Anti-angiogenic activity of inositol hexaphosphate (IP6)
Ivana Vucenik
1
2
3
Antonino Passaniti
0
1
2
Michele I.Vitolo
0
2
Kwanchanit Tantivejkul
1
2
Paul Eggleton
2
4
AbulKalam M. Shamsuddin
1
2
0
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine
,
Baltimore, MD
,
USA
1
Department of Pathology
2
Present address: Kwanchanit Tantivejkul, Cancer Center, University of Michigan
,
Ann Arbor, MI
,
USA
3
Department of Medical and Research Technology
4
Peninsula Medical School, Universities of Exeter and Plymouth and MRC Immunochemistry Unit, University of Oxford
,
Oxford
,
UK
5To whom correspondence should be addressed Email: 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 dosedependent manner (IC50 0.74 mM). The combination of IP6 and vasostatin, a calreticulin fragment with antiangiogenic 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 5 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 RT--PCR. 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.
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 mM to 1 mM (1,2). Recognized as
a strong antioxidant (3), IP6 has also been shown to play a
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 181--290); PI3K, phosphatidylinositol
30-kinase; VEGF, vascular endothelial growth factor.
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 (10--12), acting primarily via regulation of
cell growth and cell differentiation (10--14). The potential of
IP6 to reduce tumor incidence and tumor load has been shown
in rodent mammary tumors (15--18), colon tumors (19--23)
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 (30--33). 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 (...truncated)