Biochemical Pharmacology 181 (2020) 114149

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Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm

Review

Preclinical studies of a novel snake venom-derived recombinant disintegrin T

with antitumor activity: A review
⁎
Axel H. Schönthala, Stephen D. Swensonb,c, Thomas C. Chenb, Francis S. Marklandc,
a
Department of Molecular Microbiology and Immunology, Keck School of Medicine (KSOM), University of Southern California (USC), Los Angeles, CA 90089, USA
b
Department of Neurological Surgery, KSOM, USC, Los Angeles, CA 90089, USA
c
Department of Biochemistry and Molecular Medicine, KSOM, USC, Los Angeles, CA 90089, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Snake venoms consist of a complex mixture of many bioactive molecules. Among them are disintegrins, which
Brachytherapy are peptides without enzymatic activity, but with high binding affinity for integrins, transmembrane receptors
Contortrostatin that function to connect cells with components of the extracellular matrix. Integrin-mediated cell attachment is
Disintegrins critical for cell migration and dissemination, as well as for signal transduction pathways involved in cell growth.
Glioblastoma
During tumor development, integrins play key roles by supporting cancer cell proliferation, angiogenesis, and
Integrins
metastasis. The recognition that snake venom disintegrins can block integrin functions has spawned a number of
Liposomes
Ovarian cancer studies to explore their cancer therapeutic potential. While dozens of different disintegrins have been isolated,
Prostate cancer none of them as yet has undergone clinical evaluation in cancer patients. Among the best-characterized and
Snake venom preclinically most advanced disintegrins is vicrostatin (VCN), a recombinant disintegrin that was rationally
Vicrostatin designed by fusing 62 N-terminal amino acids derived from the disintegrin contortrostatin with 6 C-terminal
amino acids from echistatin, the disintegrins from another snake species. Bacterially produced VCN was shown
to target multiple tumor-associated integrins, achieving potent anti-tumor and anti-angiogenic effects in in vitro
and in vivo models in the absence of noticeable toxicity. This review will introduce the field of snake venom
disintegrins as potential anticancer agents and illustrate the translational development and cancer-therapeutic
potential of VCN as an example.

1. Introduction
Snake venoms consist of a highly complex mixture of biologically
active proteins and peptides that serve the immobilization and diges-
tion of prey, as well as the snake’s self-defense and survival. After in-
jection into prey, this mixture exerts effects on bleeding, pain and in-
flammation, and generates cytotoxic, neurotoxic, and cardiotoxic
sequelae. Many of these venom constituents harbor enzymatic activity,
whereas others do not. Examples of the most abundant components
include members of the family of phospholipase A2, metalloprotei-
nases, serine proteinases, hydrolases, lyases, endonucleases, alpha-
neurotoxins, dendrotoxins, cardiotoxins, protease inhibitors and disin-
tegrins [1–4].
Disintegrins represent a large group of low molecular weight, non-
enzymatic polypeptides that specifically bind to cell surface integrins
present on platelets and other cells. Their biological function serves to
counter blood clotting, thereby promoting the further spread of the
snake’s venom throughout the circulation of its prey. The anticoagulant
activity of disintegrins results from interference with the final step of
platelet aggregation, where disintegrins bind to integrin receptors on
platelets and as a result prevent fibrinogen binding. This potent antic-
oagulant activity of disintegrins generated medical interest toward their
use as potent anticoagulants, and eventually resulted in the develop-
ment and marketing of two therapeutic agents, eptifibatide and tir-
ofiban [5]. Eptifibatide (Integrilin) is a cyclic heptapeptide [6,7] that
represents a synthetically modified version of barbourin, a disintegrin
isolated from the venom of the pygmy rattlesnake (Sistrurus miliarius
barbouri) [8] (Fig. 1). Tirofiban (Aggrastat) is a non-peptide peptido-
mimetic [7,9] that was designed based on the snake venom disintegrin
echistatin, isolated from the venom of the saw-scaled viper (Echis car-
inatus) [10]. Both eptifibatide and tirofiban are widely used in the clinic
as antiplatelet agents during coronary interventions, including acute
coronary syndrome [11,12].
Nearly three decades have passed since eptifibatide and tirofiban
first demonstrated the compelling clinical benefit of snake venom-based
disintegrins [5]. Since then, many more venom derived disintegrins

⁎
Corresponding author.
E-mail address: markland@usc.edu (F.S. Markland).

https://doi.org/10.1016/j.bcp.2020.114149
Received 5 May 2020; Received in revised form 9 July 2020; Accepted 9 July 2020
Available online 12 July 2020
0006-2952/ © 2020 Elsevier Inc. All rights reserved.
A.H. Schönthal, et al.
have been identified and their potential use explored for a variety of
medical needs beyond their role as platelet aggregation inhibitors.
Specifically, a number of disintegrins are thought to hold significant
potential for cancer therapy in view of their ability to impede cancer
cell motility, invasion, metastasis and tumor angiogenesis. Although so
far no disintegrin-based drug has been produced for cancer therapeutic
purposes, many preclinical studies have demonstrated the great pro-
mise of this field and have provided the rationale for further explora-
tion of this fertile area of study [13,14]. In the following, we will
summarize the current status of snake venom-based disintegrin re-
search with regard to its objective of developing effective cancer ther-
apeutic agents. In particular, we will discuss native contortrostatin and
recombinant vicrostatin as exemplary representatives from among the
group of snake venom-based disintegrins.
2. Integrins as disintegrin targets
Integrins are transmembrane receptors that enable the interaction of
cells with the extracellular matrix (ECM). They function to attach the
cell cytoskeleton to specific components of the ECM, such as fi-
bronectin, vitronectin, laminin, and collagen, and in reverse initiate
signal transduction into the cell interior upon ECM binding. Integrin-
mediated cell attachment is critical for cell spreading and migration,
whereas integrin-mediated signal transduction pathways are involved
in cell cycle regulation, survival, and secretion of proteases. Not sur-
prisingly, integrins play key roles in the process of carcinogenesis by
impacting cancer cell proliferation and survival, angiogenesis, and
metastasis [15–18].
In their active conformation, integrins consist of heterodimeric
complexes of one alpha- and one beta-subunit. In mammalian cells, 18
alpha- and 8 beta-subunits with the ability to form 24 different het-
erodimers have been recognized. Their ability to interact with the ECM
or with membrane-associated proteins on other cells frequently de-
pends on a conserved tripeptide motif consisting of arginine, glycine
and aspartate, which is commonly referred to as the Arg-Gly-Asp (RGD)
motif [19–21]. It was originally discovered within the amino acid se-
quence of fibronectin [22] and since then has been confirmed in other
ECM proteins as well [23,24]. Eight of the integrin dimers are capable
of recognizing the RGD motif in natural ligands (namely: αIIbβ3, αvβ1,
αvβ3, αvβ5, αvβ6, αvβ8, α5β1, and α8β1) [24,25], but details of their
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Biochemical Pharmacology 181 (2020) 114149
Fig. 1. Examples of venomous snakes used for ob-
taining disintegrins A. The dusky pygmy rattlesnake
(Sistrurus miliarius barbouri) is endemic throughout
the southeastern U.S. and is the most commonly
encountered venomous snake in urbanized areas. Its
venom contains the disintegrin barbourin, which is
notable for its KGD motif, rather than the more
common RGD motif in many other integrins. B. The
saw-scaled viper (Echis carinatus) can be found in
parts of Central Asia and the Middle East. Its venom
contains the disintegrin echistatin. Although its
venom is lethal in < 10 percent of untreated vic-
tims, this snake is believed to cause more human
deaths than all other snake species combined, pri-
marily due to its aggressive nature and the difficul-
ties of many patients to reach hospitals with avail-
able anti-venom. C. The common green pit (or
bamboo pit) viper (Trimeresurus gramineus) is en-
demic to the southern part of India. Its venom con-
tains the disintegrin trigramin. D. The southern
copperhead viper (Agkistrodon contortrix contortrix)
is one of five copperhead subspecies that are com-
monly found in the southern U.S. Its venom contains
the disintegrin contortrostatin. Copperheads bite
more people than any other U.S. species of snake.
Although the bite is extremely painful, the venom is
not very potent and almost never fatal.
functional differences have not yet been completely established. Such
analysis is complicated by cell type-specific integrin expression profiles
that are dependent on the microenvironment within a spaciotemporal
context. Moreover, integrins are known to interact with other receptor
proteins as part of focal adhesion multiprotein networks [26] and their
expression levels can vary significantly between normal cells and tumor
cells [27–29].
Integrins exhibit structural diversity and undergo conformational
changes that are central to the regulation of their function. They exist in
three major conformational states: an inactive or low-affinity state, a
primed or activated high-affinity state, and a ligand-bound or occupied
state [30,31]. This distinction adds to their complexity and further in-
creases the challenge to target this class of receptors for therapeutic
purposes. However, the discovery of the RGD-binding ability of in-
tegrins exposed a promising target that is sought to be exploited by
interventions that harbor the RGD motif as the matching counterpart.
Among the latter are the snake venom-derived RGD-containing disin-
tegrins.
3. Snake venom disintegrins
Disintegrins are a class of disulfide-rich, non-enzymatic peptides,
originally isolated from snake venom. While many of them contain an
RGD motif, a number of disintegrins have been identified that harbor
variations of this core tripeptide domain, such as VGD, MGD, WGD, and
KGD (with preservation of glycine and aspartic acid), MLD and ECD
(with preservation of aspartic acid), and R/KTS [32–35]. As would be
expected, the specific composition of this domain (in concert with other
elements of the disintegrin peptide) impacts their high-affinity/high-
specificity interaction with select integrin targets, and thus creates a
diversity of interventional potential for different biological and patho-
logical processes [32,36].
Overall, RGD-containing disintegrins represent the largest and best-
studied family of disintegrins. While most of them are monomeric, a
few act as dimers [37]. NMR studies and crystal structures of disin-
tegrins established that their binding activity further depends on the
appropriate pairing of several cysteine residues responsible for the
disintegrin fold, as well as on a mobile 11-amino acid loop protruding
from the polypeptide core, which displays the tripeptide RGD motif
[2,13,33,38]. In addition, binding is fine-tuned by residues flanking the
A.H. Schönthal, et al.
integrin-recognition peptide and supported further by C-terminal tail
sequences, which are thought to trigger conformational changes in the
bound integrin receptor, thereby completing the selective inhibition of
integrin activity [33,34,39]. Intriguingly, disintegrins preferentially
bind to the activated conformation of integrins, which is present on
motile cells, such as cancer cells and angiogenic endothelial cells but
not generally on quiescent cells, thus making them attractive vehicles to
specifically target cancerous tissues [28]. In all, members of the RGD-
containing family of disintegrins should be able to inhibit the physio-
logical functions of α5β1, α8β1, αvβ1, αvβ3, αvβ5, αvβ6, αvβ8 and
αIIbβ3 integrins, the so-called RGD receptors [32].
Since the 1987 report on the isolation of the first disintegrin, tri-
gramin, from the venom of the common green pit viper (Trimeresurus
gramineus) [40] (Fig. 1), about 100 additional snake venom disintegrins
have been reported (see listing in ref. [32]). Many of them were char-
acterized structurally and functionally and were confirmed to interact
with and inhibit specific integrins, generating further efforts to study
their potential anticancer activities. In our own studies, the Markland
lab discovered contortrostatin (CN) in 1994 [41,42] a homodimeric
disintegrin from the venom of Agkistrodon contortrix contortrix, the
Southern copperhead snake (Fig. 1). CN has become one of the most-
studied members of this group of snake venom disintegrins. Its devel-
opment toward potential therapeutic applications was further sup-
ported by its incorporation into a newly-designed, chimeric, re-
combinant disintegrin, where it was genetically fused with the C-
terminal tail of the viperid disintegrin echistatin, resulting in the bac-
terially produced disintegrin vicrostatin (VCN) [43].
3.1. Contortrostatin and vicrostatin
CN was purified from snake venom using a multi-step chromato-
graphic approach with yields of 1–2 mg per gram of lyophilized crude
venom [42]. Its initial characterization in platelet aggregation inhibi-
tion assays showed an IC50 of 49 nM for human platelet-rich plasma,
and binding assays revealed its binding to αIIbβ3 integrin on platelets,
thereby preventing fibrinogen binding and exerting anticoagulant ac-
tivity. Molecular characterization presented its composition as a
homodimer with molecular mass of 13.5 kDa and 6750 Da for the in-
dividual subunits [42,44]. A cDNA library was made from the venom
gland of A. contortrix contortrix, which enabled cloning of the full-length
cDNA containing the CN disintegrin domain of 195 base pairs encoding
65 amino acids [45] (Fig. 2). Fusion of the active domain to thioredoxin
supported expression in a bacterial system and it was possible to con-
firm that integrin binding activity was retained. Due to the limited
amounts of disintegrins that are usually obtained from their natural
snake venom source, functional expression of a recombinant version
3
Biochemical Pharmacology 181 (2020) 114149
provided adequate material for further functional studies.
Using native CN, it could be established that this disintegrin re-
cognizes αIIbβ3, α5β1, αvβ3, and αvβ5 integrins [42,45,46]. The
physiological binding partners for these integrins consist of fibrinogen,
fibronectin, vitronectin, collagen, von Willebrand factor (vWF),
thrombospondin 1 (TSP-1), and fibroblast growth factor 2 (FGF-2), and
therefore blocking their activities by exposure to CN was expected to
have a variety of complex effects [32]. Consequently, beyond antic-
oagulation, CN exhibited inhibitory impact on tumor cell attachment to
matrix, cell migration and invasion, and angiogenesis, in both in vitro
and in vivo studies [41,47–50] (see additional details below).
Recombinant technology approaches have been applied to many
other snake venom disintegrins and helped to advance this area of re-
search (for a list of recombinant disintegrins, see [34]). Purification of
native peptides from snake venom not only is laborious, but the initial
milking of snakes is not without risks to the health of the worker.
Furthermore, yields are generally small and can represent mixtures of
different disintegrins, as some snake venoms may contain more than
one type of disintegrin [4,33]. Cloning of disintegrins supports synth-
esis of greater yields of high-quality protein product and enables de-
tailed studies of structure–function relationships, along with NMR
analysis and determination of crystal structures [2,34]. Another useful
approach is the production of chimeric peptides to create novel pro-
ducts with potentially enhanced or altered biological activities [39,51].
For example, domain swap experiments were performed [39] with two
snake venom disintegrins, echistatin from the venom of the saw-scaled
viper Echis carinatus [10] (Fig. 1) and eristostatin from the leaf-nosed
viper Eristocophis macmahoni [52]. While the former displayed high
binding activity to αvβ3 and α5β1 integrins, the latter exhibited much
lower binding to these integrins. Intriguingly, transfer of the C-terminal
HKGPAT amino acid sequence from echistatin to eristostatin resulted in
the latter’s greatly increased recognition and inhibition of both αvβ3
and α5β1 integrins [39]. Such results not only demonstrated that in-
herent specificity and activity of disintegrins can be genetically ma-
nipulated and altered, but they also contributed to the recognition that,
beside the central RGD motif, C-terminal tail epitopes represent critical
components of disintegrin-integrin interaction. Because αvβ3 integrin
in particular has been recognized as being frequently overexpressed in
cancer cells and plays a prominent role in tumor metastasis [53,54],
specific targeting of this integrin may prove especially beneficial for
cancer therapeutic purposes.
Similar to the above domain swap experiments, CN was purpose-
fully genetically modified by replacing three amino acids at its C-
terminal end with the six amino acids mentioned above (HKGPAT) from
the C-terminus of echistatin (Fig. 2). The resulting peptide represented
a sequence-engineered disintegrin of 69 amino acids and 7146 Da,
Fig. 2. Sequence alignment of contortrostatin (CN),
vicrostatin (VCN), and echistatin Native CN is a
dimer with two identical chains oriented in an anti-
parallel fashion (which is not represented by the
configuration shown in the Figure), held together by
two interchain disulfide bonds. Additional sec-
ondary structures are created by intrachain disulfide
bonds. VCN is a monomer that was created by re-
combinantly modifying the primary CN sequence by
swapping the C-terminal tail (3 aa underlined with
dotted line in CN) for 6 aa (underlined in blue font
and italics) derived from the C-terminus of echis-
tatin (underlined in blue font and italics). The ad-
ditional glycine at the N-terminus (underlined in
green font) is acquired during cleavage of the
thioredoxin-VCN hybrid protein with a specific
protease, tobacco etch virus (TEV) protease, during
the synthesis and purification process. In all se-
quences the RGD tripeptide motif is depicted in red
bold font.
A.H. Schönthal, et al.
which was called vicrostatin (VCN) [43,55]. It faithfully retained CN’s
targeting of αIIbβ3, α5β1, αvβ3, and αvβ5 integrins, but in addition
displayed greater than 10-fold higher affinity for α5β1 integrin, as was
expected based on the above described studies with echistatin and
eristostatin [39]. In comparison to the homodimeric structure of CN,
the novel chimeric disintegrin, VCN, was active as a monomer, thus
simplifying its production in larger quantities than those of several
other disintegrins that have been cloned and expressed [34]. Using the
Origami B Escherichia coli expression system, it was possible to reliably
produce 200 mg/L of purified VCN from the bacterial cell lysate. In
contrast to the notoriously difficult-to-amass native disintegrins, VCN
can be robustly produced in large quantity and without high-risk
milking of snakes. Like CN, VCN was shown to inhibit tumor cell ad-
hesion, endothelial and tumor cell invasion, and angiogenesis
[43,55,56] (see additional details below).
Given the complexity of the cancer microenvironment, in particular
in view of the propensity of cancer cells to change their integrin re-
pertoire in response to drug treatment, it would be advantageous to
target multiple integrins for optimized therapeutic impact [57]. In this
regard, the promiscuity of CN and VCN, i.e., their ability to target
multiple integrins simultaneously, is expected to provide a distinct
therapeutic benefit. A number of preclinical studies in various tumor
models have provided compelling evidence that these disintegrins in-
deed harbor highly promising anticancer activity, and some of these
studies will be presented below.
4. CN and VCN in breast cancer
Breast cancer is the leading type of cancer in women and accounted
for 627,000 deaths worldwide in 2018 [58]. It is a molecularly het-
erogeneous disease that is divided into different subtypes, based on
hormone receptor status, overexpression of human epidermal growth
factor receptor 2 (EGFR2/HER2/Neu), presence of mutations in breast
cancer genes 1 or 2 (BRCA1/2), and an increasing number of ad-
ditionally recognized molecular markers [59,60]. While early-stage
disease is curable in the majority of patients, prognosis for advanced
breast cancer becomes worse, and morbidity and mortality from breast
cancer is largely associated with metastatic disease [61]. The man-
agement of breast cancer depends on a variety of factors, including
subtype, stage, and the patient’s age. It generally involves surgery,
followed by radiation, hormone-blocking therapy in cases of hormone
receptor-positive disease, and chemotherapy for advanced stages. De-
pending on the presence of overexpressed tyrosine kinase growth fac-
tors, targeted antibodies (trastuzumab, cetuximab, etc.) or small-mo-
lecule inhibitors (lapatinib, olaparib, etc.) may be used. Newer
experimental regimens are exploring the benefit of immune checkpoint
inhibitory antibodies, antibody-drug conjugates, poly(ADP-ribose)
polymerase (PARP) inhibitors, cyclin-dependent kinase 4/6 inhibitors,
inhibitors targeting the phosphoinositide 3-kinase (PI3K)/protein ki-
nase B (AKT)/mammalian target of rapamycin (mTOR) pathway, and
other modalities [62–64]. Yet, despite considerable and continuous
progress in this field, 42,000 women in the U.S. are expected to die
from breast cancer in 2020 [65].
Several disintegrins, including CN and VCN, have been investigated
for their breast cancer therapeutic potential in various preclinical
models. A study from the Markland lab characterized the effects of CN
on adhesion, invasion, and proliferation of MDA-MB-435 human breast
carcinoma cells in vitro, as well as on these cells’ growth and metastasis
in an orthotopic nude mouse model in vivo [49]. It was reported that CN
blocked the attachment of cultured cells to ECM proteins fibronectin
and vitronectin and prevented cell invasion through an artificial Ma-
trigel basement membrane, while at the same time it did not exert
cytotoxic effects on these cells and did not inhibit proliferation in vitro.
In vivo, daily local injections of CN into the orthotopic tumor mass re-
sulted in reduced tumor growth, along with greatly reduced numbers of
pulmonary macro-metastases and micro-metastases, and inhibitory
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Biochemical Pharmacology 181 (2020) 114149
effects on tumor angiogenesis [49]. Combined with other studies that
have reported potent anti-angiogenic activity of CN and VCN
[48,55,66,67] (and of other disintegrins [68–71]), these results in-
dicated two main features of CN and VCN: indirect inhibition of pri-
mary tumor growth through prevention of tumor angiogenesis, and
direct blockage of metastasis via interruption of tumor cell migration
and invasion.
Several subsequent studies validated the anti-breast cancer potency
of CN and several other disintegrins and established their anti-meta-
static potential [45,72–74]. It was also demonstrated that inhibitory
effects on adhesion and migration were specific to breast cancer cell
lines, but did not impact normal breast cell lines, such as MCF-10A
[75]. The basis for this differential could be found in the overexpression
of cancer-specific integrins, primarily integrin αvβ3, which represents
the prime target for CN and several other disintegrins to exert their
antimetastatic function [45,71–74].
The above mentioned method of daily intratumor injection of CN,
which was performed in a mouse breast cancer model to establish proof
of principle of in vivo activity [49], would not be practical for clinical
translation. Therefore, various modifications of disintegrin delivery are
being developed in order to support future clinical applications. In the
context of CN development for breast cancer, a liposomal formulation
(called LCN) was designed for intravenous delivery and shown to retain
active protein with full retention of biological activity [67]. Char-
acterizing a variety of parameters of twice-weekly LCN administration
in an orthotopic mouse breast cancer model, the authors emphasized
four advantages of this system: (i) prolonged circulatory half-life of LCN
over native CN, (ii) passive accumulation of LCN in tumor tissue, (iii)
no platelet reactivity of LCN, and (iv) no immunogenicity of LCN; along
with potent antiangiogenic effects and tumor growth inhibition [67]. In
all, these studies established evidence that proper formulation of CN
allowed delivery via a clinically acceptable delivery method.
In yet another approach, multimeric disintegrin protein polymer
fusions targeting the tumor vasculature of murine breast cancer models
were studied [56]. In these studies, the VCN sequence was genetically
added to a cDNA encoding elastin-like polypeptide (A192) of 73.6 kDa,
resulting in a fusion protein that spontaneously self-assembled into
higher order multimers, quite likely via intermolecular bonds between
the many cysteine residues present on the VCN sequence (Fig. 2). Un-
like monomeric VCN, which is prone to efficient renal clearance due to
its small size of only 7.1 kDa, these high-molecular weight fusion
complexes were above the renal filtration cutoff and persisted much
longer in the circulation of mice, thereby allowing more time to accu-
mulate within the tumor tissue. The fusion protein retained the integrin
targeting selectivity of its parental monomeric VCN molecule, as well as
its therapeutic activity when tested in an orthotopic mouse breast
cancer model in vivo [56]. As shown before with native CN and
monomeric VCN, the anticancer effect of fused A192-VCN resulted from
a combination of inhibitory effects on tumor cells as well as on tumor-
associated endothelial cells.
5. VCN in prostate cancer
Prostate cancer (PC) is the most prevalent and the second most le-
thal cancer among American men [76]. Duration of benefit from an-
drogen-deprivation therapy, the mainstay for treatment of metastatic
disease, is highly variable and difficult to predict. Metastatic PC in-
variably progresses to a castrate-resistant state in spite of androgen-
signaling networks remaining vitally active. Several potent agents tar-
geting this network are associated with improved survival, including
androgen receptor antagonists such as enzalutamide, and agents tar-
geting androgen synthesis via the CYP17 pathway, such as abiraterone
[77,78]. The addition of multimodal chemotherapy also has generated
some limited benefit [79].
Bone metastases are common in advanced PC and are an important
cause of morbidity. Radium-223 is an alpha-emitting calcium mimetic
A.H. Schönthal, et al.
designed to decrease collateral damage to the bone marrow and can be
safely administered repeatedly [80]. Anti-angiogenic therapy is an al-
ternative strategy for long-term control of PC progression, consistent
with the association between elevated tumor vascularity and poor
prognosis [81]. Over time, however, all patients develop resistance to
these therapies [82,83].
Although ionizing radiation has a primary anti-angiogenic effect in
cancer, it also has a pro-angiogenic effect because of an effort by the
tumor to protect its own vasculature from damage due to radiation
exposure [84]. Interestingly, it was reported that endothelial cell sen-
sitivity to external beam radiotherapy is enhanced by the simultaneous
administration of an integrin antagonist in a PC animal model [85]. As
a consequence, advanced PC, irrespective of its hormonal dependence
and chemotherapy resistance status, should be susceptible to anti-in-
tegrin therapy. This is true due to the fundamentally different mole-
cular mechanisms of action between anti-integrin therapy and the
hormonal and chemotherapy agents presently utilized.
Activated integrins are not usually present in quiescent tissues, but
some, such as αvβ3 [86,87], play an important role in various neo-
plastic processes, which—in the case of prostate cancer—include bone
metastasis in particular [88–90]. The role of αvβ3 in enhancing skeletal
invasion by prostate cancer cells underscores its potential as a target for
therapeutic strategies aimed at inhibiting pathways involved in prostate
cancer progression [91–93]. Bone metastases are present in ~70–80%
of patients with prostate cancer. Preclinical studies provided convincing
evidence that increased expression of αvβ3 leads to metastasis and bone
invasion [93]. This integrin mediates cell adhesion to the bone extra-
cellular matrix through recognition of conserved RGD motifs in various
ligands, including osteopontin, vitronectin, and fibronectin. It has also
been reported that αvβ3 promotes binding to several extracellular
matrix proteins in the bone microenvironment that are needed both for
tumor cell colonization and tumor progression [93]. As well, metastasis
to remote sites, primarily to the bone, involves dissemination via the
bloodstream, entailing adhesion of cancer cells within the vasculature,
which depends on activated integrins. Integrin αvβ3 has been shown to
support PC cell attachment under blood flow conditions in an activa-
tion-dependent manner [94–96], underscoring its important role in the
development of bone metastases and presenting it as a therapeutic
target in advanced prostate cancer.
In an effort to investigate the therapeutic potential of VCN in pre-
clinical prostate cancer models, the Markland lab applied a liposomal
formulation of this disintegrin, called LVCN [55]. In general, liposomal
encapsulation can provide a variety of advantages, including prolonged
half-life and enhanced drug delivery due to increased tumor entrap-
ment. Based on some favorable structural attributes inherent in disin-
tegrin polypeptides, such as hydrophilicity and stability in solution at
low pH, in organic solvents, and at a range of temperatures, these
molecules can be readily encapsulated with high efficiency, while re-
taining full biological activity. In the case of VCN encapsulation to
create LVCN, efficiency was 70% or greater [55].
LVCN was studied in two different mouse models of prostate cancer
[55]. In the first, human PC-3 prostate cancer cells were subcutaneously
injected into the flank of nude mice. Different groups of mice were then
treated with LVCN, VCN, liposomes only, or with vehicle. LVCN exerted
profound inhibitory effects on tumor growth in this xenograft model. In
comparison, unencapsulated VCN displayed no quantifiable anti-tumor
activity, which may in part be explained by its short circulatory half-
life. In a second approach, it was investigated whether LVCN could treat
established prostate cancer bone metastases. In these studies, human
CWR22Rv1 prostate cancer cells were implanted into the tibia of mice,
followed by treatment as above. In this model as well, treatment of
animals with LVCN, but not with VCN, resulted in strong inhibition of
tumor growth. While these studies emphasized the antitumor activity of
LVCN, they also showed that these treatments were very well tolerated
by the animals and did not cause obvious toxicities [55].
The PC-3 prostate cancer mouse model was also used to evaluate the
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Biochemical Pharmacology 181 (2020) 114149
anti-angiogenic potential of LVCN in vivo. Nude mice carrying xeno-
grafted PC-3 cells were treated with LVCN or vehicle as described
above. After 5 weeks of treatment, tumor tissues were collected and
subjected to immunostaining with anti-CD31/PECAM-1 antibodies, re-
cognizing platelet endothelial cell adhesion molecule (PECAM-1) as a
marker of endothelial cells [97]. Computer-assisted quantitation of the
stained vessel areas was used to establish microvessel density (MVD) as
a surrogate marker for tumor angiogenesis [98]. Using this method, it
could be established that treatment with LVCN resulted in a pro-
nounced decrease in tumor associated MVD. Additional in vitro ex-
periments further confirmed VCN’s potent anti-angiogenic potency. For
example, using human umbilical cord vascular endothelial cells
(HUVEC) in a tubulogenesis assay [99], VCN was shown to inhibit tu-
bule formation by these endothelial cells, along with pronounced dis-
ruption of their actin cytoskeleton, at low nanomolar concentrations
[55]. In all, these experiments conclusively established the anti-angio-
genic potential of our recombinant disintegrin.
6. VCN in ovarian cancer
Advanced Ovarian Cancer (OC) is a devastating and invariably fatal
disease. High-grade serous ovarian cancer (HGSOC) is the prevalent
subtype and accounts for 70–80% of OC deaths. The overall 5-year
survival in OC is only 30%. The standard treatment is aggressive sur-
gery followed by adjuvant chemotherapy, but no current therapeutic
solution can effectively prevent disease recurrence [100–102].
Consistent molecular findings in HGSOC include the activation of
multiple components of the cellular invasion apparatus (i.e., invado-
some-associated integrins and their downstream kinases [103]) and the
presence of cancer stem cell-associated markers, which collectively
correlate with invasiveness, chemoresistance, and progression
[104–106]. The mode of OC dissemination is characteristic, but not
unique among solid tumors. Exfoliated OC cells from primary tumors
and already established peritoneal lesions assemble themselves into
free-floating multicellular aggregates (cancer spheroids) which are
further circulated by the peritoneal fluid to secondary sites of im-
plantation, where they attach, invade into the submesothelial con-
nective tissue, and form new metastases [107]. The spheroid cells are
significantly more invasive and resistant to chemotherapy [108]. OC
micrometastases usually evade detection at the time of surgery, and
failure to eradicate them drives disease recurrence.
Intravenously administered chemotherapy penetrates only poorly
into the peritoneal compartment where OC is actively spreading. As a
result, significantly higher drug concentrations would have to be ad-
ministered, but would reach levels that exceed maximum tolerated
doses (MTDs) of chemotherapy. One solution to this problem consists of
the administration of chemotherapy directly into the intraperitoneal
(IP) space, and in advanced OC a 22% decrease in the risk of death was
shown in patients who received combined IV/IP therapy versus IV
therapy alone [109]. In 2006, the NCI issued the statement: “On the
basis of the results of these randomized phase III clinical trials, a
combination of IV and IP administration of chemotherapy conveys a
significant survival benefit among women with optimally debulked
ovarian cancer, compared to IV administration alone” [110]. However,
when translating this recommendation into clinical practice over the
years that followed, the issue of tolerability became readily apparent
[110,111]. Due to the altered peritoneal fluid dynamics following de-
bulking surgery, drug clearance from the peritoneal cavity becomes not
only impeded but also more unpredictable than in normal subjects.
While it may result in prolonged exposure of residual tumors to much
higher drug concentrations, it comes at the expense of additional off-
target toxicities [112,113]. Therefore, although efficacious, the taxane-
and platinum-based IP regimens are in general poorly tolerated, which
is the main reason why a significant number of women opt out of IP
treatment despite its potential benefits [114]. Wright et al. [115] re-
ported that although the combined IP/IV chemotherapy use increased
A.H. Schönthal, et al.
significantly at many National Comprehensive Cancer Network centers
between 2003 and 2012, < 50% of eligible patients completed the IP
chemotherapy course.
The Markland lab studied potential benefits of VCN in preclinical
OC mouse models. For these studies, VCN was formulated using Oxiplex
as a delivery vehicle. Oxiplex is a hydrogel consisting of polyethylene
oxide and carboxymethyl cellulose stabilized by calcium chloride
[116]. In clinical applications, it is used as a barrier gel by placing it at
sites of tissue injury post-operatively, where it serves as a temporary
mechanical barrier separating opposing tissue surfaces to reduce the
extent of postoperative adhesions [116,117]. Adhesion prevention is
particularly relevant after gynecological surgery, which suggested the
option of formulating an anti-adhesive gel with VCN incorporated
uniformly throughout the Oxiplex gel for local delivery immediately
following OC surgery.
This idea was investigated by using nude mice implanted into their
peritoneum with multicellular spheroids of highly aggressive human
OC cells. The use of such spheroids, rather than individual cells, is
considered a physiologically more relevant model in the case of OC, in
particular because these three-dimensional clusters, which are also
present in patient-derived effusions [118], display increased radio- and
chemoresistance [119–121] and tend to harbor a more aggressive, in-
vasive phenotype [107]. Therefore, to investigate the impact of VCN in
a more clinically relevant mouse model, the tumor cells were grown as
spheroids in vitro before injection into the peritoneum [122]. As treat-
ment, the animals received VCN alone, VCN incorporated into Oxiplex
gel, or Oxiplex alone. VCN exerted pronounced anticancer activity in
this tumor model. Intriguingly, both formulations of VCN, either in
saline or mixed with Oxiplex, were highly effective, and quantitation of
bioluminescent imaging showed greater than 95% inhibition of tumor
spread. Upon dissection, control animals (without any VCN treatment)
revealed extensive and widespread macroscopic carcinomatosis
throughout the peritoneal cavity, while the VCN-treated groups showed
a significant decrease in the total number of macroscopic foci. In fact,
several of the VCN-treated animals were devoid of visible macroscopic
tumor foci by gross examination.
In vivo studies investigating VCN’s therapeutic activity were com-
plemented by toxicity studies in mice and rats. Both single-dose and
multi-dose applications were performed, and toxicity was evaluated by
a number of parameters, including signs of overt toxicity, weight loss,
or changes in physical activity of animals, microscopic and histo-
pathological analysis of organs, hematological parameters, and blood
chemistry panels [123]. None of these various assays yielded signs of
toxicological effects of VCN, even at repeat dosing as high as 500 mg/
kg. Therefore, as consistently observed throughout other in vivo ex-
periments [43,55], VCN appeared to be very well tolerated, setting a
promising stage for advancement to Phase I clinical trials.
131
7. VCN in Glioblastoma: I-VCN
Malignant glioma, particularly glioblastoma (GB, WHO grade IV
astrocytic glioma), accounts for over half of all gliomas, which in turn
account for 80% of all malignant brain and CNS tumors [124]. The
great majority of patients with GB are faced with a dismal prognosis.
Advances in neurosurgery, radiation, and chemotherapy during the past
decades have provided only small improvements in clinical outcome,
with 5-year survival remaining at about 10% [125,126]. The first-line
treatment of glioblastoma is usually surgery, both to confirm the di-
agnosis and to remove as much of the tumor as possible. Concurrent
temozolomide (TMZ) with radiotherapy, followed by adjuvant TMZ,
has produced a median survival of about 15 months, and this regimen is
now the standard of care for GB, as well as for grade III anaplastic
glioma [125,126]. An approved treatment for the recurrent setting is
the angiogenesis inhibitor bevacizumab [127]. Although it improves
outcome for most GB patients, the duration of benefits is limited and
survival after initial bevacizumab progression is poor [128,129]. The
6
Biochemical Pharmacology 181 (2020) 114149
latest therapy to be approved is the alternating electric fields generator
NovoTTF/Optune [130], which may extend median overall survival by
about 5 months [131]. Regardless of the treatment regimen, however,
GB patients continue to have dismal prognosis and novel treatments are
urgently needed.
In mouse models of GB, the utility of VCN as a brachytherapy agent
was investigated. It was shown that the sole tyrosine residue at position
51 of the VCN peptide was amenable to radiolabeling with full reten-
tion of disintegrin biological activity [132]. Consequently, the Mark-
land lab created 131I-VCN for evaluation of its potential use as a bra-
chytherapy agent. VCN was directly radiolabeled with radioiodine
(131I) using a modification of the chloramine T method [133] and
confirmation by mass spectrometry. The ensuing product was shown to
retain its ability to inhibit platelet aggregation with identical activity to
that of non-iodinated parental VCN [132]. Furthermore, fluorescence-
activated cell sorting (FACS) demonstrated avid binding of 131I-VCN to
glioblastoma cell lines overexpressing the expected target integrins, in
particular αvβ3 and αvβ5 integrins. After intravenous delivery of 131I-
VCN to nude mice with or without human GB cells implanted into their
brains, a significant portion of the injected material was found in the
brains of tumor-bearing mice, whereas radioactivity was below the
detectable threshold in the brains of mice without tumors.
Therapeutic activity of 131I-VCN was investigated in different in vivo
mouse GB models [132]. In one model, the brains of nude mice were
implanted with human U87 GB cells. Treatment with vehicle only, 131I
only, or 131I-VCN, established that only 131I-VCN treatment resulted in
significant (p < 0.04) survival benefit, whereas 131I alone did not
extend survival over that observed with vehicle treatment alone. This
encouraging result was extended in a nude mouse model with in-
tracranial human U251 GB cells. In this study, 131I-VCN confirmed its
therapeutic impact (p < 0.003 over untreated), and extended median
survival even further when combined with TMZ, the chemotherapeutic
standard of care for GB patients [134]. In an immunocompetent mouse
model, mouse brains were implanted with syngeneic murine GL216
glioma cells that had been transfected with a plasmid encoding the gene
for O6-methylguanine DNA methyltransferase (MGMT). MGMT is a
DNA repair enzyme that specifically corrects the cytotoxic O6-methyl-
guanine lesions produced by the alkylating activity of TMZ. As a result,
MGMT-overexpressing cells are profoundly resistant to killing by TMZ,
and this mechanism of resistance represents a well-recognized clinical
problem [135]. It was therefore of great interest to determine whether
131
I-VCN would be able to exert therapeutic activity in such a clinically
relevant resistance model. Mice harboring MGMT-overexpressing
glioma cells were treated with 131I-VCN or with 131I-VCN in combina-
tion with TMZ and were further compared to the clinical standard of
care, i.e., radiation combined with TMZ. All three regimens yielded
significant (p < 0.05) survival benefit over untreated control animals.
Intriguingly, the 131I-VCN plus TMZ combination performed the best
and was slightly better than radiation combined with TMZ. In ag-
gregate, these in vivo studies established very encouraging proof-of-
principle that VCN can be successfully used as a novel delivery me-
chanism to target a radioactive payload to tumor tissue.
8. Conclusions
The discovery of snake venom disintegrins has spawned a number of
studies to investigate their potential benefit for cancer therapeutic
purposes. Among the best-characterized peptides is VCN, a single chain
peptide genetically engineered based on the structure of native CN with
several modifications, in particular the replacement of the three COOH-
terminal amino acids of CN with the six amino acid sequence at the
COOH-terminus of echistatin to optimize VCN’s anticancer activity. The
complexity of the cancer microenvironment dictates that for optimal
efficacy an anti-integrin therapeutic must target at least two members
of the RGD-binding integrin class and preferably more given the ability
of cancer cells to change their integrin repertoire in response to drug
A.H. Schönthal, et al.
treatment [57]. VCN, which targets multiple integrin members in-
cluding (but not limited to) αvβ3, αvβ5, α5β1 and αIIbβ3, admirably
meets this criterion. VCN has demonstrated robust cancer-therapeutic
activity in a great variety of preclinical animal models, along with
undetectable toxicity, pointing to a fairly large therapeutic window. It
can be encapsulated in liposomes for enhanced tumor delivery, mixed
with surgical anti-adhesive gels, or chemically modified with radio-
isotopes for tumor-selective delivery of a toxic payload, all of which
present additional options for translational development and inclusion
in cancer therapeutic regimens. Among the key steps in the near future
is the submission of an investigational new drug (IND) application, to
enable phase I safety studies in cancer patients. The successful clinical
introduction and long-term market experience of two other disintegrin
derived agents, the antiplatelet compounds eptifibatide and tirofiban,
may serve as encouraging examples in this regard. It therefore appears
promising for VCN, and potentially other disintegrins currently un-
dergoing development, to advance to clinical testing in the near future.
CRediT authorship contribution statement
Axel H. Schonthal: Conceptualization, Writing - original draft,
Writing - review & editing. Stephen D. Swenson: Data curation,
Investigation, Methodology. Thomas C. Chen: Funding acquisition.
Francis S. Markland: Funding acquisition, Conceptualization, Project
administration, Supervision, Writing - original draft, Writing - review &
editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
We thank all current and former members of the Markland lab for
their valuable contributions to disintegrin research. Funding to support
research in the Markland lab has been provided by the National Cancer
Institute of the National Institutes of Health, USA, Grants R41
CA126001, R41 CA168228 and R41 CA165626, the Department of
Defense, US Army, USA, Grants PC030704 and OC060278, and the
Southern California Clinical and Translational Science Institute at the
University of Southern California, USA. The funding sources had no
influence on the assembly and writing of this manuscript. Animal re-
search performed in the Markland lab was in accordance with all ap-
plicable ethical standards and was reviewed and approved by the
Institutional Animal Care and Use Committee (IACUC) at the University
of Southern California.
References
[1] R.J. McCleary, R.M. Kini, Non-enzymatic proteins from snake venoms: a gold mine
of pharmacological tools and drug leads, Toxicon 62 (2013) 56–74.
[2] A. Munawar, S.A. Ali, A. Akrem, C. Betzel, Snake venom peptides: tools of bio-
discovery, Toxins (Basel) 10 (11) (2018) 474.
[3] P.G. Ojeda, D. Ramirez, J. Alzate-Morales, J. Caballero, Q. Kaas, W. Gonzalez,
Computational studies of snake venom toxins, Toxins (Basel) 10 (1) (2017) 8.
[4] T. Tasoulis, G.K. Isbister, A review and database of snake venom proteomes,
Toxins (Basel) 9 (9) (2017) 290.
[5] J.J. Calvete, The continuing saga of snake venom disintegrins, Toxicon 62 (2013)
40–49.
[6] K. Schror, A.A. Weber, Comparative pharmacology of GP IIb/IIIa antagonists, J.
Thromb. Thrombolysis 15 (2) (2003) 71–80.
[7] M. Hashemzadeh, M. Furukawa, S. Goldsberry, M.R. Movahed, Chemical struc-
tures and mode of action of intravenous glycoprotein IIb/IIIa receptor blockers: a
review, Exp. Clin. Cardiol. 13 (4) (2008) 192–197.
[8] R.M. Scarborough, J.W. Rose, M.A. Hsu, D.R. Phillips, V.A. Fried, A.M. Campbell,
L. Nannizzi, I.F. Charo, Barbourin. A GPIIb-IIIa-specific integrin antagonist from
the venom of Sistrurus m. barbouri, J. Biol. Chem. 266 (15) (1991) 9359–9362.
[9] E.J. Topol, T.V. Byzova, E.F. Plow, Platelet GPIIb-IIIa blockers, Lancet 353 (9148)
7
Biochemical Pharmacology 181 (2020) 114149
(1999) 227–231.
[10] Z.R. Gan, R.J. Gould, J.W. Jacobs, P.A. Friedman, M.A. Polokoff, Echistatin. A
potent platelet aggregation inhibitor from the venom of the viper, Echis carinatus,
J. Biol. Chem. 263 (36) (1988) 19827–19832.
[11] X. Zhou, X. Wu, H. Sun, J. Li, Efficacy and safety of eptifibatide versus tirofiban in
acute coronary syndrome patients: a systematic review and meta-analysis, J. Evid.
Based Med. 10 (2) (2017) 136–144.
[12] C.Y. Koh, R.M. Kini, From snake venom toxins to therapeutics–cardiovascular
examples, Toxicon 59 (4) (2012) 497–506.
[13] Y.S. Chan, R.C.F. Cheung, L. Xia, J.H. Wong, T.B. Ng, W.Y. Chan, Snake venom
toxins: toxicity and medicinal applications, Appl. Microbiol. Biotechnol. 100 (14)
(2016) 6165–6181.
[14] H. Waheed, S.F. Moin, M.I. Choudhary, Snake venom: from deadly toxins to life-
saving therapeutics, Curr. Med. Chem. 24 (17) (2017) 1874–1891.
[15] J. Cooper, F.G. Giancotti, Integrin signaling in cancer: mechanotransduction,
stemness, epithelial plasticity, and therapeutic resistance, Cancer Cell 35 (3)
(2019) 347–367.
[16] H. Hamidi, J. Ivaska, Every step of the way: integrins in cancer progression and
metastasis, Nat. Rev. Cancer 18 (9) (2018) 533–548.
[17] S. Raab-Westphal, J.F. Marshall, S.L. Goodman, Integrins as therapeutic targets:
successes and cancers, Cancers (Basel) 9 (9) (2017) 110.
[18] G. Sokeland, U. Schumacher, The functional role of integrins during intra- and
extravasation within the metastatic cascade, Mol. Cancer 18 (1) (2019) 12.
[19] X. Lu, D. Lu, M.F. Scully, V.V. Kakkar, Integrins in drug targeting-RGD templates
in toxins, Curr. Pharm. Des. 12 (22) (2006) 2749–2769.
[20] D.A. Cheresh, Structural and biologic properties of integrin-mediated cell adhe-
sion, Clin. Lab. Med. 12 (2) (1992) 217–236.
[21] E. Ruoslahti, M.D. Pierschbacher, New perspectives in cell adhesion: RGD and
integrins, Science 238 (4826) (1987) 491–497.
[22] M.D. Pierschbacher, E. Ruoslahti, Cell attachment activity of fibronectin can be
duplicated by small synthetic fragments of the molecule, Nature 309 (5963)
(1984) 30–33.
[23] Y.A. Kadry, D.A. Calderwood, Chapter 22: structural and signaling functions of
integrins, Biochim. Biophys. Acta (BBA) – Biomembr. 1862 (5) (2020) 183206.
[24] M. Nieberler, U. Reuning, F. Reichart, J. Notni, H.J. Wester, M. Schwaiger,
M. Weinmuller, A. Rader, K. Steiger, H. Kessler, Exploring the role of RGD-re-
cognizing integrins in cancer, Cancers (Basel) 9 (9) (2017) 116.
[25] R.O. Hynes, Integrins: bidirectional, allosteric signaling machines, Cell 110 (6)
(2002) 673–687.
[26] B. Geiger, J.P. Spatz, A.D. Bershadsky, Environmental sensing through focal ad-
hesions, Nat. Rev. Mol. Cell Biol. 10 (1) (2009) 21–33.
[27] S.M. Albelda, Role of integrins and other cell adhesion molecules in tumor pro-
gression and metastasis, Lab. Invest. 68 (1) (1993) 4–17.
[28] J.S. Desgrosellier, D.A. Cheresh, Integrins in cancer: biological implications and
therapeutic opportunities, Nat. Rev. Cancer 10 (1) (2010) 9–22.
[29] M.M. Zutter, S.A. Santoro, W.D. Staatz, Y.L. Tsung, Re-expression of the alpha 2
beta 1 integrin abrogates the malignant phenotype of breast carcinoma cells, Proc.
Natl. Acad. Sci. U.S.A. 92 (16) (1995) 7411–7415.
[30] M. Bachmann, S. Kukkurainen, V.P. Hytonen, B. Wehrle-Haller, Cell adhesion by
integrins, Physiol. Rev. 99 (4) (2019) 1655–1699.
[31] W. Wang, B.H. Luo, Structural basis of integrin transmembrane activation, J. Cell.
Biochem. 109 (3) (2010) 447–452.
[32] J.K. Arruda Macedo, J.W. Fox, M. de Souza Castro, Disintegrins from snake ve-
noms and their applications in cancer research and therapy, Curr. Protein Pept.
Sci. 16 (6) (2015) 532–548.
[33] J.J. Calvete, C. Marcinkiewicz, D. Monleon, V. Esteve, B. Celda, P. Juarez, L. Sanz,
Snake venom disintegrins: evolution of structure and function, Toxicon 45 (8)
(2005) 1063–1074.
[34] V. David, B.B. Succar, J.A. de Moraes, R.F.G. Saldanha-Gama, C. Barja-Fidalgo,
R.B. Zingali, Recombinant and chimeric disintegrins in preclinical research, Toxins
(Basel) 10 (8) (2018) 321.
[35] E.M. Walsh, C. Marcinkiewicz, Non-RGD-containing snake venom disintegrins,
functional and structural relations, Toxicon 58 (4) (2011) 355–362.
[36] J.J. Calvete, J.W. Fox, A. Agelan, S. Niewiarowski, C. Marcinkiewicz, The presence
of the WGD motif in CC8 heterodimeric disintegrin increases its inhibitory effect
on alphaII(b)beta3, alpha(v)beta3, and alpha5beta1 integrins, Biochemistry 41 (6)
(2002) 2014–2021.
[37] J.J. Calvete, M.P. Moreno-Murciano, R.D. Theakston, D.G. Kisiel,
C. Marcinkiewicz, Snake venom disintegrins: novel dimeric disintegrins and
structural diversification by disulphide bond engineering, Biochem. J. 372 (Pt 3)
(2003) 725–734.
[38] J.P. Xiong, T. Stehle, R. Zhang, A. Joachimiak, M. Frech, S.L. Goodman,
M.A. Arnaout, Crystal structure of the extracellular segment of integrin alpha
Vbeta3 in complex with an Arg-Gly-Asp ligand, Science 296 (5565) (2002)
151–155.
[39] I. Wierzbicka-Patynowski, S. Niewiarowski, C. Marcinkiewicz, J.J. Calvete,
M.M. Marcinkiewicz, M.A. McLane, Structural requirements of echistatin for the
recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins, J. Biol. Chem. 274
(53) (1999) 37809–37814.
[40] T.F. Huang, J.C. Holt, H. Lukasiewicz, S. Niewiarowski, Trigramin. A low mole-
cular weight peptide inhibiting fibrinogen interaction with platelet receptors ex-
pressed on glycoprotein IIb-IIIa complex, J. Biol. Chem. 262 (33) (1987)
16157–16163.
[41] M. Trikha, Y.A. De Clerck, F.S. Markland, Contortrostatin, a snake venom disin-
tegrin, inhibits beta 1 integrin-mediated human metastatic melanoma cell adhe-
sion and blocks experimental metastasis, Cancer Res. 54 (18) (1994) 4993–4998.
A.H. Schönthal, et al.
[42] M. Trikha, W.E. Rote, P.J. Manley, B.R. Lucchesi, F.S. Markland, Purification and
characterization of platelet aggregation inhibitors from snake venoms, Thromb.
Res. 73 (1) (1994) 39–52.
[43] R. Minea, C. Helchowski, B. Rubino, K. Brodmann, S. Swenson, F. Markland Jr.,
Development of a chimeric recombinant disintegrin as a cost-effective anti-cancer
agent with promising translational potential, Toxicon 59 (4) (2012) 472–486.
[44] B. Mercer, F. Markland, C. Minkin, Contortrostatin, a homodimeric snake venom
disintegrin, is a potent inhibitor of osteoclast attachment, J. Bone Miner. Res. 13
(3) (1998) 409–414.
[45] Q. Zhou, P. Hu, M.R. Ritter, S.D. Swenson, S. Argounova, A.L. Epstein,
F.S. Markland, Molecular cloning and functional expression of contortrostatin, a
homodimeric disintegrin from southern copperhead snake venom, Arch. Biochem.
Biophys. 375 (2) (2000) 278–288.
[46] Q. Zhou, M.T. Nakada, P.C. Brooks, S.D. Swenson, M.R. Ritter, S. Argounova,
C. Arnold, F.S. Markland, Contortrostatin, a homodimeric disintegrin, binds to
integrin alphavbeta5, Biochem. Biophys. Res. Commun. 267 (1) (2000) 350–355.
[47] V. Golubkov, D. Hawes, F.S. Markland, Anti-angiogenic activity of contortrostatin,
a disintegrin from Agkistrodon contortrix contortrix snake venom, Angiogenesis 6
(3) (2003) 213–224.
[48] Q. Zhou, M.T. Nakada, C. Arnold, K.Y. Shieh, F.S. Markland Jr., Contortrostatin, a
dimeric disintegrin from Agkistrodon contortrix contortrix, inhibits angiogenesis,
Angiogenesis 3 (3) (1999) 259–269.
[49] Q. Zhou, R.P. Sherwin, C. Parrish, V. Richters, S.G. Groshen, D. Tsao-Wei,
F.S. Markland, Contortrostatin, a dimeric disintegrin from Agkistrodon contortrix
contortrix, inhibits breast cancer progression, Breast Cancer Res. Treat. 61 (3)
(2000) 249–260.
[50] S. Swenson, S. Ramu, F.S. Markland, Anti-angiogenesis and RGD-containing snake
venom disintegrins, Curr. Pharm. Des. 13 (28) (2007) 2860–2871.
[51] G. Mezo, F. Hudecz, Synthesis of linear, branched, and cyclic peptide chimera,
Methods Mol. Biol. 298 (2005) 63–76.
[52] M.A. McLane, M.A. Kuchar, C. Brando, D. Santoli, C.A. Paquette-Straub,
M.E. Miele, New insights on disintegrin-receptor interactions: eristostatin and
melanoma cells, Haemostasis 31 (3–6) (2001) 177–182.
[53] J.A. Eble, J. Haier, Integrins in cancer treatment, Curr. Cancer Drug Targets 6 (2)
(2006) 89–105.
[54] S. Katsamakas, T. Chatzisideri, S. Thysiadis, V. Sarli, RGD-mediated delivery of
small-molecule drugs, Future Med. Chem. 9 (6) (2017) 579–604.
[55] R.O. Minea, C.M. Helchowski, S.J. Zidovetzki, F.K. Costa, S.D. Swenson,
F.S. Markland Jr., Vicrostatin - an anti-invasive multi-integrin targeting chimeric
disintegrin with tumor anti-angiogenic and pro-apoptotic activities, PLoS ONE 5
(6) (2010) e10929.
[56] S.M. Janib, J.A. Gustafson, R.O. Minea, S.D. Swenson, S. Liu, M.K. Pastuszka,
L.L. Lock, H. Cui, F.S. Markland, P.S. Conti, Z. Li, J.A. MacKay, Multimeric dis-
integrin protein polymer fusions that target tumor vasculature,
Biomacromolecules 15 (7) (2014) 2347–2358.
[57] H.M. Sheldrake, L.H. Patterson, Strategies to inhibit tumor associated integrin
receptors: rationale for dual and multi-antagonists, J. Med. Chem. 57 (2014)
6301–6315.
[58] F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global
cancer statistics 2018: GLOBOCAN estimates of incidence and mortality world-
wide for 36 cancers in 185 countries, CA Cancer J. Clin. 68 (6) (2018) 394–424.
[59] K. Barzaman, J. Karami, Z. Zarei, A. Hosseinzadeh, M.H. Kazemi, S. Moradi-
Kalbolandi, E. Safari, L. Farahmand, Breast cancer: biology, biomarkers, and
treatments, Int. Immunopharmacol. 84 (2020) 106535.
[60] U. Testa, G. Castelli, E. Pelosi, Breast cancer: a molecularly heterogenous disease
needing subtype-specific treatments, Med Sci (Basel) 8 (1) (2020) 18.
[61] S. Al-Mahmood, J. Sapiezynski, O.B. Garbuzenko, T. Minko, Metastatic and triple-
negative breast cancer: challenges and treatment options, Drug Deliv. Transl. Res.
8 (5) (2018) 1483–1507.
[62] J.J. Chan, T.J.Y. Tan, R.A. Dent, Novel therapeutic avenues in triple-negative
breast cancer: PI3K/AKT inhibition, androgen receptor blockade, and beyond,
Ther. Adv. Med. Oncol. 11 (2019) 1758835919880429.
[63] W. Janni, A. Schneeweiss, V. Muller, A. Wockel, M.P. Lux, A.D. Hartkopf,
N. Nabieva, F.A. Taran, H. Tesch, F. Overkamp, D. Luftner, E. Belleville, F. Schutz,
P.A. Fasching, T.N. Fehm, H.C. Kolberg, J. Ettl, Update breast cancer 2019 Part 2 -
implementation of novel diagnostics and therapeutics in advanced breast cancer
patients in clinical practice, Geburtshilfe Frauenheilkd 79 (3) (2019) 268–280.
[64] E. Mezni C. Vicier M. Guerin R. Sabatier F. Bertucci A. Goncalves New therapeutics
in HER2-positive advanced breast cancer: towards a change in clinical practices?
Cancers (Basel) 12(6) (2020) E1573.
[65] American Cancer Society. Cancer Facts & Figures 2020, American Cancer Society
(2020).
[66] F.S. Markland, K. Shieh, Q. Zhou, V. Golubkov, R.P. Sherwin, V. Richters,
R. Sposto, A novel snake venom disintegrin that inhibits human ovarian cancer
dissemination and angiogenesis in an orthotopic nude mouse model, Haemostasis
31 (3–6) (2001) 183–191.
[67] S. Swenson, F. Costa, R. Minea, R.P. Sherwin, W. Ernst, G. Fujii, D. Yang,
F.S. Markland Jr., Intravenous liposomal delivery of the snake venom disintegrin
contortrostatin limits breast cancer progression, Mol. Cancer Ther. 3 (4) (2004)
499–511.
[68] T.F. Huang, C.H. Yeh, W.B. Wu, Viper venom components affecting angiogenesis,
Haemostasis 31 (3–6) (2001) 192–206.
[69] I.C. Kang, Y.D. Lee, D.S. Kim, A novel disintegrin salmosin inhibits tumor angio-
genesis, Cancer Res. 59 (15) (1999) 3754–3760.
[70] K.Z. Olfa, L. Jose, D. Salma, B. Amine, S.A. Najet, A. Nicolas, L. Maxime,
Z. Raoudha, M. Kamel, M. Jacques, S. Jean-Marc, A.A.Lebestatin, a disintegrin
8
Biochemical Pharmacology 181 (2020) 114149
from Macrovipera venom, inhibits integrin-mediated cell adhesion, migration and
angiogenesis, Lab. Invest. 85 (12) (2005) 1507–1516.
[71] O.H. Ramos, A. Kauskot, M.R. Cominetti, I. Bechyne, C.L. Salla Pontes,
F. Chareyre, J. Manent, R. Vassy, M. Giovannini, C. Legrand, H.S. Selistre-de-
Araujo, M. Crepin, A. Bonnefoy, A novel alpha(v)beta (3)-blocking disintegrin
containing the RGD motive, DisBa-01, inhibits bFGF-induced angiogenesis and
melanoma metastasis, Clin. Exp. Metastasis 25 (1) (2008) 53–64.
[72] Z. Deng, J. Chai, Q. Zeng, B. Zhang, T. Ye, X. Chen, X. Xu, The anticancer prop-
erties and mechanism of action of tablysin-15, the RGD-containing disintegrin, in
breast cancer cells, Int. J. Biol. Macromol. 129 (2019) 1155–1167.
[73] R.S. Yang, C.H. Tang, W.J. Chuang, T.H. Huang, H.C. Peng, T.F. Huang, W.M. Fu,
Inhibition of tumor formation by snake venom disintegrin, Toxicon 45 (5) (2005)
661–669.
[74] S.E. Lucena, Y. Jia, J.G. Soto, J. Parral, E. Cantu, J. Brannon, K. Lardner,
C.J. Ramos, A.I. Seoane, E.E. Sanchez, Anti-invasive and anti-adhesive activities of
a recombinant disintegrin, r-viridistatin 2, derived from the Prairie rattlesnake
(Crotalus viridis viridis), Toxicon 60 (1) (2012) 31–39.
[75] L. Montealegre-Sanchez, S.N.C. Gimenes, D.S. Lopes, S.C. Teixeira, L. Solano-
Redondo, V. de Melo Rodrigues, E. Jimenez-Charris, Antitumoral potential of
lansbermin-i, a novel disintegrin from porthidium lansbergii lansbergii venom on
breast cancer cells, Curr. Top. Med. Chem. 19 (22) (2019) 2069–2078.
[76] T.R. Rebbeck, G.P. Haas, Temporal trends and racial disparities in global prostate
cancer prevalence, Can. J. Urol. 21 (5) (2014) 7496–7506.
[77] K. Mori, N. Miura, H. Mostafaei, F. Quhal, R. Sari Motlagh, B. Pradere, S. Kimura,
T. Kimura, S. Egawa, A. Briganti, P.I. Karakiewicz, S.F. Shariat, Sequential therapy
of abiraterone and enzalutamide in castration-resistant prostate cancer: a sys-
tematic review and meta-analysis, Prostate Cancer Prostatic Dis. (2020) March 9
[online ahead of print].
[78] X. Zheng, X. Zhao, H. Xu, X. Han, H. Xu, X. Dong, R. Peng, L. Yang, Q. Wei, J. Ai,
Efficacy and safety of abiraterone and enzalutamide for castration-resistant pros-
tate cancer: a systematic review and meta-analysis of randomized controlled trials,
Medicine 98 (44) (2019) e17748.
[79] M.J. Ferris, Y. Liu, J. Ao, J. Zhong, M. Abugideiri, T.W. Gillespie, B.C. Carthon,
M.A. Bilen, O. Kucuk, A.B. Jani, The addition of chemotherapy in the definitive
management of high risk prostate cancer, Urol. Oncol. 36 (11) (2018) 475–487.
[80] M.C. Cursano, M. Iuliani, C. Casadei, M. Stellato, G. Tonini, G. Paganelli,
D. Santini, U. De Giorgi, Combination radium-223 therapies in patients with bone
metastases from castration-resistant prostate cancer: a review, Crit. Rev. Oncol.
Hematol. 146 (2020) 102864.
[81] Z. Melegh, S. Oltean, Targeting angiogenesis in prostate cancer, Int. J. Mol. Sci. 20
(11) (2019) 2676.
[82] M. Carceles-Cordon, W.K. Kelly, L. Gomella, K.E. Knudsen, V. Rodriguez-Bravo,
J. Domingo-Domenech, Cellular rewiring in lethal prostate cancer: the architect of
drug resistance, Nat. Rev. Urol. 17 (5) (2020) 292–307.
[83] A.K. Miyahira, R.B. Den, M.I. Carlo, R. de Leeuw, T.A. Hope, F. Karzai,
R.R. McKay, S.S. Salami, J.W. Simons, K.J. Pienta, H.R. Soule, Tumor cell het-
erogeneity and resistance; report from the 2018 Coffey-Holden Prostate Cancer
Academy Meeting, Prostate 79 (3) (2019) 244–258.
[84] P. Grabham, P. Sharma, The effects of radiation on angiogenesis, Vasc. Cell 5 (1)
(2013) 19.
[85] A. Abdollahi, D.W. Griggs, H. Zieher, A. Roth, K.E. Lipson, R. Saffrich, H.J. Grone,
D.E. Hallahan, R.A. Reisfeld, J. Debus, A.G. Niethammer, P.E. Huber, Inhibition of
alpha(v)beta3 integrin survival signaling enhances antiangiogenic and antitumor
effects of radiotherapy, Clin. Cancer Res. 11 (17) (2005) 6270–6279.
[86] N.P. McCabe, S. De, A. Vasanji, J. Brainard, T.V. Byzova, Prostate cancer specific
integrin alphavbeta3 modulates bone metastatic growth and tissue remodeling,
Oncogene 26 (42) (2007) 6238–6243.
[87] S. Stucci, M. Tucci, A. Passarelli, F. Silvestris, Avbeta3 integrin: pathogenetic role
in osteotropic tumors, Crit. Rev. Oncol. Hematol. 96 (1) (2015) 183–193.
[88] R. San Martin, R. Pathak, A. Jain, S.Y. Jung, S.G. Hilsenbeck, M.C. Pina-Barba,
A.G. Sikora, K.J. Pienta, D.R. Rowley, Tenascin-C and integrin alpha9 mediate
interactions of prostate cancer with the bone microenvironment, Cancer Res. 77
(21) (2017) 5977–5988.
[89] Z. Zhao, E. Li, L. Luo, S. Zhao, L. Liu, J. Wang, R. Kang, J. Luo, A PSCA/PGRN-NF-
kappaB-integrin-alpha4 axis promotes prostate cancer cell Adhesion to bone
marrow endothelium and enhances metastatic potential, Mol. Cancer Res. 18 (3)
(2020) 501–513.
[90] S. Ziaee, L.W. Chung, Induction of integrin alpha2 in a highly bone metastatic
human prostate cancer cell line: roles of RANKL and AR under three-dimensional
suspension culture, Mol. Cancer 13 (2014) 208.
[91] H.L. Goel, J. Li, S. Kogan, L.R. Languino, Integrins in prostate cancer progression,
Endocr. Relat. Cancer 15 (3) (2008) 657–664.
[92] M.C. Juan-Rivera, M. Martinez-Ferrer, Integrin inhibitors in prostate cancer,
Cancers (Basel) 10 (2) (2018) 44.
[93] K.A. Kwakwa, J.A. Sterling, Integrin alphavbeta3 signaling in tumor-induced bone
disease, Cancers (Basel) 9 (7) (2017) 84.
[94] J.A. Nemeth, M.L. Cher, Z. Zhou, C. Mullins, S. Bhagat, M. Trikha, Inhibition of
alpha(v)beta3 integrin reduces angiogenesis, bone turnover, and tumor cell pro-
liferation in experimental prostate cancer bone metastases, Clin. Exp. Metastasis
20 (5) (2003) 413–420.
[95] M. Trikha, E. Raso, Y. Cai, Z. Fazakas, S. Paku, A.T. Porter, J. Timar, K.V. Honn,
Role of alphaII(b)beta3 integrin in prostate cancer metastasis, Prostate 35 (3)
(1998) 185–192.
[96] D.Q. Zheng, A.S. Woodard, M. Fornaro, G. Tallini, L.R. Languino, Prostatic car-
cinoma cell migration via alpha(v)beta3 integrin is modulated by a focal adhesion
kinase pathway, Cancer Res. 59 (7) (1999) 1655–1664.
A.H. Schönthal, et al.
[97] H.M. DeLisser, P.J. Newman, S.M. Albelda, Platelet endothelial cell adhesion
molecule (CD31), Curr. Top. Microbiol. Immunol. 184 (1993) 37–45.
[98] Y. Miyata, H. Sakai, Reconsideration of the clinical and histopathological sig-
nificance of angiogenesis in prostate cancer: usefulness and limitations of micro-
vessel density measurement, Int. J. Urol. 22 (9) (2015) 806–815.
[99] K.L. DeCicco-Skinner, G.H. Henry, C. Cataisson, T. Tabib, J.C. Gwilliam,
N.J. Watson, E.M. Bullwinkle, L. Falkenburg, R.C. O’Nneill, A. Morin, J.S. Wiest,
Endothelial cell tube formation assay for the in vitro study of angiogenesis, J. Vis.
Exp. 91 (2014) e51312.
[100] D.D. Bowtell, S. Bohm, A.A. Ahmed, P.J. Aspuria, R.C. Bast Jr., V. Beral, J.S. Berek,
M.J. Birrer, S. Blagden, M.A. Bookman, J.D. Brenton, K.B. Chiappinelli,
F.C. Martins, G. Coukos, R. Drapkin, R. Edmondson, C. Fotopoulou, H. Gabra,
J. Galon, C. Gourley, V. Heong, D.G. Huntsman, M. Iwanicki, B.Y. Karlan, A. Kaye,
E. Lengyel, D.A. Levine, K.H. Lu, I.A. McNeish, U. Menon, S.A. Narod, B.H. Nelson,
K.P. Nephew, P. Pharoah, D.J. Powell Jr., P. Ramos, I.L. Romero, C.L. Scott,
A.K. Sood, E.A. Stronach, F.R. Balkwill, Rethinking ovarian cancer II: reducing
mortality from high-grade serous ovarian cancer, Nat. Rev. Cancer 15 (11) (2015)
668–679.
[101] Y.L. Liu, Q. Zhou, A. Iasonos, V.N. Emengo, C. Friedman, J.A. Konner,
R.E. O'Cearbhaill, C. Aghajanian, D. Zamarin, Subsequent therapies and survival
after immunotherapy in recurrent ovarian cancer, Gynecol. Oncol. 155 (1) (2019)
51–57.
[102] T. May, R. Comeau, P. Sun, J. Kotsopoulos, S.A. Narod, B. Rosen, P. Ghatage, A
comparison of survival outcomes in advanced serous ovarian cancer patients
treated with primary debulking surgery versus neoadjuvant chemotherapy, Int. J.
Gynecol. Cancer 27 (4) (2017) 668–674.
[103] R. Pelaez, A. Pariente, A. Perez-Sala, I.M. Larrayoz, Integrins: moonlighting pro-
teins in invadosome formation, Cancers (Basel) 11 (5) (2019) 615.
[104] J. Hatina, M. Boesch, S. Sopper, M. Kripnerova, D. Wolf, D. Reimer, C. Marth,
A.G. Zeimet, Ovarian cancer stem cell heterogeneity, Adv. Exp. Med. Biol. 1139
(2019) 201–221.
[105] F. Tomao, A. Papa, M. Strudel, L. Rossi, G. Lo Russo, P. Benedetti Panici,
F.R. Ciabatta, S. Tomao, Investigating molecular profiles of ovarian cancer: an
update on cancer stem cells, J Cancer 5 (5) (2014) 301–310.
[106] M. Varas-Godoy, G. Rice, S.E. Illanes, The crosstalk between ovarian cancer stem
cell niche and the tumor microenvironment, Stem Cells Int 2017 (2017) 5263974.
[107] K.L. Sodek, M.J. Ringuette, T.J. Brown, Compact spheroid formation by ovarian
cancer cells is associated with contractile behavior and an invasive phenotype, Int.
J. Cancer 124 (9) (2009) 2060–2070.
[108] K. Shield, M.L. Ackland, N. Ahmed, G.E. Rice, Multicellular spheroids in ovarian
cancer metastases: biology and pathology, Gynecol. Oncol. 113 (1) (2009)
143–148.
[109] K. Jaaback, N. Johnson, T.A. Lawrie, Intraperitoneal chemotherapy for the initial
management of primary epithelial ovarian cancer, Cochrane Database Syst. Rev.
11 (2011) CD005340.
[110] A.G. Zeimet, D. Reimer, A.C. Radl, A. Reinthaller, C. Schauer, E. Petru, N. Concin,
S. Braun, C. Marth, Pros and cons of intraperitoneal chemotherapy in the treat-
ment of epithelial ovarian cancer, Anticancer Res. 29 (7) (2009) 2803–2808.
[111] M. Kwa, S. Edwards, A. Downey, E. Reich, R. Wallach, J. Curtin, F. Muggia,
Ovarian cancer in BRCA mutation carriers: improved outcome after in-
traperitoneal (IP) cisplatin, Ann. Surg. Oncol. 21 (5) (2014) 1468–1473.
[112] M. Kwa, F. Muggia, Ovarian cancer: a brief historical overview of intraperitoneal
trials, Ann. Surg. Oncol. 21 (5) (2014) 1429–1434.
[113] C. Marchetti, F. De Felice, G. Perniola, I. Palaia, A. Musella, V. Di Donato,
G. Cascialli, L. Muzii, V. Tombolini, P. Benedetti, Panici, Role of intraperitoneal
chemotherapy in ovarian cancer in the platinum-taxane-based era: a meta-ana-
lysis, Crit. Rev. Oncol. Hematol. 136 (2019) 64–69.
[114] M. Markman, J.L. Walker, Intraperitoneal chemotherapy of ovarian cancer: a re-
view, with a focus on practical aspects of treatment, J. Clin. Oncol. 24 (6) (2006)
988–994.
[115] A.A. Wright, A. Cronin, D.E. Milne, M.A. Bookman, R.A. Burger, D.E. Cohn,
M.C. Cristea, J.J. Griggs, N.L. Keating, C.F. Levenback, G. Mantia-Smaldone,
U.A. Matulonis, L.A. Meyer, J.C. Niland, J.C. Weeks, D.M. O'Malley, Use and ef-
fectiveness of intraperitoneal chemotherapy for treatment of ovarian cancer, J.
Clin. Oncol. 33 (26) (2015) 2841–2847.
[116] M. Metwally, Y. Cheong, T.C. Li, A review of techniques for adhesion prevention
after gynaecological surgery, Curr. Opin. Obstet. Gynecol. 20 (4) (2008) 345–352.
[117] S. Farag, P.F. Padilla, K.A. Smith, M.L. Sprague, S.E. Zimberg, Management, pre-
vention, and sequelae of adhesions in women undergoing laparoscopic gyneco-
logic surgery: a systematic review, J. Minim. Invasive Gynecol. 25 (7) (2018)
Biochemical Pharmacology 181 (2020) 114149
1194–1216.
[118] H.J. Allen, C. Porter, M. Gamarra, M.S. Piver, E.A. Johnson, Isolation and mor-
phologic characterization of human ovarian carcinoma cell clusters present in
effusions, Exp Cell Biol 55 (4) (1987) 194–208.
[119] M. Bardies, P. Thedrez, J.F. Gestin, B.M. Marcille, D. Guerreau, A. Faivre-Chauvet,
M. Mahe, C. Sai-Maurel, J.F. Chatal, Use of multi-cell spheroids of ovarian carci-
noma as an intraperitoneal radio-immunotherapy model: uptake, retention ki-
netics and dosimetric evaluation, Int. J. Cancer 50 (6) (1992) 984–991.
[120] J.L. Becker, T.L. Prewett, G.F. Spaulding, T.J. Goodwin, Three-dimensional growth
and differentiation of ovarian tumor cell line in high aspect rotating-wall vessel:
morphologic and embryologic considerations, J. Cell. Biochem. 51 (3) (1993)
283–289.
[121] I.V. Filippovich, N.I. Sorokina, N. Robillard, J.F. Chatal, Radiation-induced
apoptosis in human ovarian carcinoma cells growing as a monolayer and as
multicell spheroids, Int. J. Cancer 72 (5) (1997) 851–859.
[122] S.D. Swenson, C. Silva-Hirschberg, F.S. Markland, Methods for evaluation of a
snake venom-derived disintegrin in animal models of human cancer, Methods Mol.
Biol. 2068 (2020) 185–204.
[123] S. Swenson, R. Minea, S. Zidovetzki, C. Helchowski, F. Costa, F.S. Markland, Anti-
angiogenesis and disintegrins, in: R.M. Kini, K.J. Clemetson, F.S. Markland,
M.A. McLane, T. Morita (Eds.), Toxins and Hemostasis, Springer, New York, 2010,
pp. 301–330.
[124] Q.T. Ostrom, H. Gittleman, P. Farah, A. Ondracek, Y. Chen, Y. Wolinsky,
N.E. Stroup, C. Kruchko, J.S. Barnholtz-Sloan, CBTRUS statistical report: primary
brain and central nervous system tumors diagnosed in the United States in
2006–2010, Neuro Oncol. 15 (suppl. 2) (2013) ii1-56.
[125] R. Stupp, M.E. Hegi, W.P. Mason, M.J. van den Bent, M.J. Taphoorn, R.C. Janzer,
S.K. Ludwin, A. Allgeier, B. Fisher, K. Belanger, P. Hau, A.A. Brandes,
J. Gijtenbeek, C. Marosi, C.J. Vecht, K. Mokhtari, P. Wesseling, S. Villa,
E. Eisenhauer, T. Gorlia, M. Weller, D. Lacombe, J.G. Cairncross, R.O. Mirimanoff,
Effects of radiotherapy with concomitant and adjuvant temozolomide versus
radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-
year analysis of the EORTC-NCIC trial, Lancet Oncol. 10 (5) (2009) 459–466.
[126] R. Stupp, W.P. Mason, M.J. van den Bent, M. Weller, B. Fisher, M.J. Taphoorn,
K. Belanger, A.A. Brandes, C. Marosi, U. Bogdahn, J. Curschmann, R.C. Janzer,
S.K. Ludwin, T. Gorlia, A. Allgeier, D. Lacombe, J.G. Cairncross, E. Eisenhauer,
R.O. Mirimanoff, Radiotherapy plus concomitant and adjuvant temozolomide for
glioblastoma, N. Engl. J. Med. 352 (10) (2005) 987–996.
[127] Y. Narita, Bevacizumab for glioblastoma, Ther. Clin. Risk Manage. 11 (2015)
1759–1765.
[128] D.A. Abrams, J.A. Hanson, J.M. Brown, F.P. Hsu, J.B. Delashaw Jr., D.A. Bota,
Timing of surgery and bevacizumab therapy in neurosurgical patients with re-
current high grade glioma, J. Clin. Neurosci. 22 (1) (2015) 35–39.
[129] D.A. Reardon, J.E. Herndon 2nd, K.B. Peters, A. Desjardins, A. Coan, E. Lou,
A.L. Sumrall, S. Turner, E.S. Lipp, S. Sathornsumetee, J.N. Rich, J.H. Sampson,
A.H. Friedman, S.T. Boulton, D.D. Bigner, H.S. Friedman, J.J. Vredenburgh,
Bevacizumab continuation beyond initial bevacizumab progression among re-
current glioblastoma patients, Br. J. Cancer 107 (9) (2012) 1481–1487.
[130] K.D. Swanson, E. Lok, E.T. Wong, An overview of alternating electric fields
therapy (NovoTTF therapy) for the treatment of malignant glioma, Curr. Neurol.
Neurosci. Rep. 16 (1) (2016) 8.
[131] R. Stupp, S. Taillibert, A.A. Kanner, S. Kesari, D.M. Steinberg, S.A. Toms,
L.P. Taylor, F. Lieberman, A. Silvani, K.L. Fink, G.H. Barnett, J.J. Zhu,
J.W. Henson, H.H. Engelhard, T.C. Chen, D.D. Tran, J. Sroubek, N.D. Tran,
A.F. Hottinger, J. Landolfi, R. Desai, M. Caroli, Y. Kew, J. Honnorat, A. Idbaih,
E.D. Kirson, U. Weinberg, Y. Palti, M.E. Hegi, Z. Ram, Maintenance therapy with
tumor-treating fields plus temozolomide vs temozolomide alone for glioblastoma:
a randomized clinical trial, JAMA 314 (23) (2015) 2535–2543.
[132] S. Swenson, R.O. Minea, C.D. Tuan, T.Z. Thein, T.C. Chen, F.S. Markland, A novel
venom-derived peptide for brachytherapy of glioblastoma: preclinical studies in
mice, Molecules 23 (11) (2018) 2918.
[133] F.C. Greenwood, W.M. Hunter, J.S. Glover, The preparation of I-131-labelled
human growth hormone of high specific radioactivity, Biochem. J. 89 (1963)
114–123.
[134] R. Stupp, M.J. van den Bent, M.E. Hegi, Optimal role of temozolomide in the
treatment of malignant gliomas, Curr. Neurol. Neurosci. Rep. 5 (3) (2005)
198–206.
[135] S.Y. Lee, Temozolomide resistance in glioblastoma multiforme, Genes Dis. 3 (3)
(2016) 198–210.