"APPROVED" Customer of investigations General Director of ECOMED Research & Production Association S.A.Khvorostov /signature/ Seal: ECOMED Research & Production Association Company Limited (Moscow) Date: December 22, 1994

"APPROVED" Director of Research Institute of Children's Hematology Russian Federation Ministry of Health Protection Corresponding Member of Russian Academy of Natural Sciences Professor A.G.Rumyantsev (signature) Seal: Date: December 22, 1994

R E P O R T
Antitumor properties of autonomous electrostimulator
of alimentary canal and mucosa in cell culture

     The goal of this work was to study the direct and indirect tumoricidal activity of the autonomous electrostimulator in short-term and long-term cell cultures.

     The objects of investigation were peritoneal macrophages of rats, EL-4 ascitic limphoma, MCA/77-23 sarcoma (clone H). The tumor cell cultures for our work were kindly granted by the Jackson Laboratory (USA). The tumors were reinoculated to the mice of 8 to 10 weeks old of the C57BL/6J(H-2k) line.

     Basic methods of investigation

     Isolation of peritoneal macrophages.

     The cells of peritoneal exudate were isolated using a general technique with insignificant modifications. We made intraperitoneal injections of 5 to 10 ml of the Henx solution heated to 37°C to the rats under light ether narcosis, the abdominal wall was massaged for 3 to 5 minutes and opened with scissors. The contents of the abdominal cavity was sucked off using a Pasteur pipette and introduced into the centrifugal test tubes. Centrifuging was performed for 10 min at 150 g in order to precipitate the cell elements. The cell precipitate was washed for three times with large volumes of the cold Henx solution. Finally, the cells were resuspended in 1 ml of the Henx solution containing 10% veal serum inactivated by heating. The cells were stored at the temperature of melting ice and used for investigations during 4 hours after the isolation procedure.

     Generation of oxygen free radicals by macrophages was estimated by the intensity of liminol-dependent and lucigenin-dependent chemiluminence (spontaneous and activated by the latex particles). To carry out the analysis, we placed 500,000 macrophages into the medium containing the Henx solution and 5 micromol of luminol or lucegenin in the special polyethylene cuvette of a LKB 1251 chemiluminometer. The intensity measurements of luminol- and lucegenin-dependent chemiluminescence were carried out upon continous mixing the contents of the measuring cuvette at the constant temperature of 37°C. The conditions of incubation, measurement, and displaying the results were monitored using a built-in computer. We registered the intensity of the spontaneous luminescence in the suspension of macrophages for 3 minutes; then, without breaking the light isolation of the system, we introduced the suspension of latex particles (0.1%) and measured the maximum value of chemiluminescent response to the activator (this value was determined as the difference between the intensities of activated and spontaneous cell luminescence). Upon studying the effects of electrostimulator on generation of oxygen radicals by the phagocyting cells, we placed the electrostimulator into the culture of macrophages and performed sampling of cells for analysis in 30, 60, 120, and 180 minites. The inactive electrostimulator was placed into the control culture.

     Production of tumor necrosis factor (alpha-THF) by peritoneal macrophages was determined by the immunoenzymatic method using special kits for specific discovery of microquantities of alpha-THF. We placed the electrostimulator into the macrophage culture for 1, 2, and 1.5 hours, and measured production of alpha-THF after 24-hour incubation of cells under sterile conditions.

     Cultivation of tumor cells.

     We cultivated EL-4 and MCA/77-23 cells in 24-hole boards (Costar) in the RPMI-1640 medium containing 10% veal embryonic serum, HERES buffer, L-glutamine, indispensable aminoacids, sodium pyrivate, antibiotics, and 2-mercaptoethanol. At various times after beginning the cultivation, we determined the level of 3_H-thymidine introduction; on this purpose, we transferred the cells into 96-hole boards (NUNC) in the amount of 50,000 cells per one hole, added 1.5 microcurie of 3_H-thymidine, and cultivated for 1 hour. Then we transferred the cells to the filter using a harvester and determined the level of radiation using a beta-counter (Beckman). We found initially that the maximum amount of 3_H-thymidine is introduced into the EL-4 limphoma cells on the second day and into the MCA/77-23 sarcoma cells on the fourth or fifth day after beginning the cultivation.

     In order to determine the antitumor activity of the electrostimulator, the limphoma and sarcoma cells were cultivated in 6-hole boards containing 8 ml of cultural medium with the cell concentration of (3.5 — 4 ) x 100,000 cell/ml. In 1, 6, and 24 hours, the cells were resuspended directly in the holes, and some part of suspension was taken out to determine the parameters of their functionong and the structural integrity.

     Methods for determination of functioning parameters and structural integrity of the tumor cells.

     Vitality of the tumor cells in the culture was estimated by the color of a vital pigment (trypan blue); on this purpose, we added 5% solution of this pigment to the culture and in 5 minutes determined the number of colored nonviable cells using a microscope. We calculated the percentage of colorless (viable) cells in the culture.

     The intensity of DNA synthesis in the tumor cells was estimated by the 3_H-thymidine introduction. On this purpose, we incubated 1.5 microcurie of tritium-labeled thymidine for 1 hour, transferred the cells on the filter. The results were expressed in the form of the depression index of 3_H-thymidine introduction I, which is the ratio of the number of pulses in the test culture to the number of pulses in the control culture. Each point was measured in triplication. We performed five independent experiments. The statistical treatment of results was carried out using the Student method with 5% criterion of reliability of difference between the test and the control results.

     Clonization activity of the tumor cells was estimated in boron sillicate capillaries. The tumor cells in the amount of 500,000 cell/ml in the whole medium of RPMI 1640 with 10% veal embryonic serum were mixed with agar prepared with twice distilled water and placed into a capillary. The capillaries were incubated for 14 days at 37°C in the atmosphere containing 5% of carbon dioxide. The clusters containing at least 50 tumor cells were considered as colonies. The efficiency of colonization (EC) was estimated as the persentage of colonies with respect to the number of cells sown. Three capillaries were filled for each point. The statistical analysis was performed using the Student method.

     Results

     1. Influence of the electrostimulation in generation of oxygen radicals and tumor necrosis factor by peritoneal macrophages.

It was found from the statistics that the electrostimulator significantly strengthens the spontaneous luminol-dependent chemiluminescence (Table 1). Simultaneously we observe the depression of luminol- and lucigenin-dependent luminescence activated by the latex particles. The maximum activating influence of the electrostimulator was observed in 60 minutes of combined incubation (the amplification effect was 280%). Amplification of the luminol-dependent chemiluminescence indicates strengthened generation of oxygen radicals formed upon decomposition of hydrogen peroxide. As was demonstrated in some investigations, just these radicals have the maximum cytotoxicity with respect to the tumor cells. On the other hand, the lucigenin-dependent chemiluminescence indicates formation of superoxide radicals, which are important in physiological respect, but do not virtually affects the vitality of tumor cells. It is remarkable that the maximum amplification of generation of the tumor necrosis factor by the phagocyting cells was observed in 60 minutes after incubation of the electrostimulator with the macrophage suspension (Table 2).

     The combined cultivation of the electrostimulator with the cells of EL-4 limphoma and MCA/77-23 sarcoma for 1, 2, and 6 hours did not virtually affects the vitality of the tumor cells (Figs. 1a and 2a). Unfortunately, it was impossible to determine the cell vitality in the experimental cultures after 24-hour incubation because of the sharp changes in the incubation medium of the cells under the influence of the operating electrostimulator.

     The proliferative activity of the cells of limphoma and sarcoma was significantly desreased under the influence of the electrostimulation after 1 and 6 hours of cocultivation (Figs. 1b and 2b). Thus, after one-hour influence of the electrostimulator, the depression index of thymidine introduction into DMA was 0.74 ± 0.06 and 0.8 ± 0.05 for the cells of limphoma and sarcoma, respectively. The corresponding values after 6-hour combined cultivation were 0.5 ± 0.01 (p<0.05) and 0.6 ± 0.04.

     The clonization activity of the tumor cells under the influence of the electrostimulator was studied on the EL-4 limphoma at the stage of logarithmic growth of tumor for the maximum thymidine introduction therein. The initial number of colonies formed in the capillary was 20.0 ± 1.8, and EC was 0.04%. In one hour, the number of colonies was insignificantly desreased and achieved the value of 15.0 ± 1.6, while EC was 0.03%. After 6 hours of incubation, the number of colonies in the test capillaries was decreased to 1 or 2, and EC was 0.0002%. The number of colonies in the control capillaries did not virtually change with respect to the time of incubation and was 20.0 ± 1.7, 20.0 ± 1.4, 19.0 ± 1.5, respectively, and EC was 0.04%. Thus, we observed a reliable reduction in EC in 6 hours of combined cultivation of the limphoma cells with the electrostimulator (p<0.05).

     Conclusion

     The performed investigations demonstrated that the autonomous electrostimulator of alimentary canal and mucosa has an expressed antitumor effect in the cell cultures. This stimulator reliably decreases the proliferative activity of cells of solid and ascitic tumors in the culture that was indicated by the depressed introduction of thymidine into DNA of the quickly reproducible tumor cells. However, this depression of precursor introduction into DNA is not associated with the direct cytostatic influence of the electrostimulator, because the vitality of the tumor cells was virtually unchangeable. We can suppose that a weak electromagnetic field generated by the electrostimulator depresses metabolism and the proliferation rate of the tumor cells, but does not affect the penetrability of the cell membranes. The electrostimulation has an ability to reduce significantly colonization of the tumor cells (the rate of colonization is an indicator, which characterize the rate of metastasis of tumors and the degree of their malignancy). The electrostimulation enhances the antitumor activity of the phagocyte cells, which are the basic cell elements of the natural antitumor immunity of an organism, strengthens their generation of oxygen free radicals registered by means of luminol-dependent chemiluminescence, and increases generation of the tumor necrosis factor by the cells.

     On the basis of the results obtained we can assume that the electrostimulator can be efficient medicine, which increases the resistance of an organism with respect to tumor and enhances the therapeutic effect of known chemotherapeutic preparations. However, the molecular and cellular mechanism of this activity has not been studied and requires additional specifications and comprehensive investigations.

Supervisor of studies doctor of medical science, professor

/signature/

L.G.Korkina