Effect of Dextromethorphan and Dimemorfan on the Neutral Lipids of Tumor Cells

Shigeru KIGOSHI, Kousaku KITAJIMA and Matomo NISHIO

Department of Pharmacology, Fukui Medical School , Matsuoka, Fukui 910-11, Japan

Accepted November 16, 1983

Dextromethorphan and dimemorfan , cen dihydrocodeine phosphate were dissolved in trally acting antitussives , have recently been physiological saline (20 mM). Ehrlich shown to be cytotoxic to Ehrlich carcinoma carcinoma cells or sarcoma-180 cells were cells or sarcoma-180 cells in vitro (1 ). inoculated intraperitoneally into ddY strain However, the mechanism responsible for the female mice (7-8 weeks of age). At 10 days cytotoxicity of these drugs on tumor cells after inoculation, the tumor cell suspension remains obscure. There are several reports was prepared as described previously (1) indicating that free fatty acids and their using Hanks balanced salt solution (pH 7.4) esters are involved in the tumor cell lysis by supplemented with 2% bovine albumin cytotoxic agents (2-5). This suggests that fraction V (VVako Pure Chem. Indust.). dextromethorphan and dimemorfan might Twenty ml of the tumor cell suspension affect the contents of neutral lipids ire tumor (2X 106 cells/ml) in 50-ml test tubes was cells. This paper reports that dextrome incubated at 37 °C for 1 20 min in the presence thorphan and dimemorfan cause a significant or absence of the drugs. After incubation, the decrease of triglycerides and cholesterol total lipids were extracted from the washed esters in Ehrlich carcinoma cells cr sarcoma tumor cells with chloroform-methanol (2:1, 180 cells in vitro, whereas dihydrocodeine v/v) by the method of Folch et al. (6, 7), and has little effect on the contents of these they were analyzed quantitatively according neutral lipids in the tumor cells in vitro. to the dichromate reduction procedure of The drugs used were as follows: dextro Amenta (8). methorphan hydrobromide (Maruko Phar Table 1 shows the contents of neutral maceut. Co., 5 mg/ml solution), dimemorfan lipids in Ehrlich carcinoma cells after incu phosphate (Yamanouchi Pharmaceut. Co.) bation for 120 min with or without 1 mM and dihydrocodeine phosphate (Takeda drugs. Ehrlich carcinoma cells treated with Chem. Indust.). Dimemorfan phosphate and dextromethorphan or dimemorfan contained

Table 1. Contents of neutral lipids in Ehrlich carcinoma cells treated with antitussives small amounts of triglycerides as compared sarcoma-180 cells were examined after in with the tumor cells incubated alone. The cubation with 1 mM dextromethorphan for triglyceride contents in Ehrlich carcinoma 1 5 to 120 min. The amounts of triglycerides cells treated with these drugs were one half and cholesterol esters in the tumor cells of those in the tumor cells incubated alone. were reduced progressively during the 120 Similarly, the amounts of cholesterol esters min incubation with dextromethorphan. The and free fatty acids in Ehrlich carcinoma cells reduction of these neutral lipids in the tumor treated with dextromethorphan or dimemorfan cells treated with the drug was about 30% were smaller than those in the tumor cells after 30-min incubation and about 40% after incubated alone (20-40% reduction in 60-min incubation, respectively. However, cholesterol esters and about 40% reduction in only a slight change was observed in the free fatty acids). In contrast, no significant contents of free fatty acids and cholesterol difference was observed in the quantities of in Ehrlich carcinoma cells or sarcoma-180 neutral lipids between Ehrlich carcinoma cells cells during the 60-min incubation with treated with and without dihydrocodeine. dextromethorphan. The contents of neutral lipids in sarcoma These results indicate that dextrome 180 cells were then examined after incubation thorphan and dimemorfan cause a significant for 120 min in the presence or absence of decrease of triglycerides and cholesterol 1 mM drugs. The incubation of sarcoma-180 esters in Ehrlich carcinoma cells or sarcoma cells with dextromethorphan resulted in a 180 cells in vitro at a concentration of 1 mM. decrease of triglycerides, cholesterol esters Concerning this, histological studies on and free fatty acids in the tumor cells (Table Ehrlich carcinoma cells or sarcoma-180 cells 2). The reduction of these neutral lipids in showed that many red granules (about 30 sarcoma-180 cells treated with the drug was granules/cell) were present in the cytoplasm about 40% for triglycerides and about 50% for of th° tumor cells before and after incubation cholesterol esters and free fatty acids, respec at 37°C for 120 min when the tumor cells tively. The amounts of triglycerides, choles were stained by the cil red 0 method for terol esters and free fatty acids were also neutral lipids (9). The number of red granules decreased in sarcoma-180 cells treated with in the tumor cells was decreased after dimemorfan (about 20% reduction in trigly incubation at 37°C for 120 min with 1 mM cerides and cholesterol esters, about 40% re dextromethorphan or dimemorfan (10-15 duction in free fatty acids). However, no granules/cell). significant change was observed in the con Recently, we have reported that dextro tents of neutral lipids in sarcoma-180 cells methorphan and dimemorfan are cytotoxic to after incubation for 120 min with dihydro Ehrlich carcinoma cells or sarcoma-180 cells codeine. in vitro, but dihydrocodeine has little effect In the following experiments, the contents on the viability of these tumor cells in vitro of neutral lipids in Ehrlich carcinoma cells or (1 ). When Ehrlich carcinoma cells and

Table 2. Contents of neutral lipids in sarcoma-180 cells treated with antitussives

immunity. Adv. Lipid Res. 16, 127-165 (1978)

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Identification of lipids synthesized and released by tumor cells under attack by antibody and complement. J. Immunol. 120, 1644-1650 (1978)

sarcoma-180 cells were incubated at 37'C References for 120 min with 1 mM dextromethorphan or 1 Kigoshi, S., Nishio, M. and Kokubo, M.: Effect of dimemorfan, the proportion of viable tumor the centrally acting antitussives on ascites tumor cells decreased from 85% to less than 20% , cells. Japan. J. Pharmacol. 33, 343-348 (1983) irrespective of the tumor cells examined . In 2 Meade, C.J. and Mertin, J.: Fatty acids and addition, the incubation of Ehrlich carcinoma cells or sarcoma-180 cells with 1 mM of 3 Turnell, R.W., Clarke, L.R. and Burton, A.F.: these drugs for 180 min resulted in the tumor Studies of mechanism of corticosteroid-induced cell lysis. Thus, the decrease in the proportion lymphocytolysis. Cancer Res. 33, 203-212 of viable Ehrlich carcinoma cells or viable (1973) sarcoma-180 cells may be accompanied by 4 Schlager, S.I., Ohanian, S.H. and Boros, T.: the decrease of triglycerides and cholesterol Stimulation of the synthesis and release of esters when these tumor cells are incubated with dextromethorphan or dimemorfan . 5 Schlager, S.I., Ohanian, S.H. and Boros, T.: Many studies have revealed that fatty acids and their esters are involved in the tumor cell lysis by cytotoxic agents. Schlager et al. have indicated that tumor cells under attack by complement-dependent cytotoxic anti 6 Folch, J., Lees, M. and Sloan Stanley, C.H.: A body release considerable amounts of tri simple method for isolation and purification of glycerides and cholesterol esters (4, 5). total lipids from animal tissues. J. Biol. Chem. Turnell et al. have reported that in cor 226, 487-509 (1957) ticosteroid-induced lymphocytolysis corti 7 Kigoshi, S. and Kitajima, K.: Effect of strepto costeroids cause a significant reduction of coccal lipids on Ehrlich ascites tumor cells. triglycerides in lymphocytes (3). Therefore, Japan. J. Pharmacol. 31, 201-209 (1981) triglycerides and cholesterol esters may be 8 Amenta, J.S.: A rapid chemical method for involved in the lysis of Ehrlich carcinoma cells quantitation of lipids separated by thin-layer chromatography. J. Lipid Res. 5, 270-272 and sarcoma-180 cells by dextromethorphan or dimemorfan. (1964) 9 Pearse, A.G.E.: Histochemistry, Theoretical and Applied. 3rd edition, Vol. 1, P. 697, Churchill, London (1968)