Key Scientific Papers

_Effect of inositol hexaphosphate (IP6) on human normal and leukaemic haematopoietic cells. British J Haematology, 2002, 117, P: 577-587 GL Deliliers, F Servida, NS Fracchiolla, C Ricci, C Borsotti, G Colombo and D Soligo.

_Inositol hexaphosphate (IP6), a naturally polyphosphorylated carbohydrate, has been reported to have significant in vivo and in vitro anticancer activity against numerous tumours, such as colon, prostate, breast, liver and rhabdomyosarcomas. To confirm this activity in haematological malignancies and to characterize some of the mechanisms of IP6 action, we analysed its effects on human leukaemic cell lines and fresh chronic myelogenous leukaemia (CML) progenitor cells using a combined cellular and molecular approach. IP6 has a dose-dependent cytotoxic effect on all of the evaluated cell lines, with accumulation in the G2M phase in two out of five cell lines tested. At the molecular level, cDNA microarray analysis after IP6 exposure showed an extensive downmodulation of genes involved in transcription and cell cycle regulation and a coherent upregulation of cell cycle inhibitors. Furthermore, IP6 treatment of fresh leukaemic samples of bone marrow CD34+ CML progenitor cells significantly inhibited granulocyte-macrophage colony-forming unit (CFU-GM) formation (P=0.0062) in comparison to normal bone marrow specimens, which were not affected. No differentiating effect on HL60 cells was observed. Taken together, our results confirm the antiproliferative activity of IP6 and suggest that it may have a specific antitumour effect also in chronic myeloid leukaemias, via active gene modulation.


Inositol hexaphosphate inhibits growth, and induces G1 arrest and apoptotic death of prostate carcinoma DU145 cells: modulation of CDKI-CDK-cyclin and pRb-related protein-E2F complexes. Singh RP, Agarwal C, Agarwal R. Carcinogenesis 2003 Mar;24(3):555-63

_Cancer chemopreventive effects of inositol hexaphosphate (IP6), a dietary constituent, have been demonstrated against a variety of experimental tumors, however, limited studies have been done against prostate cancer (PCA), and molecular mechanisms are not well defined. In the present study, we investigated the growth inhibitory effect and associated mechanisms of IP6 in advanced human PCA cells. Advanced human prostate carcinoma DU145 cells were used to study the anticancer effect of IP6. Flow cytometric analysis was performed for cell cycle progression and apoptosis studies. Western immunoblotting, immunoprecipitation and kinase assay were performed to investigate the involvement of G1 cell cycle regulators and their interplay, and end point markers of apoptosis. A significant dose- as well as time-dependent growth inhibition was observed in IP6-treated cells, which was associated with an increase in G1 arrest. IP6 strongly increased the expression of CDKIs (cyclin-dependent kinase inhibitors), Cip1/p21 and Kip1/p27, without any noticeable changes in G1 CDKs and cyclins, except a slight increase in cyclin D2. IP6 inhibited kinase activities associated with CDK2, 4 and 6, and cyclin E and D1. Further studies showed the increased binding of Kip1/p27 and Cip1/p21 with cyclin D1 and E. In down-stream of CDKI-CDK/cyclin cascade, IP6 increased hypophosphorylated levels of Rb-related proteins, pRb/p107 and pRb2/p130, and moderately decreased E2F4 but increased its binding to both pRb/p107 and pRb2/p130. At higher doses and longer treatment times, IP6 caused a marked increase in apoptosis, which was accompanied by increased levels of cleaved PARP and active caspase 3. IP6 modulates CDKI-CDK-cyclin complex, and decreases CDK-cyclin kinase activity, possibly leading to hypophosphorylation of Rb-related proteins and an increased sequestration of E2F4. Higher doses of IP6 could induce apoptosis and that might involve caspases activation. These molecular alterations provide an insight into IP6-caused growth inhibition, G1 arrest and apoptotic death of human prostate carcinoma DU145 cells.

Phytic acid (IP6), novel broad spectrum anti-neoplastic agent: a systematic review. Fox CH, Eberl M. Complement Ther Med 2003 Dec;10(4):229-34

INTRODUCTION: Phytic acid or IP6 has been extensively studied in animals and is being promoted as an anti-cancer agent in health food stores. It is naturally found in legumes, wheat bran, and soy foods. It is believed to be the active ingredient that gives these substances their cancer fighting abilities. Proposed mechanisms of action include gene alteration, enhanced immunity, and anti-oxidant properties. METHODS: A Medline search from 1966 to May 2002 using the keywords phytic acid and cancer, and limiting the search to the subheadings of therapeutic uses, prevention, and adverse effects revealed 28 studies. These studies were included in the review. RESULTS: A great majority of the studies were done in animals and showed that phytic acid had anti-neoplastic properties in breast, colon, liver, leukemia, prostate, sarcomas, and skin cancer. There were no human studies. Side effects included chelation of multivalent cations, and an increase in bladder and renal papillomas. This increase in papilloma formation only occurred with the sodium salt of phytic acid. It did not occur with either the potassium or magnesium salts. CONCLUSIONS: There is a large body of animal evidence to show that phytic acid may have a role in both the prevention and treatment of many forms of cancer. There is clearly enough evidence to justify the initiation of Phase I and Phase II clinical trials in humans.

Inositol hexakisphosphate blocks tumor cell growth by activating apoptotic machinery as well as by inhibiting the Akt/NFkappaB-mediated cell survival pathway. Ferry S,

Matsuda M, Yoshida H, Hirata M. Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science and Station for Collaborative Research, Kyushu University, Fukuoka 812-8582, Japan.

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It has been reported that inositol hexakisphosphate (InsP(6), phytic acid), a natural product, has an anticancer role. However, there is inadequate information regarding the mechanism by which InsP(6) exerts anticancer actions, and the effect requires relatively high concentration of the agent, both of which hinders the usage of InsP(6) as an anticancer drug. In the present study, we investigated the mechanism by which InsP(6) acts as an anticancer agent, and tried to reduce the concentration of effective InsP(6). Treatment of HeLa cells with InsP(6) at 1 mM induced apoptosis, as assessed by counting the cell number, and by Hoechst and TUNEL staining. This is probably mediated by intracellular InsP(6) itself and/or the dephosphorylated forms of metabolized InsP(6), because incubation of HeLa cells with [(3)H]InsP(6) produces dephosphorylated forms such as InsP(4) and InsP(5). Induction of apoptosis by InsP(6) was examined in two ways: inhibition of cell survival signaling and direct induction of apoptosis. Treatment of HeLa cells with tumor necrosis factor (TNF) or insulin stimulated the Akt-nuclear factor kappaB (NFkappaB) pathway, a cell survival signal, which involves the phosphorylation of Akt and IkappaB, nuclear translocation of NFkappaB and NFkappaB-luciferase transcription activity. InsP(6) blocked all these cellular events, but phosphatidylinositol 3-kinase activity was not affected. As well as inhibiting the Akt-NFkappaB pathway, InsP(6) itself caused mitochondrial permeabilization, followed by cytochrome c release, which later caused activation of the apoptotic machinery, caspase 9, caspase 3 and poly (ADP-ribose) polymerase. When InsP(6) was applied together with histone, the effective concentration to induce apoptosis was approximately 10-fold lower. These results revealed that extracellularly applied InsP(6) directly activates the apoptotic machinery as well as inhibits the cell survival signaling, probably by the intracellular delivery followed by a dephosphorylation

Phosphorylated inositol compounds in (beta)-cell stimulus-response coupling.
Barker-C-J, Leibiger-I-B, Leibiger-B, Berggren-P-O. American Journal of Physiology Endocrinology and Metabolism 01 DEC 2002, Vol/Iss/Pg. 283/6 46-6

_Pancreatic (beta)-cell function is essential for the regulation of glucose homeostasis in humans, and its impairment leads to the development of type 2 diabetes. Inputs from glucose and cell surface receptors act together to initiate the (beta)-cell stimulus-response coupling that ultimately leads to the release of insulin. Phosphorylated inositol compounds have recently emerged as key players at all levels of the stimulus-secretion coupling process. In this current review, we seek to highlight recent advances in (beta)-cell phosphoinositide research by dividing our examination into two sections. The first involves the events that lead to insulin secretion. This includes both new roles for inositol polyphosphates, particularly inositol hexakisphosphate, and both conventional and 3-phosphorylated inositol lipids. In the second section, we deal with the more novel concept of the autocrine role of insulin. Here, released insulin initiates signal transduction cascades, principally through the activity of phosphatidylinositol 3-kinase. This new round of signal transduction has been established to activate key (beta)-cell genes, particularly the insulin gene itself. More controversially, this insulin feedback has also been suggested to either terminate or enhance insulin secretion events.


Binding of inositol hexakisphosphate (IP(6)) to Ku but not to DNA-PK(cs).
Ma-Y, Lieber-M-R. Journal of Biological Chemistry 29 MAR 2002, 277/13 (10756-10759)

The nonhomologous DNA end joining (NHEJ) pathway is responsible for repairing a major fraction of double strand DNA breaks in somatic cells of all multicellular eukaryotes. As an indispensable protein in the NHEJ pathway, Ku has been hypothesized to be the first protein to bind at the DNA ends generated at a double strand break being repaired by this pathway. When bound to a DNA end, Ku improves the affinity of another DNA end-binding protein, DNA-PK(cs), to that end. The Ku-DNA-PK(cs) complex is often termed the DNA-PK holoenzyme. It was recently shown that myo-inositol hexakisphosphate (IP(6)) stimulates the joining of complementary DNA ends in a cell free system. Moreover, the binding data suggested that IP(6) bound to DNA-PK(cs) (not to Ku). Here we clearly show that, in fact, IP(6) associates not with DNA-PK(cs), but rather with Ku. Furthermore, the binding of DNA ends and IP(6) to Ku are independent of each other. The possible relationship between inositol phosphate metabolism and DNA repair is discussed in light of these findings.


The Synthesis of Inositol Hexakisphosphate John W. Verbsky, Monita P. Wilson, Marina V. Kisseleva , Philip W. Majerus, and Susan R. Wente J. Biol. Chem., Vol. 277, Issue 35, 31857-31862, August 30, 2002

The enzyme(s) responsible for the production of inositol hexakisphosphate (InsP6) in vertebrate cells are unknown. In fungal cells, a 2-kinase designated Ipk1 is responsible for synthesis of InsP6 by phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (InsP5). Based on limited conserved sequence motifs among five Ipk1 proteins from different fungal species, we have identified a human genomic DNA sequence on chromosome 9 that encodes human inositol 1,3,4,5,6-pentakisphosphate 2-kinase (InsP5 2-kinase). Recombinant human enzyme was produced in Sf21 cells, purified, and shown to catalyze the synthesis of InsP6 or phytic acid in vitro. The recombinant protein converted 31 nmol of InsP5 to InsP6/min/mg of protein (Vmax). The Michaelis-Menten constant for InsP5 was 0.4 µM and for ATP was 21 µM. Saccharomyces cerevisiae lacking IPK1 do not produce InsP6 and show lethality in combination with a gle1 mutant allele. Here we show that expression of the human InsP5 2-kinase in a yeast ipk1 null strain restored the synthesis of InsP6 and rescued the gle1-2 ipk1-4 lethal phenotype. Northern analysis on human tissues showed expression of the human InsP5 2-kinase mRNA predominantly in brain, heart, placenta, and testis. The isolation of the gene responsible for InsP6 synthesis in mammalian cells will allow for further studies of the InsP6 signaling functions.


Specific interaction of IP6 with human Ku70/80, the DNA-binding subunit of DNA-PK. Hanakahi LA. West SC. EMBO Journal. 21(8):2038-44, 2002 Apr 15.

In eukaryotic cells, DNA double-strand breaks can be repaired by non-homologous end-joining, a process dependent upon Ku70/80, XRCC4 and DNA ligase IV. In mammals, this process also requires DNA-PK(cs), the catalytic subunit of the DNA-dependent protein kinase DNA-PK. Previously, inositol hexakisphosphate (IP6) was shown to be bound by DNA-PK and to stimulate DNA-PK-dependent end-joining in vitro. Here, we localize IP6 binding to the Ku70/80 subunits of DNA- PK, and show that DNA-PK(cs) alone exhibits no detectable affinity for IP6. Moreover, proteolysis mapping of Ku70/80 in the presence and absence of IP6 indicates that binding alters the conformation of the Ku70/80 heterodimer. The yeast homologue of Ku70/80, yKu70/80, fails to bind IP6, indicating that the function of IP6 in non-homologous end-joining, like that of DNA-PK(cs), is unique to the mammalian end-joining process.


Inositol hexakisphosphate kinase 2 sensitizes ovarian carcinoma cells to multiple cancer therapeutics. Morrison BH. Bauer JA. Hu J. Grane RW. Ozdemir AM. Chawla-Sarkar M. Gong B. Almasan A. Kalvakolanu DV. Lindner DJ. Oncogene. 21(12):1882-9, 2002 Mar 14.

We recently identified inositol hexakisphosphate kinase 2 (IP6K2) as a positive regulator of apoptosis. Overexpression of IP6K2 enhances apoptosis induced by interferon-beta (IFN-beta) and cytotoxic agents in NIH-OVCAR-3 ovarian carcinoma cells. In this study, we contrast and compare IFN-beta and radiation-induced death, and show that IP6K2 expression sensitizes tumor cells. Unirradiated NIH-OVCAR-3 cells transfected with IP6K2 formed fewer colonies compared to unirradiated vector-expressing cells. IP6K2 overexpression caused increased radiosensitivity, evidenced by decreased colony forming units (CFU). Both IFN-beta and radiation induced caspase 8. IFN-beta, but not gamma-irradiation, induced TRAIL in NIH-OVCAR-3 cells. Gamma irradiation, but not IFN-beta, induced DR4 mRNA. Apoptotic effects of IFN-beta or gamma-irradiation were blocked by expression of a dominant negative mutant death receptor 5 (DR5Delta) or by Bcl-2. Caspase-8 mRNA induction was more pronounced in IP6K2-expressing cells compared to vector-expressing cells. These data suggest that overexpression of IP6K2 enhances sensitivity of some ovarian carcinomas to radiation and IFN-beta. IP6K2 may function to enhance the expression and/or function of caspase 8 and DR4 following cell injury. Both IFN-beta and gamma-irradiation induce apoptosis through the extrinsic, receptor-mediated pathway, IFN-beta through TRAIL, radiation through DR4, and both through caspase 8. The function of both death inducers is positively regulated by IP6K2.


Effects of exogenous inositol hexakisphosphate (InsP(6)) on the levels of InsP(6) and of inositol trisphosphate (InsP(3)) in malignant cells, tissues and biological fluids.
AU Grases-F Simonet-B-M, Vucenik-I, Perello-J, Prieto-R-M, Shamsuddin- A-M. F Grases, Lab. of Renal Lithiasis Research, Faculty of Science, University of Balearic Islands, Ctra. Valldemossa Km 7.5, 07071 Palma de Mallorca, Spain Life Sciences {LIFE-SCI}, 16 AUG 2002, Vol/Iss/Pg. 71/13 (1535-1546), ISSN: 0024-3205.

AB InsP(6) is abundant in cereals and legumes. InsP(6) and lower insositol phosphates, in particular InsP(3), participate in important intracellular processes. In addition, InsP(6) possess significant health benefits, such as anti-cancer effect, kidney stones prevention, lowering serum cholesterol. Because of the insensitivity of existing methods for determination of non-radiolabelled inositol phosphates, little is known about the natural occurrence, much less on the concentrations of InsP(6) and InsP(3) in biological samples. Using gas chromatography-mass detection analysis of HPLC chromatographic fractions, we report a measurement of unlabelled total InsP(3) and InsP(6) (a) as they occur within cells culture, tissues, and plasma, and (b) their changes depending on the presence of exogenous InsP(6). When rats were fed on a purified diet in which InsP(6) was undetectable (AIN-76A) the levels of InsP(6) in brain were 3.35 0.57 (SE) (mu)molkg(-1) and in plasma 0.023 0.008 (SE) (mu) moll(-1). The presence of InsP(6) in diet dramatically influenced its levels in brain and in plasma. When rats were given an InsP(6)- sufficient diet (AIN-76A + 1% InsP(6)), the levels of InsP(6) were about 100-fold higher in brain tissues (36.8 1.8 (SE)) than in plasma (0.29 0.02 (SE)); InsP(6) concentrations were 8.5-fold higher than total InsP(3) concentrations in either plasma (0.033 0.012 (SE)) and brain (4.21 0.55 (SE)). When animals were given an InsP(6)-poor diet (AIN-76A only), there was a 90% decrease in InsP(6) content in both brain tissue and plasma (p < 0.001); however, there was no change in the level of total InsP(3). In non-stimulated malignant cells (MDA-MB 231 and K562) the InsP(6) contents were 16.2 9.1 (SE) (mu)molkg(-1) for MDA-MB 231 cells and 15.6 2.7 (SE) for K 562 cells. These values were around 3-fold higher than those of InsP(3) (4.8 0.5 (mu)molkg(-1) and 6.9 0.1 (SE) for MDA-MB 231 and K562 cells respectively). Treatment of malignant cells with InsP (6) resulted in a 2-fold increase in the intracellular concentrations of total InsP(3) (9.5 1.3 (SE) and 10.8 1.0 (SE) (mu)molkg(-1) for MDA-MB 231 and K562 cells respectively, p < 0.05), without changes in InsP(6) levels. These results indicate that exogenous InsP (6) directly affects its physiological levels in plasma and brain of normal rats without changes on the total InsP(3) levels. Although a similar fluctuation of InsP(6) concentration was not seen in human malignant cell lines following InsP(6) treatment, an increased intracellular levels of total InsP(3) was clearly observed.


Absorption and excretion of orally administered inositol hexaphosphate (IP(6) or phytate) in humans.Grases-F, Simonet-B-M, Vucenik-I, Prieto-R-M, Costa-Bauza-A, March-J- G, Shamsuddin-A-M. BioFactors 2001, VOL: 15 (1), P: 53-61, ISSN: 0951-6433.

A study of the pharmacokinetic profile (oral absorption and renal excretion) of inositol hexaphosphate or phytate (IP(6)) is presented. Seven healthy volunteers were following a IP(6) poor diet (IP(6)PD) in a first period, and on IP(6) normal diet (IP(6)ND) in a second one. When following the IP(6)PD they become deficient in IP(6), the basal levels found in plasma (0.07+/- 0.01 mg/L) being clearly lower than those found when IP(6)ND was consumed (0.26+/- 0.03 mg/L). During the restriction period the maximum concentration in plasma were obtained 4 h after the ingestion of a single dose of IP(6), observing almost the same renal excretion profiles for the three different commercial sources and doses. After the IP(6) restriction period, volunteers were on IP(6)ND, reaching normal plasma and urinary IP(6) values in 16 days. Thus, the normal plasma and urinary concentrations, can be obtained either by consumption of a IP(6)ND taking a long time or in a short period by IP(6) supplements.


G0/G1 arrest and S phase inhibition of human cancer cell lines by inositol hexaphosphate IP6).El-Sherbiny-Y-M, Cox-M-C, Ismail-Z-A, Shamsuddin-A-M, Vucenik-I. Anticancer research 2001 Jul-Aug, VOL: 21 (4A), P: 2393-403, ISSN: 0250-7005.

Inositol hexaphosphate (InsP6 or IP6) has shown a striking anti-cancer activity in both in vivo and in vitro models. In an attempt to elucidate the mechanism(s) underlying the anti- neoplastic potential of IP6, we investigated its effect on cell cycle progression of MCF-7 estrogen receptor (ER)-positive and MDA-MB 231 ER-negative human breast cancer cell lines and HT-29 human colon cancer cells.

The anti-proliferative effect of IP6 was evaluated using dual-parameter flow cytometric measurements of DNA content, versus the incorporation of 5-bromo-2-deoxyuridine (BrdU) to determine cells actively synthesizing DNA. Combined analysis of the expression of cell cycle-related proteins, proliferation marker Ki-67 and proliferating cell nuclear antigen (PCNA) versus DNA content were used to determine the amount of proliferating cells in each phase, engaged in cell cycle transit. After 3 days of treatment with 5 mM IP6, S-phase, as estimated by BrdU uptake, was significantly decreased in all three cell lines (p = 0.002). MCF-7 and HT-29 cells accumulated in the G0/G1 range of DNA contents (p = 0.002 and p = 0.001, respectively). MDA MB-231 cells transiently accumulated in G0/G1 only after 2 days (p = 0.01). There was a significant decrease in the percentage of Ki-67 expression in IP6- treated cells, from 82.8+/-3.0% to 66.8+/-4.2% in MCF-7 (p = 0.007), from 93.4+/-4.6% to 71.7+/-3.3% in MDA-MB 231 (p = 0.004), and from 95.2+/-1.2% to 73.5+/-2.5% in HT-29 cells (p = 0.002) respectively. PCNA expression levels were also significantly decreased by IP6 in all three cell lines (MCF-7 p = 0.0007; MDA-MB 231 p = 0.0006; HT-29 p = 0.0001).

These results show that IP6 controls the progression of cells through the cycle by decreasing S- phase and arresting cells in the G0/G1-phase of the cell cycle. A significant decrease in the expression of proliferation markers indicated that IP6 disengaged cells from actively cycling. Further investigations of cell cycle regulators may lead us to a better understanding of the mechanism(s) of the anti-neoplastic action of IP6.


Inhibition of skin cancer by IP6 in vivo: initiation-promotion model Ishikawa-T, Nakatsuru-Y, Zarkovic-M, Shamsuddin-A-M Anticancer research 1999 Sep-Oct, VOL: 19 (5A), P: 3749-52, ISSN: 0250-7005.

A two-stage mouse skin carcinogenesis model was used to examine the effects of IP6 on initiation and promotion phases of tumorigenesis. Seven week old ICR female mice were divided into 6 groups, each consisting of 20 animals. Initiation was performed by a single application of the carcinogen 7,12-dimethyl benz(a)anthracene (DMBA) (50 micrograms) to the back skin. Three weeks later, local application of the promoter TPA was started (2.5 micrograms, 2 x /week) and continued up to the end of the experiment (22 weeks). Mice were also administered 2% IP6 in drinking water over the entire duration of the experiment, or during the initiation (initial 3 weeks) or promotion (final 19 weeks) periods only. The animals consuming IP6 during the initiation stage showed an approximately 50% reduction in the mean number of papillomas per animal, as well as in the number of tumor bearing mice. However, no such inhibition was observed when IP6 was given during the tumor promotion stage. In a separate experiment the effects of IP6 on epithelial cell growth were assessed by BrdU labeling at several time points. Statistically significant inhibition of cell proliferation was observed during the initiation stage (one week after DMBA treatment) in the group given IP6. No inhibition was evident during the promotion stage.


Metabolism and cellular functions of IP6: a review. Shamsuddin-A-M. Anticancer research 1999 Sep-Oct, VOL: 19 (5A), P: 3733-6, Refs: 33, ISSN: 0250-7005.

Inositol hexaphosphate (IP6) has a demonstrably effective anti-cancer action against a variety of experimental tumors. However, the mechanisms of its actions are yet to be completely discerned. Studies in my laboratory have shown that IP6 is rather rapidly absorbed by rats in vivo. Ion exchange chromatography demonstrates the presence of inositol and IP1-6 in gastric epithelial cells as early as within 1 h of intragastric 3H-IP6 administration. The metabolized IP6, in the form of inositol and IP1 is transported via plasma and reaches distant organs as well as tumors. In rats, the urinary metabolites of IP6 are inositol and IP1. However, in humans 1-3% of total administered IP6 is excreted in the urine as IP6; the level shows a normal oscillation between 0.5-6 mg/L (F. Grases et al). Investigations of the uptake and metabolism by a variety of cancer cell lines in vitro also demonstrate an instantaneous absorption of IP6. The rate and pattern at which IP6 is metabolized by cancer cells varies depending on the cell type. Intracellular inositols accumulated mostly (80-97%) in the cytosol as inositol and IP1-6. IP6 treatment of all the cell lines tested so far demonstrates that it is cytostatic and not cytotoxic. Along with inhibition of cell proliferation, there is enhanced differentiation of malignant cells to a more mature phenotype, often resulting in reversion to normal. Studies of the expression of tumor suppressor gene demonstrate up- regulation of wild type p53 and down-regulation of the mutant form. Since p53-mediated cell cycle arrest may be the direct result of induction of WAF-1 gene (p21WAF-1/CIP1), our studies demonstrate that IP6 up-regulates the expression of p21WAF-1/CIP1 in a dose-dependent manner. These data strongly point towards the involvement of signal transduction pathways, cell cycle regulatory genes, differentiation genes, oncogenes and perhaps, tumor suppressor genes in bringing about the observed anti-neoplastic action of IP6.


Antiplatelet activity of inositol hexaphosphate (IP6). Vucenik-I, Podczasy-J-J, Shamsuddin-A-M. Anticancer research 1999 Sep-Oct, VOL: 19 (5A), P: 3689-93, ISSN: 0250-7005.

Platelet adhesion to endothelial cells, their aggregation and subsequent release of platelet-derived mediators are key steps in the pathogenesis of thrombosis and atherosclerosis. Using impedance technology the effect of inositol hexaphosphate (IP6) on platelet aggregation and adenosine triphosphate (ATP) release were simultaneously measured in whole blood obtained from healthy volunteers (n = 10). The platelets were activated with adenosine diphosphate (ADP) (10 microM), collagen (2 micrograms/mL), or thrombin (1 U/mL) in the presence or absence of IP6. IP6 significantly inhibited platelet aggregation induced with all agonists in a dose-response manner (p 0.0001 for ADP and collagen, p = 0.0103 for thrombin), with the IC50 values of 0.9, 1.6 and 0.8 mM. Secretion of platelet dense granule content was measured in parallel. IP6 strongly and significantly reduced agonist-induced ATP release (p = 0.00247 for ADP; p = 0.0074 for collagen; p = 0.0069 for thrombin). These data demonstrate that IP6 effectively inhibits human platelet aggregation in vitro, suggesting its potential in reducing the risk for cardiovascular disease.


Mammary tumor inhibition by IP6: a review. Shamsuddin-A-M, Vucenik-I. Anticancer research 1999 Sep-Oct, VOL: 19 (5A), P: 3671-4, Refs: 34, ISSN: 0250-7005.

While most studies of diet and breast cancer are focused on the role of fat, very few have addressed the effect of fiber. Emerging epidemiological data, and careful review of previous studies point to a negative correlation of breast cancer with high fiber cereal diets. Inositol hexaphosphate (IP6) is abundant in cereals and legumes, particularly in the bran part of mature seeds. Experimental studies using 7,12-dimethylbenz (alpha)anthracene (DMBA) and N- methylnitrosourea (NMU) in rats and mice in vivo, as well as human cell lines in vitro demonstrate a reproducible and striking anti- cancer action of IP6. It therefore appears that IP6 is one of the components, if not the most active ingredient, of high fiber cereal diet responsible for cancer inhibition. Could eating high fiber diet afford the same protection as IP6? Thus, we investigated whether dietary fiber containing high IP6 shows a dose-response inhibition of DMBA-induced rat mammary carcinogenesis, and if pure IP6 is more active as a cancer preventive agent, compared to that in diet. Our data show that supplemental dietary fiber in the form of bran exhibited a modest, statistically nonsignificant inhibitory effect. In contrast, animals given IP6 in drink showed significant reduction in tumor number, incidence and multiplicity. Therefore, pure IP6 is definitively more effective than a high fiber diet in preventing experimental mammary tumors. Thus, for cancer prevention, prophylactic intake of IP6 may be not only more effective, but also more practical than gorging on large quantities of fiber.


Proceedings of the First International Symposium on Disease Prevention by IP6, and Other Rice Components, Takaragaike, Kyoto, Japan, 8-9 June 1998: Ishikawa-T, Ogawa-S, Shamsuddin-A-M. Anticancer Research 1999, Vol/Iss/Pg. 19/5 A (3633) , ISSN: 0250-7005.

No abstract available


IP6 in treatment of liver cancer. II. Intra-tumoral injection of IP6 regresses pre-existing human liver cancer xenotransplanted in nude mice Vucenik-I, Zhang-Z-S, Shamsuddin-A-M. . Anticancer research 1998 Nov-Dec, VOL: 18 (6A), P: 4091-6, ISSN: 0250-7005.

Hepatocellular carcinoma (HCC) is a deadly malignant disease with extremely poor prognosis. Many therapeutic modalities have been proposed, but considerable uncertainty still remains about their effectiveness. Inositol hexaphosphate (IP6), a naturally occurring polyphosphorylated carbohydrate, has novel anti-cancer function both in vitro and in vivo. We have recently demonstrated that IP6 inhibits HepG2 human liver cancer cell line. The aim of this study was to assess whether IP6 can (a) inhibit tumorigenicity, and (b) suppress or regress the growth of HepG2 cells in a transplanted nude mouse model. To test the inhibition of tumorigenicity, HepG2 cells were treated with a single exposure to 5.0 mM IP6 in vitro; 48 h later they were inoculated (1 x 10(7) cells/mouse) subcutaneously. No tumor was found in mice which had received HepG2 cells pretreated with IP6 whereas 71% of mice receiving the same number of control untreated HepG2 cells developed solid tumors at the transplantation site (p 0.03). For a tumor suppression/regression study, when the transplanted tumors reached 8-10 mm in diameter, intra-tumoral injection of IP6 (40 mg/kg) was given for 12 consecutive days, after which the animals were sacrificed. At autopsy, the tumor weight in IP6-treated mice was 86% to 1180% (340% average) less than that in control mice (0.33 +/- 0.12 g versus 1.13 +/- 0.25 g, p = 0.016). These data show that IP6 inhibits the formation of liver cancer and regresses pre-existing human hepatic cancer xenograft; therefore, it has the potential for clinical use as a preventive and therapeutic agent for hepatocellular carcinoma as well.


IP6 in treatment of liver cancer. I. IP6 inhibits growth and reverses transformed phenotype in HepG2 human liver cancer cell line. Vucenik-I, Tantivejkul-K, Zhang-Z-S, Cole-K-E, Saied-I, Shamsuddin-A- M. Anticancer research 1998 Nov-Dec, VOL: 18 (6A), P: 4083-90, ISSN: 0250-7005.

Hepatocellular carcinoma (HCC) is a common tumor world-wide with extremely poor prognosis. Recent studies have shown that inositol hexaphosphate (IP6), a naturally occurring carbohydrate, has novel anti-cancer function in various in vitro and in vivo models. The aim of this study was to assess whether IP6 could inhibit the growth of human hepatocellular carcinoma. We treated HepG2, a human liver cancer cell line in vitro with IP6 and evaluated its effect on growth and differentiation. IP6 treatment of HepG2 cells caused a dose- dependent growth inhibition. Compared to other cancer cell lines, Hep G2 cells were quite sensitive to IP6, IC50 (50% inhibition of cell growth) of IP6 being 1.0 mM (0.338 mM). Treatment with IP6 decreased the ability of HepG2 cells to form colonies, as assessed in the plating efficiency assay. Morphological changes induced by IP6 were consistent with differentiation of HepG2 cells. Exposure of HepG2 cells to IP6 drastically decreased the rate of production of alpha- fetoprotein (AFP), a tumor marker of HCC, indicating also that IP6 treatment leads to differentiation of malignant liver cells. Further, IP6 treatment caused a decreased expression of mutant p53 protein in HepG2 cells, with no significant change in the expression of wild- type p53. The expression of p21WAF1 protein was increased by 1.5 fold, as determined by immunocytochemical staining and ELISA assay. These data demonstrate that IP6 inhibits the growth, and induces differentiation, and a less aggressive phenotype of HepG2 cells, suggesting a role of IP6 in the treatment of HCC.


Up-regulation of the tumor suppressor gene p53 and WAF1 gene expression by IP6 in HT-29 human colon carcinoma cell line. Saied-I-T, Shamsuddin-A-M. Anticancer research 1998 May-Jun, VOL: 18 (3A), P: 1479-84, ISSN: 0250-7005.

Inositol hexaphosphate (InsP6 or IP6) ubiquitous in various cells has a novel anti-cancer action both in vivo and in vitro. IP6 inhibits cell growth, decreases cell proliferation and also causes differentiation of various cell lines, including HT-29 human colon carcinoma cell. We hypothesize that the tumor suppressor genes such as p53 and WAF1/CIP1 may be involved in mediating the anti-neoplastic action of IP6 p53 acts as a molecular policeman prevention of genetically damaged cells; it causes the cells to arrest in the G1 phase of cell cycle, and regulates the level of p21waf1/cip1 which acts as a growth inhibitor. We therefore investigated the effects of IP6 on the expression of p53 and WAF1/p21 in HT-29 human colon carcinoma by immunocytochemistry and quantitative ELISA. Our immunocytochemical studies with anti p53 antibodies (wild type-PAb246 and PAb1620) and anti p21waf1/cip1 (EA10) antibodies demonstrated an increased level of p53 and p21waf1/cip1 after 3 and 6 days of treatment with 3.3 and 5 mM IP6. Quantitative assay for p53 and p21waf1/cip1 by ELISA did not show detectable levels in untreated control cells, while strong expression of p53 and p21waf1/cip1 protein by 3.3 and 5 mM IP6 was seen on day 3 and day 6 of treatment. This increase was dose-dependent; however, a definite time-dependent increase was not observed. These data demonstrate that IP6 up- regulates the expression of the tumor suppressor gene p53 and p21WAF1 /CIP1 gene and their modulation may be one of the mechanisms of the anti-neoplastic action of IP6. Since loss of p53 function enhances cancer cells' resistance to chemotherapeutic agents, the stimulating function of IP6 on p53 makes it an attractive adjuvant chemotherapeutic agent as well.


Novel anticancer function of inositol hexaphosphate: inhibition of human rhabdomyosarcoma in vitro and in vivo. Vucenik-I, Kalebic-T, Tantivejkul-K, Shamsuddin-A-M. Anticancer research 1998 May-Jun, VOL: 18 (3A), P: 1377-84, ISSN: 0250-7005.

Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate that has been shown to suppress the growth of epithelial cancers, including those of breast and colon. The objective of this study was to investigate whether IP6 inhibits growth of rhabdomyosarcoma (RMS), a tumor of mesenchymal origin, which is the most common soft tissue sarcoma in children. We performed both in vitro and in vivo studies to evaluate the effect of IP6 on human RD cells growth. Our results show that IP6 suppresses growth of rhabdomyosarcoma cell line (RD) in vitro in a dose- dependent fashion. A 50% inhibition of cell growth (IC50) was induced by 1.0 mM IP6. However, the removal of IP6 from the media, after 72 hours of treatment, allowed cells to recover their logarithmic growth. Exposure of RD cells to IP6 led to differentiation; cells became larger with abundant cytoplasm, expressing higher levels of muscle-specific actin. Consistent with in vitro observation, IP6 suppressed RD cell growth in vivo, in a xenografted nude mice model. When compared to controls, IP6-treated mice produced a 25 fold smaller tumors (p = 0.008), as observed after a two weeks treatment. In a second experiment, wherein the treatment period was extended to five weeks, a 49 fold (p = 0.001) reduction in tumor size was observed in mice treated with IP6. Histologically no evidence of tumor cell necrosis was observed. These data suggest a potential usefulness of this cytostatic, and non-cytotoxic, compound in novel therapeutic strategies for these types of tumor.


IP6: a novel anti-cancer agent. Shamsuddin-A-M, Vucenik-I, Cole-K-E. Life sciences 1997, VOL: 61 (4), P: 343-54, Refs: 68, ISSN: 0024-3205.

Inositol hexaphosphate (InsP6 or IP6) is ubiquitous. At 10 microM to 1 mM concentrations, IP6 and its lower phosphorylated forms (IP(1-5)) as well as inositol (Ins) are contained in most mammalian cells, wherein they are important in regulating vital cellular functions such as signal transduction, cell proliferation and differentiation. A striking anti-cancer action of IP6 has been demonstrated both in vivo and in vitro, which is based on the hypotheses that exogenously administered IP6 may be internalized, dephosphorylated to IP(1-5), and inhibit cell growth. There is additional evidence that Ins alone may further enhance the anti-cancer effect of IP6. Besides decreasing cellular proliferation, IP6 also causes differentiation of malignant cells often resulting in a reversion to normal phenotype. These data strongly point towards the involvement of signal transduction pathways, cell cycle regulatory genes, differentiation genes, oncogenes and perhaps, tumor suppressor genes in bringing about the observed anti-neoplastic action of IP6.


Comparison of pure inositol hexaphosphate and high-bran diet in the prevention of DMBA-induced rat mammary carcinogenesis. Vucenik-I, Yang-G-Y, Shamsuddin-A-M. Nutrition and cancer 1997, VOL: 28 (1), P: 7-13, ISSN: 0163-5581.

Inositol hexaphosphate (IP6), abundant in cereals and legumes, has been demonstrated to be a promising anticancer agent in different in vivo and in vitro models. Because IP6 is particularly abundant in the bran part of certain mature seeds such as wheat, we investigated whether a high-fiber bran diet containing high IP6 shows a dose- response inhibition of 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinogenesis. Starting at two weeks before DMBA intubation, rats were divided into five groups and fed AIN-76A diet only or AIN-76A diet containing 5%, 10%, or 20% Kelloggs' All Bran; the fifth group received 0.4% IP6 given in drinking water, an amount equivalent to the IP6 content in 20% bran. After carcinogen administration, the rats remained on these regimens for 29 weeks. Compared with the carcinogen control, at 29th week, tumor incidence was reduced by 16.7%, 14.6%, and 11.4% in rats fed 5%, 10%, and 20% bran, respectively (not statistically significant). However, rats given 0.4% IP6 in drinking water, equivalent to that in 20% bran, had a 33.5% reduction in tumor incidence (p 0.02) and 48.8% fewer tumors (p 0.03). These data show that supplemental dietary fiber in the form of bran exhibited a very modest, statistically nonsignificant inhibitory effect, which was also not dose dependent. In contrast, animals given IP6 showed significant reduction in tumor number, incidence, and multiplicity. Thus IP6 an active substance responsible for cereal's beneficial anticancer effect, is clearly more effective than 20% bran in the diet. In practical terms, intake of IP6 may be a more pragmatic approach than gorging enormous quantities of fiber for cancer prophylaxis.


Inositol hexaphosphate (IP6) reduces risk for cancer and cardiovascular diseases. Vucenik-I, Shamsuddin-A-M. Periodicum Biologorum 1997, Vol/Iss/Pg. 99/1 (25-30), ISSN: 0031-5362.

An etiological link exists between diet cancer, cardiovascular and some chronic diseases. Inositol hexaphosphate (IP6) is ubiquitous in plants and eukaryotic cells. Although found in a substantial amount, its intracellular functions are still not clear. Supported by experimental data, we believe that IP6 is an active compound of high fiber diet, responsible for many of the beneficial effects of fiber. Recent studies have shown that IP6 is chemopreventive and chemotherapeutic in different cancers in vivo and in vitro. Additional hypocholesterolemic and antiplatelet effects of IP6 may be important in preventing and controlling cardiovascular disorders. IP6 may bring about its beneficial action by: a) acting as an anti- oxidant and/or b) by entering into intracellular inositol phosphates pool, and affecting signal transduction, thereby controlling cellular proliferation, differentiation and other biological processes. Being a natural and a non-toxic compound, IP6 has great potential in controlling cancer and preventing cardiovascular diseases.


Novel anti-cancer functions of IP6: growth inhibition and differentiation of human mammary cancer cell lines in vitro. Shamsuddin-A-M, Yang-G-Y, Vucenik-I. Anticancer research 1996 Nov-Dec, VOL: 16 (6A), P: 3287-92, ISSN: 0250-7005.

Inositol hexaphosphate (InsP6 or IP6) is an active ingredient of high fiber diet that has anti-cancer action in both in vitro and in vivo models. Recently we have demonstrated that InsP6 significantly inhibits DMBA-induced rat mammary cancer in vivo. To test the hypothesis that InsP6 mediates its function via inhibition of cell proliferation irrespective of hormonal dependence, its effect on growth inhibition and differentiation were studied in two human mammary carcinoma cell lines with different estrogen receptor status. Cell growth was measured by MTT incorporation assay, DNA synthesis by 3H-Tdr uptake and differentiation marker lactalbumin by immunocytochemistry. Dose-dependent growth inhibition was observed in both estrogen receptor-positive (MCF-7) and receptor-negative cells (MDA-MB-231). Statistically significant growth inhibition (p 0.05) was observed starting at 1 mM InsP6 as early as after the first day of treatment and continued up to 6 days for both the cell lines. DNA synthesis in both the cell lines was suppressed by InsP6 occurring as early as 3 h after the beginning of treatment and continued up to 48 h; significant inhibition (p 0.05) started at 1 mM InsP6 after 6 h of treatment. Compared to untreated cells, a 5-fold (p 0.05) and 22-fold (p 0.01) increase in expression of lactalbumin, associated with luminal cell differentiation was identified by immunocytochemistry after 48 h of treatment with 1 and 5 mM InsP6. Our data show that the inhibition of DNA synthesis and cell growth and induction of differentiation of human mammary cancer cell lines by InsP6 is independent of the estrogen receptor status of the cells. Taken together with results from in vivo studies, InsP6 may be an important candidate for the prevention and treatment of human breast cancer.


IP6-induced growth inhibition and differentiation of HT-29 human colon cancer cells: involvement of intracellular inositol phosphates. Yang-G-Y, Shamsuddin-A-M. Anticancer research 1995 Nov-Dec, VOL: 15 (6B), P: 2479-87, ISSN: 0250-7005.

Inositol hexaphosphate (InsP6 or IP6) ubiquitous in plants and animals is not only a natural antioxidant, but may also be the precursor/storage of intracellular inositol phosphates, important for various cellular functions. A novel anti-tumor action of InsP6 was demonstrated in models of experimental colon and mammary carcinogenesis in vivo. We now show its effects on growth and differentiation of HT-29 human colon carcinoma cells in vitro. A dose- and time-dependent (0.33-20 mM InsP6 and 1-6 days treatment) growth inhibition was observed as tested by MTT- incorporation assay. The inhibition was statistically significant (p 0.05) at 1 mM concentration as early as first day after treatment and continued up to 6 days. DNA-synthesis was also suppressed by InsP6 and significantly inhibited as early as 6 h after treatment at 1 mM concentration (p 0.05) and continued to 48 h (p 0.01). The expression of proliferation marker PCNA was down-regulated (p 0.05) by InsP6 (1 and 5 mM) after 48 h of treatment. To investigate the mechanism of action of InsP6 the intracellular phosphatases (including phytase) were inhibited by F to slow down the dephosphorylation of InsP6. Ion- exchange chromatographic separation of intracellular inositol phosphates demonstrated a 84-98% decrease of Ins, InsP1 and InsP2 Ins P3 was reduced by 39% and InsP4 and InsP5 by 21% and 13% respectively, whereas intracellular InsP6 was increased by 24.6% at 5 min following 3H-InsP6. Since neither the rate of uptake of 3H-InsP6 was unaffected, nor was the efficacy of growth inhibition altered by F inhibition of phytase, data suggest that contrary to the popular misconception, phytase plays no role in influencing the anti- neoplastic action of InsP6. Alkaline phosphatase activity (brush border enzyme, associated with absorptive cell differentiation), increased following 1 and 5 mM InsP6 treatment for 1-6 days. The expression of a mucin antigen associated with goblet cell differentiation and defined by the monoclonal antibody CMU10 was augmented (p 0.0001) by InsP6. The tumor mucin marker Gal-GalNAc, expressed by precancer and cancer of colon, but not by the normal cells showed a time-dependent biphasic change by InsP6; an increased expression after 1 day of treatment followed by suppression after 2 days suggest progression of mucin synthesis and differentiation of cancer cells with reversion to normal phenotype. Because the tumor marker Gal-GalNAc is a) easily detected in rectal mucin of patients with colonic cancer and precancer with high sensitivity and specificity, and b) suppressed by InsP6 treatment, it can be used to monitor the efficacy of chemoprevention by InsP6 or other such agents. Since InsP6 a natural dietary ingredient of cereals and legumes, inhibits growth and induces terminal differentiation of HT-29 cancer cells, it is an excellent candidate for adjuvant chemotherapy and prevention of cancer.


Inositol hexaphosphate inhibits growth and induces differentiation of PC-3 human prostate cancer cells. Shamsuddin-A-M, Yang-G-Y. Carcinogenesis 1995 Aug, VOL: 16 (8), P: 1975-9, ISSN: 0143-3334.

We investigated the effects of inositol hexaphosphate (InsP6) on growth inhibition and differentiation of human prostate cancer cells PC-3 in vitro. A significant dose- and time-dependent growth inhibition was observed as tested by the MTT-incorporation assay (P 0.05 at 1 mM InsP6 after 24 h treatment, P 0.01 at 0.1 mM after 3 days). DNA synthesis as determined by (3H)thymidine incorporation assay was also suppressed by InsP6 in a dose-dependent manner, occurring as early as 3 h after treatment and continuing up to 48 h (P 0.01 at 1 mM InsP6). A 9- to 10-fold increase (P 0.01) in expression of HLA class I molecule associated with tumor immunosurveillance and cell differentiation was induced by InsP6. The marker for prostatic cell differentiation, prostate acid phosphatase, was significantly (P 0.05) increased after 48 h treatment at 0.5-5 mM InsP6. Since InsP6 strongly inhibits growth and induces differentiation in human prostate cancer cells in vitro, in vivo studies using a tumor xenograft model and a prostate carcinogenesis model are warranted to validate the efficacy of InsP6 in the treatment and prevention of prostate cancer.


Inositol hexaphosphate and inositol inhibit DMBA-induced rat mammary cancer. Vucenik-I, Yang-G-Y, Shamsuddin-A-M. Carcinogenesis 1995 May, VOL: 16 (5), P: 1055-8, ISSN: 0143-3334.

Because inositol hexaphosphate (InsP6) and inositol (Ins), contained in plants and most mammalian cells, have been demonstrated to have anti-cancer and anti-cell proliferative action in several experimental models of carcinogenesis we have examined the effect of InsP6 +/- Ins on DMBA-induced rat mammary tumor model. Starting two weeks prior to induction with DMBA, the drinking water of female Sprague-Dawley rats was supplemented with either: 15 mM InsP6, 15 mM Ins, or 15 mM InsP6 + 15 mM Ins; a control group received no inositol compounds. Animals (49-day-old) were given a single intragastric dose of DMBA (5 mg/rat) in 1 ml of corn oil administered by oral intubation. After 45 weeks of treatment, the animals in all the three treatment regimens showed a significant reduction (P 0.05) in tumor incidence. Tumor number, multiplicity and tumor burden were also significantly (P 0.05) reduced by InsP6 +/- Ins. When all the parameters were taken into consideration, the best results were obtained by the combination treatment of InsP6 + Ins. Four additional groups not receiving DMBA, but drinking tap water, InsP6, Ins, or Ins P6 + Ins of the same molarity as experimental groups were observed for the duration of the study to monitor for any toxicity following this long-term treatment; no significant toxicity as evaluated by body weight gain, serum and bone mineral levels was detected. We demonstrate that InsP6 +/- Ins reproducibly inhibits experimental mammary carcinoma, therefore having great potential as a chemopreventive and adjuvant therapeutic agent for this disease as well.


Inositol phosphates have novel anticancer function. Shamsuddin-A-M. The Journal of nutrition 1995 Mar, VOL: 125 (3 Suppl), P: 725S-732S, Refs: 34, ISSN: 0022-3166.

Inositol hexaphosphate (InsP6, phytic acid) is ubiquitous in the plant kingdom and is abundant in cereals and legumes. In much smaller amounts InsP6 and its lower phosphorylated forms (InsP1-5) are contained in most mammalian cells, where they are important in regulating vital cellular functions. Both in vivo and in vitro experiments have suggested striking anticancer potential (preventive as well as therapeutic) for InsP6 with and without inositol. In addition to reduce cell proliferation, InsP6 increases differentiation of malignant cells often resulting in reversion to the normal phenotype. InsP6 is quickly absorbed from the rat stomach and upper intestine and distributed as inositol and InsP1. In vitro it is instantaneously taken up by malignant cells undergoing variable dephosphorylation to inositol and InsP1-5, pointing toward their role in mediating the action of InsP6. Because InsP6 is high in high-fiber diets, our studies also may explain, at least in part, the epidemiologic observation showing high-fiber diets are associated with a lower incidence of certain cancers. Although further studies are needed to elucidate the mechanism(s) of this action, inclusion of InsP6 in our strategies for cancer prevention and therapy is warranted.


(3H)inositol hexaphosphate (phytic acid) is rapidly absorbed and metabolized by murine and human malignant cells in vitro. Vucenik-I, Shamsuddin-A-M. The Journal of nutrition 1994 Jun, VOL: 124 (6), P: 861-8, ISSN: 0022-3166.

To test the hypothesis that the antineoplastic activity of phytic acid (inositol hexaphosphate (InsP6)) is a result of rapid intake by the cells and its conversion to lower inositol phosphates (InsP1-5), thereby affecting the intracellular inositol phosphate pool, YAC-1 (mouse T cell leukemia), K562 (human erythroleukemia) and HT-29 (human colon adenocarcinoma) cell lines were incubated at 37 degrees C with (3H)InsP6. After 1 h, 31.3 +/- 3.1% of administered radio- activity was taken up by YAC-1 cells, 6.2 +/- 0.9% by K562 cells and 6.6 +/- 3.8% by HT-29 cells. Differential centrifugation and high resolution subcellular fractionation of cell homogenates demonstrated that within the various cellular compartments, 80% (HT-29) to 97% (YAC-1) of the total radioactivity was in the cytosol. Kinetic study showed that the peak of the total absorption was obtained after 30 min of cell exposure to radiolabeled InsP6, after with a plateau was reached. Analysis of the radioactivity accumulated within the cells showed variable proportions of myo-inositol and InsP1-6, with a preponderance of InsP1 and InsP2. The presence of (3H)myo-inositol and (3H)InsP1-6 suggests that InsP6 may, in some cells at least, be absorbed as such and that a variable degree of dephosphorylation of InsP6 takes place both extra- and intracellularly.


Inhibition of rat mammary carcinogenesis by inositol hexaphosphate (phytic acid). A pilot study. Vucenik-I, Sakamoto-K, Bansal-M, Shamsuddin-A-M. Cancer letters 1993 Dec 10, VOL: 75 (2), P: 95-102, ISSN: 0304-3835.

Since phytic acid (inositol hexaphosphate, InsP6) and inositol (Ins) have been demonstrated to have anti-tumor and anti-cell proliferative action in several experimental models of carcinogenesis, in a pilot study we have examined their effect on 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumor model. Starting a week prior to induction with DMBA, the drinking water of female Sprague-Dawley rats was supplemented with either: 15 mM InsP6, 15 mM Ins, or 15 mM InsP6 + 15 mM Ins; a control group received no inositol compounds. Animals (55-day-old) were given a single dose of DMBA (20 mg) in 1 ml of sesame oil by oral intubation. Four additional groups not receiving DMBA, but drinking tap water, InsP6, Ins, or InsP6 + Ins of the same molarity as experimental groups were observed for the duration of the study to monitor for any putative toxicity following this long-term treatment. As opposed to the DMBA-only group, rats treated with InsP6 +/- Ins showed a 48% reduction in the number of tumors/tumor bearing animal (tumor multiplicity) and a 40% reduction in the number of tumors/rat. In contrast to 20% rats in DMBA-only group, only 0-8% animals in the treatment group had 5 or more tumors. Likewise, the tumor incidence was reduced by 19% in InsP6 +/- Ins as compared to control untreated animals. The tumors in the treated groups were also 16% smaller in size. Data from this pilot study suggest that in addition to being effective against colon cancer, InsP6 +/- Ins may be protective against mammary carcinoma as well; additional studies are however warranted.


Growth inhibition and differentiation of HT-29 cells in vitro by inositol hexaphosphate (phytic acid). Sakamoto-K, Venkatraman-G, Shamsuddin-A-M. Carcinogenesis 1993 Sep, VOL: 14 (9), P: 1815-9, ISSN: 0143-3334.

Inositol hexaphosphate (InsP6 or phytic acid) has been shown to have antineoplastic action in in vivo models of colon carcinogenesis. We therefore investigated its effect on proliferation and differentiation of the human colon cancer cell line HT-29 in vitro. Proliferation was evaluated by neutral red incorporation assay, and differentiation was assessed by expression of the markers, cytokeratin, carcinoembryonic antigen (CEA) and beta-D-galactose-(1-- >3)-N-acetyl-galactosamine (Gal-GalNAc). InsP6 in the culture media (0.66-10 mM) inhibited cell proliferation in a dose-dependent manner (P 0.001), while inositol or inositol hexasulfate used as controls or

media without InsP6 did not show any suppressive effect. The expression of the tumor marker, Gal-GalNac, was augmented (100.7% increase) by low dose (0.66 mM) of InsP6 but was subsequently suppressed with higher concentrations of InsP6. The expression of cytokeratin and CEA were both augmented by either InsP6 or inositol at all concentrations tested, although the degree of augmentation was milder with inositol than with InsP6. The combination of InsP6 and inositol (both 0.66 mm) resulted in augmentation (P 0.001) of cytokeratin expression, while that of CEA remained unchanged. The inhibitory effect of InsP6 on cell proliferation was not altered by combination with additional inositol at any concentrations tested. Our results show that InsP6 inhibits cell proliferation and concomitantly increases differentiation and is therefore a candidate chemopreventive and chemotherapeutic agent for human large intestinal cancer.


(3H)phytic acid (inositol hexaphosphate) is absorbed and distributed to various tissues in rats. Sakamoto-K, Vucenik-I, Shamsuddin-A-M. The Journal of nutrition 1993 Apr, VOL: 123 (4), P: 713-20, ISSN: 0022-3166.

To understand the mechanism of antineoplastic action of phytic acid, we investigated the absorption and distribution of myo-(inositol-2-3H (N)) hexakisphosphate in rats. The radioactivity was measured in urine, feces, blood, gastrointestinal tract contents and various organs and tissues at 1 and 24 h after intragastric administration. Of the total radioactivity, 79.0 +/- 10.0% was absorbed and at least 26.6% was degraded during the 24-h period following ingestion. The absorption was rapid; 11.0 +/- 2.6% of the radioactivity was detected in the wall of the stomach (4.4 +/- 3.7%) and upper small intestine (6.6 +/- 1.9%), 6.5 +/- 2.6% in the skeletal muscle and 4.0 +/- 1.5% in the skin after 1 h. Much of the radioactivity after 24 h was in the liver (4.0 +/- 0.9%), kidneys (2.2 +/- 1.1%), muscle (18.1 +/- 3.4%) and skin (10.1 +/- 3.3%). Analysis of plasma and urine demonstrated that most of the radioactivity was due to myo-inositol and small amounts of inositol monophosphate (InsP1). Gastric epithelial cells, however, contained inositol and various inositol phosphates (InsP1-6). Our data suggest that soluble InsP6 when administered in drinking water is rapidly absorbed through the stomach and upper small intestine, becomes quickly dephosphorylated within the mucosal cells and is distributed to various organs as inositol and InsP1.


Antitumor activity of phytic acid (inositol hexaphosphate) in murine transplanted and metastatic fibrosarcoma, a pilot study. Vucenik-I, Tomazic-V-J, Fabian-D, Shamsuddin-A-M. Cancer letters 1992 Jul 31, VOL: 65 (1), P: 9-13, ISSN: 0304-3835.

We have previously reported that phytic acid (inositol hexaphosphate or InsP6), a natural constituent of cereal diet, when administered in drinking water exerts a consistent antitumor effect on experimental colon cancer in vivo. The objective of this study was to determine whether InsP6 has similar anti-neoplastic effect on other tumor models, such as murine fibrosarcoma. We report that intraperitoneal injection of InsP6 reduces growth of subcutaneously transplanted fibrosarcoma (FSA-1) in mice, prolongs survival of tumor-bearing mice and reduces the number of pulmonary metastases. Since InsP6 is a common constituent of our diet and has very little or no toxic effects, in addition to being chemopreventive, it could have potential use in therapy of cancer as well.


Effects of inositol hexaphosphate on growth and differentiation in K-562 erythroleukemia cell line. Shamsuddin-A-M, Baten-A, Lalwani-N-D. Cancer letters 1992 Jul 10, VOL: 64 (3), P: 195-202, ISSN: 0304-3835.

Inositol hexaphosphate (InsP6) has recently been shown to inhibit experimental cancers in vivo. Since the lower phosphorylated forms of InsP6 are important in cell growth in a wide variety of mammalian cells, we tested the efficacy of InsP6 in growth reduction of K-562 human erythroleukemia cells in vitro. We report that InsP6 decreases the K-562 cell population by 19-36% (P less than 0.001) concomitant to an increased differentiation as evidenced by ultrastructural morphology and increased hemoglobin synthesis. Pilot experiments to study the mechanism of action of InsP6 show that following treatment with InsP6, the concentration of intracellular (Ca2+) ((Ca2+)i) is increased by 57% (P less than 0.02). Likewise, a 41% increase (P less than 0.05) in InsP3 and a 26% decrease (P less than 0.02) in InsP2 were noted 1 h following treatment with InsP6. Contrary to the dogma that cell division is associated with increased (Ca2+)i, our data show that reduced cell growth and enhanced differentiation is associated with increased (Ca2+)i and increased InsP3 in the presence of InsP6.


Phytate and colon-cancer risk. Shamsuddin-A-M. The American journal of clinical nutrition 1992 Feb, VOL: 55 (2), P: 478, ISSN: 0002-9165.

Dose-dependent inhibition of large intestinal cancer by inositol hexaphosphate in F344 rats. Ullah-A, Shamsuddin-A-M. Carcinogenesis 1990 Dec, VOL: 11 (12), P: 2219-22, ISSN: 0143-3334.

We have previously reported that inositol hexaphosphate (InsP6) inhibits mitosis and large intestinal cancer (LIC) in F344 rats and CD1 mice when given as 1 or 2% solution in drinking water at the unadjusted pH of 11.3. The purpose of this study was to determine whether InsP6 (i) shows a dose-response inhibition of LIC, and (ii) retains its anti-neoplastic effect at physiological pH. Since InsP6 is known to be a chelator of divalent cations, in preparation for putative clinical trials in humans, we also looked at the mineral bioavailability. F344 rats were fed 0.1% (pH 10.8), 1% (pH 11.3) and 1% (pH 7.4) Na-InsP6 in drinking water. Two weeks following the beginning of InsP6 supplementation, rats were given six injections of azoxymethane (AOM) at a dose of 8 mg/kg body wt/week and were killed 30 weeks following the last injection. Compared to the untreated control rats injected with AOM, 1% InsP6 (pH 11.3) reduces tumor prevalence by 52.2% (P less than 0.01), tumor frequency by 55.8% (P = 0.001) and tumor size by 62.3% (P = 0.001); 0.1% InsP6 showed a lesser reduction in tumor prevalence (21%) but a greater reduction in tumor size 71% (P = 0.001). While there was no significant difference in tumor prevalence and frequency between the two pH groups, the tumor size following 1% InsP6 (pH 7.4) was the smallest (65% smaller than those of pH 11.3, P less than 0.005). There was no significant difference in the serum Mg2+, Ca2+, Fe2+ and Zn2+ level between control rats and those treated with 1% InsP6. We therefore demonstrate that InsP6 (i) is consistently anti-neoplastic for LIC in a dose-dependent manner, (ii) retains its anti-neoplastic activity at physiological pH and (iii) has no demonstrable toxic effect on long- term administration as evident by body wt data and serum mineral levels.


Inositol-phosphate-induced enhancement of natural killer cell activity correlates with tumor suppression. Baten-A, Ullah-A, Tomazic-V-J, Shamsuddin-A-M. Carcinogenesis 1989 Sep, VOL: 10 (9), P: 1595-8, ISSN: 0143-3334.

In recent studies, we have demonstrated that inositol hexaphosphate (InsP6) inhibits experimental colon carcinogenesis. Since natural killer (NK) cells are involved in tumor cell destruction, we investigated the effect of InsP6 on murine NK cell activity. We show that; (i) 1,2-dimethylhydrazine (DMH), a colon carcinogen, depresses NK activity; (ii) in vivo treatment of mice with InsP6 enhances baseline NK activity and reverses DMH-induced depressed NK activity with an inverse correlation (r = -0.9811) with tumor incidence, (iii) short-term in vitro treatment of spleen cells and NK-enriched fraction with InsP6 also enhances NK cytotoxicity in a dose-dependent manner, (iv) inositol potentiates the action of InsP6. Our data suggest yet another important role of inositol phosphates in the regulation of cellular activity.


Inositol and inositol hexaphosphate suppress cell proliferation and tumor formation in CD-1 mice. Shamsuddin-A-M, Ullah-A, Chakravarthy-A-K. Carcinogenesis 1989 Aug, VOL: 10 (8), P: 1461-3, ISSN: 0143-3334.

In previous studies, we have shown that inositol hexaphosphate (Ins P6), a constituent of cereal diet, inhibited azoxymethane-induced experimental large intestinal cancer (LIC) in Fischer 344 rats. We now report a similar antineoplastic action of InsP6 in CD-1 mice injected with 1,2-dimethylhydrazine (DMH). We had hypothesized that InsP6 may bring about this effect by undergoing dephosphorylation to lower phosphorylated forms; the ready availability of Ins, to react with phosphates, may increase the total amount of the lower phosphorylated Ins and potentiate the action of InsP6. LIC induced by DMH (15 mg/kg/week x 13) in mice given a mixture of 1% InsP6 + 1% Ins show a significant reduction (P less than 0.005) in LIC prevalence over InsP6 treatment. Surprisingly, Ins, an in vitro growth promoting agent also caused a significant (P less than 0.001) suppression of LIC. InsP6 +/- Ins also showed a concomitant reduction in the mitotic rate in the non-neoplastic epithelium. Body weight data did not suggest any overt toxic effect of long-term administration of InsP6, Ins or InsP6 + Ins. Since InsP6 is antineoplastic in two species of experimental animals, it should, in combination with Ins, be considered in our strategies for prevention of large intestinal cancer.


Inositol hexaphosphate inhibits large intestinal cancer in F344 rats 5 months after induction by azoxymethane. Shamsuddin-A-M, Ullah-A. Carcinogenesis 1989 Mar, VOL: 10 (3), P: 625-6, ISSN: 0143-3334.

A treatment regimen of 2% Na-InsP6 in drinking water was effective in significantly reducing large intestinal cancer in F344 rats even when the treatment was begun 5 months after carcinogenic induction with azoxymethane (AOM 8 mg/kg/wk X 6). Compared to untreated (AOM-only) rats, animals on InsP6 had 27% fewer tumors (P less than 0.02). The tumors were approximately two-thirds smaller in size (P less than 0.01) and percentage mitotic rate in the non-neoplastic epithelium was less than half (1.0 +/- 0.1, compared to 2.3 +/- 0.2 of AOM-only animals, significant at P less than 0.001). We postulate that InsP6 may exert its antineoplastic effect by way of regulating cellular proliferation even after effective carcinogenic stimuli and thus may be an important candidate for chemointervention.


Suppression of large intestinal cancer in F344 rats by inositol hexaphosphate. Shamsuddin-A-M, Elsayed-A-M, Ullah-A. Carcinogenesis 1988 Apr, VOL: 9 (4), P: 577-80, ISSN: 0143-3334.

Epidemiological data demonstrate correlations between dietary factors and the incidence of large intestinal cancer (LIC). Certain high- fiber diets are associated with a lower risk of LIC; these high-fiber diets are also rich in inositol hexaphosphate (IP6 or phytic acid). In a pilot study, we have used F344 rats to investigate the effect of sodium inositol hexaphosphate (Na-IP6) prior to (experiment I) and following injections of the carcinogen azoxymethane (AOM) (experiment II). In experiment I, rats started on 1% Na-IP6 in drinking water 1 week prior to the carcinogen treatment showed a 34.7% decrease (P less than 0.01) in LIC compared to control carcinogen treatment group. A similar reduction in the incidence of LIC was also observed in experiment II, wherein Na-IP6 supplementation was started 2 weeks following the last dose of the carcinogen. Comparison of the incidence of mitosis in the colonic crypts of the animals in different groups show that animals on AOM + IP6 demonstrate a significantly lower (P less than 0.001) mitotic rate than those receiving AOM only. Pilot studies of free radical generation demonstrate a reduction in .OH radical formation by Na-IP6. Further studies to expand this pilot data and to understand the mechanism of IP6 mediated LIC suppression are needed for it may have significance in our strategies for LIC control.