DOI:http://dx.doi.org/10.7314/APJCP.2012.13.10.4855
Snake Venom as an Anticancer Agent
4855
Asian
Pacific Journal of Cancer Prevention, Vol 13, 2012
MINI-REVIEW
Snake Venom: A Potent Anticancer Agent
Deepika Jain, Sudhir Kumar*
IGNOU-I2IT
Centre of Excellence for Advanced Education and Research, Pune, Maharashtra,
India *For correspondence: sudhirk@isquareit.ac.in
Deepika Jain and
Sudhir Kumar
Asian
Pacific Journal of Cancer Prevention, Vol 13, 2012
Abstract
Since cancer is one
of the leading causes of death worldwide, and there is an urgent need to find
better treatment. In recent years remarkable progress has been made towards the
understanding of proposed hallmarks of cancer development and treatment.
Treatment modalities comprise radiation therapy, surgery, chemotherapy,
immunotherapy and hormonal therapy. Currently, the use of chemotherapeutics
remains the predominant option for clinical control. However, one of the major
problems with successful cancer therapy using chemotherapeutics is that
patients often do not respond or eventually develop resistance after initial
treatment. This has led to the increased use of anticancer drugs developed from
natural resources. The biodiversity of venoms and toxins makes them a unique
source from which novel therapeutics may be developed. In this review, the
anticancer potential of snake venom is discussed. Some of the included molecules
are under clinical trial and may find application for anticancer drug
development in the near future.
Keywords: Radiation
therapy - chemotherapy - immunotherapy - hormonal therapy - venoms- toxins
Asian
Pacific J Cancer Prev, 13 (10),
4855-4860
Introduction
Cancer is the major public burden in
all developed and developing countries. A total of 1,638,910 new cancer cases
and 577,190 deaths from cancer are projected to occur in year 2012 (Siegel et
al., 2012). Currently, 1 in 4 deaths in U.S. is due to cancer. It’s a
multi-genic and multi-cellular disease that can arise from all cell types and
organs with a multi-factorial etiology (Baskar et al., 2012). In all types of
cancer, genetic alterations give rise to changes in expression, activation or localization
of regulatory proteins in the cells, affecting the signaling pathways that
alter their response to regulatory stimuli and allow the unrestricted cell
growth.
Since cancer is the leading cause of
death worldwide, there is an urgent need of finding a better way to treat it.
Various therapies have been used for treating cancer such as chemotherapy,
radiotherapy, immunotherapy and gene therapy (Baskar et al., 2012).
Out of the therapies being used for
treatment, chemotherapy remains the predominant option. One of the main
obstacle in chemotherapy is that patients eventually gets resistant after some
time (Lai et al., 2012). Radiotherapy/radiation therapy being an important part
of cancer treatment, contributes to almost 40% of curative/successful treatment
for cancer. Its main aim is to decline the multiplication potential of cancer
cells (Baskar et al., 2012). But challenge in using radiotherapy for cancer
treatment is to increase/maximize effect of radiation doses on cancer cells,
while minimizing its effect on surrounding normal cells. Since there are
several cases documenting either acute, or late radiation toxicity, therefore,
it limits the usage of radiation therapy (Barnett et al., 2009).
Immunotherapy for cancer treatment has
become a more promising approach in the past decades (Kruger et al., 2007). It
is used in the early stage of the tumor development (Geissler and Weth, 2002).
Immune targets don’t play a significant role in the life or death of the cancer
cells since they serve only to direct immune effectors to the tumor cells
(Orentas et al., 2012). It mainly focuses on empowering the immune system to
overcome the tumor rather than producing widespread cyto-toxicity to kill tumor
cells. Many anti-cancer immuno-therapies use tumor-associated antigens as
vaccines in order to stimulate immune response against cancer cells
(Hammerstrom et al., 2011). Since, the tumor invokes multiple
immune-suppressive mechanism to defend itself, hence, we need to overcome it so
as to make immunotherapy a suitable option for treating cancer (Berzofsky et
al., 2012).
Surgery, chemotherapy and radiotherapy
provide inadequate effect or affect normal cells along with the diseased one.
It leads to search for cancer cure from natural products. Anticancer drug developments
from natural biological resources are ventured throughout the world. The
biodiversity of venoms or toxins made it a unique tool from which new
therapeutic agents may be developed. Snake venom has been shown to possess a
wide spectrum of biological activities. Snakes use their venom to alter
biological function and that’s what a medicine does too. Therefore, venoms have
always been the topic of interest to most medical researchers.
Types of Snake Venom
Venom is nothing but a secretion of
venomous animals, which are synthesized in a specific part of their body,
called venom gland. It’s a modified saliva containing a mixture of different
bioactive proteins and polypeptides used by an animal for defense or to
immobilize its prey (Gomes et al., 2010). Not only the venom of every snake is
different but a subtle difference exists between different species, between
juveniles and adults, even among the snake of same species but of different
geographical regions. Approximately 90-95% of venom’s dry weight is composed of
protein. These proteins may be toxic or non-toxic. Venoms are sub-divided into
cytotoxins, cardiotoxins, neurotoxins, and hemotoxins (Ferrer, 2001).
Neurotoxins have an adverse effect on
central nervous system resulting in heart failure and/or breathing problems.
They have the ability to inhibit ion movement across the cell membrane or
communication between neurons across the synapse (Bradbury and Deane, 1993).
This toxin attacks the cholinergic neurons mimicking the shape of acetylcholine
and therefore fits into its receptor site, blocking the binding of
acetylcholine.
Toxins that cause destruction of RBCs
are collectively known as Hemotoxins. It targets the circulatory system and
muscle tissue of the host causing scarring, gangrene.
Cardiotoxins are those compounds which
are toxic specifically to heart. It binds to particular sites on muscle cells
of the heart preventing muscle contraction (Yang et al., 2005).
Cobras, mambas, sea snakes, kraits and
coral snakes contain neurotoxic venom whereas viperidae family members such as
rattle snake, copper heads, and cotton heads have hemotoxic venoms. Some snakes
contain combinations of both neurotoxins and hemotoxins.
Basic Composition of Snake
Venom
As said earlier, venom is not composed
of a single substance but it’s a cocktail of hundreds, or even thousands of
different peptides, proteins, enzymes, and chemicals. There are approximately
20 different type of toxic enzymes known to us till now found to be present in
snake venom in varying combinations and concentrations. Most common snake venom
enzymes include acetylcholinesterases, L-amino acid oxidases, serine proteases,
metalloproteinases, and phospholipases-A(2). Higher catalytic efficiency,
thermal stability, and resistance to proteolysis make these enzymes attractive
models for every researcher (Kang et al., 2011).
Cholinesterase
It attacks the nervous system,
relaxing the muscles to the point where the victim has very little or no
control. It plays a lead role in the cholinergic system where it functions in
the rapid termination of nerve impulse transmission. Its high reactivity
towards organophosphorus compound suggests that exogeneous cholinesterases can
serve as an effective therapeutic agent in the treatment of prophylaxis and organophosphorus
poisoning (Cohen et al., 2001).
L-amino acid oxidase (LAAO)
It is a dimeric flavoprotein which
contains a non-covalently bound FAD as a co-factor. It constitutes 1-9% of the
total venom protein and is responsible for the light yellowish color of the
venom and catalyzes the stereospecific de-amination of an L-amino acid
substrate to an alpha-keto acid along with the production of ammonia and
hydrogen peroxide. It has been found that LAAO from snake venom can induce
apoptosis in mammalian endothelial cells possible due to the production of high
concentration of hydrogen peroxide (Pawelek et al., 2000).
Hyaluronidase
It is actually an endogycosidase as it
degrades the beta-N-acetyl-glucosaminidic linkages in HA polymer (Lokeshwar and
Selzer, 2008). It is virtually present in all snake venom and has been known as
“spreading factor”. It damages the extra cellular matrix at the site of bite
leading to the severe morbidity. It helps in rapid spreading of other toxins by
destroying the integrity of the extra cellular matrix of the tissue. Inspite of
its role as a spreading agent, it is required to explore its function as a
therapeutic agent for inhibiting the systemic distribution of venom and also
for minimizing local tissue destruction at the site of bite (Kemparaju and
Girish, 2006).
Phospholipases A2
PLA(2) plays an important role in many
biological events such as cell signaling and cell growth, generation of
pro-inflammatory lipid mediators such as prostaglandin, and leukotrienes
(Rodrigues et al., 2009). These are the enzymes that hydrolyze the sn-2 acyl
ester bond of various phospholipids to produce free fatty acids and
lysophospholipids. Mammalian PLA (2) plays important role in various biological
processes such as phospholipid metabolism, and remodeling, homeostasis of
cellular membrane, host defense, and mediator production as well as signal
transduction (Gao et al., 2005). Whereas, snake venom are chemically complex
mixture of various active proteins or peptides belonging to Ca2+ dependent
secretory PLA (2), which serve not only as digestive enzyme but also plays
important role as a defense weapon by immobilizing the prey (Wei et al., 2009).
It has other pharmacological properties as anti-platelet, anticoagulant,
hemolytic, neurotoxic, myotoxic. It has been classified into two broad groups,
1PLA (2), found mainly in the venom of cobras, kraits, and sea snakes, and 2PLA
(2), found in venom of vipers and pit vipers (Armugam et al., 2009).
Metalloproteinase
This enzyme belong to the family of zinc
endopeptidase that degrades protein of extra cellular matrix and components of
hemostatic system (Panfoli et al., 2010). It has ability to disrupt
microvessels, which is then responsible for provoking local and systemic
hemorrhagic and also contribute to other pathways that lead to local tissue damage. It might also
prove cytotoxic to endothelial cells
(Escalante et al., 2011).
Other enzymes
Proteinases, adenosine triphosphate,
phosphodiesterases, etc. proteinases are important in digestion and they break
down victim’s tissue at an accelerated rate. Adenosine triphosphate when enters
victim’s body, it results in deep shock, and phosphodiesterases are responsible
for negative cardiac reaction in victim and also a rapid drop in blood
pressure.
Anticancer Activity of
Snake Venom
Claude Bernad, father of physiology, was the first one to realize the
involvement of some components of snake venom in different therapeutic
potential. Use of venom for the treatment of cancer in laboratory animal was first reported by Calmette, 1993.
It was found that the snake venom toxin from Vipera lebentina turnica induces apoptotic cell death of ovarian cancer cells through the
inhibition of NF-kB and STAT3 signal accompanied by inhibition of p50 and p65
translocation into nucleus. This toxin increases the expression of
pro-apoptotic protein Bax and Caspase-3 but down-regulates the anti-apoptotic
protein Bcl-2 (Song et al., 2012).
The anticarcinogenic activities of
crude venom of Indian monocellate Cobra (Naja kaouthia) and Russell’s
viper (Vipera russelli) were studied on carcinoma, sarcoma and leukemia
models. Under in vivo experiments, it was observed that life span of EAC
(Ehrlich ascites carcinoma) mice got increased with the strengthening of
impaired host anti-oxidant system. In case of in vitro study, venom showed potent cytotoxic and
apoptogenic effect on human leukemic cells (U937/K562) by reducing cell proliferation rate and produced morphological alterations
(Debnath et al., 2007).
From past few decades, research has
been undertaken on isolation and characterization of the snake venom cytotoxin.
Cytotoxins exhibit various physiological effects as cytotoxicity, inhibition of
platelet aggregation, cardiac arrest, hemolysis, etc. Cytotoxin or Cardiotoxin
are polypeptide of 60-70 amino acid residues long found in snakes of elapid
family having various pharmacological effects such as depolarization of
muscles, and haemolysis (Ferrer, 2001). Cardiotoxin-3 (CTX-3), a basic
polypeptide of 60 amino acid residue present in Naja naja atra venom has been reported to possess anti cancer
property. It induces apoptotic cell
death accompanied by upregulation of both Bax and endonuclease G, and down
regulation of Bcl-x in K562 cells which was confirmed by DNA fragmentation
(Yang et al., 2006). In a study carried out by different group of scientists on
the same cell line, CTX-3 was reported to show apoptotic cell death through activation of Caspase-12 and JNK
pathway which then triggered Ca2+ influx
because of rapid increase in cytosolic Ca2+ concentration
(Yang et al., 2008). Two different studies were carried on HL-60 cells using
CTX-3. It has been reported that anti-proliferative property of CTX-3 mediated
through apoptosis by a significant
increase in sub G1 population and the activation of c-JUN-N-terminal kinase
(Chien et al., 2008). According to
another study, apoptosis was induced by activation of both endoplasmic
reticulum pathway of apoptosis and mitochondrial death pathway, indicated
by increased level of Ca2+ and glucose-related
protein 78 (GRP 78) (Chien et al., 2008). When MDA-MB-231 (Human breast cancer)
cells were exposed by CTX-3, it induces apoptosis which was confirmed by
accumulation of sub-G1 population and loss of mitochondrial membrane potential
(Lin et al., 2010). CTX-3 down regulates NF-kB in MCF-7 (human breast cancer)
cells leading to the suppression of
proliferation and induction of apoptosis which was confirmed by sub-G1
population, phosphotidylserine externalization, and poly (ADP-ribose)
polymerase (Chiu et al., 2009). Later on, it was found that CTX-3 induces
apoptosis in A549 cells by inactivating the EGFR, P13-K/Akt and JAK/STAT3
signaling pathways (Su et al., 2010).
drCT-1 is a heat stable, 7.2 kDa
protein toxin isolated from Indian russell’s viper (Daboia russelli russelli)
venom and is supposed to possess
anti-proliferative, cytotoxic, and apoptotic property. In vivo and in vitro
experiments were done using drCT-1 on EAC mice and human leukemic cells
(U937/K562) respectively. It showed decrease in EAC cell count, cell viability,
and an increased survival time of diseased mice and showed a dose, and time
dependent inhibition of U937 and K562 cells because of apoptosis through G1
phase arrest of the cell cycle (Gomes et al., 2007).
Disintegrins also possess the ability
to inhibit tumor behavior both in vitro and in vivo. RGD containing
disintegrins are non-enzymatic proteins that inhibit cell-cell interactions,
cell-matrix interactions, and signal transduction. Salmosin, a disintegrin
isolated from Korean snake venom, effectively
suppressed growth of metastatic tumor as well as solid tumor in mice (Kang
et al., 1999) This antimetastatic activity was resulted from blockage of
integrin-mediated adherence of αvβ3 integrin mediated proliferation of the
melanoma cells (Bradbury and Deane, 1993). Contortrostatin (CN) is a
homodimeric disintegrin found in southern copperhead snake venom. Its
anti-cancer effect was studied on OVCAR-5 (human epithelial carcinoma cell line
of ovary) cells. CN effectively blocks the adhesion of OVCAR-5 cells to several
extracellular matrix proteins and inhibits tumor cell invasion through an
artificial basement membrane (Markland et al., 2001).
Condrostatin, a homodimeric
disintegrin, isolated from copper head snake venom, was found to be a potent inhibitor of in vitro
beta1integrin-mediated cell adhesion and in vivo lung colonization (Bradbury
and Deane, 1993).
Snake venom containing cystatin
(sv-cyst), a member of cysteine protease family inhibitors, has been reported
to play an important role in tumor invasion and metastasis. In a study carried
out on MHCC97H (liver cancer) cells, sv-cyst has shown inhibition of tumor cell invasion and metastasis through the reduction
of the proteinases activity and epithelial-mesenchymal transition (EMT)
with a decreased activity of cathespin B, MMP-2 and 9, and EMT change index,
and increased acitivity of E-cadherin, and decrease in the activity of
N-cadherin and twist activity (Tang et al., 2011).
Phospholipases A (2) is the enzyme
that hydrolyzes the sn-2 acyl ester bond of various phospholipids to produce
free fatty acids and lysophospholipids (Gao et al., 2005). Snake venom is a
chemically complex mixture of various active proteins or peptides belonging to
Ca2+ dependent 025.050.075.0100.0Newly diagnosed without treatment
secretory PLA (2) (Arimura et al.,
1989). A group of scientists reported that the PLA (2) from Macrovipera
lebentina venom exhibits anti-integrin activity. In their study, done on
HMEC-1 (human micro vascular endothelial) cells, MVL-PLA (2) has shown
inhibition of cell adhesion and migration; also the actin cytoskeleton and
distribution of αvβ3 integrin were disturbed. MVL-PLA (2) also reported
increase in microtubule dynamicity by 40% (Bazaa et al., 2010).
Since, we are all aware of high
cytotoxic property of snake venom or toxins, its effect on non-cancerous cell
line is still controversial with some groups suggesting it is harmless to
non-cancer cell line while other mentioning its cytotoxic effect on non-cancer
cell line also. But now, people have found out solution to this also by
combining the components obtained from snake venom with nano-particle and allow
it for targeted delivery to the diseased site. According to recent study, snake
venom extracted from Walterinessia aegyptia (WEV), alone or in
combination with silica nano-particles can
decrease the proliferation of human breast carcinoma cell line
(MDA-MB-231). In this study, decreased expression of Bcl-2 and enhanced
activation of caspase-3 has been found when breast cancer cell line was treated
with WEV along with nano-particle and also showed significant reduction in
actin polymerization and cytoskeletal rearrangement but it was not the case
with non-cancer cell line (Al-Sadoon et al., 2012).
Crotoxin is a cytotoxic PLA2 compound
isolated from a South American snake, Crotalus durissus terrificus venom
(Faure et al., 1993). Crotoxin displays cytotoxic activity against a variety of
murine and human tumor cell line in vitro (Rudd et al., 1994). Crotoxin induced
cytotoxic effects appear to be highly selective towards cell line expressing
high density of epithelial growth factor receptor. Antitumor efficacy in vivo
using daily intra muscular administration of crotoxin has been demonstrated on
Lewis lung carcinoma (Newman et al., 1993) with 83% growth inhibition, and MX-1
human mammary carcinoma with 69% growth inhibition. Lower activity was observed
in HL-60 leukemia cells with 44% growth inhibition suggesting that crotoxin
might have high specificity towards solid tumor. In phase I clinical trial,
crotoxin was administered intra-muscular for 30 days in patients with solid
tumor refractory to conventional therapy at doses ranging from 0.03-0.22 mg/kg.
A total number of 35 cycles of crotoxin administration was evaluated in 23
patients. No death was observed in this study. Patients with different types of
carcinomas responded in different way resulting in the reduction of disease.
The therapeutic response obtained in some patients was quite promising and
deserved additional development of this compound under phase 2 clinical trial
with a recommended dose of 0.14 mg/m2 (Cura
et al., 2002).
VRCTC-310 is a natural product
produced by combining two purified snake venom, a non-covalent heterodimer
crotoxinn, and a basic amphipathic peptide cardiotoxin. A phase I study was performed
to evaluate the mechanism tolerated dose (MTD), safety profile, and
pharmacokinetic data with VRCTC-310. 15 patients with refractory malignancies
were given intramuscular injection daily for 30 days continuously. MTD was
found to 0.017 mg/kg and recommended for phase II studies with dose range of
0.017 mg/kg (Costa et al., 1997).
According to a news published in a
journal (Popular science), an Irish company is using American rattle snake
venom to test its anti-cancer potential. They isolated a protein from rattle
snake venom that causes malignant cancer cell to commit suicide. This company
has developed a venom-derived drug called CB24 and started testing it on humans
in October’11. That drug has already been tested in mice and human cell lines with
great success.
Conclusion and Future Prospects
Above description makes it clear that
different components of the venom are being used for clinical trial and they
can be used as a natural therapeutic agent against cancer. Since there is
controversy about the cytotoxic effect of the venom on normal cells, therefore
its effect on normal cells should be evaluated. Tagging of the venom with
nanoparticles for targeting the cancer cells can be one of the best therapeutic
approach for the treatment of cancer.
Acknowledgements
We thank SoBT, IGNOU-I2IT Centre of
Excellence for Advanced Education and Research, Pune, for their support. The
authors declare no conflict of interest.
