Biochimica et Biophysica Acta 1647 (2003) 127 – 130

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Review
Antitumor effect of vitamin B6 and its mechanisms
S. Komatsu a, N. Yanaka a, K. Matsubara b, N. Kato a,*
a
Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan
b
Faculty of Health and Welfare Science, Okayama Prefectual University, Okayama, 719-1197, Japan

Received 13 July 2002; received in revised form 24 September 2002; accepted 22 January 2003

Abstract

Epidemiological studies have reported an inverse association between vitamin B6 intake and colon cancer risk. Our recent study
has been conducted to examine the effect of dietary vitamin B6 on colon tumorigenesis in mice. Mice were fed diets containing 1, 7,
14 or 36 mg/kg pyridoxine for 22 weeks, and given a weekly injection of azoxymethane (AOM) for the initial 10 weeks. Compared
with the 1 mg/kg pyridoxine diet, 7, 14 and 35 mg/kg pyridoxine diets significantly suppressed the incidence and number of colon
tumors, colon cell proliferation and expressions of c-myc and c-fos proteins. Supplemental vitamin B6 lowered the levels of colonic
8-hydroxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE, oxidative stress markers) and inducible nitric oxide (NO) synthase
protein. In an ex vivo serum-free matrix culture model using rat aortic ring, supplemental pyridoxine and pyridoxal 5V-phosphate
(PLP) had antiangiogenic effect. The results suggest that dietary vitamin B6 suppresses colon tumorigenesis by reducing cell
proliferation, oxidative stress, NO production and angiogenesis.
D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Vitamin B6; Colon cancer; Cell proliferation; Oxidative stress; Nitric oxide; Angiogenesis

1. Introduction azoxymethane (AOM)-treated mice [10]. The results

There is growing evidence that vitamin B6 has a new role
as a chemopreventive agent. High levels of vitamin B6 have
been reported to suppress growth of animal or human cancer
cells in vitro [1– 4]. Animal research also indicated that a
high dietary intake of vitamin B6 suppresses herpes simplex
virus type 2 transformed cell-induced tumor growth in mice
[5]. From these studies, supraphysiological doses of vitamin
B6 have been believed to have potential use in antineoplastic
therapy [6].
Recent case-control studies have indicated an inverse
association between vitamin B6 intake and colon cancer
risk [7,8]. An inverse relationship between vitamin B6
intake and prostate cancer incidence has also been
reported [9].Our group has postulated that colorectal
cancer risk might be reduced by moderate levels of dietary
vitamin B6 [10]. To test this hypothesis, animal experi-
ment was conducted to examine effect of dietary level of
vitamin B6 on the development of colon tumorigenesis in
* Corresponding author.
E-mail address: nkato@hiroshima-u.ac.jp (N. Kato).
showed that colon tumorigenesis was significantly sup-
pressed by moderate doses of dietary vitamin B6, support-
ing our hypothesis [10]. This review describes these recent
studies on the antitumor effect of vitamin B6 and its
mechanisms.
2. Colon tumorigenesis
Growing male ICR mice were fed the diets containing 1, 7,
14 or 35 mg/kg pyridoxine for 22 weeks, and given a weekly
injection of AOM for the initial 10 weeks [10]. One hour
before termination, 5-bromo-2V-deoxyuridine (BrdU) was
given by injection for immunohistochemical analysis of cell
proliferation. A 1 mg/g pyridoxine diet has been reported to
be the minimum level required for preventing growth depres-
sion caused by vitamin B6 deficiency [11]. Food intake and
growth were unaffected by dietary level of vitamin B6.
Supplemental dietary vitamin B6 markedly suppressed the
incidence of colon tumors (Table 1) and the number of colon
tumors compared with the 1 mg/kg pyridoxine diet group.
Compared with the 1 mg/kg pyridoxine diet, the minimum
level for preventing colon tumorigenesis was 7 mg/kg pyr-

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128 S. Komatsu et al. / Biochimica et Biophysica Acta 1647 (2003) 127–130

Table 1 4. Oxidative stress and nitric oxide (NO)

Effect of dietary vitamin B6 on the incidence of colon tumors in AOM-
treated mice1,2
It has been reported that c-myc and c-fos expression can
Pyridoxine HCl Mice with colon tumors (n)
be induced by oxidative stress [21,22]. In vitro study has
(mg/kg diet) Total Adenoma Adenocarcinoma indicated that vitamin B6 has a strong antioxidative effect
a a
1 13 10 4 [23]. It has been reported that vitamin B6 deficiency leads to
7 5b 2b 3 greater lipid peroxidation in plasma and liver when the
14 2b 2b 0
animal consumes a high fat diet [24]. Thus, Komatsu et al.
35 1b 1b 0
1
[25] postulated that the suppression effect of vitamin B6 on
Thirty-four mice per diet group were examined for tumorigenesis [10].
2 the cell proliferation might be mediated through reduced
Means in a column not sharing a superscript letter are significantly
different; v2 test, P < 0.05. oxidative stress. As oxidative stress markers, colonic 8-
hydroxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (4-
HNE) were examined in the AOM-treated mice fed graded
levels of vitamin B6. The immunohistochemical analysis
idoxine diet. In general, the greatest suppression of tumori- showed that supplementation of vitamin B6 to low-vitamin-
genesis by dietary vitamin B6 was observed in the mice fed 14 B6 diet (1 mg/kg diet) significantly suppressed the levels of
and 35 mg/kg pyridoxine diets. The recommended level in 8-OHdG and 4-HNE (Table 2) [25]. The alteration of these
the AIN-93 diet is 7 mg/kg diet [12]. This result implies that oxidative stress markers significantly correlated with the
colon carcinogenesis can be reduced by moderate levels of expression of c-myc and c-fos proteins. Thus, reduced
dietary vitamin B6, being close to the recommended level. oxidative stress might be partially involved in the mecha-
The optimum requirement of pyridoxine appears to be 14 – 35 nism of the preventive effect of vitamin B6 on tumori-
mg/kg for suppression of colon tumorigenesis. genesis.
NO has been considered to play an important role in
colon carcinogenesis by elevating cyclooxygenase-2 and
3. Cell proliferation angiogenesis [26,27]. Some studies have demonstrated that
AOM-induced colon tumors have increased expression and/
Komatsu et al. [10] have found that supplemental vitamin or activity of iNOS when compared to levels in adjacent
B6 reduced the BrdU-labeling index (index of cell prolifer- colonic tissue [28]. Administration of iNOS inhibitor sup-
ation) in the colon of mice (Table 2), whereas vitamin B6 presses the development of colon aberrant crypt foci, early
had no significant effect on colonic epithelium apoptosis. preneoplastic lesions, in AOM-treated rats [29]. The pro-
The labeling index in all of the colon epithelium of the 14 duction of NO and the expression of iNOS mRNA are
and 35 mg/kg pyridoxine diet groups was significantly elevated by oxidative stress [30]. Komatsu et al. [25] have
lower than that of the 7 mg/kg pyridoxine diet group. The further examined the effect of dietary vitamin B6 on colonic
labeling index of proliferating cells in all of the colon expression of iNOS protein in AOM-treated mice. The
epithelium significantly correlated with the number of result showed a significant depression in the colonic iNOS
tumors. The labeling indices of c-myc and c-fos proteins
(oncogene products relating to cell proliferation) in the
colonic crypts were also significantly reduced by supple-
mental vitamin B6 (Table 2). The results imply that lowered Table 2
cell proliferation by dietary vitamin B6 leads to its antitumor Effect of dietary vitamin B6 on the indices of colonic cell proliferation and
effect. oxidative stress in AOM-treated mice1,2
Pyridoxal 5V-phosphate (PLP) has been found to be Pyridoxine HCl 1 7 14 35
effective inhibitor of many enzymes that have binding (mg/kg diet)
sites for phosphate-containing substrates or effectors, Labeling index (%)
including RNA polymerase [13,14], reverse transcriptase BrdU-labeled cells 8.2 F 0.2a 6.4 F 0.2b 5.0 F 0.2c 4.7 F 0.3c
[15] and DNA polymerase [16,17]. Oka et al. [18,19] c-myc expression 9.7 F 0.3a 7.6 F 0.2b 5.7 F 0.2c 5.1 F 0.3c
cells
reported that vitamin B6 deficiency generally enhances c-fos expression 12.3 F 0.2a 9.9 F 0.2b 7.3 F 0.2c 6.8 F 0.3c
gene expression in rat liver, including that of glycogen cells
phosphorylase. Recently, they have demonstrated that PLP 8-OHdG expression 6.7 F 0.1a 5.4 F 0.1b 5.0 F 0.1c 5.0 F 0.2c
first binds to Lys-197 of HNF1 protein, a tissue-specific cells
transcription factor, and modulates DNA binding activity, 4-HNE expression 10.5 F 0.3a 8.4 F 0.2b 7.7 F 0.3b 7.5 F 0.3b
cells
which in turn suppresses expression of the albumin gene iNOS expression 11.5 F 0.2a 10.8 F 0.2b 9.9 F 0.3c 9.2 F 0.3c
in rat liver [20]. The possibility that the inhibitory effect cells
of dietary vitamin B6 on colon cell proliferation is 1
These parameters were examined in all the colon epithelium [10,25].
mediated through alternation of gene expression remains 2
Values are means F S.E. (n = 17). Means in a row not sharing a superscript
to be examined. letter are significantly different; Duncan’s multiple-range test, P < 0.05.
 S. Komatsu et al. / Biochimica et Biophysica Acta 1647 (2003) 127–130
Fig. 1. Effect of pyridoxine and PLP on angiogenesis in rat aortic ring
model. The length of microvessels was measured on day 7, and the average
values ( F S.E.) of six cultures are shown [32].
protein by elevating dietary vitamin B6 (Table 2). The
expression of iNOS protein was associated with the levels
of 8-OHdG and 4-HNE. Thus, the lower NO production by
vitamin B6 might also partially lead to reduced colon
tumorigenesis. Consistent with this finding, our recent study
has demonstrated that higher intake of vitamin B6 lowered
hepatic iNOS activity and lipid peroxide level in LPS-
treated rats (Komatsu et al., unpublished results).
5. Angiogenesis
Angiogenesis is forming new blood vessels and is
involved in tumor growth and metastasis [31]. Matsubara
et al. [32] have studied the effect of vitamin B6 on angio-
genesis using an in vitro model, in which rat aortic ring was
cultured in collagen gel [33,34]. Microvessels appeared
from the edge of aortic ring and elongated in the absence
of vitamin B 6 . The elongation of microvessels was
inhibited at high concentrations of pyridoxine and PLP
(Fig. 1). PLP showed stronger inhibitory effect than pyr-
idoxine. PLP suppressed the elongation of microvessels in
a dose-dependent manner within the range of 25– 500 AM.
They further studied the effect on the growth of human
umbilical vein endothelial cells, being considered to be
associated with angiogenesis. The results showed that PLP
suppressed the proliferation of endothelial cells, but did not
affect the tube formation on reconstituted membrane (Mat-
subara et al., unpublished results). The antiangiogenic
effect appears to be ascribed to antiproliferation activity
for endothelium cells. The mechanism of the effect on the
proliferation of endothelial cells is unknown at present, and
further in vivo study is necessary to test whether dietary
129
vitamin B6 suppresses colon carcinogenesis by reducing
angiogenesis.
6. Conclusions
Our recent studies provided the first evidence for the
preventive effect of dietary vitamin B6 against colon tumori-
genesis in the mice that received AOM. Interestingly, its
antitumor effect appeared to be apparent by moderate doses
of vitamin B6. Recent epidemiological studies also support
an inverse association between vitamin B6 intake and colon
cancer risk. The preventive effect of vitamin B6 against
colon tumorigenesis in mice might be mediated through
suppressing cell hyperproliferation, oxidative stress and NO
synthesis in the colon. In vitro studies have further provided
evidence for antiangiogenic effect of vitamin B6. These
studies imply that dietary vitamin B6 is an important factor
suppressing colon tumorigenesis. Since elevating dietary
vitamin B6 improves immune function [4,35], the possibility
that the antitumor effect of vitamin B6 is ascribed to an
improved immune function remains to be examined.
References
[1] D.M. DiSorbo, G. Litwack, Nutr. Cancer 3 (1982) 216 – 222.
[2] D.M. DiSorbo, L. Nathanson, Nutr. Cancer 5 (1983) 10 – 15.
[3] D.M. DiSorbo, R. Wagner Jr., L. Nathanson, Nutr. Cancer 7 (1985)
43 – 52.
[4] T. Molina, S.M. Oka, M. Munoz, M. Chikamori-Aoyama, M.
Kuwahata, Y. Natori, Nutr. Cancer 28 (1997) 206 – 211.
[5] D.S. Grindley, D.R. Stickney, R.L. Nutter, J.M. Slater, T.D. Shultz,
J. Natl. Cancer Inst. 78 (1987) 951 – 959.
[6] T. Oka, Nutr. Res. Rev. 14 (2001) 257 – 265.
[7] M.L. Slattery, J.D. Potter, A. Coates, K.N. Ma, T.D. Berry, D.M.
Duncan, B.J. Caan, Cancer Causes Control 8 (1997) 575 – 590.
[8] M.C. Jansen, H.B. Bueno-de-Mesquita, R. Buzina, F. Fidanza,
A. Menotti, H. Blackburn, A.M. Nissinen, F.J. Kok, D. Kromh-
out, Int. J. Cancer 81 (1999) 174 – 179.
[9] T.J. Key, P.B. Silcocks, G.K. Davey, P.N. Appleby, D.T. Bishop,
Br. J. Cancer 76 (1997) 678 – 687.
[10] S. Komatsu, H. Watanabe, T. Oka, H. Tsuge, H. Nii, N. Kato, J. Nutr.
131 (2001) 2204 – 2207.
[11] S.P. Coburn, Vitam. Horm. 48 (1994) 259 – 300.
[12] G.P. Reeves, H.F. Nielsen, C.G. Fahey Jr., J. Nutr. 123 (1993)
1939 – 1951.
[13] J. Martial, J. Zaldivar, P. Bull, A. Venegas, P. Valenzuela, Biochem-
istry 14 (1975) 4907 – 4911.
[14] A. Venegas, J. Martial, P. Valenzuela, Biochem. Biophys. Res. Com-
mun. 55 (1973) 1053 – 1059.
[15] A. Basu, R.S. Tirumalai, M.J. Modak, J. Biol. Chem. 264 (1989)
8746 – 8752.
[16] M.J. Modak, Biochemistry 15 (1976) 3620 – 3626.
[17] J.F. Diffley, J. Biol. Chem. 263 (1988) 14669 – 14677.
[18] T. Oka, N. Komori, M. Kuwahata, I. Suzuki, M. Okada, Y. Natori,
Experientia 50 (1994) 127 – 129.
[19] T. Oka, N. Komori, M. Kuwahata, T. Sassa, I. Suzuki, M. Okada,
Y. Natori, FEBS. Lett. 331 (1993) 162 – 164.
[20] T. Oka, H. Sugitatsu, H. Nordin, M.K. Thakur, M. Aoyama, T. Sa-
sagawa, I. Suzuki, H. Tsuji, Biochim. Biophys. Acta 1568 (2001)
189 – 196.
130 S. Komatsu et al. / Biochimica et Biophysica Acta 1647 (2003) 127–130
[21] M. Jang, J.M. Pezzuto, Cancer. Lett. 134 (1998) 81 – 89.
[22] L.M. Crosby, K.S. Hyder, A.B. DeAngelo, T.B. Kepler, B. Gaskill,
G.R. Benavides, L. Yoon, K.Y. Morgan, Toxicol. Appl. Pharmacol.
169 (2000) 205 – 221.
[23] S.K. Jain, G. Lim, Free Radic. Biol. Med. 30 (2001) 232 – 237.
[24] V. Ravichandran, R. Selvam, Biochem. Int. 21 (1990) 599 – 605.
[25] S. Komatsu, H. Watanabe, T. Oka, H. Tsuge, N. Kato, J. Nutr. Sci.
Vitaminol. 48 (2002) 65 – 68.
[26] L.M. Landino, B.C. Crews, M.D. Timmons, J.D. Morrow, L.J. Mar-
nett, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 15069 – 15074.
[27] V. Chiarugi, L. Magnelli, O. Gallo, Int. J. Mol. Med. 2 (1998)
715 – 719.
[28] M. Takahashi, K. Fukuda, T. Ohata, T. Sugimura, K. Wakabayashi,
Cancer Res. 57 (1997) 1233 – 1237.
[29] C.V. Rao, C. Indranie, B. Simi, P.T. Manning, J.R. Connor, B.S.
Reddy, Cancer Res. 62 (2002) 165 – 170.
[30] P.C. Kuo, K.Y. Abe, Gastroenterology 109 (1995) 206 – 216.
[31] J. Folkman, Nat. Med. 1 (1995) 27 – 31.
[32] K. Matsubara, M. Mori, Y. Matsuura, N. Kato, Int. J. Mol. Med. 8
(2001) 505 – 508.
[33] M. Mori, Y. Sadahira, S. Kawasaki, T. Hayashi, K. Notohara, M.
Awai, Acta Pathol. Jpn. 38 (1988) 1503 – 1512.
[34] S. Kawasaki, M. Mori, M. Awai, Acta Pathol. Jpn. 39 (1989)
712 – 718.
[35] R.E. Hodges, W.B. Beam, M.A. Ohlson, R.E. Bleiler, Am. J. Clin.
Nutr. 11 (1962) 180 – 186.