Snake Venom Protein: A Wonderful Medicine In Cancer Treatment

Swarnali Das

Contortrostatin (CN) (Mr 13,500 Da) is a novel
homodimeric disintegrin isolated from the venom of Agkistrodon contortrix
contortrix (Southern Copperhead) snake and displays two RGD motifs (one on each
chain), which modulate its interaction with integrins on tumor cells and
angiogenic vascular endothelial cells

⁴
.

Angiogenesis is a process
of  the development of new blood vessels,
which is exploited by a tumour to obtain a supply of nutrients and growth
factors, a disposal route for its wastes, and a route for cancerous cells to
spread.The copperhead protein acts by inhibiting the development of new blood
vessels to nourish the tumours and by putting tumour cells into a "suspended
state of animation",the dual action helped prevent the spread of cancer, a
process called metastasis³.

CN is cytostatic rather than cytotoxic. This means it
does not kill tumour cells but "freezes" them in a long lasting state
of "suspended animation". The cancer cells are prevented from
adhering to and invading normal surrounding cells². Because the protein does
not kill cells directly it has none of the side effects of powerful
chemotherapy drugs, such as severe nausea².

There are several approaches which are used or in
trial to deliver CN efficiently in human body. CN administered in a liposomal
formulation exhibits potent anti-angiogenic and tumor growth inhibitory
activities in mice

⁶

. Liposome formulations used to deliver CN contain protein at


concentrations greater than 1.5 mg/ml with encapsulation efficiency


of
greater than 80% as determined by BCA protein assay

¹²

.


 Mass production of contortrostatin using
recombinant DNA technology is also in trial.

Introduction :

Snakes use venom to alter biological functions, and
that's what medicine does too, this is why venoms have always been of interest
to medical researchers¹. Venoms are exquisitely complex, composed of as many as
a hundred different peptides, enzymes, and toxins. Not only are the venoms of
every snake species different, there are also subtle variations within each
species. There are differences between
[venoms of] juveniles and adults, and even among different geographic
regions.These differences may be due to different evolutionary pressures, like
different ancestry, prey, and environments¹. Of the nearly 3,000 species of
snakes in the world, about 650 are venomous.

The Copperhead, Agkistrodon
contortrix, grows to about three feet long and has a distinctive copper head
and reddish brown bands. Although poisonous, its bite is rarely fatal. The
protein extracted from the venom of the Southern Copperhead viper slowed the
growth of tumours cells by up to 70%². It is still unknown the role of
contortrostatin in the snake venom is and what is does to the victim. The best
guess is that contortrostatin in the snake venom keeps platelets from
aggregating and thereby helps to maintain the blood fluid. Then the more
damaging components of the venom can move quickly through the body and rapidly
immobilize and/or kill the prey

¹¹

.

CN belongs to a class of proteins known as
disintegrins, so named because they disrupt the function of certain other
proteins, called integrins, on the surface of cells that enable them to stick
together³.
Disintegrinsare small, disulfide-rich proteins containing an R/KGD (Arg/Lys-Gly-Asp)
sequence at the tip of a flexible loop protruding from the main polypeptide
chain. Integrins are a family of cell surface proteins found on many cell types
that mediate interactions between cells, and between cells and their
surroundings

¹¹

. Contortrostatin binds to integrins on the surface of
cancer cells and inhibits tumor growth and metastasis. Contortrostatin is
unique from all other disintegrins described to date in that it is a homodimer,
which means it has two identical peptide chains held together by
covalent disulfide bonds.

¹¹

CN is stable to extremes


of pH and to
organic solvents (like most disintegrins) as evidenced


by its
purification by reverse-phase high-performance liquid


chromatography

^14-16

.
Thus, CN is a small, very stable


protein that is uniquely suited for
liposomal encapsulation. The size of
liposomes with encapsulated


CN is

100–150 µm. Thus, liposomes leak out of the

newly
growing angiogenic vessels and deliver CN into the tumor.

Once the
liposomes exit the "leaky" vessels and form depots

in the
tumor, presumably lipases and other extracellular enzymes


derived
from tumor or stromal cells aid in the slow degradation


of the
liposome, releasing the entrapped protein

¹³

. On release


from
the liposome, CN binds to integrins on both tumor and endothelial


cells
thereby blocking both tumor growth and angiogenesis. The


development
of the liposomal delivery system eliminates the


problems of how to
overcome the short circulatory half-life


of CN and repress or
eliminate the potential immunogenicity


of the snake venom protein.

¹⁷

Wonderful
mechanism of anticancer action of Contortrostatin :

CN inhibited migration and invasion, and significantly
altered Matrigel-induced tube formation of human umbilical vein endothelial
cells (HUVEC), but did not affect cell viability, or MMP-2 and MMP-9 activity.
Immunocytochemistry of HUVEC revealed that CN disrupted actin cytoskeleton and
altered VE-cadherin distribution at cell-cell contacts. CN down regulated focal
adhesion kinase (FAK) and paxillin tyrosine phosphorylation in adherent HUVEC.
There was also significant inhibition of angiogenesis in vivo by CN as assessed
by implanting Matrigel plugs in C57 mice and measuring in growth of blood vessels
using either factor VIII staining or hemoglobin determination.

⁵

contortrostatin blocks
several critical steps in tumor metastasis and is, therefore, more potent than
other agents which only block a single step In addition, contortrostatin
significantly inhibits invasion of breast cancer cells through an artificial
barrier similar to the tissue surrounding blood vessels. This action was most
likely due to the ability of contortrostatin to inhibit cell motility.

¹¹

Interestingly,
contortrostatin is not cytotoxic to human breast cancer cells. A metastatic
breast cancer model is studied by implanting human breast cancer cells into the
mammary fat pads of immunodeficient mice. Contortrostatin, or saline, was
injected daily into tumors in several different groups of mice .Observation was
that the size of the tumor masses in the contortrostatin-treated mice were
significantly smaller than those in saline-treated mice. Even more exciting,
the contortrostatin-treated group showed 65% to 85% inhibition of lung metastasis,
as compared to the saline-treated group. In order for a tumor mass to grow
beyond 1-2 mm, the development of a vascular network is required. Since growth
of new blood vessels is essential for progressive growth of cancer, therapies
that block new blood vessel growth into the tumor will also inhibit tumor
growth.  Chick embryo membrane also used
to investigate the effect of contortrostatin on new blood vessel growth. The
chick embryo membrane serves as an immunosuppressed surface on which the human
breast cancer cells can grow effectively. It was found that contortrostatin
inhibited new blood vessel growth induced by the breast cancer cells.

¹¹

Conclusion
:

Investigations also going
to quantify the anti-angiogenic and anti-tumor effects of contortrostatin in
several other tumor systems including prostate and bladder cancers, and glioma.
Future research will be aimed at developing a large-scale expression system for
mass production of contortrostatin using recombinant DNA technology

¹¹

.Targeted
delivery system that will enable intravenous administration of the venom
protein is also in trial.Clinical development of this novel therapy should
benefit patients with various forms of cancer.

Current isolation of the protein from crude venom is
difficult and prohibitively expensive for translation into the clinic.
large-scale production of a soluble monomeric form of recombinant CN with
biologic activity will revolutionize this problem. Here  the protein is expressed directly in the
cytoplasm of an engineered bacterial system with an expression yield of
approximately 20 mg/l of culture

⁴

. It potentially transform one of
nature's deadliest toxins into a curative agent.

In conclusion, the present findings confirm that CN
possess strong anti-angiogenic activity.It also has minimum side effects unlike
other anticancer drugs because it has no direct action on cell. CN would need
to be administered over a period of time in the hope of shrinking a tumour to a
size where treatment could be scaled back or stopped³. So these therapies may
provide a unique and practical way for long term control of  various forms of cancer.

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3.
Roger Highfield (Daily Telegraph), “Snake's venom may help treat breast cancer”

Web address-  snake_venom_in_cancer.zip\snake venom in
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