Journal of Nutrition OpenSOurce Diets- www.ResearchDiets.com

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maeda, Y.
Right arrow Articles by Kimura, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maeda, Y.
Right arrow Articles by Kimura, Y.
© 2004 The American Society for Nutritional Sciences J. Nutr. 134:945-950, April 2004

Nutrition and Cancer

Antitumor Effects of Various Low-Molecular-Weight Chitosans Are Due to Increased Natural Killer Activity of Intestinal Intraepithelial Lymphocytes in Sarcoma 180–Bearing Mice1

Yasunori Maeda and Yoshiyuki Kimura*,2

Laboratory of Maeda Kampo Medicine, Kure-city, Hiroshima 737-0889 and * Second Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan

2To whom correspondence should be addressed. E-mail: yokim{at}m.ehime-u.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Various low-molecular-weight chitosans such as the 21-kDa, 46-kDa, and 120-kDa chitosans obtained by enzymatic hydrolysis of a high-molecular-weight chitosan (average molecular weight, 650 kDa) had low viscosity and were water soluble. We examined the antitumor activity of various water-soluble chitosans with different molecular weights in sarcoma 180–bearing mice. A 21-kDa water-soluble chitosan and oligochitosan (100 and 300 mg/kg body) administered by i.g. intubation reduced the tumor growth and final tumor weight in sarcoma 180–bearing mice. A 46-kDa water-soluble chitosan at a dose of 100 mg/kg body reduced the tumor growth and final tumor weight, but had no effect at 300 mg/kg. On the other hand, a 130-kDa water-soluble chitosan had no effect on tumor growth. The 21- and 46-kDa chitosans (10 mg/L) enhanced the natural killer (NK) activity in intestinal intraepithelial lymphocytes (IELs) or splenic lymphocytes. The NK activity of low-molecular-weight chitosan (21- and 46-kDa chitosans)-treated IELs or splenic lymphocytes was stronger than that of high-molecular-weight chitosan (130- and 650-kDa chitosans)-treated IELs or splenic lymphocytes. In addition, low-molecular-weight chitosan-treated IELs or splenic lymphocytes also enhanced the cytotoxic activity against sarcoma 180 cells. In an in vivo study, although low-molecular-weight chitosan-treated IELs had cytotoxic activity against tumor cells, splenic lymphocytes treated with chitosans had no effect. These findings suggest that the antitumor activity of low-molecular-weight chitosans (12- and 46-kDa chitosans) and oligochitosan might be due in part to the enhancement of NK activity in IELs. Thus, the low-molecular-weight chitosans or oligochitosan might be useful in preventing tumor growth through the activation of intestinal immune functions.

KEY WORDS: • water-soluble chitosan • low-molecular-weight chitosan • antitumor activity • intestinal intraepithelial lymphocytes • natural killer activity

Chitin and chitosan are polymers that have molecular weights of ~1000 kDa and contain >5000 acetylglucosamine and glucosamine units, respectively. Chitin is widely distributed in natural products such as the protective cuticles of crustaceans and insects, as well as in the cell walls of some fungi and microorganisms; it is usually prepared from the shells of crabs and shrimp. Chitin is converted to chitosan by deacetylation with 450 g/L NaOH at 100°C for 2 h. Chitosan is commercially produced in different parts of the world (e.g., Japan, North America, Poland, Italy, Russia, Norway, and India) on a large scale (1). A large amount of literature exists regarding the effects of water-insoluble chitosans with a high molecular weight on the growth of Meth A tumor, adjuvant activity, and stimulation of cytokine production in mice (2–4). High-molecular-weight chitosans have high viscosity and are water-insoluble. It was reported that chitosan with a high molecular weight augments the natural killer (NK)3 activity of mouse lymphocytes (5). Recently we reported that a high-molecular-weight chitosan (average molecular weight, 650 kDa) prevented the adverse effects (myelotoxicity, gastrointestinal toxicity, immunocompetent organic toxicity, and reduction of body weight) induced by the administration of the cancer chemotherapeutic drugs, 5-fluorouracil, cisplatin, and doxorubicin, without interfering with the antitumor activity of these drugs (6–8), but it had no direct antitumor activity in sarcoma 180–bearing mice (6). Furthermore, high-molecular-weight chitosans exhibit myriad biological actions, namely, hypocholesterolemic, antimicrobial, wound healing, and anti-obesity properties (9–12). There is little doubt that such properties would influence absorption in the human intestine because most animal intestines, especially the human gastrointestinal tract, do not possess enzymes such as chitinase and chitosanase, which directly degrade the ß-glucosidic linkage in chitosan (13).

Low-molecular-weight chitosans, obtained by chemical or enzyme hydrolysis of high-molecular-weight chitosan, have lower viscosity and are soluble in water. Subsequently, they seem to be readily absorbed in vivo. A low-molecular-weight chitosan was shown to reduce blood glucose and serum triglyceride levels in obese diabetic KK-Ay mice (14). It was reported that oligochitosans such as N-acetylchitohexaose and chitohexaose increase NK activity in tumor-bearing mice (15), and that oligochitosans (molecular weight: 1–3 kDa and 3–5 kDa) prevent oxidative stress in mice (16). In a clinical study, a high-molecular-weight chitosan was used for the prevention of hypercholesterolemia, diabetes, obesity, and cancer (1,17). The normal intake of high-molecular-weight chitosans in humans is 3–5 g 3 times/d. Although it was suggested that water-soluble chitosans may have antitumor activities in clinical use, such effects are as yet unproven. In this study, we examined the antitumor activities and mechanism(s) of action of various water-soluble chitosans with a range of molecular weights in sarcoma 180–bearing mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Materials. Three kinds of water-soluble chitosans were supplied by Hatanaka Mind Ace. The average molecular weight of each of these chitosans was 21, 46, and 130 kDa, respectively, judging from the viscosity. Oligochitosan (oligoglucosamine) was supplied by Kouzukyu Honten and consisted of a mixture of chitobiose (159 g/kg), chitotriose (286 g/kg), chitotetraose (279 g/kg), chitopentaose (189 g/kg), and chitohexaose (87 g/kg). DMEM and RPMI 1640 media were obtained from Nissui Pharmaceutical. Fetal bovine serum (FBS) and antibiotic and antimycotic solution (times 100) were purchased from Gibco BRL and Sigma Chemical, respectively. 3'-O-Acetyl-2',7'-bis(carboxyethyl)-4- or 5-carboxytlfluorescein acetoxymethylester (BCECF-AM) was purchased from Dojin. The 6-, 12-, 24-, and 96-well plates were purchased from Corning Glass Works. The laboratory diet was purchased from oriental Yeast. Other chemicals were of reagent grade.

    Cells. Sarcoma 180 cells are generally used as the first choice for evaluating the antitumor effects of various drugs, and they have a high response to immune system. Therefore, in this study, we used the sarcoma 180 cells to evaluate the antitumor effects of various water-soluble chitosans and oligochitosans through immune function. The sarcoma 180 cells and YAC-1 cells (natural-killer-cell-sensitive target cells) were maintained in DMEM and RPMI 1640 supplemented with 100 g/L FBS, penicillin (1 x 105 U/L), streptomycin (100 mg/L) and amphotericin B (0.25 mg/L).

    Animals. Male ICR strain mice (6 wk old) and C57 BL/6 mice (5 wk old) were obtained from Clea Japan. These mice were housed for 1 wk in a room maintained at 25 ± 1°C with 60% relative humidity and provided with free access to laboratory standard diet [per kg of diet: cornstarch, 380 g; casein, 210 g; {alpha}-starch, 100 g; cellulose powder, 80 g; corn oil 60, g; sugar, 50 g; mineral mixture (AIN-76) (18), 60 g; and vitamin mixture (AIN-76) (18), 20 g; 3328 kcal/kg, oriental Yeast] and water. The room lights were on for 12 h/d starting at 0700 h. Mice were treated according to the ethics guidelines of the Animal Center, School of Medicine, Ehime University. The experimental protocol was approved by the Animal Studies Committee of Ehime University.

    Treatment of sarcoma 180–bearing mice. Solid-type sarcoma 180 was prepared by subcutaneous transplantation of 2 x 106 cells into the backs of mice on d 0. Various water-soluble chitosans or oligochitosan were dissolved in distilled water at a concentration of 10 or 30 g/L. The above solutions (21-kDa chitosan, Expt. 1; 46-kDa chitosan, Expt. 2; oligochitosan, Expt. 3; 130-kDa chitosan, Expt. 4; 10 or 30 g/L) were administered orally by i.g. intubation at 0.1 mL/10 g body weight (corresponding to 100 or 300 mg/kg) twice daily at 0700 and 1900 h for 20 consecutive days, starting 12 h after the implantation of tumor cells. Control mice were also given distilled water on the same schedule. The tumor volume was determined by direct measurement with calipers and calculated as follows: [length (mm) x width (mm2)]/2 every 2 or 3 d starting 5 d after the tumor implantation. On d 21, blood was obtained by venous puncture in mice under diethyl ether anesthesia. Subsequently, the tumor, epididymal adipose tissue, spleen, and thymus were removed and weighed for evaluation of antitumor activity and immunocompetent organ functions. Blood samples were chilled in test tubes containing heparin, and the numbers of leukocytes and RBC were measured using a Coulter Counter. The hemoglobin concentration in the blood was determined using Hemoglobin-Test kits (Wako Pure Chemical).

    Cytotoxicity against sarcoma180 cells (in vitro). Sarcoma 180 cells were placed in DMEM supplemented with 100 g/L FBS at 1 x 104 cells/well in 24-well culture plates. After the cells were cultured overnight, the medium was changed to fresh DMEM with 100 g/L FBS, and the cells were exposed to the indicated amounts of various water-soluble or -insoluble chitosans for 24 h. After the incubation period, the cytotoxicity against sarcoma 180 cells was assessed using the Cell Counting kit (WST-1 assay; Wako Pure Chemical).

    Isolation of splenic lymphocytes or intestinal intraepithelial lymphocytes (IELs). Splenic lymphocytes were isolated using methods described previously (19). IELs were isolated by the method of Ishikawa et al. (20). Briefly, the inverted intestinal 4 segments were added to 45 mL of HBSS supplemented with 50 g/L FBS, penicillin (1 x 105 U/L), streptomycin (100 mg/L), and amphotericin (0.25 mg/L) and shaken at 150 rotations per min and 37°C for 45 min. The resultant cell suspension was collected and passed through a glass-wool column to remove cell debris and sticky cells, and was then subjected to Percoll (Pharmacia) gradient centrifugation. IELs were isolated at the interphase between the 440 and 700 g/L Percoll solutions.

    Preparation of BCECF-labeled YAC-1 (natural killer cell sensitive target cells) or sarcoma 180 cells. Loading of BCECF into the YAC-1 or sarcoma 180 cells was carried out using a modification of the method described previously (19,21,22). Briefly, 3 µmol/L BCECF-AM was added to the YAC-1 cell suspension (1 x 109 cells/L) in RPMI 1640 medium supplemented with 100 g/L FBS and 1 mmol/L EDTA; the cells were incubated for 30 min at 37°C with gentle agitation in a water bath. After the incubation period, the cells were then washed twice with RPMI 1640 medium supplemented with 100 g/L FBS.

    Chitosan content in small intestine after orally administered 21-kDa chitosan. The upper small intestine (~1 cm) was removed 2 h after oral administration of a water-soluble 21-kDa chitosan at 100 mg/kg, and the chitosan contents in the upper small intestine (1 cm) were determined by the Phenol-Sulfate assay.

    Cytotoxic activity of IELs or splenic lymphocytes against YAC-1 or sarcoma 180 cells (in vitro). Isolated IELs or splenic lymphocytes were placed in RPMI 1640 medium containing 100 g/L FBS at 5 x 105 cells in 96-well culture plates and exposed to the indicated amounts of various chitosans for 24 h. After the incubation period, the IELs or splenic lymphocytes were washed twice with fresh RPMI 1640 medium containing 100 g/L FBS. BCECF-labeled YAC-1 cells or sarcoma 180 cells (target cells; 5 x 103 cells) were added to the effector cells and incubated with them for 2 h; then these cell mixtures were centrifuged at 410 x g for 10 min. The fluorescence intensity of the supernatant was measured by fluorimetry (FP-777, JASCO) with excitation at 500 nm and emission at 540 nm. The total fluorescence intensity of the target cells (BCECF-labeled YAC-1 or sarcoma 180 cells) was determined after solubilizing the cells by adding 2.5 g/L Triton X-100. The specific cytotoxic activity was calculated as follows: % specific cytotoxicity = (total fluorescence intensity of target cell plus experimental group IELs or splenic lymphocytes - fluorescence intensity of spontaneous release)/(total fluorescence intensity of target cells plus control group IELs - fluorescence intensity of spontaneous release) x 100.

    Cytotoxic activity of IELs or splenic lymphocytes against YAC-1 in sarcoma 180–bearing mice (in vivo). Sarcoma 180 cells (2 x 106 cells) were implanted subcutaneously into the back of mice on d 0. Various water-soluble chitosans or oligochitosan (100 mg/kg) were administered orally twice daily for 7 d starting on d 11 to tumor-bearing mice. After overnight food deprivation, the mice were killed by cervical dislocation and the small intestine or spleen was quickly removed. The measurements of cytotoxic activity of IELs or splenic lymphocytes against tumor cells were performed using the methods described above.

    Statistical analysis. All values are expressed as means ± SE. Because the same experiments were conducted separately, the data from each experiments were analyzed by one-way ANOVA at each time point, for each concentration or each chitosan; differences among means of each experiment were analyzed using Fisher’s protected least-significant difference (LSD) multiple-comparison test. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Antitumor activities. Chitosan of molecular weight 21 kDa (chitosan-21) significantly reduced the tumor growth and final tumor weight at 100 and 300 mg/kg (Expt. 1; Fig. 1a and Table 1). Chitosan of molecular weight 46 kDa (chitosan-46) significantly reduced the tumor growth on d 15 and 19 at a dose of 100 mg/kg, but not at 300 mg/kg (Expt. 2; Fig. 1b). The final tumor weight was also significantly reduced by the oral administration of chitosan-46 at 100 mg/kg but not at 300 mg/kg (Expt. 2; Table 1). Oligochitosan at 100 and 300 mg/kg significantly reduced the tumor growth and final tumor weight (Expt. 3; Fig. 1c and Table 1). In contrast, chitosan of molecular weight 130 kDa (chitosan-130) had no effect on the tumor growth or final tumor weight (Expt. 4; Fig. 1d and Table 1). Oral administration of chitossan-130 was stopped on d 15 due to the severe loss of body weight.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 1 Effects of oral administration of 21-kDa chitosan (Expt.1) (a), 46-kDa chitosan (Expt. 2) (b), oligochitosan (Expt. 3) (c) and 130-kDa chitosan (Expt. 4) (d) on tumor volume at 20 d in sarcoma 180–bearing mice. Values are means ± SEM, n = 10. Values at the same time not sharing a letter differ, P < 0.05.

 

View this table:
[in this window]
[in a new window]
  		TABLE 1 Effects of oral administration of various chitosans at 100 and 300 mg/kg body on final tumor weight in sarcoma 180-bearing mice1

 
    Body and tissue weights, and blood count. The oral administration of chitosan-21 (100 or 300 mg/kg) had no effect on body weight, adipose tissue, or thymus in sarcoma 180–bearing mice (Expt. 1; Table 2). The weight of the spleen (229.2 ± 40.9 mg, n = 10) in sarcoma 180–bearing mice was significantly greater than that of normal mice (96.0 ± 5.43 mg, n = 10). The increase in spleen weight in sarcoma 180–bearing mice was significantly reduced (to the normal level) by the oral administration of chitosan-21 at 300 mg/kg (Expt. 1; Table 2). The blood cell numbers (leukocytes and RBC) and blood hemoglobin concentration were not affected by chitosan-21 (Expt. 1; Table 2). Chitosan-46, at 300 mg/kg, had no effect on body weight, spleen, thymus, or adipose tissue, or on the blood cell numbers (leukocytes and RBC) or blood hemoglobin concentration in sarcoma 180–bearing mice (Expt. 2; Table 2). However, at the lower dose of 100 mg/kg, chitosan-46 significantly reduced the increase in spleen weight caused by the implantation of sarcoma 180. The weights of other tissues (thymus and adipose tissue) were not affected by chitosan-46 (data not shown). Chitosan-46 significantly increased the RBC number and blood hemoglobin concentration compared with those of sarcoma 180–bearing mice (Expt. 2; Table 2). Neither the oral administration of oligochitosan for 20 d nor of chitosan-130 for 14 d affected the weights of the spleen, thymus, or adipose tissue (data not shown) in sarcoma 180–bearing mice (Expts. 3 and 4; Table 2). Oligochitosan (100 and 300 mg/kg) reduced the leukocyte number compared with that in sarcoma 180–bearing mice, but did not affect the RBC number or blood hemoglobin concentration (Expt. 3; Table 2). On the other hand, chitosan-130 (100 and 300/mg kg) significantly reduced the body weight on d 15 (Expt. 4; Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2 Effects of oral administration of various chitosans at 100 and 300 mg/kg body on the weights of body, thymus, spleen, and adipose tissue, the numbers of leukocytes and RBC and the hemoglobin concentration in sarcoma 180-bearing mice1

 
    Cytotoxic activity against YAC-1 or sarcoma 180 cells (in vitro). The chitosan contents in the upper small intestine 2 h after oral administration of 21-kDa chitosan at 100 mg/kg were 30–100 µg/cm small intestine (data not shown). On the other hand, chitosan was not detected in blood or spleen (data not shown). On the basis of a preliminary experiment, we examined the cytotoxic activity against YAC-1 or sarcoma 180 cells by IELs or splenic lymphocytes treated with various chitosans. Treatment of IELs with oligochitosan (10–1000 mg/L), chitosan-21 (10–1000 mg/L), or chitosan-46 (10–1000 mg/L) enhanced their cytotoxic activity against YAC-1 cells compared with that of untreated IELs (Fig. 2; Expt. 5). The cytotoxic activity of chitosan-21-treated IELs was similar to that of oligochitosan-treated IELs, and stronger than that of chitosan-46–treated IELs, at 500 or 1000 mg/L. On the other hand, treatment of IELs with chitosan-130 at 10 mg/L augmented their cytolytic activity against YAC-1 cells compared with that of untreated IELs, but treatment of IEL with chitosan-130 at 100, 500, or 1000 mg/L had no effect. Treatment of IELs with a high-molecular-weight (650 kDa) chitosan (water-insoluble chitosan) at 10 mg/L also enhanced their cytolytic activity against YAC-1 cells, but treatment at 100, 500 or 1000 mg/L had no such effect.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 2 Enhancing effects of various chitosans on the cytotoxic activity against YAC-1 cells of IELs (in vitro) (Expt. 5). Isolated IELs were assayed for lytic activity against BCECF-labeled YAC-1 target cells in the presence or absence of various chitosans. Lytic activities elicited at effector to target ratios of 100:1 are presented. Values are means ± SEM, n = 4. Values at a given concentration not sharing a letter differ, P < 0.05.

 
Treatment of IELs with oligochitosan, chitosan-21, or chitosan-46 enhanced their cytotoxic activity against sarcoma 180 cells compared with that of untreated IELs, at 10 mg/L, but neither chitosan-130 nor chitosan-650 had an effect (Expt. 6; Fig. 3). The cytotoxic activity of various chitosan-treated IELs against YAC-1 cells was stronger than that against sarcoma 180 cells. Furthermore, treatment of splenic lymphocytes with oligochitosan, chitosan-21, chitosan-46, or chitosan-130 enhanced the cytotoxic activity against YAC-1 and sarcoma 180 cells compared with that of untreated splenic lymphocytes at 10 mg/L (Fig. 3). Oligochitosan and various water-soluble or -insoluble chitosans did not affect the direct cytotoxicity against sarcoma180 cells (data not shown).



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 3 Enhancing effects of various chitosans on cytotoxic activity against YAC-1 or sarcoma 180 target cells of IELs or splenic lymphocytes (in vitro) (Expt. 6). Isolated IELs or splenic lymphocytes were assayed for lytic activity against BCECF-labeled YAC-1 or sarcoma 180 target cells in the presence or absence of various chitosans at a concentration of 10 mg/L. Lytic activities elicited at effector to target ratios of 100:1 are presented. Values are means ± SEM, n = 4. Values for the same treatment not sharing a letter differ, P < 0.05.

 
    Cytotoxic activity against YAC-1 cells (In vivo). IELs isolated from mice orally administered oligochitosan, chitosan-21, or chiotosan-46 at 100 mg/kg enhanced the cytotoxic activity against YAC-1 cells, but the IELs isolated from chitosan-130–treated mice had no effect (Expt. 7; Table 3). On the other hand, splenic lymphocytes from mice orally administered various chitosans at 100 mg/kg did not affect the cytotoxicity against YAC-1 cells (data not shown).


View this table:
[in this window]
[in a new window]
  		TABLE 3 Cytotoxic activity against YAC-1 target cells by IELs isolated from sarcoma 180-bearing mice killed on d 19 after in vivo treatment with various chitosans (100 mg/kg body) administered orally twice daily for 7 d starting on d 11 (Experiment 7)1

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
It was reported that water-insoluble chitosan with a high molecular weight (650 kDa) prevents the adverse reactions such as myelotoxicity, gastrointestinal toxicity, immunotoxicity, and body weight loss induced by the cancer chemotherapeutic drugs, 5-FU, cisplatin, and doxorubicin, without reducing the antitumor activities of these drugs (6–8), although water-insoluble chitosan with a high molecular weight has no direct antitumor effects (6). The normal intake of high-molecular-weight chitosans in humans is 3–5 g 3 times/d. In this study, the doses of various low-molecular-weight water-soluble chitosans were administered orally 2 times/d at 100 or 300 mg/kg body. Therefore, the dose (ml/kg body weight) administered to mice was approximately two- to fourfold the dose (ml/kg body weight) in humans. Although it was suggested that clinical treatment with water-soluble chitosan with a low molecular weight may have antitumor effects, these effects have not been confirmed. In this study, the antitumor activities of various water-soluble chitosans with a range of molecular weights in sarcoma 180–bearing mice were examined. Although the water-soluble chitosans with a low molecular weight (oligochitosan, chitosan-21, and chitosan-46) had antitumor activity, the water-soluble (130 kDa) or -insoluble chitosan (650 kDa) with a high molecular weight had no effect. However, oral administration of a 46-kDa chitosan at 300 mg/kg had no effect on tumor growth. The viscosity of a 30 g/L water solution of 46 kDa chitosan is higher than that of a 10 g/L solution. Therefore, it seems likely that the rate of small intestinal absorption after oral administration of a 46-kDa chitosan at 300 mg/kg may be lower than that at 100 mg/kg; consequently it did not affect antitumor activity at 300 mg/kg. Further studies are required to clarify the above hypothesis.

Chitosan with a molecular weight of 130 kDa had no effect on tumor growth; rather it caused a reduction in body weight compared with tumor-bearing mice. The conversion of high-molecular-weight chitosans resulted in their becoming very soluble in water and having potent antitumor activities, suggesting that the antitumor activity of low-molecular-weight chitosans is stronger than that of high-molecular-weight chitosans. It was shown clinically that the increase in natural killer (NK) cell activity was significantly higher in the group treated with oligochitoisan than in the placebo group, 6 and 9 h after administration of oligochitosan or placebo in a crossover double-blind test (23). Seo et al. (24) reported that the synergism between the effects of interferon (IFN)-{gamma} and water-soluble chitosan on nitric oxide (NO) synthesis and tumoricidal activity was dependent mainly on the increased secretion of tumor necrosis factor-{alpha} induced by water-soluble chitosan. Shibata et al. (25) reported that C57BL/6 mice pretreated with monoclonal antibodies against mouse IFN-{gamma} or NK.1.1 had a markedly decreased level of alveolar macrophage priming after injection of chitin particles (10 µm). They suggested that the alveolar macrophage priming mechanism of chitin was due to direct activation of macrophages by IFN-{gamma}, which is produced by NK1.1+ and CD4- T cells in the spleen (25). In the preliminary experiment, the chitosan contents in the upper small intestine 2 h after oral administration of 21-kDa chitosan at 100 mg/kg were 30–100 µg/cm small intestine. Therefore, it seems likely that the concentrations of various water-soluble chitosans of IELs used in vitro may be closely associated with the doses used in vivo. In this study, intestinal intraepithelial lymphocytes (IELs) treated with low-molecular-weight water-soluble chitosan (21 or 46 kDa) or oligochitosan enhanced the cytotoxic activity against tumor cells compared with untreated IELs in both in vitro and in vivo experiments. Although splenic lymphocytes treated with the low-molecular-weight water-soluble chitosans (21 kDa, 46 kDa, or oligochitosan) in vitro enhanced the cytotoxic activity against tumor cells, the splenic lymphocytes after oral administration of low-molecular-weight water-soluble chitosans had no effect in vivo. These findings suggest that water-soluble chitosan with a low molecular weight (21 or 46 kDa) may act as an immunomodulator in the intestinal immune systems of animals. They further suggest that the antitumor activity of low-molecular-weight water-soluble chitosans (21 and 46 kDa) and oligochitosan might be due in part to an enhancement of the cytotoxic activityof IELs against tumors. Furthermore, it seems likely that low-molecular-weight water-soluble chitosans (21 and 46 kDa) and oligochitosan may induce the activation of macrophages through the production of cytokines such as IFN-{gamma}, IL-12, and IL-18 from the IELs; consequently, their chitosans may have antitumor activity. Experiments are now in progress to clarify the activation of macrophages through the alteration of immune function in IELs by treatment with various low-molecular-weight water-soluble chitosans in tumor-bearing mice.


   FOOTNOTES
 
1 Supported by research grants from Hatanaka Mind Ace Company, Ltd. (Miyazaki, Japan) and Kozukyu Honten Company (Osaka, Japan). Back

3 Abbreviations used: BCECF, 3'-O-acetyl-2',7'-bis(carboxyethyl)-4- or 5-carboxyfluorescein; BCECF-AM, 3'-O-acetyl-2',7'-bis(carboxyethyl)-4- or 5-carboxyfluorescein acetoxymethylester; FBS, fetal bovine serum; IEL, intestinal intraepithelial lymphocyte; IFN, interferon; NK, natural killer. Back

Manuscript received 22 November 2003. Initial review completed 17 December 2003. Revision accepted 8 January 2004.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

1. Singla, A. K. & Chawla, M. (2001) Chitosan: some pharmaceutical and biological aspects—an update. J. Pharm. Pharmacol. 53:1047-1067.[Medline]

2. Nishimura, K., Nishimura, S., Nishi, N., Saiki, I., Tokura, S. & Azuma, I. (1984) Immunological activity of chitin and its derivatives. Vaccine 2:93-99.[Medline]

3. Nishimura, K., Nishimura, S., Nishi, N., Mumata, F., Tone, Y., Tokura, S. & Azuma, I. (1985) Adjuvant activity of chitin derivatives in mice and guinea pigs. Vaccine 3:379-384.[Medline]

4. Nishimura, K., Ishihara, C., Ueki, S. & Tokura, I. (1986) Stimulation of cytokine product in mice using deacetylated chitin. Vaccine 4:151-156.[Medline]

5. Zhou, A., Matsuura, Y. & Okuda, H. (1994) Chitosan augments cytolytic activity of mouse lymphocytes. J. Tradit. Med. 11:62-64.

6. Kimura, Y. & Okuda, H. (1999) Prevention by chitosan of myelotoxicity, gastrointestinal toxicity and immunocompetent organic toxicity induced by 5-flurouracil without loss of antitumor activity in mice. Jpn. J. Cancer Res. 90:765-774.[Medline]

7. Kimura, Y., Onoyama, M., Sera, T. & Okuda, H (2000) Antitumor activity and side effects of combined treatment with chitosan and cisplatin in sarcoma 180-bearing mice. J. Pharm. Pharmcol. 52:883-890.[Medline]

8. Kimura, Y., Sawai, N. & Okuda, H. (2001) Antitumor activity and adverse reactions of combined treatment with chitosan and doxorubicin in tumor-bearing mice. J. Pharm. Pharmacol. 53:1373-1378.[Medline]

9. Sugano, M., Watanabe, S., Kishi, A., Izume, M. & Ohtakara, A. (1988) Hypocholesterolemic action of chitosans with different viscosity in rats. Lipids 23:187-191.[Medline]

10. Chen, C. S., Liau, W. Y. & Tsai, G. J. (1998) Antibacterial effects of N-sulphonated and N-sulphobenzoyl chitosan and application to oyster preservation. J. Food Prot. 61:1124-1128.[Medline]

11. Okamoto, Y., Shibazaki, K., Minami, S., Matsuhashi, A., Tanioka, S. & Shigemasa, Y. (1995) Evaluation of chitin and chitosan in open wound healing in dogs. J. Vet. Med. Sci. 57:851-854.[Medline]

12. Han, L.-K., Kimura, Y. & Okuda, H. (1999) Reduction in fat storage during chitin-chitosan treatment in mice fed a high-fat diet. Int. J. Obes. 23:174-179.

13. Fukamizo, T. & Brzezinski, R. (1997) Chitonase from Streptomyces sp. strain N174: a comparative review of its structure and function. Biochem. Cell Biol. 75:687-696.[Medline]

14. Hayashi, K. & Ito, M. (2002) Antidiabetic action of low molecular weight chitosan in genetically obese diabetic KK-Ay mice. Biol. Pharm. Bull. 25:188-192.[Medline]

15. Kobayashi, M., Watanabe, T., Suzuki, S. & Suzuki, M. (1990) Effects of N-acetylchitohexaose against Candida albicans infection of tumor-bearing mice. Microbiol. Immunol. 34:413-426.[Medline]

16. Shon, Y.-H., Park, I.-K., Moon, I.-S., Chang, H. W., Park, I.-K. & Nam, K.-S. (2002) Effects of chitosan oligosaccharide on 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced oxidative stress in mice. Biol. Pharm. Bull. 25:1161-1164.[Medline]

17. Tai, T.-S., Sheu, W.H.-H., Lee, W.-J., Yao, H.-T. & Chaiang, M.-T. (2000) Effects of chitosan on plasma lipoprotein concentrations in type 2 diabetic subjects with hypercholesterolemia. Diabetes Care 23:1703-1704.

18. American Institute of Nutrition (1977) Report of American Institute of Nutrition and hoc committee on standards for nutritional studies. J. Nutr. 107:1340-1348.

19. Kimura, Y. & Okuda, H. (2001) Resveratrol isolated from Polygonum cuspidateum root prevents tumor growth and metastasis to lung and tumor-induced neovascularization in Lewis lung carcinoma-bearing mice. J. Nutr. 131:1844-1849.[Abstract/Free Full Text]

20. Ishikawa, H., Ki, Y., Abellivich, A., Yamamoto, S., Kaufmann, S.H.F. & Tonegawa, S. (1993) Cytotoxic and interferon {gamma}-producing activities of {gamma}{delta} T cells in the mouse intestinal epithelium are strain dependent. Proc. Natl. Acad. Sci. U.S.A. 90:8204-8208.[Abstract/Free Full Text]

21. Kimura, Y. & Okuda, H. (2000) Effects of naturally occurring stilbene glucosides from medicinal plants and wine, on tumor growth and lung metastasis in Lewis lung carcinoma-bearing mice. J. Pharm. Pharmacol. 12:1287-1295.

22. Kimura, Y. (2002) Carp oil or oleic acid, but not linoleic acid or linolenic acid, inhibits tumor growth and metastasis in Lewis lung carcinoma-bearing mice. J. Nutr. 132:2069-2075.[Abstract/Free Full Text]

23. Kajimoto, O., Sakamoto, K., Miwa, T., Matahira, Y., Yoshida, I., Kiyotake, T. & Takahashi, T. (1999) A study of the immunoactivation effect of oral oligoglucosamine in humans, elevated by NK-cell activation. Results from a cross-over double blind study with placebo in healthy subjects (in Japanese). Nippon Rinshou Eiyougaku Zasshi 21:41-47.

24. Seo, W.-G., Pae, H.-O., Kim, N.-Y., Oh, G.-S., Park, I.-S., Kim, Y.-H., Kim, Y.-M., Lee, Y.-H., Jun, C.-D. & Chung, H. T. (2000) Synergistic cooperation between water-soluble chitosan oligomers and interferon-{gamma} for induction of nitric oxide synthesis and tumoricidal activity in murine peritoneal macrophages. Cancer Lett. 159:189-195.[Medline]

25. Shibata, Y., Foster, L. A., Metzger, W. J. & Myrvik, Q. N. (1997) Alveolar macrophages priming by intravenous administration of chitin particles, polymers of N-acetyl-D-glucosamine, in mice. Infect. Immun. 65:1734-1741.[Abstract]




This article has been cited by other articles:


Home page
 Journal of Bioactive and Compatible PolymersHome page
Y.-H. Uen, Y.-J. Wang, T.-M. Hsu, M.-H. Chen, H.-Y. Chan, Y.-S. Ho, and S.-Y. Lin
Effects of Fungal-derived High Molecular Weight Chitosan on 5-Fluorouracil-induced Adverse Reactions
Journal of Bioactive and Compatible Polymers, September 1, 2008; 23(5): 458 - 472.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maeda, Y.
Right arrow Articles by Kimura, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maeda, Y.
Right arrow Articles by Kimura, Y.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Copyright © 2004 by American Society for Nutrition