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Evaluation of active hexose correlated compound (AHCC) in combination with pegylated liposomal doxorubicin for treatment of ovarian cancer

Authors:

Abstract

The objective was to define the mechanism of the growth inhibition of active hexose correlated compound (AHCC) alone and evaluate its activity in combination with pegylated liposomal doxorubicin (PLD). Scientific Methods: In vitro growth inhibition assays were completed with AHCC alone and in combination with PLD in panel of human cancer cell lines and findings confirmed in vivo in an ovarian cancer xenograft mouse model. AHCC mechanism of action was evaluated with immunoblotting and flow cytometry studies. Major Findings: The in vitro growth inhibition assays demonstrated additive activity when AHCC is co-administered with PLD. The combination of AHCC with PLD demonstrated a 64.1% reduction in tumor growth compared to the untreated group (p value = 0.03) and a 31.2% improvement in tumor response with combination regimen compared to PLD alone. No difference in toxicity was observed in the control or treatment groups. An increased expression of Bcl-2 was observed and induction of apoptosis confirmed in presence of AHCC. Conclusions: There is potential improvement in PLD activity when co-administered with AHCC and decrease side effects of PLD. A clinical study to evaluate of the combination of AHCC plus PLD in the treatment of ovarian cancer is being pursued. Industrial Relevance: This study presents an example of the successful integration of a well- known herbal supplement, AHCC, with traditional western medicine cytotoxic agent, pegylated liposomal doxorubicin, for the treatment of recurrent ovarian cancer. In addition to providing evidence of the efficacy of AHCC, the mechanism of improved activity was also investigated. Using a traditional approach this study provides pre-clinical data to support the benefits previously observed and reported in the clinical setting and supports future endeavors to integrate AHCC into standard of care to be given with chemotherapy.. These finding are particularly beneficial in the treatment of recurrent cancer when maintaining a good quality of life during chemotherapy is a priority and allows patients to have a natural, nutritional approach to preventing and managing chemotherapy adverse effects.
International Journal of Applied Research in Natural Products
Vol. 4 (3), pp. 6-14, Sep-Oct 2011
Directory of Open Access Journals
©2011. IJARNP-HS Publication
______________________
*Corresponding Author:
E-mail: jasmith@mdanderson.org
Tel: 713-500-6408
Available online http://www.doaj.org/doaj?func=openurl&issn=19406223&genre=journal
Original Article
Evaluation of active hexose correlated compound
(AHCC) in combination with pegylated liposomal
doxorubicin for treatment of ovarian cancer
Hunter RJ
1,2
, Fujii H
3
, Wakame K
3
, Gaikwad A
4
, Wolf JK
4
, Smith JA
1,4,5*
1
The University of Texas M.D. Anderson Cancer Center, Division of Pharmacy;
2
Texas Southern
University College of Pharmacy and Health Sciences;
3
Amino Up Chemical Company, Ltd,
Sapporo, Japan;
4
The University of Texas M.D. Anderson Cancer Center, Department of
Gynecologic Oncology, Houston, TX;
5
The University of Texas Health Science Center,
Department of Obstetrics, Gynecology and Reproductive Sciences
Summary: The objective was to define the mechanism of the growth inhibition of active hexose correlated compound (AHCC)
alone and evaluate its activity in combination with pegylated liposomal doxorubicin (PLD). Scientific Methods: In vitro growth
inhibition assays were completed with AHCC alone and in combination with PLD in panel of human cancer cell lines and
findings confirmed in vivo in an ovarian cancer xenograft mouse model. AHCC mechanism of action was evaluated with
immunoblotting and flow cytometry studies. Major Findings: The in vitro growth inhibition assays demonstrated additive
activity when AHCC is co-administered with PLD. The combination of AHCC with PLD demonstrated a 64.1% reduction in
tumor growth compared to the untreated group (p value = 0.03) and a 31.2% improvement in tumor response with combination
regimen compared to PLD alone. No difference in toxicity was observed in the control or treatment groups. An increased
expression of Bcl-2 was observed and induction of apoptosis confirmed in presence of AHCC. Conclusions: There is potential
improvement in PLD activity when co-administered with AHCC and decrease side effects of PLD. A clinical study to evaluate
of the combination of AHCC plus PLD in the treatment of ovarian cancer is being pursued.
Industrial Relevance: This study presents an example of the successful integration of a well- known herbal supplement, AHCC,
with traditional western medicine cytotoxic agent, pegylated liposomal doxorubicin, for the treatment of recurrent ovarian cancer.
In addition to providing evidence of the efficacy of AHCC, the mechanism of improved activity was also investigated. Using a
traditional approach this study provides pre-clinical data to support the benefits previously observed and reported in the clinical
setting and supports future endeavors to integrate AHCC into standard of care to be given with chemotherapy. . These finding
are particularly beneficial in the treatment of recurrent cancer when maintaining a good quality of life during chemotherapy is a
priority and allows patients to have a natural, nutritional approach to preventing and managing chemotherapy adverse effects.
Keywords: AHCC, Doxil, ovarian, cancer, drug resistant
Introduction
Ovarian cancer is the fifth leading cause of cancer among women and the number one gynecologic fatality with an
estimated 21,880 women will be newly diagnosed and 13,850 deaths will occur in 2011.(American Cancer Society,
2011) Ovarian cancer is difficult to diagnose early and patients often initially present with advanced disease with a
poor prognosis.(Barnes & Grizzel 2002) The current standard of care for first line treatment includes surgery and
cytotoxic chemotherapy with 70 % of patients achieving an initial clinical complete response.(Partiridge & Barnes
1999) However, over 50% of patients will have recurrence within the first two years from completion on first line
treatment which is often associated with taxane/platinum-resistant disease with a poor prognosis..(Partiridge &
Barnes 1999, Martin 2002; Barnhill & Kurman 1995) The response rates with second-line agents such as
gemcitabine, topotecan, or pegylated liposomal doxorubicin have not been impressive ranging from 15 to 30% in
platinum resistant ovarian cancer. (Martin 2002; Barnhill & Kurman 1995).
Hunter et al.
7
Pegylated liposomal doxorubicin (PLD) (Doxil
®
, Ortho Biotech Products, L.P., Bridgewater, NJ) which was
formulated in an attempt to further reduce cardiac toxicities associated with doxorubicin but still has significant
dermatologic and hematologic toxicity. PLD has demonstrated clinical activity in the treatment of recurrent
platinum resistant ovarian cancer patients with response rates of 17-26% with a median progression free survival of
5-6 months.( Thigpen & Aghajanian 2005, Muggia & Hainsworth 1997) While PLD has clinical activity the
toxicities are often challenging for this patient population who is typically of advanced age and may have other co
morbid conditions. (Thigpen & Aghajanian 2005)
Active Hexose Correlated Compound (AHCC, Amino Up Chemical Co, Ltd., Sapporo, Japan), is mixture of
polysaccharides, amino acids, lipids and minerals extracted from the culture the basidiomycete mushroom Lentinula
edodes (shiitake) that has been proposed to have many health benefits including both immunomodulatory and anti-
tumor effects.(Miura & Kitadta 2010, Kidd 2000) In animal studies AHCC has demonstrated benefit to decrease the
side effects associated with anticancer chemotherapy and to have a role in treatment and prevention of
cancer.(Hirose & Sato 2007, Shigama & Nakaya 2009, Gao & Zhang 2006) The functions of particular interest in
the oncology arena are AHCC’s immunomodulating and potential restorative effects on natural killer (NK) cells,
macrophages and cytokines after anti-cancer chemotherapy as well as the potential to integrate AHCC with
cytotoxic regimens for the treatment of cancer.(Gao& Zhang 2006, Sun & Wakame 1997)
Since there has been limited success with traditional cytotoxic chemotherapy agents in the persistent and recurrent
ovarian cancer, alternative approaches need to be considered, including the integration of natural supplements with
potential to improve growth inhibitory activity and adverse effects. This preclinical study was designed to evaluate
the in vitro growth inhibition and confirm the in vivo activity of active hexose correlated compound (AHCC) alone
and when given in combination with pegylated liposomal doxorubicin (PLD).
Materials and Methods
Supplies: All human cancer cell lines- ES-2, SKOV
3
, MES-SA, MCF-7 and HeLa were obtained from the
American Type Culture Collection (ATCC, Manassas, VA) and the SKOV
3
-IP1 ovarian cancer cell line was
generously provided by Dr. Isaiah J. Fidler at The University of Texas M.D. Anderson Cancer Center (UTMDACC).
All cell lines were maintained for less than fifteen passages. Pegylated liposomal doxorubicin (PLD) (Doxil
®
, Ortho
Biotech Products, L.P., Bridgewater, NJ) and doxorubicin HCL (Bedford Laboratories
TM
, Bedford, OH) was
purchased from UTMDACC Division of Pharmacy (Houston, Texas). The active hexose correlated compound was
generously provided by Amino Up Chemical Company, Ltd (Sapporo, Japan). Fetal bovine serum (FBS) and
trypsin-EDTA were purchased from GIBCO Invitrogen Co (Carlsband, CA). ((3-(4,5-cimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) and dimethyl sulphoxide (DMSO), and tris base were purchased from Sigma-
Aldrich Co (St. Louis, MO).
Chemicals and reagents: The BCA protein estimation kit was obtained from Pierce (Rockford, IL). All primary
antibodies, including Bcl-2, Parp-1/2, and Caspase-3 and Goat and Mouse anti-rabbit IgG purified AB were
obtained from Calbiochem-Novabiochem Company (San Diego, CA). The buffer solutions including: 40%
Acrylamide/Bis Solution, 19:1, N,N,N’,N’– Tetra-methylethylenediamine (TEMED), 10% SDS, 10x
Tris/Glycine/SDS buffer, 10x Tris/Glycine buffer, 10x Tris-Buffered Saline (TBS), Tween 20, Blotting Grade
Blocker- Non fat dry milk, Immun-Blot PVDF Membrane for protein blotting (0.2 µM) were all purchased from
Bio-Rad Laboratories (Hercules, CA). Finally, the ECL plus Western Blotting Detection Reagents were obtained
from Amersham Biosciences Co. (Piscataway, NJ).
Standard Solutions: In the in vitro studies a 100 mg/mL stock solution of AHCC was prepared by dissolving
1000mg of AHCC in 10 mL sterile distilled water. Doxorubicin was used in the in vitro studies was prepared by
dissolving doxorubicin HCL 90 mg in 3 mL of sterile water for injection to achieve final concentration of 5 mg/mL.
All further dilutions were prepared using respective cell culture media for each cell line. The MTT stock solution
was prepared by dissolving 54 mg of MTT in phosphate buffered saline (PBS) to achieve a final concentration of 0.3
mg/mL.
For the animal studies the AHCC oral suspension was prepared once every seven days with 150 mg of AHCC
powder of polysorbate-80 which achieved a final concentration of 5 mg/mL and stored in the 4
o
C. The intravenous
doses of PLD were prepared by drawing up diluting 2 mg/mL solution with dextrose 5% water to achieve a final
concentration of 0.25 mg/0.15 mL.
Cell Culture: The human uterine sarcoma cell line (MES-SA) was propagated in medium consisting of McCoy’s
5A medium with 10% FBS. The MCF-7 human breast cancer cell line was propagated in a media consisting of
Eagles minimum essential medium (EMEM), 0.01 mg/mL bovine insulin and 10% FBS. The ES-2 clear cell
ovarian carcinoma cell line and both the SKOV
3
-IP1 and SKOV
3
adenocarcinoma ovarian cancer cell lines were
both propagated with media consisting of McCoy’s5a medium
with 2mM L-glutamine and 10% FBS. Finally, the
AHCC/Doxil for treatment of ovarian cancer
8
HeLa adenocarcinoma cervical cancer cell line was propagated in a media consisting of EMEM with 2 mM L-
glutamine and Earl’s BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0
mM sodium pyruvate and 10% FBS.
Growth Inhibition Assay: Growth inhibition assays were conducted as previously described. (Smith & Brown
2004). Briefly, cells were plated at 2,500-5,000 cells per well in 96-well and incubated at 37°C for 24-hours. Each
of the cancer cell lines were treated with AHCC or doxorubicin at concentrations ranging from 1 x 10
-6
µg/mL to
1000 µg/mL. Control wells had either no drug, media alone or were blank wells (no cells, drug or media). After a
72-hour incubation period, 25 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was
added to each well and 96-well plates incubated for two hours. Plates were then centrifuged and the supernatant was
removed and then 50 µL of DMSO was added to each well and absorbance measured at 563 nm by FL600 Dual-
Band plate reader from BioTek Instruments, Inc. (Winooski, VT).
The inhibitory concentration to achieve 20% and 50% cell death (IC
20
and IC
50
) for each drug and each cell line
were calculated. The IC
20
concentration was selected and use in combination AHCC + doxorubicin studies to
ensure that enough viable cells were available completing for immunoblotting and flow cytometry experiments. All
experiments were done in quadruplicate.
SKOV
3
-IP1 Xenograft Mouse Model: The animal protocol was approved by the institutional animal care and
utilization committee (IACUC) prior to initiating any animal work. All mice were
handled according to the Guide
for the Care and Use of Laboratory
Animals. Although in vitro activity in the MTT assays also demonstrated
synergistic activity in the MCF-7 cell line, only the SKOV
3
-IP1 cell line was selected to evaluate the combination of
AHCC with PLD in this animal study because the FDA approved indication of PLD is for the treatment of recurrent
ovarian cancer with long term toxicities being the major treatment limitation hence the priority for translation to
human correlative studies. On day -10, SKOV
3
-IP1 cells (9 x 10
6
) were injected intraperitoneal (IP) into each
athymic mice for all treatment groups. Ten days after inoculation, mice were randomized to receive 0.25 mL of
oral vehicle by gastric gavage once daily, single 0.15 mL dose of intravenous (IV) vehicle (0.9% sodium chloride
solution), AHCC (50 mg/kg) via gastric gavage once daily, a single dose PLD (10 mg/kg) IV, or the combination of
AHCC (50 mg/kg) via gastric gavage once daily and a single dose PLD (10 mg/kg) IV.
To assess IP tumor growth in the study the body weight, abdominal circumference, and accumulation of ascites
were utilized as surrogates to reflect tumor burden were quantified three times a week. . Abdominal circumference
measurements were obtained at the body’s widest point throughout the experimental period while the animal was
held in a prone position. In addition, mice were monitored daily for evidence of signs/symptoms of moriboundity
(weight loss, anorexia, hunching, listlessness, extensive enlarge abdominal cavity, lethargy, etc.). Ascites was
removed and the volume of ascites measured as appropriate to relieve abdominal bloating up to three times then
animals were sacrificed. At the end of the experiment, all remaining mice were euthanized via CO
2
inhalation
followed by cervical dislocation as per the institutional protocol. Post-mortem, macroscopic evaluation was
completed to evaluate extent of tumor burden and all tumors were harvested, weighed and then stored for evaluation
of molecular markers.
Immunoblotting: Evaluation of apoptosis included assessment of three commonly observed up-regulated proteins
in ovarian cancer pathogenesis were evaluated including Bcl-2, Parp-1/2, and caspase-3. Samples were obtained
from tumors in each of the respective treatment arms of the animal study and protein extracts were prepared by
lysing cells on ice in 300-400 µL of NP40 lysis buffer. Pierce Micro BCA Protein Assay Kit (Pierce: Rockford, IL)
was used to determine the protein concentration. For each series of protein determinations, a standard curve was
constructed with known concentrations of bovine serum albumin (BSA). For direct immunoblotting, 50 µg protein
were run on 10% SDS-PAGE gels, transferred to polyvinylidene difluoride (PVDF) membranes and probed with the
appropriate antibodies using manufacturer’s protocol (Calbiochem- Novabiochem Co. San Diego, CA).
Flow analysis of cell cycle: Flow cytometry was performed as confirmation of apoptosis observed in the
immunoblotting experiments. Following the 24 hour in vitro treatment of SKOV
3
cells with IC
20
concentration of
AHCC alone, IC
20
concentration of PLD alone, or combination of IC
20
concentration of AHCC and PLD, all cells
were pooled and washed in ice-cold PBS then fixed in ice-cold 1% paraformaldehyde (PFA) in PBS for 30 minutes
on ice. Next, cells were washed again with ice-cold PBS then were fixed in 70% ethanol for overnight. After
overnight incubation, the cells washed in ice-cold PBS then stained with propidium iodide (PI) in presence of RNase
at 37° C for fifteen minutes. Cell cycle distribution was analyzed on 10,000 cells for each experimental condition.
Data analysis was performed using Flow Cytometry and Cellular Imaging Core Facility of UTMDACC.
Quantitative data from cell cycle analysis were graphed to illustrate the accumulation of percentage of cells in
various phases of cell cycle.
Hunter et al.
9
Data Analysis: In the growth inhibition assay the interaction index to determine antagonism, additive or
synergistic activity as described by Tallarida and colleagues was determined with following equation: γ = a/A + b/B.
Whereas A= dose of drug A alone that gives the specified effect; B= dose of drug B lone that gives specified effect;
a= dose of drug A used in combination to achieve specified effect; and b= dose of drug B used in combination to
achieve specified effect. (Tallarida 1996) The interaction index measures drug combination as follows: γ value
equal to one it is additive; γ value that is less than one is synergistic and γ value greater than one is antagonistic.
(Tallarida 1996) Descriptive statistics were utilized to summarize in vitro study results. Data was analyzed using a
paired two tailed Student’s t-test for comparison between the treatment groups. Differences between groups were
considered statistically significant at P < 0.05.
In the animal study, each control, single chemotherapy treatment arm included ten mice in which tumor growth
was measured and independently compared. A total of 60 mice were utilized for completion of the study. Power
calculations were completed to determine that a sample size of ten mice is required to detect a 10% increase in
inhibition of tumor growth of each of the respective study arms. An independent sample t-test was used in the data
analysis to evaluate differences in tumor growth inhibition. For these experiments the α was set at 5% and the β
was set at 20% (power = 80%). The Holm’s methods were employed for evaluating differences between the
multiple treatment agents and combination regimens. (Aickin & Gensler 1996)
Results
MTT studies employed determined the concentration to achieve IC
20
for doxorubicin was 0.54 ng/mL, 0.1 ng/mL,
0.1 ng/mL, 0.005 ng/mL, and 0.004 ng/mL in the ES-2, SKOV
3
, MES-SA, MCF-7 and HeLa cancer cell lines,
respectively. AHCC did not demonstrate any cytotoxic activity as a single agent at clinically relevant
concentrations and however IC
20
was achieved at concentrations of 0.478 mg/mL, 0.42 mg/mL, 0.823 mg/mL, 0.77
mg/mL, and 0.75 mg/mL in the ES-2, SKOV
3
, MES-SA, MCF-7 and HeLa cancer cell lines, respectively. The
interaction index was determined for each cell line determined that AHCC plus doxorubicin was at least additive and
potentially synergistic in the panel of human cancer cell lines. (Table 1)
Table 1 Summary of the interaction index measure of drug synergism between PLD and AHCC
ES-2 MES-SA MCF-7 HeLa SKOV3
A
0.478 0.823 0.75 0.77 0.42
a
0.25
0.433
0.354
0.363
0.22
B
0.54 0.1 0.004 0.005 0.1
b
0.28
0.05
0.001
0.002
0.05
Interaction Index
1.0 1.0 0.7 0.9 1.0
Type of Interaction additive additive synergistic synergistic additive
This table represents the interaction index (γ = a/A + b/B) measure of drug synergism between PLD in plus AHCC in a
panel of selected human cancer cell lines. A= dose of AHCC alone that achieved specified effect (IC
20
); a= dose of
AHCC used in combination to achieve same effect (IC
20
); B= dose of PLD alone that achieved specified effect (IC
20
);
b= dose of PLD used in combination to achieve same effect (IC
20
). γ = 1 it is additive; γ < 1 is synergistic and γ > is
antagonistic.
AHCC/Doxil for treatment of ovarian cancer
10
These in vitro findings were confirmed with in vivo ovarian cancer xenograft mouse model study to determine if
the combination of AHCC plus PLD would have improved efficacy compared to PLD alone. The combination of
AHCC plus PLD reduced tumor growth by 15.2% and 33.9% more than PLD or AHCC alone, respectively. The
percent tumor reduction observed in the PLD plus AHCC and PLD group alone were reduced by 64.1% and 48.9%,
respectively, compared to the untreated group. (Figure 1)
Figure 1 Mean percent reduction in tumor burden after completion of 28 day treatment cycle
In a mouse xenograft model of SKOV
3
IP1 60 mice were randomly divided into six treatment groups (N=10) and received
AHCC once daily alone, PLD once alone, or combination of AHCC and PLD, vehicle, or were untreated controls. The mean
percent reduction was determined by calculating difference from untreated control with each respective treatment group. Data
was analyzed using a paired two tailed Student’s t-test for comparison between the study groups. Differences between groups
were considered statistically significant at P < 0.05.
Postmortem macroscopic evaluation of all mice was performed to confirm all results of tumor location and
adjacent organ involvement. Tumors were present on the surface of the peritoneum cavity, uterus, liver, lung,
intestines, and mesentery in all mice except the AHCC plus PLD treatment group the tumor was restricted to
peritoneum cavity in 9 of 10 mice. The mean total tumor weight was reduced by 29.8% in the AHCC plus PLD
treatment group compared to the PLD alone group. (Figure 2).
The safety and tolerability of AHCC was also evaluated during the study. There were no significant differences in
the baseline total body weight nor any differences in the mean total body weight distribution throughout the
treatment period between any of the treatment groups. No toxicity or morbidity observed during the study associated
with the AHCC or PLD alone or in combination.
64.1%
48.9%
30.2%
32.7%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Vehicle alone AHCC Treatment Doxil Treatment AHCC + Doxil
Percent Tumor Reduction (%)
*
p < 0.05 compared to
PLD alone
Hunter et al.
11
Figure 2 Summary of the mean tumor weight after completion of 28 day treatment cycle
In a nude mouse xenograft model of SKOV
3
IP1 60 mice were randomly divided into six treatment groups (N=10) and received
AHCC once daily alone, PLD, once alone, or combination of AHCC and PLD, vehicle, or were untreated controls. At
postmortem examination, tumors were found on the surface of the peritoneum, uterus, liver, lung, intestines, and mesentery in all
treatment and control groups. Data was analyzed using a paired two tailed Student’s t-test for comparison between the study
groups. Differences between groups were considered statistically significant at P < 0.05.
Following the completion of the in vivo ovarian cancer xenograft mouse study demonstrating AHCC plus PLD
demonstrated improved efficacy compared to PLD alone, molecular studies were embarked to define the mechanism
of additive cytotoxicity activity of this combination. Down regulation of unphosphorylated Bcl-2 was observed
only in the AHCC treatment group and AHCC plus PLD which was suggestive of increase in apoptosis via the Bax-
Bcl-2 pathway. (Figure 3)
Figure 3 AHCC down regulation of unphosphorylated Bcl-2 in tumor samples
Blot represent the protein expression analysis on six tumor samples collected it completion of the mouse study. 1- protein
standard (+); 2- AHCC (A) + pegylated liposomal doxorubicin (D); 3- pegylated liposomal doxorubicin (D); 4- AHCC
alone(A); 5- PO vehicle control; 6- IV vehicle control; 7- untreated
AHCC/Doxil for treatment of ovarian cancer
12
Apoptosis was confirmed by flow cytometry studies that illustrated with a larger relative proportion of cells in the
M1 phase of the cell cycle in the AHCC alone and combination AHCC plus PLD tumors compared to the control
groups. (Figure 4)
Figure 4 Confirmation of increase cytotoxicity observed with AHCC plus PLD
Flow cytometry experiments were performed on in vitro samples collected from SKOV
3
treated with AHCC, pegylated
liposomal doxorubicin, and combination of both at concentrations at the IC
20
. PI staining was utilized to quantify DNA content
in samples.
Discussion
The integration of nutritional supplements and herbal products with traditional cytotoxic chemotherapy regimens
has become increasingly of more interest in clinical practice based on patient preferences. (Navo & Phan 2004)
However, there is growing evidence that herbal products, nutritional supplements and functional foods are not
completely benign when combining such agents with other medications. Although a time consuming process, each
combination should be evaluated for potential drug interactions specifically if antagonistic, additive or synergistic.
When increase activity is observed in vitro, then appropriate in vivo studies should be conducted to confirm findings
and ultimately studies should be conducted to define the mechanism of the improved cytotoxicity activity.
This study systematically evaluated the potential activity of AHCC alone and in combination with PLD. First the
in vitro data demonstrated potential additive or synergistic activity, the SKOV
3
-IP1 xenograft which then was
confirmed in the combination of AHCC plus PLD limited the spread of the tumor growth to the peritoneal cavity as
well as reduced tumor volume close to one third compared to PLD alone. The combination regimen was well
tolerated as well. The potential mechanism of improved activity with the addition of AHCC to PLD was also
evaluated to determine AHCC has role in induction of apoptosis via the Bcl-2/BAX pathway achieved with doses of
AHCC of 50 mg/kg in mice which is approximate equivalent to the recommended supplement dose of 3 g/day for
humans.
There has been clinical benefit in patients with hepatocellular carcinoma, gastric cancer or colon cancer with use
of single agent AHCC.(Matsui & Uhara 2002, Kawaguchi 2009) In the clinical study by Matsui and colleagues of
269 hepatocellular cancer patients, 113 received AHCC (3g/day) orally following curative hepatocellular surgery
while the other 157 patients were just observed, and the AHCC treatment group had a significantly longer
Hunter et al.
13
progression free survival (HR, 0.639; CI 95%, 0.429-0.952; P = 0.0277) and overall survival (HR, 0.421; 95% CI,
0.253-0701; P = 0.0009) compared to the observation group.( Matsui & Uhara 2002)
There is a growing body of literature that has demonstrated the immunomodulatory activity of AHCC. (Miura &
Kitadte 2010, Gao & Zhang 2006, Matsui & Uhara 2002, Uno & Kosuna 2000) Since an athymic mouse has no
immune function, the immunomodulatory pathways could be eliminated as potential mechanisms of additive
cytotoxicity activity observed in this study. Multiple potential pathways for mechanisms of cytotoxicity were
investigated with only the positive finding suggesting induction of apoptosis via down regulation of
unphosphorylated Bcl-2 was observed in this study. This is one mechanism but is unlikely the only mechanism that
has contributed to the anti-tumor activity of AHCC that has been demonstrated in this study as well as multiple
clinical studies.( Miura & Kitadte 2010, Gao & Zhang 2006, Matsui & Uhara 2002, Uno & Kosuna 2000) This
study only evaluated the non-immunomodulatory activity of AHCC in the xenograft mouse model. There is a
potential for additional activity to be observed with the combination of AHCC to PLD in humans with functional
immune systems both improved antitumor activity as well as decreasing side effects similar to what has been
observed in AHCC single agent studies. A confirmatory clinical study is warranted to further evaluate the activity
of PLD when given in combination with AHCC in recurrent ovarian cancer patients.
Acknowledgement
Research supported by an unrestricted research grant from Amino Up Chemical Co., Ltd., Sapporo, Japan.
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AHCC/Doxil for treatment of ovarian cancer
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... No durable and effective systemic treatment regimen has been established for high-risk HPV infections [9]. Created in Japan in 1992, Active hexose correlated compound (AHCC) is created in Japan in 1992, compound (AHCC) is a standardized and proprietary extract of grown lentinula edodes mycelia made up of α-glucanicomponents (AHCC ®, AminoUp, Ltd.Sapporo,iJapan) [10][11][12][13][14][15]. Many human and animal investigations in the literature report various therapeutic effects of AHCC (e.g., preventing mechanisms that cause bacterial and viral infections, having an antioxidant effect, showing anticancer activity, and modulating the immune system) [10][11][12][13][14][15]. ...
... Created in Japan in 1992, Active hexose correlated compound (AHCC) is created in Japan in 1992, compound (AHCC) is a standardized and proprietary extract of grown lentinula edodes mycelia made up of α-glucanicomponents (AHCC ®, AminoUp, Ltd.Sapporo,iJapan) [10][11][12][13][14][15]. Many human and animal investigations in the literature report various therapeutic effects of AHCC (e.g., preventing mechanisms that cause bacterial and viral infections, having an antioxidant effect, showing anticancer activity, and modulating the immune system) [10][11][12][13][14][15]. Clinical studies report the effectiveness of AHCC in reducing the infection risks and improving symptoms of current diseases [10][11][12][13][14][15]. ...
... Many human and animal investigations in the literature report various therapeutic effects of AHCC (e.g., preventing mechanisms that cause bacterial and viral infections, having an antioxidant effect, showing anticancer activity, and modulating the immune system) [10][11][12][13][14][15]. Clinical studies report the effectiveness of AHCC in reducing the infection risks and improving symptoms of current diseases [10][11][12][13][14][15]. However, there are no studies investigating the effect of AHCC as an adjuvant therapy on the recurrence of genital warts after treatment. ...
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Introduction One of the most prevalent sexually transmittedxillnesses is considered to be Humanipapillomavirus (HPV). HPV is responsible for genital condyloma lesions. A durable and effective systemic treatment regimen has not been established for HPV-related infections. In the present study, our purpose was to analyse by evaluating the role of activexhexose correlatedxcompound (AHCC) in preventing relapse in patients who underwent cauterization for condyloma accuminata. Materials and Methods A total of 244 individuals who were hospitalized to our hospital in the interval from January 2019 to June 2022, were diagnosed as having condyloma acuminata, and underwent condyloma cauterization were evaluated retrospectively In this study; 133 individuals who met the criteria were taken in the investigation. In our study, patients who received AHCC were scheduled for follow-up examinations at regular intervals. For a year, they were scheduled for follow-up appointments every three months. Patients who did and did not use AHCC were divided into two groups and analyzed. Results The age average of AHCC non-users was significantly greater than AHCC users (p < 0.01). The number of condylomas and the maximum condyloma diameter of AHCC users before treatment were found to be significantly higher than in AHCC non-users (p = 0.006 and p = 0.004, respectively). In participants with recurrence, the number of condylomas and the condyloma diameter in AHCC users were significantlyilower than in AHCC non-users (p = 0.019 and p = 0.042, respectively). Conclusion Although the usage of AHCC is not expected to help prevent recurrence after cauterization of condylomata acuminate in all patients, physicians may consider AHCC as a nutritional supplement and supportive therapy in the absence of other systemic treatments. Consequently, the duration of AHCC support necessary to optimize the effect of AHCC use on relapse prevention requires further evaluation on the basis of both target IFN-βilevels and HPV infectionistatus.
... AHCC is a proprietary, standardized extract of cultured lentinula edodes mycelia (AHCC ® , Amino Up, Ltd., Sapporo, Japan) that was developed in Japan in 1992; the compound is primarily composed of a-glucan components. Several animal and human studies have reported a variety of therapeutic effects, including antioxidant and anticancer activities and modulation of the immune system to prevent the infectious processes of both viral and bacterial infections (9)(10)(11)(12)(13)(14). In clinical studies, AHCC has demonstrated the benefit to decrease the risk of infection and ameliorate symptoms of existing infections (11). ...
... The human immune system begins to weaken at about 25 years of age, which perhaps could be the contributing factor to why persistent HPV infections are most often observed in women over the age of 30 or with other immunosuppressive conditions. This phase II study confirmed findings from the two previous pilot studies that demonstrated that AHCC supplementation will modulate the host immune system to eliminate persistent highrisk HPV infections (14). While it may not help all patients, in the absence of other systemic treatments, clinicians can recommend AHCC, which is a readily available nutritional supplement that offers a good chance of clearance of persistent HPV infections. ...
Article
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Objective To determine the efficacy, safety, and durability of the use of AHCC supplementation for 6 months to support the host immune system to clear high-risk human papillomavirus (HPV) infections. The AHCC supplement is a proprietary, standardized extract of cultured lentinula edodes mycelia (AHCC®, Amino Up, Ltd., Sapporo, Japan) that has been shown to have unique immune modulatory benefits. Study Design This was a randomized, double-blind, placebo-controlled study (CTN: NCT02405533) in 50 women over 30 years of age with confirmed persistent high-risk HPV infections for greater than 2 years. Patients were randomized to placebo once daily for 12 months (N = 25) or AHCC 3-g supplementation by mouth once daily on empty stomach for 6 months followed by 6 months of placebo (N = 25). Every 3 months, patients were evaluated with HPV DNA and HPV RNA testing as well as a blood sample collected to evaluate a panel of immune markers including interferon-alpha, interferon-beta (IFN-β), interferon-gamma (IFN-γ), IgG1, T lymphocytes, and natural killer (NK) cell levels. At the completion of the 12-month study period, patients on the placebo arm were given the option to continue on the study to receive AHCC supplementation unblinded for 6 months with the same follow-up appointments and testing as the intervention arm. Results Fifty women with high-risk HPV were enrolled, and 41 completed the study. Fourteen (63.6%) of the 22 patients in the AHCC supplementation arm were HPV RNA/HPV DNA negative after 6 months, with 64.3% (9/14) achieving a durable response defined as being HPV RNA/HPV DNA negative 6 months off supplementation. On the placebo arm, two (10.5%) of 19 patients were HPV negative at 12 months. In the twelve placebo arm patients who elected to continue on the unblinded study, 50% (n = 6) were HPV RNA/HPV DNA negative after 6 months of AHCC supplementation. At the time of completion of the study, there were a total of 34 patients (22 blinded and 12 unblinded) who had received AHCC supplementation with an overall response rate of 58.8% that cleared HPV persistent infections. At the time of enrollment, the mean IFN-β level was 60.5 ± 37.6 pg/ml in women with confirmed persistent HPV infections. Suppression of IFN-β to less than 20 pg/ml correlated with an increase in T lymphocytes and IFN-γ and durable clearance of HPV infections in women who received AHCC supplementation. Conclusion Results from this phase II study demonstrated that AHCC 3 g once daily was effective to support the host immune system to eliminate persistent HPV infections and was well tolerated with no significant adverse side effects reported. The duration of AHCC supplementation required beyond the first negative result needs more evaluation to optimize success for durable outcomes. The suppression of the IFN-β level to less than 20 pg/ml correlated with clearance of HPV infections and merits further evaluation as a clinical tool for monitoring patients with HPV infections. Clinical Trial Registration clinicaltrials.gov/ct2/, identifier NCT02405533
... It is worth mentioning that previous evidence coming from other research groups highlights no cytotoxic effect that could be ascribed to AHCC even at clinically relevant concentrations. 25 The above findings demonstrated that BAIC has an antiproliferative impact on the adenocarcinoma cell lines tested (MCF-7 and Panc02) with differences that are probably associated to the nature of the cells used. According to our study, Chen et al also reported a great reduction in human pancreatic cancer cells viability following the exposure to the natural Wasabi compound, the 6-MITC, and its chemical derivatives. ...
... 34 On the other hand, no significant differences have been noticed in cell cycle distribution following the treatment with AHCC, which is consistent with its role as coadjutant of antitumorigenic therapies than as single agent. 25,35,36 Since the mode of action of natural compounds inducing the cell cycle arrest has been closely related to the activation of the apoptotic cascade, 37 we investigated whether the reduction of viable cells following the treatment with BAIC could also be ascribed to apoptosis in both cell lines. Following the treatment, a statistically significant increase in the percentage of apoptotic Panc02 and MCF-7 cells was demonstrated (up to 24% for apoptotic cells for Panc02 and 50% for MCF-7). ...
Article
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The Standardized Cultured Extract of Lentinula edodes Mycelia (also known as Active Hexose Correlated Compound, AHCC) and Wasabia japonica (Wasabi) are natural nutritional supplements known for their immunomodulatory and anticancer potential. The aim of this study was to evaluate the combinatorial effect of the bioactive immunomodulatory compound (BAIC), obtained by combining Wasabi and AHCC, on human breast (MCF-7) and pancreatic (Panc02) adenocarcinoma cell lines. Data obtained revealed that BAIC determines a striking decline in cancer cell growth at minimal concentrations compared with the use of Wasabi and AHCC as single agents. A significant increase in the G 0 /G 1 subpopulation together with a marked augmentation in the percentage of apoptotic cells was demonstrated by flow cytometry, together with a significant upregulation in the expression of genes associated to the apoptotic cascade in both cell lines. The inhibitory role BAIC plays in mammospheres formation from MCF-7-derived cancer stem cells was shown with a marked reduction in size and number. Interestingly, when BAIC was exposed to monocytic cells, no cytotoxic effects were observed. A monocytes-to-macrophages differentiation was rather observed with the concomitant acquisition of an anti-inflammatory phenotype. Taken together, our findings suggest that BAIC could be used as a potential integration of standard chemotherapy treatments because of the improved inhibitory activity on cancer cell proliferation and reduced potential adverse effects.
... In clinical studies AHCC has demonstrated benefit to decrease risk of infection and ameliorate symptoms of existing infections (9)(10)(11)13). Since studies have demonstrated AHCC induces apoptosis (12)(13)(14), it is possible that AHCC may also prevent/delay tumor growth regardless of the role of HR-HPV. ...
... Growth inhibition assays were conducted as previously described (12). The IC 20 (inhibitory concentration to achieve 20% cell death), and IC 50 for AHCC and each cell line were calculated ( ...
Article
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Objective: There is currently no effective medicine or supplement for clearance of high risk- human papillomavirus (HR-HPV) infections. We have taken a systematic approach evaluating the potential use of AHCC supplementation to support clearance of HR-HPV infections. The primary objective of this research was to evaluate AHCC supplementation to modulation of the host immune system to clear HR-HPV infections from bench to bedside. Methods: Cervical cancer cells, CaSki (HPV16⁺), HeLa(HPV18⁺), SiHa(HPV16/18⁺), and C-33A(HPV⁻), were treated in vitro with AHCC 0.42 mg/mL daily x7 days then observed x7 days with daily sample collection. A confirmatory study in cervical cancer mouse models, SiHa(HPV16/18⁺) and C-33A(HPV⁻), was conducted: mice were divided into three groups per cell line then dosed with AHCC 50 mg/kg/d (N = 10), or vehicle alone (N = 10), or no supplementation (N = 10) for a total of 90 days followed by 30 days of observation. Tumors were measured 3x/week and blood samples collected bi-weekly to evaluate interferon (IFN) alpha(α), beta(β), and gamma(γ) and immunoglobulin G(IgG) by immunoassays. Tumors were evaluated for HR-HPV expression by PCR. Two pilot studies of 10 patients each were conducted in women with confirmed persistent HR-HPV+ infections. The 1st study evaluated AHCC 3g from 5 weeks up to 6 months and 2nd study evaluated AHCC 1g < 8 months. HR-HPV DNA status and the immune panel were monitored at each visit. Results: HR-HPV clearance was observed in vitro and confirmed in the animal studies as a durable response. Four of six (66.7%) patients had confirmed HR-HPV clearance after 3–6 months of AHCC 3g. Similarly, 4 of 9 (44%) patients had confirmed HR-HPV clearance after 7 months of AHCC 1g. Suppression of IFNβ <25 pg/mL was observed in those clearing the HR-HPV infection. Conclusion: Pre-clinical in vitro and in vivo studies demonstrated durable clearance of HR-HPV infections. The preliminary data from the two pilot studies suggested that AHCC supplementation supports the host immune system for successful clearance of HR-HPV infections. A confirmatory phase II randomized, double-blinded, placebo-controlled study is ongoing.
... Data are expressed as mean ± standard error (SE) of the target protein ratio to actin protein. (8)(9)(10)(11)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) Statistical analysis. Statistical analysis was performed on a database using IBM SPSS Statistics v22 (IBM Corp.). ...
Article
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AHCC®, a standardized extract of cultured Lentinula edodes mycelia, enhances the therapeutic effects and reduces the adverse effects of chemotherapy. Our previous study reported that treatment with AHCC® downregulated the expression levels of tumor-associated proteins in the gemcitabine-resistant pancreatic cancer cell line, KLM1-R. However, to the best of our knowledge, the role of AHCC® in the inhibition of cell migration remains unexplored. Cortactin (CTTN), an actin nucleation-promoting factor, has been reported to be upregulated and correlated with migration, invasion and metastasis in pancreatic cancer cells. The present study aimed to investigate the effects of AHCC® on cell migration and the protein expression level of CTTN in KLM1-R cells. The Gene Expression Profiling Interactive Analysis (GEPIA2), an online bioinformatics platform, was used to analyze CTTN mRNA expression levels in pancreatic cancer tissues compared with normal pancreatic tissues. CTTN mRNA expression and its association with clinicopathological characteristics were assessed by using the GEPIA2 platform. Next, the effects of AHCC® on KLM1-R cell migration were investigated by in vitro wound-healing assay. The KLM1-R cells were treated with AHCC® at a concentration of 10 mg/ml for 48 h. Western blotting was performed on of cell lysates with anti-CTTN or anti-actin antibodies to assess the protein expression levels of CTTN. Bioinformatics analysis indicated that the mRNA expression level of CTTN increased in pancreatic cancer tissues. The increased mRNA expression levels of CTTN were inversely associated with clinicopathological characteristics, including disease stages and prolonged patient survival times. The administration of 10 mg/ml AHCC® significantly inhibited KLM1-R cells migration compared with controls. The protein expression levels of CTTN were significantly reduced in AHCC®-treated KLM1-R cells, whereas actin expression was not affected. The downregulation of CTTN indicated the anti-metastatic potential of AHCC® in pancreatic cancer cells. Overall, AHCC® may have the potential to be a complementary and alternative therapeutic approach in treating pancreatic cancer.
... These beneficial effects of AHCC on reducing adverse events caused by 5-FU are consistent with previous reports by others [29][30][31]. These results also indicated that AHCC is safe when administered at an effective dose [32]. Indeed, in a 90-day study with Sprague-Dawley rats, the no-observed-adverse-effect level of AHCC was considered to be as high as 3000 mg/kg body weight/day [33]. ...
Article
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A variety of immunomodulators can improve the efficacy of low-dose chemotherapeutics. Active hexose correlated compound (AHCC), a mushroom mycelia extract, has been shown to be a strong immunomodulator. Whether AHCC could enhance the antitumor effect of low-dose 5-fluorouracil (5-FU) via regulation of host immunity is unknown. In the current study Hepatoma 22 (H22) tumor-bearing mice were treated with PBS, 5-FU (10 mg·kg(-1)·d(-1), i.p), or AHCC (360 mg·kg(-1)·d(-1), i.g) plus 5-FU, respectively, for 5 d. CD3(+), CD4(+), CD8(+), and NK in peripheral blood were detected by flow cytometry. ALT, AST, BUN, and Cr levels were measured by biochemical assay. IL-2 and TNFα in serum were measured using the RIA kit and apoptosis of tumor was detected by TUNEL staining. Bax, Bcl-2, and TS protein levels were measured by immunohistochemical staining and mRNA level was evaluated by RT-PCR. Diet consumption and body weight showed that AHCC had no apparent toxicity. AHCC could reverse liver injury and myelosuppression induced by 5-FU (P < 0.05). Compared to mice treated with 5-FU, mice treated with AHCC plus 5-FU had higher thymus index, percentages of CD3(+), CD4(+), and NK cells (P < 0.01), and ratio of CD4(+)/CD8(+) (P < 0.01) in peripheral blood. Radioimmunoassay showed that mice treated with AHCC plus 5-FU had the highest serum levels of IL-2 and TNFα compared with the vehicle group and 5-FU group. More importantly, the combination of AHCC and 5-FU produced a more potent antitumor effect (P < 0.05) and caused more severe apoptosis in tumor tissue (P < 0.05) compared with the 5-FU group. In addition, the combination of AHCC and 5-FU further up-regulated the expression of Bcl-2 associated X protein (Bax) (P < 0.01), while it down-regulated the expression of B cell lymphoma 2 (Bcl-2) (P < 0.01). These results support the claim that AHCC might be beneficial for cancer patients receiving chemotherapy.
Article
The purpose of this study was to investigate the antiproliferative effect of active hexose correlated compound (AHCC), derived from basidiomycete mushroom culture, on ovarian cancer cell lines. An in vitro growth inhibition assay was performed using AHCC in ovarian cancer cell lines. Western blotting was performed to investigate the mechanism of the observed antiproliferative effect of AHCC. We identified that ovarian cancer cell viability was significantly reduced through treatment with AHCC compared to that in the control. AHCC inhibited constitutive signal transducer and activator of transcription 3 (STAT3) phosphorylation in ovarian cancer cell lines. In contrast, treatment with pervanadate, a protein tyrosine phosphatase inhibitor, reversed AHCC-induced STAT3 suppression. AHCC treatment induced the expression of SHP-1, a protein tyrosine phosphatase, and suppressed the expression of cyclin D1, Bcl-2, Mcl-1, survivin, and VEGF, which are STAT3-regulated gene products that are associated with cell proliferation or apoptosis. These results suggest that AHCC has an antiproliferative effect on ovarian cancer cell lines, via STAT3 phosphorylation; thus, this compound has the potential to be a complementary and alternative anticancer therapy for the treatment of ovarian cancer.
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Objective: To determine the impact on antitumor activity when active hexose correlated compound (AHCC) in combination with anticancer hormonal agents in orthotopic mouse models of human estrogen receptor positive breast cancer and evaluate impact of AHCC on aromatase activity. Methods: The study consisted of 7 treatment arms (n=10) conducted in 2 breast cancer mouse models: MCF-7 and ZR-75. Treatment groups included untreated, vehicle, AHCC 50 mg/kg, AHCC 50 mg/kg + tamoxifen 10 mg/kg, tamoxifen 10 mg/kg, AHCC 50 mg/kg + letrozole 10 µg/mouse, or letrozole 10 µg/mouse. All treatments were administered daily by oral gavage for 12 weeks. Tumors were measured 3 times a week. In vitro estrone and 17β-estradiol enzyme immunoassay was used to evaluate aromatase activity. Results: There was no difference in the activity with the combination of AHCC + tamoxifen compared with tamoxifen ( P = 0.29). In the ZR-75 model (catechol- O-methyltransferase [COMT] wild-type), there was no difference in activity with the letrozole + AHCC compared with letrozole. However, in the MCF-7 model (COMT variant), AHCC + letrozole resulted in a decrease in activity compared with letrozole ( P < 0.01). Immunoassay data suggested that AHCC is a potential inducer of aromatase activity. In both tumor models, there was cytotoxicity observed with AHCC compared with untreated ( P < 0.02). Conclusion: AHCC did not change the activity of tamoxifen. AHCC may have some interaction with letrozole in patients with COMT variant genotype. AHCC had cytotoxicity that warrents additional studies to evaluate its potential role for consolidation/prevention of breast cancer.
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An evidence-based systematic review of active hexose correlated compound (AHCC) by the Natural Standard Research Collaboration consolidates the safety and efficacy data available in the scientific literature using a validated, reproducible grading rationale. This article includes written and statistical analysis of clinical trials, plus a compilation of expert opinion, folkloric precedent, history, pharmacology, kinetics/dynamics, interactions, adverse effects, toxicology, and dosing.
Article
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Immunoceuticals can be considered as substances having immunotherapeutic efficacy when taken orally. More than 50 mushroom species have yielded potential immunoceuticals that exhibit anticancer activity in vitro or in animal models and of these, six have been investigated in human cancers. All are non-toxic and very well tolerated. Lentinan and schizophyllan have little oral activity. Active Hexose Correlated Compound (AHCC) is poorly defined but has shown early clinical promise. Maitake D-Fraction has limited proof of clinical efficacy to date, but controlled research is underway. Two proteoglycans from Coriolus versicolor - PSK (Polysaccharide-K) and PSP (Polysaccharide-Peptide - have demonstrated the most promise. In Japanese trials since 1970, PSK significantly extended survival at five years or beyond in cancers of the stomach, colon-rectum, esophagus, nasopharynx, and lung (non-small cell types), and in a HLA B40-positive breast cancer subset. PSP was subjected to Phase II and Phase III trials in China. In double-blind trials, PSP significantly extended five-year survival in esophageal cancer. PSP significantly improved quality of life, provided substantial pain relief, and enhanced immune status in 70-97 percent of patients with cancers of the stomach, esophagus, lung, ovary, and cervix. PSK and PSP boosted immune cell production, ameliorated chemotherapy symptoms, and enhanced tumor infiltration by dendritic and cytotoxic T-cells. Their extremely high tolerability, proven benefits to survival and quality of life, and compatibility with chemotherapy and radiation therapy makes them well suited for cancer management regimens.
Article
Active hexose correlated compound (AHCC), a phyto-polysaccharide extract, is known to show biological response modifier (BRM)-like activity. Because interleukin-12 (IL-12) and interferon-γ (IFN-γ) negatively modulate tumor growth, we evaluated the possible effect of AHCC on the production of IL-12 and IFN-γ from peripheral blood mononuclear cells (PBMC) as well as on NK cell activity, which also plays a critical role in cancer immunity. Thirty-eight patients with solid tumors were given AHCC orally for 6 months, and their peripheral blood was taken every two months to verify the effects of AHCC on their immune function. PBMCs (2x105/200 μ l) resuspended in RPMI-1640 with 10% FCS were stimulated with 20 μ g/ml of phytohemagglutinin (PHA) in microtiter plates for 24 hours at 37 °C. Supernatant was collected for cytokine assay. Both IL-12 and IFN-γ were measured by the enzyme-linked immunosorbent assay (ELISA) kit. For the assay of NK cell activity, 51Cr- sodium chromate-labeled target cells (K-562; 1x104/10 μ l) were mixed with effector cells (1x106/200 μ l) and incubated for 3.5 hours at 37°C. Supernatant fluid was collected and radioactivity was measured. Performance status (PS) as an indicator of QOL was also evaluated before and after the intake of AHCC. The basal levels of two cytokines and NK activity in patients with tumors were lower than those in normal controls. All three immunological parameters of the patients increased to normal levels after intake of the compound. PS of patients also improved after intake of the compound. These results demonstrate that AHCC improves both immunological abnormalities and clinical conditions.
Article
Active Hexose Correlated Compounds (AHCC) extracted from cultured broth of Basidiomycotina were examined for their protective effects on carbon tetrachloride (CCl4)-induced liver injury in mice. The AHCC treatment obviously suppressed the changes of several physiological and biochemical parameters in the mice injected with CCl4 for 5 days: the decrease of body weights and the increases of liver weights, the sGPT (serum glutamate pyruvate transaminase) level and the level of LPO (lipid peroxide) induced by CCl4 were significantly reduced by the AHCC treatment. The AHCC treatment also induced detoxication enzymes - GST (glutathione S-transferase) and UDP- GT (uridine diphosphate glucuronyl transferase) activities. AHCC also showed some effects on the CCl4-induced suppression of phase I drug-metabolizing enzymes. The anti-oxidant effect and the activity modifying-effect on drug- metabolizing enzymes might be related to the AHCC protection of liver from CCl4-induced liver injury.
Article
Effects of Active Hexose Correlated Compound (AHCC) on the onset of diabetes were studied in rats treated with streptozotocin (STZ). AHCC was given to male rats at 4% in drinking water. A single i.v. injection of STZ (40 mg/kg body weight) to rats resulted in an increase in blood glucose levels, a decrease in serum insulin levels, suppression of body weight gain, and an increase in serum GOT and GPT activities and serum levels of lipid peroxides. Treatment of AHCC restored these parameters to normal. Insulin immunoreactive β-cells in Langerhans islets reduced in number after treatment with STZ, while insulin immunoreactivity in the islets was normalized when AHCC was administered to STZ-treated rats. These results show that AHCC treatment is effective on the prevention of diabetes onset induced by STZ.
Article
Active hexose correlated compound (AHCC) is an extract of a basidiomycete mushroom that is used as a supplement by some cancer patients undergoing chemotherapy; it is thought to enhance the therapeutic effects and reduce the side effects of select anticarcinogenic agents. AHCC has been reported to strengthen the anticancer effects of cisplatin (CDDP) and ameliorate its side effects in female BALB/cA mice inoculated with Colon-26 tumor cells. In this study, the role of AHCC in alleviating the side effects induced by several other anticancer drugs was explored in non-tumor-bearing mice receiving monotherapy with paclitaxel (TAX), or multi-drug chemotherapy with TAX plus CDDP, 5-fluorouracil (5FU) plus irinotecan, CDDP plus 5FU, or doxorubicin plus cyclophosphamide. Outcomes from the drug treatment groups with and without AHCC supplementation were compared to controls that received vehicle alone. The multi-drug treatments significantly reduced bone marrow cell viability in all groups and leukocyte count in all groups except for TAX+CDDP; these myelosuppresive effects were generally alleviated by AHCC. Hepatotoxicity and nephrotoxicity caused by the treatments that included TAX and CDDP were also significantly improved by AHCC. The death rate was 20 to 30 percent in all treatment groups except TAX+CDDP, and supplementation with AHCC greatly reduced or eliminated mortality. These results support the concept that AHCC can be beneficial for cancer patients receiving chemotherapy.
Article
From December 1983 through February 1992, a prospective study designed to determine the clinical course of patients with ovarian tumors of low malignant potential (LMP) was conducted by the Gynecologic Oncology Group (GOG). This protocol was developed to evaluate the following (1) the biologic behavior of ovarian LMP tumors, (2) the effectiveness of melphalan chemotherapy in patients with clinically detectable residual disease after surgical staging and in patients whose tumors progress or recur after surgical therapy, and (3) the response rate to cisplatin in those who failed to respond to melphalan therapy. The study group consisted of 146 assessable patients with stage I serous LMP tumors. All of these women had the affected ovary (or ovaries) removed, and a complete staging operation was performed in each case. While 123 patients had a total abdominal hysterectomy (TAH) and bilateral salpingo-oophorectomy (BSO), 21 retained the uterus and one normal-appearing ovary and fallopian tube. No adjuvant chemotherapy or radiation therapy was administered to any patients in the stage I study group. The median follow-up time was 42.4 months (range, 1.6 to 108). Thus far, no patient with a stage I ovarian serous LMP tumor has developed recurrent disease. Stage I ovarian serous LMP tumors rarely, if ever, recur. Limited resection, after meticulous surgical exploration, is adequate therapy for women of reproductive age.
Article
A phase II study of liposomal doxorubicin was conducted in patients with ovarian cancer who failed to respond to platinum- and paclitaxel-based regimens. Liposomal doxorubicin was selected as a result of its superior activity against ovarian cancer xenografts relative to free doxorubicin and activity in refractory ovarian cancer patients that was noted during the phase I study. Thirty-five consecutive patients were accrued in two institutions (22 in one and 13 in the other). All had progressive disease after either cisplatin or carboplatin and paclitaxel, or at least one platinum-based and one paclitaxel-based regimen. Patients received intravenous (I.V.) liposomal doxorubicin 50 mg/m2 every 3 weeks with a dose reduction to 40 mg/m2 in the event of grade 3 or 4 toxicities, or a lengthening of the interval to 4 weeks (and occasionally to 5 weeks) with persistence of grade 1 or 2 toxicities beyond 3 weeks. Nine clinical responses (one complete response [CR], eight partial responses [PRs]) were observed in 35 patients (25.7%), with seven of these having been confirmed by two consecutive computed tomographic (CT) measurements. The median progression-free survival was 5.7 months with an overall survival of 1.5 to 24+ months (median, 11 months). Although 13 patients experienced grade 3 or 4 nonhematologic skin and mucosal toxicities (either hand-foot syndrome or stomatitis), with dose modifications, the treatment was very well tolerated. Nausea that was clearly attributable to the drug, hair loss, extravasation necrosis, or decreases in ejection fraction did not occur. Liposomal doxorubicin has substantial activity against ovarian cancer refractory to platinum and paclitaxel. The responses achieved with liposomal doxorubicin were durable and maintained with minimal toxicity. This liposomal formulation should be evaluated further in combination with other drugs in less refractory patients.
Article
Synergistic effects of active hexose correlated compound (AHCC) extracted from mushroom on the treatment with UFT against mammary adenocarcinoma, SST-2 cells, in congenitally T cell-depressed spontaneously hypertensive rats (SHR) were observed. AHCC plus UFT had slight but significant effects on the growth of primary tumors. Pulmonary metastases were not inhibited by the treatment with AHCC plus UFT, whereas metastases to axillary lymph nodes (LN) were obviously inhibited. Combination of AHCC plus UFT showed similar synergistic anti-metastatic effects in SHR rats with accelerated pulmonary metastases following the surgical removal of the primary tumors. In vitro studies demonstrated that AHCC plus UFT enhanced the NK cell activity in tumor-bearing rats, whereas UFT alone depressed the NK cell activity. AHCC plus UFT also enhanced the NO production and cytotoxicity of peritoneal macrophages. In addition, AHCC restored the suppressed mRNA expression of interleukin-1alpha and tumor necrosis factor-alpha induced by the chemotherapy. Taken together, the combination of AHCC plus UFT brought about good therapeutic effects not only on primary tumor growth but also on reducing metastasis and these effects were mediated by host immunity which was restored or activated by AHCC. AHCC may be a good candidate for a biological response modifier.
Article
The leading cause of death from gynecologic malignancies in the United States is epithelial ovarian cancer. The significant risk factor for development of ovarian cancer is advancing age, although there is clearly a genetic predisposition--often associated with the BRCA1 and BRCA2 genes--in at least 5% to 10% of all epithelial ovarian cancers. Oral contraceptives are known to reduce the risk for development of ovarian cancer and should be considered as a method of birth control in women at increased risk. Currently, there is no acceptable method of screening for this disease, although measurement of CA-125 level and transvaginal ultrasound have been utilized. Ovarian cancer is a surgically staged disease. In apparent early-stage disease, complete surgical staging is critical for the selection of adjunctive therapy. In advanced-stage disease, the goal is primary cytoreduction. Standard postoperative therapy for advanced-stage ovarian cancer includes platinum-based chemotherapy with the substitution of paclitaxel for cyclophosphamide occurring in the last decade. Despite these advances in chemotherapy, ovarian cancer continues to be fatal in far too many cases.