See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/311565812

Anti-angiogenic potential of trypsin inhibitor purified from Cucumis melo

seeds: Homology modeling and molecular docking perspective

Article  in  International Journal of Biological Macromolecules · December 2016

DOI: 10.1016/j.ijbiomac.2016.12.027

CITATIONS READS

20 298

6 authors, including:

Rasouli H. Shahram Parvaneh

55 PUBLICATIONS   466 CITATIONS   
Kermanshah University of Medical Sciences
11 PUBLICATIONS   47 CITATIONS   
SEE PROFILE
SEE PROFILE

Mohsen Rastegari-Pouyani Kamran Mansouri

Shahid Beheshti University of Medical Sciences Kermanshah University of Medical Sciences
14 PUBLICATIONS   107 CITATIONS    154 PUBLICATIONS   1,270 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Mesenchymal stem cell therapy (in vitro) View project

Protein and peptide engineering View project

All content following this page was uploaded by Rasouli H. on 09 March 2020.

The user has requested enhancement of the downloaded file.

 International Journal of Biological Macromolecules 96 (2017) 118–128

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Anti-angiogenic potential of trypsin inhibitor purified from Cucumis

melo seeds: Homology modeling and molecular docking perspective
Hassan Rasouli a , Sharham Parvaneh a , Azadeh Mahnam a , Mohsen Rastegari-Pouyani b ,
Zohreh Hoseinkhani a , Kamran Mansouri a,∗
a
Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
b
Student Research Committee, Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Melons have a good source of protease inhibitors. Its fruit and seeds have been used as a traditional
Received 22 October 2016 medicine. However, its effects on angiogenesis and mechanism of its action remain elusive.
Received in revised form 7 December 2016 Herein trypsin inhibitor from aqueous extract of C. melo seeds (TICMS) was purified. Its effects on differ-
Accepted 9 December 2016
ent steps of angiogenesis were evaluated. Also, we examined its effects on migration and angiogenesis of
Available online 10 December 2016
endothelial cells. Three dimensional model of TICMS protein was accurately built in which TICMS docked
to ␣V ␤3 integrin and VEGFR1.
Keywords:
Electrophoresis analysis of the purified protein revealed a single band with a molecular mass of about
Migration
Tumor metastasis
3 kDa. Treatment with TICMS at six doses resulted in a significant decrease of endothelial cell proliferation
Traditional medicine with an IC50 value of about 20 ␮g/ml. Tubulogenesis assay revealed that a dose dependent anti-angiogenic
activity of TICMS (5–40 ␮g/ml). Also, TICMS had inhibitory effects on VEGF, MMP-2 and MMP-9 secretion.
Our docking result speculated that TICMS could bind to the cleft between the ␣V ␤3 integrin and it able
to decrease the activity of this receptor. The TICMS was also able to interact with VEGFR1 receptor, but
with low probability. Based on our study, TICMS could be used as a specific angiogenesis inhibitor.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction and attractiveness as an adjuvant to conventional therapy [4,6,7].

Angiogenesis, the process of new blood vessel formation from
pre-existing ones, is important under physiologic conditions such
as embryonic development, wound healing, and placenta forma-
tion [1–5]. It is also necessary for pathological processes of different
disorders, including tumor growth and metastasis [2,5]. Scien-
tists focus on anti-angiogenesis research due to its importance
Abbreviations: BSA, bovine serum albumin; DMEM, Dulbecco’s modified
eagle’s medium; BBI, Bowman-Birk inhibitor; ELISA, enzyme-linked immunosor-
bent assay; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cells;
LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-
tetrazolium bromide; PBS, phosphate buffered saline; PBST, PBS containing tween
20; PDB, protein data bank; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis; TICMS, trypsin inhibitor from aqueous extract of C. melo seeds;
VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; MMP, matrix met-
alloproteinases; ERK, extracellular signal–regulated kinase; EC, endothelial cell; FCS,
fetal calf serum.
∗ Corresponding author at: Medical Biology Research Center, Kermanshah Uni-
versity of Medical Sciences, Kermanshah 6714967346, Iran.
E-mail addresses: kmansouri@kums.ac.ir, kamranmansouri@gmail.com
(K. Mansouri).
According to former studies, plant-derived compounds specifically
inhibit tumor cell proliferation and new vessel formation in tumors
without significant toxicity to normal tissues and major side effects
[8,9]. Plant-derived foods are able to prevent one third of cancers
[9,10]. Therefore, a diet rich in plants can inhibit progression of
chronic diseases, such as malignant solid tumors relying on angio-
genesis [1,9,11,12].
The most common name used for Cucumis melo is melon [13].
It is native to Iran, Anatolia, Armenia, and adjacent areas on the
west and the east which are believed to be its center of origin
and development, with a secondary center including the northwest
provinces of Afghanistan and India. In those regions several related
wild species have been noted however, truly wild forms of C. melo
have not been found [13].
Several studies regarding cytotoxic, antioxidant/anti-
inflammatory, antifungal and immunomodulatory effects of
this fruit extract have already been performed. Proteases are
enzymes which catalyze the hydrolysis of the peptide bonds
forming the primary structure of proteins [14]. They are common
to organisms, from micro-organisms to plants and animals [15].
Protease inhibitors are greatly abundant in tubers and plant seeds

http://dx.doi.org/10.1016/j.ijbiomac.2016.12.027
0141-8130/© 2016 Elsevier B.V. All rights reserved.
 H. Rasouli et al. / International Journal of Biological Macromolecules 96 (2017) 118–128
[14]. Protease enzymes, which are crucial for disease propagation,
selectively catalyze the hydrolysis of peptide bonds and protease
inhibitors are emerging as promising therapeutic agents for
treatment of diseases such as cancer. Inhibitors of these proteases
can suppress several stages of carcinogenesis including initiation,
promotion and progression; although their mechanisms of action
are not yet fully clear. Studies have suggested that the protease
inhibitors which prevent carcinogenesis affect processes in the
early stages of carcinogenesis; though, they can be effective after a
long-term carcinogen exposure in both in vitro and in vivo systems.
Strong evidence indicates that protease inhibitors can affect both
the initiation and promotion stages of carcinogenesis but they
have no effect on already transformed cells [16–18].
Protease inhibitors derived from dietary sources are widely
distributed among living organisms and their antitumor activi-
ties have been thoroughly investigated. Plant seeds contain two
major families of protease inhibitors, the kunitz and Bowman-Birk
inhibitors. Bowman-Birk inhibitors include peptides with 71 amino
acids and 7 disulfide linkages. Kunitz inhibitors with two disulfide
linkages have been earlier studied as anticancer agents [19–21].
Some protease inhibitors from other sources have also been studied
for cancer control. Since the most common cause of cancer death in
humans is angiogenesis-mediated metastasis of the primary tumor
(not growth of the primary tumor), modulation of angiogenesis can
be a promising approach to treat cancer. [22,23].
In this study, we purified trypsin inhibitor from C. melo seeds
using a simple and efficient procedure and the effectiveness of its
anti-angiogenic activity on three dimensional cultures of HUVECs
was determined. The C. melo seeds are rich in urease, lipase, lipoxy-
genase and trypsin inhibitors [24] which they considered for their
enzyme inhibitor activities [20]. VEGF, MMP-2 and MMP-9 secre-
tion by HUVECs were assessed quantitatively. Since, angiogenesis
and angiogenic factor secretion specially VEGF and MMP-2,9 are
closely linked to ␣V␤3 integrin receptors and VEGFR1 activities,
understanding the mechanism through which TICMS interferes
with the activities of these receptors are of great importance. Thus,
3D structure of TICMS was constructed and TICMS was docked to
␣V␤3 integrin and VEGFR1.
2. Methods
2.1. Materials
Rat tail collagen 2 mg/ml in 0.5 M acetic acid (Collagen R,
SERVA), 10X minimum essential medium (10X MEM) (Gibco),
sodium bicarbonate 1.4% (W/V) solution, sterile NaOH solution
(1 M), DMEM (Gibco), FBS (Gibco), dextran-coated cytodex 3 micro-
carriers (Amersham Pharmacia Biotech, Piscataway, NJ), HUVEC,
lactate dehydrogenase LDH assay kit (Roche), and All other reagents
were from Sigma-Aldrich (Taufkirchen, Germany) and R&D Sys-
tems.
2.2. Affinity chromatography using trypsin ligand
At first C. melo seeds were ground to produce fine flour and the
flour was defatted by adding methanol in two steps as described
earlier [25]. In each step, four volumes of methanol were added
to one weight of the flour and mixed for 24 h. Then, it was cen-
trifuged at 5000g for 30 min at room temperature. After this step,
the pellet was dried at room temperature. One weight of the dried
powder was mixed with four volumes of ethanol 60% (V/V) for 24 h.
The mixture was centrifuged at 5000g for 30 min at room tempera-
ture. The supernatant was collected and dried at 37 ◦ C. The powder
of the extraction was dissolved in 20 mM sodium acetate buffer
(pH 4.5) and precipitated with 70% saturated ammonium sulfate at
119
4 ◦ C. It was centrifuged at 5000g for 30 min at 4 ◦ C and the pellet
was collected. In order to prepare affinity medium, CNBr activated
Sepharose 4B (Amersham Pharmacia Biotech) was washed three
times with 1 mM HCl. After resin swelling, bovine trypsin (Sigma,
St. Louis, MO, USA) in 0.1 M carbonate buffer, pH 8.5 containing
0.5 M NaCl was added and the mixture was stirred for 2 h at room
temperature. Residual active sites were blocked with 0.2 M glycine
containing 0.5 M NaCl. The resin was washed three times with alter-
nate buffers, then loaded onto a column and equilibrated with
deionized water with pH 6. The ammonium sulfate precipitated
fraction was dissolved in 50 mM Tris-HCl buffer (pH 8) and cen-
trifuged at 5000g for 10 min at room temperature. The supernatant
was loaded onto the column, and the column was washed with
deionized water till the absorbance of fractions at 280 nm came to
zero. Three column volumes of deionized water with pH 2.5 were
used to elute bound proteins from the column (deionized water
was adjusted to pH 2.5 with 0.1 N HCl).
2.3. Analytical methods
The SDS-PAGE was performed as described by Schagger and Von
Jagow [26]. A 16.5% separating gel, 10% spacer gel, and a 4% stacking
gel were used. Protein samples were heated in boiling water for
5 min. Electrophoresis was carried out at 30 V for 60 min and 100 V
for 3 h. After electrophoresis, the gel was fixed with glutaraldehyde
(10%) for 30 min and protein bands were visualized by staining with
silver, and the molecular mass was estimated in comparison with
standard marker proteins (low MW marker, Pharmacia).
2.4. Determination of protein concentration
In the final step and also during the purification procedure, the
protein concentration was determined [27] using BSA as standard.
2.5. Measurement of trypsin inhibition activity
Inhibitory activity of the obtained protein was measured using
the method offered by the American Association of Cereal Chemists
(AACC) [28]. In this method, firstly, different concentrations of
TICMS were prepared in 2 ml volumes. Then, 2 ml of trypsin solu-
tion in a concentration of 20 ␮g/ml was added to each tube and the
contents of the tubes were gently mixed for a few minutes. After-
wards, 5 ml of trypsin substrate (BAPA) with the concentration of
0.4 mg/ml was added to each tube and the tubes were incubated at
37 ◦ C for 10 min. The reaction between trypsin and its substrate was
stopped by adding 1 ml of glacial acetic acid 30%. Light absorbance
corresponding to the produced dye level in each tube was measured
at 410 nm. Activity unit of this inhibitory protein was calculated
based on the reduction of absorbance value (inhibition of trypsin
activity). Each activity unit of TICMS was defined as the reduction
of absorbance by 0.01.
2.6. Cytotoxicity assays
Cell viability was determined using the LDH assays. In brief,
HUVECs were seeded at a density of 1 × 104 cells per well
into 96-well plates. After 24 h, TICMS at different concentrations
(5–120 ␮g/ml) was added and the cells were cultured for additional
48 h. The experimental procedure for LDH assay was done accord-
ing to Linford’s method [29]. A group of wells was treated with 1%
Triton X-100 solution for 45 min for maximum LDH release. Plates
were centrifuged at 200g, afterwards 100 ␮l of the LDH assay mix-
ture was added and the plates were incubated at 37 ◦ C for 30 min
and the LDH release was estimated at 495 nm. All experiments
were done in triplicates and percentage of specific cytotoxicity was
determined.
120 H. Rasouli et al. / International Journal of Biological Macromolecules 96 (2017) 118–128

2.7. Cell proliferation assay

Cell proliferation was measured using MTT (Sigma). HUVECs

were seeded at 2 × 103 cells/well in a medium supplemented
with 10% FBS in 96-well culture plates, after 24 h of incubation at
37 ◦ C and 5% CO2 , different concentrations of TICMS (5–120 ␮g/ml)
were added and the cells were incubated for additional 48 h. All
concentrations were tested in triplicate, and results were gath-
ered from three sets of experiments. After incubation for 48 h
(37 ◦ C and 5% CO2), 20 ␮l of 3-(4,5-dimethylthiazolyl-2)-2, 5-
diphenyltetrazolium bromide (MTT, Sigma, USA) solution (5 mg/ml
in PBS) was added to each well and re-incubated for 4 h at 37 ◦ C. To
dissolve formazan crystal, MTT-containing supernatant was elim-
inated and replaced by 100 ␮l of dimethyl sulfoxide (DMSO) and
kept at room temperature for 30 min. Finally, the absorbance was
read at 570 nm, with background subtraction of 630 nm using the
plate reader (Space fax 2100, Awareness, USA). MTT (5 mg/ml in
PBS) was added to each well and the plates were incubated for 4 h
at 37 ◦ C and 5% CO2 .

2.8. Scratch motility (wound healing) assay

The scratch assay is a useful and a straightforward method for

evaluation of cell migration in vitro. HUVECs were seeded into 6-
well plates. The next step involved scratching the confluent cell
monolayer to create a cell free zone by a 1000 ␮l pipette tip during
the following day. After washing the detached cells, the cells were
incubated in medium supplemented with indicated concentrations
of TICMS (5–120 ␮g/ml). The images were captured immediately
after wounding and again after 24 h. In order to compare the migra-
tion rate, scratches were photographed in five separated fields and
then images were analyzed by NIH Image J software. The percent-
age of wound closure was estimated as previously described [5] by
the following equation:
Wound closure (%) = [1 − (wound area at t1 /wound area at
t0 ) × 100%], where t1 is the time after 24 h of wounding and t0 is
the time immediately after wounding.

2.9. Preparation of collagen-cytodex model

In this study, type I collagen was obtained by dissolution of iso-

lated tendons from rat tail in 0.02 M acetic acid for 48 h in 4 ◦ C [5].
Cytodex particles were purchased from Pharmacia and for prepa-
ration of collagen-cytodex model; cytodex particles were dissolved
in PBS buffer and incubated overnight at 4 ◦ C. Then turgid particles
were autoclaved and finally PBS buffer was replaced with DMEM.
For attachment of cells to cytodex particles, isolated endothelial
cells were mixed with turgid cytodex particles and shaken every
30 min in 37 ◦ C for 8 h. Afterwards, cell-covered micro-carriers
were divided in a 24-well culture plate and incubated overnight in
CO2 incubator for growth and proliferation of cells on the surface of
cytodex particles in order to have fully cell-covered micro-carriers
for the experiment. The day after, contents of the wells were
collected in a 50 ml falcon and cell-covered micro-carriers were
allowed to precipitate. Then the supernatant containing culture
medium was aspirated. Finally, for preparation of collagen-cytodex
model, as described previously [4,30], 7 vol of cold collagen solu-
tion, 1 vol of culture medium (10×) and 2 vol of sodium bicarbonate
(11.76 mg/ml) were added to the falcon incubated in ice and the
falcon contents were mixed.
Then, this mixture was divided in 24-well culture plates and the
plates were incubated for 30 min in CO2 incubator. After this time,
a collagen matrix is formed around the cells.
Fig. 1. Reducing SDS-PAGE of purified trypsin inhibitor.
2.10. Examination of anti-angiogenesis effects of TICMS
After preparation of collagen-cytodex model, 600 ␮l of fresh
medium with 10% FCS, and indicated concentrations of TICMS
(5–120 ␮g/ml) were added to the wells and the plates were incu-
bated at CO2 incubator for 72 h. Finally, the anti-angiogenesis
effects were examined using inverted microscope and photogra-
phy. The test was carried out in triplicate for each dose.
2.11. ELISA assay for VEGF, MMP-2 and MMP-9
VEGF, MMP-2 and MMP-9 are key mediators of angiogenesis
and metastasis processes. We assessed these factors using ELISA
method [5]. HUVECs in logarithmic growth phase were cultured
in 24 well plates at a density of 1 × 105 in DMEM medium sup-
plemented with 5% FBS for 24 h. Afterwards, cells were treated
with different concentrations of TICMS (5–120 ␮g/ml) in serum-
free medium for an additional 18 h. Cell culture supernatants were
collected and total protein levels were measured by Bradford pro-
 H. Rasouli et al. / International Journal of Biological Macromolecules 96 (2017) 118–128 121

Fig. 2. A) TICM have not cytotoxic effect on HUVEC cells at 5–120 ␮g/ml B) TICMS inhibits the growth of HUVEC cells. At 5–120 ␮g/ml, TICMS decreased cell proliferation.
Each data point was presented as mean ± SD from 3 to 4 independent experiments.

Fig. 3. Decreasing the migration capacity of HUVEC in response to TICMS. (a) After creating a scratch in a confluent monolayer of HUVECs, cells were incubated for 24 h with
different concentration of TICMS. Results were monitored under the invert microscope. (b) Control, (c) 5 ␮g/ml, (d) 10 ␮g/ml, (e) 20 ␮g/ml, (f) 40 ␮g/ml, (g) 80 ␮g/ml, (h)
120 ␮g/ml. The number of migrated cells from the edge of the scratch could in five separate fields for each well and compared in treated well with control. Each point or
column represents the mean ± standard error of mean (SEM, n = 3). **P < 0.01 ***P < 0.001.

tein assay [27]. Then, MMP2, MMP9 and VEGF concentrations were Z-score using the ProSA web server. ProSA is a tool widely used to
assessed using a quantitative ELISA kit (R&D Systems). check 3D models of protein structures for potential errors [33].

2.12. Homology modeling 2.13. Molecular docking

Homology modeling method was used to build the 3D struc-
ture of TICMS. The aligned sequences between the target protein
(CMeTI-B accession number: 2116324B) and the two templates
(Eeti-II protein, PDB ID: 1H9I and Mcoti-II, PDB ID: 1HA9) were
used for the model constructing using MODELLER 9v17 program.
MODELLER is a computer program for comparative protein struc-
ture modeling [31]. The stereo-chemical and structural properties
of model were evaluated using the PROCHECK program [32]. The
sequence-structure compatibility of the models was evaluated on
Molecular docking is a functional tool in bioinformatics analysis
[34]. The goal of ligand-protein docking is to predict the predom-
inant binding condition(s) of a ligand with a protein of known
three-dimensional structure [35]. Molecular docking assays were
carried out in the present study by using the web-based server
ClusPro 2.0.
ClusPro 2.0 is a web server that filters docked conformation with
good surface complementarity, therefor selects complexes with
lowest desolvation and electrostatic energies [34,36].
122 H. Rasouli et al. / International Journal of Biological Macromolecules 96 (2017) 118–128

Fig. 4. Inverted microscopy of cultured HUVEC after 3 days in collagen gel. A): Part a shows angiogenesis of the untreated endothelial cells (negative control) endothelial
cells attached to particles have been proliferated and migrated through the collagen matrix. B–E): Test wells treated with 5–40 ␮g/ml of trypsin inhibitor respectively. Each
point or column represents the mean ± standard error of mean (SEM, n = 3). ***P < 0.001.

Table 1
Ramachandran plot summary.

Most favored Additionally Additionally Disallowed

Regions allowed regions allowed regions regions

TICMS model 88% 12% 0% 0%

The crystal structure of ␣V ␤3 (PDB ID: 3IJE [37]) and VEGFR1
(PDB ID: were 3HNG [38]) were used as receptor for docking with
prepared 3D model of CMeTI-B. The electrostatic potential surfaces
were generated using Adaptive Poisson-Boltzmann Solver (APBS)
[39]. The images of docking results were generated using Molegro
Virtual Docker software.
ac.uk/thornton-srv/databases/pdbsum/Generate.html) web-based
server [40] and CFinder (http://bioinf.modares.ac.ir/software/
cfinder/) server. Position prediction and distance of hydrogen bonds
among amino acids were analyzed by UCSF Chimera [41].

2.14. Data analysis

2.13.1. Assessment of interaction between ligand and receptors
All amino acid interactions between ligand and recep- All experiments were repeated at least three times. Results were
tors were calculated by PDBsum Generate (http://www.ebi. analyzed using ANOVA test and Tukey HSD test as post-hoc test by
 H. Rasouli et al. / International Journal of Biological Macromolecules 96 (2017) 118–128 123

GraphPad Prism software; p values < 0.05 were considered statisti-

cally significant.

3. Results

3.1. Protein purification and electrophoresis

Electrophoresis analysis of purified protein showed a single

band with a molecular mass of about 3 kDa (Fig. 1). The activity
of TICMS was 765 CIU/mg (chymotrypsin inhibitory unit per mg of
protein).

3.2. Anti-proliferative effect of TICMS

The results speculated that TICMS was not cytotoxic up to

120 ␮g/ml as assessed by LDH cytotoxicity assays (Fig. 2A). TICMS
induced a significant decrease in the proliferation of HUVECs which
was dose dependent with an IC50 value of about 20 ␮g/ml (Fig. 2B).

3.3. TICMS inhibits cell motility and invasion

Tumor cells exposed to normoxic and hypoxic conditions have

shown an increased tendency to spread to distant organs, and these
cells have been characterized by increased migration and inva-
sive potentials. The effect of TICMS on the migration potential of
HUVECs was studied in vitro. Wound healing assay was used to
determine whether TICMS could inhibit motility of HUVECs. After
24 h, cell monolayers were wounded and following another 24 h
HUVECs completely filled the cleared zone in control wells, while
different concentrations of TICMS inhibited motility of HUVECs
(Fig. 3).

3.4. Anti-angiogenic activity of TICMS

The effects of TICMS at different doses on HUVEC migration

and angiogenesis are shown in Fig. 4. Anti-angiogenic activity of
TICMS was assessed at the doses 5–120 ␮g/ml. TICMS was able to
completely inhibit angiogenesis in HUVEC capillary tube formation
model at 40 ␮g/ml. In these conditions, migration and tubuloge-
nesis of the endothelial cells through the collagen matrix were
inhibited in a dose dependent manner.

3.5. TICMS inhibits secretion of MMP-2, 9 and VEGF

TICMS inhibits secretion of MMP-2 and MMP-9 as confirmed

by ELISA analysis (Fig. 5A,B). VEGF secretion was also decreased by Fig. 5. TICMS inhibits MMP-2, MMP-9 and VEGF secretion. (A–C) Effects of TICMS
TICMS in the above mentioned doses as confirmed by ELISA analysis on MMP-2, MMP-9 and VEGF secretion were assayed by ELISA method. Each data
(Fig. 5C). point was presented as mean ± SD from 3 to 4 independent experiments.

3.6. Structural analysis and model quality

The primary structure of trypsin inhibitor was generated by
MODELLER 9v17 software [31]. MODELLER implements compara-
tive protein structure modeling by satisfaction of spatial restraints.
The MODELLER was designed to apply as many different types of
data about the target sequence as possible of the 100 models gen-
erated with MODELLER, the one corresponding to the lowest value
of energy and Dope score was selected for further analysis (Fig. 6).
In order to check for quality of model, PROCHECK program [32]
and ERRAT server [42] were used. Ramachandran plot and sum-
maries of trypsin inhibitor structure are shown in Fig. 7 and Table 1.
In the ERRAT plots black bars identify the regions that can be
rejected at the 99% level, gray bars demonstrate the error region
between 95% and 99%, and white bars indicate the region with a
lower error rate for protein folding. * On the error axis, two lines
are drawn to indicate the confidence with which it is possible to
reject regions that exceed that error value. ** Expressed as the per-
centage of the protein for which the calculated error value falls
below the 95% rejection limit. Good high resolution structures gen-
erally produce values around 95% or higher. For lower resolutions
(2.5–3 Å) the average overall quality factor is around 91%.
Ramachandran plot analysis showed that the main-chain con-
formations for 100% of amino acids residues are within most
favored or additionally allowed regions. In general, score close to
100% implies good stereochemical quality of the structure [32,43].
Therefore, these PROCHECK results suggest that predicted model
has a good quality (Fig. 7A). The ERRAT score for templates (PDB
ID: 1H9I and 1HA9) and trypsin inhibitor models were calculated
to be 94.783, 100 and 70, respectively (Fig. 7 B, C and D). The gener-
ally accepted range is >50 for a high quality model [42,44]. Thus, this
analysis revealed that the backbone conformation and non-bonded
interactions of TIPCS fit well within the range of a high quality
124 H. Rasouli et al. / International Journal of Biological Macromolecules 96 (2017) 118–128

Fig. 8. Protein Structure Assessment server (ProSA-web) result of the trypsin

inhibitor homology model; the black dot represents the model z-score.

Fig. 6. Cartoon representation of trypsin inhibitor protein. Cartoon model displayed

with UCSF Chimera software.

uses only the C˛ atoms of the input structure, hence it can also
model. For final quality validation quality of the generated TICMS be applied to low resolution structures and approximate models
model, ProSA web server was employed (Fig. 8). ProSA-web server obtained early in the structure determination process [33].

Fig. 7. Ramachandran plot for (A) and ERRAT plots for (B) trypsin inhibitor protein, (C) Eeti-II protein and (D) Mcoti-II protein.
 H. Rasouli et al. / International Journal of Biological Macromolecules 96 (2017) 118–128 125

Table 2
Scores of the docked structures shown in Fig. 9.

Ligand Receptor Cluster No. No. of representative Score

Trypsin inhibitor ␣V␤3 4 57 Center −859.7

Lowest energy −859.7
VEGFR1 1 117 Center −617.6
Lowest energy −621.2

Table 3 ing pathways [48]. Naturally, protease inhibitors are essential to

Interaction between trypsin inhibitor and docked receptors.
regulate the activity of their corresponding proteases within these
Receptor residues Ligand residues Distance (Å) Type of bond pathways. At least, varieties of 12 families of serine protease
␣V␤3 Glu637 a
OE1(A ) 1
Arg NH2 2.70 H-bond
inhibitors have been isolated from plants. The protease inhibitors
Arg379 NH2(A) Pro4 O 2.71 H-bond are small proteins, which can be found in plants tissues and they
379
Arg NH1(A) Lys5 O 2.62 H-bond probably comprise about 10% of the total protein content of the
Pro387 O (A) Lys9 NZ 2.89 H-bond plants. The plant leaves facing insect pathogenic microorganisms
Thr672 N(A) Asp19 OD2 3.26 H-bond
may induce production of these inhibitors [49–51]. The protease
Arg530 NH2(B) Ser14 O 2.80 H-bond
inhibitors found in plants seeds are classified into three general cat-
VEGFR1 His1020 O (A) Arg1 NH2 2.58 H-bond egories: A) serine protease inhibitors, B) metallocarboxy protease
Asp1040 OD1 (A) Arg1 NH1 2.61 H-bond
Glu871 0E2 (A) Lys5 NZ 2.53 H-bond
inhibitors, and C) cysteine protease inhibitors [49,52,53]. They have
Arg1021 NH1 (A) Asp13 OD1 2.75 H-bond been increasingly considered in pharmacology, medicine, and also
Lys1074 NZ (A) Ser14 O 2.61 H-bond for sanitary and cosmetic uses. In several studies, the anti-cancer
Lys1074 NZ (A) Asp15 OD1 2.56 H-bond role of protease inhibitors has been determined, but the exact
Arg1021 NH1(A) Cys20 O 2.72 H-bond
mechanisms have not been understood yet [54,55]. It is believed
a
A: subunit ␣; B: subunit ␤. that different mechanisms are involved such as: I) selectively killing
cancer cells II) induction of super oxide anions and hydrogen per-
In Fig. 8, obtained from ProSA-web server, groups of structures oxide release from white blood cells and tumor stimulated cells III)
solved by X-ray or NMR are shown in distinctive colors, and the it is hypothesized that these compounds have anti-inflammatory
obtained model Z-score was −7.49, which is placed within the range properties and due to the strong relationship between inflamma-
of scores typically found for native proteins of a similar size [33]. tion and cancer, they can prevent cancer formation and finally it is
We conclude that all results are acceptable and consistent, and the believed that these compounds cause cancer prevention by affect-
model has good quality and can be used for further work. ing proto-oncogenes [55–57].
Because tumor growth critically depends on new blood ves-
sel formation, angiogenesis inhibition is an important approach to
3.7. Molecular docking analysis
treat cancer and prevent cancer progression [4]. Through epidemi-
ological and animal studies, it has been indicated that consumption
Many biological functions involve the formation of protein-
of protease inhibitors reduces the risk of cancer development [58].
protein complexes [45]. Investigation of protein-protein interac-
Proteases produced by vascular endothelial and smooth muscle
tions by molecular docking is an increasingly accepted tool for drug
cells are expected to play significant roles in the angiogenic pro-
discovery [45]. The molecular docking approach can be used to
cesses as well as intravasation and extravasation processes of
model the interaction between a small molecule and a protein at the
metastatic tumor cells [59–61].
atomic level, which allow us to find the behavior of small molecules
This study was the first to explore anti-angiogenic potential
in the binding site of target proteins as well as to elucidate fun-
of C. melo seeds. We showed that TICMS by affinity chromatog-
damental biochemical process [46]. The goal of protein-protein
raphy inhibits angiogenesis in vitro. TICMS inhibited proliferation
docking is to determine the 3D structure of a protein-protein com-
of HUVECs without any cell toxicity at the doses used in this
plex formed by interaction of two or more proteins without need
study. So its anti-proliferative effect may not be due to EC apopto-
for experimental measurement [45,46]. Herein, molecular docking
sis or necrosis. In addition, the potential mechanisms underlying
was performed to investigate the binding mode of trypsin inhibitor
the anti-angiogenic effect of TICMS were investigated. The results
protein with ␣V ␤3 integrin and VEGFR1 receptors (Fig. 9)
of HUVECs migration assay revealed that TICMS shows signifi-
The molecular docking results show high tendencies for trypsin
cant inhibitory effect on EC migration. Additionally, TICMS strongly
inhibitor to bind the ␣V ␤3 integrin within the cleft between the ␣
inhibited EC morphogenic differentiation into capillary-like struc-
and ␤ subunits (Fig. 9). The trypsin inhibitor protein was also able to
tures.
interact with VEGFR1 receptor, but with low probability (Table 2).
Angiogenesis is a multistep process involving: degradation of
List of the most relevant interactions between residues of ligand
basement membrane and extracellular matrix components, prolif-
and receptors is shown in Table 3.
eration, migration and tubulogenesis of endothelial cells and finally
According to the molecular docking results Arg1 and Ser14
maturation of the neovasculature [62,63]. As shown by our study
residues of TICMS are would interact with both receptors. The
TICMS exerted suppressive effects on HUVEC proliferation, migra-
involvement of these two residues in the both ligand-receptor
tion as well as MMP-2, MMP-9 secretion which could account for its
dockings is suggestive of their possible important roles in the inter-
anti-angiogenic activity in this in vitro model. On the other hand,
action of trypsin inhibitor with the mentioned receptors.
TICMS inhibited secretion of VEGF from HUVECs as indicated by
ELISA method. VEGF, as the most important angiogenic factor, plays
4. Discussion a key role during the angiogenesis process which involves induction
of endothelial cell proliferation, migration and also MMP secretion
Proteases and their specific inhibitors are present in animals, [64]. Thus, TICMS’s suppressive effect on the secretion of VEGF,
plants, and microorganism kingdoms [47], with mainly regula- which affects other important events during angiogenesis, could be
tory roles in many biological processes, such as apoptosis, blood considered as the main mechanism of its anti-angiogenic activity.
coagulation system, complement cascade, and hormone process-
126 H. Rasouli et al. / International Journal of Biological Macromolecules 96 (2017) 118–128

Fig. 9. Representation of trypsin inhibitor (colored in red) docked to ␣V (blue) ␤3 (green) integrin (at the right) and VEGFR1 (at the left) receptors. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 10. Inhibition of angiogenesis by TICMS.

Previous studies have shown that protease inhibitors such as
soybean BBI induce cell cycle arrest at G1/S stage. The mechanism of
this effect is suggested to be related to inhibition of ubiquitination-
proteasome machinery by BBI which is associated with MKP-1
(a cyclin dependent kinase inhibitor) upregulation and cyclin D1,
E1 and phospho-ERK1/2 downregulation [65,66]. We suggest here
that the same mechanism of action could be attributable to TICMS.
Therefore, anti-proliferative effect of TICMS in this study could be
due to cell cycle arrest induction by TICMS at G1/S stage associ-
ated with downregulation of phospho-ERK1/2. On the other hand,
as phospho-ERK1/2 is one of the mediators downstream of VEGF
signaling pathway, and due to suppressive effects of TICMS on the
secretion of VEGF, and hence its signaling pathway (Fig. 10), explor-
ing the effect of TICMS on intracellular phospho-ERK1/2 would be
of great interest for future studies helping us understand TICMS’s
mechanisms of action in more detail.
During angiogenesis, ␣v integrins are overexpressed on the
surface of endothelial cells to facilitate their survival and induce
angiogenesis. Therefore, disrupting ligand binding which blocking
␣v integrin function can produce an antiangiogenic effect [67].
Our results speculate that trypsin inhibitor ligand apparently
link the mobile domain of the integrin ␣ subunit to the fixed domain
of the ␤ subunit. This kind of interaction may impair the mobility of
the chains and therefore inhibit integrin activation (Fig. 10). Inte-
grins are extra cellular matrix receptors involved in tumor invasion
and angiogenesis [68]. In vertebrates, integrins also play impor-
tant roles in certain cell-cell adhesions [69]. Several studies have
showed that integrins and their ligands play key roles in develop-
ment, immune responses, leukocyte traffic, hemostasis, and cancer
and are at the center of many human diseases [68,69]. Based on our
molecular docking results, it is suggested that TICMS is able to bind
the ␣V ␤3 integrin and VEGFR1 receptors and inhibit their activity.
 H. Rasouli et al. / International Journal of Biological Macromolecules 96 (2017) 118–128
Based on our experimental results, TICMS successfully inhibited
angiogenesis of HUVECs in the 3D collagen −cytodex model.
Taken together, we demonstrated that the anti-tumor effect of
TICMS is due to inhibition of several key events involved in tumor
progression, such as endothelial cell proliferation, migration, and
tubulogenesis and TICMS could be considered as ␣v an integrin
antagonist that having the potential to disrupt multiple aspects of
angiogenesis and tumor progression.
Conflict of interest statement
Authors certify that no actual or potential conflict of interest
in relation to this article exists. All of them read and checked the
manuscript and they were agreed for publication.
Author contributions
K.M and H.R conceived the main idea of this manuscript; H.R
wrote the first draft of the paper; All experimental assays were done
by S.P, A.M, Z.H, M.R and H.R; Homology modeling and molecular
docking were performed by H.R; Statistical analysis was evaluated
by K.M and H.R; Final revision was done by K.M and H.R
Acknowledgement
The authors specially thank Prof. Reza Khodarahmi, Ms. Fatemeh
Noroznezhad and Dr. Hamid Reza Mohammadi-Motlagh for their
helpful assistances. We also thank the Research Council of Kerman-
shah University of Medical Sciences (KUMS) for financial support
of this investigation (Grant Number: 95396). Effective, instructive
and invaluable comments, provided by the respectful editor and
reviewers are gratefully acknowledged.
References
[1] G. Tortora, F. Ciardiello, D. Melisi, Angiogenesis: a target for cancer therapy,
Curr. Pharm. Des. 10 (1) (2004) 11–26.
[2] O. Hashimoto, A. Nakamura, T. Nakamura, H. Iwamoto, M. Hiroshi, K. Inoue, T.
Torimura, T. Ueno, M. Sata, Methylated-(3  )-epigallocatechin gallate analog
suppresses tumor growth in Huh7 hepatoma cells via inhibition of
angiogenesis, Nutr. Cancer 66 (4) (2015) 728–735.
[3] N. Bhatia, P. Gupta, B. Singh, A.K. Koul, Lycopene enriched tomato extract
inhibits hypoxia, angiogenesis, and metastatic markers in early stage
N-nitrosodiethylamine induced hepatocellular carcinoma, Nutr. Cancer
(2015) 1–8.
[4] K. Mansouri, R. Khodarahmi, A. Foroumadi, A. Mostafaie, H.M. Motlagh,
Anti-angiogenic/proliferative behavior of a 4-aryl-4H-chromene on blood
vessel’s endothelial cells: a possible evidence on dual anti-tumor activity,
Med. Chem. Res. 20 (7) (2011) 920–929.
[5] K. Mansouri, A. Mostafi, D. Rezazadeh, M. Shahlaei, M. Hossein Modarressi,
New function of TSGA10 gene in angiogenesis and tumor metastasis: a
response to a challengeable paradox, Hum. Mol. Genet. 25 (2) (2016) 233–244.
[6] D. Ribatti, B. Nico, E. Crivellato, A. Roccaro, A. Vacca, The history of the
angiogenic switch concept, Leukemia 21 (1) (2007) 44–52.
[7] A.S. Mousa, S.A. Mousa, Anti-angiogenesis efficacy of the garlic ingredient
alliin and antioxidants: role of nitric oxide and p53, Cancer Nutr. 53 (1) (2005)
104–110.
[8] S. Sagar, D. Yance, R. Wong, Natural health products that inhibit angiogenesis:
a potential source for investigational new agents to treat cancer—part 1, Curr.
Oncol. 13 (1) (2006) 14–26.
[9] H. Rasouli, M.H. Farzaei, K. Mansouri, S. Mohammadzadeh, R. Khodarahmi,
Plant cell cancer: may natural phenolic compounds prevent onset and
development of plant cell malignancy? A literature review, Molecules 21 (9)
(2016) 1–26.
[10] A.R. Khuda-Bukhsh, S. Das, S.K. Saha, Molecular approaches toward targeted
cancer prevention with some food plants and their products: inflammatory
and other signal pathways, Cancer Nutr. 66 (2) (2013) 194–205.
[11] H.-R. Mohammadi-Motlagh, K. Mansouri, A. Mostafaie, Plants as useful agents
for angiogenesis and tumor growth prevention, Physiol. Pharmacol. 14 (3)
(2010) 302–317.
[12] K. Ramawat, S. Goyal, Natural Products in Cancer Chemoprevention and
Chemotherapy, Herbal Drugs: Ethnomedicine to Modern Medicine, Springer,
2009, pp. 153–171.
[13] D.J. Mabberley, The Plant-book: a Portable Dictionary of the Higher Plants
Utilising Cronquist’s An Integrated System of Classification of Flowering
127
Plants (1981) and Current Botanical Literature Arranged Largely on the
Principles of Editions 1–6 (1896/97-1931) of Willis’s A Dictionary of the
Flowering Plants and Ferns, Cambridge University Press, 1993.
[14] X.M. Fan, B.C.Y. Wong, W.P. Wang, X.M. Zhou, C.H. Cho, S.T. Yuen, S.Y. Leung,
M.C.M. Lin, H.F. Kung, S.K. Lam, Inhibition of proteasome function induced
apoptosis in gastric cancer, Int. J. Cancer 93 (4) (2001) 481–488.
[15] L.A. Dunn, K.T. Andrews, J.S. McCarthy, J.M. Wright, T.S. Skinner-Adams, P.
Upcroft, J.A. Upcroft, The activity of protease inhibitors against Giardia
duodenalis and metronidazole-resistant Trichomonas vaginalis, Int. J.
Antimicrob. Agents 29 (1) (2007) 98–102.
[16] A.R. Kennedy, The Bowman-Birk inhibitor from soybeans as an
anticarcinogenic agent, Am. J. Clin. Nutr. 68 (6) (1998) 1406S–1412S.
[17] A.R. Kennedy, Chemopreventive agents: protease inhibitors, Pharmacol. Ther.
78 (3) (1998) 167–209.
[18] S. Rakashanda, S. Amin, Proteases as targets in anticancer therapy using their
inhibitors, J. Life Sci. 5 (2013) 133–138.
[19] M.B. Barać, S.P. Stanojević, M.B. Pešić, Biologically active components of
soybeans and soy protein products: a review, Acta Periodica Technol. 36
(2005) 155–168.
[20] E.F. Fang, T.B. Ng, A trypsin inhibitor from rambutan seeds with antitumor,
anti-HIV-1 reverse transcriptase, and nitric oxide-Inducing properties, Appl.
Biochem. Biotechnol. 175 (8) (2015) 3828–3839.
[21] E.F. Fang, J.H. Wong, T.B. Ng, Thermostable Kunitz trypsin inhibitor with
cytokine inducing, antitumor and HIV-1 reverse transcriptase inhibitory
activities from Korean large black soybeans, J. Biosci. Bioeng. 109 (3) (2010)
211–217.
[22] F. Doñate, Antiangiogenic therapy in cancer, Drugs Future 30 (2005) 695–707.
[23] R.N. Eskander, K.S. Tewari, Incorporation of anti-angiogenesis therapy in the
management of advanced ovarian carcinoma—mechanistics, review of phase
III randomized clinical trials, and regulatory implications, Gynecol. Oncol. 132
(2) (2014) 496–505.
[24] S. Samant, D. Rege, Some enzymes and enzyme inhibitors from cucurbit
seeds, J. Sci. Food Agric. 47 (3) (1989) 383–385.
[25] M.N. Gupta, S. Jain, I. Roy, Immobilized metal affinity chromatography
without chelating ligands: purification of soybean trypsin inhibitor on zinc
alginate beads, Biotechnol. Prog. 18 (1) (2002) 78–81.
[26] H. Schägger, G. Von Jagow, Tricine-sodium dodecyl sulfate-polyacrylamide
gel electrophoresis for the separation of proteins in the range from 1 to
100 kDa, Anal. Biochem. 166 (2) (1987) 368–379.
[27] M.M. Bradford, A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding,
Anal. Biochem. 72 (1) (1976) 248–254.
[28] A.A.o.C.C.A.M. Committee, Approved Methods of the American Association of
Cereal Chemists, Amer Assn of Cereal Chemists, 2000.
[29] N.J. Linford, D.M. Dorsa, 17␤-Estradiol and the phytoestrogen genistein
attenuate neuronal apoptosis induced by the endoplasmic reticulum
calcium-ATPase inhibitor thapsigargin, Steroids 67 (13) (2002) 1029–1040.
[30] S.S. Shahangian, R.H. Sajedi, S. Hasannia, S. Jalili, M. Mohammadi, M. Taghdir,
A. Shali, K. Mansouri, R. Sariri, A conformation-based phage-display panning
to screen neutralizing anti-VEGF VHHs with VEGFR2 mimicry behavior, Int. J.
Biol. Macromol. 77 (2015) 222–234.
[31] N. Eswar, B. Webb, M.A. Marti-Renom, M.S. Madhusudhan, D. Eramian, M.Y.
Shen, U. Pieper, A. Sali, Comparative protein structure modeling using
Modeller, Curr. Protoc. Bioinformatics 5 (6) (2006) 1–30.
[32] R.A. Laskowski, M.W. MacArthur, D.S. Moss, J.M. Thornton, PROCHECK: A
program to check the stereochemical quality of protein structures, J. Appl.
Crystallogr. 26 (1993) 283–291.
[33] M. Wiederstein, M.J. Sippl, ProSA-web: interactive web service for the
recognition of errors in three-dimensional structures of proteins, Nucleic
Acids Res. 35 (2007) W407–W410.
[34] A. Rakhmetov, S.P. Lee, D. Grebinyk, L. Ostapchenko, H.Z. Chae, Simulation of
peroxiredoxin II and brain-type creatine kinase protein-protein interaction
using the on-line docking server ClusPro 2.0, J. App. Pharm. Sci. 5 (2015)
8011–8016.
[35] M. Awasthi, S. Singh, V.P. Pandey, U.N. Dwivedi, Molecular docking and
3D-QSAR-based virtual screening of flavonoids as potential aromatase
inhibitors against estrogen-dependent breast cancer, J. Biomol. Struct. Dyn. 33
(4) (2014) 804–819.
[36] S.R. Comeau, D.W. Gatchell, S. Vajda, C.J. Camacho, ClusPro: an automated
docking and discrimination method for the prediction of protein complexes,
Bioinformatics 20 (1) (2004) 45–50.
[37] J.-P. Xiong, B. Mahalingham, J.L. Alonso, L.A. Borrelli, X. Rui, S. Anand, B.T.
Hyman, T. Rysiok, D. Müller-Pompalla, S.L. Goodman, M.A. Arnaout, Crystal
structure of the complete integrin ␣V␤3 ectodomain plus an ␣/␤
transmembrane fragment, J. Cell Biol. 186 (4) (2009) 589–600.
[38] L. Tresaugues, A. Roos, C.H. Arrowsmith, H. Berglund, C. Bountra, R. Collins,
A.M. Edwards, S. Flodin, A. Flores, S. Graslund, M. Hammarstrom, I. Johansson,
T. Karlberg, T. Kotenyova, M. Moche, T. Nyman, C. Persson, T. Kragh-Nielsen,
A. Kotzch, J. Sagemark, H. Schueler, P. Schutz, M.I. Siponen, L. Svensson, A.G.
Thorsell, S. Van der Berg, J. Weigelt, M. Welin, M. Wisniewska, P. Nordlund,
Crystal Structure of VEGFR1 in Complex with
N-(4-Chlorophenyl)-2-((pyridin-4-ylmethyl)amino)benzamide, 2009 (http://
www.rcsb.org/pdb/explore.do?structureId=3hng).
[39] T.J. Dolinsky, P. Czodrowski, H. Li, J.E. Nielsen, J.H. Jensen, G. Klebe, N.A. Baker,
PDB2PQR: Expanding and upgrading automated preparation of biomolecular
128 H. Rasouli et al. / International Journal of Biological Macromolecules 96 (2017) 118–128
structures for molecular simulations, Nucleic Acids Res. 35 (2007)
W522–W525.
[40] R.A. Laskowski, PDBsum new things, Nucleic Acids Res. 37 (2008) D355–D359.
[41] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C.
Meng, T.E. Ferrin, UCSF Chimera: a visualization system for exploratory
research and analysis, J. Comput. Chem. 25 (13) (2004) 1605–1612.
[42] C. Colovos, T.O. Yeates, Verification of protein structures: patterns of
nonbonded atomic interactions, Protein Sci. 2 (9) (1993) 1511–1519.
[43] C.S. Reddy, K. Vijayasarathy, E. Srinivas, G.M. Sastry, G.N. Sastry, Homology
modeling of membrane proteins: a critical assessment, Comput. Biol. Chem.
30 (2) (2006) 120–126.
[44] M.W. MacArthur, R.A. Laskowsk, J.M. Thornton, Knowledge-based validation
of protein structure coordinates derived by X-ray crystallography and NMR
spectroscopy, Curr. Opin. Struct. Biol. 4 (5) (1994) 731–737.
[45] D. Kozakov, D. Beglov, T. Bohnuud, S.E. Mottarella, B. Xia, D.R. Hall, S. Vajda,
How good is automated protein docking? Proteins 81 (2013) 2159–2166.
[46] H.A. Gabb, R.M. Jackson, M.J.E. Sternberg, Modelling protein docking using
shape complementarity, electrostatics and biochemical information, J. Mol.
Biol. 272 (1997) 106–120.
[47] C. Shee, A.K. Sharma, Purification and characterization of a trypsin inhibitor
from seeds of Murraya koenigii, J. Enzyme Inhib. Med. Chem. 22 (1) (2007)
115–120.
[48] R.F. Qi, Z.W. Song, C.W. Chi, Structural features and molecular evolution of
bowman-birk protease inhibitors and their potential application, Acta
Biochim. Biophys. Sin. 37 (5) (2005) 283–292.
[49] V. da Silveira Ramos, G.n. de Souza Silva, M. das Graças Machado Freire, O.
Lima Tavares Machado, J.R. Postali Parra, M.L.g. Rodrigues Macedo,
Purification and characterization of a trypsin inhibitor from Plathymenia
foliolosa seeds, J. Agric. Food Chem. 56 (23) (2008) 11348–11355.
[50] M.L.R. Macedo, C.F.R. de Oliveira, P.M. Costa, E.C. Castelhano, M.C. Silva-Filho,
Adaptive mechanisms of insect pests against plant protease inhibitors and
future prospects related to crop protection: a review, Protein Pept. Lett. 22 (2)
(2015) 149–163.
[51] V.D. Parde, Inhibition of Helicoverpa Armigera Gut Zymogen Activation by
Plant Protease Inhibitors, Dr. Babasaheb Ambedkar Marathwada University,
2009.
[52] P.K. Lawrence, K.R. Koundal, Plant protease inhibitors in control of
phytophagous insects, Electron. J. Biotechnol. 5 (1) (2002) 5–6.
[53] C.A. Ryan, Proteolytic enzymes and their inhibitors in plants, Annu. Rev. Plant
Physiol. 24 (1) (1973) 173–196.
[54] T. Koltai, Nelfinavir and other protease inhibitors in cancer: mechanisms
involved in anticancer activity, F1000Research 4 (2015).
[55] C. López-Otín, L.M. Matrisian, Emerging roles of proteases in tumour
suppression, Nat. Rev. Cancer 7 (10) (2007) 800–808.
View publication stats
[56] Y. DeClerck, S. Imren, Protease inhibitors: role and potential therapeutic use
in human cancer, Eur. J. Cancer 30 (14) (1994) 2170–2180.
[57] M. Hidalgo, S.G. Eckhardt, Development of matrix metalloproteinase
inhibitors in cancer therapy, J. Natl. Cancer Inst. 93 (3) (2001) 178–193.
[58] P.K. Mills, W.L. Beeson, D.E. Abbey, G.E. Fraser, R.L. Phillips, Dietary habits and
past medical history as related to fatal pancreas cancer risk among adventists,
Cancer 61 (12) (1988) 2578–2585.
[59] G. Marone, F. Granata, Angiogenesis, lymphangiogenesis and clinical
implications, Respiration 86 (5) (2013) 353–440.
[60] J.G. Rasmussen, O. Frøbert, L. Pilgaard, J. Kastrup, U. Simonsen, V. Zachar, T.
Fink, Prolonged hypoxic culture and trypsinization increase the
pro-angiogenic potential of human adipose tissue-derived stem cells,
Cytotherapy 13 (3) (2011) 318–328.
[61] Y. Shakiba, K. Mansouri, A. Mostafaie, Anti-angiogenic effect of soybean
kunitz trypsin inhibitor on human umbilical vein endothelial cells, Fitoterapia
78 (7) (2007) 587–589.
[62] D.C. Achê, M.S.R. Gomes, D.L.N. de Souza, M.A. Silva, M.I.H. Brandeburgo,
K.A.G. Yoneyama, R.S. Rodrigues, M.H. Borges, D.S. Lopes, V. de Melo
Rodrigues, Biochemical properties of a new PI SVMP from Bothrops
pauloensis: inhibition of cell adhesion and angiogenesis, Int. J. Biol.
Macromol. 72 (2015) 445–453.
[63] H. Rasouli, K. Mansouri, L. Mahamed-Khosroushahi,
Anti-angiogenic/inflammatory behavior of mushroom Ganoderma Lucidum
extract could be effective for treatment of corneal neovascularization: a
hypothesis, J. Rep. Pharm. Sci. 3 (1) (2014) 14–18.
[64] H. Tao, Z.-W. Chen, J.-J. Yang, K.-H. Shi, MicroRNA-29a suppresses cardiac
fibroblasts proliferation via targeting VEGF-A/MAPK signal pathway, Int. J.
Biol. Macromol. 88 (2016) 414–423.
[65] Y.-W. Chen, S.-C. Huang, S.-Y. Lin-Shiau, J.-K. Lin, Bowman–Birk inhibitor
abates proteasome function and suppresses the proliferation of MCF7 breast
cancer cells through accumulation of MAP kinase phosphatase-1,
Carcinogenesis 26 (7) (2005) 1296–1306.
[66] T. Saito, H. Sato, N. Virgona, H. Hagiwara, K. Kashiwagi, K. Suzuki, R. Asano, T.
Yano, Negative growth control of osteosarcoma cell by Bowman-Birk protease
inhibitor from soybean; involvement of connexin 43, Cancer Lett. 253 (2)
(2007) 249–257.
[67] S.M. Weis, D.A. Cheresh, ␣v integrins in angiogenesis and cancer, Cold Spring
Harbor Perspect. Med. 1 (1) (2011).
[68] S. Monferran, N. Skuli, C. Delmas, G. Favre, J. Bonnet, E.
Cohen-Jonathan-Moyal, C. Toulas, Alphavbeta3 and alphavbeta5 integrins
control glioma cell response to ionising radiation through ILK and RhoB, Int. J.
Cancer 123 (2008) 357–364.
[69] O.M. Hynes, Integrins: Bidirectional, Allosteric signaling machines, Cell 110
(6) (2002) 673–687s.