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[CANCER RESEARCH 31, 501-504, May 1971] Effects of Some Fatty Acid Esters on the Viability and Transplantability of Ehrlich Ascites Tumor Cells Akiko Kato, Kunio Ando, Gakuzo Jamura, and Kei Arima Laboratory of Microbiology, Department of Agricultural Chemistry, University of Tokyo, Tokyo, Japan SUMMARY Fatty acids, monoglycerides, and some esters of fatty acids show antitumor activity against Ehrlich ascites tumor in mice. For investigation of the in vivo mode of action of these lipids, the effects of those fatty acid esters on animal cells in vitro were studied in sheep red blood cells and Ehrlich ascites tumor cells. Throughout these studies, we used fatty acid sucrose esters because they are soluble in water, so that we could obtain reproducible results without interference from the solubility of the samples. Most of the fatty acids and their esters showed hemolytic action in sheep red blood cells and tumor cell-killing activity in vitro. The tumor cells treated with such agents in vitro lost their transplantability in mice. The sucrose monoester of lauric acid, which showed the strongest in vivo antitumor activity, exhibited a strong in vitro activity, but polyoxyethylene sorbitan monolaurate (Tween 20), which had no antitumor activity, showed the stronger hemolytic and tumor cell-killing activities. These results indicate that antitumor activity of some fatty acid esters cannot be explained only by their physical attack of the tumor cells. INTRODUCTION In our screening for antitumor antibiotics, we found that fatty acids and monoglycerides isolated from acetone extracts of some fungal mycelia show antitumor activity in vivo against Ehrlich ascites tumor in mice (3, 5). Chemical study of the fatty acids and the monoglycerides indicated that they are the mixture of the fatty acids or monoglycerides of fatty acids with carbon numbers of 16 and 18 (2, 6). Antitumor activity of fatty acids was first reported by Nakahara (10-13). He reported that olive oil and fatty acids were effective in increasing the resistance of mice to several forms of transplantable tumors. Townsend et al. (21,22) found that lO-hydroxy-2-decenoic acid from royal jelly suppressed the development of a transplantable mouse leukemia and the formation of ascitic tumors in mice. They successively investigated the in vitro antitumor activity of mono- and dicarboxylic acids and their hydroxy- and keto- derivatives (9, 17-20), and it was found that most of the mono- and dicarboxylic acids, when mixed with 3 different ascites tumor cells at pH values below 5.0 and prior to inoculation into mice, completely suppressed the development of the ascites tumor but when acids were mixed Received February 2, 1970; accepted December 21, 1970. at higher pH values, only 2-decenoic, linoleic, and linolenic acids showed the activity. Unsaturated fatty acid fraction from the liver of X-ray-irradiated rabbit has been reported to have in vivo antitumor activity against the Brown-Pearce sarcoma of rabbit, Ehrlich ascites tumor in solid form, and several human carcinomas. The study on the chemical analysis of the fatty acid fraction was carried out by Seno and Yamamota (16), and they found that the main components were hexadecanoic, octadecanoic, octadecenoic, and octadecadienoic acids. Through the in vivo experiments with Ehrlich ascites tumor in mice, we studied the antitumor activity of each fatty acid (1), each monoglyceride (7), and other fatty acid esters (8). Our results indicated that the antitumor activity of the fatty acids depended upon the number of the carbon chain and the degree and the position of unsaturation. It was also found that some esters were active while others were not. In the course of these studies, we realized that the insolubility of the fatty acids and their esters in water might affect the results, or at least it was a problem to solve in carrying out the experiments. Tolnai and Morgan (20), meeting with the same problem, studied the amino acid-fatty acid salts, which were water soluble, and found that L-lysine and L-arginine laurate were effective against the Ehrlich and TN3 ascites tumor cells in vitro. We examined the sucrose ester of fatty acids that were freely soluble in water. The antitumor activity of sucrose esters of 7 fatty acids was studied and reported in a previous paper (8). As summarized in Table 1, all the sucrose esters inhibit the tumor growth. This finding of tumor inhibition by fatty acid sucrose esters enabled us to study the mechanism of action of fatty acids and their esters on tumor cells. Most of the works on the activity of fatty acids and their esters thus far reported were done with water-insoluble materials. All the works by Townsend et al. (21, 22), Morgan et al. (9), and Tolnai et al. (17-19) were carried out with water-insoluble fatty acids themselves, as was the work of Bennett et al. (4), who studied the effect of fatty acids on plasma membrane of Ehrlich ascites tumor cells and mouse erythrocytes. In these experiments, we used sucrose esters of fatty acids as in vivo antitumor-active materials, and Tweens as fatty acid derivatives with water solubility, which are inactive against the tumor in vivo. Hemolytic activity and in vitro attack of the tumor cells were studied, and results were compared with in vivo activity. In the study of the in vitro attack of the tumor cells, we utilized a differential staining method for dead and live cells and transplantability assay. MAY 1971 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1971 American Association for Cancer Research. 501 Akiko Kato, Kunio Ando, Gakuzo Jamura, and Kei Arima Table I Antitumor activity of some esters of ¡auricacid against Ehrlich ascites tumor in mice About 1 million of the tumor cells were implanted i.p. into 5-week-old ddY mice. A solution or suspension of the samples in 0.86% NaCl solution was administered once daily for 5 successive days. Six mice were used for each group. Control mice were given injections of 0.2 ml of the NaCl solution. 7daysBody CompoundsMonolaurinMethyl weight Tumor (g)+1.3+4.0+2.0+2.9++ gain time (days)>2926>272417182221>30>30>3026>30262528 laurateSorbitan laurateTween 20Sucrose +3.7+++ +4.2+++ +6.6+++ +6.9+2.9+ laurateSucrose 1.4+1.8+3.0+1.5+2.9+1.4+1.2+++ myristateSucrose palmitateSucrose linoleateControlDose(mg/mouse/day)10.02.520.05.010.05.010.02.516.04.020.05.010.02.518.04.5After +8.4Survival Hemolytic 012345678 Activity Agents with strong antitumor Ci2-sucrose Cm-sucrose Cie-sucrose C|'e-sucrose activity MATERIALS AND METHODS -I—I—I—I—i—\—I Sucrose Monoesters of Fatty Acids. These were synthesized according to the method of Osipow et al. (15). About 3 moles of sucrose are dissolved in dimethylformamide, and 1 mole of methyl ester of fatty acid is added with 0.1 mole of potassium carbonate as an alkaline catalyst. The reaction mixture is maintained at 90-95° under 80 to 100 mm Hg pressure. After 6 to 9 hr, the solvent is distilled off and the residue is dried in a vacuum to obtain sucrose ester, monoester being the main component. Other Esters of Fatty Acids. These were all available as chemicals or industrial detergents. Assay for Antitumor Activity in Vivo. Antitumor activity was assayed with the use of Ehrlich ascites tumor in mice. About 1 million of the tumor cells were implanted i.p. in ddY mice, 5 weeks old, weighing 18 to 22 g. A solution or suspension of the samples in 0.86% NaCl solution was administered once daily for 5 successive days, and the tumor growth and body weight gain after 7 days and life-span up to 30 days were observed. The life-span was shown as the average life-span of 10 mice in each group. Assay for Hemolytic Activity. Hemolytic activity of the esters of fatty acids was assayed with the use of sheep red blood cells. Sheep red blood cells were collected from the commercial sheep red blood cell suspension (Toshiba Kagaku, Tokyo, Japan) by centrifugation at 1500 rpm for 5 min, washed with 0.86% NaCl solution 3 times, and suspended in 100 volumes of the NaCl solution. Samples were dissolved or suspended in the 0.86% NaCl solution at the serial dilutions, and an equal volume of the red cell suspension was added. After incubation at 37°,for 4 hr, the hemolytic activity was determined. The activity was expressed by the highest dilution in which hemolysis was observed visually. 502 1—f- Cfe-sucrose C,4-PG Microbio! MG Agents with weak antitumor activity Ciu-MG Cio-sucrose dz-FA Ci2-Me ester Agents without antitumor Tween 20 Tween 40 activity I 2345678 Chart 1. Hemolytic activity of fatty acid esters. Samples were dissolved or suspended in 0.86% NaCl solution at serial dilutions starting with 1 mg/ml, and an equal volume of the sheep red blood cell suspension in the NaCl solution was added. After the incubation at 37° for 4 hr, the hemolytic activity was determined. The activity was expressed by the highest dilution in which hemolysis was observed. PC, propylene glycol; AfC, monoglyceride;FA, fatty acid;A/e, methyl. Assay for Viability of Tumor Cells. Ascitic tumor cells of Ehrlich carcinoma maintained by weekly i.p. transplantation were washed 3 times with PBS1 and suspended at a 1The abbreviation used is: PBS, phosphate-buffered saline. CANCER RESEARCH VOL. Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1971 American Association for Cancer Research. 31 An titumor Activity of Some Fatty Acids concentration of 1 X IO6 cells/ml. Equal volumes of the the mixture was determined by counting the number of sample in PBS and the tumor cell suspension were mixed and cells microscopically. incubated at 37°for 1 hr. The viability of the treated tumor Assay for Transplantability of Tumor Cells. Ehrlich ascites cells was determined by staining the cells with safranin dye tumor cells were treated as described for assay for viability according to the method described by Oda (14). To the except that the tumor cells were suspended at a concentration of 1 X IO7 cells/ml. After the incubation at 37°for 1 hr, 0.2 reaction mixture, safranin solution in PBS was added to a final concentration of 0.05%. The percentage of the dead cells in ml of the mixture was implanted i.p. into a ddY mouse. The survival time and tumor growth of the each mouse were ob Table 2 served. Ten mice were used for each group, and the control Effect of some fatty acid esters on Ehrlich ascites tumor cells mice were given injections of untreated tumor cells. Figures indicate the percentage of cells stained with safranin. To 0.5 ml of the tumor cell suspension (10* cells/ml), 0.5 ml of the sample suspension or solution was added. After incubation at 37°for 1 hr, safranin was added at a final concentration of 0.05%. The percentage of dead cells was determined by counting the stained cells microscopically. C, „,C,,, C14, C,0,, and C,28> indicate the esters of capric, lauric, myristic, stearic, and linoleic acids, respectively. Percent at 500 Mg/ml Compounds Agents activityC, with strong antitumor sucroseC, -, esterFungal 4 propylene glycol monoglycerideAgents activityC, with weak antitumor monoglycerideC, j sucroseC, „ esterAgents 2 methyl activityC,°8 without antitumor monoglycerideC,% esterTween methyl 20Control100100100100994310010063100 50 Mg/ml 5 Mg/ml 3010052891003 RESULTS In vivo antitumor activity of some of the fatty acid esters is shown in Table 1. While monolaurin and sucrose monolaurate show activity, sorbitan laurate and polyoxyethylene sorbitan monolaurate (Tween 20) have no activity. Studies on the activity of other esters (8) showed that most of the sucrose esters of fatty acids and the propylene glycol ester of myristic acid were the most effective against the tumor tested and that Tweens, sorbitan esters, and most of the methyl esters were ineffective. Hemolytic activity of some of the fatty acid esters was studied by the method described in "Materials and Methods." The purpose of this experiment was to examine whether there would exist a parallel relationship between hemolytic activity and in vivo antitumor activity. The result is presented in Chart 1. Hemolytic activity was expressed by the highest dilution in which hemolysis was observed. As can be seen from the chart, some of the esters with strong in vivo activity show strong hemolytic activity while others show very weak activity. Table 3 Viability and transportability of tumor cells treated by some esters at 2500 or 500 ng/W cells Equal volumes of tumor cell suspension (2 X IO7 cells/ml) and the sample suspension or solution (5 mg/ml or 1 mg/ml) were mixed and incubated at 37°for 1 ht. Viability was determined by staining the cells with safranin, and transplantability was determined by implanting 0.2 ml of the incubated mixture into a ddY mouse. Ten mice were used for each group. The figures after the compounds indicate the final concentration of the compound in mg/107 cells. CompoundsNoneLauric without cells(%)980880940850670870580102152Mice time tumor after 7 days (%)0100010001000100010001000100808040Survival (days)18.8>30.020.8>29.920.1>28.422.0>30.020.8>29.3 esterPalmitic sucrose esterOleicsucrose esterLinoleic sucrose esterElaidic sucrose esterPropylene sucrose myristateTween glycol 20Tween 802.50.52.50.52.50.52.50.52.50.52.50.52.50.52.50.5Viable MAY 1971 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1971 American Association for Cancer Research. 503 Akiko Kato, Kunio Ando, Gakuzo Tamura, and Kei Arima Tween 20, which showed no in vivo activity, demonstrated strong hemolytic activity. Thus there is no relationship between antitumor activity and hemolytic activity. Next, we examined in vitro action of those lipids on the tumor cells, thinking that each lipid might have specific affinity to the tumor cells and that those with strong affinity attack the cell and show antitumor activity. In vitro attack of the cell was measured by viability and transplantability of the treated tumor cells. The method is described in "Materials and Methods." In Table 2, viability of the tumor cells treated with some esters at 3 doses is presented with the reference to their antitumor activity. Here again, we see no strict relationship between in vitro and in vivo activities. Table 3 shows the viability and transplantability of the tumor cells treated with sucrose and propylene glycol esters of fatty acids and Tweens at concentrations of 2500 and 500 Mg/107 cells. At the higher concentration, none of the treated cells were viable; when inoculated into mice, these cells did not produce tumors. When the concentration was reduced to 500/ug/107 cells, none of the sucrose esters and propylene glycol ester showed any cytotoxic effects on the tumor cells. However, 90% of the cells treated with Tween 20 at the same concentration, 500/ug/107 cells, lost their transplantability. viability and consequently 4. 5. 6. 7. 8. 9. their 10. DISCUSSION It is significant that fatty acid esters with strong in vivo activity were less effective in vitro than Tween 20, which did not show antitumor activity in vivo. Thus antitumor activity shown by sucrose monoesters and some other esters of fatty acids cannot be explained only by their physical attack of the tumor cells. Differences in physiological and biochemical properties of the esters should differentiate their antitumor activity. Further studies are under way. This is the first report in which sucrose esters of fatty acids are used in biological studies. Our results suggest the possibility that sucrose esters of fatty acids could be used as water-soluble derivatives of fatty acids in many experiments. 11. 12. 13. 14. 15. 16. 17. ACKNOWLEDGMENTS We are thankful to Mr. Seikichi Suzuki for his kind advice and discussions. 18. 19. REFERENCES 1. Ando, K., Kalo, A., Suzuki, S., Tamura, G., and Arima, K. Antitumor Activity of Fatty Acids and Their Esters. I. Activity of Fatty Acids. In: H. Umezawa (ed.), Progress in Antimicrobial and Anticancer Chemotherapy, Proceedings of the Sixth International Congress of Chemotherapy, Vol. 2, p. 136. Tokyo, Japan: University of Tokyo Press, 1970. 2. Ando, K., Kato, A., Tamura, G., and Arima, K. Chemical Study of Fatty Acids with Antitumor Activity Isolated from Fungal Mycelia. J. Antibiotics (Tokyo), 22: 23-26, 1969. 3. Ando, K., Suzuki, S., Suzuki, K., Kodama, K., Kato, A., Tamura, G., and Arima, K. Isolation of Fatty Acids with Antitumor 504 20. 21. 22. Activity from Fungal Mycelia. J. Antibiotics (Tokyo), 22: 18-22, 1969. Bennett, L. R., and Connon, F. E. Effect of Lytic Agents on Plasma Membrane of Ehrlich Ascites Tumor Cells and Mouse Erythrocytes. J. Nati. Cancer Inst., 19: 999-1011, 1957. Kato, A., Ando, K., Kodama, K., Suzuki, S., Suzuki, K., Tamura, G., and Arima, K. Production, Isolation and Purification of Antitumor Active Monoglycerides and Other Antibiotics from Sepedonium ampullosporum. J. Antibiotics (Tokyo), 22: 67-72, 1969. Kato, A., Ando, K., Kodama, K., Tamura, G., and Arima, K. Identification and Chemical Properties of Antitumor Active Monoglycerides from Fungal Mycelia. J. Antibiotics (Tokyo), 22: 73-78, 1969. Kato, A., Ando, K., Suzuki, S., Tamura, G., and Arima, K. Antitumor Activity of Monoglycerides and Other Esters of Fatty Acids. J. Antibiotics (Tokyo), 22: 83-84, 1969. Kato, A., Ando, K., Suzuki, S., Tamura, G., and Arima, K. Antitumor Activity of Fatty Acids and Their Esters. II. Activity of Monoglycerides and Other Esters. In: H. Umezawa (ed.), Progress in Antimicrobial and Anticancer Chemotherapy, Proceedings of the Sixth International Congress of Chemotherapy, Vol. 2, p. 142. 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Chemical Analysis and Biological Activities of Fatty Acids from the Liver of X-ray Irradiated Rabbit, the Antitumor Agent So-called OX. Acta Med. Okayama, 19: 59-72, 1965. Tolnai, S., and Morgan, J. F. Studies on the in Vitro Antitumor Activity of Fatty Acids. III. Saturated Monocarboxylic Acids. Can. J. Biochem. Physiol., 39: 713-719, 1961. Tolnai, S., and Morgan, J. F. Studies on the in Vitro Antitumor Activity of Fatty Acids. V. Unsaturated Acids. Can. J. Biochem. Physiol., 40: 869-875, 1962. Tolnai, S., and Morgan J. F. Studies on the in Vitro Antitumor Activity of Fatty Acids. VI. Derivatives of Mono- and Di-carboxylic and Unsaturated Fatty Acids. Can. J. Biochem. Physiol., 40: 1367-1373, 1962. Tolnai, S., and Morgan, J. F. Studies on the in Vitro Antitumor Activity of Fatty Acids. VII. Effect of Amino Acid-Fatty Acid Salts. Can. J. Biochem., 44: 979-981, 1966. Townsend, G. F., Morgan, J. F., and Hazlett, B. Activity of 10-Hydroxydecenoic Acid from Royal Jelly against Experimental Leukemia and Ascitic Tumors. Nature, 183: 1270-1271, 1959. Townsend, G. F., Morgan, J. F., Tolnai, S., Hazlett, B., Morton, H. J., and Shuel, R. W. Studies on the in Vitro Antitumor Activity of Fatty Acids. I. lO-Hydroxy-2-decenoic Acid from Royal Jelly. Cancer Res., 20: 503-510, 1960. CANCER RESEARCH VOL. Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1971 American Association for Cancer Research. 31 Effects of Some Fatty Acid Esters on the Viability and Transplantability of Ehrlich Ascites Tumor Cells Akiko Kato, Kunio Ando, Gakuzo Tamura, et al. Cancer Res 1971;31:501-504. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/31/5/501 Sign up to receive free email-alerts related to this article or journal. 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