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Anti-cancer function of phytic acid
Abulkalam M. Shamsuddin
Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201-1192, USA
Summary Inositol hexaphosphate (InsP
6
a.k.a. phytic acid or IP
6
) is ubiquitous. In the plant
kingdom it is particularly abundant in cereals and legumes; in much smaller amounts IP
6
and its lower phosphorylated forms (IP
1)5
) are contained in most mammalian cells, where
they are important in regulating vital cellular functions. Both in vivo and in vitro
experiments have demonstrated striking anticancer (preventive as well as therapeutic)
effects of IP
6
. Inositol also is anti-carcinogenic, albeit to a lesser extent; it acts
synergistically IP
6
in inhibiting cancer. In addition to reduction in cell proliferation, IP
6
increases differentiation of malignant cells often resulting in reversion to the normal
phenotype. IP
6
is quickly absorbed from the rat stomach and upper intestine and
distributed as inositol and IP
1
.In vitro, it is instantaneously taken up by malignant cells
undergoing variable dephosphorylation to inositol and IP
1)5
, pointing towards their role in
mediating the action of IP
6
. In humans, IP
6
has recently been detected in urine, plasma and
other biological fluids; the levels fluctuating with ingestion or deprivation of IP
6
or IP
6
-rich
diet. As IP
6
is high in high-fibre diets, these also may explain, at least in part, the
epidemiological observation showing the association of ingesting high-fibre diets with a
lower incidence of certain cancers. Along with safety, the reproducible efficacy of IP
6
and
inositol in the prevention of cancer in laboratory animals warrant their inclusion in our
strategies for cancer prevention and perhaps therapy in humans. Aside from the anticancer
action, IP
6
and inositol also have numerous other health benefits. All these facts of normal
physiological presence of IP
6
in our body the level of which fluctuates with intake,
association of an IP
6
-rich diet with low incidence of several diseases and vice versa, and
finally reversal of some of these conditions, at least in part, by IP
6
supplementation
strongly argue in favour of its inclusion as an essential nutrient or perhaps a vitamin.
Keywords Chemoprevention, differentiation, inositol hexaphosphate, IP
6
.
Introduction
myo-Inositol hexaphosphate (InsP
6
or IP
6
)isa
simple ringed carbohydrate with six phosphate
groups attached to each carbon. In a pH range of
0.5–9.0, it adopts the sterically stable 1ax/5eq (one
phosphate at carbon position 2 in the axial
position and five phosphates in the equatorial
position) and sterically hindered 5ax/1eq confor-
mation over pH 9.5 (Barrientos & Murthy, 1996).
IP
6
is contained in substantial amounts in cereals
and legumes (0.4–6.4%), primarily existing as a
form of salt with monovalent and divalent cations,
e.g. Ca
2+
,Mg
2+
and K
+
(Harland & Oberleas,
1987). Since its discovery in the mid-nineteenth
century, the popularity of IP
6
stemmed mostly
from the fact that it is the chief storage form of
phosphorous for the germinating seeds. Interest in
IP
6
in the mid-1980s was rekindled partly due to
its antioxidant function as a result of its ability to
chelate divalent cations. This property however,
has been marred by concerns expressed during the
past half-a-century about an alleged mineral defi-
ciency that resulted from intake of foods high in
IP
6
. It was, and unfortunately to some extent,
thought that IP
6
reduces the bioavailability of diet-
ary minerals; hence its infamy as an anti-nutrient,
Correspondent: Fax: +1 410 706 8414;
e-mail: research@ip-6.net
International Journal of Food Science and Technology 2002, 37, 769–782 769
2002 Blackwell Science Ltd
notwithstanding the reports to the contrary that
even long-term intake of IP
6
in food (Walker
et al., 1948; Cullumbine et al., 1950) or in pure
form (Henneman et al., 1958) did not cause such a
deficiency in humans.
Biochemists and cell biologists on the other
hand, have been interested in the phosphorylation
and dephosphorylation of IP
6
, and how this might
affect cellular functions. Lower inositol phosphates
(IP
1)4
) are recognized as intracellular messengers.
The second messenger role of inositol 1,4,5-tris-
phosphate [Ins(1,4,5)P
3
] in bringing about a host of
cellular functions including mitosis via mobilizing
intracellular Ca
2+
is well recognized. Its cousin–
inositol 1,3,4,5-tetrakisphosphate (IP
4
), has also
been shown to induce Ca
2+
sequestration. Higher
forms of IP, inositol 1,3,4,5,6-pentakisphosphate
(IP
5
) and inositol hexakisphosphate (IP
6
) are also
abundant and represent the bulk of IP content of
mammalian cells. IP
5&6
are present in virtually all
mammalian cells in substantial amounts, between
10 and 100 lm(Szwergold et al., 1987), much
higher than any other IP. Why should there be an
intracellular abundance of these compounds which
were presumed toxic by nutritionists or inert at best
by biochemists (Menniti et al., 1993)? Moreover,
we know very little of their purpose. Recent studies
provide an increased understanding of the func-
tional roles of IP
5&6
. Physiological functions of IP
5
include regulation of the affinity of avian haemo-
globin for oxygen, and along with IP
6
it may be
involved in neuronal excitation (Menniti et al.,
1993). Recent demonstrations that IP
5&6
are
precursors of several derivatives that turnover
rapidly suggest that these forms of IP are not inert
or metabolically lethargicand that they play a
more dynamic role has been previously appreciated
(Menniti et al., 1993). As described below, one
reason why IP
6
has been receiving increased
attention is its anticancer action.
As IP
6
undergoes dephosphorylation to IP
1–5
and IP
3
is central in cellular signal transduction
and intracellular function, I hypothesized that IP
6
exerts its anticancer function through lower inos-
itol phosphates by entering into the intracellular
IP pool (Shamsuddin, 1992; Shamsuddin et al.,
1988, 1989). It was also hypothesized that the
addition of inositol (Ins), a precursor of IP
and also an innocuous natural carbohydrate, to
IP
6
may enhance the anticancer function of IP
6
(Shamsuddin, 1992; Shamsuddin et al., 1989).
Furthermore, because inositol phosphates are
ubiquitous and are common molecules involved
in signal transduction in most mammalian cell
systems, I further hypothesized that the anticancer
action of inositol phosphates would be observed
in different cells and tissue systems (Shamsuddin,
1992; Shamsuddin et al., 1992).
Cancer chemoprevention
Because there were no prior data on the effect of
IP
6
on cancer, my first experiment was essentially
ashot in the dark. Although experts in the field of
nutrition and cancer would like such an experi-
ment to begin by adding IP
6
to the diet, conven-
tional wisdom from pharmacology suggested that
the administration of IP
6
solution via drinking
water would be the preferred one. Pilot studies to
test the palatability of the Na-IP
6
solution quickly
revealed that rats only drink it up to a concentra-
tion of 5% (by the way, I tasted it prior to giving it
to the rats, as it didn’t taste bad to me, I thought
that the rats wouldn’t mind!). However, it ap-
peared that a solution of 1–2% Na-IP
6
was the
most desirable, beyond which their water intake
was so reduced that higher levels would have been
self-defeating. Furthermore, it is well known that
IP
6
readily binds with proteins and other compo-
nents of the diet, rendering itself less readily
available for absorption. So our experiments were
designed to test the efficacies at a maximum dose
of 2% in drinking water. Different species (rats
and mice) and carcinogens (1,2-dimethylhydra-
zine, azoxymethane, dimethylbenzan-thracene)
were used to examine its effectiveness across
species and agents. In the colon cancer model,
Na-IP
6
treatment was begun 1–2 weeks prior to
the beginning of carcinogen administration (pre-
initiation phases) to (i) give the treatment an
advantage of time and (ii) to see if treatment
would directly or indirectly nullify the carcino-
genic action (chemically). Six months after the
beginning of the experiment, animals receiving IP
6
had fewer neoplasms in their colon than the
control animals which were not treated with IP
6
,
and the tumours were approximately two-thirds
smaller (Table 1). Early during this experiment I
also observed that the rate of cell division in the
non-tumorous colonic epithelium of IP
6
-treated
Anti-cancer function of phytic acid A. M. Shamsuddin770
International Journal of Food Science and Technology 2002, 37, 769–782 2002 Blackwell Science Ltd
animals was similar to that of the normal control
animals; in other words, the carcinogen-induced
increase in mitotic rate was normalized, confirm-
ing my hypothesis that the anticancer action of IP
6
lies in controlling cell division. Most interestingly,
the mitotic rate of the animals receiving IP
6
treatment but not carcinogen remained at normal
rate (Shamsuddin et al., 1988, 1989; Shamsuddin &
Ullah, 1989; Ullah & Shamsuddin, 1990). An
interesting point of note is that adding a much
higher amount of IP
6
to the diet, Jariwalla et al.
(1988) in a rat fibrosarcoma model have reported
similar results; this points to the earlier argument I
made for not using IP
6
mixed in food which
renders it less efficient.
Subsequently my co-workers and I showed that
the inhibition of large intestinal cancer was not
only dose-dependent but also related to the pH of
the solution; Na-IP
6
solution has a pH of approxi-
mately 11, neutralization gave better results (Ullah
& Shamsuddin, 1990).
The lack of a dramatic decrease in cancer
incidence implied that IP
6
was not a direct
antagonist to the carcinogen. Thus, coupled with
the observation that the tumour size in IP
6
-treated
animals was smaller, these facts led me to believe
that IP
6
might be effective even after the beginning
of cancer induction (post-initiation phase). Con-
sequently, IP
6
was administered to the drinking
water of rats as early as 2 weeks or as late as
5 months following carcinogen. Eight months
after four doses of the carcinogen azoxymethane
(8 mg kg
)1
), only 10% of the animals on IP
6
developed colon cancer compared with 43% in
the control group. Animals showed significantly
lower tumour number and size even when given
IP
6
5 months after initiation, a time when most
of the animals are expected to have cancers
(Shamsuddin & Ullah, 1989) (Table 2). These
findings pointed towards its possible therapeutic
use (vide infra).
To test the hypothesis that the anti-tumour
action of IP
6
may be mediated via lower phos-
phorylated forms of inositol – IP, which are
important in cell division, and that the addition
of inositol may enhance the anti-proliferative and
thus the anti-cancer action of IP
6
, additional
experiments were conducted. It was indeed noted
that inositol potentiated both the anti-proliferative
and anti-neoplastic effect of IP
6
in vivo. A signi-
ficantly greater suppression of both cell prolifer-
ation and colorectal cancer was noted when
inositol was added to IP
6
(Shamsuddin et al.,
1989). Similar potentiation was seen in the mam-
mary and metastatic tumour models (Vucenik
et al., 1992, 1993).
Inositol
As regards the relationship between inositol and
IP
6
, the phrase chicken or the eggcould best
describe it. Inositol deficiency within cells in
patients with diabetes is implicated in the devel-
opment of various complications of diabetes, such
as altered sensations (peripheral neuropathy),
cataract and retinal damage, early derangements
of kidney functions (diabetic nephropathy), etc. In
experimentally induced diabetes mellitus, free
inositol level in the peripheral nerve is reduced
which correlates with a decreased motor nerve
Table 1 Colon tumour inhibition by IP
6
after high-dose (twelve injections) of carcinogen
Treatments No. of tumour/rat Tumour volume (cm
3
)Percentage of cells with mitosis
AOM 4.6 ± 0.61* 1.8 ± 0.74 1.71 ± 0.14
AOM + IP
6
3.0 ± 0.42* 0.68 ± 0.19 0.87 ± 0.09
†
Control None 0 0.20 ± 0.05
*Compared with the carcinogen-only treatment, the reduction in the number of tumours/rat by IP
6
treatment is statistically
significant at P< 0.01.
† The reduction in the rate of mitosis by IP
6
treatment is statistically significant at P< 0.001.
‡ A group of rats were given 1% IP
6
without any carcinogen to see the effect of IP
6
alone. The rate of mitosis in these rats were the
same as the negative control group (rats on regular tap water without carcinogen or IP
6
treatment) shown here suggesting that IP
6
simply brings down the elevated rate of cell division during cancer formation, but does not affect the normal rate in otherwise
healthy animals.
Anti-cancer function of phytic acid A. M. Shamsuddin 771
2002 Blackwell Science Ltd International Journal of Food Science and Technology 2002, 37, 769–782
conduction velocity. Inositol, ranging from 0.5 g
twice a day to 3 g has been given to patients with
diabetic neuropathy; results to date support that
oral supplementation of inositol may be of benefit
in the prevention and treatment of neural compli-
cations of diabetes mellitus (Holub, 1986).
Several psychiatric diseases have been treated
with inositol. These include clinical depression,
panic disorder and obsessive compulsive disorder.
When twenty-eight depressed patients were given
12 g of inositol a day, a statistically significant
overall benefit was found for inositol treatment as
compared with the placebo control as early as
week 4 on the Hamilton Depression Scale, the
standard measure for assessing the effectiveness of
an antidepressant substance (Levine, 1997). Pati-
ents with obsessive compulsive disorder were
treated with 18 g of inositol or placebo per day
for 6 weeks in a double-blind controlled crossover
trial. Here too, inositol reduced the scored (Yale-
Brown Obsessive Compulsive Scale) symptoms of
obsessive compulsive disorder with good statistical
significance when compared with the placebo
control.
Anti-cancer action of inositol
My colleagues and I were also the first to
demonstrate that myo-inositol alone or in combi-
nation with IP
6
can prevent the formation and
incidence of several cancers in experimental ani-
mals in soft tissue, colon, metastatic lung cancer
and mammary cancers (Shamsuddin et al., 1989;
Vucenik et al., 1992, 1995, 1997). Dr Hecht and
Dr Wattenberg and their colleagues have shown
that inositol prevents carcinogen-induced lung
tumour formation in mouse models (Estensen &
Wattenberg, 1993; Wattenberg & Estensen, 1996;
Hecht et al., 1999).
Combined IP
6
+ inositol
At the beginning I alluded to my hypothesis that
since IP
6
undergoes dephosphorylation to IP
1)5
and IP
3
is central in cellular signal transduction
and other functions such as cell division, IP
6
could
enter into the intracellular inositol phosphate pool
and cause tumour suppression through lower
inositol phosphates such as IP
3
. I had also
hypothesized that inositol could also become
phosphorylated to the higher inositol phosphates
such as IP
1)6
, but most importantly IP
3
.
Thus addition of inositol to IP
6
and adminis-
tering the cocktail as therapeutic agent would
allow the nascently released phosphates from IP
6
to be captured by the inositol that is pretty much
standing by. Therefore, delivering IP
6
+ inositol
may cause increased availability of lower inositol
phosphates, most importantly IP
3
as per the
following reaction:
IP6þinositol 2IP
3
If indeed IP
3
is involved in the transmission of
signals from the growth factors in the cell’s
exterior to the nucleus and cell division, too much
of this signal may somehow cause a negative
feedback and therefore shut down cell prolifer-
ation and growth. Furthermore, because inositol
phosphates are ubiquitous and are common mol-
ecules involved in signal transduction in most
mammalian cell systems, I also hypothesized that
the anticancer action of inositol phosphates would
be observed in different cells and tissue systems.
The anticancer function of IP
6
against various
tumours was already discussed; the following are
examples of how the combination treatment is also
better than inositol or IP
6
in different cancers.
Table 3 shows the results from the colon cancer
study (Shamsuddin et al., 1989); similar results
Table 2 Colon cancer inhibition after 5 months
Treatments No. of tumour/rat Tumour volume (mm
3
)Mitotic rate (%)
AOM only 7.1 ± 0.6 570 ± 110 2.3 ± 0.2
AOM + IP
6
5.2 ± 0.6 200 ± 60 1.0 ± 0.1
Significance* P< 0.02 P< 0.01 P< 0.001
*The reduction in the number of tumour/rat, tumour size and the rate of cell division by IP
6
treatment are statistically significant at
the Pvalues given for each parameter. Note that IP
6
treatment has resulted in two-thirds smaller tumours than the untreated
controls.
Anti-cancer function of phytic acid A. M. Shamsuddin772
International Journal of Food Science and Technology 2002, 37, 769–782 2002 Blackwell Science Ltd
were seen in the mammary cancer (Vucenik et al.,
1992, 1995, 1997) and metastatic lung cancer
(Vucenik et al., 1992) models.
It is of interest to note that the response of
various cancer models to either IP
6
or inositol vary
depending on the parameters tested. For instance
in the mammary tumour model, while IP
6
caused a
reduced incidence of tumours, the tumour size was
larger than the control! But when all parameters of
tumour assessments are taken the most consistent
anticancer results were obtained from the combi-
nation of IP
6
+ inositol.
Cancer therapy
The fact that colorectal cancer inhibition was
observed when IP
6
treatment was begun as late as
5 months following initiation suggested to us that
the beneficial action of IP
6
was not restricted to the
prevention of tumour development, but perhaps
treatment of existing cancers as well. That IP
6
normalizes cell division rate provides additional
rationale for such experiments; inasmuch as cellu-
lar proliferation is integral to fully developed
cancers as it is to early lesions. Thus, studies in
my laboratory were extended to test the therapeutic
properties of IP
6
in a mouse transplantable tumour
model (Vucenik et al., 1992). Daily treatment of
syngeneic host mice with intraperitoneal injections
0.25% Na-IP
6
(1 mL every other day) following
subcutaneous inoculation of mouse fibrosarcoma
FSA-1 cells, resulted in both a significant inhibition
of tumour size and improvement of survival over
the untreated controls. Intravenous injection of
FSA-1 cells developed experimental lung metasta-
sis, and similar treatment of host mice with IP
6
resulted in a significant reduction in the number of
metastatic lung colonies (Vucenik et al., 1992).
Effect on cancer cell lines
In vitro studies of both human and rodent cancer
cell lines in my laboratory demonstrate that IP
6
reduces cell proliferation rate in all of the cell lines
tested, including MCF-7 human mammary carci-
noma cells. Interestingly, unlike most other anti-
cancer agents, the cells do not show an
overwhelming evidence of cytotoxicity; rather,
the growth of malignant cells slows and they
mature and die. The reduced cell growth and
enhanced differentiation of cancer cells to the
point of reversion back to normal phenotype is
seen in different cell lines. For instance K-562
human erythroleukaemia cells are relatively small
compared with their normal counterpart, the
erythrocytes; and they are devoid of haemoglobin.
A striking growth inhibition can be achieved with
6–10 mmol L
)1
of Na-IP
6
and few cells remain
viable after 48 h. However, at lower concentra-
tions (e.g. 75 lmol L
)1
), the cells can be maintained
for a longer period, albeit at reduced numbers
wherein the cells become large and accumulate
haemoglobin, akin to the more mature cells
(Shamsuddin et al., 1992).
Reversion of malignant to normal phenotype
has also been observed in the HT-29 human colon
carcinoma cell line. The disaccharide tumour mar-
ker b-d-galactose-[1fi3]-N-acetyl-galactosamine is
expressed by malignant and premalignant cells of
the colon and other epithelia, but not by the
Table 3 Synergistic cancer inhibition by IP
6
when combined with inositol
Experimental group Tumour prevalence Tumour frequency*Mitotic rate (%)
DMH 63%
†
22/19 1.92 ± 0.17
12/19 (1.16)
DMH + IP
6
47%
13/21 1.48 ± 0.15
10/21 (0.62)
DMH + inositol 30% 9/20 1.01 ± 0.14
6/20 (0.45)
DMH + IP
6
+ inositol 25% 4/16 1.06 ± 0.13
4/16
§
(0.25)
*Tumour frequency is represented as number of tumours (gross + microscopic cancers) per mouse.
†
The difference in tumour
prevalence between DMH-only (carcinogen control group) and DMH + IP
6
+ inositol§is significant at P< 0.001, and between
DMH + IP
6
and
§
DMH + IP
6
+ inositol at P< 0.005. Thus the combination of IP
6
+ inositol treatment is significantly better than
either one alone.
Anti-cancer function of phytic acid A. M. Shamsuddin 773
2002 Blackwell Science Ltd International Journal of Food Science and Technology 2002, 37, 769–782
normal cells. Following IP
6
treatment, along with
a decreased rate of cell proliferation, tumour
marker expression is markedly suppressed. In
most cells no expression whatsoever could be seen
although the cells produced the parent mucopoly-
saccharide (Sakamoto et al., 1993a). Thus while
IP
6
suppresses the malignant phenotype, it never-
theless allows the progression of normal mucin
synthesis and maturation of human colon cancer
cells to structurally and behaviourally resemble
normal cells.
It is to be noted that the doses or the concen-
tration required to achieve IC
50
(50% inhibition of
cell number) varies for the different cell lines.
While the cells of haematopoietic lineage (e.g.
K-562, YAC-1, HL-60, etc.) are highly sensitive to
IP
6
, those of the epithelial and mesenchymal lines
require higher concentrations. Furthermore,
whereas Ins alone has a modest anticancer effect
in vivo (Shamsuddin et al., 1989; Estensen &
Wattenberg, 1993; Vucenik et al., 1993) when
added to IP
6
there is a synergistic effect.
Using BALB/c mouse 3T3 fibroblasts and
midpoint cytotoxicity assay by neutral red method,
Babich et al. (1993) compared the effects of several
chemopreventive agents and demonstrated a mod-
erate efficacy of IP
6
. This modest result is not
surprising because extensive experiments with
human cancer cell lines in vitro in my laboratory
have shown little cytotoxicity.
Tumour abrogation
While in vitro studies using human cancer cell lines
lend support to the data obtained from in vivo
animal models, preparation for a human trial
require further investigations. Towards that goal,
we tested the efficacy of IP
6
against human rhab-
domyosarcoma – a highly malignant tumour of
skeletal muscle origin, common in children and
young adults. We first tested in vitro cell growth
inhibition which not surprisingly was striking as in
all other cell lines. Consistent with in vitro obser-
vation, IP
6
also suppressed the growth of rhabdo-
myosarcoma cells in vivo in a xenografted nude
mice model. NIH athymic male nude mice were
used to determine the effect of IP
6
on the tumour
forming capacity of rhabdomyosarcoma cells.
Each mouse received 10
7
viable rhabdomyosar-
coma cells, injected subcutaneously into the lower
dorsal region. Two days later, IP
6
(40 mg kg
)1
in 0.1 mL PBS) was injected around the tumour,
and was continued every other day for 2 weeks
(expt 1) or for 5 weeks (expt 2). When compared
with controls, IP
6
-treated mice produced 25-fold
smaller tumours (P¼0.008), as observed after a
2-week treatment. In the second experiment,
wherein the treatment period was extended to
4 weeks, a 49-fold (P¼0.001) reduction in
tumour size was observed in mice treated with IP
6
.
Histologically no evidence of tumour cell necrosis
was observed, confirming all the previous studies
that IP
6
does not kill tumour cells (cytostatic,
and non-cytotoxic), yet suppresses the tumour by
25–49-fold (Vucenik et al., 1998a) (Table 4).
As exciting as the results may be, this experi-
ment only showed inhibition of tumour formation
and not reduction of a preformed tumour, as the
injection was begun before a visible tumour has
formed. Therefore the next logical experiment was
to test whether IP
6
could shrink an existing
tumour. Using a similar xenotransplantation
model, this time for liver cancer, we addressed
this question. However, prior to this we tested the
ability of IP
6
to inhibit growth of a human liver
cancer cell line and then tested the effect on
tumour formation and growth of human hepato-
cellular carcinoma HepG2 cell line in a transplan-
ted nude mouse model (Vucenik et al., 1998b,c). A
solid tumour was observed in 71% of nude mice
after subcutaneous injection of HepG2 cells
(1 ·10
7
/mouse) during a period of 18–41 days
following transplantation, but no tumour was
found in the mice which had received the same
number of HepG2 cells pretreated in vitro with
5mmIP
6
for 48 h (P< 0.03). When transplanted
tumours reached 8–10 mm in diameter, an intra-
tumoral injection of IP
6
(40 mg kg
)1
) was given
for 12 consecutive days, after which the animals
Table 4 Reduction of rhabdomyosarcoma tumour volume
(mm
3
)byIP
6
Expt 1
(2 weeks treatment)
Expt 2
(5 weeks treatment)
Controls
(n¼5)
818 ± 377 1666 ± 1122
IP
6
-treated 33 ± 63 34 ± 47
Significance P¼0.008 P¼0.001
Data presented as mean ±standard deviation.
Anti-cancer function of phytic acid A. M. Shamsuddin774
International Journal of Food Science and Technology 2002, 37, 769–782 2002 Blackwell Science Ltd
were killed. At autopsy, the tumour weight in IP
6
-
treated mice was 3.4-fold less than that in con-
trol mice (0.333 ± 0.270 g vs. 1.130 ± 0.423 g,
P¼0.016).
The most intriguing finding in this experiment is
that a single treatment of hepatocellular cancer
cells by IP
6
resulted in the complete loss of
the ability of these cells to form tumour when
inoculated subcutaneously in nude mice (tumori-
genicity); on the other hand untreated cells did.
Moreover, then when the pre-existing liver cancers
themselves were treated directly with IP
6
, they
regressed (Vucenik et al., 1998b,c).
Effect on host defence mechanism
Natural killer (NK) cells play a pivotal role in
various aspects of the host’s defence system. As
NK cells are important in tumour abrogation, my
co-workers and I investigated whether IP
6
medi-
ates its anti-neoplastic action via NK cells. Using
YAC-1 target cells labelled with
51
Cr, the cyto-
toxicity of murine spleen NK cells was measured.
Mice with carcinogen-induced tumours, treated
in vivo with IP
6
, showed augmented NK activity
over the untreated controls and NK activity
correlated with tumour suppression (Baten et al.,
1989). Similar enhancement of NK activity was
also demonstrated when splenocytes from normal
mice were treated with IP
6
in vitro (Baten et al.,
1989).
For bacterial killing by polymorphonuclear
leucocytes (or neutrophils) the stimulation of
respiratory burst associated with free radical
generation is a critical event. Eggleton et al.
(1991) demonstrated that the preincubation of
human polymorphonuclear leucocytes with IP
6
results in substantially enhanced production of
reactive oxygen intermediates following stimula-
tion by phagocytic particles, or chemicals. Johnson
et al. (2000) investigated the effect of IP
6
on the
proliferation and viability of RAW 264.7 trans-
formed macrophages and the role of IP
6
as a free
radical scavenger. Their results suggested that IP
6
may have an excitatory effect on inflammatory cell
secretions. These investigators also demonstrated
a reduction of maliondialdehyde (MDA) by IP
6
indicating its effectiveness as an antioxidant. Thus
IP
6
may enhance host defence via these mecha-
nisms as well.
Mechanisms of action
Because cancer is a major public health issue, the
dramatic anti-cancer effect of IP
6
has resulted in
our quest for understanding its mechanism of
action. As a first step, studies in my laboratory
have demonstrated that contrary to popular mis-
conception,
3
H-IP
6
when intragastrically adminis-
tered to rats is very quickly absorbed from the
stomach and upper small intestine and distributed
to various organs as early as 1 h following admin-
istration (Sakamoto et al., 1993b). The radioactiv-
ity isolated from the gastric epithelium at this time
is associated with inositol and IP
1)6
, and that in the
plasma and urine with inositol and IP
1
indicating a
very rapid metabolism of the compound. The
presence of IP
6
in the gastric epithelium suggests
that the intact molecule is perhaps transported
inside the cell wherein it is rapidly dephosphoryl-
ated. Could IP
6
be dephosphorylated extracellular-
ly and then be absorbed as inositol and IP
1)5
with
some rephosphorylation back to IP
6
intracellular-
ly? This is unlikely, as there is no mucosal phytase
activity in the stomach and we administered IP
6
in
drinking water 2 h following fasting avoiding the
action of dietary phytase. In any event, as earlier
time point studies were not conducted, this study
could not beyond doubt, demonstrate the transport
of intact IP
6
. Transport of intact IP
6
across the cell
membrane is not only a normal process via various
binding proteins to transport it across cell mem-
branes (e.g. clathrin adaptor complex AP2, AP180,
coatomer of COP I coat), but IP
6
may also regulate
vesicle budding and fusion in the membrane.
Indeed, IP
6
itself interacts with AP2 and inhibits
downstream events in the cell.
Our studies of the absorption of IP
6
by malig-
nant cells in vitro also demonstrate that the cells
almost instantaneously begin to accumulate IP
6
intracellularly, the rate of accumulation varying for
the different types of cells (Vucenik & Shamsuddin,
1994). For instance, the uptake of
3
H-IP
6
by mouse
YAC-1 lymphoma cell line reached a plateau as
early as 10 min after incubation. The ability and
the rate at which the cells metabolized IP
6
also
varied; YAC-1 and K-562 cells contained only the
lower IP’s whereas HT-29 human colon carcinoma
cells had inositol and IP
1)6
.
A central pathway of cancer inhibition by IP
6
is
via control of cell division; and IP
6
reduces the
Anti-cancer function of phytic acid A. M. Shamsuddin 775
2002 Blackwell Science Ltd International Journal of Food Science and Technology 2002, 37, 769–782
rate of cellular proliferation both in vivo and
in vitro. Experiments in my laboratory with
3
H-
thymidine incorporation also demonstrated a
reduction in DNA synthesis. It is possible that
IP
6
may exert its control by chelating cations
because the metalloproteins are important in gene
regulations (O’Halloran, 1993). However, as we
used a dodecasodium salt of IP
6
rather than
inositol hexaphosphoric acid in our experiments, it
is difficult to envision how Na
12
-IP
6
would be able
to exert any, much less substantial chelation.
Insofar as the steps of carcinogenesis are con-
cerned, our studies showed the effectiveness of IP
6
both prior to and following initiation in colon
carcinogenesis models. But in a mouse skin
carcinogenesis through the classical initiation fi
promotion model, Ishikawa et al. (1999) found
that IP
6
is effective in the prevention of skin
papillomas when given during the initiation phase,
but not during promotion.
From the earlier discussion, one can surmise
that the cellular mechanism of cancer inhibition is
one of reduction of cell proliferation rate (rather
normalization of mitotic rate) and induction of
cellular differentiation. Further studies in my
laboratory showed a suppression of DNA syn-
thesis as measured by
3
H-thymidine incorpor-
ation and down-regulation of proliferation
marker PCNA by IP
6
(Yang & Shamsuddin,
1995). A marked decrease in the expression of
proliferation markers indicated that IP
6
disen-
gaged cells from active cycling. Using dual
parameter flow cytometry and combined analysis
of the expression of cell cycle-related proteins, El-
Sherbiny et al. (2001) demonstrated that IP
6
controls the progression of the cells through the
cell cycle by significantly decreasing the S-phase
and arresting the cells (human colon and breast
cancer cell lines) in the G
0
/G
1
phase. Studies of
human leukaemia cells at Professor Lambert-
enghi-Deliliers’ laboratory at the University of
Milan demonstrate that not only does IP
6
show a
dose-dependent cytotoxic effect on human
leukaemia cell lines, but also the IP
6
-treated
leukaemia cells accumulate in G
2
M phase of cell
cycle, once again arresting cells in the cycle, albeit
in a different phase (Lambertenghi-Deliliers et al.,
2002). cDNA micro-array analysis showed an
extensive down-modulation of genes involved in
transcription and cell-cycle regulation (c-myc,
HPTPCAAX1, FUSE, cyclin H) and an
up-regulation of cell cycle inhibitors such as
CKS2, p57 and Id-2. Genes such as STAT-6
and MAPKAP, involved in important signal
transduction pathways were also down regulated.
Besides the above cellular mechanism, the
following biochemical pathways may also be
operational for the various functions of IP
6
:
Chelation of Fe
3+
and suppression
of
Æ
OH formation
In the plant kingdom IP
6
functions as an anti-
oxidant, protecting and preserving the seeds,
which may remain viable for a long time. The
1,2,3 (equatorial–axial–equatorial) phosphate
grouping in IP
6
has a conformation that uniquely
provides a specific interaction with iron to com-
pletely inhibit its ability to catalyze hydroxyl
radical formation. Protection against cancer and
a multitude of other applications may be based at
least in part on this antioxidant function.
The anti-oxidative function of IP
6
, occurs by
chelating iron by occupying all the available Fe co-
ordination sites thus inhibiting
•
OH generation
from the Fenton reaction and the subsequent lipid
peroxidation and DNA damage. IP
6
has the
unique ability to remove O
2
without generating
oxy-radicals. Thus it inhibits
•
OH production by
chelating iron in the presence of O
2
,O
2
•–
, or any
reducing agent; can maintain the redox potential
of iron by accelerating both reduction of Fe
3+
by
ascorbic acid (AH
2
) and oxidation of Fe
3+
by O
2
through following reaction:
Fe3þþAH2!
IP6
O2;eFe2þþAþH2O2
4Fe2þþO2þ4HþIP6!4Fe3þþ2H2O
Therefore, IP
6
could reduce the active oxygen
species-mediated carcinogenesis and cell injury via
its anti-oxidative function (Graf & Eaton, 1990).
Chelation of divalent cation
Mg
2+
has been implicated in the second messen-
ger system within the cell. Thymidine kinase, an
enzyme essential for DNA synthesis and cell
division, is sensitive to zinc depletion. Within
6 days of zinc depletion, there is a decrease in
Anti-cancer function of phytic acid A. M. Shamsuddin776
International Journal of Food Science and Technology 2002, 37, 769–782 2002 Blackwell Science Ltd
thymidine kinase activity and thymidine incorpor-
ation into DNA. On the other hand the activity
of ribonuclease increases with zinc deficiency
(Harland & Oberleas, 1987). Inasmuch as Mg
2+
and Zn
2+
are essential for cell proliferation
including that in tumours, deprivation of these
cations may cause a decrease in tumour growth.
Jariwalla et al. (1988) and Thompson & Zhang
(1991) favour the theory that IP
6
brings about its
anticancer effect by removing these cations. Our
pilot experiments using
45
Ca in cell culture
medium, done by G.-Y. Yang and myself (unpub-
lished observation, 1993) showed that Na-IP
6
did
not significantly alter the availability of divalent
minerals in vitro. This is not totally unexpected
considering the fact that we used salts of IP
6
for all
our experiments, both in vitro and in vivo. Thus the
proposed mechanism remains inexplicable at the
moment and additional experiments are needed to
understand this. Furthermore, the observed rapid
dephosphorylation of IP
6
by both normal rats
in vivo and malignant cells in vitro to lower IPs,
particularly IP
1)3
, make the above two hypothesis
less credible; they are both based on the ability of
six phosphate groups to chelate divalent cations.
Finally, that tumour inhibition and even abroga-
tion by IP
6
was observed long after carcinogenic
stimuli suggest that other mechanisms are involved.
Participation in intracellular inositol
phosphate pool
In support of the hypothesis that the anti-tumour
action of IP
6
is mediated via lower IPs, my
co-workers and I have demonstrated that following
IP
6
treatment of K-562 human erythroleukaemia
cells, there is a 41% increase (P< 0.05) in intra-
cellular IP
3
and a 26% (P< 0.02) decrease in IP
2
(Shamsuddin et al., 1992). The fact that these
measurements were done by administration of
3
H-Ins and ion exchange chromatography of the
IP and measuring the radioactivity, indicates that
conversion of Ins to IP
1)3
does take place, and that
this occurs as early as within 1 h. The data from in
vivo and in vitro experiments showing absorption of
IP
6
described earlier point to its rapid conversion
to Ins and IP
1)5
. Thus it is clear that administration
of IP
6
results in alterations in the cellular inositol
phosphate pool. What specifically brings about the
observed biological effects are yet to be elucidated.
A pilot study demonstrated that 3 h following
0.05% IP
6
treatment, intracellular Ca
2+
increases
by 57% (P< 0.02) (Shamsuddin et al., 1992).
While the current dogma is that an increased
intracellular Ca
2+
concentration secondary to an
increase in IP
3
may be responsible for mitosis, our
data on the other hand corresponds to a decrease in
cell division. Such a paradoxical relationship
between increased intracellular Ca
2+
and biologi-
cal events is not unique to our study and has also
been observed by others. Further studies of time-
dependent changes in intracellular Ca
2+
concen-
tration are warranted. Because inositol can be
converted to IP
1)3
and perhaps IP
4)6
, it is quite
plausible that the observed anticancer effect of
inositol given alone is perhaps owing to its
conversion to metabolically active IP’s.
Cellular and nuclear signalling pathways
Studies by several investigators are beginning to
lead us to an understanding that IP
6
may be
involved in the pathways involved in cell signal-
ling. There are other ways by which IP
6
could
influence the various activities within the cells.
For instance, repair of double-strand breaks in
DNA is essential for maintaining the stability of
the genome, failure to repair may result in loss
of genetic information, chromosomal transloca-
tion and even cell death. Two mechanisms for
this repair have been described – homologous
recombination or non-homologous end-joining.
IP
6
has been demonstrated to stimulate non-
homologous end-joining; it has been proposed to
be brought about by the binding of IP
6
to the
DNA-dependent protein kinase DNA-PK
cs
(Hanakahi et al., 2000). A more recent study
reported that it is not DNA-PK
cs
(a large protein
of 3500 amino acids, M
w
465 kDa), but the
DNA end binding protein Ku (consists of Ku70 –
70 kDa, and Ku86 – 83 kDa) that binds to IP
6
(Ma & Lieber, 2002). Be that as it may, these
studies, in spite of differences in their specific
findings, clearly show a very important role of IP
6
in DNA repair mechanism.
Once the assault on the cell has gone past the
scope of DNA repair, the otherwise normal cell is
likely to transform to a malignant (cancer) cell.
Insofar as the transformation of cells from normal
to malignancy is concerned, there are various
Anti-cancer function of phytic acid A. M. Shamsuddin 777
2002 Blackwell Science Ltd International Journal of Food Science and Technology 2002, 37, 769–782
models and pathways, one of these pathways is the
activation of transcription factors activating pro-
tein-1 (AP-1) and nuclear factor NFjB via
phosphotidylinositol 3-kinase (PI-3 kinase). Using
tumour promoter-induced cell transformation of
human skin JB6 cells, Huang et al. (1997) have
demonstrated that IP
6
blocks epidermal growth
factor-induced PI-3 kinase and AP-1 activity. Zi
et al. (2000) demonstrated similar results on
DU145 human prostate cancer cells, along with
a concomitant inhibition of cell growth. Upstream
of these pathways lies the mitogen-activated pro-
tein kinases (MAPK) which are serine/threonine
kinases that are rapidly activated upon extracel-
lular stimulation. This family of kinases include
Erks (extracellular signal-regulated kinases, JNKs
(c-Jun N-terminal kinases) and p38 kinases. IP
6
inhibited the activities of Erks and JNKs, but not
of the p38 kinases in human skin, prostate and
breast cancer cells (Huang et al., 1997; Vucenik
et al., 1999a; Zi et al., 2000; Chen et al., 2001).
Thus, given the commonality shared by these three
divergent cell types, the blocking of this cellular to
nuclear signalling pathway appears as an import-
ant mechanism of anticancer action of IP
6
.
IP
6
controls nuclear export of mRNA
The transcription of DNA to messenger RNA
(mRNA) is carried out within the nucleus, being
segregated from the cytoplasm by the nuclear
envelope; the mRNA is then transported by a
complex series of events through pores into the
cytoplasm. York et al. (1999) demonstrated that
the enzyme phospholipase C and two proteins that
influence the generation of IP
6
are required for
proper and efficient export of mRNA from the
nucleus to the cell.
The enhancing effect of IP
6
on NK cells, already
mentioned, could be an additional mode of its
anti-neoplastic action. As NK-cells play an import-
ant role in host defence against neoplasia, it is
quite possible that the contribution of IP
6
via
boosting NK-cell cytotoxicity may be important.
Other biological effects and applications
of IP
6
There is evidence that it plays important roles in
various other conditions and holds promise for use
therein.
Nearly half a century ago, Henneman et al.
(1958) successfully used pure Na-IP
6
to treat the
idiopathic hypercalciuria which is associated with
a high frequency of kidney stones. A diet contain-
ing high IP
6
has also been used to treat hypercal-
ciuria and kidney stones (Ohkawa et al., 1984). IP
6
has a hypocholesterolemic effect and may find
potential use in the clinical management of hyp-
erlipidaemia and diabetes (Jariwalla et al., 1990).
A strong in vitro anticoagulant activity of Na-IP
6
has been demonstrated in the blood of various
animals (Borgo, 1983). Additionally, agonist-
induced platelet aggregation is markedly inhibited
by IP
6
(Vucenik et al., 1999b). Moreover, admin-
istration of IP
6
efficiently protects the myocardium
from ischaemic damage and reperfusion injury
(Rao et al., 1991).
Finally, Otake et al. (1989) demonstrated that
IP
6
inhibited the cytopathic effect of human
immunodeficiency virus (HIV) and HIV-specific
antigen expression in MT-4 cells; inositol hexasul-
phate showing an even stronger inhibition. Cou-
pled with the facts that IP
6
enhances NK-cell
activity and polymorphonuclear cell priming func-
tion, it is possible that IP
6
may have uses in the
management of HIV infection and associated
immunodeficiency-related problems.
Safety
For quite some time now, IP
6
has been blamed,
albeit wrongly (as discussed below) for causing
mineral, particularly Ca
2+
deficiency, hence it
had been castigated as an anti-nutrient. As Dr
June Kelsay wrote in her critical review (Kelsay,
1987), unfortunately much of that stemmed from
studies done on 2–6 people. In addition to the
very small sample size, many of those studies
suffered from poor study design; for instance, the
only two subjects in the study by Walker et al.
(1948) were consuming a lower than recommended
amount of Ca
2+
in the diet during the study
period. The three subjects studied by Cullumbine
et al. (1950) were also on a low Ca
2+
diet who
developed a negative balance during the first few
weeks, but the balance reversed to a positive one
after 8–9 weeks on the same diet! There were also
studies (see review by Kelsay, 1987) that indi-
cated that if mineral intakes are adequate and
cereal or bran intakes are held at a moderate
Anti-cancer function of phytic acid A. M. Shamsuddin778
International Journal of Food Science and Technology 2002, 37, 769–782 2002 Blackwell Science Ltd
level, there are no adverse effects on mineral
bioavailability. Ohkawa et al. (1984) did a fol-
low-up study of subjects ingesting 10 g rice bran
(containing high IP
6
) for up to 2 years and
reported no decrease, significant or otherwise, in
serum Ca
2+
,PO
4
3–
,Mg
2+
and uric acid levels.
Kelsay (1987) proposed that oxalic acid (rich in
spinach and rhubarb) in the diet complicated the
interpretation of results in studies of fruits and
vegetables; there seems to be an interaction of
fibre and oxalic acid resulting in increased faecal
excretion of some minerals, and hence decreased
mineral balances.
Direct studies of the effects of IP
6
should
resolve this lingering and rather vexing argument,
for administration of the isolated pure compound
enjoys the benefit of non-confounding varia-
bles. Indeed those studies both in experimental
animals and in humans (some done nearly half a
century ago) have conclusively refuted the negat-
ive mineral balance stigma. Studies in my
laboratory on experimental animals showed no
significant toxic effect on body weight, serum
mineral content or any pathological changes of
consequence in either male F344 or female
Sprague–Dawley rats for 40 weeks (Ullah &
Shamsuddin, 1990; Vucenik et al., 1993). Studies
by Grases et al. (1998) not only confirmed our
findings, but also report that abnormal calcifica-
tion is prevented in rats given IP
6
.
As regards toxicity and its direct relevance to
humans, of particular note is that Henneman
et al. (1958) administered pure Na-IP
6
orally to
thirty-five patients at a dose of 8.8 g day
)1
(in
divided doses) for many months. Not only did
they reduce the episodes of urinary stones in these
patients, a long-term follow-up study of ten
patients (average of 24 months) showed no unto-
ward toxicity; the reduction in hypercalciuria and
prevention of stone recurrence were rather the
benefits.
Epidemiological correlates
There are very few, if any, well controlled epide-
miological studies of the relationship of diet
containing high IP
6
content and cancer incidence.
However, the consistent negative correlation
between those high-fibre diets rich in IP
6
, and
cancers of the colon, breast and perhaps prostate
are interesting (Englyst et al., 1982; Yu et al.,
1991; Willett, 1994). As breast cancer incidence is
significantly lower in Hispanic and black women
than in non-Hispanic White, Zang et al. (1994)
studied the dietary pattern of these three groups
and showed that beans (in Hispanics), and fruits
and vegetables (in Blacks) may be responsible for
the lower incidence of mammary cancer. You may
recall that beans are rather rich in IP
6
.Yuet al.
(1991) have demonstrated that compared with
Chinese in Shanghai, Americans have fourfold
higher age-adjusted rates of colon cancer and
a twofold higher rates of rectal cancer. The rates
of prostate and postmenopausal breast cancer
were 26-fold and 10-fold higher in Americans;
corresponding to a 2.6-fold lower cereal consump-
tion in the USA. A negative correlation between
the incidence of breast cancer and cereal and fibre
consumption was also reported by Morales &
Llopis (1992).
A vitamin?!
Much of our knowledge of inositol phosphates
and their metabolism comes from studies using
radio-labelled inositol. Conversion of inositol to
IP
6
has been demonstrated in mammalian cells in
vitro (Larsson et al., 1997). However, to demon-
strate such a conversion in humans in vivo is
not permissible nowadays. Enter Professor Felix
Grases of the University of Balearic Islands in
Palma de Majorca: using a novel method of gas
chromatography – mass detection analysis of high
pressure liquid chromatography (HPLC) frac-
tions, Grases et al. (2000, 2001a) were not only
the first to identify IP
6
in human urine and
plasma, but also that the levels fluctuate depend-
ing on the intake of IP
6
by the subjects. Healthy
human volunteers become deficient in IP
6
(as
demonstrated by a low plasma level of IP
6
)in
about 2 weeks. It takes about the same time
period (2 weeks) for the plasma and urinary IP
6
levels to return to normal once the subjects are on
an IP
6
-rich diet; but it takes only 4 h if the subjects
take the pure IP
6
as a supplement (Grases et al.,
2001a). Grases et al. (2001b, 2002) were the first to
detect IP
6
and its lower phosphorylated forms
(IP
3)5
) in mammalian cells and body fluids as they
occur naturally, again demonstrating changes in
the IP
6
and IP
5
levels that depended on dietary
Anti-cancer function of phytic acid A. M. Shamsuddin 779
2002 Blackwell Science Ltd International Journal of Food Science and Technology 2002, 37, 769–782
intake of IP
6
. These data, taken together with the
above mentioned health benefits, argue most
strongly in favour of inclusion of IP
6
as an
essential nutrient (perhaps a vitamin) and shed-
ding its bad name as an anti-nutrient.
Final comments
The preceding discussion has highlighted the many
potential beneficial actions of IP
6
. The mechan-
ism(s) of how IP
6
works need to be elucidated, but
it seems unlikely that all these divergent functions
are mediated through a single pathway. Given the
numerous health benefits, its participation in im-
portant intracellular biochemical pathways, nor-
mal physiological presence in our cells, tissues,
plasma, urine, etc., the levels of which fluctuate
with intake, epidemiological correlates of defici-
ency with disease and reversal of those conditions
by adequate intake, and safety – all strongly
suggest for its inclusion as an essential nutrient,
or perhaps a vitamin. Meanwhile, inclusion of
IP
6
+ inositol in our strategies for prevention and
therapy of various ailments, cancer in particular is
warranted. Of course, eating a healthy diet rich in
IP
6
would always be a prudent thing too.
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