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Journal of Venomous Animals and Toxins

Print version ISSN 0104-7930On-line version ISSN 1678-4936

J. Venom. Anim. Toxins vol.5 n.2 Botucatu  1999 

Original paper






1 Ophidia Products Inc.USA



ABSTRACT: Cancer cell inhibitors, named Atroporin and Kaotree, having molecular weights of 35 kDa and 6 kDa have been isolated from the venoms of Crotalus atrox and Naja naja kaouthia, respectively, by fractionation on high pressure liquid chromatography. The purified Atroporin and Kaotree showed killing effects on various types of human (breast, colon, liver, ovary, etc.) and animal cancer cells in concentrations as low as 0.5µg/ml, and having no effect on normal mouse kidney, liver, spleen, and erythrocytes up to 5.0µg/ml. Both Atroporin and Kaotree prevent the formation of ascitic tumors caused by myeloma cells in Balb/C mice. In addition, both Atroporin and Kaotree showed regression of ascitic tumors formed by myeloma cells. Atroporin and Kaotree complement each other, as in combination they showed elevated anti-cancer activity in vitro and in vivo systems. However, Atroporin and Kaotree are immunologically distinct proteins showing no cross reactivity. Atroporin and Kaotree, individually or in combination, have the potential for cancer biotherapy.
 KEY WORDS: cancer, biotherapy, venom proteins.




In prosperous countries, roughly 20% or one in five people will die of cancer. The most frequently occurring cancers worldwide in a descending order are: stomach, lung, breast, colon/rectum, cervix, and mouth/pharynx (3). The cytolytic activity of most available anti-tumor chemicals is due to the inhibition of the synthesis and replication of DNA in tumor cells. Surgery, chemotherapy, and radiation show limited success, and furthermore, remove or destroy normal cells along with cancer cells. Therefore, it is of interest to search for new anti-cancer agents having a different mode and site of activity. Moreover, cancer therapy using toxic chemicals causes serious adverse side effects and other unpleasant results, such as alopecia (hair loss), which is one of the most distressing among others (5).

The search for a cure for cancer has been vigorously pursued for over half a century, and the use of chemicals to treat cancer continues. The treatment of cancer needs to be changed from chemotherapy to biotherapy using biologicals, having minimum or no adverse effects. Monoclonal antibodies alone or coupled with ricin or other toxins and interferons are the only biologicals used until now for cancer therapy on a limited and experimental basis. Although, the fundamental understanding of cancer is progressing rapidly, there is still no academic breakthrough in therapy. There is a lot of scope for the discovery of naturally occurring biologicals that can be therapeutic and generally important to the science of oncology.

Snake venom is a complex mixture of many substances such as toxins, enzymes, growth factors, activators, and inhibitors with a wide spectrum of biological activities. It has been reported (14) that venoms from the snake families Elapidae, Viperidae, and Crotalidae, but not Hydrophidae, caused lysis of Yoshida sarcoma and KB cells. There are reports showing the cytotoxic activity of various snake venoms in vitro and in vivo, using melanoma and chondrosarcoma cells (2). It was further demonstrated that a purified protein from cobra venom was selectively cytotoxic to cancer cells (1). Subsequently, the same investigators (16) isolated the cytotoxic factor having a molecular weight of 10,500 daltons, while others (6) studied the cytolytic activity of a cytotoxin isolated from Indian cobra venom against experimental Yoshida tumor cells. They observed that the cytolytic activity on tumor cells was far stronger than on exudate cells such as, spleen cells and erythrocytes from rat. Silva et al., (13) evaluated the action of the venoms of Crotalus durissus terrificus and Bothrops jararaca on Ehrlich ascitic tumors and found that both venoms act directly on tumor cells. They further postulated the various mechanisms involved in the anti-tumor activity.

Thus, crude snake venoms as well as purified snake venom proteins have been reported to have cytolytic activity on KB and Yoshida tumor cells. There are few reports on the use of snake venoms or purified venom enzymes as anti-cancer in vivo system, by studying extensively the enzyme crotalase, purified from the venom of Crotalus adamanteus for its anti-tumor activity, using B16 melanoma cells in C57BL/6 mice (10,11). The same authors observed the inhibition as well as the regression of tumors in mice after treatment using crotalase. The anticoagulant Ancrod is the only snake venom derived component that has been tried in humans to treat malignant disease with inconclusive results (7,15).

At Ophidia, cancer cell inhibitors, Atroporin and Kaotree have been isolated from the venoms of Crotalus atrox and Naja n. kaouthia, having molecular weights of 35 kDa and 6 kDa, respectively, by high pressure liquid chromatography (HPLC) fractionation. The purified Atroporin and Kaotree showed killing effects on various types of human (breast, colon, liver, ovary, etc.) and animal cancer cells in concentrations as low as 0.5µg/ml, and having no effect on normal mouse kidney, liver, spleen, and erythrocytes as high as 5.0µg/ml (9).



SNAKE VENOMS. Venom from several snakes of Crotalus atrox, family Crotalidae, and Naja n. kaouthia, family Elapidae, was collected at various times and frozen at -20°C until used.

MEDIA. Dulbecco's Modified Eagle's Medium (DMEM, Sigma) was enriched with 10% fetal bovine serum (FBS) for cell growth and 2% for study of the cytolytic effects in cell culture.

CELL LINES - The cell lines used in these studies were purchased from American Type Culture Collection (ATCC), Rockville Maryland. Human Cancer Cell Lines - HBL-100, BT-20, and ZR-75-1 of breast cancer, HT-29 and Diji (M. D. Anderson Hospital) of colon cancer, Sk-ov-3 and HBT 77 of ovary cancer, and Chang liver CCL-13 of liver cancer. Animal Cancer Cell Lines - SP/2 mouse myeloma CRL 1581, PC12 Pheochromocytoma, rat adrenal PC 12 CRL 1721, and African green monkey kidney Vero CCL 81.

PRIMARY CELL LINES - The mice used for this research were in compliance with US Public Health Service policy on humane and care use of animals. Mouse spleen, kidney, liver, and erythrocyte cultures were made in house. Aspirated monodispersed spleen cells were washed once with medium and seeded into 24 well culture plates. All cell counts were done manually on a hemocytometer and cells were dispersed each well receiving approximately 5 x 10 cells/ml.

Mouse kidney cells were monodispersed by treating them with a 1% mixture of trypsin and EDTA (Sigma). The cells were washed twice with medium in order to remove traces of trypsin/EDTA. Mouse liver tissue was processed similarly to obtain monodispersed liver cells. Mouse erythrocytes were collected directly into normal saline washed once with medium.

VENOM FRACTIONATION - Approximately 25 mg of venom diluted with 0.01 M phosphate buffer saline (PBS), pH 7.4, was fractionated on a HPLC from Toso Co. Japan, and the anion exchange column from Polymer Laboratories UK maintained at 20°C. A plurality of fractions were eluted according to relative ionic charge using gradient buffer at pH 7.3 and at a flow rate of 1.8 ml/min. The Toso HPLC automatically mixes water and 1.0 molar buffer to yield gradient buffer in the range 0.01 molar to 1.0 molar.

IDENTIFICATION OF ANTI-CANCER COMPONENTS. The venom of C. atrox resolved into 10 major fractions and that of Naja n. kaouthia into 7 fractions by HPLC. Each fraction was dialyzed against water, the protein concentration of each fraction was adjusted to 100µg/ml with sterile PBS, and filtered through 0.2µm pores to attain sterility.

Initially each fraction was tested on 5x10 cells/ml of SP/2 mouse cancer cells from 5µg to 0.1µg/ml. The results were read after 48 hours and 100% cell death was considered as the end point. We found that one fraction of Crotalus atrox and one of Naja n. kaouthia venoms showed the most cytolytic activity on SP/2 mouse myeloma cells, named Atroporin and Kaotree, respectively. These anti-proliferative active fractions of Atroporin and Kaotree were concentrated and dialyzed simultaneously, using a dialysis apparatus from the Spectrum Co., to 1/20 volume and refractionated by HPLC under identical conditions, gradient buffer, temperature, etc. Thus, purified Atroporin and Kaotree, resolved into a single band by electrophoresis with mol. wts. of 35.0 and 6.0 kDa, respectively, (Figure 1) were used for in vitro and in vivo experiments.


FIGURE 1. Electrophoretic profile of Atroporin and Kaotree. 1. Kaotree (6 kDa); 2. Markers; 3. Kaotree (6 kDa); 4. Markers; 5. Atroporin (35 kDa) and 6. Atroporin (35 kDa).


PRODUCTION OF POLYCLONAL ANTIBODIES TO ATROPORIN AND KAOTREE. Adult Balb/C mice were immunized for the production of antibodies. The first injection consisted of a mixture of 10µg of Atroporin or Kaotree in 0.1ml mixed with equal volume of Freund's complete adjuvant per mouse. The subsequent injections consisted of similar concentration of the proteins mixed with equal volume of Freund's incomplete adjuvant. The mice were injected intramuscularly (IM) three times, two weeks apart. At the end of immunization, the mice were bled through ophthalmic veins and serum was separated.

ENZYME-LINK IMMUNOSORBENT ASSAY (ELISA) AND IMMUNODIFFUSION TEST (IP) FOR ANTI-ATROPORIN AND ANTI-KAOTREE. ELISA tests were performed using a 96 well microtiter plate and all reagents were purchased from Sigma Chemical Co. The procedure for ELISA and immunodiffusion tests was as described by Lipps (8).

CYTOLYTIC EFFECTS OF ATROPORIN AND KAOTREE, INDIVIDUALLY AND IN COMBINATION, ON HUMAN AND ANIMAL CANCER CELLS AT VARIOUS CONCENTRATIONS. Cell monolayers were monodispersed and seeded into 24-well culture plates, each well receiving 5x10 cells/ml. The purified Atroporin and Kaotree were tested for their cytolytic activity ranging from 5µg/ml to 0.1µg/ml, using five wells per concentration. The results were read after 48 hours of incubation at 37°C in a humid CO2 incubator. The cut off point was taken to be 100% killing, as established by microscopic examination. Similarly, the cell cultures of normal mouse spleen, liver, kidney, and erythrocytes were tested. No quantitation was made for partial killing between 100% and 0% (Table 1 and Table 2).


TABLE 1. Cytolytic effects of Atroporin and Kaotree, individually and in combination, on human cancer cells in various concentrations.




TABLE 2. Effects of Atroporin and Kaotree, individually and in combination, on normal and cancer cells.


PREVENTION OF ASCITIC TUMOR FORMATION IN MICE. Two million SP/2 mouse myeloma cells were injected intraperitoneally (IP) to produce ascites in twenty 5-6 week old Balb/C mice. The injected mice were divided into four groups of 5 mice each. Starting on day 0, group I was given 0.2 ml PBS to serve as control. Group II was given 0.2ml containing 5µg of Atroporin, group III was given 5µg of Kaotree, and group IV was given 5µg of a combination of Atroporin and Kaotree for five consecutive days. Thus, each group received 25 µg of total protein and mice were observed for four months (Table 3).


TABLE 3. Prevention and regression of ascitic tumors in mice by Atroporin and Kaotree, individually and in combination.



REGRESSION OF ASCITIC TUMORS IN MICE. Two million SP/2 mouse myeloma cancer cells were injected IP into twenty 5-6 week old Balb/C mice to produce ascitic tumors. When the mice started showing bulging stomachs, which was after 60 days, they were divided into four groups of five each. The mice in group I were given PBS to serve as controls. The mice in groups II and III were treated with Atroporin and Kaotree, respectively. The mice in group IV were treated with a combination of Atroporin and Kaotree. In each case, 5µg of protein was administered for five consecutive days totaling 25µg (Table 3).



The results in Table 1 show that both Atroporin and Kaotree are cytolytic to a variety of human cancer cells in concentrations as low as 0.5µg/ml. Kaotree is more cytolytic to ZR-75, breast cancer cells, than Atroporin 0.5µg/ml versus 1.0µg/ml. On the other hand, Atroporin is more cytolytic to Sk-ov-3, ovary cancer cells, than Kaotree. However, the combination of Atroporin and Kaotree showed elevated cytolytic activity, which is 0.5µg/ml despite the concentration of each being reduced by half. Atroporin and Kaotree are derived from venoms of snakes belonging to different families, having different molecular weights, and therefore, complementing each other for cytolytic activity showing elevated activity.

The results in Table 2 clearly show that Atroporin and Kaotree, individually or in combination, have no cytolytic effect on normal mouse spleen, liver, kidney, and red blood cells in concentrations as high as 5µg/ml, which is 10 times higher than required to elicit cytolytic effect on cancer cells.

Atroporin and Kaotree showed similar toxicity for mouse myeloma SP/2 cells, 0.5µg/ml individually, but in combination it increased to 0.1µg/ml. Elevated toxicity was also observed for monkey Vero cells.

The results in Table 3 show that after 60-70 days mice in the control group developed ascites tumors and died in the next 10-20 days. All mice treated with Atroporin and Kaotree failed to develop bulging stomachs of ascitic tumors and survived until the experiments were terminated after four months. These experiments illustrate that tumor formation was prevented by treatment with Atroporin or Kaotree, individually and in combination.

The results of tumor regression in Table 3 show that the mice in group I died in the next 10-20 days with ascitic tumors. In groups II and III, 60% and 80%, respectively, survived and for those that died, the incubation period was longer than the controls. None of the mice in group IV died. These experiments prove that the treatment with Atroporin or Kaotree caused regression of ascitic tumors in mice. Atroporin and Kaotree complement each other, therefore, the combination provided 100 % tumor regression, which was better than either agent alone.

The immunology of anti-Atroporin and anti-Kaotree were studied by ELISA and Ouchterloney's (12) immunoprecipitation tests, as described under Materials and Methods. The results are shown in Table 4.


TABLE 4. Cross reactivity of Atroporin and Kaotree by ELISA and immuno precipitation (IP).



The results in Table 4 show that anti-Atroporin and anti-Kaotree reacted well to themselves and to venoms from which they were derived by both immunological tests. Antibodies versus the proteins Atroporin and Kaotree do not show immunological cross reactivity by ELISA and immunoprecipitation tests; therefore, they are individually distinct proteins. Atroporin and Kaotree were derived from different species of snakes belonging to the families Crotalidae and Elapidae, respectively. Therefore, they are immunologically distinct proteins.



Currently, numerous chemotherapeutic drugs are in use for cancer treatment. Unfortunately, most cancer therapy is associated with adverse side effects, such as hair loss, skin rash, and diarrhea simply because the chemicals used to kill cancer cells are toxic to normal cells. Snake venom derived Atroporin and Kaotree are novel proteins, by no means are they toxins. These proteins may show some amino acid sequence homology with toxins. Nevertheless, they are altered in biological characteristics by our unique procedure of separation, losing toxicity and retaining biological anti-cancer activity. Atroporin and Kaotree have the property of selectively killing cancer cells without harming the normal cell population. The introduction of such biologics for the treatment of cancer will change the practice of chemotherapy to biotherapy.

The mechanism behind the selective cancer cell killing ability of snake venom derived Atroporin and Kaotree is not yet elucidated, although it has been demonstrated that such inhibitory effect of the antibiotic herbimycin is greater than 40% on seven different colon cancer cell lines and only 12% in normal colonic mucosa cells (4).

The cell culture experiments show that 5x10 /ml cells of various types of cancer were killed by incorporating 0.5µg/ml of Atroporin or Kaotree. In mouse experiments, two million SP/2 cells were injected, which is 4 times greater than in vitro testing, and in theory should require 2 µg of these anti-cancer proteins to achieve a similar effect. But in vitro and in vivo systems can not strictly be compared, therefore, 25µg were used for mouse experiments. The total 25µg of venom derived proteins was tolerated by a 20g mouse, which illustrates the non-toxicity of these proteins.

Immunologically, Atroporin and Kaotree are two different proteins having no cross reactivity to their antisera by ELISA and IP tests. Because the combinations of Atroporin and Kaotree provide enhanced killing effect on cancer cells, such combination can be proposed for human therapy. Based on the in vitro test, a dose of 1 ml will be proposed for biotherapy treatment, which will kill about 1000 million or 10 cancer cells.

During a normal healthy life, cancer cells are formed due to mutation and are removed at the same rate by the natural killer cells. If, for whatever reason, the cancer cell number overrides the natural killer cell population, the cancer cells establish a region/s in body organs. A tumor is an aggregation of cancer cells due to the excessive rapid growth property of cancer cells. By the time the patient is diagnosed for cancer, the cancer may be metastasized. Diagnosis generally leads to surgery to remove cancer growth or tumor followed by chemotherapy. To date, there is no preventive therapy for cancer. We strongly believe that the venom derived non-toxic anti-cancer proteins Atroporin and Kaotree should fill this gap. People having predisposition to cancer, due to hereditary or other reasons should be given this treatment once a year. Of course, the necessary controlled studies will require large populations and long periods of time along with placebos. The venom derived Atroporin and Kaotree will function like a vaccine for prevention of cancer, although unlike vaccines, their activity is not due to the antibody production, but due to the selective killing effect.

Atroporin and Kaotree were tested by the National Cancer Institute (NCI), Bethesda, MD. The screening procedure showed positive results similar to ours. However, the NCI did not test these proteins on any type of normal cells.



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Received 05 August 1998
Accepted 09 December 1998

B. V. LIPPS - 11320 South Post Oak, # 203 Houston, Texas 77035, USA.

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