyme (1, 5), and sialic acid of the glyco-chain increases the affinity to the biotin-amide substrate in human serum biotinidase (unpublished observation). Previously, it was reported that serum biotinidase activity significantly decreased in patients with liver cirrhosis (LC) and hepatocellular carcinoma (HCC) (6); however, its significance in the pathogenesis of LC and HCC have not yet been determined. Since the biotin concentration is higher in cancerous tumors than that of normal tissue (7, 8), biotinidase should become resistant to the increased product (biotin) concentration in order to perform better substrate handling. A new biotinidase assay method has recently been developed using high-performance liquid chromatography (4), and this method has been applied to determine the biotinidase kinetics in HCC patients in this text.
Fucoidan, a sulfated poly-fucose, has become a matter of great interest for cancer therapy. The mechanisms by which fucoidan exhibits anticancer activity are related to its ability to suppress the proliferation of cancer cells, modulate the immune responses, inhibit tumor angiogenesis and to induce the apoptosis of tumor cells (9-15). More recently, it was reported that fucoidan exhibits antitumor activity toward Huh 7 hepatoma cells through down-regulation of CXCR12 expression (16).
In addition, Mekabu fucoidan (me-Fucoidan) had a higher binding affinity for basic proteins such as histone H2B, and inhibited A-kinase-mediated phophorylation in vitro (17). Fucoidan is a negatively charged polysaccharide, and biotinidase has a negatively charged sialic acid in the glycol chains. Therefore, the effects of fucoidan on the biotinidase kinetics in liver samples obtained from HCC patients and in hepatoma cell lines were also investigated.
Materials and Methods
Chemicals and reagents. Biotinyl-6-aminoquinoline (BAQ), bovine serum albumin (BSA), carrageenan lota (type V; from Eucheuma spinosa) and diethylnitrosamine (DEN) were purchased from Sigma (St. Louis, MO, USA). 6-Aminoquinoline was from Aldrich (Milwaukee, WI, USA). Polyoxyethylene cetyl ether (Brij 58) was from Nacalai Tesque (Kyoto, Japan). Chondroitin sulfate C sodium salt, dextran (Mr 200000~300000), heparin (from porcine small intestine), hyaluronic acid sodium salt (from rooster comb), L(-)-fucose, and D-biotin were from Wako (Osaka, Japan). Fetal bovine serum (FBS) was from Moregate BioTech (Bulimba, Australia). Fucoidan was prepared from Cladosiphon okamuranus as described previously (18). Purified fucoidan contained less than 0.1% of contaminated proteins as determined by the microsequencing method (19).
Liver samples. HCC samples and corresponding non-tumorous tissues were obtained from 14 patients (12 male and 2 female) ranging in age from 49 to 66 years during surgery (9 samples) or autopsy (5 samples) at Gunma University Hospital and Nishi-Gunma Hospital. The liver samples were immediately stored at -80°C. The grade of differentiation of HCC was determined pathologically. Patient data are summarized in Table I. Liver cirrhosis was found in adjacent liver tissues except for one patient (No.10, chronic active hepatitis, CAH). Normal liver samples were obtained from four patients with metastatic liver tumor and one hepatolithiasis patient during surgery (Controls 3, 5) or autopsy (Controls 1, 2, 4). Non-cancerous liver tissues demonstrating normal histological findings served as a control (Table I). Informed consent was obtained from all patients and/or their relatives. This study was approved from the institutional committee for the study of human rights.
Cell culture. HuH-6 cells (hepatoma from a 1-year-old Japanese male) were purchased from Rikagaku Kenkyusho (Tsukuba, Ibaragi, Japan). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, penicillin G (100 U/ml) and streptomycin (100 U/ml) in a humidified 37°C/5% CO2 incubator. The enzyme kinetics of biotinidase were analyzed using cell homogenates containing 59 μg/ml of protein in the final reaction buffer.
Protein concentration. Protein concentrations of the human livers and cultured cells were all determined by SEC protein determination method (20).
Enzyme kinetic analysis. Liver homogenate and cultured cell homogenate were prepared as described previously (5, 19, 20). Supernatant and membrane (nuclear membrane) fractions were prepared by ultracentrifuging the homogenate at 100000 xg for 90 min at 4°C. Biotinidase kinetic analysis was performed as described by high-performance liquid chromatography (HPLC) with fluorometric detection (5). The molecular mass and number of active centers of biotinidase were determined to be 66,000 and ones, respectively. Amo (kcat/Km), Km, Kip, Rep (kcat x Kip x 10-3), and Cap ((Amo x Rep)1/2) were defined as follows, Amo is the affinity to the biotinyl substrate, Km is also the affinity to the biotinyl substrate (BAQ), Kip is the competitive inhibition constant by biotin or product inhibition, Rep is the repulsion power against the biotin product and Cap is the total capacity of the enzyme to handle the biotinyl substrate per second per enzyme molecule, respectively (5).
Fucoidan test. A fucoidan test was carried out using 2 HCC tissue samples (No. 11, 12), 3 LC samples (No. 11, 12, 13) and 2 normal livers (Controls 2, 5). Fucoidan (100 or 200 μg/ml) was added to fractions and homogenates of liver samples. To compare the effect on biotinidase kinetics, other polysaccharides such as chondroitin sulfate, hyaluronic acid, carrageenan, dextran, heparin and L(-)-fucose were evaluated.
Characteristics of HCC and normal livers used in this study.
In addition, HuH-6 cells were subjected to the fucoidan test. Fucoidan was added to the culture medium at a concentration of 200 μg/ml, and the biotinidase kinetics were calculated.
Determination of biotin in diethylnitrosamine (DEN)-induced HCC in LEW rat. Male LEW rats (weighing 200 g; 7 weeks of age) were purchased from Sankyo Labo Service Co., Tokyo, Japan. They were kept in the animal facility of National Institute for Child Health and Development, with a 12-h light/dark regime, and were maintained on a standard CE-2 feed purchased from Japan Clea (Tokyo, Japan).
Rats were randomized into 2 experimental groups of 3 animals each. Group 1, which served as the control, had access to regular drinking water ad libitum. The control group received no diethylnitrosamine (DEN). Group 2 was injected intraperitoneally with DEN at 50 mg/kg (body weight) once a week for 16 weeks by the procedure of Schiffer et al. (21). Two weeks after the last injection (to allow recovery from acute necrosis), all rats were anesthetized with ether. The tumor lesion and the adjacent liver tissues were surgically removed and weighed. A random piece of liver from each lobe was fixed in 10% buffered formalin and embedded in a paraffin for histological analysis. In addition, the liver was frozen immediately in liquid nitrogen for the analysis of biotin and biotinidase. Amounts of total and free biotin in the rat livers were determined by a newly developed high-performance affinity chromatographic (HPAC) method (22).
All the protocols were carried out in accordance with ethical guidelines for laboratory animals of the National Research Institute for Child Health and Development.
Typical biotinidase kinetics of human livers.
Statistics. Non-parametric statistical analysis was performed. The Wilcoxon test was used to compare the kinetic values of LC and HCC samples. Mann-Whitney's U-test was also used to compare differences between two groups. For all tests, p<0.05 was regarded as significant.
Results
Biotinidase kinetics of human livers (Table II). All HCC patients' livers and 4 normal livers (except Control 5) showed Kip values of biotin, and Rep and Cap were then calculated. The mean Kip values were highest in HCC tissues, followed by normal livers and LC tissues. The Kip value was consistently higher in the HCC tissue than in the LC (plus CAH) tissue in each patient (Figure 1). The mean values of Kip were significantly (p<0.01) higher in the HCC tissues than in LC (plus CAH) tissues. The Kip values were not significantly different between normal liver and LC and HCC tissues. Rep and Cap were significantly (p<0.05) higher in HCC tissues than LC (plus CAH) tissues. However, Amo, Km and V did not differ among normal liver, LC (plus CAH) and HCC tissues.
Effect of several polysaccharides on biotinidase kinetics in HCC tissue (Table III). In the supernatant fraction of two HCC tissues, the Kip, Rep and Cap values were markedly reduced by fucoidan. Heparin also reduced the Kip, Rep and Cap values, while L-fucose, chondroitin sulfate, hyaluronic acid and carrageenan did not alter these values.
Effect of fucoidan on biotinidase kinetics in HCC tissue. The effect of fucoidan on biotinidase kinetics in the supernatant of one HCC tissue is shown in Table IV. Although a slight increase of Amo and a decrease of Km was observed, the capacity of biotinidase (Kip, Rep and Cap) was significantly reduced by fucoidan (p<0.05).
Effect of fucoidan on biotinidase kinetics in LC tissues. The effect of fucoidan on the biotinidase kinetics in the supernatant of three LC tissues was studied (Table V). Fucoidan activated LC biotinidase, i.e. although a decrease of Amo was observed, biotinidase capacities (Kip, Rep and Cap) all significantly increased in the LC tissues (p<0.05).
Typical differences in the Kip values between LC and HCC tissues of each patient's liver. The longitudinal axis shows Kip values (μM). The Kip value is consistently higher in the HCC tissue than in the LC (plus CAH) tissue in each patient.
Effect of fucoidan on biotinidase kinetics in normal liver. The effect of fucoidan on the biotinidase kinetics of two normal livers was tested (Table VI). Although not sufficient for statistical analysis, fucoidan did not seem to affect the normal livers.
Effect of fucoidan on biotinidase kinetics in HuH-6 cells. The effect of fucoidan on HuH-6 hepatoma cells was tested, and the results are summarized in Table VII. Fucoidan in DMEM reduced the biotinidase capacities relating to the repulsion of biotin product (Kip, Rep and Cap).
Effect of several polysaccharides on biotinidase kinetics in HCC tissues.
Increased free biotin (product of biotinidase) in the HCC tissue of rat. Using LEW rat, HCC was induced by diethylnitrosamine (DEN) and the adjacent liver tissues diffusely showed degenerative nodules in accordance with LC (21). Biotin was determined by a newly developed HPAC method (22). The results are summarized in Table VIII. Free biotin significantly increased by about 10-fold in the HCC tissue as compared to the normal rat liver.
Discussion
The present study clearly demonstrated that biotinidase affinity to the substrate (Amo, Km) and specific activity (enzyme amount; V) were unchanged in the HCC tissues but that the product repulsion power (Kip and Rep) and enzyme capacity (Cap) increased. It is of interest that the Kip value was consistently higher in the HCC tissue than in the LC (plus CAH) tissue in each patient (Figure 1). If the biotin concentration is higher in HCC (7, 8), biotinidase should become resistant to the increased product (biotin) concentration in order to perform better substrate handling. As shown in Table VIII, a 10-fold increase of free biotin (biotinidase product) in the HCC tissue in the DEN-treated rats was demonstrated. Furthermore, the increase of biotin in the medium of HeLa cells was reported previously by Keränen (23), and the increase of biotin in the DMEM medium using cultured liver cells was confirmed (data not shown). Thus, it is considered that biotinidase capacities in the HCC tissues must change to enhance handling of biotin (product: increased Kip and Rep) and the biotinyl substrate (substrate: increased Cap).
Effect of fucoidan on the supernatant of HCC tissue of No. 12.
Fucoidan has been reported to exhibit antitumor and anti-metastatic activities in vivo and in vitro (9-17); therefore, whether fucoidan and other polysaccharide reagents counteracted the increased Kip of HCC biotinidase or not was tested. It was found that fucoidan had the expected effect of reducing the Kip values in the HCC tissues. Conversely, fucoidan activated LC biotinidase, i.e. biotinidase capacities (Kip, Rep and Cap) all significantly increased in the LC tissues. The results indicate that fucoidan reduced the capacity of biotinidase in HCC, and conversely increased the capacity of biotinidase in LC. The vitamin biotin serves as a covalently bound coenzyme for four major carboxylases (1, 2) and a growth factor in tissue cells (23-25). In addition, conjugation of biotin to histones (DNA-binding proteins) may be mediated by biotinidase, i.e. biotinylation of histones may play a role in cell proliferation (24), gene silencing (26) and cellular response to DNA damage (27). Theoretically, fucoidan may reduce the biotin supply in HCC (reducing cellular growth in HCC), but it may increase the supply in LC (increasing cellular growth in LC) by changing the biotinidase kinetics inversely.
Effect of fucoidan on the supernatant of LC portion of No. 11, 12, and 13.
Effect of fucoidan on biotinidase kinetics of normal liver.
Effect of fucoidan on biotinidase kinetics in HuH-6 hepatoma cells.
Increase of biotin in the HCC tissue in the DEN-treated LEW rat.
Other oligosaccharides such as L-fucose, chondroitin sulfate, hyaluronic acid and carrageenan failed to affect the biotinidase kinetics in HCC and LC tissues. Although heparin showed a similar kinetic effect, it seemed to have a side-effect on normal livers (data not shown). The biological activity of fucoidan is thought to be due to the similarity in structure between fucoidan and fucosylated glycans of certain extracellular membrane glycoproteins such as selectin (28), antistasin (29), proacrosin (30), bindin (31), GMP-140 (32) and annexin II (33). It is possible that the putative fucoidan- and heparin-binding sites are in a similar region of biotinidase. In support of this suggestion is the previous report that fucoidan and heparin bind to a similar region of annexin II (33). Taken together, the effect of fucoidan is probably mediated by a lectin-polysaccharide type of interaction with biotinidase.
As expected, fucoidan in the DMEM reduced the biotinidase capacities in the hepatoma HuH-6 cells (Table VII). Even at a concentration of 200 μg/ml, no cytotoxic effect from fucoidan was observed morphologically. In support of this finding is the fact that the concentration of albumin was unchanged by treatment with fucoidan (data not shown). The effect of fucoidan on the biotinidase kinetics of normal livers was tested; although not sufficient for statistical analysis, fucoidan did not seem to affect the normal livers.
The present study suggests that fucoidan has the potential to cure HCC without apparent side-effects. The mechanism of this specific effect only on livers with cancer is difficult to understand at this time, hence further studies are necessary to elucidate the exact role of fucoidan in the pathogenesis of HCC.
Acknowledgements
This work was partially supported by the Ministry of Welfare, Labor and Health, Japan. This work was also partially supported by the scientific inquiry subsidy of the Ministry of Education, Culture, Sports, Science and Technology, Japan. Authors are grateful to Dr. Fujio Makita (Nishi-Gunma Hospital, Shibukawa, Gunma, Japan) for his kind donation of human liver samples.
Footnotes
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Dedication: In memory of the usual encouragement of my beloved daughter Reiko Hayakawa (21 November 1979 - 1 February 2007).
- Received November 26, 2008.
- Revision received January 15, 2009.
- Accepted February 13, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
