SNAKE VENOM PROTEIN: A WONDERFUL MEDICINE IN CANCER TREATMENT

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Abstract:

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 cells4. 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 mice6. 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 assay12. Mass production of contortrostatin using recombinant DNA technology is also in trial.

Full Text:

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 prey11.
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. Disintegrins are 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 surroundings11. 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 chromatography14-16. 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 protein13. 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 technology11.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 culture4. 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.
References :
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