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Research Communication

Vitamin B-6–Supplemented Diets Compared with a Low Vitamin B-6 Diet Suppress Azoxymethane-Induced Colon Tumorigenesis in Mice by Reducing Cell Proliferation

Shun-ichiro Komatsu, Hiromitsu Watanabe^*, Tatsuzo Oka, Haruhito Tsuge^**, Hironori Nii^** and Norihisa Kato

Faculty of Applied Biochemistry, Hiroshima University, Higashi-Hiroshima 739-8528, Japan; ^* Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan; Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan; and ^** Faculty of Agriculture, Gifu University, Gifu 501-

¹To whom correspondence should be addressed. E-mail: nkato{at}hiroshima-u.ac.jp.

	ABSTRACT

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Male ICR mice were examined for the effect of vitamin B-6 [pyridoxine (PN) HCl] on azoxymethane-induced colon tumorigenesis. Mice were fed the diets containing 1, 7, 14 or 35 mg PN HCl/kg for 22 wk, and given a weekly injection of azoxymethane (5 mg/kg body) for the initial 10 wk. Compared with the 1 mg PN HCl/kg diet, 7, 14 and 35 mg PN HCl/kg diets significantly suppressed the incidence and number of colon tumors, colon cell proliferation and expressions of c-myc and c-fos proteins. For some variables, 14 and 35 mg PN HCl/kg diets were more effective than the 7 mg/kg diet. Supplemental vitamin B-6 had no influence on the number of colon apoptotic cells. The results suggest that elevating dietary vitamin B-6 suppresses colon tumorigenesis by reducing cell proliferation.

KEY WORDS: • vitamin B-6 • pyridoxine • colon tumorigenesis • cell proliferation • mice

	INTRODUCTION

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High levels of pyridoxine (PN)² or pyridoxal (PL) have been reported to suppress growth or to be cytotoxic to animal or human cancer cells in vitro (1 2 3) . Gridley et al. (4) showed that a high dietary intake of vitamin B-6 suppresses herpes simplex virus type 2 transformed cell-induced tumor growth and enhances immune status in BALB/c mice. These studies indicate that supraphysiologic doses of vitamin B-6 may have potential use in antineoplastic therapy, although the underlying mechanism by which vitamin B-6 exerts its inhibitory effects remains unknown.

Recently, a population-based case-control study in the United States indicated that intake of vitamin B-6 was associated inversely with colon cancer (5) . The current study was designed to examine the effect of the dietary level of vitamin B-6 on colon tumorigenesis in mice.

	MATERIALS AND METHODS

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Animals and diets.

Male CD-1 (ICR): Crj mice (4 wk old, Charles River Japan, Hino, Japan) were housed in groups of 3 or 4 in a metal cage in a room with controlled temperature (24 ± 1°C) and a 12-h light:dark cycle (lights on, 0800–2000h). They had free access to diets and deionized water. The mice were maintained according to the Guide for the Care and Use of Laboratory Animals established by Hiroshima University. After consuming a commercial stock diet (MF, Oriental Yeast, Tokyo, Japan) for 1 wk, the mice (mean weight, 26 g) were divided into four groups of 34. The basal diet was composed of the following components (g/kg diet): -cornstarch, 402; casein, 200; sucrose, 200; corn oil, 100; cellulose, 50; AIN-93G mineral mixture, 35; AIN-93 vitamin mixture (PN free), 10; and L-cystine, 3 (6) . Vitamin B-6 (PN HCl, Nacalai Tesque, Kyoto, Japan) was supplemented to the basal diet at the levels of 1, 7, 14 and 35 mg/kg diets. The level of PN HCl/kg diet recommended in the AIN-93 diet is 7 mg(6) , and a 1 mg PN HCl/kg diet has been reported to be the minimum level required for preventing growth depression caused by vitamin B-6 deficiency (7) . The experimental feeding period was 22 wk. Food intake and body weight were measured daily. Mice were given azoxymethane (AOM, 5 mg/kg body, Sigma Chemical, St. Louis, MO) diluted by saline by subcutaneous injection once a week for the initial 10 wk of the experiment. One hour before termination (1300–1600 h), 5-bromo-2'-deoxyuridine (BrdU, Sigma Chemical) was given by intraperitoneal injection (100 mg/kg body) for immunohistochemical analysis of cell proliferation. Blood was collected from the hearts in mice under anesthesia with diethyl ether. Major organs were immediately observed and weighed.

Visualization and histological examination.

At the termination of the studies, the colon was removed, slit open longitudinally from cecum to anus, placed on a paper towel and fixed in neutral formalin for 24 h. Tumor-bearing areas and volumes were observed with a microscope and embedded in paraffin. In addition to tumor-bearing areas, areas of flat mucosa with no visible tumors were embedded in paraffin. The colon was examined histologically after staining with hematoxylin and eosin, and tumors in the colon were classified into two types, i.e., adenomas and adenocarcinomas. Adenomas were tumors with no evidence of invasion of muscularis mucosa. Adenocarcinomas were tumors with evidence of invasion of muscularis mucosa. Immunohistochemical analysis of BrdU labeling was performed for the normal colonic mucosa. The BrdU staining method was described elsewhere (8) . BrdU-positive cells in the colonic mucosal epithelium were counted under the microscope at a magnification of X200 in rectum, distal colon and proximal colon. Immunohistochemical analysis of apoptosis labeling was examined by the TUNEL method. This method is based on TdT-mediated dUTP-biotin nick end labeling of fragmented DNA (9) . After deparaffinization, they were stained by the Apoptosis in situ Detection Kit (Wako Pure Chemical, Osaka, Japan). Apoptosis-positive cells in the normal colonic mucosal epithelium were counted under the microscope at a magnification of X200. Immunohistochemical analysis of c-myc and c-fos proteins was performed for the normal colonic mucosa. The c-myc and c-fos staining was as follows. After deparaffinization, rabbit polyclonal anti-c-myc antibody (Santa Cruz Biochemistry, Santa Cruz, CA) and rabbit polyclonal anti-c-fos antibody (Oncogene Research Product, Cambridge, MA) were applied to the specimens, and they were stained by Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA). The c-myc and c-fos-positive cells in the colonic mucosal epithelium were counted under the microscope at a magnification of X200.

Hepatic vitamin B-6 concentration.

The liver was immediately excised and stored at -80°C until analysis of vitamin B-6 contents by HPLC (10) . Vitamin B-6 from liver was extracted using 1 mol/L perchloric acid and measured by HPLC with a fluorometric detector. PL 5'-phosphate was converted to pyridoxic acid 5'-phosphate and measured separately.

Statistical analysis.

Values are presented as means ± SEM. Statistical significance of difference among means was estimated at P < 0.05 according to ² test, one-way ANOVA and Duncan’s multiple-range test (11) . Wilcoxon rank sum test was used for the analysis of the difference in the number of colorectal tumors among the groups (12) . Some data underwent regression analysis and the correlation coefficient was calculated.

	RESULTS

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Body weight and food intake.

Food intake [g/(22 wk · mouse)] in the 1, 7, 14 and 35 mg PN HCl/kg diet groups was 622, 622, 632 and 628 (pooled SEM = 5, P > 0.05), respectively. Final body weights (g) in the 1, 7, 14 and 35 mg PN HCl/kg diet groups were 50, 51, 53 and 52 (pooled SEM = 0.4, P > 0.05), respectively.

Colonic tumors.

Supplemental dietary vitamin B-6 suppressed the incidence of tumors (P < 0.05, Table 1 ) and the number of tumors (P < 0.05, Fig. 1 ) compared with the 1 mg PN HCl/kg diet group. The number of mice with colon tumors was significantly higher in that group than in the other groups, which did not differ from one another. The tumor volumes (mm³) did not differ among the 1, 7, 14 and 35 mg PN HCl/kg diet groups [1.40, 1.35, 1.35 and 1.20 (pooled SEM = 0.08, respectively]. Colonic tumors were located mainly in the distal colon in all groups. The majority of the tumors were identified as adenomas. Four and three adenocarcinomas were observed in the 1 and 7 mg PN HCl/kg diet groups, respectively (Table 1) . Most adenomas and adenocarcinomas were well differentiated, and histological type was not affected by supplemental vitamin B-6.

View larger version (19K): [in this window] [in a new window]   Figure 1. Effect of dietary vitamin B-6 on the number of colorectal tumors per mouse treated with azoxymethane. Values are means ± SEM, n = 34. Means not sharing a superscript letter are significantly different (Wilcoxon rank sum test, P < 0.05).

The BrdU labeling index was significantly reduced by supplemental vitamin B-6 (P < 0.05, Table 2 ). The labeling index of proliferating cells in all of the colon epithelium correlated with the number of tumors (r = 0.70, P < 0.01). Compared with the 1 mg PN HCl/kg diet, the minimum level of PN HCl required for the suppression of cell proliferation was 7 mg PN HCl/kg diet in all areas of the colon. The labeling index in the rectum and proximal colon of the 14 and 35 mg PN HCl/kg diet groups was significantly lower than that of the 7 mg PN HCl/kg diet group. Cell proliferation in the distal colon did not differ among the three supplemented groups (P > 0.05).

Expression of c-myc and c-fos.

The labeling indices of c-myc and c-fos proteins in the colonic crypts were significantly reduced by supplemental vitamin B-6 (P < 0.05, Table 3 ). The 7 mg PN HCl/kg diet group had significantly lower labeling indices that the 1 mg PN HCl/kg diet group in all areas of the colon. The 14 and 35 mg PN HCl/kg diet groups had significantly lower expressions of c-myc and c-fos proteins in all areas of the colon compared with the 7 mg PN HCl/kg diet group.

The concentration of total vitamin B-6 (PL 5'-phosphate and pyridoxamine 5'-phosphate) did not differ among the 4 groups after 22 wk of dietary treatment [1, 7, 14 and 35 mg PN HCl/kg diet groups; 7.61, 7.04, 7.06 and 7.50 nmol/g liver (pooled SEM = 0.32), respectively]. The concentrations of PL 5'-phosphate and pyridoxamine 5'-phosphate were also unaffected by supplemental vitamin B-6 (P > 0.05, data not shown). The concentrations of PN 5'-phosphate were too low to be measured.

	DISCUSSION

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In this study, we have demonstrated for the first time that elevating dietary vitamin B-6 significantly suppressed colon tumorigenesis induced by AOM. Compared with the 1 mg PN HCl/kg diet, the minimum level for preventing colon tumorigenesis was found to be 7 mg PN HCl/kg diet. In general, the greatest suppression of tumorigenesis by dietary vitamin B-6 was observed in mice fed 14 and 35 mg PN HCl/kg diet. The recommended level in the AIN-93 diet is 7 mg PN HCl/kg diet (6) and appears to be close to the level in human diets (13) . The optimum requirement of PN HCl appears to be 14–35 mg/kg for suppression of colon tumorigenesis and cell proliferation, although the differences between the 14 and 35 mg PN HCl/kg diets and the 7 mg PN HCl/kg diet were slight. Vitamin B-6 has gained widespread acceptance as being nontoxic, with large doses being given to treat various disease states (14) . In the mice fed the 35 mg PN HCl/kg diet, no toxicity symptoms were observed throughout the entire feeding period.

The supplemental vitamin B-6 reduced cell proliferation in the colonic epithelium, whereas it had no significant effect on colonic epithelium apoptosis. The preventive effect of dietary vitamin B-6 was closely associated with reduced cell proliferation, suggesting that reduced cell proliferation is, at least in part, responsible for the protective effect of vitamin B-6 against colon tumorigenesis. The position of the highest BrdU labeled cell was also significantly lowered by the 7, 14 and 35 mg PN HCl/kg diets compared with the 1 mg PN HCl/kg diet in the rectum and distal colon, suggesting that supplemental vitamin B-6 reduced the region in which cell proliferation occurred. We also found a suppression in the protein expression of the proliferation-related genes, c-myc and c-fos by vitamin B-6. Some studies have shown that culturing some rodent and human tumor cell lines in a PN-supplemented medium inhibited cell growth (1 2 3 ,15) . PL 5'-phospate has been found to be an effective inhibitor of many enzymes that have binding sites for phosphate-containing substrates or effectors, including RNA polymerase (16 ,17) , reverse transcriptase (18) and DNA polymerase (19 ,20) . Oka et al. (21 ,22) showed that vitamin B-6 deficiency generally enhances gene expression in rat liver, including that of glycogen phosphorylase. The effects of vitamin B-6 on colon cell proliferation might also be mediated through alternations in these genes.

In vitamin B-6 deficiency, antibody production may be indirectly impaired (23) . Gridley et al. (4) showed that high dietary intake of vitamin B-6 (74.3 mg PN/kg diet) suppresses herpes simplex virus type 2 transformed cell-induced tumor growth and enhances immune status compared with a 1.2 mg PN/kg diet in BALB/c mice. Thus, it is possible that the preventive effect of vitamin B-6 against colon tumorigenesis is in part mediated by alterations in immune function.

Of interest was the observation that hepatic vitamin B-6 concentration in AOM-treated mice was not affected by dietary vitamin B-6. Thanassi et al. (24) reported that the concentration of PL 5'-phosphate was lower in the hepatomas than in the livers of host rats. Tumor-bearing rats had a reduced concentration of PL 5'-phosphate in the livers compared with control rats fed a vitamin B-6–sufficient diet (24) . In our study, the hepatic concentration of vitamin B-6 might have been affected by colon tumorigenesis, leading to no differences in the concentration of vitamin B-6. Further study is required to examine the concentration of vitamin B-6 in the colon and liver of mice fed different levels of vitamin B-6 with and without AOM treatment.

In conclusion, our study has provided the first evidence that supplementing vitamin B-6 to a low vitamin B-6 diet suppresses AOM-induced colon tumorigenesis by reducing cell proliferation. Because our study was conducted with mice injected with AOM for the initial 10 wk, the possibility that supplemental vitamin B-6 affects the metabolism of AOM and the initiation stage in colon tumorigenesis remains to be studied.

	FOOTNOTES

 
² Abbreviations used: AOM, azoxymethane; BrdU, 5-bromo-2'-deoxyuridine; PL, pyridoxal; PN, pyridoxine.

Manuscript received October 16, 2000. Initial review completed May 22, 2001. Revision accepted May 22, 2001.

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