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Japanese Association for Integrative Medicine, Tokyo, Japan
Address all correspondence to Tetsuro Ikekawa, Japanese Association for Integrative Medicine,, Sanshin Bldg, 3F, 2-15-14, Uchikanda, Chiyoda-ku, Tokyo 101-0047, Japan; e-mail Ikekawa@aol.com
ABSTRACT; The present study demonstrated the antitumor effect of treatment combining EA6 isolated from culinary-medicinal mushroom Flammulina velutipes with surgical excision (SE) in mice using Meth-A fibrosarcoma. Oral administration of EA6 alone exhibited no inhibitory effect on the growth of intradermally inoculated Meth-A tumor cells, although it was effective for increasing the lifespan in mice bearing Lewis lung carcinoma or B-16 melanoma. In Meth-A fibrosarcoma, EA6, when administered orally, displayed a marked antitumor effect against the same tumor rechallenged after SE. When oral administration of EA6 (10 mg/kg/day) was begun on post excision day 1, the growth of the secondary tumor inoculated on post excision day 7 was significantly inhibited. When mice shown this inhibitory effect for the second solid tumor challenged, immune activity of the mice was tested. EA6 was found to enhance both humoral (i.e., antibody response to sheep red blood cells) and cell-mediated (i.e., anti-Meth-A delayed-type hypersensitivity response) immunity. In tumor-neutralization tests, the spleen cells from mice treated with both EA6 and SE markedly inhibited the growth of admixed Meth-A tumor cells. The antitumor immunity of the spleen cells was eliminated by treatment with anti-CD4 monoclonal antibody plus complement, but not with anti-CD8 monoclonal antibody plus complement. These results suggest that EA6 augments the anti-tumor immunity in combination with SE, and the effect is mediated by CD4-positive T cells.
KEYWORDS: Flammulina velutipes (Curt. Ex Fr.) Sing., EA6, antitumor effect, protein-bound polysaccharide, immunopotentiator, surgical excision, combination T
therapy
ABREVIATIONS
APC: antigen-presenting cells; C: complement; DTH: delayed-type hypersensitivity; ID: intradermally;MMC: Mitomaycin C; PBS:phosphate-buffered saline; PFC: plaque-forming cells; PO: peroral administration; SE: surgical excision; SRBC: sheep red blood cells
INTRODUCTION
The aqueous extracts of edible mushrooms showed extremely high growth-inhibitory activity against solid Sarcoma 180 (Ikekawa et al., 1969). Many antitumor polysaccharides were isolated from the mushrooms, and among them, a β-(1-3)-glucan isolated from Lentinus edodes (Berk.) Singer (shiitake mushroom) was named lentinan and used clinically as an injection by a narrow adaptation in Japan (Ikekawa, 2001).
From another popular cukinary-medicinal enokitake mushroom, Flammulina velutipes (W. Curt.;Fr.) P.Karst., which possesses high antitumor activity, the polysaccharides EA3 and EA5 and a low-molecular weight protein-bound polysaccharide, EA6 were isolated (Ikekawa et ai., 1973, 1982, 1985; Yosioka etal., 1973).. EA6 was active against a solid tumor of Sarcoma 180 by oral administration,but was not so effective whenadministered by intraperitoneal injection (Ohkuma et al., 1982,1983; Otagiri et al., 1983).
On one hand, EA6 was effective for syngeneic tumors--that is, it showed an increase in the lifespan of mice bearing Lewis lung carcinoma and B-16 melanoma (Ikekawa , 2000). On the other hand, an epidemiological survey in Nagano Prefecture, Japan indicated that the cancer death rate of farmers producing F. velutipes as a main occupation and consuming these mushrooms in their diets was remarkably lower than that of other people in the Prefecture (Ikekawa, 2001). Therefore, another epidemiological study was performed, A hospital-based case-control study indicated that intake of the edible mushrooms, including F. velutipes, had the possibility of reducing the risk of stomach cancer.(Hara et al., 2003).
In this study, the antitumor effect of EA6 in combined treatment with surgical excision (SE) on murine Meth-A fibrosarcoma and the mechanism of this effect were investigated.
MATERIALS AND METHODS
Reagents
EA6 was fractionated and isolated from the hot water extract of the fruit bodies F. velutipes. EA6-PII was a main component of EA6, obtained by purification of Sephadex column. It was dissolved in distilled water just before use and administered orally. MMC (Mitomycin C) was purchased from Kyowahakko Co. (Tokyo). Rabbit low cytotoxic complement (C) was purchased from Cedarlane Laboratories Ltd. (Ontario, Canada). Monoclonal antibodies to Thy-1, CD4, CD8, FITC-conjugated Thy-1, PE-conjugated CD4 and FITC-conjugated CD8 were purchased from BD PharMingen (San Diego, Calfornia,USA).
Mice
Specific pathogen-free female BALB/c and BDF1 mice were obtained from Charles River Japan, Inc. (Atsugi, Japan) and were maintained in a barrier system. Mice at 5-6 weeks old were used for experiments
Tumor
Meth-A fibrosarcoma cells used were maintained by serial intraperitoneal transplantation in syngeneic BALB/c mice. Lewis lung carcinoma and B-16 melanoma were also maintained by serial subcutaneous transplantation in BDF1.
Antitumor Effect of EA6 Against Lewis Lung Carcinoma and B-16 Melanoma
Lewis lung carcinoma cells (1.5 X 105cells/mouse, minced and made viable cell-counting ) were subcutaneously inoculated into female BDF1 mice. Twenty-four hours after inoculation, the test sample was dissolved in distilled water and administered orally for mice of the treated group at doses of 10 mg/kg or 5mg/kg every other day from Day 1 to Day19; for the control group, the same amount of distilled water was orally administered by the same schedule and volume. Increase in the life span (%) was calculated from the average value of survival days of mice in each group.
In B-16 melanoma the viable tumor cells (5~8 X 105 cells/mouse) was also subcutaneously inoculated into female BDF1 mice. The sample, dissolved in distilled water, was orally administered for mice of the treated group once a day Day 1 to Day 10.
Another experimental method was identically carried out with case of Lewis lung carcinoma described above.
Antitumor Effect of EA6 Alone Against Meth-A Tumor Cells
Meth-A tumor cells (1.25 X 105 cells/mouse) were intradermally (ID) inoculated into the left flank of each mouse. EA6 was orally administered at a dose of 10 mg/kg by three kinds of regimens; 7 consecutive days before the tumor cell inoculation, 7 consecutive days after the tumor cell inoculation, and both before and after the inoculation.
Antitumor Effect of Combined Treatment with EA6 and SE
In preliminary tests, we determined an inoculum size at the first and second inoculations to avoid autochthonous tumor regression. Meth-A tumor cells (2.5 X 104 cells/mouse) were inoculated ID into the right flank of BALB/c mice, and 7 days later the established solid tumors were surgically excised. After another 7 days, Meth-A tumor cells (1.25 X 105 cells/mouse) were reinoculated ID into the left flank of the same mice; tumor size was measured on the indicated days, and the lifespan of the mice was observed. EA6 was orally administered once a day from post-excision Day 1. Tumor growth was serially measured as the square root of long tumor diameter times short diameter in millimeters. There were six to seven mice in each experimental group.
Plaque-Forming Cells Assay
Plaque-forming cells (PFC) were assayed according to the method of Cunningham and Szenberg (1968). The mice were immunized by intravenous injection of 1.0 X 108 sheep red blood cells (SRBC) and removed 4 days after the immunization. To prepare spleen cell suspension, the minced spleen fragments were tapped with a rubber rod on a stainless 200-mesh sheet and mixed 0.1 mL of the suspension (5.0 X 106 cells/mL) with 20% SRBC and Hemo-lo guinea pig complement. The cell mixture was poured into a Cunningham's chamber and cultured it at 37°C for 1 hour under a 5% CO2 atmosphere, and then the number of plaques were counted.
Assay for Delayed-Type Hypersensitivity Responses
With a footpad assay we examined the specific delayed-type hypersensitivity (DTH) responses to tumor cells. Meth-A tumor cells (5.0 X 106 cells/mL) were treated with MMC (50 ?g/mL) at 37°C for 1 hour, then washed 3 times with cold calcium- and magnesium-free Dulbecco's phosphate-buffered saline (PBS). The prepared cells described above were inoculated into the footpads of the mice (1.0 X 106 cells/mouse), and 24 hours later the increase in footpad volume was measured using a plethysmometer (UNICOM, Chiba, Japan).
Treatment of Spleen Cells with Antibody Plus Complement
Spleen cells (1.0 X 108 cells/mL) incubated at 4°C for 60 minutes with a monoclonal antibody at a dilution of 1:50 for Thy-1, CD4, and CD8 were washed and incubated at 37°C for 45 minutes with rabbit low cytotoxic complement (C) at a final dilution of 1:10 (at this concentration C by itself had no non-specific cell lysis).
Tumor-Neutralization Test
The Winn assay method was used to measure tumor-neutralizing activity (Winn, 1961). Spleen cells (1.0 X 107 cells) were admixed with 1.0 X 105 viable Meth-A tumor cells. The mixed cell suspension (0.1 mL) was inoculated ID into the right flank of syngeneic BALB/c recipient mice; tumor growth was observed, and size was measured by its diameter.
Statistical Analysis
Each value is shown as mean ± standard error (SE). The statistical significance of differences was determined by Student's t-test. A probability (p) value less than 0.05 was considered to be significant.
RESULTS
Antitumor Effect of EA6 Against Lewis Lung Carcinoma and B-16 Melanoma
As shown Tables 1 and 2, oral administration of EA 6 was effective for increase in the lifespan of mice bearing syngeneic tumors, Lewis lung carcinoma and B-16 melanoma.
Antitumor Effect of Combined Treatment with EA6 and SE
Oral administration of EA6 did not inhibit markedly the growth of solid Meth-A fibrosarcoma, as shown in Figure 1. Likewise, EA6 administered by two other regimens had almost no effects on the growth of the tumor. The antitumor effect of EA6 alone was not found against Meth-A fibrosarcoma. However, EA6 showed a significant growth inhibitory effect against rechallenged Meth-A firosarcoma after SE. Figure 2 presents the antitumor effect of combined treatment with EA6 and SE. Seven days after Meth-A fibrosarcoma was ID transplanted into the right flank of BALB/c mice, the solid tumor of each mouse was surgically dissected out; seven days after the surgery, the second challenge with the same tumor cells, Meth-A was ID transplanted into the other flank of each mouse and the rechallenged tumor growth was observed.
EA6 was orally administered before, after, or before and after the reinoculation of Meth-A tumor cells. Although the pretreatment of EA6 slightly inhibited the tumor growth, the post-treatment was remarkably effective for the growth inhibition (Ikekawa, 2001). In this study, a dose response was investigated in post-treatment. Complete regression of the tumor could be seen in 2 out of 7 mice and 3 out of 6 in post-treatment at doses of 10 mg/kg and 30 mg/kg , respectively, as shown in Figure 2. It can be concluded that treatment of mice with both EA6 and SE in the mice significantly inhibited tumor growth and prolonged average survival time compared to treatment SE alone.
Effect of EA6 on Humoral and Cellular Immunity
The production of antibody to heterologous antigen, SRBC, was studied in mice treated with EA6 and SE using the same protocol as in the tumor inhibition test with the experiment in Figure 2. In the mice underwent SE, the number of PFC was lower than that in normal mice. But the PFC number returned to the normal level when mice were treated with both EA6 and SE (Fig. 3). Production of the antibody against SRBC in tumor-bearing mice was reduced by surgical operation. This reduction was reproducible, although it was not significant when compared with normal mice. Oral administration of EA6 enhanced antibody production and restored the reduction caused by surgery.
However, DTH response was tested in cases of SE alone or combined with EA6 .
The footpad volume in the SE group was 17.3 ± 2.5 ?L in mice footpad inoculated with MMC-treated Meth-A tumor cells, and 40.6 ± 6.4 ?L in the group treated in combination of EA6 after SE (Fig. 4). EA6 had augmented cellular rather than humoral immunity when treated with surgery.
Tumor-Neutralizing Activity of Spleen Cells from Mice Treated with EA6 and SE
We investigated whether or not combined treatment with EA6 and SE was capable of augmentation in vivo of tumor-neutralizing activity. Spleen cells isolated from mice at 1 week after tumor reinoculation were used as effector cells in a Winn assay. As shown in Figure 5, the spleen cells from mice treated with both EA6 and SE strongly inhibited the growth of admixed Meth-A tumor cells. But such an effect was not observed in spleen cells from mice treated with SE alone, and a significant difference was found in the SE-only group and the group of EA6 and SE combined.
Phenotype of Tumor-Neutralizing Cells Induced by EA6 and SE
To determine the cellular phenotype of tumor-neutralizing effector cells, spleen cells from mice treated with both EA6 and SE were given anti-Thy-1 antibody, anti-CD4 antibody, or anti-CD8 antibody plus C, and the augmentation effect of tumor-neutralizing activity was tested and compared to each group. Treatment of cells with anti-Thy-1 or anti-CD4 antibody plus C resulted in almost complete elimination of tumor-neutralizing activity (Fig. 6). In contrast, tumor-inhibiting effect was not eliminated by treatment with anti-CD8 antibody plus C.
DISCUSSION
The mechanism by which EA6 exerts its antitumor effects and the efficacy of EA6 on sinecomitant immunity in a mouse model was studied. EA6 had no cytotoxic effect in vitro on the tumor cells used (Ikekawa et al., 1973). Furthermore, although oral administration of EA6 showed antitumor effect against syngeneic tumors of Lewis lung carcinoma and B-16 melanoma (i.e., effective for increase in the lifespan of mice bearing the tumors), it was not active for another syngeneic tumor, Meth-A tumor. Specifically, EA6 alone was ineffective in inhibiting the growth of transplanted Meth-A tumor in vivo, as shown in Figure 1. However, EA6 showed strong antitumor activity when combined with surgical treatment (Figs. 2 and 5). Excision of the first implanted solid tumor slightly induced the antitumor activity against the second implanted tumor in the host mice, but EA6 significantly enhanced this activity, suggesting that the host mice acquired recognition of tumor antigens by surgical treatment.
According to tumor-neutralization assay (Fig. 6), the spleen cells from surgically treated mice with anti-Thy-1 plus C abolished the antitumor activity of those cells, and elimination of CD4-positive cells among T-cell populations abrogated antitumor activity.
However, of interest may be that elimination of CD8-positive cells did not diminish the antitumor effect enhanced by EA6. Therefore, the enhanced antitumor activity by EA6 was suggested to be mainly mediated not by CD8-positive cells but by CD4-positive cells. It is known that CD4-positive helper T cells are related to the recognition of tumor antigens and, subsequently, to the generation of CD8-positive cytotoxic T lymphocytes that are responsible for tumor eradication (Fujisawa et al., 1988). CD4-positive cells, however, are also reported to work as final effector cells in syngeneic tumor eradication (Yamamoto et al., 1987). Our present results are consistent with the latter report. DTH effector cells are included in CD4-positive T cells and associated with tumor regression (Mukai et al., 1987). As shown in Figure 4, EA6 also markedly enhanced the DTH responses in which Meth-A tumor cells treated with MMC were used as antigens. Recognition of tumor antigens by CD4-positive cells in antitumor immune responses requires the participation of Ia-positive adherent cells as antigen-presenting cells (APC) (Kosugi et al., 10987), although a study to clarify the effect of EA6 on APC in the protocol of tumor-bearing mice used here remains to be done.
Because EA6, when used without SE, showed weak activity in direct cytotoxicity tests and barely inhibited the tumor growth of Meth-A fibrosarcoma in this study, EA6 could not be used to trigger immune responses in the syngeneic tumor model tested, but could enhance established antitumor immunity (Ikekawa et al., 1973). These results suggest that EA6 can enhance cellular immunity, mainly through its effect on CD4-positive cells (helper T and DTH effector cells), and can restore the suppression of antibody formation in surgically treated mice. Considering all the data, it can be concluded that EA6 may be useful for cancer patients receiving chemotherapy and/or surgical tumor excision as a complementary therapy.
REFERENCES
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Legends to Figures
Fig. 1. Antitumr effect of EA6 alone against Meth-A fibrosarcoma in mice. Meth-A tumor cells (1.25 X 105) were inoculated i.d. into BALB/c mice. EA6 (10 mg/kg/day) was administered p.o. for 7 consecutive days after the tumor inoculation: ●, Control; ○, EA6.
Fig. 2. Antitumor effect of combined treatment with EA6 and SE.
SE was performed on day 7 after the first inoculation of Meth-A tumor cells (2.5 X 104) into BALB/c mice, and the second inoculation (1.25 X 105) was made in the same mice on post-surgery day 7, followed by measurement of tumor size. However, control mice first received tumor inoculation at the second inoculation time. Oral administration of EA6 was begun from the day following SE and continued throughout the experiment. Each experimental group is shown as follows: ●, control; ▲, SE alone; ○, EA6 and SE (3 mg/kg); △,EA6 and SE (10 mg/kg); □, EA6 and SE (30 mg/kg).
*Significantly different from SE alone at p<0.05.
Fig. 3. Effect of EA6 combined with SE on antibody production against SRBC.
Three days after the second inoculation of Meth-A tumor cells, the mice were immunized by intravenous injection of 108 SRBC. Four days later, PFC assay was performed. The result is expressed as the number of PFC per 106 spleen cells.
*Significantly different from SE alone at p<0.01. EA6 (10 mg/kg/day) was administered p.o. for 7 consecutive days after the tumor inoculation.
Fig. 4. DTH response to Meth-A tumor cells in mice treated with EA6 and SE.
On day 6 after the second inoculation, 106 MMC-treated Meth-A tumor cells were injected into the footpad of the right hind leg, and 24 h later the increase of footpad volume was measured. EA6 (10 mg/kg/day) was administered p.o. for 7 consecutive days after the tumor inoculation.
*Significantly different from SE alone at p<0.01.
Fig. 5. Tumor-neutralizing activity of spleen cells from mice treated with EA6 and SE.
Spleen cells were obtained from mice receiving various treatments on the 7 days following the second inoculation of Meth-A tumor cells. The spleen cells (107) were admixed with viable Meth-A tumor cells (105) and then inoculated into BALB/c mice. Each group is shown as follows: ●, normal mice; ▲, tumor-bearing mice; ■, SE-treated mice; ○, EA6 (10 mg/kg)- and SE-treated mice.
*Significantly different from SE alone at p<0.05.
Fig. 6. Cellular phenotype of effector cells mediating augmented tumor-neutralizing activity.
The experiment was performed exactly as described in Fig. 5 except that spleen cells were treated with various antibodies and C. Each group is shown as follows: ●, spleen cells obtained from normal mice; spleen cells obtained from mice receiving both EA6 and SE were not treated (○), or were treated with anti-Thy-1 and C (▲), anti-CD8 and C (△), or anti-CD4 and C (■).
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