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International Journal of Clinical Medicine, 2012, 3, 361-367
http://dx.doi.org/10.4236/ijcm.2012.35069 Published Online September 2012 (http://www.SciRP.org/journal/ijcm) 1
Active Hexose Correlated Compound (AHCC) Alleviates
Gemcitabine-Induced Hematological Toxicity in
Non-Tumor-Bearing Mice*
Daisuke Nakamoto, Kota Shigama, Hiroshi Nishioka#, Hajime Fujii
Amino Up Chemical Co., Ltd., Sapporo, Japan.
Email: #nishioka@aminoup.jp
Received May 16th, 2012; revised June 17th, 2012; accepted July 16th, 2012
ABSTRACT
Active hexose correlated compound (AHCC) is known as a dietary supplement derived from an extract of a basidiomy-
cete mushroom. The present study was conducted to evaluate the role of AHCC in alleviating the side effects, particu-
larly hematological toxicity, in non-tumor-bearing mice receiving monotherapy with gemcitabine (GEM). The results
from the GEM treatment groups with and without AHCC administration were compared to control group that received
vehicle alone. The GEM alone treatment reduced peripheral leukocytes and hemoglobin, and bone marrow cell viability
in spite of no influence on body weight, food consumption, and renal and hepatic parameters. Supplementation with
AHCC significantly alleviated these side effects. The colony forming assay of bone marrow cells revealed that AHCC
improved reduction of colony forming unit-granulocyte macrophage (CFU-GM) and burst forming unit-erythroid
(BFU-E) related to GEM administration. However, when mRNA expression of granulocyte-macrophage colony-stimu-
lating factor (GM-CSF) and erythropoietin (EPO) was examined using a quantitative reverse transcription polymerase
chain reaction (RT-PCR), AHCC showed no effect for the mRNA levels of their hematopoietic growth factors. These
results support the concept that AHCC can be beneficial for cancer patients with GEM treatment through alleviating the
hematotoxicity.
Keywords: Anticancer Drug; Bone Marrow Suppression; Colony Formation; Hemoglobin; Mushroom Extract; White
Blood Cells
1. Introduction
Gemcitabine (2’,2’-difluoro-2’-deoxycytidine, GEM), a
pyrimidine based nucleoside analog, [1] is metabolized
to gemcitabine diphosphate and triphosphate inside the
cell by nucleoside kinases [2]. Gemcitabine diphosphate
is a potent inhibitor of ribonucleotide reductase, which is
associated with deoxyribonucleotide pools [3]. A reduce-
tion of deoxyribonucleotide concentration leads to the
inhibition of DNA synthesis. Gemcitabine triphosphate
competes with deoxycytidine triphosphate (dCTP) in
binding to replicating DNA polymerases and then is in-
corporated into DNA to prevent further elongation of the
replicating strand, resulting from increase in the ratio of
cellular concentrations of gemcitabine triphosphate to
dCTP [4]. Thus, the major mechanism of action of GEM
is the direct or indirect inhibition of DNA synthesis.
In cancer therapy, GEM is commonly used as a com-
ponent of adjuvant chemotherapy for advanced pancre-
atic cancer [5]. Additionally GEM is also used for the
treatment of various other carcinomas such as non-small
cell lung cancer [6], ovarian cancer [7], breast cancer [8],
and biliary tract cancer [9]. The limited toxicity associ-
ated with GEM therapy compared to other cytotoxic
anticancer drugs is one of the major reasons for the
widespread use in chemotherapy. Although hematologi-
cal toxicity and flu-like symptoms caused by GEM are
the most common side effect, they are mild and short-
lived [10]. However, these toxicities related to GEM can
lower the quality of life in cancer patients and often trig-
ger reductions in the dosage, frequency and duration of
chemotherapy, ultimately decreasing potential for opti-
mal therapeutic outcomes.
An approach to relieve the side effects of anticancer
drugs including GEM leads to the use of complementary
and alternative medicine (CAM) that has attracted great
attention. Many cancer patients are currently using CAM
in order to reduce the side effects and obtain additional
chemotherapeutic effects through boosting the immune
system [11]. In Japan, 44.6 percent of cancer patients
*No competing financial interests exist.
#Corresponding author.
Copyright © 2012 SciRes. IJCM
Active Hexose Correlated Compound (AHCC) Alleviates Gemcitabine-Induced
Hematological Toxicity in Non-Tumor-Bearing Mice
362
reported using CAM with the most frequently used treat-
ment being dietary supplements of mushrooms such as
agaricus (Agaricus blazei Murill) and active hexose cor-
related compound (AHCC) [12].
AHCC is a mixture of polysaccharides, amino acids,
lipids and minerals derived from mycelial culture of the
basidiomycete, Lentinula edodes. The predominant com-
ponent of AHCC is oligosaccharides, which contain
-1,4 glucans and partially acetylated
-1,4 glucans with
a molecular weight of around 5000 Daltons. AHCC has
been shown to increase the number and function of den-
dritic cells in healthy adult humans [13] and enhance
both the activation and proliferation of CD4+ and CD8+ T
cells in tumor-bearing mice [14]. AHCC also strength-
ened the chemotherapeutic effects of UFT (tegafur and
uracil in a 4:1 molar concentration) for mammary ade-
nocarcinoma SST-2 cells in rats [15] and cisplatin for
Colon-26 tumor cells in mice [16]. Furthermore, two
human clinical studies in liver cancer patients showed a
significant increase in survival rate among those taking
AHCC. [17,18] Several studies have explored the allevi-
ating effects of AHCC for chemotherapy-related side
effects. In cisplatin-treated tumor-bearing mice, AHCC
improved food consumption, renal damage and myelo-
suppression [16]. The role of AHCC in attenuating vari-
ous side effects was also explored in non-tumor-bearing
mice receiving monotherapy with paclitaxel, or multi-
drug chemotherapy including cisplatin plus paclitaxel,
cisplatin plus 5-fluorouracil, 5-fluorouracil plus irinote-
can, cyclophosphamide plus doxorubicin, and 6-mercapto-
purine plus methotrexate [19,20]. In newborn rats, the
AHCC-treated group was protected from cytosine arabi-
noside-caused hair loss [20].
We investigated the influence of AHCC on some of
the side effects associated with GEM as an initial study
in preparation for a human clinical trail. Non-tumor-
bearing mice, but not tumor-bearing mice, were chosen
so that the intrinsic alterations related to the anticancer
agent could be assessed independent of oncological
variables. In the present study, we focused on GEM-in-
duced hematotoxicity including bone marrow suppres-
sion, which is a dose-limiting toxicity.
2. Materials and Methods
2.1. Reagents
Active hexose correlated compound (AHCC; Amino Up
Chemical Co., Ltd., Sapporo, Japan) was produced from
the mycelia culture of Lentinula edodes in a manufactur-
ing process according to Good Manufacturing Practice
(GMP) standards for dietary supplements, and ISO9001:
2008 and ISO22000: 2005 criteria [16]. After pre-culti-
vation in flasks, the basidiomycete was cultured in 15-ton
large tanks for 45 days, and then AHCC was obtained
through filtration, sterilization, concentration and freeze-
drying. Gemcitabine (GEM) is a commercially available
anticancer drug as Gemzar Injection (Eli Lilly Japan K.
K., Kobe, Japan), and the drug was obtained from JUN-
SEI CHEMICAL CO., LTD. (Tokyo, Japan).
2.2. Animals
Specific pathogen-free male ddY mice were purchased
from Japan SLC, Inc. (Hamamatsu, Japan) and studied at
six weeks of age. Animals were maintained in a tem-
perature- and humidity-controlled room at 23˚C ± 1˚C
and 55% - 60%, respectively, under a 12-hour light-dark
cycle (lights on 08:00 to 20:00), fed a standard pelleted
rodent chow (CE-2; CLEA Japan Inc., Tokyo, Japan),
and given water ad libitum. Mice were divided into three
groups: control (untreated), GEM alone, and GEM plus
AHCC. Each group consisted of ten mice.
2.3. Treatments
The GEM solution was injected intraperitoneally at a
dose of 400 mg/kg (1200 mg/m2) once a week for three
weeks (days 7, 14 and 21). The treatment was similar to
the regimen actually used in clinical practice (1000
mg/m2 of weekly drip infusion three times followed by
one week cessation of the drug). AHCC was prepared as
a solution at a dosage of 1 g/kg and administered daily by
gavage to mice seven days before the first injection of
GEM and throughout the experiment (day 1 to day 28).
The control group received a vehicle (saline) instead of
GEM and AHCC. All animals were killed under anesthe-
sia, and blood, bone marrow (BM) cells, spleen and kid-
ney were harvested at day 28.
Since the effect of AHCC was assessed at a dosage
range from 100 mg/kg to 1 g/kg in previous studies,
[14-16,19,20] a dose of 1 g/kg of AHCC was chosen in
the current study. The experimental protocol was ap-
proved by the Animal Care Committee of Amino Up
Chemical Co., Ltd.
2.4. Evaluation of Parameters
The following parameters were assessed: body weight,
food consumption, liver function (serum aspartate ami-
notransferase; AST), kidney function (blood nitrogen
urea; BUN), hematological toxicities (peripheral total
white blood cell count and hemoglobin content), and
myelosuppression. Body weight and food consumption
were measured twice a week. Serum AST and BUN were
assessed using Transaminase CII-test WAKO and Urea
Nitrogen B-test WAKO assay kits (Wako Pure Chemical
Industries Limited, Osaka, Japan), respectively. Cardiac
blood samples were diluted to 1:10 with Turk solution
(Wako Pure Chemical Industries Limited) to determine
Copyright © 2012 SciRes. IJCM
Active Hexose Correlated Compound (AHCC) Alleviates Gemcitabine-Induced
Hematological Toxicity in Non-Tumor-Bearing Mice 363
the number of total white blood cells in accordance with
the Nageotte chamber counting procedure, [21] and he-
moglobin content in blood was measured using a Hemo-
globin B-test kit (Wako Pure Chemical Industries Lim-
ited). Myelosuppression was determined by measuring
BM cell viability and by evaluating the responses to col-
ony forming unit granulocyte-macrophage (CFU-GM)
and burst forming unit erythroid (BFU-E).
BM cell viability was determined by collecting BM
cells from the femur, which were first suspended in
0.83% NH4Cl solution and incubated at 37˚C for ten
minutes to hemolyze red blood cells. After centrifugation,
the cells were prepared at a concentration of 1 × 107
cells/mL in DMEM supplemented with 10% FBS. A
100-L aliquot of the suspension was cultured in a
96-well plate for three days, and the viability (percent of
control group) of BM cells was estimated by a MTT as-
say. The detection of CFU-GM and BFU-E was per-
formed using a colony forming cell assay kit, MethoCult
(StemCell Technologies, Vancouver, Canada). Briefly,
BM cells were suspended in Iscove’s MDM (IMDM)
with 2% FBS and the suspension (2 × 105 cells/mL) was
mixed with methylcellulose medium containing rmSCF,
rmIL-3 and rhIL-6 (MethoCult 3534) at a 1:9 ratio. The
prepared BM cells (2 × 104 cells/mL) were plated onto a
35-mm dish and incubated at 37˚C for eight days to form
CFU-GM colony. For a mature BFU-E assay, after
IMDM with 2% FBS was added at a 1:9 ratio to methyl-
cellulose medium containing rhEpo (MethoCult 3334),
BM cells (2 × 106 cells/mL) in IMDM with 2% FBS
were mixed with the diluted methylcellulose medium at a
1:9 ratio. The prepared BM cells (2 × 105 cells/mL) were
plated onto a 35-mm dish and incubated at 37˚C for four
days. Following the individual incubation time, CFU-
GM and mature BFU-E colonies were counted under a
microscope to quantify murine hematopoietic progenitor
cells.
2.5. Reverse Transcription Polymerase Chain
Reaction
Expression of granulocyte-macrophage colony-stimulating
factor (GM-CSF), erythropoietin (EPO) and beta-2-
microglobulin (B2M) mRNA was determined using a
quantitative reverse transcription polymerase chain re-
action (RT-PCR). Total RNA was extracted from 100 mg
of spleen and kidney with TRIzol reagent (Invitrogen
Corp., Carlsbad, CA, USA) according to the manufac-
turer’s protocol. First-strand cDNA was obtained by in-
cubation of 1.6 g of total RNA with PrimeScript 2 1st
strand cDNA Synthesis kit (Takara Bio Inc., Otsu, Japan),
and the RT product was then diluted to 10 g/L and
subjected to PCR using TaKaRa ExTaq (Takara Bio Inc.).
Forty cycles of amplification were carried out for GM-
CSF mRNA, and EPO mRNA and B2M mRNA were 37
and 22 cycles, respectively. The condition of each cycle
was denaturing at 94˚C for 30 seconds, annealing at 59˚C
(GM-CSF and B2M) and 65˚C (EPO) for 45 seconds,
and extension at 72˚C for 30 seconds. The primers are
described as follows; GM-CSF:
5’-GGCCTTGGAAGCATGTAGAG-3’ (sense) and 5’-
ATGAAATCCGCATAGGTGGT-3’ (antisense); EPO:
5’-CCACCCTGCTGCTTTTACTC-3’ (sense) and 5’-
GGCCTTGCCAAACTTCTATG-3’ (antisense); B2M:
5’-TAGCTGTGCTCGCGCTACT-3’ (sense) and 5’-
AGTGGGGGTGAATTCAGTGT-3’ (antisense). The gene
bands in each sample were normalized to the corre-
sponding B2M band using Alpha Innotech redTM (Alpha
Innotech Corp., San Leandro, CA, USA).
2.6. Statistical Analysis
Experimental data are shown as mean ± standard error of
the mean (SEM). Data were analyzed by one-way analy-
sis of variance (ANOVA). Fisher’s Protected Least Sig-
nificance Difference (PLSD) was used as a post hoc test,
and values of p less than 0.05 were determined to be sta-
tistically significant.
3. Results
3.1. Peripheral Hematological Toxicity
To determine whether AHCC is capable of protecting
against GEM-related hematotoxicity, peripheral total
white blood cell count and hemoglobin content in blood
were monitored. As shown in Figures 1(a) and (b), GEM
treatment was significantly associated with reductions of
leukocyte count and hemoglobin content (p < 0.01), and
supplementation with AHCC completely ameliorated
both hematological toxicities (p < 0.01). The values of
white blood cells (×106 cells/mL) in the control, GEM,
and GEM + AHCC groups were 3.05 ± 0.13, 2.01 ± 0.12,
and 3.03 ± 0.13, respectively. Hemoglobin content (g/dL)
was 14.4 ± 0.2 (control), 13.1 ± 0.3 (GEM), and 14.7 ±
0.3 (GEM plus AHCC).
3.2. Bone Marrow (BM) Suppression
To elucidate the alleviating effect of AHCC for GEM-
induced myelosuppression, BM damage was assessed by
BM cell viability and the colony forming ability of hema-
topoietic progenitor cells. The viability of BM cells iso-
lated from GEM-treated mice was lower than that of the
control group (p < 0.01; Figure 2), and AHCC admini-
stration significantly reversed the decline although it did
not achieve complete recovery (p < 0.01 vs GEM, con-
trol). Treatment with GEM alone significantly lowered
Copyright © 2012 SciRes. IJCM
Active Hexose Correlated Compound (AHCC) Alleviates Gemcitabine-Induced
Hematological Toxicity in Non-Tumor-Bearing Mice
364
(a)
(b)
Figure 1. Alleviating effect of AHCC for GEM-related he-
maotopoietic toxicity. Blood was collected from mice on the
final day of the experiment (day 28), and GEM-induced
hematotoxity was evaluated using two parameters, which
were peripheral total white blood cell count (a) and hemo-
globin content in blood (b). Blood samples were diluted to
1:10 with Turk solution to determine the number of total
white blood cells based on the Nageotte chamber counting
procedure. Hemoglobin content was analyzed by a Hemo-
globin B-test WAKO assay kit. The values show the mean ±
SEM. *p < 0.01 vs control, GEM plus AHCC.
Figure 2. Ameliorative effect of AHCC on bone marrow
(BM) cell viability. On day 28, BM cells were isolated from
femurs of mice with or without GEM injection, and the
hemolyzed BM cells (1 × 107 cells/mL) were cultured in a
96-well plate for 3 days. The viability (% of control group)
of BM cells was estimated by a MTT assay. The values (%
of control; mean ± SEM) in the control, GEM alone, and
GEM plus AHCC groups were 100.0 ± 1.5, 77.5 ± 1.3 and
89.0 ± 0.9, respectively. *p < 0.01 vs control, GEM plus
AHCC, **p < 0.01 vs control.
both CFU-GM and BFU-E forming abilities (p < 0.01;
Table 1), while the lowering was entirely recovered to
control level by AHCC administration.
3.3. Expression of GM-CSF and EPO mRNA
Expression of GM-CSF and EPO mRNA in spleen and
kidney, respectively, was compared among control, GEM
alone, and GEM plus AHCC groups. The expression
level was calculated as a percent of control after each
band of GM-CSF and EPO was normalized to the corre-
sponding B2M band (Table 2). The mRNA levels of
both GM-CSF and EPO in the GEM alone group were
significantly higher than those of the control and the
GEM plus AHCC groups (p < 0.05). In contrast, the ex-
pression levels in AHCC-treated mice were identical to
control.
3.4. Other Toxicities
No changes in body weight, food consumption, and liver
and renal functions were noted at the completion of the
study. The average of body weight (g) was 35.4 ± 1.0,
36.3 ± 0.7, and 35.9 ± 0.6 in the control, GEM alone, and
GEM+AHCC groups, respectively. Serum AST and
BUN values were also normal and did not change during
the course of the study (data not shown), which was con-
sistent with previous data [10].
4. Discussion
Gemcitabine (GEM) has shown activity in a variety of
solid tumors [22]. The drug has been approved for the
treatment of non-small cell lung cancer, pancreatic can-
Table 1. Colony forming responses of CFU-GM and BFU-E.
Group CFU-GM BFU-E
Control 88.3 ± 1.7 119.3 ±6.1
GEM 64.3 ± 8.6* 29.0 ± 0.6**
GEM+AHCC 104.0 ± 3.0 109.0 ± 8.1
All values (colony counts) represent the mean ± SEM. *p < 0.05 vs control,
p < 0.01 vs GEM + AHCC, **p < 0.01 vs control, GEM + AHCC. CFU-GM:
colony forming unit-granulocyte macrophage, BFU-E: burst forming unit-
erythroid.
Table 2. mRNA levels of GM-CSF and EPO.
Group GM-CSF EPO
Control 100.0 ± 27.4 100.0 ± 6.6
GEM 323.3 ± 74.1* 161.9 ± 19.4*
GEM+AHCC 92.9 ± 20.0 99.6 ± 3.5
All values (% of control) show the mean ± SEM. *p < 0.05 vs control, GEM
+ AHCC. GM-CSF: granulocyte-macrophage colony-stimulating factor,
EPO: erythropoietin.
Copyright © 2012 SciRes. IJCM
Active Hexose Correlated Compound (AHCC) Alleviates Gemcitabine-Induced
Hematological Toxicity in Non-Tumor-Bearing Mice 365
cer and biliary tract cancer in Japan, and non-small cell
lung, pancreatic, ovarian and breast cancers in the United
States. Although GEM is generally well tolerated and has
a good toxicity profile, myelosuppression is the most
common side effect, which can limit dose and thus po-
tentially its therapeutic efficacy. This study was designed
to investigate the impact of AHCC in terms of side ef-
fects, particularly hematological toxicity attributable to
GEM injection, in non-tumor-bearing mice.
The treatment with GEM caused reduction of white
blood cell count and hemoglobin content, respectively
leading to leukopenia and anemia. Occurrence of leuko-
penia often induces infectious complications, which may
compromise treatment efficacy. Opportunistic infections
are a major cause of morbidity and mortality in cancer
patients receiving myelotoxic chemotherapy, resulting
from invasive fungal infections, particularly invasive
aspergillosis, and an increasing spread of Gram-positive
pathogens such as methicillin-resistant Staphylococcus
aureus and vancomycin-resistant enterococci [23]. In
current clinical practice, colony-stimulating factors such
as granulocyte colony-stimulating factor (G-CSF) and
granulocyte-macrophage colony-stimulating factor (GM-
CSF) are increasingly used to recover white blood cell
counts or increase dose-density [24]. In addition, hemo-
globin reduction results in anemia, which is associated
with a significant decrease in the quality of life and may
limit the applicability and efficacy of anticancer drugs
[25]. The treatment with recombinant human erythro-
poietin (rHu Epo) has been shown to improve anticancer
drug-induced anemia in rats [26], and alleviating anemia
with rHu Epo in humans has improved the quality of life
of cancer patients [27].
Although G-CSF and GM-CSF are generally safe, well
tolerated and have favorable outcomes, several reports of
serious G-CSF and GM-CSF associated side effects exist,
[28,29] including enhanced bone tumor growth by G-
CSF in mice in an osteoclast-dependent manner [30].
Treatment with rHu EPO also has risks such as the po-
tential to promote cellular proliferation and migration in
melanoma and breast cancer cells expressing the Epo
receptor [31,32]. AHCC exerted no influence on mRNA
levels of GM-CSF and EPO in our study when the
mRNA levels were measured. However, given the ame-
liorating effects of AHCC for GEM-associated BM cell
viability, AHCC might be useful to complement the
properties of G-CSF and GM-CSF as well as Epo. The
beneficial effects of AHCC on hematotoxicities caused
by other anticancer drugs, such as cisplatin, paclitaxel,
5-fluorouracil and irinotecan, have been reported [16,19],
although the mechanism of action is not yet clear.
AHCC supplementation was significantly associated
with an improvement in the levels of colony forming unit
granulocyte-macrophage (CFU-GM) and burst forming
unit erythroid (BFU-E), which were severely depressed
as a result of GEM treatment. AHCC might therefore
alleviate chemotherapy-related hematological toxicity
through protecting hematopoietic progenitor cells. This
result is consistent with other studies demonstrating that
Maitake
-glucans promoted bone marrow cell viability
and protected the bone marrow stem cell colony forma-
tion unit from doxorubicin-induced hematological toxic-
ity, [33] as well as induced hematopoietic stem cell pro-
liferation and differentiation [34].
Despite the side effects, GEM may be a useful agent
for tumor immunotherapy since it possesses significant
immunomodulatory activity independent of its cytotoxic
effects as shown in murine tumor models [35]. Other
agents with potentially harsh side effects, such as cis-
platin, have also been to increase the susceptibility of
tumor cells to tumor-infiltrating lymphocytes or natural
killer cells [36]. Therefore, AHCC may offer promise
when used in conjunction with chemotherapy since
AHCC may help reduce side effects of drugs like GEM
or cisplatin and enable a full chemotherapeutic regimen
to be administered. Furthermore, the previous study de-
monstrated that AHCC enhanced chemotherapeutic ef-
fect of cisplatin in tumor-bearing mice [16], suggesting
an adjuvant action of AHCC.
The safety of AHCC in cancer patients and healthy
volunteers has been previously reported [13,17,18,37].
The current and previous studies suggest that AHCC
consumption may be safe in combination with GEM and
perhaps other chemotherapy agents that are not metabo-
lized via the CYP450 2D6 pathway [38] and clinical
studies are warranted.
The present study was conducted to assess whether
AHCC reduces GEM-induced side effects, particularly
hematological toxicity that is a dose limiting factor for
GEM, in non-tumor-bearing mice. As a consequence,
AHCC significantly ameliorated reduction of peripheral
total white blood cell count and hemoglobin content, and
further resulted in recovering CFU-GM and BFU-E
forming abilities. If these results are extended to humans,
AHCC might contribute to improved quality of life and
well-being of cancer patients undergoing chemotherapy
including GEM treatment.
5. Acknowledgements
We are deeply grateful to Dr. Robert M. Hackman, Uni-
versity of California-Davis, and Dr. Judith A. Smith,
University of Texas, MD Anderson Cancer Center, for
their suggestions and editorial comments.
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