Effects of Traditional Chinese Medicine, Ekki-Youketsu-Fusei-Zai,
on Survival and NK Cell Function of Tumor-Bearing Mice and on Cytokine Production
by Murine Macrophage Cell Line J774.1
Department of Microbiology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan
(Supervisor: Prof. Dr. Toshitaka Akatsuka)
医学博士 乙第792号 平成13年12月21日 (埼玉医科大学)
Materials and Methods
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(RPMI1640 with 2.0 g/L NaHCO3, 1.0 g/L HEPES, 0.6 g/L L-glutamine, and 0.25 mg/L Kanamycin) containing 10 % fetal bovine serum (FBS). 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), Angelica radix(8.0 g), Cervi parvum cornu(5.0 g), and Rehmanniae radix (10.0 g) was added to 200 ml distilled water (DW) and soaked for 20 min at room temperature. Then, it was boiled for 30 min for extraction of effective substances, and the solution was centrifuged at 3000 rpm for 20 min. Finally, the supernatant was condensed to 43 ml (1 g crude drugs/ml) by heating evaporation, and diluted with DW to desired concentrations for in vivo and in vitro experiments, and orally administered to mice at a dose of 716.7 mg crude drugs/kg. This dose was considered appropriate because oral administration of EYFZ into human is traditionally 43 g crude drugs/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, and then size was estimated by using the formula ab2/231).
Assay of splenic NK cell activity The splenic NK cell activity of tumor-bearing mice was determined by lactate dehydrogenase(LDH)assay32,33). 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 2 hr 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
J774.1 cell line culture The murine macrophage-like cell line, J774.1, was obtained from Riken Cell Bank (RCB, Japan). The cells were maintained in 75 cm2 plastic culture flasks (Falcon) in basal medium containing 5 % fetal bovine serum. Cells were incubated at 37℃ in a humidified atmosphere containing 5 % CO2. Cells harvested by gentle scraping were passaged every 3-4 days by diluting 1: 10 with fresh medium.
RT-PCR J774.1 cells were suspended in culture medium at a cell concentration of 5×104 cells/ml, and 5 ml of the cell suspension was plated in a 25 cm2 plastic culture flask (Falcon). Cells were cultured for 24 and 48 hrs in the presence of 1.2 mg crude drugs/ml of EYFZ, 10 ng/ml LPS or culture medium only, respectively, and after incubation, cells were harvested by scraping and quickly frozen and stored at −80℃. Total cellular RNAs were extracted from 1 x 106 J774.1 cells by the use of Rneasy Mini Kit (QIAGEN). The first strand cDNA synthesis was performed by incubating 0.5μg of either RNA sample in a total reaction volume of 20μl containing 2μl of 10 mM dNTP mixture, 2μl of 10 mM oligo-(dT), 10X reaction buffer for AMV reverse transcriptase, and 4 units (U) of AMV reverse transcripase XL (TOYOBO, Japan) at 42℃ for 1hr. The sequence of oligo-(dT) was 5'-GCTCTAGATTTTTTTTTTTTTTTTTTTTTT-3'. Twoμl of the first strand synthesis production and 2μl of each of 10 mM oligonucleotide primers were added to the reaction mixture (100μl ) containing 8μl of 10 mM dNTP mixture, 8μl of 25 mM MgCl2, 10μl 10X Ex TaqTM buffer, and 4 units (U) of Ex TaqTM (TAKARA, Japan). Each reaction was carried out as follows: IL-1β; denaturing at 94℃ for 5 min, 30 cycles of amplification (94℃ for 1 min, 58℃ for 2 min, and 72℃ for 3 min) and extension at 72℃ for 10 min. IL-12p40 and IFN-γ; denaturing at 94℃ for 5 min, 30 cycles of amplification (94℃ for 40 sec, 60℃ for 20 sec, and 72℃ for 49 sec) and extension at 72℃ for 5 min. IL-12p35, TNF-α and β-actin; denaturing at 94℃ for 5 min, 30 cycles of amplification (94℃ for 1 min, 55℃ for 1 min, and 72℃ for 2 min) and extension at 72℃ for 7 min.
Measurements of IL-12 IL-12 production by J774.1 cells was assayed by using ELISA kits OptEIATM Mouse IL-12 (p70) Set (PHARMINGEN) according to the protocols by manufacturer. Cells were cultured for 24, 48 and 72 hrs in the presence of 0.15 mg/ml, 1.2 mg/ml and 3.6 mg crude drugs/ml of EYFZ, 10 ng/ml LPS or culture medium only, respectively, and after incubation, the culture supernatants were collected and stored at −80℃ until use.
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).
Effect of EYFZ on a life-prolongation, tumor size and the body weight of
tumor bearing mice
We first examined the effect of oral administration of EYFZ on the survival of tumor-bearing mice. When EYFZ (716.7 mg crude drugs/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. 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.
Second, we compared the tumor size of EYFZ-administrated 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).
Third, 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 (p＜0.05).
Effect of EYFZ on NK cell activity of tumor-bearing mice 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).
Effect of EYFZ on the expression of cytokine mRNAs in J774.1 cells The cytokine mRNA expression of J774.1 cells treated with or without EYFZ was investigated. The gel electrophoretic patterns of the RT-PCR products of IL-1β, IL-12p35, IL-12p40, IFN-γ, TNF-αand β-actin are presented in Fig. 5. The expression level of IL-12p35 and IL-12p40 was induced in J774.1 cells after 12hrs and 24hrs of the treatment with EYFZ, though the expression of β-actin, a house keeping gene, was almost constant in each sample. We could not find any differences of other cytokine expressions between treated and untreated cells.
Effect of EYFZ on cytokine production in J774.1 cells We next examined whether or not this increased expression of IL-12 induced by EYFZ occurs at the protein level. After J774.1 cells were incubated with 0.15 mg/ml, 1.2 mg/ml and 3.6 mg crude drugs/ml of EYFZ for 24, 48 and 72 hrs, IL-12 in the culture supernatants was assayed by ELISA. As shown in Fig. 6, IL-12 secreted from J774.1 cells stimulated with EYFZ in the culture supernatants was significantly higher in concentration compared to those of the control.
In this experiment, we examined whether or not 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 cachexia31,34). 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
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-span35,36). Colon-26 cell line has been successfully used as the model of such cachexia by tumor growth31). 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 initially lower than that of the EYFZ-treated mice, it finally reached the similar level to that of the EYFZ-treated mice. This means the tumor growth in the control mice leading to the increase of body weight. We think the difference of the body weight between the two groups of mice on day-14 is much more important than that on the later days, and it reflects the 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 necessary to further examine the effect of oral-administration of EYFZ on 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)35-38). Some papers showed that some kinds of crude drugs and TCMs exert anti-tumor effects by activation of NK cells13,39-43). 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. 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.
To elucidate the relationship between the macrophage and anti-tumor effects of EYFZ, we also tried to examine the effects of oral administration of EYFZ on the functions of peritoneal macrophage in tumor-bearing BALB/c mice; however, it was difficult to get the some effects of EYFZ on macrophage functions. First, for example, mice got weak after the implantation of colon-26 cell, and it was difficult to collect enough cells of resting macrophage without thioglycollate-stimulation. Second, since the thioglycollate itself strongly stimulated the resting peritoneal macrophage in tumor-bearing mice, we could not find any difference between the EYFZ-administrated and control mice on functions of peritoneal macrophage. Third, J774.1 macrophage-like cell line was originated from the BALB/c mice and can be used for the study of macrophage function in vitro because this cell line was reported to show the same cytokine production as the macrophages44-47). Thus, in the present study, we examined the effect of EYFZ on cytokine production of J774.1 in vitro to elucidate the mechanisms underlying the EYFZ action on immune responses.
Macrophages are involved in almost all stages of the immune responses and play a role in the initial response to microbial infection before T- and B cell immunity are evoked. Mechanisms by which macrophages act as effector cells in a host defense system include both intracellular and extracellular cytokine secretion activities. Thus, we investigated here whether or not EYFZ could stimulate the expression and the secretion of cytokine from J774.1.
As shown in this study, EYFZ was able to induce IL-12 expression and production by J774.1. IL-12 has recently been brought into focus as an effector molecule to enhance immunity by murine macrophages48-50). Production of IL-12 by macrophages can be induced by interaction with activated T cells, which provides costimulatory signals via molecules such as CD40 ligand. These signals appear to be essential, because their inhibition can abrogate IL-12 production. IL-12 exerts multiple effects on T and NK cells including the augmentation of IFN-γ production, proliferation, and cytotoxic activity, and also plays an important role to determine a Th1/Th2 balance51). IL-12 is a heterodimeric cytokine composed of disulfide linked p40 and p35 subunits; both subunits have to be expressed within the same cell to produce biologically active p70 heterodimer52). It has been shown that p40 mRNA expression is up-regulated in the cells producing IL-12, whereas p35 mRNA is constitutively expressed in various cells53). As shown in Fig.4, the expressions of IL-12p35 and IL-12p40 were induced similarly as assessed at 12hrs and 24hrs after the treatment with EYFZ. Moreover, our results of ELISA show that this increased expression of IL-12p35 and IL-12p40 induced by EYFZ occurs at the protein level.
It was reported that IL-12 has anti-tumor effect54-57) and a powerful anti-tumor activity in mice against 17 different lines of transplantable murine tumors, including carcinomas, sarcomas, melanomas and lymphomas58). IL-12 is much more effective in anti-tumor effect than other cytokines such as IL-2 and IFN-α, and is effective at the doses with much lower toxicity. In our study, relationship is unclear between the effects of EYFZ on anti-tumor and enhancement of NK function in vivo and activation of macrophage-like cell line J774.1 in vitro. However, some papers showed that components of crude drugs of EYFZ include polysaccharide59-64), which can activate the function of macrophage. Thus, whether or not oral administration of EYFZ can induce macrophages to produce endogenous IL-12 is not clear now. So, this elucidation needs more study in the future.
In conclusion, we have found that EYFZ has the anti-tumor effects on colon-26 implanted mice via augmentation of NK cell activity and markedly activates J774.1 to produce IL-12, which is potentially an immune enhancer. Therefore, it would be important to investigate further the effective mechanism of EYFZ on the immune system.
I would like to thank Drs. Prof. Toshitaka Akatsuka and Prof. Haruhisa Wago
for their kind help and valuable suggestions in this experiment and Ms Kaori
Nakajima and Ms Xinling Ma for kind assistance with cell culture and mice-rearing.
I also appreciate Dr. Kenichiro Hasumi for critical reading of this manuscript
and financial help in this investigation.
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