ENHANCING EFFECT OF COENZYME Q10 ON IMMUNORESTORATION WITH MYCOBACTERIUM BOVIS BCG IN TUMOR-BEARING MICE*1

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1 ENHANCING EFFECT OF COENZYME Q10 ON IMMUNORESTORATION WITH MYCOBACTERIUM BOVIS BCG IN TUMOR-BEARING MICE*1 Ichiro KAWASE, Hisanobu NIITANI, Nagahiro SAIJO, Haruo SASAKI, and Tatsuhide MORITA National Cancer Center Hospital*2 Effect of the additional treatment with coenzyme Q10 on immunorestoration with Mycobacterium bovis BCG in tumor-bearing mice was investigated. Cellmediated cytotoxicity in tumor-bearing mice against alloantigenic tumor cells was determined by 51Cr release assay using spleen cells of C57BL/6N mice which had been inoculated subcutaneously with syngeneic melanoma-b16 and immunized intraperitoneally with alloantigenic mastocytoma P815-X2 cells. The cell-mediated cytotoxicity against mastocytoma P815-X2 cells was gradually depressed with the growth of melanoma-b16. The depressed, cell-mediated cytotoxicity in tumor-bearing mice recovered slightly by the treatment with BCG. The recovery effect of BCG on the depressed, cell-mediated cytotoxicity was significantly enhanced by the additional treatment with coenzyme Q10. Coenzyme Q10 did not have an apparent effect on the depressed, cell-mediated cytotoxicity in tumor-bearing mice. These results show that coenzyme Q10 enhances the immunorestoration with BCG in tumor-bearing mice. There have been many attempts to improve the depressed immune responsiveness in tumor-bearing host. Taniyama et al.9) have reported an encouraging result that the depressed, cell-mediated immune response in tumor-bearing mice can be significantly restored by the treatment with oil-attached BCG cell-wall skeleton (BCG-CWS). Bliznakov et al.1) have reported that mice treated with coenzyme Q became resistant to tumorigenesis with 3, 4, 9, 10-dibenzopyrene and leukemogenesis with Friend leukemia virus. They stated that the protective effect of coenzyme Q is based on the stimulation of the reticuloendothelial system1,2) (RES) and that coenzyme Q10 produces a more stimulative effect on RES than other coenzyme Q homologs.4) We have investigated the effect of coenzyme Q10 on immunorestoration with nonspecific immunopotentiators in immunosuppressed animals. We have already reported that the depressed IgM antibody-forming capacity of cyclophosphamide-treated mice is significantly restored by the combined treatment with Propionibacterium acnes and coenzyme Q10, while it is not apparently restored by the treatment with Propionibacterium acnes or coenzyme Q10 alone.6) The present work was undertaken to investigate the effect of coenzyme Q10 on immunorestoration with Mycobacterium bovis BOG in tumor-bearing mice. This paper describes the experiment to determine whether the additional treatment with coenzyme Q10 can enhance the recovery effect of BCG on the depressed, cell-mediated immune response in tumor-bearing mice. *1 This research was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture.

2 T. KAWASE, ET AL. MATERIALS AND METHODS Animals and Tumors Eight-week-old inbred female C57BL/6N and DBA/2 mice were used. Mastocytoma P815-X2 cells (mastocytoma cells) from DBA/2 mice were serially passaged in an ascites form through DBA/2 mice. Melanoma-B16 (melanoma cells) from C57BL/6N mice was maintained serially by subcutaneous passage in C57- BL/6N mice. Cell suspension of melanoma-b16 was obtained by mincing of solid tumor in minimum essential medium (MEM). Viability of tumor cells was determined by the Trypan Blue dye exclusion test. As reported by Taniyama et al.,9) we also confirmed by in vitro cytotoxicity test using cultured melanoma cells that there was no cross-reactivity in antigenicity between melanoma cells and mastocytoma cells. BCG Lyophilized Mycobacterium bovis of Japan strain (Japan BCG Laboratory, Tokyo) was suspended in saline before use. Coenzyme Q10 Coenzyme Q10, purified from beef heart by the method of Crane et al.,5) was kindly provided by Dr. T. Kawachi, National Cancer Center Research Institute. Four milligrams of coenzyme Q10 was dissolved in 0.2ml of ethanol in a water bath at 60 and emulsified with 1.8ml of saline just before use. Experimental Procedure The experiment was carried out according to the method of Taniyama et al.9) C57BL/6N mice were inoculated subcutane- Chart 1. Schedule of inoculation with melanoma cells, immunization with mastocytoma cells, and administration with BCG or coenzyme Q10 in each experimental group Gann

3 IMMUNORESTORATION WITH BCG AND COENZYME Q10 Fig. 1. Depression of cell-mediated cytotoxicity in tumor-bearing mice Release with spleen cells -spontaneous release Maximum release was determined by freezing and thawing of the tumor cell suspension three times. Spontaneous release was measured by incubation of tumor cells without spleen cells. The results were analyzed for statistical significance by Student's t-test. RESULTS Cell-mediated Cytotoxicity in Tumorbearing Mice Fig. 1 shows cell-mediated cytotoxicity of spleen cells against alloantigenic mastocytoma cells determined by 51Cr release assay in C57BL/6N mice inoculated subcutaneously with melanoma cells and immunized intraperitoneally with mastocytoma cells. The cell-mediated cytotoxicity gradually decreased with the growth of melanoma-b16, and was markedly depressed in the last stage of tumor-bearing mice. Effect of BCG and Coenzyme Q10 on Cell-mediated Cytotoxicity in Tumorbearing Mice Table I shows the effect of BCG, coenzyme Q10, or their combination on the cell-mediated cytotoxicity in mice inoculated subcutaneously with melanoma cells 19 days before the cytotoxicity test (19- day tumor-bearing mice). The cell-mediated cytotoxicity in 19-day tumor-bearing mice (Group 2) was one-half of that in control mice (Group 1). The depressed, cellmediated cytotoxicity was not apparently restored by the treatment with BCG (Group 3) and was significantly restored to the normal level by the combined treatment with BCG and coenzyme Q10 (Group 4). Coenzyme Q10 did not have an apparent effect by itself on the depressed cell-mediated cytotoxicity in 19-day tumor-bearing mice (Group 5). The enhancing effect of coenzyme Q10 on immunorestoration with BCG was also observed in mice inoculated subcutaneously with melanoma cells 26 days before the cytotoxicity test (26-day

4 I. KAWASE, ET AL. Table I. Recovery Effect of BCG and Coenzyme Q10 on Depressed, Cell-mediated Cytotoxicity in 19-day Tumor-bearing Mice Table II. Recovery Effect of BCG and Coenzyme Q10 on Depressed, Cell-mediated Cytotoxicity in 26-day Tumor-bearing Mice tumor-bearing mice). As shown in Table II, the cell-mediated cytotoxicity was markedly depressed in the last stage of tumorbearing mice (Group 6), compared with that in control mice (Group 1). The markedly depressed, cell-mediated cytotoxicity was slightly restored by the treatment with BCG (Group 7), and was restored more by the combined treatment with BCG and coenzyme Q10 (Group 8), while it was not apparently restored by the treatment with coenzyme Q10 alone (Group 9). There was no significant differance in tumor weight among Groups 2, 3, 4, and 5 of 19-day tumor-bearing mice, and among Groups 6, 7, 8, and 9 of 26-day tumorbearing mice (Tables I and II). From these results, it is evident that the additional treatment with coenzyme Q10 enhances the recovery effect of BCG on the depressed, cell-mediated cytotoxicity in tumor-bearing mice. DISCUSSION The results of this study demonstrate that the additional treatment with coenzyme Q10 enhances the immunorestoration with BCG in tumor-bearing mice. However, the combined effect of BCG and coenzyme Q10 in 26-day tumor-bearing mice was not so powerful, compared with that in 19- day tumor-bearing mice, showing difficult recovery of the markedly depressed immune responsiveness in the advanced stage of tumor-bearing host. Gann

5 IMMUNORESTORATION WITH BCG AND COENZYME Q10 Bliznakov et al.2, 4) reported that coenzyme Q10 stimulates RES because of its enhancing effect on the phagocytic activity in rats and on the humoral immune response to sheep erythrocytes in mice. On the other hand, Sugimura et al.8) reported that coenzyme Q10 does not have an apparent adjuvant effect on the humoral immune response to bacterial a-amylase in mice, on the helper function of the carrier-primed, thymus-derived lymphocytes (T-cells) in mice, and on the development of the delayed type hypersensitivity to ABA-N-acetyltyrosine in guinea pigs. In the present experiment, coenzyme Q10 did not show an apparent effect by itself on the depressed, cell-mediated cytotoxicity in tumorbearing mice. Therefore, the mechanisms involved in the enhancing effect of coenzyme Q10 on the immunorestoration with BCG in tumor-bearing mice cannot be explained only by the stimulation of RES. We reported that oligomycin-sensitive ATPase activity of spleen lymphocytes was significantly depressed in the advanced stage of tumor-bearing rats.7) This fact suggests that the energy-producing capacity of lymphocytes is depressed in tumorbearing animals. Coenzyme Q is one of the essential components of the electron transport system in mitochondria and is therefore of extreme importance to the energy-producing system of the cell. One of the possibilities is that administration with coenzyme Q10 might restore the depressed energy-producing capacity of lymphocytes resulting in the improvement of the hyporesponsiveness of lymphocytes against BCG in tumor-bearing mice. The other possibility to be considered is that coenzyme Qlo might act as an adjuvant on the immunorestoration with BCG because of lipophilic nature of coenzyme Q. Further investigation of these possibilities is now under way. We express our thanks to Dr. T. Sugimura, Director of the National Cancer Center Research Institute, for his encouragement throughout this research, to Prof. Y. Yamamura of Osaka University Medical School, for the kind supply of mastocytoma P815-X2 cells, and to Dr. S. Tsukagoshi, Cancer Chemotherapy Center, Tokyo, for the kind supply of melanoma-b16. (Received January 28, 1978) REFERENCES