埼玉医科大学雑誌

Anti-Tumor Activity of Traditional Chinese Medicine, Ekki-Youketsu-Fusei-Zai, and Its Effects on Immunocyte Functions
1. A Life-Prolonging Effect and Enhancement of NK Cell Function in Tumor-Bearing Mice

Hong Deng^a,b, Kaori Nakajima^a, Xinling Ma^a, Kenichiro Hasumi^a,Toshitaka Akatsuka^b and Haruhisa Wago^c

^a)TCM laboratory, Electro-Chemical and Cancer Institute, 5-45-6 Kokuryo, Chofu, Tokyo 182-0022, Japan, ^b)Department of Microbiology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan, ^c)Laboratory of Immunology, Department of Medical Technology, Saitama Medical School Junior College, Moroyama, Iruma-gun, Saitama 350-0495, Japan

　　Some kinds of crude drugs of Traditional Chinese Medicine (TCM) have been so far useful for cancer patients as both an anti-tumor drug and an adjuvant, which greatly enhances the immunological functions of cancer patients^1,2). Ekki-Youketsu-Fusei-Zai (EYFZ), one of the TCMs, is a mixture composed of six kinds of crude drugs, which have been used for cancer patients in China. Recent studies have shown that each crude drug has various biological activities. For example, Astragali radix has immunologically enhancing and anti-tumor effects^3-5), while Angelicae radix has anti-inflammatory, analgesic, interferon inducing, and immunopotentiating effects^6-9). Cervi parvum cornu is known to show anti-tumor and immuno-enhancing effects by inhibiting the monoamine oxidase (MAO) activity^10-12), and Zizyphi fructus augments the function of natural killer cell as well as ciliary motility in the airway^13,14). Finally, Rehmanniae radix is reported to have anti-tumor activity, enhance cytotoxic T lymphocytes (CTL) activity and induce IL-2 production by T cells^15,16).
　　Though each component of EYFZ has shown various biological activities and EYFZ has been used for cancer patients, its anti-tumor effects and immuno-enhancing effects have not been investigated. In this study, we examined the effects of oral administration of EYFZ on the survival, tumor size, body weight and natural killer (NK) cell activity of tumor-bearing mice in order to elucidate the relationship between immuno-enhancing effects of EYFZ and its anti-tumor effects.

Animals and tumor cell line Specific-pathogen-free BALB/c female mice at the age of 5 weeks, originally purchased from Japan CLEA Co, were used throughout this experiment and approved by institutional animal care committee. Colon-26 cell line, colon-adenocarcinoma from BALB/c mice, was generously provided by Prof. Kikuo Nomoto, Kyushu University, and cultured in vitro with the basal medium (RPMI-1640) containing 10 % fetal bovine serum (FBS), 2.0 g/L NaHCO₃, 1.0 g/L HEPES, 0.6 g/L L-glutamine, and 0.25 mg/L Kanamycin. By a strong pipetting procedure, we obtained a single cell suspension and 5×10⁵ cells were implanted subcutaneously into the back of mouse. Murine lymphoma YAC-1 cell was maintained in the same culture medium as shown above.
Preparation of EYFZ EYFZ is a mixture composed of six kinds of crude drugs as shown in Table 1. All of the crude drugs were obtained from Tochimoto, Ltd., Osaka, and EYFZ was prepared as follows. First, a mixture of Astragali radix (10.0 g), Zizyphy fructus (5.0 g), Amomi semen (5.0 g), Angelicae radix (8.0 g), Cerve parvum cornu (5.0 g), and Rehamnniiae radix (10.0 g) was added to 200 ml water and soaked for 20 min. Then, it was boiled for 30 min for extraction of effective substances, and filtered. Finally, the extracted solution was condensed to 43 ml (1 g crude drug/ml) by heating evaporation, and orally administered to mice at a dose of 716.7 mg crude drug/kg. This dose was considered appropriate because oral administration of EYFZ into human is traditionally 43 g crude drug/60 kg/day.

Evaluation of survival, tumor size, and body weight of tumor-bearing mice Colon-26 (5×10⁵) cells were subcutaneously implanted into 6-week-old mouse at once, and simultaneously the oral administration of EYFZ was started. Control mice received the same volume of saline instead of EYFZ. Thereafter, the survival of these mice was monitored everyday for evaluating the life-prolonging effect. The tumor size and body weight were examined 2-3 times a week. The major axis (a) and minor axis (b) of the tumor were measured, size was estimated by using the formula ab²/2¹⁷⁾.
Assay of splenic NK cell activity The splenic NK cell activity of tumor-bearing mice was determined by lactate dehydrogenase (LDH) assay^18,19). The spleen cell suspension was prepared by squeezing the spleen between two glass slides. The distilled water was added to the spleen cell suspension for provoking lysis of red blood cells. After washing three times with serum-free basal medium, the cells were incubated in a 25 cm² culture flask (FALCON, Becton Dickinson) in serum-free basal medium at 37 ℃ in a 5 % CO₂ incubator for 2hr to remove adherent cells. The non-adherent cells were collected as effector cells. Effector cells (5×10⁶ cells/ml) were incubated with NK-sensitive target cells, YAC-1 (5×10⁴ cells/ml), in a total volume of 0.2 ml/well using 96 well round bottomed microplate (IWAKI Glass Co, Ltd.). An effector-target ratio of 100:1 was considered optimum. The plate was incubated for 4 hr at 37 ℃ in a 5 % CO₂ incubator. After incubation, 0.05 ml of the supernatant from each well was collected, then used for LDH assay to determine the cytotoxic activity using the LDH cytotoxic Kit (Wako Pure Chemical Industries, Ltd.). The percentage of specific release was calculated according to the following formula: % specific lytic activity ＝(experiment release - spontaneous release) / (maximum release - spontaneous release) × 100.
Statistical analysis Survival curve was determined using the method of Kaplan and Meier, and the log rank test was used to calculate the significance. Other data were statistically analyzed based on the Student's t test, and the differences were recognized significant with p value less than 0.05. The results were expressed as mean ±standard deviation (SD).

　　We first examined the effect of oral administration of EYFZ on the survival of tumor-bearing mice. When EYFZ (716.7 mg crude drug/kg/day) was continuously administrated to the mice which had been implanted subcutaneously with colon-26 for 28 days, the life-prolonging effect was found as shown in Fig. 1. All of tumor-bearing mice in the control group that received only the saline died within 59 days after the onset of this experiment (Fig. 1). On the other hand, the tumor-bearing mice treated with EYFZ showed a significant life-prolonging effect as compared to the control (p＜0.01), and died in 70 days on average.

　　Secondly, we compared the tumor size of EYFZ-administered group mice with that of control group mice. The tumor size, as described in Materials and Methods, was calculated by measuring the major (a) and minor (b) axis of formed tumor tissue based on the formula ab²/2. Result obtained from observation for 34 days revealed that the tumor size in EYFZ-administrated mice was smaller than that in the control mice (Fig. 2). Although the tumor size on day-13 was almost similar to that in the control, those on day-20 and on day-27 were clearly smaller than that in the control group (p＜0.5 and p＜0.1, respectively).

　　Thirdly, we examined the effect of oral administration of EYFZ on the body weight in tumor-bearing mice. When we observed the body weight of mice in the EYFZ-treated or control mice successively for 28 days after the onset of this experiment, it was shown that the body weight in the EYFZ-treated mice was much larger than that in the control mice (Fig. 3). Particularly, the body weight in the treated mice was significantly larger than that in the control mice on day-14 (pl0.05).

　　Finally, we examined the effect of oral administration of EYFZ on splenic NK cell cytotoxicity in tumor-bearing mice. We found that the NK cytotoxic activity of splenic cells in orally EYFZ-administrated mice was significantly higher than that in control mice on the day-17 and day-24 after the onset of transplantation and oral administration, as shown in Fig. 4 (p＜0.01)

　　In this experiment, we examined whether EYFZ, one of the TCMs, affected the anti-tumor activity in mice into which the murine colon-26 carcinoma cell line was subcutaneously implanted. This colon-26 cell line, an undifferentiated carcinoma induced by the carcinogen N-nitroso-N-methylurethan, has been successfully used as the model of tumor-bearing mice and cachexia^17,20). In our study, colon-26 cells could grow well after subcutaneous implantation into the normal BALB/c mice. Oral administration of EYFZ caused a statistically significant prolonging effect on survival in tumor-bearing mice as compared with the control mice. We also examined both the tumor size and body weight. The tumor size in EYFZ-administrated mice was shown to be much more decreased than that in the control mice. Particularly, the tumor size in the treated mice after 27 days became significantly smaller than that in the control. Hence, it is suggested that the oral administration of EYFZ was more effective compared with oral administration of saline for both the survival and the decrease of a tumor size in tumor-bearing mice.
　　The cachexia, an exhaustive state with severe weight loss, is a serious problem in cancer patients affecting their morbidity and mortality. It lowers their quality of life and shortens their life-span^21,22). Colon-26 cell line has been successfully used as the model of such cachexia by tumor growth¹⁷⁾. Thus, we observed the body weight of tumor-bearing mice everyday following the subcutaneous implantation of colon-26 cells into both the EYFZ-treated and the control mice. Results clearly showed that oral administration of EYFZ led to the better increase of body weight as compared to the control without EYFZ. Though the body weight of the control mice was lower than that of the EYFZ-treated mice, initially, it finally reached the similar level to that of the EYFZ-treated mice. This means the tumor growth in the control mice leads to the increase of body weight. We think the difference of the body weight between two groups of mice on day-14 is much more important that on the later days, and it reflects improvement of cachexia by EYFZ. However, since there was a possibility that EYFZ influenced the increase of the body weight of cancer-bearing mice from the nutritious viewpoints, it will be necesarry to further examine the effect of oral-administration of EYFZ on the body weight of the normal mice without cancer cell in the future.
　　NK cells exhibit spontaneous cytotoxic activity in a non-major histocompatibilty complex (MHC) restricted manner against virus-infected cells and cancer cells in vivo and their activity can be augmented by administration of interferon-γ(IFN-γ)^21-24). Some papers showed that some kinds of crude drugs and TCMs exert anti-tumor effects by activation of NK cells^13,25-29). Therefore, we also tried to examine the cytotoxic effect of oral administration of EYFZ on splenic NK cell activity in tumor-bearing mice on day-10, day-17, day-24 and day-31 following the subcutaneous implantation of colon-26 cells. Results clearly showed that the NK activity of spleen cells in orally EYFZ-administrated mice was significantly higher than that in control mice on the day-17 and day-24 after transplantation and oral EYFZ administration. Those results suggest the possibility that EYFZ has the anti-tumor effect on colon-26 implanted mice via augmentation of NK cell activity. In our present experiment, it was hard to investigate the cytokine production of NK cells in the spleen due to the insufficient number of splenic NK cells. However, as the NK activity was clearly augmented in the EYFZ-treated mice, further evaluation for EYFZ in anti-tumor activity seems to be needed by examining the effect of oral-administration of EYFZ on the cytokine production of NK cells in the spleen.
　　EYFZ does not show the direct cytotoxicity on some tumor cell lines including colon-26, A549 and Kato III (data not shown). This greatly suggests that the efficacy of EYFZ on survival and tumor-growth in tumor-bearing mice is attributable to the enhancement of host defense or immune system such as NK cell activity but not to the direct cytotoxicity against tumor cells. The efficacy of biological response modifier (BRM) in cancer therapy has been attributed to its activating functions on immune system including neutrophils, macrophages, NK cells, and cytotoxic T cells as well as on various cytokines. Therefore, it would be important to investigate further the effect of EYFZ on the immune system. This point is currently being pursued in detail in our laboratory.

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中国の伝統的漢方薬「益気養血扶正剤」の抗腫瘍活性と免疫細胞機能に及ぼす影響
1. 担癌マウスにおける延命効果とNK細胞機能の促進
Deng Hong^{1, 2)}，中島かおり¹⁾，Ma Xinling¹⁾，蓮見賢一郎¹⁾，赤塚俊隆²⁾，和合治久³⁾

¹⁾蓮見癌研究所中医学研究室， ²⁾埼玉医科大学微生物学教室，³⁾埼玉医科大学短期大学臨床検査学科
〔平成13年4月24日受付〕

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