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2005;65:8339-8349. Cancer Res
Tania Maffucci, Enza Piccolo, Albana Cumashi, et al.
Antitumor Effects andby Inositol Pentakisphosphate Results in Antiangiogenic
Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway
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Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway
by Inositol Pentakisphosphate Results in Antiangiogenic
and Antitumor Effects
Tania Maffucci,1Enza Piccolo,3Albana Cumashi,3Manuela Iezzi,3Andrew M. Riley,5
Adolfo Saiardi,2H. Yasmin Godage,5Cosmo Rossi,3Massimo Broggini,6
Stefano Iacobelli,3Barry V.L. Potter,5Paolo Innocenti,4and Marco Falasca1
1Department of Medicine, The Sackler Institute; 2Medical Research Council Cell Biology Unit and Laboratory for Molecular Cell Biology,
Department of Biochemistry and Molecular Biology, University College London, London, United Kingdom; 3Center of Excellence on Aging,
G. D’Annunzio Foundation; 4Department of Surgical Sciences, G. D’Annunzio University, Chieti, Italy; 5Wolfson Laboratory of Medicinal
Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom; and 6Laboratory of Molecular
Pharmacology, Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy
Abstract
The purpose of this study was to investigate the antiangio-
genic and in vivo properties of the recently identified
phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor Inosi-
tol(1,3,4,5,6) pentakisphosphate [Ins(1,3,4,5,6)P
5
]. Because
activation of the PI3K/Akt pathway is a crucial step in
some of the events leading to angiogenesis, the effect of
Ins(1,3,4,5,6)P
5
on basic fibroblast growth factor (FGF-2)–
induced Akt phosphorylation, cell survival, motility, and
tubulogenesis in vitro was tested in human umbilical vein
endothelial cells (HUVEC). The effect of Ins(1,3,4,5,6)P
5
on
FGF-2-induced angiogenesis in vivo was evaluated using s.c.
implanted Matrigel in mice. In addition, the effect of
Ins(1,3,4,5,6)P
5
on growth of ovarian carcinoma SKOV-3
xenograft was tested. Here, we show that FGF-2 induces Akt
phosphorylation in HUVEC resulting in antiapoptotic effect
in serum-deprived cells and increase in cellular motility.
Ins(1,3,4,5,6)P
5
blocks FGF-2-mediated Akt phosphorylation
and inhibits both survival and migration in HUVEC.
Moreover, Ins(1,3,4,5,6)P
5
inhibits the FGF-2-mediated cap-
illary tube formation of HUVEC plated on Matrigel and the
FGF-2-induced angiogenic reaction in BALB/c mice. Finally,
Ins(1,3,4,5,6)P
5
blocks the s.c. growth of SKOV-3 xenografted
in nude mice to the same extent than cisplatin and it
completely inhibits Akt phosphorylation in vivo . These data
definitively identify the Akt inhibitor Ins(1,3,4,5,6)P
5
as a
specific antiangiogenic and antitumor factor. Inappropriate
activation of the PI3K/Akt pathway has been linked to the
development of several diseases, including cancer, making
this pathway an attractive target for therapeutic strategies.
In this respect, Ins(1,3,4,5,6)P
5
, a water-soluble, natural
compound with specific proapoptotic and antiangiogenic
properties, might result in successful anticancer therapeutic
strategies. (Cancer Res 2005; 65(18): 8339-49)
Introduction
Angiogenesis, the formation of a mature vasculature from a
primitive vascular network, is required for different physiologic
events such as development, growth, tissue remodeling, and wound
healing (1). On the other hand, angiogenesis is involved in
pathologies such as arteriosclerosis and tumor growth. Angiogenesis
consists of a multistep process involving capillary endothelial cell
migration and proliferation and formation of three-dimensional
structures capable of carrying blood (1). Endothelial cell migration
and proliferation is induced by growth factors such as vascular
endothelial growth factor (VEGF or VEGF-A) and fibroblast growth
factor (FGF; ref. 2). Basic FGF (FGF-2) was the first proangiogenic
molecule to be identified. FGF-2 and VEGF stimulate survival,
proliferation, migration, and differentiation of endothelial cells,
although the efficiencies of transduction of these responses are
dependent on the cell types (2). Binding of VEGF and FGF-2 to their
respective receptors leads to receptor phosphorylation and subse-
quent activation of signaling proteins such as phosphatidylinositol
3-kinase (PI3K) and phospholipase C g(2). A recent report showed
that stimulation of PI3K by FGF receptor is mediated by coordinated
recruitment of multiple docking proteins (3).
PI3K catalyzes the phosphorylation of inositol phospholipids at
the D3 position to generate 3V-phosphorylated phosphoinositides
(4). The 3V-phosphorylated phosphoinositides act by recruiting
specific signaling proteins to the plasma membrane in a mechanism
mediated by their interaction with some structural protein domains,
among which the pleckstrin homology domain is the most studied
(5). One of the best-characterized PI3K downstream targets is the
serine/threonine protein kinase B, also known as Akt (6), and all
studies concerning the role of PI3K in angiogenesis focus their
attention on the PI3K/Akt pathway (7). Three members of the Akt
family (Akt1, Akt2, and Akt3) have been identified and they are, in
general, broadly expressed. Akt activation requires its translocation
to the plasma membrane via interaction of its pleckstrin homology
domain with 3V-phosphorylated phosphoinositides (8–11) and
subsequent phosphorylation at residues Thr
308
and Ser
473
. Phos-
phorylation at residue Thr
308
is catalyzed by the enzyme phosphoi-
nositide-dependent kinase-1 (12, 13), which itself is recruited to the
plasma membrane via the interaction of its pleckstrin homology
domain with 3V-phosphorylated phosphoinositides (14). The kinase
responsible for phosphorylation at residue Ser
473
is yet to be
definitively identified. Once activated, Akt can phosphor ylate several
signaling proteins, which, in turn, lead to the choice of cellular
proliferation or apoptosis (15–17). Therefore, Akt activation is
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Requests for reprints: Marco Falasca, Department of Medicine, The Sackler
Institute, University College London, Rayne Building, 5 University Street, London,
WC1E 6JJ, United Kingdom. Phone: 44-0-20-7679-6167; Fax: 44-0-20-7679-6219; E-mail:
m.falasca@ucl.ac.uk.
I2005 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-0121
www.aacrjournals.org 8339 Cancer Res 2005; 65: (18). September 15, 2005
Regular ArticleResearch Article
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considered both necessary and sufficient for cell survival. In
particular, Akt promotes survival through inactivation of caspase-9
(18), activation of the transcription factor nuclear factor-nB (19, 20),
and phosphorylation of different components of the apoptotic ma-
chinery, such as BAD and forkhead transcription factor (FKHRL-1;
ref. 21). Overexpression of Akt may therefore contribute to tumor
development and progression. The crucial role of the PI3K/Akt
pathway in cancer is further supported by the fact that the tumor
suppressor PTEN, whose gene is deleted or mutated in a wide variety
of human cancers, possesses a 3V-phosphoinositide-phosphatase
activity inactivating the PI3K/Akt pathway (22).
Very recently, we have evaluated the effect of different inositol
polyphosphates on Akt activation and we have shown that
Inositol(1,3,4,5,6)pentakisphosphate [Ins(1,3,4,5,6)P
5
] is specifically
able to inhibit Akt phosphorylation and kinase activity, whereas
other inositol polyphosphates tested have no effect (23, 24).
Consequently, Ins(1,3,4,5,6)P
5
specifically promotes apoptosis in
lung, ovarian, and breast cancer cell lines (24). In this report, we
describe the antiangiogenic properties and the in vivo antitumor
effects of Ins(1,3,4,5,6)P
5
. We show that Ins(1,3,4,5,6)P
5
inhibits
FGF-2-induced Akt phosphorylation and consequently blocks FGF-
2-mediated survival in human umbilical vein endothelial cells
(HUVEC). In addition, Ins(1,3,4,5,6)P
5
specifically inhibits the FGF-
2-mediated cell migration and capillary tube formation of HUVEC
plated on Matrigel. Moreover Ins(1,3,4,5,6)P
5
inhibits the FGF-2-
induced angiogenic reaction in BALB/c mice, indicating that
Ins(1,3,4,5,6)P
5
is an antiangiogenic factor both in vivo and in vitro.
Finally, we report that Ins(1,3,4,5,6)P
5
reduces the s.c. growth of the
human ovarian carcinoma, SKOV-3, xenografted in nude mice and
blocks the Akt phosphorylation in vivo . These data definitively
identify the Akt inhibitor Ins(1,3,4,5,6)P
5
as a specific antiangio-
genic and antitumor factor.
Materials and Methods
Materials
Ins(1,4,5,6)P
4
(25) and Ins(1,3,4,5,6)P
5
(24) were synthesized as previously
reported. Each compound was purified to homogeneity by ion-exchange
chromatography on Q-Sepharose Fast Flow resin and used as the
triethylammonium salt, which was fully characterized by
31
P and
1
H
spectroscopy, and accurately quantified by total phosphate assay. For the
in vivo experiments, Ins(1,3,4,5,6)P
5
was synthesized on a larger scale via
2-O-benzoyl-myo-inositol (26), purified as before, and then converted into
the hexasodium salt by treatment with Dowex 50WX2-100 ion-exchange
resin followed by addition of sodium hydroxide (6 equivalents) and lyoph-
ilization. Ins(1,2,3,4,5,6)P
6
was obtained from Sigma (St. Louis, MO, phytic
acid). Anti-pSer
473
Akt, anti-pThr
308
Akt, and anti-Akt were from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-CD-31 was from PharMingen (San
Diego, CA). FGF-2 was from Peprotech (London, United Kingdom). ‘‘Akt
inhibitor’’ (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octade-
cylcarbonate) was from Calbiochem (La Jolla, CA; ref. 27). SH5 (D-3-deoxy-2-
O-methyl-myo -inositol 1-[(R)-2-methoxy-3-(octadecyloxy)propyl-hydrogen
phosphate) was from Alexis (Nottingham, United Kingdom; ref. 28).
Cell Culture
HUVECs were purchased from TCS CellWorks (Buckingham, United
Kingdom) and grown in EBM medium (Bio Whittaker, Walkersville, MD)
containing 10% fetal bovine serum (FBS) and supplemented with hEGF,
hydrocortisone, bovine brain extract, and gentamicin sulfate-amphotericin-
B, as suggested (EGM kit, Bio Whittaker). Cells were used at passages 2-6.
Cell Survival
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
HUVECs were left untreated (control) or pretreated with the indicated
concentrations of inositol polyphosphates or Akt inhibitors for 30 minutes
in M199. FGF-2 (100 ng/mL) was added for a further 48 hours in the
absence or presence of the inhibitors. Serum was added in some wells as a
positive control. 3-(4,5-Dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide solution in M199 (500 Ag/mL final concentration) was added to each
well for the last 4 hours. After washing, DMSO was added to the wells for 15
minutes, collected, and absorbance (570-650 nm) was determined by using
a spectrophotometer.
Acridine orange/ethidium bromide staining. HUVECs were left
untreated or pretreated with 50 Amol/L of inositol polyphosphates for 30
minutes in M199. FGF-2 (100 ng/mL) was added for further 48 hours in the
absence or presence of the inhibitors. Serum was added in some wells as a
positive control. Apoptosis was assessed by adding an acridine orange (100
Ag/mL)/ethidium bromide (100 Ag/mL; 1:1 v/v) mixture as described (24).
DNA laddering assay. HUVECs grown in Petri dishes were left untreated
(control) or pretreated with 50 Amol/L Ins(1,3,4,5,6)P
5
for 30 minutes in
M199. FGF-2 (100 ng/mL) was then added in the absence or presence of the
inhibitor. Cells kept in medium supplemented with serum were used as a
positive control. After 48 hours, cells were detached and DNA laddering
assay was done as described (24).
Migration Assay
Cell migration was done in Transwell chambers (tissue culture treated,
10 mm diameter, 8-Am pores; Nunc, Rochester, NY) coated with 100 Ag/mL
gelatin (0.1% in acetic acid) or 10 Ag/mL fibronectin. HUVECs were serum
starved in M199 containing 0.5% FBS overnight; pretreated with 100 nmol/L
wortmannin, 10 Amol/L LY294002, or 50 Amol/L of the indicated inositol
polyphosphates for 30 minutes; and detached. Cells were then resuspended
in M199 containing 1% bovine serum albumin Fwortmannin, LY294002, or
the inositol polyphosphates, added (25,000 cells/150 AL) to the top of each
migration chamber, and allowed to migrate in the presence of 100 ng/mL
FGF-2 Fwortmannin, LY294002, or the inositol polyphosphates in the
lower chamber. After 4 hours, cells that had not migrated were removed by
using a cotton swab, whereas migrated cells were fixed with 4%
paraformaldehyde, stained with 1% crystal violet, and counted.
Angiogenesis In vitro (Capillary Tube Formation)
Matrigel (Becton Dickinson, Bedford, MA) was added to an eight-
chamber slide and allowed to gel for 2 hours at 37jC. Cells were serum
deprived overnight in serum-free medium before detaching. HUVECs (5
10
4
) were preincubated with 50 Amol/L of the different inositol
polyphosphates or 1 Amol/L of SH5 for 20 minutes at 37jC; then, before
plating, FGF-2 (100 ng/mL) was added to the cells where necessary.
Endothelial cell migration and rearrangement was visualized after 4 to 6
hours and the number of branching points counted. Only points generating
at least three tubules were counted. Representative fields were photo-
graphed using a Nikon microscope.
Angiogenesis In vivo
BALB/c mice were injected s.c. dorsolaterally with 0.4 mL of Matrigel alone
or in combination with 5 Ag/mL FGF-2 and/or 50 Amol/L of the different
inositol polyphosphates. The injected Matrigel rapidly formed a solid gel that
persisted for at least 10 days in mice. The mice were sacrificed after 6 days, the
mass of Matrigel was removed along with overlying skin and fixed with 10%
formaldehyde for 24 hours before it was embedded in paraffin. The paraffin
blocks were then cut into 4-Am-thick sections and processed for
immunohistochemistry by using a modification of the avidin-biotin
peroxidase complex technique. Briefly, 4-Am tissue sections were deparaffi-
nized, rehydrated, and placed in 3% hydrogen peroxide to inhibit endogenous
peroxidase. The tissue sections were trypsinized with 0.1% trypsin and 0.1%
CaCl
2
for 30 minutes at 37jC to expose the antigenic sites masked by formalin
fixation, blocked for 1 hour with 3% normal goat serum, and subsequently
incubated with anti-CD-31 for 60 minutes at a dilution of 1:1,000. The sections
were then treated with biotinylated secondary antibody for 30 minutes at
room temperature followed by avidin biotin complex reagent for 30 minutes
and diaminobenzidine for 1 minute. Counterstaining was done with
hematoxylin. For a quantitative analysis, capillary density was calculated,
in the area immediately below the skin, as mean of the total number of vessels
in five independent fields in three sections.
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Xenotransplantation of Human Cell Culture in Nude Mice
SKOV-3 cells (3 10
6
) were injected s.c. (0.2 mL per mouse). Twelve days
after tumor implantation, mice were randomized in groups of nine animals
each. Ins(1,3,4,5,6)P
5
and Ins(1,2,3,4,5,6)P
6
were dissolved in saline and
injected i.p. at the dose of 1 mg per mouse per 0.2 mL. Control mice were
treated with 0.2 mL of saline. Cisplatinum was given i.v. on days 15 and 22
(q7dx2) at the dose of 4 mg/kg. The drug was dissolved in saline
immediately before use. On day 23 [24 hours following the last cisplatin
dose and 6 hours after the last dose of Ins(1,3,4,5,6)P
5
or Ins(1,2,3,4,5,6)P
6
],
two mice per group were killed and the tumor removed for monitoring Akt
phosphorylation. The experiment was ended 40 days following tumor
implant. Procedures involving animals and their care were conducted in
conformity with the institutional guidelines that are in compliance with
national and international laws and policies.
High-performance Liquid Chromatography Analysis of
Inositol Phosphates
[
3
H]Ins(1,3,4,5,6)P
5
was obtained by phosphorylation of [
3
H]Ins(1,3,4,5)P
4
(American Radiolabelled Chemicals, Inc., St. Louis, MO). [
32
P]Ins(1,3,4,5,6)P
5
was obtained by phosphorylation of Ins(1,4,5)P
4
in the presence of g[
32
P]ATP.
Phosphorylation was obtained by using yeast Inositol Polyphosphate Multi
Kinase (29). All the radiolabeled inositol phosphates synthesized were
purified by high-performance liquid chromatography (HPLC) and desalted as
described (30). SKOV-3 cells were incubated with medium containing the
radioactive compound for different times before washing with ice-cold PBS.
Alternatively, cells were incubated with the radioactive compound for 30
minutes and medium was then removed and replaced with normal medium.
After further incubation for the specified times, cells were washed with ice-
cold PBS. Inositol polyphosphates were extracted using 0.6 mL of ice-cold
acidic buffer [0.6 mol/L perchloric acid, 0.1 mg/mL Ins(1,2,3,4,5,6)P
6
, 2 mmol/
L EDTA]. The acidic extracts were neutralized with the neutralization
solution (1 mol/L potassium carbonate and 5 mmol/L EDTA) for 2 hours
in ice. Inositol polyphosphates were resolved by HPLC using a 4.6 125 mm
Partisphere SAX column (Whatman, Inc., Florham Park, NJ). The column was
eluted with a gradient generated by mixing Buffer A (1 mmol/L Na
2
EDTA)
and Buffer B [Buffer A + 1.3 mol/L (NH
4
)
2
HPO
4
(pH 3.8)] as follows: 0 to
5 minutes, 0% B; 0 to 60 minutes, 0% to 100% B; 60 to 85 minutes, 100% B.
Fractions (1 mL) were collected and counted using 4 mL of Ultima-Flo AP
LCS-cocktail (Packard, Downers Grove, IL).
Results
Ins(1,3,4,5,6)P
5
inhibits FGF-2-induced Akt phosphorylation
and survival of endothelial cells. We have recently described the
proapoptotic properties of the natural compound Ins(1,3,4,5,6)P
5
(24) and defined its mechanism of action through inhibition of the
PI3K/Akt pathway. To determine whether Ins(1,3,4,5,6)P
5
might
have antiangiogenic properties, we first tested whether
Ins(1,3,4,5,6)P
5
was able to inhibit Akt activation in endothelial
cells. FGF-2 induced Akt phosphorylation in HUVEC with a rapid
peak of activation after 10 minutes of stimulation (Fig. 1A). No Akt
phosphorylation was detected at 15 minutes of FGF-2 stimulation
(Fig. 1B), whereas a second peak of activation was observed at
longer times of stimulation (Fig. 1B). When we tested the effect of
different inositol polyphosphates, we observed that Ins(1,3,4,5,6)P
5
but not other inositol polyphosphates (Supplementary Fig. 1)
inhibited FGF-2-induced Akt phosphorylation (Fig. 1A). Once the
efficiency of Ins(1,3,4,5,6)P
5
in inhibiting the PI3K/Akt pathway in
endothelial cells was assessed, we tested the effect of this
compound in several processes associated with angiogenesis.
Figure 1. Ins(1,3,4,5,6)P
5
inhibits Akt
phosphorylation and survival in HUVEC.
A, Western blotting analysis of lysates
from serum-starved HUVEC untreated
or treated with 50 Amol/L Ins(1,3,4,5,6)P
5
for 30 minutes before stimulation with
100 ng/mL FGF-2 for 10 minutes. Top,
phosphorylation of Akt at residue Ser
473
was assessed by using a specific antibody.
Filter was then stripped and reprobed with
an anti-Akt antibody. Bottom, densitometry
analysis was carried out and the levels
of pSer
473
were normalized to the
corresponding total amount of Akt.
B, Western blotting analysis of lysates
from serum-starved HUVEC untreated or
treated with 50 Amol/L Ins(1,3,4,5,6)P
5
for 30 minutes before stimulation with
100 ng/mL FGF-2 for the indicated
times. Phosphorylation of Akt at residue
Ser
473
and total amount of Akt were
assessed as above. Bottom, densitometry
analysis was carried out and the levels
of pSer
473
were normalized to the
corresponding total amount of Akt.
C, 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl
tetrazolium bromide assay in HUVEC
treated with the indicated inhibitors in the
absence or presence of 100 ng/mL FGF-2.
The effect of Ins(1,3,4,5,6)P
5
by its own
is also shown. Columns, mean of at
least three independent experiments
done in duplicate; bars, FSE.
D, 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl
tetrazolium bromide assay in HUVEC
treated with 50 Amol/L of the indicated
inositol polyphosphates in the presence
of 100 ng/mL FGF-2. Columns, mean of
three independent experiments done in
duplicate; bars, FSE.
Antitumor Properties of Inositol Pentakisphosphate
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Angiogenesis is a process involving cellular survival, migration,
proliferation, and differentiation. Therefore, we tested the effect of
Ins(1,3,4,5,6)P
5
on FGF-2-induced cell survival in HUVEC. As shown
in Fig. 1C, FGF-2 was able to induce survival in serum-starved
HUVEC. Pretreatment of HUVEC with Ins(1,3,4,5,6)P
5
inhibited
FGF-2-mediated survival in a dose-dependent manner (Fig. 1C).
Treatment with Ins(1,3,4,5,6)P
5
in the absence of FGF-2 did not
affect survival, indicating that Ins(1,3,4,5,6)P
5
had no toxic effects
(Fig. 1C). The observation that two commercially available Akt
inhibitors (‘‘Akt inhibitor’’ from Calbiochem and SH5 from Alexis)
had a similar inhibitory effect confirmed that the PI3K/Akt
pathway is involved in FGF-2-induced cell survival (Fig. 1C). The
effect of Ins(1,3,4,5,6)P
5
was very specific because treatment with
50 Amol/L of other inositol polyphosphates, such as
Ins(1,2,3,4,5,6)P
6
and Ins(1,4,5,6)P
4
, was inactive (Fig. 1D). Taken
together, these data indicate that Ins(1,3,4,5,6)P
5
inhibits survival of
endothelial cells by blocking Akt activation.
To better characterize the mechanism of action of Ins
(1,3,4,5,6)P
5
, we checked whether it may have a proapoptotic effect
in endothelial cells, as we have reported for different cancer cell
lines (24). We therefore did apoptosis assays in HUVEC in the
absence or presence of Ins(1,3,4,5,6)P
5
and FGF-2. Acridine orange/
ethidium bromide assays confirmed that FGF-2 protected HUVEC
from apoptosis induced by serum deprivation and its effect was
comparable with that of serum (Fig. 2Aand B). Ins(1,3,4,5,6)P
5
prevented the FGF-2-mediated inhibition of apoptosis in serum-
deprived HUVEC, whereas no effect was observed in cells
treated with Ins(1,2,3,4,5,6)P
6
(Fig. 2Aand B). The proapoptotic
effect of Ins(1,3,4,5,6)P
5
was confirmed by analyzing cell morphol-
ogy (Fig. 2C) and by DNA laddering assays (Fig. 2D). Taken together,
these data indicate that Ins(1,3,4,5,6)P
5
has proapoptotic effects
in HUVEC and can overcome the FGF-2-mediated cell survival.
Ins(1,3,4,5,6)P
5
inhibits FGF-2-induced cell migration. We
next tested the effect of Ins(1,3,4,5,6)P
5
on cell migration in HUVEC.
FGF-2-induced cell migration in HUVEC was assessed by using
gelatin-coated (Fig. 3Aand B) or fibronectin-coated (see below)
Transwell chambers. Pretreatment with Ins(1,3,4,5,6)P
5
specifically
reduced the FGF-2-mediated cell migration on gelatin similarly to
Figure 2. Ins(1,3,4,5,6)P
5
prevents
FGF-2-mediated inhibition of apoptosis in
serum-deprived HUVEC. A-B, acridine
orange/ethidium bromide assays done in
serum-starved HUVEC. A, representative images
of cells: cells alive are green (acridine orange)
and dead cells are orange (ethidium bromide).
B, quantitative analysis. Columns, means of two
independent experiments done in duplicate; bars,
FSE. C, representative images of HUVEC serum
starved for 24 hours (serum free) or treated with
100 ng/mL FGF-2, 50 Amol/L Ins(1,3,4,5,6)P
5
+
FGF-2, or serum. D, electrophoresis analysis
of DNA laddering assay done in serum-starved
HUVEC as described in Materials and Methods.
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the PI3K inhibitors wortmannin and LY294002 (Fig. 3A). No
inhibition was observed when we used Ins(1,2,3,4,5,6)P
6
and
Ins(1,4,5,6)P
4
(Fig. 3A). Ins(1,3,4,5,6)P
5
on its own had no effect
on cell migration (Fig. 3B). Inhibition of Akt activation was
observed by monitoring the phosphorylation state of Akt in parallel
experiments (Fig. 3C). The observation that pretreatment with
wortmannin and LY294002 (Fig. 3A) had a similar inhibitory effect
on FGF-2-dependent migration suggested that this process is
mediated by activation of a PI3K-dependent pathway. It is
important to notice that in these particular experiments, a slight
inhibition of Akt phosphorylation was observed in cells treated
with Ins(1,2,3,4,5,6)P
6
(Fig. 3C) and this is likely due to a partial
conversion of Ins(1,2,3,4,5,6)P
6
to Ins(1,3,4,5,6)P
5
at such long
time of treatment as recently proposed (see Discussion). Never-
theless, the minimal inhibition of Akt activation induced by
Ins(1,2,3,4,5,6)P
6
did not seem sufficient to affect the FGF-2-
mediated migration (Fig. 3A), whereas the more efficient blockade
of Akt phosphorylation induced by Ins(1,3,4,5,6)P
5
was able to
reduce the FGF-2-mediated migration. Interestingly, Ins(1,3,4,5,6)P
5
completely inhibited the FGF-2-induced migration on fibronectin
[n
migrated cells
(% control): FGF-2, 261.3 F18.7; Ins(1,3,4,5,6)P
5
+
FGF-2, 92.0 F26.1; n= 3].
Ins(1,3,4,5,6)P
5
inhibits angiogenesis in vitro and in vivo .We
then tested the effect of Ins(1,3,4,5,6)P
5
on tubulogenesis in vitro.
FGF-2 induced capillary tube formation in HUVEC cultured on
diluted Matrigel and this effect was completely inhibited by
pretreatment with 50 Amol/L Ins(1,3,4,5,6)P
5
,whereas
Ins(1,4,5,6)P
4
and Ins(1,2,3,4,5,6)P
6
used at the same concentra-
tion had no effect (Fig. 4). The involvement of Akt in this
process was confirmed by a similar inhibition obtained using the
Akt inhibitor SH5 (Fig. 4). Representative fields are shown in
Fig. 4A, whereas a quantitative analysis of the same experiment
is shown in Fig. 4B. Neither Ins(1,3,4,5,6)P
5
nor SH5 on their
own had some effect on tubulogenesis (Supplementary Fig. 2)
confirming that they were specifically inhibiting FGF-2-mediated
effect. To test whether Ins(1,3,4,5,6)P
5
inhibits not only
morphogenesis (in vitro) but also angiogenesis (in vivo), we
injected BALB/c mice with FGF-2 along with Matrigel with or
without several different inositol polyphosphates. After 5 days,
the gels were removed, embedded, sectioned, and stained with
anti-CD-31 antibody for the presence of blood vessels. As shown
in Fig. 5A, Matrigel supplemented with FGF-2 induced an
angiogenic reaction associated with the chemotactic response.
Interestingly, in the presence of 50 Amol/L Ins(1,3,4,5,6)P
5
,we
observed a clear inhibition of the FGF-2-induced angiogenic
response, whereas under the same conditions, no effect was
observed in the presence of Ins(1,2,3,4,5,6)P
6
, Ins(1,4,5,6)P
4
,
Ins(1,3,4,5)P
4
, or Ins(3,4,5,6)P
4
(Supplementary Fig. 3). No blood
vessel formation was detected in the presence of the different
inositol polyphosphates but in the absence of FGF-2 (Supple-
mentary Fig. 3). Capillary density was calculated, in the area
immediately below the skin, as mean of the total number of
vessels in five independent fields in three sections. Taken
together, these data indicate that Ins(1,3,4,5,6)P
5
specifically has
antiangiogenic properties in both in vitro and in vivo assays.
Ins(1,3,4,5,6)P
5
inhibits growth of human ovarian carcinoma
SKOV-3 xenograft. The proapoptotic and antiangiogenic effects
of Ins(1,3,4,5,6)P
5
strongly suggested that this compound might
have antitumor properties. We therefore tested the antineoplastic
activity of Ins(1,3,4,5,6)P
5
on SKOV-3 human ovarian carcinoma
implanted s.c. in nude mice. Twelve days after inoculation of cells,
one group of mice was treated with Ins(1,3,4,5,6)P
5
, a second one
with Ins(1,2,3,4,5,6)P
6
, a third one with cisplatin, a drug
commonly used in treatment of ovarian carcinoma, and a control
group was treated with vehicle alone. Measurements of tumors
volume are graphed in Fig. 6A. Very interestingly, we observed
that Ins(1,3,4,5,6)P
5
clearly reduced tumor growth (Fig. 6A) to the
same extent than cisplatin (Fig. 6A). On the contrary,
Ins(1,2,3,4,5,6)P
6
only minimally slowed tumor growth during
the first days of treatment but had no further effect during the
Figure 3. Ins(1,3,4,5,6)P
5
inhibits the FGF-2-induced migration of HUVEC. A, Transwell assays on gelatin-coated chambers were done on cells pretreated with
100 nmol/L wortmannin, 10 Amol/L LY294002, or 50 Amol/L of the indicated inositol polyphosphates in the presence of 100 ng/mL FGF-2. Columns, mean of at least
five independent experiments and are expressed as percentage of each control; bars, FSE. *, P< 0.05; **, P< 0.01. B, Transwell assays on gelatin-coated chambers
were done on cells pretreated with 50 Amol/L of Ins(1,3,4,5,6)P
5
in the presence of 100 ng/mL FGF-2. Columns, mean of at least three independent experiments
and are expressed as the number of migrated cells per field; bars, FSE. In such experimental conditions, 300 F25 cells per field migrated upon FGF-2 stimulation:
this represented a 2-fold increase of migration over basal (143 F16 cells per field). *, P< 0.01. C, Western blotting analysis of lysates from HUVEC pretreated with
50 Amol/L of the indicated inositol polyphosphates. After detachment, cells were plated on gelatin-coated wells and allowed to adhere for 4 hours in the presence of 100
ng/mL FGF-2 alone or in combination with the indicated inositol polyphosphates. Top, phosphorylation of Akt at residue Ser
473
was assessed by using a specific
antibody. Filter was then stripped and reprobed with an anti-Akt antibody. Arrow points the band corresponding to total Akt. Bottom, densitometry analysis was
carried out and the levels of pSer
473
were normalized to the corresponding total amount of Akt.
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following days (Fig. 6A). No sign of toxicity, as judged by parallel
monitoring body weight, was observed in Ins(1,3,4,5,6)P
5
-treated
mice. A reduction in tumor weight was clearly appreciable in
Ins(1,3,4,5,6)P
5
-treated mice at the end of the experiment (Fig. 6B).
Importantly, a complete inhibition of Akt phosphorylation at
both residue Ser
473
(Fig. 6C) and Thr
308
(Fig. 6D) was observed in
Ins(1,3,4,5,6)P
5
-treated mice after 12 days of treatment. Strikingly,
the Ins(1,3,4,5,6)P
5
-induced inhibition was even higher than the
cisplatin-induced reduction (Fig. 6Cand D). A slight inhibition of
Akt phosphorylation was observed in Ins(1,2,3,4,5,6)P
6
-treated mice
(Fig. 6Cand D), although such effect was clearly not sufficient
to completely block tumor growth (Fig. 6A-B). This is the first
demonstration that Ins(1,3,4,5,6)P
5
can inhibit Akt activation and
block the tumor growth in vivo.
Ins(1,3,4,5,6)P
5
internalization and dephosphorylation in
SKOV-3 cells. Data thus far did not rule out the possibility that
Ins(1,3,4,5,6)P
5
might be converted into different metabolites that
were ultimately responsible for the observed antitumor effect.
Therefore, we decided to check the intracellular stability of
exogenously added Ins(1,3,4,5,6)P
5
over time and to check whether
Ins(1,3,4,5,6)P
5
was converted intracellularly to different phosphor-
ylated metabolites. SKOV-3 were then incubated with
[
32
P]Ins(1,3,4,5,6)P
5
for different times and inositol phosphates
were extracted and analyzed by HPLC to detect the different
radioactive inositol compounds. First of all, we observed that
Ins(1,3,4,5,6)P
5
was indeed able to enter SKOV-3 in a time-
dependent manner (Fig. 7A) and this represents a clear
demonstration that, although highly negatively charged, inositol
polyphosphates can cross the plasma membrane and be internal-
ized by cells as we reported for the first time (23) and was later
confirmed by other groups (31). Furthermore, we observed that the
turnover of Ins(1,3,4,5,6)P
5
was quite slow, because only a 5.0% of
the total Ins(1,3,4,5,6)P
5
was converted into different metabolites
after 30 minutes of incubation and only a 6.2% was converted after
1 hour of incubation (Fig. 7B). The slow turnover of Ins(1,3,4,5,6)P
5
was confirmed by pulse-chase experiments done by incubating
SKOV-3 with [
3
H]Ins(1,3,4,5,6)P
5
for 30 minutes followed by
incubation in the absence of the radioactive compound for
different times. Parallel control experiments were done by using
[
3
H]Ins(1,3,4,5)P
4
. Inositol phosphates were extracted at different
times of incubation and analyzed by HPLC. As shown in Fig. 7C,
the incorporated Ins(1,3,4,5,6)P
5
was very stable and a 84.6% of
total [
3
H]Ins(1,3,4,5,6)P
5
was still detectable intracellularly even
after 5 hours of incubation (Fig. 7C). On the contrary,
[
3
H]Ins(1,3,4,5)P
4
was rapidly and almost completely metabolized
with only a 7% of total still detectable after 5 hours of incubation
(Fig. 7C).
These data clearly indicate that Ins(1,3,4,5,6)P
5
is rapidly and
efficiently internalized by cells and is only minimally converted
into different metabolites strongly suggesting that the observed
antitumor effects were due to its activity and were not mediated by
conversion to different phosphorylated forms.
Figure 4. Ins(1,3,4,5,6)P
5
inhibits the
FGF-2-induced capillary tube formation of
HUVEC. A, representative images of HUVEC
on Matrigel in the absence or presence of
FGF-2 along with the indicated inhibitors (50
Amol/L inositol polyphosphates, 1 Amol/L SH5).
B, quantitative analysis of the experiments
shown in (A) obtained by counting the number
of branching points from five fields. *, P< 0.01.
Representative of at least three independent
experiments.
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Discussion
Regulation of cell proliferation and cell survival in cancer
involves a complex interplay among steroid hormones, growth
factors, and their receptors. Understanding the signaling pathways
involved in these processes may help in finding predictive factors
for tumor aggressiveness and therapy resistance. Among the
different pathways activated by growth factor receptors, signals
transmitted by PI3K and Akt have proven important for cell
survival in many cell types, and genetic and biochemical evidence
suggest that inappropriate activation of the PI3K/Akt pathway is
linked to the development of cancer (32–35). Altered expression or
mutation of several components of this pathway has been
implicated in the development and progression of human cancer
(36). In particular, it has been reported that PI3K mutations
identified in human cancer are oncogenic (37). Indeed, amplifica-
tion of the gene coding for the p110 catalytic subunit of PI3K has
been observed in different tumor types (34, 38), and activating
somatic mutations in its regulatory subunit have been found in
primary ovarian and colon tumors (39). In addition, amplification
of Akt2 can occur in about 40% of breast cancer specimens as well
as ovarian and pancreatic cancers (40–42), and Akt phosphoryla-
tion is frequently detected in ovarian cancer (43). The most
compelling evidence for the involvement of the PI3K/Akt pathway
in human cancer comes from studies of the PTEN tumor
suppressor gene that encodes a dual specificity protein phospha-
tase, which also possesses a phosphoinositide 3-phosphatase
activity (22). In several human cancers, the PTEN gene has been
found to be deleted or mutated, indicating that PTEN loss occurs
in a wide spectrum of human cancers and that this is correlated,
among other effects, with a constitutive activation of the PI3K/Akt
pathway. Interestingly, an elevated Akt activity is associated with
increased cellular resistance to treatment with chemotherapeutic
agents (44), and recent data suggest that Akt may be a potential
target for enhancing the response to radiotherapy in patients with
breast cancer (45). Indeed, inhibition of this pathway has been
shown to facilitate apoptosis and to sensitize cells to cytotoxic
drugs in experimental studies (24, 46).
The link between activation of the PI3K/Akt pathway and cancer
makes this pathway an attractive target for therapeutic intervention
strategies (47). Indeed, it has been reported that the PI3K inhibitors
wortmannin and LY294002 possess antitumor activity in vitro and
in vivo, although their general toxicity and lack of selectivity,
together with the instability of wortmannin and the insolubility of
LY294002, mean that neither has very promising pharmaceutical
potential (48–50). In addition, other compounds have emerged over
the past few years as potential inhibitors of the PI3K/Akt pathway
such as alkylphospholipids or phosphoinositide ether lipid ana-
logues (49, 50). Limitations to the use of these compounds as
chemotherapeutic agents include difficulties with solubility, chem-
ical stability, and toxicity (49, 50). Given that administration of
standard chemotherapy agents is usually associated with at least
mild toxicity, the possible use of nontoxic, natural compounds to
target the PI3K/Akt pathway and prevent cancer is attractive.
Recently, we have successfully used an alternative strategy to
specifically block the PI3K/Akt pathway based on the mechanism of
membrane targeting mediated by the interaction of pleckstrin
homology domains with the lipid products of PI3K (23, 24). In
particular, we have used inositol polyphosphates, the water-soluble
head groups of phosphoinositides, to specifically block the re-
cruitment of Akt to the plasma membrane and therefore inhibit
its activation. This strategy was based on the hypothesis that, by
binding to the Akt pleckstrin homology domain, specific exogen-
ous inositol polyphosphates can compete with phosphatidylin-
ositol(3,4,5)trisphosphate [PI(3,4,5)P
3
] and therefore prevent
PI(3,4,5)P
3
-dependent recruitment to the plasma membrane (51).
The use of inositol polyphosphates as potential anticancer
agents has been supported by several studies indicating that
Ins(1,2,3,4,5,6)P
6
possesses antitumor activity in vitro and in vivo
(52). However, the very high concentrations required for
Ins(1,2,3,4,5,6)P
6
to be active (1-5 mmol/L) suggest a lack of
selectivity of this compound, although it is noteworthy that, even at
these concentrations, inositol polyphosphates do not seem to have
toxic effects (52). Although several mechanisms have been
proposed including through PI3K inhibition, little is still known
about the mechanisms by which Ins(1,2,3,4,5,6)P
6
exerts its
anticancer actions. Interestingly, it has been recently reported that
Ins(1,2,3,4,5,6)P
6
enters HeLa cells and is dephosphorylated to
lower forms, mainly Ins(1,3,4,5,6)P
5
, that in turn is able to induce
Figure 5. Ins(1,3,4,5,6)P
5
has antiangiogenic properties in vivo.
A, representative images of tissue sections in the presence of FGF-2 alone
or FGF-2 along with 50 Amol/L Ins(1,3,4,5,6)P
5
.B, quantitative analysis:
capillary density was calculated, in the area immediately below the skin,
as mean of the total number of vessels in five independent fields in three
sections. *, P< 0.01.
Antitumor Properties of Inositol Pentakisphosphate
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apoptosis (31). Indeed, among the different inositol polyphosphates
tested in HeLa cells, Ins(1,3,4,5,6)P
5
specifically inhibited Akt
phosphorylation and activity, showed proapoptotic properties, and
was more active than Ins(1,2,3,4,5,6)P
6
. These data are in agreement
with our previous work (24) and suggest that the anticancer activity
of Ins(1,2,3,4,5,6)P
6
is actually due to its dephosphorylation to lower
forms that are potent in inducing apoptosis. It is noteworthy that
in this report, we observed that Ins(1,2,3,4,5,6)P
6
does not seem to
have an effect on Akt phosphorylation when tested in short-time
experiments (Supplementary Fig. 1), whereas it seems to slightly
inhibit Akt at longer incubations (Fig. 3C; Fig. 6Cand D). The
observation that such an effect is visible only at longer incubation
strongly suggests that it is likely due to a slight conversion of such
inositol polyphosphate to different forms, likely Ins(1,3,4,5,6)P
5
.
Indeed, a degradation of Ins(1,2,3,4,5,6)P
6
to lower phosphorylated
forms has been already observed in cancer cells although with a
slow kinetic because the bulk of such compound seems to be in the
hexakisphosphate form even after 6 hours of incubation (31). Such
a slow conversion of Ins(1,2,3,4,5,6)P
6
to lower phosphorylated
forms may easily explain the only minimal effect of
Ins(1,2,3,4,5,6)P
6
on Akt inhibition and, more important, the lack
of effect on both the FGF-2-induced migration and tumor cell
growth; that is, the amount of active compound derived from
degradation of Ins(1,2,3,4,5,6)P
6
is not sufficient to mediate a
physiologic effect. This might also explain the requirement of very
high concentrations for Ins(1,2,3,4,5,6)P
6
to be active. In this
respect, our data corroborate the reported anticancer properties of
Ins(1,2,3,4,5,6)P
6
that are possibly due to its conversion to
Ins(1,3,4,5,6)P
5
. Indeed, Ins(1,3,4,5,6)P
5
seems the most stable
among the lower phosphorylated forms of inositol polyphosphates.
In fact, the accurate HPLC analysis done in this report shows that
the turnover of Ins(1,3,4,5,6)P
5
is very slow, because we observed
that only 5.0% of the total [
32
P]Ins(1,3,4,5,6)P
5
was converted into
different metabolites after 30 minutes of incubation and only 6.2%
was converted after 1 hour of incubation (Fig. 7B). The slow
turnover of Ins(1,3,4,5,6)P
5
was confirmed by pulse-chase experi-
ments showing that the incorporated [
3
H]Ins(1,3,4,5,6)P
5
was very
stable and 84.6% of total [
3
H]Ins(1,3,4,5,6)P
5
was still detectable
intracellularly even after 5 hours of incubation (Fig. 7C). On the
contrary, [
3
H]Ins(1,3,4,5)P
4
was almost completely metabolized
with only 7% of the total still detectable after 5 hours of incubation
(Fig. 7C). This is consistent with our reported data obtained in
SCLC where conversion of [
3
H]Ins(1,3,4,5)P
4
was even more rapid
(23). Taken together, these data clearly indicate that Ins(1,3,4,5,6)P
5
is rapidly and efficiently internalized by cells. The observation that
the inhibitory effects of Ins(1,3,4,5,6)P
5
were observed already at
short times of incubation and that only a small percentage of the
compound is further converted into different metabolites even
Figure 6. Ins(1,3,4,5,6)P
5
inhibits growth of
SKOV-3 xenograft by blocking Akt activation.
SKOV-3 cells were implanted s.c. in nude mice
as described in Materials and Methods. A-B,
columns, mean of tumor volume measured
at the indicated days after implant; bars, FSE.
‘‘Control’’ group (black line) was treated
with vehicle alone; ‘‘Ins(1,3,4,5,6)P
5
’’ group
(turquoise line) was treated daily with 50 mg/kg
Ins(1,3,4,5,6)P
5
for a further 12 days
(horizontal blue line); ‘‘Ins(1,2,3,4,5,6)P
6
’’
group (pink line) was treated daily with
50 mg/kg Ins(1,2,3,4,5,6)P
6
for a further 12
days (horizontal blue line ); ‘‘cisplatin’’ group
(red line) was treated with 4 mg/kg cisplatin
at the indicated days (vertical green lines ).
For both Ins(1,3,4,5,6)P
5
and cisplatin group: *,
P< 0.05 and **, P< 0.01 versus control.
B, tumor weight measured at the end of the
experiment. C-D, Western blotting analysis
of homogenates from tumors at day 12.
Phosphorylation of Akt at residue Ser
473
(C)
or Thr
308
(D) was assessed by using a
specific antibody. Filter was then stripped and
reprobed with an anti-Akt antibody. Bottom,
corresponding densitometry analysis where the
levels of phosphorylated Akt were normalized
to the corresponding total amount of Akt.
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after long time of incubation strongly suggest that the observed
antitumor effects are directly attributable to Ins(1,3,4,5,6)P
5
and are
not mediated by conversion to different phosphorylated forms.
Several pieces of evidence indicate that Ins(1,3,4,5,6)P
5
promotes
apoptosis through inhibition of the PI3K/Akt pathway. In fact, the
specificity of Ins(1,3,4,5,6)P
5
activity is confirmed by our experimen-
tal evidence indicating that this compound is active in human
cancer cell lines characterized by an elevated PI3K/Akt activity,
whereas it is inactive in other cell lines tested (23, 24). Our previous
work confirmed a strict correlation between the Ins(1,3,4,5,6)P
5
-
dependent apoptotic rate and the degree of inhibition of Akt
signaling and indicated that Ins(1,3,4,5,6)P
5
was a very specific
inhibitor of this pathway in vitro resulting in proapoptotic effects in
cancer cell lines. We then aimed at investigating the properties of
this compound in vivo and further characterizing its function as an
antitumor agent. Therefore, in this study, we checked whether
Ins(1,3,4,5,6)P
5
might have antiangiogenic effects, together with its
established proapoptotic properties. Angiogenesis consists of a
multistep process involving cell survival, migration, and differenti-
ation. Therefore, we first examined the effects of Ins(1,3,4,5,6)P
5
in
such different processes associated with angiogenesis. We observed
that Ins(1,3,4,5,6)P
5
was specifically able to inhibit FGF-2-induced
Akt phosphorylation and survival in HUVEC. The observation that
other commercially available Akt inhibitors induced a similar
inhibitory effect suggested that Akt is involved in the FGF-2-
mediated cell survival and therefore that the Ins(1,3,4,5,6)P
5
-
mediated effect on survival occurs through inhibition of the PI3K/
Akt pathway. More specifically, we observed that Ins(1,3,4,5,6)P
5
promoted apoptosis, assessed both by acridine orange/ethidium
bromide and by DNA laddering assays. In addition, we observed that
Ins(1,3,4,5,6)P
5
inhibited FGF-2-induced cell migration and angio-
genesis, as assessed both in vitro and in vivo . This is the first study
reporting an antiangiogenic activity for Ins(1,3,4,5,6)P
5
. Although the
antiangiogenic effect of Ins(1,3,4,5,6)P
5
can be partly explained by
the observed proapoptotic properties, it is likely that the complete
inhibition of angiogenesis is the result of the Ins(1,3,4,5,6)P
5
-
mediated reduction of FGF-2-induced migration as well. Therefore,
the antiangiogenic properties reported represent a clear advance in
understanding the properties of Ins(1,3,4,5,6)P
5
.
The proapoptotic and antiangiogenic properties of Ins
(1,3,4,5,6)P
5
led us to the hypothesis that this compound might
have antitumor effects. Indeed, when we examined its potential
antineoplastic activity on SKOV-3 human ovarian carcinoma
implanted s.c. in nude mice, we found that Ins(1,3,4,5,6)P
5
blocked
tumor growth. These data make a significant advance compared
with our previous work in particular because they validate the
effects of Ins(1,3,4,5,6)P
5
in an in vivo model. Strikingly, not only did
we observe that Ins(1,3,4,5,6)P
5
inhibited the growth of the human
ovarian carcinoma SKOV-3 xenografts but also that the effect of
Ins(1,3,4,5,6)P
5
was comparable to that of cisplatin, the drug
commonly used for ovarian cancer treatment. Furthermore, a clear
and total inhibition of Akt phosphorylation at both residues Ser
473
and Thr
308
was observed in Ins(1,3,4,5,6)P
5
-treated mice. This
represents the first demonstration of an in vivo effect of
Ins(1,3,4,5,6)P
5
on Akt activation.
Inhibition of the Akt pathway may prove highly effective in
future treatment regimens, although likely in combination with
canonical cytotoxic drugs. Indeed, our previous work showed that
in vitro Ins(1,3,4,5,6)P
5
enhances the proapoptotic effects of
cisplatin and etoposide in ovarian and lung cancer cells,
respectively (24), supporting a role for Ins(1,3,4,5,6)P
5
as a
compound able to sensitize cancer cells to the action of commonly
used anticancer drugs. It is important to underline that inositol
polyphosphates, including Ins(1,3,4,5,6)P
5
, are naturally occurring
substances that are present in most legumes, and in wheat bran
and nuts (53). Therefore, our study reveals a new pharmacologically
active nutrient (‘‘nutraceutical’’) and underlines the importance of
the use of certain foods in the prevention of cancer. Furthermore,
the observation that inositol polyphosphates are easily absorbed by
oral administration (52) makes Ins(1,3,4,5,6)P
5
even more promis-
ing in terms of therapeutic potential.
Figure 7. Ins(1,3,4,5,6)P
5
internalization and dephosphorylation in
SKOV-3 cells. A-B, SKOV-3 cells were incubated with medium containing
[
32
P]Ins(1,3,4,5,6)P
5
obtained as described in Materials and Methods. Cells were
incubated for the indicated minutes (1-60 minutes, legends ) before inositol
phosphates extraction. A, results of the HPLC analysis [total cpm (Yaxis up to
150,000 cpm) versus the different retention times (Xaxis)]. The peak
corresponds to Ins(1,3,4,5,6)P
5
.B, magnification of the same graph: total cpm
on Yaxis are shown up to 3,000 cpm only. This allows to observe small
degradation products of Ins(1,3,4,5,6)P
5
.C, SKOV-3 cells were incubated with
medium containing [
3
H]Ins(1,3,4,5)P
4
or [
3
H]Ins(1,3,4,5,6)P
5
obtained as
described in Materials and Methods. Cells were incubated with the radioactive
compound for 30 minutes and medium was then removed and replaced with
normal medium. After further incubation for the specified times (Xaxis), inositol
polyphosphates were extracted and analyzed by HPLC as described in Materials
and Methods. Percentage of the total labeled compound incorporated.
Antitumor Properties of Inositol Pentakisphosphate
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Taken together, these data indicate that specific blockade of the
PI3K/Akt pathway by Ins(1,3,4,5,6)P
5
results in proapoptotic and
antiangiogenic effects, and they show for the first time that
Ins(1,3,4,5,6)P
5
is able to inhibit tumor growth in vivo . It is note-
worthy that the concentration of Ins(1,3,4,5,6)P
5
used in our in vivo
experiment is very low (50 mg/kg) and that we do not detect any
toxic effects, as judged by monitoring body weight. These properties,
together with the fact that Ins(1,3,4,5,6)P
5
is a water-soluble, natural
compound, suggest that Ins(1,3,4,5,6)P
5
may overcome problems
concerning solubility, chemical stability, and toxicity that are
currently limiting the use of other potential inhibitors of the PI3K/
Akt pathway. The identification of novel properties of Ins(1,3,4,5,6)P
5
together with the in vivo data shown in this article not only are
consistent with our previous work (23, 24) but also represent a huge
step forward in validating Ins(1,3,4,5,6)P
5
as a potent and specific
functional inhibitor of the PI3K/Akt pathway that may be useful in
the treatment of human cancers whose progression is driven by PI3K
activation or PTEN gene alterations. Therefore, we believe that
Ins(1,3,4,5,6)P
5
may be a very promising agent to develop rapidly and
bring to clinical testing.
Acknowledgments
Received 1/14/2005; revised 6/2/2005; accepted 7/11/2005.
Grant support: Compagnia di San Paolo Programma Oncologia (P. Innocenti and
M. Falasca), British Heart Foundation grant PG/04/033/16906 (M. Falasca), Wellcome
Trust Programme grant 060554 (B.V.L. Potter), Italian Ministry of Health (M. Broggini),
Caripe Fondazione Cassa di Risparmio di Pescara (cost of equipment and
instruments), Diabetes UK RD L awrence Fellowship grant BDA:RD0 4/0002884
(T. Maffucci), and Dr. Mortimer and Mrs. Theresa Sackler Trust (M. Falasca).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Prof. I. Zachary (University College London, United Kingdom) and Dr. R.
Giavazzi (Mario Negri Institute for Pharmacological Research, Bergamo, Italy) for
critical readings of the article.
Cancer Research
Cancer Res 2005; 65: (18). September 15, 2005 8348 www.aacrjournals.org
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