Supplement: International Research Conference on Food, Nutrition,
*Department of Medical and Research Technology and Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201
3 To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
KEY WORDS: • prevention • treatment • differentiation • phytic acid
Cancer remains a major health problem in the United States and in other developed countries (1). In our continuing effort to reduce the public health burden of cancer, there is a constant search for more effective cancer treatment, and increased interest in the concept of prevention, as a promising approach to the control of cancer (2).
A novel anticancer function of inositol hexaphosphate (IP6;4 also InsP6 and phytic acid) has been shown both in vivo and in vitro (3–5). IP6 is a polyphosphorylated carbohydrate, contained in high concentrations (0.4–6.4%) in cereals and legumes (6). Myo-inositol is a parent compound of IP6. Only myo-inositol hexaphosphate has been found in plants; neo-, chiro-, and scyllo-inositol hexaphosphates have been isolated from soil (7). The phosphate grouping in positions 1, 2, and 3 (axial-equatorial-axial) is unique for IP6, providing a specific interaction with iron to completely inhibit its ability to catalyze hydroxyl radical formation, making IP6 a strong antioxidant, probably still the only role of IP6 that is widely recognized and accepted.
Almost all mammalian cells contain IP6 and much smaller amounts of its forms with fewer phosphate groups (IP1-5), which are important for regulating vital cellular functions. Inositol occurs ubiquitously in cell membranes in conjugation with lipids, as phosphatidylinositol. Recently, inositol phospholipids in the plasma membrane have received much attention because of their biological significance for signal transduction systems. Phosphatidylinositol 4,5-bisphosphate (PIP2), a phosphoinositide, is a precursor for several informational molecules in signal transduction—inositol 1,4,5-P3 (IP3), 1,2-diacylglycerol, and phosphatidylinositol 3,4,5-trisphosphate—linking receptor stimulation to Ca2+ mobilization (8). A second messenger role in intracellular Ca2+ homeostasis for IP4 was also shown. It is now recognized that subsequent to PIP2 hydrolysis a cascade of inositol phosphate metabolites are formed and that these multiple isomers show a complex pattern of interconversion (8–10). Inositol phosphates are versatile molecules with important roles in controlling diverse cellular activities (9,10). IP6 may serve as a natural antioxidant (11) and possibly as a neurotransmitter (10). Different binding proteins for inositol polyphosphates have been isolated, indicating their importance for the cellular functions (12) such as effects on ion channels and protein trafficking (13,14), endocytosis (15), exocytosis (16), and efficient export of mRNA from the nucleus to the cell (17).
How can exogenously administered IP6 affect tumor growth? Pioneering experiments showing this novel anticancer feature of IP6 were performed by Shamsuddin et al. (18–20), who were intrigued by the epidemiologic data indicating that only diets containing a high IP6 content (cereals and legumes) showed a negative correlation with colon cancer. Almost 15 y ago, Shamsuddin et al. hypothesized that IP6 can be internalized by the cells and dephosphorylated to IP1-5 and then can enter into the intracellular inositol phosphate pool and inhibit tumor growth. It was also hypothesized that the addition of inositol, a precursor of inositol phosphates and also a natural carbohydrate, to IP6 may enhance the anticancer function of IP6 (18–20). Because inositol phosphates are common molecules involved in signal transduction in most mammalian cell systems, it was further hypothesized that the anticancer action of inositol phosphates would be observed in different cells and tissue systems (18–20). All these proposed hypotheses have been confirmed.
Contrary to the dogma and skepticism at that time, we showed that IP6 is taken up by malignant cells (21) and that orally administered IP6 can reach target tumor tissue distant from the gastrointestinal tract (22). Because of the highly charged nature of IP6, it was a common misconception that it could not be transported into the cells. Analyzing absorption, intracellular distribution, and metabolism of IP6 in HT-29 human colon carcinoma and cells of hematopoietic lineage (K-562, human erythroleukemia and YAC-1, mouse lymphoma cells), we found that IP6 is rapidly taken up by mechanisms probably involving pinocytosis or receptor-mediated endocytosis, transported intracellularly, and dephosphorylated into inositol phosphates with fewer phosphate groups (21). Similar data were obtained when MCF-7 human breast cancer cells were incubated with [3H]-IP6 (SA 444 GBq/mmol, 370 Bq/106 cells): as early as 1 min after incubation, 3.1% of IP6-associated radioactivity was taken up by MCF-7 cells, and 9.5% after 1 h. By differential centrifugation 86% radioactivity was recovered from the cell cytosol. Anion-exchange chromatography showed that 58% of the absorbed radioactivity was in IP6 form. When [3H]-IP6 was administered intragastrically to rats, it was quickly absorbed from the stomach and upper intestine and distributed to various organs as early as 1 h after administration (22). Although the radioactivity isolated from gastric epithelium at this time was associated with inositol and IP1-6, the radioactivity in the plasma and urine was associated with inositol and IP1. These data indicate that the intact molecule was transported inside the gastric epithelial cells, wherein it was rapidly dephosphorylated, and that the metabolism of IP6 was very rapid. In our preliminary studies, [3H]-IP6 was given via oral gavage to rats bearing 7,12-dimethylbenz[a]anthracene-induced mammary tumors. A substantial amount of radioactivity (19.7% of all radioactivity recovered in collected tissues) was found in tumor tissue as early as 1 h after administration, providing at least partial explanation for the antineoplastic activity of IP6 at sites distant from the gastrointestinal tract. In this study only 50% of the radioactivity was excreted in urine within 72 h after administration; in addition feces accounted for another 10% of radioactivity, suggesting that at least 40% of the IP6-associated radioactivity was distributed within the animal tissues. These data indicate that IP6 can reach and concentrate at cellular targets. Chromatographic analysis of tumor tissue revealed the presence of inositol and IP1, similar to plasma.
Using a novel and highly sensitive method combining gas chromatography–mass spectrometry analysis and HPLC, Grases et al. (23,24) were able to identify IP6 in human urine and plasma and detect IP6 and its less-phosphorylated forms (IP3-5) in mammalian cells and in body fluids as they occur naturally. They also showed that the levels of IP6 and its less phosphorylated forms fluctuate depending on the intake of IP6.
That the extracellularly applied IP6 enters the cell and that this intracellular delivery is followed by a dephosphorylation of IP6 was recently confirmed by Ferry et al. (25).
Anticancer action of IP6
As hypothesized, it was demonstrated that IP6 is a broad-spectrum antineoplastic agent, affecting different cells and tissue systems. In vitro studies with IP6 are summarized in Table 1.
The cancer preventive activity of IP6 in vitro was first tested in a benzo[a]pyrene-induced transformation in the rat tracheal cell culture transformation assay (30) and then was tested in a model using BALB/c mouse 3T3 fibroblasts (37) with modest efficacy. The observation that IP6 impaired the transformation induced by epidermal growth factor or phorbol ester in JB6 (mouse epidermal) cells (35) strongly suggested the potential role of IP6 as a cancer preventive agent, because this model has been a well-characterized cell system for studying the tumor promotion and molecular mechanisms of antitumor agents. Furthermore, IP6 reduced 12-O-tetradecanoylphorbol-13-acetate–induced ornithine decarboxylase activity, an essential event in tumor promotion in HEL-30 cells, a murine keratinocyte cell line (36).
A summary of in vivo studies using IP6 and inositol is shown in Table 2. Although experts in the field of nutrition and cancer have been performing in vivo experiments by adding IP6 to the diet, in all our cancer prevention studies, IP6 was given via drinking water in concentrations ranging from 0.4% to 2.0%. We were able to obtain comparable or even stronger tumor inhibition with much lower concentrations of IP6 when it was given in drinking water. For example, much stronger tumor inhibition was achieved with 0.4% IP6 in drinking water compared with the same amount given in a 20% high fiber diet (52).
Myo-inositol itself was also demonstrated to have anticancer function, albeit modest. It inhibited pulmonary adenoma formation in mice (49,50). We found that inositol alone or in combination with IP6 can prevent the formation and incidence of several cancers in experimental animals: in soft tissue, colon, metastatic lung, and mammary cancers. Additionally, we showed that inositol potentiates both the antiproliferative and antineoplastic effects of IP6 in vivo (3–5,19,39,51,52). Synergistic cancer inhibition by IP6 when combined with inositol was observed in colon cancer (Table 3) (19) and mammary cancer studies (Table 4) (51,52). Similar results were seen in the metastatic lung cancer model (39). Thus, the combination of IP6 and inositol was significantly better in different cancers than was either one alone.
IP6 can also modulate cellular response at the level of receptor binding. IP6, after sterically blocking the heparin-binding domain of basic fibroblast growth factor, disrupted further receptor interactions (58). This modulation in binding and the activity of basic fibroblast growth factor is thought to be due to the chair conformation of IP6 mimicking that of the pyranose ring structure in heparin (58).
The observed anticancer effect of inositol compounds could be mediated through several other mechanisms. The antioxidant role of IP6 is known and widely accepted; this function of IP6 occurs by chelation of Fe3+ and suppression of ·OH formation (11). Therefore, IP6 can reduce carcinogenesis mediated by active oxygen species and cell injury via its antioxidative function. This activity seems to be closely related to its unique structure. The phosphate grouping in positions 1,2,3 (axial-equatorial-axial) is unique to IP6, specifically interacting with iron to completely inhibit its ability to catalyze hydroxyl radical formation, making IP6 a strong antioxidant. This anticancer action of IP6 may be further related to mineral binding ability; IP6 by binding with Zn2+ can affect thymidine kinase activity, an enzyme essential for DNA synthesis, or remove iron, which may augment colorectal cancer (3–5,41,46).
Besides affecting tumor cells, IP6 can act on a host by restoring its immune system. IP6 augments natural killer cell activity in vitro and normalizes the carcinogen-induced depression of natural killer cell activity in vivo (59).
Value of IP6 as a therapeutic and preventive agent for cancer
Safety. IP6 is a natural compound and an important dietary component. Some concerns have been expressed regarding the mineral deficiency that results from an intake of foods high in IP6 that might reduce the bioavailability of dietary minerals. However, recent studies demonstrate that this antinutrient effect of IP6 can be manifested only when large quantities of IP6 are consumed in combination with a diet poor in oligoelements (60–63). A long-term intake of IP6 in food (60,61) or in a pure form (64) did not cause such a deficiency in humans. Studies in experimental animals showed no significant toxic effects on body weight, serum, or bone minerals (Table 5) or any pathological changes in either male F344 or female Sprague-Dawley rats for 40 wk (40,51,52). Grases et al. (65) confirmed our findings and also reported that abnormal calcification was prevented in rats given IP6.
IP6 affects principal pathways of malignancy. Our goal is to identify agents that can target tumors at vulnerable sites and interrupt specific pathways of carcinogenesis. From the behavior and characteristics of malignant cells, several principal pathways of malignancy have been established, such as proliferation, cell cycle progression, metastases and invasion, angiogenesis, and apoptosis; interestingly, IP6 targets and acts on all of them.
Uncontrolled proliferation is a hallmark of malignant cells, and IP6 can reduce the cell proliferation rate of many different cell lines of different lineage and of both human and rodent origin (3–5,26,28,31–33,38). Although normal cells divide at a controlled and limited rate, malignant cells escape from the control mechanisms that regulate the frequency of cell multiplication and usually have lost the checkpoint controls that prevent replication of defective cells. IP6 can regulate the cell cycle to block uncontrolled cell division and force malignant cells either to differentiate or go into apoptosis. IP6 induces G1 phase arrest and a significant decrease of the S phase of human breast (68,69), colon (69), and prostate (34) cancer cell lines. However, IP6 causes the accumulation of human leukemia cells in the G2M phase of the cell cycle; a cDNA microarray analysis showed a down-modulation of multiple genes involved in transcription and cell-cycle regulation by IP6 (27).
One important characteristic of malignancy is the ability of tumor cells to metastasize and infiltrate normal tissue. A significant reduction in the number of lung metastatic colonies by IP6 was observed in a mouse metastatic tumor model using FSA-1 cells (39). Using highly invasive MDA-MB 231 human breast cancer cells, we demonstrated that IP6 inhibits metastasis in vitro through effects on cancer cell adhesion, migration, and invasion (70,71). Tumor cells emit substances known as matrix metalloproteinases that allow metastatic cells to pass into the blood vessels; IP6 significantly inhibited secretion of MMP-9 from MDA-MB 231 cells (70).
Tumors depend on the formation of new blood vessels to support their growth and metastasis. Many tumors produce large amounts of vascular endothelial growth factor, a cytokine that signals normal blood vessels to grow. IP6 inhibited the growth and differentiation of endothelial cells (66,72) and inhibited the secretion of vascular endothelial growth factor from malignant cells (27,66,72). IP6 can also adversely affect angiogenesis as antagonist of fibroblast growth factor (58).
Apoptosis is a hallmark of action of many anticancer drugs. It has been reported that IP6 induces apoptosis in vivo (45) and in vitro in prostate (34) and cervical cancer (25) cell lines, involving cleavage of caspase 3, caspase 9, and poly ADP-ribose polymerase, an apoptotic substrate, in a time- and dose-dependent manner.
Effectiveness of IP6 as a cancer preventive agent. Possible mechanisms of the cancer preventive action of IP6 include carcinogen blocking activities, antioxidant activities, and antiproliferation and antiprogression activities (73). Therefore, the strategy of chemoprevention is to use agents that will inhibit mutagenesis, induce apoptosis, induce maturation and differentiation, and inhibit proliferation (74). The antioxidant activity of IP6 is widely accepted and indisputable (11), and IP6 possesses antiproliferative and antiprogression activities. Its induction of terminal differentiation (26,28,29,32,33,38), restoration of immune response (59), modulation of growth factors (58), modulation of signal transduction pathways (15,16,35,57), induction of apoptosis (25,34,45), and possibly inhibition of oncogene activity and restoration of tumor suppressor function are well documented. IP6 not only inhibits the activities of some liver enzymes (75,76) but also significantly increases the hepatic levels of glutathione S-transferase (44,77), both of which indicate its possible role in carcinogen-blocking activities and cancer protection.
Although IP6 may belong to almost all previously mentioned categories of cancer preventive drugs, affecting almost all phases of cancer prevention, it still appears that IP6 is not a direct antagonist to the carcinogen because of its moderate efficacy in vitro when tested and compared with other chemopreventive agents (30) and a lack of dramatic decrease in cancer incidence when tested in vivo. However, because cancer prevention is a long process, a long administration of cancer preventive agent is generally needed, requiring usually 10–40 y of continuous treatment (2,73), and, therefore, it is very important that cancer preventive agents have low or almost no toxicity. IP6, a natural compound with virtually no toxicity, can satisfy this special and very important requirement for cancer prevention.
IP6 plus inositol and patients
An enhanced antitumor activity without compromising the patient's quality of life was demonstrated in a pilot clinical trial involving six patients with advanced colorectal cancer (Dukes C and D) with multiple liver and lung metastasis (78). IP6 plus inositol was given as an adjuvant to chemotherapy according to Mayo protocol. One patient with liver metastasis refused chemotherapy after the first treatment, and she was treated only with IP6 plus inositol; her control ultrasound and abdominal computed tomography scan 14 mo after surgery showed a significantly reduced growth rate. A reduced tumor growth rate was noticed overall and in some cases a regression of lesions was noted. Additionally, when IP6 plus inositol was given in combination with chemotherapy, side effects of chemotherapy (drop in leukocyte and platelet counts, nausea, vomiting, alopecia) were diminished and patients were able to perform their daily activities (78). Further controlled randomized clinical trials are necessary to confirm these observations.
Other biological effects of IP6
In humans, IP6 not only has almost no toxic effects, but it has many other beneficial health effects such as inhibition of kidney stone formation and reduction in risk of developing cardiovascular disease. IP6 was administered orally either as the pure sodium salt or in a diet to reduce hypercalciuria and to prevent formation of kidney stones, and no evidence of toxicity was reported (64,65,79,80). A potential hypocholesterolemic effect of IP6 may be very significant in the clinical management of hyperlipidemia and diabetes (75,76,81). IP6 inhibits agonist-induced platelet aggregation (82) and efficiently protects myocardium from ischemic damage and reperfusion injury (83), both of which are important for the management of cardiovascular diseases.
Many potential beneficial actions of IP6 have been described. The inclusion of IP6 plus inositol in our strategies for prevention and treatment of cancer as well as other chronic diseases is warranted. However, the effectiveness and safety of IP6 plus inositol need to be determined in Phase I and Phase II clinical trials in humans.
2 Studies from the authors' laboratories were supported by the American Institute for Cancer Research, the Susan Komen Breast Cancer Foundation, the University of Maryland Designated Research Initiative Fund, and the University of Maryland Women Health Research Foundation.
4 Abbreviations used: Ins, inositol; IP6, inositol hexaphosphate; IP3, inositol 1,4,5-P3; KS, Kaposi's sarcoma; PIP2, phosphatidylinositol 4,5-bisphosphate.
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