AHCC

Effects of an only AHCC treatment in tumor-bearing animals.

AHCC(100mg/ml) was injected(i.v.) for 7days to ascites rats(WKAH rat) induced KMT-17 cells. AHCC treatment was effective for tumor-bearing rats induces 10 KMT-17 cells. This condition meant early stage. AHCC treatment was not effective for tumor-bearing rats induces 10 KMT-17 cells.

AHCC was injected intraperitoneally at a dose of 300mg/kg in every other day for 8 times to MM-46 cells-induced(s.c.) mice(C3H/He).
Tumor weights were measured at 4 weeks after AHCC administrations. Inhibitory effects were shown in AHCC treated group compared with in PSK treated group

	Tumor weights(g)	Ratio(%)
PS-K	1.96+-0.34	71
AHCC	1.54+-0.34	56
Control	2.76+-0.64	100

AHCC was administered orally or by peritoneal injections (20mg/kg, times) for 1*106 of Sarcoma-180 cells transplanted ICR mice(s.c.).
Tumor weights were measured at 25 days after tumor cells were transplanted.
Tumor progression was inhibited by treated with AHCC peritoneal injection signigicantly, and tended to be inhibited by oral administration of AHCC.

AHCC was administered orally at a dose of 1000mg/kg in 9 times or injected intraperitoneally at a dose of 20mg/kg in 5 times for 104 of Colon-26 cells transplanted in the spleen. PSK was administered orally at a dose of 1000mg/kg in 9 times or injected intraperitoneally at a dose of 10mg/kg in 5 times for the same model. Moreover, AHCC and PSK were coadministered orally or co-injected intraperitoneally. Body weights, original tumor weights, liver weights and number of metastatic hepatoma were measured at 14 days after transplanted tumor cells.

Intraperitoneal injection of AHCC reduced the number of metastatic hepatoma from spleen. Coadministration and co-injection of AHCC and PSK were more effective for metastasis.

Effects of coadministration of AHCC and CPA(cyclophosphamide)

1. AHCC(100mg/kg) or CPA(50mg/kg) were injected intravenously at 2 days intervals in 5 times form 2 days agter tumor cells were transplanted. Moreover, AHCC and CPA were co-injected in the same manner.

   AHCC and CPA coadministration showed prolongation of survival term and reduced the decrease of body weights. But these effects were not shown in an only AHCC treated mice.

2. AHCC were injected intraperitoneally for consecutive 7 days at a dose of 100mg/kg for 103 of KMT-17 cells transplanted rats. CPA were injected intravenously at a dose of 50 mg/kg at 3 days after tumor cells were transplanted. AHCC and CPA were coadministered in the same manner.

   AHCC and CPA coadministration showed prolongation of average survival term significantly while this effect did not show in an only AHCC administration. There was no differences on the changes of body weight but decrease of body weight shown in CPA administration was not show in the coadministration group.

Effect of coadministration of AHCC and bleomycin

For the 106 of cKDH-8/11(hepatocarcinoma cell line) tumor cell hypodermic injected rat, AHCC was taken by drinking freely at a dose of 100mg/kg/day, blemycin were injected intraperitpneally at 14 days after tumor cells transplanted, and AHCC and bleomycin were coadministered in the same manner.

The coadministration showed prolongation of survival term. But there were not effective for tumor cell progression in AHCC treated group, and NK cell activity did not enhance in AHCC treated group, too.

Coadministration of AHCC and UFT

AHCC was taken by drinking freely at a dose of 100mg/kg/day and UFT were administered orally at a dose of 15mg/kg from 3 days to 35 days after 106 of SST-2 breast cancer cells were transplanted(s.c.) to SHR rats. This model would die for lung metastasis. In another group, only UFT was administered orally at a same dose. Then, tumor progressions and lung metastasis were observed at 38 days after tumor cells were transplanted, and after original tumor were rejected at 21 days after rumor cell were transplanted, survival term were observed till rats died for lung metastasis.

The coadministration and UFT administration showed a reduction of tumor size at 38 days after tumor transplanted significantly. And coadministration of AHCC and UFT inhibited metastasis to the armpit lymph nodes.

Coadministration prolonged the survival term and there was significant difference on average survival rate in coadministration group.

Effect of coadministration of AHCC and AcD, MMC, and Adr

Masatoshi Yamazaki(Teikyo University)

AHCC(20mg/mouse), Actinomycin D(0.2μg/mouse), Mitonicin C(15μg/mouse) and Adriamycin (15μg/mouse) were administered respectively at 4 days, 7 days and 10 days after 2 * 105 of solid tumor cell(MH134 cell) were transplanted(s.c.) to C3H/He mice. Moreover, AHCC and Actinomycin C, AHCC and mitomycin C, and AHCC and Adriamycin were coadministered respectively. Tumor diameter was measured at 28 days and number of survival mice were counted at 60 days. A dose of chemotherapeutics was not effective one in only administration. < p/> Reduction of tumor size and prolongation of survival term were observed in only administration group a liitle. However, coadministration of AHCC and AcD, MMC, and Adr showed a reduction of tumor size and prolonged the survival term. Furthermore, 1~2mice were alive at 60 days in coadministration group.

Discussions

Coadministration of AHCC and chemotherapeutics which dose was not effective on were effective for reduction of tumor, prolongation of average survival rate and suvival term, and metastasis. Moreover, These coadministrations were effective for some side-effects, for example, decrease of body weight.

Furthermore, the dose of chemotherapeutics might be reduced by AHCC coadministration with chemotherapeutics, and these administration might be useful for treatment and decrease of side-effects.

Effect of AHCC on decrease of side-effects of antitumor agents.

Effect of AHCC on decrease of side-effects induced by CPA

Masatoshi Yamazaki(Tikyo University)

1. AHCC was injected intarperitoneally at a dose of 20mg/kg to normal mice. At 5 days after first administration of AHCC, AHCC(20mg/kg) and CPA(200mg/kg) were injected intraperitoneally. In other group, only AHCC or CPA were injected in the same manner, respectively. At 3 days after second AHCC administration, the weight of spleen were measured.

   AHCC coadministration inhibited the loss of body weights and the reduction of spleen induced by CPA.

2. 0.05%, 0.2% and 0.5% of AHCC were taken by drinking freely for 10 days in normal mice. CPA was injected intraperitoneally at a dose of 200mg/kg at 7 days. At 3 days after administration of CPA, tumor and body weight were measured.

   Coadministration of AHCC with CPA inhibited the loss of body weights induced by CPA. However, the reduction of spleen induced by CPA was not inhibit by AHCC coadministration.

Effect of AHCC on decrease of side-effects induced by CPA or 5-FU ( in normal mice)

Buxiang Sun, Koji Wakame and Tomomi Mukoda (Amino Up Chemical Co.,Ltd)

5-FU(50mg/kg) or CPA(100mg/kg) were injected intraperitpneally for 14 consecutive days, and 5-FU and CPA were coadministered in the same manner in normal ddY mice. AHCC were taken freely from 5% of AHCC contained fodder. Number of peripheral blood cells and indexes of sera and bone marrow were measured at 14 days.

AHCC reduced the decrease of erythrocytes resulted by antitumor agents. And only AHCC administration increased the erythrocytes, significantly. The increase of micronucleous erythrocytes induced by antitumor agents were suppressed by coadministration with AHCC. So, AHCC reduced or suppressed the mutagenicity of antitumor agents. 5-FU and CPA administrations suppressed the bone marrow according to the decrease of polychromatic erythrocyte/erythrocyte rates, nevertheless, coadministration of AHCC with these agents augmented significantly this rates. AHCC augmented the function of hematopoietic bone marrow significantly. AHCC might be useful for anemia.

Effect of AHCC on Ara-C induced alopecia

Tomomi Mukoda and Buxiang Sun (Amino Up Chemical Co.,Ltd)

AHCC protects against Ara-C-induced alopecia in the new born rat animal model 8-day-old SD rats were divided 5 groups. Control group was non-treated one, Ara-C group was that Ara-C was administered at a dose of 30mg/day for 7 consecutive days, and AHCC groups were that AHCC were treated orally; at a dose of 500mg/day, intraperitoneally; at a dose of 500mg/day, or onto skin for 7 consectuive days with Ara-C treatments at a dose of 30mg/day. The grade of alopecia was observed on day 9 of the experiment.

Ara-C treated mice were shown severe alopecia. However, the treatments of AHCC protected from alopecia induced by Ara-C. Especially, oral administration of AHCC showed protective effects from alopecia.

Moreover, the decreases and loss of heir follicles were shown in Ara-C treated mice, but coadministration of AHCC reduced these side-effects induced by Ara-C.

Alopecia grade 1 : 0~25% 2 : 25~50% 3 : 50~70% 4 : 75~100% hair loss

Group	n	Alopecia grade
		1	2	3	4
Control	3	3	0	0	0
Ara-C	7	0	1	1	5
Ara-C+AHCCp.o.	9	4	1	2	1
Ara-C+AHCCi.p.	10	1	5	1	2
Ara-C+AHCC swabbing	10	2	4	2	2

Discussions

AHCC reduced side-effects induced by chemotherapeutics. Especially, recover of hematopoiesis and the inhibition of loss of weight were observed.

Moreover, alopecia induced by chemotherapeutics were inhibited by coadministration of AHCC. Furthermore, AHCC suppressed mutagenesities of chemotherapeutics. The effects of AHCC on reducing of side-effects might be activation of hematopoiesis.

Effects of AHCC on immune system

The activities of NK and LAK cells

Yong-Qing Li, Kazuhiro Matsushita and Masuo Hosokawa (Hokkaido University, School of Medicine)

Effects of AHCC on NK cell activity

1. The groups AHCC was administered for C57BL/6 mice for 1 month, CPA was administered at 3 and 7 days before AHCC treatment, and AHCC and CPA were coadministered.
   After administrations of AHCC, NK cell activity of spleen cells was measured.
2. NK cell activity at effector phase was measured. AHCC was added to the phase when NK cells attacked target cells at doses of 1μg/ml, 10μg/ml, 100μg/ml.
   NK cell activity was enhanced by AHCC administration. NK cell activity was reduced by CPA administration, but coadministration of AHCC activates the NK cell activity.
   AHCC did not enhance the NK cell activity at an effector phase.
3. NK cell activity in SST-2 breast cancer model.
   AHCC was taken by drinking freely at a dose of 100mg/kg/day and UFT were administered orally at a dose of 15mg/kg from 3 days to 35 days after 106 of SST-2 breast cancer cells were transplanted(s.c.) to SHR rats. This model would die for lung metastasis. In another group, only UFT was administered orally at a same dose. Then, the spleen was removed at 38days after tumor cells were transplanted, and NK cell activity were measured by 51 Cr-release method at 38 days after tumor cells were transplanted.
   NK cell activity was depressed when UFT alone was administration. However, administration of AHCC in combination with UFT restored NK cell activity.

Effects of AHCC on LAK cell activity

AHCC was co-cultured with LAK cells for 5 days at the LAK cell induction phase. And the effects of AHCC on LAK cell induction of LAK cells was needed IL-2, effects of AHCC on LAK cell activity under the presence or absence of IL-2 were observed.

Co-culture of AHCC and LAK cell showed the enhance of LAK activity in 2 or 3 times. Concerned with the effects of IL-2, LAK activity was not enhanced under absense of IL-2 by administration of AHCC. LAK cell activity was enhanced under existence of IL-2 when AHCC added to the LAK cell inductive phase.

Discussions

AHCC administrations enhanced the NK cell and LAK cell activities in normal and tumor-bearing mice. The NK cell activity were depressed by administration of chemotherapeutics, but administration of AHCC in combination with these agents recovered NK cell activity. The enhancements of NK and LAK cell activities on effector phases were not observed in spite of AHCC administration.
These activities were not always enhanced by administration of AHCC, and the antitumor effects by AHCC administration can not be explained by only enhancements of these effectors.
AHCC administration enhanced the activities of NK cells in normal mice and recovered NK activities from UFT-induced reduction in SST-2 breast cancer cell transplanted rats.
Moreover, AHCC augmented the growth and activities of LAK cells under the presence of IL-1, but did not show any significant effect on P2 leukemia cells-transplanted animals.

Effects of AHCC on macrophages and productions of cytokines.

1. Akikuni Yagita (Kyorin University)
   AHCC administrations indeced the production of IL-12 in colon-26 tumor cells transplanted balb/c mice obviously though PS-K administrations did not show any remarkable changes of IL-12 level in the same model.
2. Yong-Qing Li, Kazuhiro Matsushita and Masuo Hosokawa (Hokkaido University, School of Medicine)
   AHCC were injected intraperitoneally for consecutive 7 days at a dose of 100mg/kg for 103 of MKT-17 cells transplanted rats. CPA were injected intravenously at a dose of 50mg/kg at 3 days after tumor cells were transplanted. AHCC and CPA were coadministered in the same manner.

   AHCC administration or coadministration with CPA increased peritoneal exudates cells (PEC). In these cells, macrophages were increased by AHCC administrations in spite of no change in T or B cells. Cytotoxities of these macrophages in peritoneal cavity against KMT-17 tumor cells as targets significantly enhanced compared with control group.

   AHCC administration induced IL-1 and THF α productivities of macrophages in peritoneal cavity, but no changes showed the levels of IFN γ and IL-10.

3. Coadministration of AHCC and UFT
   AHCC was taken by drinking freely at a dose of 100mg/kg/day and UFT were administered orally at a dose of 15mg/kg from 3 days to 35 days after 106 of SST-2 breast cancer cells were transplanted (s.c.) to SHR rats. This model would die for lung metastasis. In another group, only UFT was administered orally at a same dose. Then, OK-432(1 KE) were injected intraperitoneally at 36 days after tumor cells were transplanted, and at 2 days after injection of OK-432 macrophages were collected and measured NO production after cultured with SST-2 tumor cells.

   Coadministration of AHCC with UFT for SST-transplanted rats enhanced the production of nitric oxide (NO) and cytotoxities against tumor cells which decreased in non-treated rats.

   Administration of UFT decreased the appearance of IL-1 and TNF α but coadministration with AHCC recovered the production of IL-1 and TNF α, and iNOS.

   AHCC increased the numbers of peritoneal exudates cells and macrophages and enhanced the productivities of IL-1 and TNF α from these macrophages.

Effects of AHCC on differenciation and induction abilities in omental milky spots.

Nobuo Takemori (Asahikawa Kosei General Hospital)

The mouse omentum contains peculiar lymphoid tissues termed omental milky spots. They are composed of abundant lymphocytes/plasma cells with macrophages/monocytes, neutrophils, eosinophils, and various stromal cells. Lymphocytes in milky spots are known to incrude B- and T-cells. AHCC were dissolved in saline at doses of 0.5 , 5 and 50mg/0.5ml. These solutions were injected intraperitoneally in ddY mice for 7 consecutive days, respectively. Omental milky spots obtained one day after the last injection were examined by light and electron microscopy.

AHCC treatments induced and differenciated monocytes and macrophages in/into omental milky spots. And clustered macrophages appeared in omental milky spots in 50mg injection group. These macrophages had PAS-positive cytoplasm, which were deduced to be glycogen. Megakaryocytes appeared in omental milky spots by AHCC injections. They had also PAS-positive cytoplasm. Blood vessels were well developed in AHCC treatment groups. Moreover, the productions of granulocytes were also well developed in AHCC treatment group. Furthermore, clustered lymphocytes were observed in omental milky spots by AHCC treatments. They were distinctly positive for IgM, IgG and IgA. Then, lymphocytes induced by AHCC treatments were mainly B cell lines.

Discussions

Macrophages excruded into peritoneal cavity when AHCC was administered intraperitoneally. The productions of IL-1, IL-12 and TNF α, and cytotoxities of these macrophages were enhanced. It is suggested that macrophages activated by AHCC treatments are important for antitumor effects. Clustered macrophages were observed in omental milky spots by AHCC treatments. AHCC has the effects of differenciations and inductions of macrophages/monocytes.

Effects of AHCC on neutrophils

Masatoshi Yamazaki (Teikyo University)

AHCC concentrated neutrophils in peritoneal cavity which AHCC was injected. This effects is basic property of BRMs. AHCC were injected intraperitoneally at a dose of 20mg. Concentrated neutrophils were collected at 6 hours after administration of AHCC, and obtained the carprotectin included fraction. This fraction lead to apoptosis to EL-4 cells (mouse lymphoma cell) and MOLT-4 cells. As a results, these cancer cells were died fro apoptosis by AHCC ingredients.

Discussions

We knew that AHCC concentrated neutrophils. And in this study, these netrophils included the proteins which have anticancer effects.

Active substances in sera from mice treated with AHCC in short term

Buxiang Sun, Koji Wakame and Tomomi Mukoda (Amino Up Chemical Co.,Ltd )

1. AHCC added the to the media of cancer cell at doses of 1μg/ml and 10μg/ml. However, AHCC did not influence the growth of cancer cells.
2. C57BL/6 male mice were administered with diet containing 1% or 10% of AHCC for 10 days, and then the sera and their supernatants (6.5g/ml of protein contained) were added to the media of 3LL (Lowis lung carcinoma) cells at 5% concentration. The 3LL cells were incubated in 5% CO2 at 37C for 72 hours. The number of living cells were measured with MTT assay.
   Moreover, that supernatants which the proteins were precipitated with 0.5NPCA were added to the media of 3LL cells at the same concentration.
   The control sera from normal mice had no effects on the growth of 3LL cells. On the other hand, the AHCC-treated sera inhibited the growth of the cells and showed a dose-response.
   Furthermore, AHCC-treated sera which were treated with PCA inhibited the cell growth, too. These inhibited rates were stronger than those of non-PCA treated sera. The levels of cytokines, for example, TNF α, INF and IL-12 did not change in the sera treated with AHCC from 1 to 10 days.
3. AHCC was incubated with microsomal fraction (abbreviated as S9), which was prepared from ICR mice treated with Phenobarbital (80mg/kg weight in PBS) and 3-methylcholanthrene (20mg/kg in live oil) for 4 days, in the presence of HADPH-generating system at 37C for 1 hours. This "metabolite AHCC" and "original AHCC" were added to the media of 3LL. The number of living cells were counted after 72 hours.
   The "original AHCC" did not influence the growth of 3LL tumor cells during the concentrations of 1 to 100μg/ml. However, the "metabolite AHCC" reduced the number of the cells sbviously and showed a dose-response.
4. Satoru Yui and Masatoshi Yamazaki (Teikyo University)
   There are cell-mediated immunity and humoral immunity in host degence-system. In this time, the active substance in blood as humoral immunity was studied. AHCC were injected in C3H/He mice intraperitoneally at doses of 1mg or 10mg. The sera which were obtained at 24 hours after injection were added to the media of 3LL cells. After cultivation for two days, the progressed cell were counted using MTT-assay.
   Moreover, AHCC were injected to C3H/He mice at doses 10mg intraperitoneally. The sera at 5, 24 and 48 hours after injections of AHCC and 24 hours after injection of saline were collected. There sera were added to the media of 3LL cells.
   Furthermore, 5% aquous, AHCC were taken freely in mice for 2 days. The sera collected from mice were added to the media of 3LL cells. The number of living cells were 2 days after addition of the sera.
   The sera which obtained from AHCC-treated mice inhibited the growth of 3LL cells both at doses of 1mg and 10mg. Moreover, Concerning with morphology, 3LL cells cultured with sera from AHCC-treated mice showed round form, however, those from saline-treated mice extended spreadly.
   The sera at 5,24 and hours after injections of AHCC inhibited the proliferation of 3LL cancer cell compared with the sera at 24 hours after injection of saline.
   Furthermore, the sera obtained from oral administration of AHCC in mice also inhibited the proliferation of 3LL cells.

Discussions

There are various mechanisms in AHCC which boost the host immune system. Many effectors against cancer cells were activated and then AHCC shows anticancer effects. However, in the clinical study, other mechanisms not immune boosting effects might be suggested.
Since AHCC showed antitumor effects by oral administration, it is suggested that the components of AHCC might be changed by metabolite, and so on. This experiments showed the addition to the media of cancer cell proliferation. Moreover, the present study disclosed that a short administration of AHCC produced some active substances which inhibit the growth of tumor cells. These active substances differed from cytokines or lymphokines. We are now searching for them.

Effects of AHCC administration in cancer patients on NK activities

Takafumi Omori (Usa central internal Hospital)

NK activities in the sea which obtained from patients administered AHCC at doses of 3g/day were not always enhanced. Negative correlation between number of lymphocytes and NK activities were observed in liver and lung cancer patients.

The case of hepatocellar carcinoma

In this patients the chemotherapy had not been effective, and then the immunotherapy was started. Moreover, AHCC was administered at a dose of 3g/day. As a results, the level of α-fetoprotain decreased, and the number of leukocytes decreased but NK cell activity was enhanced.

The case of lung cancer

As two times of chemotherapies had no effects, administration of AHCC was started. After administration of AHCC, the number of lymphocytes increased but NK cell activity went down.

NK activities of effective cases in the patients administered AHCC at a dose of 3g/day

*Akiku Yagita (Kyorin University Hospital)

Biochemical and immunological indexes in the sera which obtained from CR and PR cases were measured. In these indexes, NK activities administered AHCC at doses of 3g/day or 6g/day were not always enhanced. However, the levels of TNF α and IL-12 increased. There were many cases the tumors disappeared by the enhancement of production of IL-12 and TNF α, and so on.
There were two paterns in the dissapearance of tumor were observed by the enhancements of NK activities.
The other cases were dissapearance of tumor were observed by the enhancements of the activities of killar T cells induced by IL-12.

Discussion

We had said that AHCC enhanced the NK activity, but from clinical studies, NK cell activity did not always enhanced. Moreover, there are many cases the relationship between the number of lymphocytes and NK cell activity did not found. In recent studies, there were many repots that AHCC enhanced the levels of cytokines such as TNF α, IL-12, and IFN and suppressed immunosuppresor such as IAP and IL-10.

Basic and clinical studies in AHCC-immunotherapy

Akikuni Yagita (Kyorin University)

AHCC were administered about 300 patients at doses of 3g/day till August in 1996. There were 46 CR or PR cases out of 58 effective ones. And 11 cases treated with AHCC were reported in detail.

cases		judgment
1. esophagus cancer	CR	Surgery was not operable. Administration of AHCC, tumor decreased and then disappeared. There was a improvement of appetite.
2. colon cancer with lung metastasis	PR	Tumor makers normalized from two months after treatment of AHCC. 5 tumors out of 6 disappeared.
3. breast cancer	CR	Tumor at metastasis disappeared.
4. hepatocellar carcinoma with lung metastasis		Tumor decreased from 1.5 months after administered AHCC and disappeared after treatment of AHCC.
5. hepatocellar carcinoma	CR	Tumor disappeared after treatment of AHCC.
6. colon cancer with liver metastasis	CR	Tumor disappeared after treatment of AHCC.
7. cecum cancer with carcinometis peritonetis	CR	Tumor disappeared after maker normalized after treatment of AHCC.
8. gastric cancer with liver metastasis		Tumor disappeared after treatment of AHCC.
9. gastric cancer	PR	Tumor disappeared after treatment of AHCC.
10. testicular carcinoma with lymph metastasis	CR	Hormone normalized and lymph metastasis disappeared after treatment of AHCC.
11. thyloid carcinoma and tumor at trachea		Repapse occurred after surgery. But after treatment of AHCC, tumor desappered.

Effective cases of treatment of AHCC-immunotherapy

	CR	CR~PR	PR	NC
lung cancer		1	4	4
hepatocellar carcinoma	1	2	5
breast cancer		1	4	3
gastric cancer	4		2	1
colon cancer	2	1	2	2
esophagus cancer	2			2
testicular carcinoma	2		1
ovarian cancer	3			1
thyloid carcinoma	1		2
prostatic cancer			2
renal cancer			1
uterus cancer			1
earlymph carcinoma				1
	15	5	26	12