Anti-angiogenic activity of inositol hexaphosphate (IP6)
Ivana Vucenik1,2,
Antonino Passaniti2,3,
Michele I. Vitolo3,
Kwanchanit Tantivejkul2,
Paul Eggleton4 and
AbulKalam M. Shamsuddin2,5
1 Department of Medical and Research Technology, 2 Department of Pathology and 3 Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA and 4 Peninsula Medical School, Universities of Exeter and Plymouth and MRC Immunochemistry Unit, University of Oxford, Oxford, UK
5 To whom correspondence should be addressed Email: shams{at}som.umaryland.edu
 |
Abstract
|
---|
A significant anticancer activity of the naturally occurring carbohydrate inositol hexaphosphate (IP6) has been reported against numerous cancer models. Since tumors require angiogenesis for growth and metastasis, we hypothesize that IP6 reduces tumor growth by inhibiting angiogenesis. Because angiogenesis depends on the interaction between endothelial and tumor cells, we investigated the effect of IP6 on both. IP6 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 (IC50 = 0.74 mM). The combination of IP6 and vasostatin, a calreticulin fragment with anti-angiogenic activity, was synergistically superior in growth inhibition than either compound. IP6 inhibited human umbilical vein endothelial cell (HUVEC) tube formation (in vitro capillary differentiation) on a reconstituted extracellular matrix, Matrigel, and disrupted pre-formed tubes. IP6 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 IP6 for 8 h, resulted in a dose-dependent decrease in the mRNA levels of vascular endothelial growth factor (VEGF), as assessed by RTPCR. IP6 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, IP6 has an inhibitory effect on induced angiogenesis.
Abbreviations: BAECs, bovine aortic endothelial cells; bFGF, basic fibroblast growth factor; CI, combination index; HIF, hypoxia-inducible factor; HRVT, HUVEC retrovirus telomerized; HUVEC, human umbilical vein endothelial cell; IP6, hexaphosphate; MBP, maltose-binding protein; PDN, P-domain of calreticulin (residues 181290); PI3K, phosphatidylinositol 3'-kinase; VEGF, vascular endothelial growth factor
 |
Introduction
|
---|
Inositol hexaphosphate (IP6, InsP6) is ubiquitous in plants, such as cereal and legumes, and is found in most mammalian cells at concentrations of 10 µM to 1 mM (1,2). Recognized as a strong antioxidant (3), IP6 has also been shown to play a critical role in neurotransmission (4), regulation of vesicle trafficking and recycling (5), and endo- and exocytosis (6). These activities are mediated by protein kinase C (6), modulation of calcium influx coupled with the inhibition of phosphatases (7), and efficient messenger RNA export (8,9). A novel anticancer function of IP6 has been demonstrated both in vivo and in vitro (1012), acting primarily via regulation of cell growth and cell differentiation (1014). The potential of IP6 to reduce tumor incidence and tumor load has been shown in rodent mammary tumors (1518), colon tumors (1923) and in other experimental models, including transplanted and metastatic fibrosarcoma (24), rhabdomyosarcoma (25) and hepatoma (26,27). IP6 is present abundantly in mammalian cells, is a component of a regular diet, is safe and efficiently absorbed from the gastrointestinal tract (1,28,29), and, therefore, represents an excellent preventive and therapeutic candidate for various tumors. However, all studies addressing the anticancer action of IP6 have been performed with malignant epithelial cells, with no published reports of the effect of IP6 on vascular endothelial cells and angiogenesis.
Angiogenesis is the process of blood vessel formation that occurs under physiological and pathological conditions; in particular, angiogenesis is associated with progressive growth and metastasis of solid tumors, which depend on recruitment of new blood vessels (3033). The induction of tumor vascularization is mediated by the release of angiogenic factors. Among these factors, vascular endothelial growth factor (VEGF) is thought to be the most specific (34). VEGF is secreted by tumor cells, and promotes the proliferation of endothelial cells and remodeling of neo-vessels. Since endothelial cells can communicate directly with tumor cells by producing growth-promoting factors, the inter-relationship between endothelial and tumor cells and the imbalance between angiogenic factors and angiogenic inhibitors can promote tumor vascularization. Identification of novel angiogenic inhibitors that target both the proliferating endothelial and tumor cell compartments may, therefore, lead to therapeutic regulation of tumor growth.
Several angiogenic inhibitors that regulate vascular endothelial cell proliferation by targeting angiogenic factors or their receptors have been developed (34,35). Thalidomide (3638) and TNP-470 (39,40), inhibitors of endothelial cell growth, are in clinical trial. VEGF, its receptor VEGFR-2, and novel small-molecule inhibitors of VEGFR-2, such as SU5416, are currently the targets of intense efforts to inhibit dysregulated blood vessel formation in cancer (35). Angiostatin, an internal fragment of plasminogen containing four kringle domains (41) and endostatin, a C-terminal fragment of collagen XVIII from hemangioendothelioma (42), are endogenous inhibitors of angiogenesis. Additionally, a number of compounds with anti-angiogenic activity have been derived from plants, such as genistein, isoliquitrin and ginsenoside. Recently, the anti-angiogenic potential of sylmarin, a polyphenolic flavonoid antioxidant isolated from milk thistle, Silybum marianum (L.) Gaertn (43), torilin, isolated from Torilis japonica (44) and catechins from green tea extracts (45) have been reported.
In our study, we evaluated the anti-angiogenic activity of IP6. We hypothesize that IP6 inhibits tumor angiogenesis by affecting both vascular endothelial cells and tumor cells. We report that IP6 reduced angiogenesis in vivo and inhibited proliferation and differentiation of endothelial cells in vitro. The development of more successful anti-angiogenic therapy requires combined treatment directed to different cellular compartments. Therefore, we further tested IP6 in combination with vasostatin, a small calreticulin fragment, with anti-angiogenic properties directed primarily to endothelial cells (4648), and found that the combination of IP6 and vasostatin was synergistically superior in growth inhibition than each compound alone. In addition, we report an inhibitory action of IP6 on the secretion of the primary angiogenic cytokine, VEGF, by a human liver cancer cell line.
 |
Materials and methods
|
---|
Cell lines and cell culture
Human umbilical vein endothelial cells (HUVECs), obtained from Clonetics (San Diego, CA), were maintained and propagated in Medium 199(E) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 µg/ml bovine pituitary extract (Gibco BRL Life Techonologies, Gaithersburg, MD), 10 ng/ml of EGF (Gibco BRL) and 50 µg/ml of gentamycin (Gibco BRL). HUVECs were used between passages 4 and 10. HUVEC retrovirus telomerized (HRVT) cells were created in our laboratory from the human dermal microvascular endothelial cells as described previously (49). HRVT cells were used between passages 20 and 30, and were maintained and grown in Medium 199(E) (Gibco BRL). These cells are equivalent to the early passages of HUVECs. Bovine aortic endothelial cells (BAECs), from Coriell (Camden, NJ) and HepG2, human hepatoblastoma cell line, obtained from the American Type Culture Collection (Rockville, MD) were grown in DMEM medium (Gibco BRL) supplemented with 10% FBS, 2 mM L-glutamine, 50 U/ml of penicillin and 50 mg/ml of streptomycin. Cell cultures were maintained in a humidified 37°C incubator with 5% CO2.
IP6
IP6, as a dodecasodium salt from rice (Sigma Chemical, St Louis, MO), was prepared as a 100 mM stock solution in distilled water at pH 7.4. This stock solution was further diluted to the desired concentration with culture medium. Fresh IP6 stock solution was prepared before use.
Vasostatin
Vasostatin was purified and expressed as described before (50). In brief, a bacterial maltose-binding protein (MBP) fusion system was used to express recombinant protein; bacterial strain was Escherichia coli BL21 (
DE3), and plasmid pMAL-c2. Vasostatin is a N-domain of human calreticulin (residues 1180). The P-domain of calreticulin (residues 181290) (PDN) and MBP2 (MBP) (New England Biolabs, Hitchin, Hertfordshire, UK) were tested as control proteins. All proteins were dialysed into endotoxin-free water containing 10 mM NaCl and freeze-dried.
Growth inhibition assay
To evaluate the effect of IP6 on the proliferation of BAECs, a colorimetric MTT assay was used as described previously (13,25,26). This assay measures the reduction of a tetrazolium salt, MTT ([4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) (Sigma), by living cells to a blue-colored formazan product, evaluating mitochondrial as well as the anti-proliferative activity. Cells were plated in 96-well microtiter plates (Sarstedt, Newton, NC) at a density of 2 x 103 cells/well/100 µl and treated either with various concentrations of IP6, ranging from 0.1 to 3.0 mM, or vasostatin, ranging from 0.1 to 10 µg/ml, or their combinations. After 1, 3 and 5 days of treatment, 50 ml of MTT (1.0 mg/ml) was added, and the incubation continued for 4 h at 37°C. The precipitated formazan was solubilized with 150 ml of DMSO (Sigma) and the absorbance was determined at 540 nm. The MTT assay was performed with BAECs grown in the presence or absence of basic fibroblast growth factor (bFGF) (10 mg/ml). Mean and standard deviations were from triplicate data points. Independent experiments were repeated three times.
Plating efficiency assay
BAECs were seeded on 10-cm tissue culture dishes (Sarstedt) at a density of 5 x 103 cells/dish and incubated at 5% CO2 for 10 days. The growth medium contained various concentrations of either IP6 (ranging from 0.1 to 3.0 mM) or vasostatin (ranging from 0.1 to 10 µg/ml) or their combinations; for control, BAECs of the same density were plated in dishes containing growth medium only. BAECs were seeded in triplicates, and this experiment was repeated thrice.
Capillary tube formation on Matrigel (in vitro angiogenesis)
In experiments assessing the inhibitory effect of IP6 on capillary tube formation, HUVECs and HRVT cells were cultured in 24-well plates on a 0.2 ml layer of previously polymerized Matrigel (Becton Dickinson, Bedford, MA) with or without IP6, as described previously (51). HUVECs were plated in triplicate at a density of 105 cells/well, while HRVT cells were seeded between 7 and 8 x 104 cells/well. In this assay, cells attach within 30 min, become growth arrested, migrate, secrete proteases to invade into the gel, and form inter-connecting networks within 6 h. Complete differentiation is usually achieved after 1824 h. The networks are stable for several days, after which regression and aggregation occurs. Changes of cell morphology influenced by the presence of IP6, were captured after 4 and 22 h through a phase contrast microscope (x40) (Zeiss Axiovert 10, Germany) and photographed with a CCD video camera. To investigate the effect of IP6 on pre-formed tubes, HUVECs and HRVT cells were seeded onto Matrigel for 6 h to form rudimentary tubes, then the medium was replaced and IP6 was added. Tube morphology was observed over time, and representative pictures were taken 24 h after the initiation of IP6 treatment. Independent experiments were repeated twice.
In vivo mouse Matrigel plug assay
The mouse Matrigel plug assay was performed as described previously by Passaniti et al. (52). IP6 (2.0 mM) and bFGF (400 ng/ml) in PBS were mixed with Matrigel (Becton Dickinson, Bedford, MA) in proportions not exceeding 1% of the total volume of Matrigel. A mixture of 0.5 ml Matrigel with IP6 or vehicle and/or bFGF was injected s.c. into C57BL/6 J mice. The study was approved by the University of Maryland School of Medicine Animal Care and Use Committee, and performed in accordance with current regulations and standards of the NIH and our institutional guidelines for animal care. Since IP6 is a water soluble compound that might diffuse easily from the plugs, to ensure adequate exposure to IP6, the experimental groups were also given IP6 in drinking water (2% w/v, 15 mM), as described previously (1517,1921). After 57 days, mice were killed, and the excised Matrigel plugs were fixed and stained using the Masson-Trichrome method. The experiment was repeated twice using 5 mice/data point. For quantitative analysis of angiogenesis in Matrigel plugs, a computerized digital system and NIH Image-1 software was used on images captured as TIF files. The relative angiogenic response was evaluated from the cellular density of each slide after background subtraction.
VEGF secretion by cancer cells
The production of VEGF angiogenic factor in response to IP6 treatment was measured in HepG2 cells. To study the effect of IP6 on production and expression of VEGF mRNA, we used reverse transcriptionpolymerase chain reaction (RTPCR) (53). Total RNA was isolated from HepG2 cells treated with IP6 after 8 and 24 h using RNAzol (Tel-Test, Friendswoods, TX) extraction procedure according to the manufacturer's protocol. For a positive control, total RNA was extracted from the prostate carcinoma cell line, LNCaP. RTPCR was used to amplify cDNAs. After the RT reaction with Superscript II reverse transcriptase (Gibco BRL), PCR was used to identify the presence of VEGF isoforms in the RNA samples. Primers used for the amplification assay were a slight modification of those published previously. Because these primers initiate within the first exon and terminate within the eighth exon of VEGF-A, they enable amplification of all the known VEGF splice variants. The oligonucleoide sequences of the primers used were: 5' human start of exon 1, 5' TGC ACC CAT GGC AGA AGG AGG 3'; 3' human end of exon 8, 5' TCA CCG CCT CGG CTT GTC ACA 3'.
PCR was carried out with Taq DNA polymerase (Boehringer Mannheim Biochemicals, Indianapolis, IN) with a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) as follows: 94°C for 7 min, then 35 cycles of denaturation at 94°C for 1 min, annealing and extension at 72°C for 1 min each. The PCR products were analyzed by electrophoresis on a 2% agarose gel that was subsequently stained with ethidium bromide for visualization of DNA bands. A 100-bp ladder DNA molecular weight marker (Boehringer Mannheim Biochemicals) was used to provide a size reference for the test reaction. The samples were run in duplicates for each time point. The experiment was performed twice independently.
To investigate the effect of IP6 on the secretion of VEGF protein from cancer cells, HepG2 cells were grown in T25 flasks in complete medium until confluent. The medium was removed, cells were washed thrice with PBS and then treated in serum-free medium with increasing concentrations of IP6. Serial 1-ml aliquots were removed from the culture media for VEGF analysis after 1, 3, 6 and 24 h. In a second set of experiments, cells were pre-treated with increasing concentrations of IP6 for 24 h, media were replaced with serum-free media, and then 1-ml aliquots were collected. Samples were analyzed for VEGF protein content by an ELISA kit following manufacturer's instructions (R&D Systems, Minneapolis, MN). To express the protein levels of VEGF in tissue culture media, we determined the protein levels by the Bio-Rad protein determination assay kit, based on Bradford method, and normalized the results relative to the protein levels. The experiment was repeated. Each sample was measured in triplicate.
Statistical analysis
The mean and standard deviations were calculated. The results were evaluated either by Student's t-test or ANOVA for multiple group comparisons using Prism (GraphPad Software, San Diego, CA). Synergism was detected by the median effect equation as described by Chou and Talalay (54). The extent of interaction between the compounds was determined by using the combination index (CI), where CI <1.0 signified synergism, CI = 1.0 additive effect, and CI >1.0 antagonism.
 |
Results
|
---|
Inhibition of endothelial cell proliferation by IP6 in vitro
As angiogenesis critically depends on the ability of endothelial cells to proliferate we investigated the anti-angiogenic activity of IP6 by first studying its effect on the proliferation of endothelial cells by monitoring the growth and plating efficiency of endothelial cells treated with IP6. Different doses of IP6 ranging 03.0 mM were applied to BAECs and the effect on their growth was determined by MTT assay. IP6 inhibited the growth of BAECs in a dose-dependent manner, with an IC50 of 0.74 mM on day 3 (Figure 1A). While lower concentrations of IP6 (0.1 and 0.3 mM) had no effect on the growth compared with the control, the growth was significantly inhibited by 0.6 mM (P < 0.01; 84% on day 3, 62% on day 5), and completely suppressed at concentrations
1.0 mM (Figure 1A). Similar results were obtained when the growth of BAECs was stimulated by bFGF (10 µg/ml) (data not shown). However, in the presence of bFGF, cells grow faster, reaching a plateau earlier, not allowing good evaluation of a growth rate. It is important to note that these IP6 concentrations are typically found in mammalian cells (1,2), used for in vitro experiments, and that 2.0 mM inositol hexasulphate, a structural analog of IP6, used as additional control, had no effect on growth of either endothelial cells (data not shown) or epithelial cells (14), indicating the specific effect of IP6.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1. IP6 inhibits the proliferation of endothelial cells. IP6 inhibited growth of BAECs proliferation in a dose-dependent manner, with IC50 being 0.74 mM on day 3 (A). While a concentration-dependent inhibition of proliferation of BAECs was obtained with vasostatin with IC50 of 2.33 µg/ml (B), IP6 in combination with vasostatin exhibited a synergistic effect in growth inhibition of BAECs, with CI of 0.22 for day 3 (note that CI <1.0 identifies synergism) (C). Simultaneously, there was no growth inhibition by MBP and PDN (not shown) proteins used as control for vasostatin (D).
|
|
Since vasostatin directly targets endothelial cells, we further tested IP6 in combination with vasostatin. As expected, a strong, concentration-dependent growth inhibition of BAECs by vasostatin was observed: 65% inhibition with concentration of 3 µg/ml, and with IC50 of 2.33 µg/ml on day 3 (Figure 1B). The specificity of this response was confirmed by the inability of control proteins, MBP and PDN (not shown), to suppress the growth of BAECs at concentrations ranging 0.110 µg/ml (Figure 1D). However, when BAECs were treated with the combination of IP6 and vasostatin a synergistic response was obtained with a CI of 0.22 at day 3, and 0.33 at day 5 [note that CI <1.0 signifies synergism (54)] (Figure 1C). All combinations of all concentrations of IP6 and vasostatin were performed, also in the presence and absence of bFGF. Although observed with 0.3 mM IP6, the most dramatic combination effect was obtained with 0.6 mM IP6.
Additionally, in a plating efficiency assay, IP6 or vasostatin inhibited colony formation of BAECs (Figure 2AC). The concentration of 1 µg/ml of vasostatin exhibited a stronger ability to prevent BAECs colony formation than 0.6 mM IP6 (Figure 3C). Reduced plating efficiency can result from both decreased proliferation and reduced attachment of cells in the presence of IP6 and vasostatin. Actually, the ability of IP6 to reduce adhesion of human breast cancer cells was reported recently from our laboratory (55). Furthermore, it appears that vasostatin at this concentration exhibited a stronger ability to prevent BAEC colony formation than to inhibit BAECs growth, as judged from the MTT assay (Figure 1B), suggesting that vasostatin inhibits attachment of cells. However, similar to MTT, a synergistic response of IP6 in combination with vasostatin was observed in this assay, achieving almost a complete inhibition of BAECs colony formation (Figure 2D). In contrast, control proteins MBP and PDN, did not affect the ability of BAEC to form colonies when tested alone or in combination with IP6 (data not shown).

View larger version (97K):
[in this window]
[in a new window]
|
Fig. 2. IP6 inhibits the colony formation of endothelial cells. BAECs were seeded in 10-cm tissue culture dishes at a density of 5 x 103 cells/dish for 10 days; the growth medium contained either 0.6 mM IP6, or 1 µg/ml vasostatin, or their combination. Although both IP6 (B) and vasostatin (C) inhibited colony formation of BAECs, assessed by plating efficiency assay, a synergism between these two compounds is shown by a complete block of BAECs colonies (D).
|
|

View larger version (78K):
[in this window]
[in a new window]
|
Fig. 3. IP6 inhibits the differentiation of endothelial cells. The inhibitory effect of IP6 on capillary tube formation (in vitro differentiation) was assessed by exposure to IP6 of HUVECs and HRVTs on Matrigel at the time of seeding. After 22 h, changes of cell morphology were captured with an inverted microscope. While both HUVECs and HRVT cells normally form hollow tubes on Matrigel, IP6 almost completely inhibited capillary tube formation of HUVECs and HRVT cells on Matrigel in a dose-dependent manner (A). The effect was seen with 0.3 mM IP6 on HUVECs, and with only 0.05 mM IP6 on HRVT cells. To assess the effect of IP6 on pre-formed tubes, HUVECs and HRVT cells were seeded into Matrigel pre-coated 24-well plate for 6 h to allow tube formation. The pre-formed tubes were treated with IP6, and fixed 24 h after treatment. 1.0 mM IP6 caused the retraction of cells and disintegration of pre-formed tubes (B).
|
|
Inhibition of differentiation of endothelial cells by IP6 in vitro
To determine whether IP6 had an inhibitory effect on differentiation and tube formation of vascular endothelial cells, we conducted the experiments in an in vitro angiogenesis model using HUVECs and HRVT cells seeded on Matrigel (a reconstituted extracellular matrix preparation of EHS mouse sarcoma). These cells undergo rapid in vitro differentiation into capillary-like structures, a complex process that requires cellmatrix interaction, intercellular communication and cell motility. IP6 exhibited a striking inhibitory effect on capillary tube formation in this model. The treatment was initiated simultaneously with the seeding of cells on Matrigel. As shown in Figure 3A, both HUVECs and HRVT cells formed inter-connected networks and hollow tubes on Matrigel; in control cells, these structures became stronger, more robust and longer with time. In contrast, IP6 inhibited tube formation of HUVECs in a time- and dose-dependent fashion (data after 4 h not shown). After 22 h of exposure to 0.3 mM IP6 there was near total inhibition of network formation by both cell lines, reflecting the inhibition of attachment, migration and invasion of cells by IP6 (Figure 3A). Interestingly, HRVT cells were more sensitive, showing inhibition of tube formation at 0.05 mM IP6. Representative fields from three independent experiments are shown (Figure 3A). To evaluate whether IP6 could also disrupt pre-existing tubes, HUVECs and HRVT cells were treated with IP6 6 h after seeding on Matrigel. Exposure of these pre-formed tubes to 1.0 mM IP6 for 24 h resulted in the retraction of cells and disintegration of tubes (Figure 3B). It is important to note that these cells were alive. In doses used in the majority of in vitro studies, IP6 appears to be a cytostatic, not a cytotoxic agent. No cytotoxicity of endothelial cells was observed up to 3.0 mM IP6, a dose higher than those used in the differentiation assay, tube formation and tube regression assay, as reported previously for colon and breast cancer cells (56).
Inhibition of angiogenesis by IP6 in an in vivo mouse model
To further assess the anti-angiogenic potential of IP6, we used an in vivo assay, in which Matrigel plugs were impregnated with bFGF, as an inducer of neovascularization (57). In addition to 2 mM IP6 incorporated into plugs, mice were given 2% IP6 in drinking water (w/v, 15 mM), because IP6, as a water-soluble compound, could diffuse easily from the plugs. IP6 in the plugs was able to inhibit neovascularization induced by bFGF. However, this effect was much more pronounced when in addition to plugs, IP6 was given in drinking water. In these assays the tissue response to bFGF includes endothelial cell proliferation and migration and increased density within the gel plug (Figure 4A). Red blood cells were abundant within the lumen of numerous vessels (arrows, Figure 4A). However, IP6 in combination with bFGF almost completely inhibited vessel induction, indicating that IP6 potently suppressed the bFGF-stimulated angiogenesis in mice (Figure 4B). As a result of IP6-suppressed cellular infiltration and functional neovessel formation within the Matrigel plugs, the relative cellular density of the plugs was 75% smaller when compared with the control (P < 0.001), equal to the response observed in the absence of bFGF (Figure 4C).

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 4. IP6 suppresses the bFGF-induced angiogenesis in mouse model in vivo. Matrigel mixed with 200 ng/ml bFGF without (A) and with 2 mM IP6 in RPMI (B) was injected s.c. into C57BL/6 J mice. Arrows indicate the area of cellular infiltration and functional neovessel formation within Matrigel in cross-section after 7 days. This area is wider with new vessels filled with red blood cells in plugs of Matrigel in combination with bFGF (positive control) (A). However, IP6 in the presence of bFGF almost completely prevented the vessel induction, indicating that IP6 potently suppressed the bFGF-stimulated angiogenesis in mice (B) (M = Matrigel plug). As a result of IP6-suppressed cellular infiltration and functional neovessel formation within the Matrigel plugs, the relative cellular density of plugs was significantly smaller, with a 75% reduction in the IP6-treated group compared with control (P < 0.001) (C). Each data point represents the vascular density from up to five different slides and five regions per slide (n = 1015; P < 0.001 for bFGF + IP6 relative to bFGF).
|
|
Inhibition of VEGF expression and secretion by IP6 in HepG2 cells
Having established the anti-angiogenic activity of IP6 on endothelial cells, we conducted studies to explore the effects of IP6 on the expression and secretion of a primary angiogenic cytokine VEGF by HepG2 cells, a human liver cancer cell line, because VEGF is expressed in these cells (57), and because IP6 inhibited tumor growth leading to regression of the HepG2 tumors in a transplanted nude mouse model (26,27). We showed previously that IP6 treatment of HepG2 cells caused a dose-dependent growth inhibition (26). When inoculated into athymic nude mice, IP6 significantly reduced the growth of these human hepatoma tumors, leading to tumor regression (27). To test whether IP6 has the ability to reduce the expression and secretion of VEGF by HepG2 cells in vitro, first we evaluated its effect on VEGF mRNA expression, using RTPCR. In a second set of experiments, we evaluated the ability of IP6 to reduce the secretion of VEGF protein from the cancerous HepG2 cells.
HepG2 cells showed strong expression of three mRNA isoforms: 122, 165 and 189 (Figure 5A). IP6 treatment of HepG2 cells for 8 h induced a marked down-regulation of mRNA expression of all VEGF isoforms in a dose-dependent fashion (P < 0.001) (Figure 5A). To determine whether IP6 would down-regulate the expression of the VEGF protein, two sets of experiments were performed: the secretion of VEGF protein from HepG2 in the presence of various concentrations of IP6 (Figure 5B), and the secretion of VEGF from cancer cells that were pre-treated with several doses of IP6 for 24 h, and then cultured without IP6 for an additional 24 h (Figure 5C). IP6 decreased the secreted VEGF contents in the conditioned medium after 24 h by
50% (P = 0.012) (Figure 5B) in a concentration-dependent manner. This inhibitory effect on VEGF expression was more dramatic in HepG2 cells pre-treated with IP6. These cells continued to grow in serum-free media without IP6. After 24 h, 1.0 mM IP6 induced a 79% reduction in VEGF secretion (P = 0.02), while an 86% reduction was observed with 2.0 mM IP6 (P = 0.015) (Figure 5C).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5. IP6 inhibits VEGF expression and secretion. HepG2 cells were treated with 0, 0.5, 1 and 2 mM IP6 for 8 h, and VEGF mRNA levels were assessed by RTPCR; a doseresponse down-regulation of VEGF was observed. Total RNA was analyzed for VEGF and GAPDH mRNA. The VEGF primers identified three isoforms of VEGF mRNA transcripts, and all three isoforms were markedly down-regulated by IP6 relative to GAPDH (P < 0.001). Note that smaller amount of mRNA from control cells than those from experimental cells were used for RTPCR because of too high density for VEGF (A). Representative time course of IP6 effects on VEGF protein secretion by HepG2 cells in serum-free media (B). Confluent cells in T25 flasks were treated with increasing concentrations of IP6. At designated time points, 1 ml aliquots were taken for VEGF protein determination by ELISA. IP6 significantly decreased secreted VEGF after 24 h (P = 0.012) (B). HepG2 cells pre-treated with 2 mM IP6 for 24 h exhibited an 86% reduction in VEGF secretion after 48 h (P = 0.015) (C).
|
|
 |
Discussion
|
---|
Angiogenesis plays a central role in a variety of physiologic and pathologic conditions, such as cancer, vascular, rheumatoid and other disease. Because the presence of an adequate blood supply is required for the growth and metastasis of malignant tumors, the inhibition of tumor-induced angiogenesis represents a promising target for anti-neoplastic therapy; thus, angiogenesis-suppressing agents may provide a new modality of antitumor treatment (35). These novel agents can treat not only cancer, but any other disease involving hyper-angiogenesis. Although a number of pharmacological inhibitors are under investigation, the possible toxicities and a need for chronic dosing dictate a search for novel agents with low toxicity that are effective and safe for long-term use. Recently it was observed that a series of substances proposed as possible cancer preventive agents show anti-angiogenic properties; therefore, a novel concept angioprevention was proposed (58).
IP6, a natural carbohydrate that is abundant in the diet, possesses significant cancer preventive and therapeutic activity (1027). Along with inhibition of cell proliferation, IP6 enhances differentiation of malignant cells to more mature cells, often resulting in reversion to normal phenotype (1,1012,25,26). In this study, we investigated the anti-angiogenic potential of IP6, which may critically contribute to its cancer preventive and therapeutic efficacy. We show that IP6 can target both endothelial and cancer cells. Our data demonstrate that IP6 inhibited the growth and induced differentiation of vascular endothelial cells, and inhibited the secretion of the angiogenic factor VEGF by malignant epithelial cells.
IP6 inhibited FGF-stimulated angiogenesis in vivo, which indicates that IP6 may suppress abnormal angiogenesis. In vitro inhibition of endothelial cell growth by IP6 additionally implies that IP6 inhibits excessive angiogenesis via this route as well, since proliferation of endothelial cells is vital for capillary sprouting. Furthermore, IP6 inhibited and prevented vascular network formation of HUVECs on Matrigel beds in vitro. It is important to note that the inhibitory effect of IP6 on tube formation was observed whether the treatment was initiated simultaneous with seeding cells on the Matrigel or after the tubes had formed. The ability of IP6 to disrupt the integrity of pre-formed tubes indicates that IP6 may not only prevent, but also regress new blood vessels. Capillary formation is a complex process requiring cellmatrix interactions, inter-cellular communications, as well as cell motility; therefore, the reduced network formation may indicate inhibition of attachment, migration and invasion of endothelial cells. Work is in progress to elucidate the effect of IP6 on these processes. Furthermore, the results of the in vivo Matrigel angiogenesis assay, showing significantly smaller plugs, containing markedly less vascular cell infiltration, suggest that the inhibition of endothelial cell proliferation may be central to suppression of angiogenesis and tumor growth by IP6. Although no toxicity or distinct morphology associated with apoptotic endothelial cells could be detected, we are not excluding the possibility that at higher doses IP6 might induce apoptosis (59) of endothelial cells, as well.
In addition to these inhibitory effects on the responses of endothelial cells, and in vivo and in vitro angiogenesis, IP6 exerted an inhibitory action on the secretion of angiogenic cytokines. Angiogenesis can be inhibited, and consequently tumor growth may be reduced, by blocking the induction of VEGF or FGF (34). VEGF induces angiogenesis in both pathologic and physiologic situations; it has a narrow specificity, and acts with specific mitogenic and chemotactic effects on vascular endothelial cells, thereby promoting their proliferation and permeability.
Here we have investigated the effects of IP6 on secretion of VEGF from HepG2 cells, human liver cancer cells and found that IP6 reduces the expression of VEGF mRNA and protein. Very recently, Singh et al. (60) also reported the inhibition of VEGF by IP6 in a prostate tumor model. FGF is another factor implicated in tumor growth and angiogenesis. Morrison et al. (61) investigated the effect of IP6 on FGF and found that IP6 was a potent antagonist of FGF cellular binding and activity. They attributed this activity to the chair conformation of IP6, which mimics that of the pyranose ring structure of heparin. Interestingly, VEGF is also a heparin-binding angiogenic factor.
We have shown previously that IP6 was effective in the treatment of liver cancer in vitro and in vivo in nude mice (26,27). A dose-dependent growth inhibition of HepG2 cells was induced by IP6 in vitro (26), and a reduction of transplanted hepatoma tumor growth in mice given intra-tumoral injections of IP6 (27). In a model of human rhabdomyosarcoma, IP6-treated nude mice produced 2549-fold smaller tumors when compared with controls (25). Therefore, it is possible that IP6, by depriving tumor endothelium of VEGF and FGF, may disrupt the formation of new vessels by inhibiting the secretion of angiogenic factors leading to tumor growth inhibition. Higher levels of basal VEGF mRNA have been detected in hepatocellular carcinoma than in normal tissues, including the major VEGF isoforms 121 and 165 (62). Three mRNA VEGF isoforms were expressed in HepG2 cells. Increased expression of VEGF and VEGF 121, 165 and 189 mRNA isoforms is associated with tumor progression in hepatocellular carcinoma (62) and the formation of liver metastases with poor prognosis (62,63). It is important to note that IP6 dramatically down-regulated the expression of all three VEGF mRNA isoforms. The data further raise questions about the possible effects of IP6 on VEGF steady-state levels and under experimental conditions associated with over-expression of VEGF, such as hypoxia. Hypoxia can be detected in the central regions of tumors, and is an important stimulus for new vessel formation. It is also critically involved in the process of metastasis. Hypoxia up-regulates VEGF expression partly through increasing mRNA transcription: within tumors, VEGF mRNA co-localizes to hypoxic regions (57). The VEGF gene and several other genes regulated by hypoxia are under the control of the hypoxia-inducible factor (HIF)-1, a transcription factor (64). Therefore, it will be important to investigate the effect of IP6 on HIF-1 expression and function in hypoxia.
Several molecular mechanisms can be suggested for the observed anti-angiogenic effect of IP6 and its inhibition of VEGF expression. IP6 may down-regulate VEGF acting directly, or indirectly by inhibiting the expression of factors up-stream of VEGF. Various signal transduction pathways have been implicated in angiogenesis, and in the regulation of angiogenic factor expression. Phosphatidylinositol 3'-kinase (PI3K)/Akt is a common signaling pathway for oncogenes and tumor suppressor genes and is involved in VEGF and HIF-1 regulation (64). Activation of Erk1 and Erk2 has also been shown to be an important mediator for up-regulation of VEGF (65,66). Although both pathways have been shown to be inhibited by IP6 (59,6769), further studies are required to define more precisely the mechanism of anti-angiogenic action of IP6 related to these pathways. The potential of IP6 to inhibit PI3K may be related to its structure, because IP6 is similar to D-3-deoxy-3-fluoro-PtdIns, a PI3K inhibitor (67). However, IP6 is also known to be a strong metal ion chelator (15), and may inhibit metalloproteinases, which is necessary for capillary sprouting (70). Furthermore, as some receptor kinases depend on divalent cations for their activity, IP6 could inhibit the activity of these kinases by chelating the divalent cations, affecting both Erks and PI3K/Akt pathways. Higher levels of basal mRNA and protein of both VEGF and insulin-like growth factor-II (IGF-II), another angiogenic factor, can be detected in hepatocellular carcinoma relative to normal tissue (62). Kar et al. (71) demonstrated an interaction between IP6 and IGF-II receptor binding sites in rat brain. It has been reported that the combination of hypoxia and IGF-II induces VEGF mRNA (72), and that IGF-II can up-regulate HIF-1 protein; therefore, it is possible that IP6 interfering with IGF-II may further contribute to the observed down-regulation of VEGF.
Cancer therapy that combines distinct inhibitors of angiogenesis is a novel, effective strategy for the experimental treatment of cancer. To develop more successful anti-angiogenic therapy, combined treatment, directed to different cellular compartments may be important. It is hoped that the combination of these angiogenesis inhibitors with cytotoxic and differentiating chemotherapeutic agents will significantly improve survival and quality of life for not just cancer patients, but also of others affected by enhanced angiogenesis.
In conclusion, this study shows that IP6 has inhibitory action on several angiogenic responses, including growth and differentiation of endothelial cells, as well as inhibitory effects on the secretion of VEGF, a key angiogenic cytokine. This anti-angiogenic activity, combined with the previously known potential of IP6 as a strong anti-proliferative and differentiation-inducing agent, provides evidence that IP6 is useful for prevention and therapy of cancers and other conditions of excessive angiogenesis in humans, and merits further investigation.
 |
Notes
|
---|
Present address: Kwanchanit Tantivejkul, Cancer Center, University of Michigan, Ann Arbor, MI, USA
 |
Acknowledgments
|
---|
This work was supported in part by the American Institute for Cancer Research Grant 02A099 (to I.V.).
 |
References
|
---|
- Shamsuddin,A.M. (1999) Metabolism and cellular functions of IP6: a review. Anticancer Res., 18, 37333736.
- Poyner,D.R., Cooke,F., Hanley,M.R., Reynolds,D.J.M. and Hawkins,P.T. (1993) Characterization of metal ion-induced [3H]-inositol hexakisphosphate binding to rat cerebellar membranes. J. Biol. Chem., 268, 10321038.[Abstract/Free Full Text]
- Graf,E. and Eaton,J.W. (1990) Antioxidant functions of phytic acid. Free Radical Biol. Med., 8, 6169.[CrossRef][ISI][Medline]
- Sasakawa,N., Sharif,M. and Hanley,M.R. (1995) Metabolism and biological activities of inositol pentakisphosphates and inositol hexakisphosphate. Biochem. Pharmacol., 50, 137146.[CrossRef][ISI][Medline]
- Shears,S.B. (1996) Inositol pentakis- and hexakisphosphate metabolism adds versatility to the actions of inositol polyphosphates: novel effects on ion channels and protein traffic. Sub-Cell. Biochem., 26, 187226.[Medline]
- Efanov,A.M., Zaitsev,S.V. and Berggren,P.O. (1997) Inositol hexakisphosphate stimulates non-Ca2+-mediated and primes Ca2+-mediated exocytosis of insulin by activation of protein kinase C. Proc. Natl Acad. Sci. USA, 94, 44354439.[Abstract/Free Full Text]
- Larsson,O., Barker,C.J., Sjöholm,Å. et al. (1997) Inhibition of phosphatases and increased Ca2+ channel activity by inositol hexakisphosphate. Science, 278, 471474.[Abstract/Free Full Text]
- York,J.D., Odom,A.R., Murphy,R., Ives,E.B. and Wente,S.R. (1999) A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science, 285, 96100.[Abstract/Free Full Text]
- Odom,A.R., Stahlberg,A., Wente,S.R. and York,J.D. (2000) A role for nuclear inositol 1,4,5-trisphosphate kinase in transcriptional control. Science, 287, 20262029.[Abstract/Free Full Text]
- Shamsuddin,A.M., Vucenik,I. and Cole,K.H. (1997) IP6: a novel anticancer agent. Life Sci., 61, 343354.[CrossRef][ISI][Medline]
- Shamsuddin,A.M. (2002) Anti-cancer function of phytic acid. Int. J. Food Sci. Technol., 37, 769782.[CrossRef][ISI]
- Vucenik,I. and Shamsuddin,A.M. (2003) Cancer inhibition by inositol hexaphosphate (IP6): from laboratory to clinic. J. Nutr., 133, 3778S3784S.[Abstract/Free Full Text]
- Shamsuddin,A.M., Yang,G.-Y. and Vucenik,I. (1996) Novel anti-cancer functions of IP6: growth inhibition and differentiation of human mammary cancer cell lines in vitro. Anticancer Res., 16, 32873292.[ISI][Medline]
- Sakamoto,K., Venkatraman,G. and Shamsuddin,A.M. (1993) Growth inhibition and differentiation of HT-29 cells in vitro by inositol hexaphosphate (phytic acid). Carcinogenesis, 14, 18151819.[Abstract]
- Vucenik,I., Sakamoto,K., Bansal,M. and Shamsuddin,A.M. (1993) Inhibition of mammary carcinogenesis by inositol hexaphosphate (phytic acid). A pilot study. Cancer Lett., 75, 95102.[CrossRef][ISI][Medline]
- Vucenik,I., Yang,G.-Y. and Shamsuddin,A.M. (1995) Inositol hexaphosphate and inositol inhibit DMBA-induced rat mammary cancer. Carcinogenesis, 16, 10551058.[Abstract]
- Vucenik,I., Yang,G.-Y. and Shamsuddin,A.M. (1997) Comparison of pure inositol hexaphosphate (IP6) and high-bran diet in the prevention of DMBA-induced rat mammary carcinogenesis. Nutr. Cancer, 28, 713.[ISI][Medline]
- Shamsuddin,A.M. and Vucenik,I. (1999) Mammary tumor inhibition by IP6: a review. Anticancer Res., 19, 36713674.[ISI][Medline]
- Shamsuddin,A.M. and Ullah,A. (1989) Inositol hexaphosphate inhibits large intestinal cancer in F344 rats 5 months after induction by azoxymethane. Carcinogenesis, 10, 625626.[Abstract]
- Shamsuddin,A.M., Ullah,A. and Chakravarthy,A. (1989) Inositol and inositol hexaphosphate suppresses cell proliferation and tumor formation in CD-1 mice. Carcinogenesis, 10, 14611463.[Abstract]
- Ullah,A. and Shamsuddin,A.M. (1990) Dose-dependent inhibition of large intestinal cancer by inositol hexaphosphate in F344 rats. Carcinogenesis, 11, 22192222.[Abstract]
- Pretlow,T.P., O'Riordan,M.A., Somich,G.A., Amini,S.B. and Pretlow,T.G. (1992) Aberrant crypts correlate with tumor incidence in F344 rats treated with azoxymethane and phytate. Carcinogenesis, 13, 15091512.[Abstract]
- Challa,A., Rao,D.R. and Reddy,B.S. (1997) Interactive suppression of aberrant crypt foci induced by azoxymethane in rat colon by phytic acid and green tea. Carcinogenesis, 18, 20232026.[Abstract]
- Vucenik,I., Tomazic,V.J., Fabian,D. and Shamsuddin,A.M. (1992) Antitumor activity of phytic acid (inositol hexaphosphate) in murine transplanted and metastatic fibrosarcoma, a pilot study. Cancer Lett., 65, 913.[CrossRef][ISI][Medline]
- Vucenik,I., Kalebic,T., Tantivejkul,K. and Shamsuddin,A.M. (1998) Novel anticancer function of inositol hexaphosphate: inhibition of human rhabdomyosarcoma in vitro and in vivo. Anticancer Res., 18, 13771384.[ISI][Medline]
- Vucenik,I., Tantivejkul,K., Zhang,Z.S., Cole,K.E., Saied,I. and Shamsuddin,A.M. (1998) IP6 in treatment of liver cancer I. IP6 inhibits growth and reverses transformed phenotype in HepG2 human liver cancer cell line. Anticancer Res., 18, 40834090.[ISI][Medline]
- Vucenik,I., Zhang,Z.S. and Shamsuddin,A.M. (1998) IP6 in treatment of liver cancer II. Intra-tumoral injection of IP6 regresses pre-existing human liver cancer xenotransplanted in nude mice. Anticancer Res., 18, 40914096.[ISI][Medline]
- Grases,F., Simonet,B.T., Vucenik,I., Prieto,R.M., Costa-Bauzá,A., March,J.G. and Shamsuddin,A.M. (2001) Absorption and excretion of orally administered inositol hexaphosphate (IP6 or phytate) in humans. Biofactors, 15, 5361.[ISI][Medline]
- Sakamoto,K., Vucenik,I. and Shamsuddin,A.M. (1993) [3H]-Phytic acid (inositol hexaphosphate) is absorbed and distributed to various tissues in rats. J. Nutr., 123, 713720.[ISI][Medline]
- Folkman,J. (1995) Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Med., 1, 2731.[ISI][Medline]
- Folkman,J. (1995) Clinical applications of research on angiogenesis. N. Engl. J. Med., 333, 17571763.[Free Full Text]
- Griffioen,A.W. and Molema,G. (2000) Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases and chronic inflammation. Pharmacol. Rev., 52, 237268.[Abstract/Free Full Text]
- Folkman,J. (1986) How is blood vessel growth regulated in normal and neoplastic tissue? Cancer Res., 46, 467473.[ISI][Medline]
- Cross,M.J. and Claesson-Welsh,L. (2001) FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. Trends Pharmacol. Sci., 22, 201207.[CrossRef][ISI][Medline]
- Rosen,L.S. (2001) Angiogenesis inhibition in solid tumor. Cancer J., 7 (suppl. 3), S120S128.[ISI][Medline]
- D'Amato,R.J., Loughnan,M.S., Flynn,E. and Folkman,J. (1994) Thalidomide is an inhibitor of angiogenesis. Proc. Natl Acad. Sci. USA, 91, 40824085.[Abstract]
- Motzer,R.J., Berg,W., Ginsberg,M., Russo,P., Vuky,J., Yu,R., Bacik,J. and Mazumdar,M. (2002) Phase II trial of thalidomide for patients with advanced renal cell carcinoma. J. Clin. Oncol., 20, 302306.[Abstract/Free Full Text]
- Steins,M.B., Padro,T., Bieker,R., Ruiz,S., Kropff,M., Kienast,J., Kessler,T., Buechner,T., Berdel,W.E. and Mesters,R.M. (2002) Efficacy and safety of thalidomide in patients with acute myeloid leukemia. Blood, 99, 834839.[Abstract/Free Full Text]
- Ingber,D., Fujita,T., Kishimoto,S., Sudo,K., Kanamaru,T., Brem,H. and Folkman,J. (1990) Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumor growth. Nature, 348, 555557.[CrossRef][ISI][Medline]
- Moore,J.D., Dezube,B.J., Gill,P., Zhou,X.J., Acosta,E.P. and Sommadossi,J.P. (2000) Phase I dose escalation pharmacokinetics of O-(chloroacetylcarbamoyl) fumagillol (TNP-470) and its metabolites in AIDS patients with Kaposi's sarcoma. Cancer Chemother. Pharmacol., 46, 173179.[CrossRef][ISI][Medline]
- Cao,Y., Ji,R.W., Davidson,D., Schaller,J., Marti,D., Söhndel,S., McCance,S.G., O'Reilly,M.S., Llinás,M. and Folkman,J. (1996) Kringle domains of human angiostatin. Characterization of the anti-proliferative activity on endothelial cells. J. Biol. Chem., 271, 2946129467.[Abstract/Free Full Text]
- O'Reilly,M.S., Boehm,T., Shing,Y., Fukai,N., Vasios,G., Lane,W.S., Flynn,E., Birkhead,J.R., Olsen,B.R. and Folkman,J. (1997) Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell, 88, 277285.[ISI][Medline]
- Jiang,C., Agarwal,R. and Lü,J. (2000) Anti-angiogenic potential of a cancer chemopreventive flavonoid antioxidant, sylmarin: inhibition of key attributes of vascular endothelial cells and angiogenic cytokine secretion by cancer epithelial cells. Biochem. Biophys. Res. Commun., 276, 371378.[CrossRef][ISI][Medline]
- Kim,M.S., Lee,Y.M., Moon,E.-J., Kim,S.E., Lee,J.J. and Kim,K.-W. (2000) Anti-angiogenic activity of torilin, a sesquiterpene compound isolated from Torilis japonica. Int. J. Cancer, 87, 269275.[CrossRef][ISI][Medline]
- Jung,Y.D., Kim,M.S., Chay,K.O., Ahn,B.W., Liu,W., Bucana,C.D., Gallick,G.E. and Ellis,L.M. (2001) EGCG, a major component of green tea, inhibits tumour growth by inhibiting VEGF induction in human colon carcinoma cells. Br. J. Cancer, 84, 844850.[CrossRef][ISI][Medline]
- Pike,S.E., Yao,L., Jones,K.D. et al. (1998) Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth. J. Exp. Med., 188, 23492356.[Abstract/Free Full Text]
- Pike,S.E., Yao,L., Setsuda,J. et al. (1999) Calreticulin and calreticulin fragments are endothelial cell inhibitors that suppress tumor growth. Blood, 94, 24612468.[Abstract/Free Full Text]
- Yao,L., Pike,S.E., Setsda,J., Parekh,J., Gupta,G., Raffeld,M., Jaffe,E.S. and Tosato,G. (2000) Effective targeting of tumor vasculature by the angiogenesis inhibitors vasostatin and interleukin-12. Blood, 96, 19001905.[Abstract/Free Full Text]
- Yang,J., Nagavarapu,U., Relloma,K., Sjaastad,M.D., Moss,W.C., Passaniti,A. and Herron,G.S. (2001) Telomerized human microvasculature is functional in vivo. Nat. Biotechnol., 19, 219224.[CrossRef][ISI][Medline]
- Kishore,U., Sontheimer,R.D., Sastry,K.N., Zaner,K.S., Zappi,E.G., Hughes,G.R., Khamashta,M.A., Strong,P., Reid,K.B. and Eggleton,P. (1997) Release of calreticulin from neutrophils may alter C1q-mediated immune functions. Biochem. J., 322, 543550.[ISI][Medline]
- Pili,R., Chang,J., Partis,R.A., Mueller,R.A., Novick,T., Chrest,F.J. and Passaniti,A. (1995) The
-glucosidase I inhibitor castanospermine alters endothelial cell glycosylation, prevents angiogenesis and inhibits tumor growth. Cancer Res., 55, 29202926.[Abstract]
- Passaniti,A., Taylor,R.M., Pili,R., Guo,Y., Long,P.V., Haney,J.A., Pauly,R.R., Grant,D.S. and Martin,G.R. (1992) A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin and fibroblast growth factor. Lab. Invest., 67, 519528.[ISI][Medline]
- Burchardt,M., Burchardt,T., Chen,M.-W., Shabsigh,A., de la Taille,A., Buttyan,R. and Shagsigh,R. (1999) Expression of messenger ribonucleic acid splice variants for vascular endothelial growth factor in the penis of adult rats and humans. Biol. Reproduct., 60, 398404.[Abstract/Free Full Text]
- Chou,T.-C. and Talalay,P. (1987) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul., 22, 2755.
- Tantivejkul,K., Vucenik,I. and Shamsuddin,A.M. (2003) Inositol hexaphosphate (IP6) inhibits key events of cancer metastasis: In vitro studies of adhesion, migration and invasion of MDA-MB 231 human breast cancer cells. Anticancer Res., 23, 36713680.[ISI][Medline]
- El-Sherbiny,Y., Cox,M.C., Ismail,Z.A., Shamsuddin,A.M. and Vucenik,I. (2001) G0/G1 arrest and S phase inhibition of human cancer cell lines by inositol hexaphosphate (IP6). Anticancer Res., 21, 23932404.[ISI][Medline]
- Park,S.H., Kim,K.W., Lee,Y.S., Beak,J.H., Kim,M.S., Lee,Y.M., Lee,M.S. and Kim,Y.J. (2001) Hypoglycemia-induced VEGF expression is mediated by intracellular Ca2+ and protein kinase C signaling pathway in HepG2 human hepatoblastoma cells. Int. J. Mol. Med., 7, 9196.[ISI][Medline]
- Tossetti,F., Ferrari,N., De Flora,S. and Albini,A. (2002) Angioprevention: angiogenesis is a common and key target for cancer chemopreventive agent. FASEB J., 16, 214.[Abstract/Free Full Text]
- Singh,R.P., Agarwal,C. and Agarwal,R. (2003) Inositol hexaphosphate inhibits growth and induces G1 arrest and apoptotic death of prostate carcinoma DU145: modulation of CDKI-CDK-cyclin and pRb-related protein-E2F complexes. Carcinogenesis, 24, 555563.[Abstract/Free Full Text]
- Singh,R.P., Sharma,G., Mallikarjuna,G.U., Dhanalakshmi,S., Agarwal,C. and Agarwal,R. (2004) In vivo suppression of hormone-refractory prostate cancer growth by inositol heaxaphosphate: induction of insulin-like growth factor binding protein-3 and inhibition of vascular endothelial growth factor. Clin. Cancer Res., 10, 244250.[Abstract/Free Full Text]
- Morrison,R.S., Shi,E., Kan,M., Yamaguchi,F., McKeehan,W., Rudnicka-Nawrot,M. and Palczewski,K. (1994) Inositolhexakisphosphate (InsP6): an antagonist of fibroblast growth factor receptor binding and activity. In vitro Cell. Dev. Biol., 30A, 783789.[ISI]
- Torimura,T., Sata,M., Ueno,T., Kin,M., Tsuji,R., Suzaku,K., Hashimoto,O., Sugawara,H. and Tanikawa,K. (1998) Increased expression of vascular endothelial growth factor is associated with tumor progression in hepatocellular carcinoma. Hum. Pathol., 29, 986991.[ISI][Medline]
- Tokunaga,T., Oshika,Y., Abe,Y. et al. (1998) Vascular endothelial growth factor (VEGF) mRNA isoform expression pattern is correlated with liver metastasis and poor prognosis in colon cancer. Br. J. Cancer, 77, 9981002.[ISI][Medline]
- Blancher,C., Moore,J.W., Robertson,N. and Harris,A.L. (2001) Effects of ras and von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1a, HIF-2a and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3'-kinase/Akt signaling pathway. Cancer Res., 61, 73497355.[Abstract/Free Full Text]
- Milanini,J., Vinals,F., Pouyssegur,J. and Pages,G. (1998) p42/p44 MAP kinase module plays a key role in the transcriptional regulation of the vascular endothelial growth factor gene in fibroblast. J Biol. Chem., 273, 1816518172.[Abstract/Free Full Text]
- Jung,Y.D., Nakano,K., Liu,W., Gallick,G.E. and Ellis,L.M. (1999) Extracellular signal-regulated kinase activation is required for up-regulation of vascular endothelial growth factor by serum starvation in human colon carcinoma cell. Cancer Res., 59, 48044807.[Abstract/Free Full Text]
- Huang,C., Ma,W.-Y., Hecht,S.S. and Dong,Z. (1977) Inositol hexaphosphate inhibits cell transformation and activator protein 1 activation by targeting phosphatidylinositol-3' kinase. Cancer Res., 57, 28732878.
- Zi,X., Singh,R.P. and Agarwal,R. (2000) Impairment of erbB1 receptor and fluid-phase endocytosis and associated mitogenic signaling by inositol hexaphosphate in human prostate carcinoma DU145 cells. Carcinogenesis, 21, 22252235.[Abstract/Free Full Text]
- Ferry,S., Matsuda,M., Yoshida,H. and Hirata,M. (2002) Inositol hexakisphosphate blocks tumor cell growth by activating apoptotic machinery as well as by inhibiting the Akt/NF
B-mediated cell survival pathway. Carcinogenesis, 23, 20312041.[Abstract/Free Full Text]
- Vihinen,P. and Kahari,V.M. (2002) Matrix metalloproteinases in cancer: prognostic markers and therapeutic targets. Int. J. Cancer, 99, 157166.[CrossRef][ISI][Medline]
- Kar,S., Quirion,R. and Parent,A. (1994) An interaction between inositol hexakisphosphate (IP6) and insulin-like growth factor II receptor binding sites in the rat brain. Neuroreport, 5, 625628.[ISI][Medline]
- Kim,K.W., Bae,S.K., Lee,O.K., Bae,M.H., Lee,M.J. and Park,B.C. (1998) Insulin-like growth factor II induced by hypoxia may contribute to angiogenesis of human hepatocellular carcinoma. Cancer Res., 58, 348351.[Abstract]
Received April 8, 2004;
revised June 12, 2004;
accepted July 6, 2004.