Synthetic curcuminoids modulate the arachidonic acid metabolism of human platelet 12-lipoxygenase and reduce sprout formation of human endothelial cells.

Abstract

Platelet 12-lipoxygenase (P-12-LOX) is overexpressed in different types of cancers, including prostate cancer, and the level of expression is correlated with the grade of this cancer. Arachidonic acid is metabolized by 12-LOX to 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], and this biologically active metabolite is involved in prostate cancer progression by modulating cell proliferation in multiple cancer-related pathways inducing angiogenesis and metastasis. Thus, inhibition of P-12-LOX can reduce these two processes. Several lipoxygenase inhibitors are known, including plant and mammalian lipoxygenases, but only a few of them are known inhibitors of P-12-LOX. Curcumin is one of these lipoxygenase inhibitors. Using a homology model of the three-dimensional structure of human P-12-LOX, we did computational docking of synthetic curcuminoids (curcumin derivatives) to identify inhibitors superior to curcumin. Docking of the known inhibitors curcumin and NDGA to P-12-LOX was used to optimize the docking protocol for the system in study. Over 75% of the compounds of interest were successfully docked into the active site of P-12-LOX, many of them sharing similar binding modes. Curcuminoids that did not dock into the active site did not inhibit P-12-LOX. From a set of the curcuminoids that were successfully docked and selected for testing, two were found to inhibit human lipoxygenase better than curcumin. False-positive curcuminoids showed high LogP (theoretical) values, indicating poor water solubility, a possible reason for lack of inhibitory activity or/and nonrealistic binding. Additionally, the curcuminoids inhibiting P-12-LOX were tested for their ability to reduce sprout formation of endothelial cells (in vitro model of angiogenesis). We found that only curcuminoids inhibiting human P-12-LOX and the known inhibitor NDGA reduced sprout formation. Only limited inhibition of sprout formation at approximately IC(50) concentrations has been seen. At IC(50), a substantial amount of 12-HETE can be produced by lipoxygenase, providing a stimulus for angiogenic sprouting of endothelial cells. Increasing the concentration of lipoxygenase inhibitors above IC(50), thus decreasing the concentration of 12(S)-HETE produced, greatly reduced sprout formation for all inhibitors tested. This universal event for all tested lipoxygenase inhibitors suggests that the inhibition of sprout formation was most likely due to the inhibition of human P-12-LOX but not other cancer-related pathways.

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Synthetic curcuminoids modulate the arachidonic acid
metabolism of human platelet 12-lipoxygenase and
reduce sprout formation of human endothelial cells
Jerzy Jankun,1,2Ansari M. Aleem,1
Sylvia Malgorzewicz,1Maria Szkudlarek,1
Maria I. Zavodszky,3David L. DeWitt,3
Michael Feig,3Steven H. Selman,1,2
and Ewa Skrzypczak-Jankun1
1Urology Research Center, Department of Urology;2Physiology
and Molecular Medicine, Medical University of Ohio, Toledo, Ohio;
and3Department of Biochemistry and Molecular Biology,
Michigan State University, East Lansing, Michigan
Abstract
Platelet 12-lipoxygenase (P-12-LOX) is overexpressed in
different types of cancers, including prostate cancer, and
the level of expression is correlated with the grade of this
cancer. Arachidonic acid is metabolized by 12-LOX to
12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], and
this biologically active metabolite is involved in prostate
cancer progression by modulating cell proliferation in
multiple cancer-related pathways inducing angiogenesis
and metastasis. Thus, inhibition of P-12-LOX can reduce
these two processes. Several lipoxygenase inhibitors are
known, including plant and mammalian lipoxygenases, but
only a few of them are known inhibitors of P-12-LOX.
Curcumin is one of these lipoxygenase inhibitors. Using a
homology model of the three-dimensional structure of
human P-12-LOX, we did computational docking of
synthetic curcuminoids (curcumin derivatives) to identify
inhibitors superior to curcumin. Docking of the known
inhibitors curcumin and NDGA to P-12-LOX was used to
optimize the docking protocol for the system in study.
Over 75% of the compounds of interest were successfully
docked into the active site of P-12-LOX, many of them
sharing similar binding modes. Curcuminoids that did not
dock into the active site did not inhibit P-12-LOX. From a
set of the curcuminoids that were successfully docked and
selected for testing, two were found to inhibit human
lipoxygenase better than curcumin. False-positive curcu-
minoids showed high LogP (theoretical) values, indicating
poor water solubility, a possible reason for lack of
inhibitory activity or/and nonrealistic binding. Additionally,
the curcuminoids inhibiting P-12-LOX were tested for their
ability to reduce sprout formation of endothelial cells
(in vitro model of angiogenesis). We found that only
curcuminoids inhibiting human P-12-LOX and the known
inhibitor NDGA reduced sprout formation. Only limited
inhibition of sprout formation at fIC50concentrations has
been seen. At IC50, a substantial amount of 12-HETE can
be produced by lipoxygenase, providing a stimulus for
angiogenic sprouting of endothelial cells. Increasing the
concentration of lipoxygenase inhibitors above IC50, thus
decreasing the concentration of 12(S)-HETE produced,
greatly reduced sprout formation for all inhibitors tested.
This universal event for all tested lipoxygenase inhibitors
suggests that the inhibition of sprout formation was
most likely due to the inhibition of human P-12-LOX but
not other cancer-related pathways. [Mol Cancer Ther
2006;5(5):1371–82]
Introduction
Several studies have implicated the role of dietary fatty
acids, especially arachidonic acid, in prostate cancer
formation and progression (1, 2). Three types of enzymes
[cyclooxygenases, epoxygenases (cytochrome P450), and
lipoxygenases] can metabolize this acid. Most cancer-
related research has been done on cytochromes and
cyclooxygenases, but much less is known about lipoxy-
genases. Human lipoxygenases (f670 amino acids) are
divided into several major categories [5-lipoxygenase
(5-LOX), 8-LOX, 11-LOX, 12-LOX, and 15-LOX] depending
on the outcome of arachidonic acid peroxidation (3). A
growing body of evidence points to the crucial role of
12-LOX involvement in prostate cancer.
Originally, platelet-type 12-LOX (P-12-LOX) was believed
to be expressed solely in platelets, HEL cells, and umbilical
vein endothelial cells (4). However, P-12-LOX expression
has been detected in various cell lines (DU-145, LnCAP, and
PC-3) and tumor tissues, including the prostate (5). Gao et al.
(6) found that P-12-LOX mRNA expression was significantly
higher in prostate adenocarcinoma tissue compared with
matched normal prostate epithelium, and that this increased
expression is correlated with advanced stage and grade of
adenocarcinomas. In their study, tissues from >130 patients
were examined with 38% showing elevated P-12-LOX
mRNA in malignant tissue compared with normal matched
tissue. The level of elevation of P-12-LOX expression among
Received 1/13/06; revised 2/20/06; accepted 3/17/06.
Grant support: NIH grants CA90524 and CA109625 and Frank D.
Stranahan Endowment Fund for Oncological Research.
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.
Note: The present address for S. Malgorzewicz is Department of Clinical
Nutrition, Institute of Internal Medicine, Medical University of Gdansk,
80-211 Gdansk, Poland.
Requests for reprints: Jerzy Jankun, Urology Research Center, Medical
University of Ohio, 3065 Arlington, Toledo, OH 43614-5807.
Phone: 419-383-3691; Fax: 419-383-3785. E-mail: jerzy@meduohio.edu,
http://golemxiv.dh.mco.edu/~jerzy/
Copyright C 2006 American Association for Cancer Research.
doi:10.1158/1535-7163.MCT-06-0021
1371
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high-grade prostatic adenocarcinomas compared with that
of low- and intermediate-grade prostatic adenocarcinoma
proved statistically significant. Some studies suggest an
association among prostate cancer progression, metastasis,
and an elevated expression of P-12-LOX (6, 7). Furthermore,
it was suggested that prostate cancer cells express several
megakaryocytic genes (adhesion receptors a Iib, b3, thrombin
receptor, and PECAM/CD31 and/or P-12-LOX) mimicking
platelet cells, which help in cancer hematogenous dissemi-
nation (8).
Arachidonic acid is metabolized by 12-LOX to 12(S)-
hydroxyeicosatetraenoic acid [12(S)-HETE], and this bio-
logically active metabolite has been reported to be
potentially involved in prostate cancer development by
modulating cell proliferation (1, 9, 10). 12(S)-HETE has also
been shown to play a significant role in the processes of
tumor-induced angiogenesis and metastasis. 12(S)-HETE
possesses mitogenic properties for microvascular endothe-
lial cells (11) and can promote endothelial cell migration
(12). Surface expression of integrin avh3, a tumor-induced
angiogenic vasculature–related endothelial cell integrin, is
up-regulated by 12(S)-HETE, promoting integrin transloca-
tion from intracellular pools (13). Furthermore, 12(S)-HETE
can induce endothelial cell cytoskeletal rearrangement,
resulting in endothelial cell retraction (14), a necessary step
for tumor cell extravasations. In addition, 12(S)-HETE can
stimulate tumor cell motility (15) and augment the invasive
potential of AT2.1 rat prostate tumor cells (16). Through a
protein kinase C–dependent pathway, 12-HETE has been
reported to modulate the release of the lysosomal enzyme
cathepsin B in MCF10AneoT human mammary carcinoma
cells and murine B16a melanoma cells (10). Our own
studies show that P-12-LOX overexpression in human
prostate cancer (PC3) cells promotes the increased accu-
mulation of 12(S)-HETE and vascular endothelial growth
factor in culture media, leading to constitutive extracellular
signal-regulated kinase 1/2 phosphorylation. This process
is driven by 12(S)-HETE that stimulate extracellular signal-
regulated kinase 1/2 phosphorylation via a pertussis
toxin–sensitive G-protein–coupled receptor and mitogen-
activated protein/extracellular signal regulated kinase
kinase (17).
Recent studies have verified the significant role that
12(S)-HETE plays in tumor related angiogenesis. Nie et al.
(12) used nude mice injected with human prostate PC-3
cancer cells overexpressing P-12-LOX to show that P-12-
LOX-transfected cells grow faster in vivo and form larger
tumors, and that there was a positive correlation between
tumor size and increased tumor angiogenesis. In a similar
study, Connolly and Rose (18) injected P-12-LOX over-
expressing human breast MCF-7 cancer cells into nude
mice and showed that P-12-LOX could accelerate the
growth rate and the tumor volume due to increased
angiogenic-stimulating properties. Furthermore, Pidgeon
et al. (1) showed that treatment of PC-3 and DU145 human
prostatic cancer cells with P-12-LOX inhibitors baicalein
and N-benzyl-N-hydroxy-5-phenylpentamine resulted in
significant apoptosis of these prostate cancer cells. In
addition, PC-3 cells showed a decrease in phosphorylated
retinoblastoma protein and inhibition of other retinoblas-
toma-associated proteins (p107 and p130). Of significance
in this study was that treatment with baicalein blocked the
loss of phosphorylated retinoblastoma protein; however,
the addition of 12(S)-HETE induced phosphorylated
retinoblastoma protein expression. In addition, the addition
of 12(S)-HETE reversed baicalein-induced apoptosis,
whereas other lipoxygenase metabolites, 5(S)-HETE, or
15(S)-HETE did not. The authors suggest that these results
stress the critical role of the 12-LOX pathway in the
regulation of prostate cancer progression and apoptosis.
They also strongly endorse the idea that inhibitors of 12-
LOX are potential therapeutic agents in the treatment of
prostate cancer (1). We have found that baicalein reduces
sprout formation and tumor size of human prostate
xenografts (PC3 and DU145) in experimental animals (19).
India is the one of the countries with the most diverse
populations and diets in the world. Rates for colorectal,
prostate, and lung cancers in that country (despite popula-
tion and diet diversity) are one of the lowest in the world. Of
particular interest for cancer prevention in India is the role of
turmeric (curcumin), one of the most common Indian spices
(20). Curcumin is also used in Indian traditional medicine for
various ailments and through different routes of adminis-
tration, including topical, oral, and by inhalation (21). This
chemical is a naturally occurring polyphenolic phytochem-
ical isolated from the powdered rhizome of the plant
Curcuma longa. Curcumin has known anti-inflammatory
properties and was used for generations in folk medicine for
that purpose. Traditionally, two possible mechanisms of
curcumin (diferuloyl methane) for protection against cancer
have been postulated: (a) antioxidant property and (b)
antioxidant-dependent induction of detoxifying enzymes
(22). However, curcumin can down-regulate the expression
and activity of some other enzymes important in cancero-
genesis, including cyclooxygenases and lipoxygenases
(23–25). Limiting factors in the therapeutic use of curcumin
are its relatively low IC50and bioavailability. By employing
homology modeling to predict the structure of the human
P-12-LOX and using this structure as target for docking, we
were able to predict a possible binding mode of curcumin in
the active site of human P-12-LOX that is identical to
soybean lipoxygenase determined by X-ray experiment (26).
Using the same target, we then screened a variety of
curcumin derivatives in search of better and novel human
lipoxygenase inhibitors.
Materials and Methods
Homology Modeling of P-12-LOX
The structure of P-12-LOX is unknown. However, a
model has been created using an automated protein
modeling server, Swiss Model (27, 28), which is based
mainly on the homology to the known structure of rabbit
lipoxygenase, PDB entry 1-LOX (29). Additional structures
used in modeling included soybean lipoxygenases 2SBL
(30), 1NO3 (31), 1JNQ (32), and 1IK3 (33) and human
autocrine motility factor 1JIQ (34).
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The model was visually examined, manually corrected to
avoid unfavorable conformations and steric constrains,
meet the commonly used validation criteria, and minimize
potential energy using the programs CHAIN, (35), Mod-
eller (36, 37), and CHARMM (38). Subsequently, short
molecular dynamics simulations were done with
CHARMM and the MMTSB Tool Set (39).
Docking of Small Organic Molecules to P-12-LOX
Using SLIDE
SLIDE is a docking/screening tool using distance
geometry techniques to match ligand interaction points to
template points describing the binding site of the target
protein (40). The template consists of points identified as
the most favorable positions for ligand atoms to form
hydrogen bonds or make hydrophobic interactions with the
neighboring protein atoms (41). After the initial matching
step, SLIDE uses full atom representation of both the ligand
and the target protein to model induced fit upon binding
and score the complex based on hydrophobic complemen-
tarity and the number of protein-ligand hydrogen bonds.
Residues within 9.0 A˚of the binding site cavity of P-12-
LOX were used as the target for the docking.
Evaluation of Ligand-Protein Complex Formation
In addition to the built-in scoring function of SLIDE,
DrugScore was used to score the dockings. Although SLIDE
evaluates the predicted protein-ligand complex based on
geometric and chemical complementarity, DrugScore will
estimate the binding affinity based on the statistical
preferences ofligand atoms tobefound nearvarious protein
atomsobservedinknowncrystalcomplexes(40–42).Bothof
these scoring functions were trained on experimental data
and then tested on an independent set of diverse enzymes,
with statistical analysis done to evaluate the correlation
between predicted scores and experimentally measured
binding affinities (42). Once they were validated this way, it
is not necessary to perform statistical analysis for every
system the scoring function is applied to. The ligand
candidates were ranked based on their consensus score
computed as the sum of their normalized DrugScores and
SLIDE scores, and that was the most important single
criteria used to select the best candidates to inhibit P-12-
LOX. In addition, we have visually inspected the docked
orientations to exclude docked ligand orientations with
parts of the ligand exposed to the solvent and/or unoccu-
pied cavities left in the binding site.
Molecular Graphics
SwissPDB, Chain v.7, and PyMOL viewers were used to
display the three-dimensional structures of P-12-LOX and
to generate POV-Ray scenes (43).
Expression and Purification of P-12-LOX
Human P-12-LOX with a 6-His tag on the NH2terminus
inserted into the pFastBac1 vector (Life Technologies,
Gaithersburg, MD) was a generous gift of Dr. Holman
(University of California, Santa Cruz, CA; ref. 44).
Expression and purification were done basically as de-
scribed before (44). In pFastbac vector, the expression of the
gene is controlled by the Autographa californica multiple
nuclear polyhedrosis virus (AcMNPV) polyhedrin or p10
promoter for high-level expression in insect cells. The
plasmids were then transposed into a recombinant bacmid
with the help of DH10Bac Escherichia coli cells (Invitrogen,
Carlsbad, CA), which contain a baculovirus shuttle vector
(Bacmid) with a min-attTn7 target site and a helper
plasmid. Transposition occurs between the mini-Tn7
element on the pFastBac vector and the mini-attn7 target
site on the bacmid to generate a recombinant bacmid. This
transposition reaction occurs in the presence of transposi-
tion proteins supplied by the helper plasmid. This high
molecular weight recombinant bacmid DNA was isolated
from the white colonies grown for 48 hours at 37jC on a
Luria-Bertani agar plate containing 50 Ag/mL kanamycin,
7 Ag/mL gentamicin, 10 Ag/mL tetyracycline, 100 Ag/mL
X-gal, and 40 Ag/mL isopropyl-L-thio-B-D-galactopyrano-
side. Recombinant bacmid DNA was used to transfect Sf9
cells derived from Spodoptera frugiperda (Fall armyworm)
using cellfectin reagent (Invitrogen) and following the
instruction provided. The virus generated was P1 viral
stock. The virus was subsequently amplified to f2 ? 107
plaque forming units/mL. This virus was then added to Sf9
cells (f2 ? 106/mL) at a concentration of f2 ? 107plaque
forming units/mL in 6- or 24-well tissue culture plates. The
plates were incubated at 27jC in a humidified chamber for
different time intervals. The cells were harvested and lysed
in 62.5 mmol/L Tris-HCl (pH 6.8), 2% SDS and analyzed by
SDS-PAGE and Western blot using an anti-histidine tag
antibody (no anti-P-12-LOX antibody is available).
Nonreducing Gel Electrophoresis
The electrophoresis was done at room temperature in
gradient gels with 4% to 12% polyacrylamide, in the
absence of 2-mercaptoethanol. Gels were stained with
Colloidal Coomassie Blue (Invitrogen).
In-Gel Digestion withT rypsin
The protein band was excised from a 4% to 12% gradient
SDS-PAGE gel and destained with 30% methanol for 3
hours at room temperature. In-gel proteolysis was done
with sequencing grade trypsin (Promega, Madison, WI)
and was carried out as described previously (45). Briefly,
a gel slice was washed with 150 AL of 50% acetonitrile in
0.1 mol/L ammonium bicarbonate buffer (pH 8) for
30 minutes. The gel slice was then diced into small cubes
and dried under vacuum. Trypsin (0.5 Ag) was added in a
minimal volume of 0.1 mol/L ammonium bicarbonate
buffer, and digestion was carried out for 16 hours at 37jC
with an additional aliquot of trypsin (0.25 Ag) added after
12 hours. Peptides were extracted once with 150 AL of 60%
acetonitrile, 0.1% trifluoroacetic acid for 30 minutes
followed by a further extraction with 100 AL of the same
solution. All extracts were pooled and concentrated using
Vacufuge to a final volume of 10 AL.
Protein Identification by Peptide Sequencing Using
Liquid Chromatography
Tandem mass spectrometry (liquid chromatography
tandem mass spectrometry) was done at Proteomics
Laboratory, Program in Bioinformatics and Proteomics/
Genomics at the Medical University of Ohio (45). Two
microliters of the digest were separated on a reverse-phase
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column (Aquasil C18, 15 Am tip ? 75 Am id ? 5 cm Picofrit
column; New Objectives, Woburn, MA) using acetonitrile/
1% acetic acid gradient system (5–75% acetonitrile over 35
minutes followed by 95% acetonitrile wash for 5 minutes) at
aflowrateof250nL/min.Peptidesweredirectlyintroduced
into an ion-trap mass spectrometer (LCQ, ThermoFinnigan)
equipped with a nanospray source. The mass spectrometer
was set for analyzing the positive ions and acquiring a full
massspectrometryscanandacollision-induceddissociation
spectrum on the most abundant ion from the full mass
spectrometry scan (relative collision energy f30%). Dy-
namic exclusion was set to collect three collision-induced
dissociation spectra on the most abundant ion and then
exclude it after 2 minutes. Collision-induced dissociation
spectra were manually verified by comparing against an
in silico tryptic digest of P-12-LOX sequence using the MS-
Digest and MS-Product provisions of Protein Prospector.4
Iron Contentin P-12-LOX
The iron content was determined independently by two
different methods. First, it was measured by atomic
absorption spectroscopy (spectrometer Varian AA-1275).
The second measurement was done by inductively coupled
plasma optical emission spectroscopy (Shimadzu Trace TOC
Analyzer at Galibraith Laboratories, Inc., Knoxville, TN).
Inhibitors of P-12-LOX
The curcuminoids were a generous gift from Dr. Richard
Hart and were synthesized and purified as described
before (46).
Determination of IC50
The enzyme activity was determined as described before
(44). The inhibitory activity of curcuminoids was deter-
mined by direct measurement of the 12(S)-HETE formation
as measured by the increase of absorbance at 234 nm
[25 mmol/L HEPES (pH 8), 3 Amol/L arachidonic acid].
The reaction was done in a buffer and 200 nmol/L of
enzyme stirred with a rotating stir bar in the beginning of
the assay (23jC). IC50values were determined by measur-
ing the enzymatic rate at a variety of inhibitor concen-
trations (depending on the inhibitor strength) and plotting
their values versus inhibitor concentration. The
corresponding data were fitted to a simple saturation
curve, and the inhibitor concentration at 50% activity was
determined (IC50). The inhibitors were typically dissolved
in DMSO or ethanol at a concentration of 1 mg/mL (44).
P-12-LOX pHActivity Dependence
Enzyme activity was done as described above in pH 7.0
to 8.0 (in 0.2 increments) and additionally at pH 8.5.
Sprout Formation Assay
Human umbilical vascular endothelial cells (HUVEC)
were grown to confluence in an EGM-2 growth medium.
Next, the cells were trypsinized and seeded onto 0.5%
agarose-coated culture dishes. This procedure resulted in
cell aggregate formation after 24 hours of incubation at
37jC. HUVEC aggregates were decanted by allowing the
cells to stand for 30 minutes at room temperature. The
old medium supernatant was decanted, and HUVEC
aggregates were suspended in 5 mL of fresh EGM-2 growth
medium. Three-dimensional fibrin gels were prepared by
mixing the following in 12-well culture plates: 960 AL of
human fibrinogen (type III, 60% of protein clotable; 2.50
mg/mL concentration in RPMI 1640), 40 AL of HUVEC
aggregate suspension, and 12.5 AL of human thrombin
(25 IU/mL concentration in RPMI 1640). The mixture was
gently mixed and allowed to gel for about 4 minutes at
37jC before adding EGM-2 growth medium over the gel.
The sprout formation assay was done as described by
Pepper et al. (47). Briefly, HUVEC aggregates were
suspended in fibrin gel containing P-12-LOX inhibitors;
1 mL of EGM-2 growth medium was later added over the
fibrin gel. After 3 days of cell incubation, cultures were
fixed in situ for 24 hours with 2 mL of 10% formalin
solution and photographed under a phase-contrast micro-
scope. Measurements were carried out in duplicate for
three to six independent HUVEC aggregates.
Statistical Analysis
The Kruskal-Wallis test was done for normality with
multiple comparisons between all groups (Mann-Whitney
test). The differences were considered significant for P <
0.05 (11.5.1 SPSS for Windows).
Results and Discussion
Modeling of the Human P-12-LOX Molecular Struc-
ture
Although f50 sequences of different lipoxygenases have
been determined for plant and mammalian enzymes,
structural data are available for only three enzymes:
soybean LOX-1 and LOX-3 and rabbit 15-LOX. Despite the
differences in size (LOX-3, 857 residues; rabbit 15-LOX, 663
residues; human P-12-LOX, 662 residues), these proteins
have a 62% homology, and plant and rabbit enzymes show
the same topology. In addition, the rabbit reticulocyte
15-LOX exhibits the best overall alignment to the human
genesequencewithBLAST(48).Theonlyknownstructureof
the mammalian enzyme lacks structural information about
the crucial fragments near the active site (see broken ends
pointed to by the magenta arrows in Fig. 1A). An automatic
routine cannot provide reliable model for the missing
part, and it was obvious that upper fragment, depicting a
stretched coil and a pin-like structure (Fig. 1B, red), was
unrealistic because predictions based on sequence call for
the formation of the helical structure there. In addition, such
model can and often does contain steric constraints and
bumpsin thewhole model. Therefore, thistheoreticalmodel
was carefully examined; the main chain and side chains
were corrected to avoid collisions and improve the torsion
angles to better fit the common acceptance criteria and
possible hydrogen bonding network; and the model was
validated using PDB validation tools (Fig. 1B, light green).
Independently, the fragments missing in rabbit lipoxyge-
nase and those of a questionable quality in the theoretical
model were examined by performing short, restrained
molecular dynamics simulations, resulting in two alternate
4http:/ /prospector.ucsf.edu
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models (see Fig. 1C, silver and yellow/green models). All
considered models differ substantially in the relative
orientation and structure of the 175 to 195 fragment while
showing high correlation in the molecule core. This upper
fragment above the active site shows greater flexibility than
the core of the molecule in soy and rabbit enzyme; hence, it
is possible that it might be a common feature in other
lipoxygenases aswell. Thedocking procedure that was used
to test binding of curcuminoids allows flexibility for the
protein, and the defined receptor site does not encompass
the above fragment. Therefore, we feel that our carefully
examined, predicted molecule of P-12-LOX (Fig. 1B, light
green) provides a sufficiently accurate approximation to
serve well the purpose of this research.
Molecular Modeling of P-12-LOX Inhibition
Commercial curcumin isolated from the rhizome of the
plant Curcuma longa contains three major curcuminoids:
f77% curcumin, 17% demethoxycurcumin, and 3% bisde-
methoxycurcumin (49). In the literature, these chemicals
are referred as natural curcuminoids (50), as opposed to
synthetic curcuminoids, which are related to curcumin but
undergo significant chemical modification (50, 51). Because
natural curcuminoids show a consistently lower activity
than curcumin in many different assays, our search for
better inhibitors of P-12-LOX was limited to synthetic
curcuminoids (52–54).
Initially, a three-dimensional database of known inhib-
itors of various lipoxygenases was created. Low-energy
conformers of these ligand candidates were generated with
Omega (OpenEye Scientific Software, Inc., Santa Fee, NM).
From the total of 106 compounds, 80 where docked into the
cavity containing the active site of P-12-LOX. The results
from docking were scored independently by SLIDE and
DrugScore. The ligand candidates were ranked based on
their consensus score computed as the sum of their
normalized DrugScores and SLIDE scores. It has been
Figure 1.
oxygenase (light brown), rabbit (light
blue), the automatic model of human P-12-
LOX from Swiss-Model Repository (red),
corrected and verified model (light green),
iron cofactor (orange sphere). A, alignment
of soybean lipoxygenase (1JNQ) over rabbit
15-LOX (1LOX). B, alignment of the auto-
matic model over corrected model of human
P-12-LOX, most differences in loop 175 to
195, that according to theoretical predic-
tions should be helical in nature. C, align-
ment of the fragments 174 to 198 for the
automatic model (red), two calculated mod-
els (silver and yellow-green), and manually
verified model shown (light green). D, align-
ment of active site P-12-LOX model over
rabbit 1LOX and soybean 1JNQ. E, E22C
docked into the active site of h-P-12-LOX:
keto form carbons (green), enol form car-
bons (magenta). F, E26C docked into the
active site of h-P-12-LOX, colors as in E.
Ribbon models of soybean lip-
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Table 1. Structure and properties of compounds tested for P-12-LOX inhibition
Name StructureDocking rank
AB
Log P keto*/enolc
C
IC50(Amol/L)
Curcumin
19*61 1.72.30 66.0 F 4.6
NDGA
1530 0.83.50 1.7 F 5.0
E22C
22* 266 0.2 3.6012.3 F 2.6
E26C
1*253.36.62b ,x
17.0 F 1.0
E16C
NDNDND4.82b
>100
E17C
23*172.92.51k
>100
E19C
70*4 8.76.82b ,x
>100
E25C
2c
9 6.7 7.32b ,x
>100
E27C
4*8 2.84.20 >100
E35C
ND NDND1.91b
>100
E57C
NDND ND2.01b
>100
NOTE: A, number of docked orientations/molecule. B, distance from geometric center of the molecule to the center of the binding site (shortest distance listed
in A˚). C, number of Lipinsky rule violations.
Abbreviation: ND, not docked.
*Keto form:.
cEnol form:.
bMolecular weight >500.
xLog P > 5.
kNumber of O and N atoms >10.
Human P-12-LOX and Angiogenesis
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Page 7

shown repeatedly that consensus scoring improves hit rates
in computational screening (55–57). To test our theoretical
predictions, we determined the inhibitory activity of all
curcuminoids using recombinant human P-12-LOX. The
ranks of the experimentally tested ligand candidates
together with their log Ps calculated with Interactive
logP calculator are listed in Table 1.5
Given the known limitations of existing scoring func-
tions in correctly predicting binding affinities, additional
features were also considered and computed to help
discriminate true positive hits from false positives. One of
these features is the number of docked orientations
per molecule (Table 2), which in case of docking with
SLIDE is proportional to the number of possible matches
between different ligand interaction point triplets and
template triangles. The more similar the shape and
chemistry of the ligand to the template describing the
binding site, the more docked orientations can result.
Another feature describing how well the docked ligand is
buried in the binding site is the distance between the
geometric center of the docked ligand and the geometric
center of the template (Table 2). The shorter this distance,
the smaller the part of the docked ligand only partially
buried in the binding site or completely exposed. True
positive inhibitors were found to dock with a larger
number of orientations and tended to be well buried and
closer to the center of the binding site, than false positives.
Such relationships between geometric and chemical fea-
tures of the modeled protein-ligand complex, even if not
generally valid across various systems are valuable for
identifying additional new inhibitors for P-12-LOX. The
top scoring binding orientations of the compounds that
we confirmed to have P-12-LOX inhibitory activity exhibit
some common binding motifs. One of the aromatic rings
is stacked invariantly between the plane of the side
chain carboxylic acid of Glu355and the side chain of Ile592,
with two other aromatic rings (Phe351and Phe413) posi-
tioned in a perpendicular way around it. These residues
form an ideal pocket for binding an aromatic ring. The
other aromatic ring docked next to His364into the
hydrophobic pocket lined by Leu360, Ile398, Leu406, Ala402
in the case of NDGA, or, alternatively, in the pocket defined
by Trp143, Leu407, Leu360, Leu365in the case of larger
ligands. Thus, two hydrophobic groups, at least one of
them aromatic, connected by a flexible linker seems to be
necessary for binding strongly enough to inhibit the
enzyme. Some of the molecules we tested have hydropho-
bic groups that are too bulky; thus, they were docked with
only one half buried into the binding site (E19C and E25C)
or could not be docked at all (E35C and E57C). These
compounds turned out not to have inhibitory activity in
experimental testing.
Enzyme Characterization
Histidine-tagged human P-12-LOX with a 6-His tag on
the NH2terminus yielded f95% pure protein in single step
purification using 6xHis affinity column as determined
by PAGE gel densitometry (Fig. 2). A Western blot with
anti-6-His antibody showed a band exactly in the same
position as standard P-12-LOX. P-12-LOX was produced
and purified in f20 mg/L of cell culture. In the absence
of the h-P-12-LOX antibody, protein identity was con-
firmed by mass spectroscopy (Table 3; Fig. 3). Collision-
induced dissociation spectra were manually verified by
comparing against an in silico tryptic digest of P-12-LOX
sequence using the MS-Digest and MS-Product provi-
sions of Protein Prospector.4At dominant band, only
P-12-LOX peptides were found confirming the identity of
this protein.
The enzyme was found to be active, showing Km= 15.6
Amol/L, and Vmax = 1.5 Amol/L/min. The measured
values for P-12-LOX were very similar to values found by
others for the same enzyme (Kmf 10 Amol/L, Vmaxf 2
Amol/L/min; refs. 44, 58). The maximum activity was
observed at pH 8 (Fig. 4), and this is also consistent with
Table 2.Number of docked orientations and center distance for the compounds tested for P-12-LOX inhibition
Name Docking RankNo. docked orientations Center distance (A˚)True inhibitor
Curcumin
NDGA
E22C
E26C
E16C
E17C
E19C
E25C
E27C
E35C
E57C
19*
15*
22*
1*
—
23*
70*
2c
4*
—
—
61
30
266
25
1.7
0.8
0.2
3.3
—
2.9
8.7
6.8
2.8
—
—
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
0
17
4
6
8
0
0
NOTE: Only the higher-ranking form (keto or enol) is listed for each compound.
*Keto.
cEnol.
5http:/ /www.molinspiration.com/cgi-bin/properties
Molecular Cancer Therapeutics 1377
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Page 8

previous reports (44, 58). The iron content was measured
by atomic absorption spectroscopy and was determined
as 0.45 F 0.10 mol of iron per 1 mol of enzyme. A second
independent measurement was done by inductively
coupled plasma optical emission spectroscopy at <9
ppm, which translates into a molecular ratio of 0.7. This
method required a very large amount of protein for
analysis (20 mg), and for this reason, only one measure-
ment was done. Theoretically, the stoichiometric ratio is
expected to be 1:1, but in practice, the iron cofactor can be
easily washed out; therefore, its content in a protein
sample is usually lower. This is a common finding for
lipoxygenases: Matsuda et al. cited 0.7 for porcine
leukocyte 12-LOX (59), and Segraves and Holman have
quoted 0.35 for human P-12-LOX (60).
Synthetic Curcuminoids Inhibit Human P-12-LOX
As shown in Table 1, P-12-LOX was inhibited by
curcumin, NDGA, E22C, and E26C. NDGA is a known
lipoxygenases inhibitor, and its IC50reported by Amagata
et al. is identical with the value determined by us (44).
Curcumin is the known inhibitor of other lipoxygenase
types, and it is no surprise that we found it inhibits P-12-
LOX as well (23, 61). In general, we have found that
computational predictions (e.g., high rank of docked
ligands, low log Ps) agreed with the ability of the
compounds to inhibit P-12-LOX.
Synthetic Curcuminoids Inhibit Sprout Formation
The significance of cancer-related neovascularization has
been characterized over the past two decades (62).
Angiogenesis is a prerequisite of tumor growth and is the
target of drug development in many preclinical and
clinical trials. Angiogenesis is a multistep progression in
physiologic and pathologic processes. It involves endo-
thelial cell sprouting from the parent vessel followed by
migration, proliferation, tube formation, and connecting
to other vessels (63). Several in vitro models have
attempted to recreate this complex sequence of events
with varying degrees of success. Angiogenic sprouting
and capillary lumen formation in fibrin gel is one of the
commonly accepted models of angiogenesis in vitro and
provides a powerful tool for analysis of this complex
phenomenon.
When HUVEC aggregates were treated (Fig. 5) with
synthetic E22C and E26C curcuminoids with NDGA as a
control, a significant reduction in sprout length and sprout
number was observed. Sprouting ability of endothelial cells
is related to stimulation by vascular endothelial growth
factor. Nie et al. showed that endothelial cells synthesize
various eicosanoids, including the 12-LOX product 12(S)-
HETE, and that endogenous 12-LOX is involved in
endothelial cell angiogenic responses. They have showed
that 12-LOX inhibitors reduced endothelial cell prolifera-
tion by down-regulation of vascular endothelial growth
factor (64). That phenomenon could explain reduction in
number of sprouts formed in our experiments. It has been
reported by Rondeau et al. that NDGA down-regulates
urokinase plasminogen activator mRNA level and uroki-
nase plasminogen activator biosynthesis via protein kinase
C and/or lipoxygenases pathways also (65). Urokinase
plays a major role in extracellular proteolytic events
associated with angiogenesis (66), and reduced urokinase
Figure 2.
column, (C) wash, (D) elutant 1 times, E 1.5 times, F 2.5 times, G 15
times higher than (D). Arrow indicates P-12-LOX. Photograph was
electronically enhanced to show potential contaminants (visible on F and
G only). Purity of P-12-LOX was determined as +95%.
Coomassie blue stain of (A) cell lysate, (B) flow thought from
Table 3. Sequence of peptides extracted from dominant band of PAGE gel
Access no.Protein namesTheoretical mass Observed massPeptide Sequence
P18054Arachidonate 12-LOX, 12(S)-type (12-LOX; P-12-LOX)1,770.90
1,364.65
1,155.63
1,207.64
919.49
1,800.94
1,784.94
1,043.48
1,293.66
1,636.91
1,523.80
847.47
1,771.02
1,364.24
1,155.48
1,207.44
919.44
1,801.18
1,784.63
1,043.18
1,293.28
1,636.82
1,523.30
847.84
98-113 WVQGEDILSLPEGTAR
114-125 LPGDNALDmFQK
145-155 EGLPLTIAADR
169-177 RLDFEWTLK
178-187 AGALEmALK
249-265 LVLPSGmEELQAQLEK
249-265 LVLPSGMEELQAQLEK
394-401 YTmEINTR
404-415 TQLISDGGIFDK
449-465 GLLGLPGALYAHDALR
473-484 YVEGIVHLFYQR
621-627 AVLNQFR
Abbreviation: m, oxidized methionine.
Human P-12-LOX and Angiogenesis
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Page 9

plasminogen activator activity of HUVECs by lipoxygenase
inhibitors would reduce length in sprout formation assay,
which to propagate must hydrolyze fibrin gel.
Results are presented in Fig. 6 as a percentage relative to
untreated control sprouts. These results are statistically
significant starting at concentrations higher than IC50for
all inhibitors tested. The ability of curcumin to affect gene
transcription and to induce apoptosis is likely to be of
particular significance in cancer chemoprevention and
chemotherapy in patients. However, curcumin’s low
systemic bioavailability following oral administration may
be a limiting factor to assure sufficient concentrations for
pharmacologic effect in certain tissues. Furthermore,
curcumin and natural curcuminoids possess anti-inflam-
matory and anticancer properties following oral or topical
administration. Separately from antioxidant properties of
these compounds, the mechanisms of action include
inhibition of enzymes, such as lipoxygenases, cyclooxyge-
nases, inducible nitric oxide synthase, and xanthine
dehydrogenase/oxidase (67). Curcumin is also a potent
inhibitor of the protein kinase C, epidermal growth factor
receptor tyrosine kinase, and InB kinase. Additionally,
curcumin inhibits the activation of NFnB and the expres-
sion of c-jun, c-fos, and c-myc (68, 69). NDGA is a phenolic
compound isolated from the creosote bush Larrea divaricatta
that has been reported to inhibit lipoxygenases and has
anti-cancer activities as well. These are attributed to the
Figure 4.
and pH measured as an increase of absorbance at 234 nm. Similar
dependence was observed when different P-12-LOX inhibitors where
used.
Increase of concentration of 12(S)-HETE as a function of time
Figure 3.
12-LOX. Amino acids shown in bold
were detected by mass spectroscopy
as indicated in Table 3. Arrows,
potential trypsin cleavage site.
Sequence of human P-
Figure 5.
treaded with 30 Amol/L E26C.
Sprout formation of human endothelial cells: (A) control,
Molecular Cancer Therapeutics 1379
Mol Cancer Ther2006;5(5). May 2006

Page 10

ability of NDGA to directly inhibit the function of
important in carcinogenesis receptors: tyrosine kinases,
insulin-like growth factor, and c-erbB2/HER-2/neu receptors
(70).
Inhibition of any of these proteins could be of therapeutic
significance. What is important in our experiments is the
limited inhibition of sprout formation at concentrations
fIC50 for human P-12-LOX of inhibitors tested. Even
under this condition (IC50), a substantial amount of 12-
HETE can be produced by lipoxygenase, providing a
stimulus for angiogenic sprouting of endothelial cells.
Increasing the concentrations of lipoxygenase inhibitors
above IC50 greatly reduces sprout formation for all
inhibitors tested. It should be noted that this phenomenon
was observed in different concentrations. For example,
NDGA inhibited sprout formation in a concentration of
10 Amol/L (>IC50), whereas E26C at a concentration of
17 Amol/L (IC50) did not. This universal event for all tested
lipoxygenase inhibitors suggests that inhibition of sprout
formation was most likely due to the inhibition of human
P-12-LOX but not other cancer-related pathways.
Although this is still not an exhaustive demonstration
of a specific inhibition of P-12-LOX by curcuminoids,
we conclude that protein structure-based ligand
selection supported by theoretical log P determination
and structural analysis of ligands binding to human
P-12-LOX is in a good agreement with in vitro effects
of lipoxygenase inhibition by different curcuminoids.
Furthermore, successful selection of two novel lipoxyge-
nase inhibitors by combination of computational and
biochemical methods provides template for future search
of novel P-12-LOX inhibitors from very large database of
three-dimensional structures.
Acknowledgments
We thank Dr. R. Hart (President of American Diagnostica, Inc., Stamford,
CT) for his support and the chemicals used in this study, Dr. Gerhard Klebe
(University of Marburg, Germany) for providing DrugScore, and OpenEye
Scientific Software for providing Omega for our use.
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