A significant anticancer activity of the naturally occurring carbohydrate inositol hexaphosphate (IP(6)) has been reported against numerous cancer models. Since tumors require angiogenesis for growth and metastasis, we hypothesize that IP(6) reduces tumor growth by inhibiting angiogenesis. Because angiogenesis depends on the interaction between endothelial and tumor cells, we investigated the effect of IP(6) on both. IP(6) inhibited the proliferation and induced the differentiation of endothelial cells in vitro; the growth of bovine aortic endothelial cells (BAECs) evaluated by MTT proliferation assay was inhibited in a dose-dependent manner (IC(50) = 0.74 mM). The combination of IP(6) and vasostatin, a calreticulin fragment with anti-angiogenic activity, was synergistically superior in growth inhibition than either compound. IP(6) inhibited human umbilical vein endothelial cell (HUVEC) tube formation (in vitro capillary differentiation) on a reconstituted extracellular matrix, Matrigel, and disrupted pre-formed tubes. IP(6) significantly reduced basic fibroblast growth factor (bFGF)-induced vessel formation (P < 0.01) in vivo in Matrigel plug assay. Exposure of HepG2, a human hepatoma cell line, to IP(6) for 8 h, resulted in a dose-dependent decrease in the mRNA levels of vascular endothelial growth factor (VEGF), as assessed by RT-PCR. IP(6) treatment of HepG2 cells for 24 h also significantly reduced the VEGF protein levels in conditioned medium, in a concentration-dependent manner (P = 0.012). Thus, IP(6) has an inhibitory effect on induced angiogenesis.

BACKGROUND:

We have shown that inositol hexaphosphate (IP6), a natural compound and a potent anti-cancer agent, inhibited cancer cell adhesion to the extracellular matrix (ECM) proteins, thereby leading to inhibition of cell migration and invasion. Cell adhesion to ECM is mediated by specific cell surface integrins, which transduce intracellular signals through their interaction and activation of other proteins that are recruited to the focal adhesion. We hypothesize that IP6 decreases cell adhesion by suppressing the integrin receptors and their subsequent signaling pathway.

MATERIALS AND METHODS:

We analyzed integrin expressions of the highly invasive estrogen receptor-negative human breast cancer MDA-MB 231 cells exposed to IP6 by flow cytometry. The expression of focal adhesion proteins was investigated by immunocytochemistry and Western blotting.

RESULTS:

IP6 treatment caused a significant (P < 0.005) decrease in the expression of integrin heterodimers alpha 2 beta 1 (collagen receptor), alpha 5 beta 1 (fibronectin receptor) and alpha v beta 3 (vitronectin receptor); flow cytometry showed that it was the alpha 5 subunit that was down-regulated ( < 0.001). However, the expression of the alpha 2, alpha v, beta 1 and beta 3 subunits were not affected by IP6 treatment. When the expression of integrins on the cell surface was assessed, there was a dramatic 82% decrease in the expression of alpha 5 beta 1 on IP6-treated cells (P < 0.0001), indicating a decrease in cell surface expression of the heterodimers. No effect was seen when inositol hexasulfate (IS6), an analogue of IP6, was used as a control. Immunocytochemistry showed a lack of clustering of paxillin; tyrosine-phosphorylated proteins in IP6-treated cells were discontinuous and scattered around the cell periphery, whereas the patterns were more dense and localized in control cells. Consistent with these observations, focal adhesion kinase (FAK) autophosphorylation at tyrosine-397 residue was suppressed, albeit modestly, by IP6 treatment, suggesting a down-regulation in the integrin-mediated signaling pathway.

CONCLUSION:

The results of this study indicate that IP6-induced inhibition of cancer cell adhesion, migration and invasion may be mediated through the modulation of integrin dimerization, cell surface expression and integrin-associated signaling pathway.

BACKGROUND:

The anti-cancer agent inositol hexaphosphate (IP6) is an abundant intrinsic component of both plant and mammalian cells. In addition to inducing differentiation and inhibiting growth of numerous cancer cell lines in vitro, IP6 has been demonstrated to prevent and abrogate both primary tumor and metastasis in vivo.

MATERIALS AND METHODS:

Using MDA-MB 231 human breast cancer cells, we studied the potential of IP6 to inhibit cell adhesion, migration and invasion, the key steps in cancer metastasis, utilizing the extracellular matrix (ECM) proteins, a culture wounding assay, modified Boyden chambers, immunocytochemistry and zymography.

RESULTS:

IP6 treatment caused a 65% reduction of cell adhesion to fibronection (p = 0.002) and a 37% reduction to collagen (p = 0.005). To determine whether a decrease in cell adhesion leads to a decrease in cell motility, migration assays were performed; IP6 decreased both the number of migrating cells and the distance of cell migration into the denuded area by 72% (p < 0.001). Haptotatic cell migration in a modified Boyden chambers was also reduced in a dose-dependent manner. While cell migration on fibronectin was inhibited by 65% (p < 0.001), migration on collagen and laminin was decreased by 32% (p < 0.01) and 13% (p < 0.05), respectively. Immunocytochemistry revealed the absence of lamellipodia structure in IP6-treated cells as compared to untreated cells, corresponding to a diminished ability of cancer cells to form cellular network as determined by Matrigel outgrowth assay. Likewise, cell invasion also was decreased (by 72% after IP6 treatment, p = 0.001) in a dose-dependent fashion. Additionally, IP6 significantly (p = 0.006) inhibited the secretion of matrix metalloproteinase (MMP)-9 as assessed by zymography.

CONCLUSION:

The results of this study show that IP6 inhibits the metastasis of human breast cancer cells in vitro through effects on cancer cell adhesion, migration and invasion.

Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate that is present in substantial amounts in almost all plant and mammalian cells. It was recently recognized to possess multiple biological functions. A striking anticancer effect of IP6 was demonstrated in different experimental models. Inositol is also a natural constituent possessing moderate anticancer activity. The most consistent and best anticancer results were obtained from the combination of IP6 plus inositol. In addition to reducing cell proliferation, IP6 increases differentiation of malignant cells, often resulting in a reversion to normal phenotype. Exogenously administered IP6 is rapidly taken into the cells and dephosphorylated to lower-phosphate inositol phosphates, which further interfere with signal transduction pathways and cell cycle arrest. Enhanced immunity and antioxidant properties can also contribute to tumor cell destruction. However, the molecular mechanisms underlying this anticancer action are not fully understood. Because it is abundantly present in regular diet, efficiently absorbed from the gastrointestinal tract, and safe, IP6 holds great promise in our strategies for the prevention and treatment of cancer. IP6 plus inositol enhances the anticancer effect of conventional chemotherapy, controls cancer metastases, and improves the quality of life, as shown in a pilot clinical trial. The data strongly argue for the use of IP6 plus inositol in our strategies for cancer prevention and treatment. However, the effectiveness and safety of IP6 plus inositol at therapeutic doses needs to be determined in phase I and phase II clinical trials in humans.

The current treatment of breast carcinomas recognizes the importance of combination therapy in order to increase efficacy and decrease side effects of conventional chemotherapy. Inositol hexaphosphate (IP6), a naturally occurring polyphosphorylated carbohydrate, has shown a significant anti-cancer effect in various in vivo and in vitro models, including breast cancer. In this study, we investigated the in vitro growth inhibitory activity of IP6 in combination with adriamycin or tamoxifen, against three human breast cancer cell lines: estrogen receptor (ER) alpha-positive MCF-7, ER alpha-negative MDA-MB 231 and adriamycin-resistant MCF-7 (MCF-7/Adr) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Much lower concentrations of IP6 were required after 96 h of treatment to inhibit the growth of MCF-7/Adr cells than MCF-7 cells; the IC50 for MCF-7/Adr cells was 1.26 mM compared to 4.18 mM for MCF-7 cells. The ER-negative MDA-MB 231 cells were also highly sensitive to IP6 with IC50 being 1.32 mM. To determine the effects of IP6 in combination with either adriamycin or tamoxifen, the median effect principle and Webb’s fraction method were used to determine the combination index (CI) and the statistical differences. Growth suppression was markedly increased when IP6 was administered prior to the addition of adriamycin, especially against MCF-7 cells (CI = 0.175 and p < 0.0001). Synergism was also observed when IP6 was administered after tamoxifen in all three cell lines studied (CI = 0.343, 0.701 and 0.819; p < 0.0001, p = 0.0003 and 0.0241 for MCF-7/Adr, MCF-7 and MDA-MB 231, respectively). The growth of primary culture of breast cancer cells from patients was inhibited by IP6 with LC50 values ranging from 0.91 to 5.75 mM (n = 10). Our data not only confirm that IP6 alone inhibits the growth of breast cancer cells; but it also acts synergistically with adriamycin or tamoxifen, being particularly effective against ER alpha-negative cells and adriamycin-resistant cell lines.

BACKGROUND:

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.

METHODS:

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.

RESULTS:

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).

CONCLUSION:

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.

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.

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.

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.

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.

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.

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, HepG2 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.

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.

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.

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.

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 (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.

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 InsP3 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 natúral 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.