G0/G1 arrest and S phase inhibition of human cancer cell lines by inositol hexaphosphate (IP6).

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.

Absorption and excretion of orally administered inositol hexaphosphate (IP(6) or phytate) in humans.

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.

Usefulness of galactose oxidase-Schiff test in rectal mucus for screening of colorectal malignancy.

Based on a "field-effect" theory in colon carcinogenesis, and the expression of the disaccharide tumor marker D galactose-beta-[1-->3]-N-acetyl-D-galactosamine (Gal-GalNAc) in the rectal mucus of patients with cancer and precancer of the colon, Shamsuddin developed a simple, accurate, inexpensive, easy to perform and rapid (< or = 15 min) screening test for colonic cancer and precancerous lesions. In this study we examined 137 rectal mucus samples of randomly selected patients with colorectal malignancy or other colorectal diseases to confirm the sensitivity and specificity of this test in Croatia. Additionally, to test the validity of the "field-effect" theory, that the mucosa away from the obvious cancer will show abnormalities as a result of the generalized effect of the carcinogen throughout the entire field of the target tissue, we also monitored a subset of 53 patients post-operatively. Individuals free of colonic or any other malignancies served as control (n = 31). The rectal mucin was smeared on membrane filter and developed by a sequential reaction of galactose oxidase (GO) and Schiff's reagent. The test results were correlated to the findings from colonoscopy/surgery and histopathology. The sensitivity of the test was shown to be 100% and the specificity was 96.8% (p < 0.001). Interestingly, the test was positive in 60% (32 of 53) of the samples collected from patients after tumor resection, showing the persistence of the biochemical changes even though malignant tumors were removed, hence supporting the field-effect phenomenon of carcinogenic stimuli. Five patients out of these 32 (16%) postoperative cases with positive GO test had a tumor recurrence within a year (0.05 < p < 0.10), suggesting that a persistently positive GO test in this population may serve as a predictor of tumor recurrence. We conclude that Gal-GalNAc is an early and intermediate biomarker, suitable not only for the detection of malignancy in its inception, but also for monitoring of people at high risk for cancer, and the efficacy of the cancer therapy as well as secondary prevention by this technology.

Comparison of different techniques for detection of Gal-GalNAc, an early marker of colonic neoplasia.

The tumor marker, D-galactose-beta [1-3]-N-acetyl-D-galactosamine (Gal-GalNAc, also known as T-antigen) can be identified by a very simple galactose oxidase-Schiff's (GOS) reaction either on tissues or on rectal mucus samples from patients with colorectal neoplasms. Gal-GalNAc is expressed in the neoplastic mucosa as well as the remote non-neoplastic mucosa. It is, however, not expressed in colonic mucosa of normal subjects. We studied the expression of Gal-GalNAc by GOS reaction, lectin reactivity and immunocytochemistry in 10 normal, .45 precancerous [5 Crohn's disease, 15 ulcerative colitis (5 without dysplasia and 10 with dysplasia), 25 tubular adenomas], and 25 adenocarcinoma cases. Normal mucosa remote from tubular adenoma and adenocarcinoma was also studied. The GOS method was compared with reactivity of the lectin jacalin and immunostaining with antibody to T antigen (Anti-Tag Ab). GOS reaction was negative in all of the 10 normal specimens. Of the 5 Crohn's disease specimens, 2 were positive and 3 negative. In the 5 ulcerative colitis cases without dysplasia, positive reaction was seen in 2 cases and negative in 3. Of the 10 cases of ulcerative colitis with dysplasia, 5 showed positivity in dysplastic areas, and 3 of these were also positive in remote non dysplastic mucosa. Twenty of 25 tubular adenomas yielded a positive reaction in the adenoma, 14 of them showing positivity also in remote mucosa; 3 cases showed a positive reaction only in remote mucosa. Of the 25 adenocarcinomas, 21 showed a positive reaction in the adenocarcinoma as well as the remote mucosa. GOS reaction was intense in well differentiated adenocarcinoma and weak in poorly differentiated adenocarcinoma. Intense reaction was also seen in the intracellular mucus of some aberrant crypts and morphologically normal crypts remote from adenocarcinoma and tubular adenoma. GOS reaction showed an overall sensitivity of 75.7% and specificity of 100% for cancer and precancerous lesions. Jacalin reactivity was slightly more sensitive (84.3%) but less specific (80%) and Tag Ab reactivity even less sensitive (50%) but as specific (100%) for neoplastic and dysplastic mucosa. We conclude that the detection of the carbohydrate moiety Gal-GalNAc varies with the technique used. Compared to other techniques, GOS reaction is extremely simple and has a high degree of sensitivity and specificity. It can be used for detection of this tumor marker in remote non-neoplastic mucosa of patients with neoplasia or at risk of developing neoplasia. It, therefore, could be used as a cost effective screening test in rectal biopsy specimens of such patients.

Mammary tumor inhibition by IP6: a review.

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.

Antiplatelet activity of inositol hexaphosphate (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.

Metabolism and cellular functions of IP6: a review.

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.

Inhibition of skin cancer by IP6 in vivo: initiation-promotion model.

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.

Novel anticancer function of inositol hexaphosphate: inhibition of human rhabdomyosarcoma in vitro and in vivo.

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.

Up-regulation of the tumor suppressor gene p53 and WAF1 gene expression by IP6 in HT-29 human colon carcinoma cell line.

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.

IP6 in treatment of liver cancer. I. IP6 inhibits growth and reverses transformed phenotype in HepG2 human liver cancer cell line.

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.

IP6 in treatment of liver cancer. II. Intra-tumoral injection of IP6 regresses pre-existing human liver cancer xenotransplanted in nude mice.

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: a novel anti-cancer agent.

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.

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.

Novel anti-cancer functions of IP6: growth inhibition and differentiation of human mammary cancer cell lines in vitro.

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.

Gal-GalNAc: a biomarker of colon carcinogenesis.

The disaccharide tumor marker Gal-GalNAc visualized by galactose oxidase-Schiff sequence is commonly present in cancer cells and in rectal mucous of patients with colon cancer. The expression of this marker on tissue sections taken during experimental colon carcinogenesis shows excellent correlation with human precancerous lesions and even higher percentage of colon cancers express this marker, whereas, no expression is seen in the normal human large intestine. Multifocal expression of the marker is seen throughout the entire colon of patients with precancer and cancer; these include dysplasia, dilated and distorted crypts, regenerative dysplasia and hyperplastic crypts, as well as the morphologically normal crypts remote from cancer. Nearly identical pattern of Gal-GalNAc expression throughout the entire colon also appear during rat colon carcinogenesis induced by azoxymethane including non-expression by the normal and regenerative epithelia during wound healing following mechanical injury. Thus, Gal-GalNAc detected by the simple technique of galactose oxidase-Schiff sequence, is a biomarker that appears during the very early stages of progression of carcinogenesis. The expression pattern supports the field effect theory of carcinogenesis and also explains the basis for mass screening for cancer and precancerous conditions. Chemoprevention strategy using Gal-GalNAc as an intermediate marker detected by accurate and cost-effective rectal mucus test may have great potential.

A simple mucus test for cancer screening.

Comparative and correlative studies of the pathology and pathogenesis of colon cancer in animal models and human disease have resulted in conceptualization of 'field effect" theory and identification of markers that are expressed early during carcinogenesis. This assimilated body of knowledge has resulted in development of a simple rectal mucus test for colon cancer screening. The marker galactose-N acetylgalactosamine (Gal-GalNAC) is expressed in the rectal mucus of patients with colonic cancer or precancerous lesions and is detected by enzymatic oxidation (10 minutes) followed by color reaction (1 minute). The high sensitivity, specificity, positive predictive value and negative predictive value, as well as the cost-effectiveness of this test makes it a great tool in our strategies for early detection, hence control of colon cancer. Because of its high accuracy (as opposed to the fecal occult blood tests), it would reduce the number of unnecessary colonoscopies, thereby decreasing the total national health-care cost to the society. Similar expression of this marker in cancers of breast, lungs, prostate, pancreas, makes it a potentially useful general cancer screening test.

IP6-induced growth inhibition and differentiation of HT-29 human colon cancer cells: involvement of intracellular inositol phosphates.

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.

Inositol hexaphosphate inhibits growth and induces differentiation of PC-3 human prostate cancer cells.

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.

A new murine monoclonal antibody, CMU10, as a marker for colonic carcinoma and precancerous conditions.

OBJECTIVE: To investigate the usefulness of a cancer-associated mucin antigen using a new monoclonal antibody, CMU10, as a tumor marker. Expression was assessed in normal adult, fetal, hyperplastic, preneoplastic, and neoplastic tissues of colon. DESIGN: CMU10 monoclonal antibody recognizes a mucin antigen that is not expressed in normal fetal and adult large intestine, but is rather commonly expressed in cancerous and precancerous lesions. Immunocytochemical analysis was performed on human tissues obtained at surgery or at autopsy. RESULTS: Expression of this mucin antigen was observed in 27 (98.4%) of 28 cases of colorectal carcinoma, 24 (96%) of 25 cases of adenomatous polyps, 9 (75%) of 12 cases of ulcerative colitis, and 9 (100%) of 9 cases of hyperplastic polyps. In cancerous specimens, the antigen distribution was mainly localized in the secretory mucin and surface membrane, whereas in precancerous lesions it was located in dysplastic, dilated, and distorted crypts. CONCLUSIONS: Because CMU10 differentially recognizes an antigen in cancerous and precancerous tissues, but not in normal tissues, it may be useful as a tumor marker for immunodiagnosis and, hence, early detection.