Carcinogenesis vol.20 no.1 pp.59–63, 1999
Inhibition of hepatocellular carcinoma by glycyrrhizin in
diethylnitrosamine-treated mice
Goshi Shiota2, Ken-ichi Harada, Masato Ishida,
Yasushi Tomie, Michiko Okubo, Shunsuke Katayama,
Hisao Ito1 and Hironaka Kawasaki
Second Department of Internal Medicine, Tottori University School of
Medicine, Yonago 683-8504 and 1First Department of Pathology,
Tottori University School of Medicine, Yonago 683-8504, Japan
2To
whom correspondence should be addressed
Glycyrrhizin (GL) is widely used in Japan as a therapeutic
agent for chronic active liver diseases. However, its action
on hepatocarcinogenesis remains to be elucidated. To clarify
its effect, mice treated with diethylnitrosamine (NDEA)
with or without GL were analyzed. Five-week-old male
BALB/c mice were divided into two groups, GL (n J 50)
and C (n J 47). Mice in the GL group intramuscularly
received 2 mg of GL 3 days a week, and mice in the C
group received the same volume of saline in the same way.
After 2 weeks, the mice were treated with an i.p. injection
of 75 mg/kg body wt of NDEA weekly for 3 weeks and
100 mg/kg body wt of NDEA weekly for the following 3
weeks. Thirty additional mice that did not receive NDEA
treatment were divided into two groups, GC (n J 15) and
SC (n J 15). They received GL or saline, respectively.
Mice in the 4 groups were killed every 5 weeks after the
last injection of NDEA from 7 weeks to 32 weeks. Liver
function tests such as AST and albumin were significantly
improved in the GL group compared with the C group
(P < 0.05, each). Although liver nodules appeared in the C
group at 22 weeks, they were not observed until 32 weeks
in the GL group. At 32 weeks, the mean number of
liver tumors, composed of adenoma and hepatocellular
carcinoma (HCC), in the GL group was 0.71, which was
significantly decreased compared with 1.64 of the C group
(P < 0.05). The mean number of HCC in the GL group
was 0.29/liver, which was lower than 0.82/liver in the C
group (P < 0.05). The incidence rate of HCC at 32 weeks
was 64% in the C group and 21% in the GL group (P <
0.05, C versus GL group). Our results suggest that GL
treatment inhibits the occurrence of HCC.
Introduction
Hepatocellular carcinoma (HCC) is one of the most common
cancers with a poor prognosis (1). There are ~500 000–
1 000 000 new HCC cases per year (2). Major risk factors for
HCC include cirrhosis, aflatoxin exposure, hepatitis B virus
(HBV) and hepatitis C virus (HCV) infection (3). Studies on
the prevalence of anti-HCV antibody in patients with HCC
Abbreviations: AFP, α-fetoprotein; ALP, alkaline phosphatase; ALT, alanine
aminotransferase; AST, aspartate aminotransferase; GA, glycyrrhetic acid;
GL, glycyrrhizin; GOT, glutamic oxaloacetic transaminase; GPT, glutamic
pyruvic transaminase; HBC, hepatitis C virus; HBV, hepatitis B virus; HCC,
hepatocellular carcinoma; IFN, interferon; LW/BW, liver weight/body weight;
NDEA, diethylnitrosamine.
© Oxford University Press
indicate that the highest prevalence of anti-HCV positivity is
found in Japan (66–94%), that a relatively high prevalence
(57–79%) is found in Africa, Italy and France, and that a
lower prevalence (,40%) is typical in the USA, Switzerland,
Austria, China and Hong Kong (2). HCC can develop late in
the course of viral infection, especially of chronic HCV
infection, after the development of cirrhosis (4). HCV carriers
demonstrate higher rates of developing HCC than HBV carriers
(5); the incidence of HCC in HCV carriers is 4.2-fold higher
than in HBV carriers, and it is 572-fold higher than in normal
individuals (6). Therefore, prophylaxis of HCC occurrence
from chronic viral hepatitis is likely as important as the
progress of HCC therapy.
Glycyrrhizin (GL), a molecule of glycyrrhetic acid (GA)
and two molecules of glucuronic acid, is an aqueous extract
from licorice root (7). This compound is well known for its
anti-inflammatory properties in Chinese medicine (8). GL and
the GA derivatives exhibit anti-ulcerous (9), anti-viral (10–
12) and interferon (IFN)-inducing activity (13). GL was proven
to be useful for therapy of chronic hepatitis (14). Indeed,
double blind controlled trials showed that GL improved hepatic
function tests such as GOT, GPT and γ-GTP in patients with
chronic active hepatitis (15,16). Thereafter, GL has often been
used in treatment for chronic active liver diseases in Japan.
We examined the effect of GL on hepatocarcinogenesis in
mice treated with diethlynitrosamine (NDEA). An NDEA
model was used because nitrate and nitrosamine synthesis is
increased in viral hepatitis (17,18). Therefore, a NDEA model
may be useful as a model of hepatocarcinogenesis due to viral
hepatitis.
Materials and methods
Chemicals
NDEA was purchased from Wako Pure Chemical Industries (Osaka, Japan).
GL was kindly provided from Minophagen Pharmaceutical (Tokyo, Japan).
Animals
Five-week-old male BALB/c mice, weighing 22–23g, were purchased from
Charles River Japan (Yokohama, Japan). The mice were fed a CE-2 diet
(CLEA Japan, Tokyo, Japan) and were given water ad libitum. Light and dark
alternated at 12 h intervals. All animals received humane care in compliance
with the guidelines of our university.
Experimental procedure
Ninety-seven BALB/c mice were divided into two groups, GL (n 5 50) and
C (n 5 47). The experimental schedule is shown in Figure 1. In brief, mice
in the GL group intramuscularly received 2 mg/mouse of GL 3 days a week,
and mice in the C group received the same volume (0.1 ml) of saline in the
same way (Figure 1A). Two weeks after the start of administration of GL or
saline, an i.p. of 75 mg/kg body wt of NDEA was given weekly for 3 weeks
(19). Then, 100 mg/kg body wt of NDEA was weeky given for the following
3 weeks. Thirty additional mice without NDEA treatment were divided into
two groups, GC (n 5 15) and SC (n 5 15). Mice in the GC group
intramuscularly reveived 2 mg/mouse of GL 3 days a week, and mice in the
SC group received 0.1 ml of saline in the same way (Figure 1B). Mice in the
four groups were killed every 5 weeks from 7 to 25 weeks. The numbers of
mice killed in the GL group at 12, 17, 22, 27 and 32 weeks were 9, 9, 9, 9
and 14, respectively, and those in the C group at 12, 17, 22, 27 and 32 weeks
were 9, 9, 9, 9 and 11, respectively. The number of mice in the GC and SC
59
G.Shiota et al.
Fig. 1. Experimental schedule of the NDEA model. (A) Experimental
schedules of the GL and C groups are shown. (B) Experimental schedules
of the GC and SC groups are shown.
groups at each time point was three. Animals were killed under ether
anesthesia, weighed, necropsied and examined for the presence of grossly
visible lesions. The livers were removed in total, weighed and sectioned into
1–2 mm strips to examine the presence of tumors. Strips of liver were fixed
in 4% paraformaldehyde for 24 h, and then embedded in paraffin. Sectioned
paraffin blocks were stained with hematoxylin & eosin.
Histology
The morphology of hepatocellular neoplasms was classified by cell size,
tinctorial properties of the cytoplasm, pattern of growth and other morphological features as previously described (20). Two independent investigators
performed the histological assessment in a blind manner. The liver sections
from the four groups were randomly shown to the investigators.
Liver function tests
Liver function was examined since HCC develops from chronic liver diseases
(21,22). Blood parameters were used since they were useful to assess liver
function of chronic liver diseases (23). Serum bilirubin was measured by the
azobilirubin method, using Kurimate BIL-2 (Daiichi Kagaku Pharmaceutical,
Tokyo, Japan). Serum albumin was measured by the bromcrezol green method,
using Alb Jisseiken (Jisseiken, Japan). Serum aspartate aminotransferase
(AST) and alanine aminotransferase (ALT) were measured by the ultraviolet
spectrometry method, using Quickauto II, glutamic oxaloacetic transaminase
[GOT(S)] Shinotest (Shinotest, Japan) and Quickauto II glutamic pyruvic
transaminase [GPT(S)] Shinotest (Shinotest, Japan), respectively. Serum alkaline phosphatase (ALP) was measured by the SSCC method, using ALP
Jisseiken (Jisseiken, Japan). These were analyzed using a Biochemical
Autoanalyzer (AU5232; Olympus, Tokyo, Japan). Measurement of α-fetoprotein (AFP) was performed by a radioimmunoassay, using AFP Eiken
(Eiken Kagaku, Tokyo, Japan), and followed by counting by γ-counter (ARC950; Aroka, Tokyo, Japan).
Statistics
All data were expressed as means 6 SEM. The difference between the two
groups was determined by the Mann–Whitney U-test and the χ2 test.
Significance was set at P , 0.05.
Table I. Effect of glycyrrhizin on liver function tests in mice
Groups
Bilirubina
(mg/ml)
Albumina
(g/dl)
ASTa
(IU/l)
ALTa
(IU/l)
ALPa
(IU/l)
GLb
12
17
22
27
32
weeks
weeks
weeks
weeks
weeks
0.1
0.18
0.16
0.13
0.24
0.05
0.02
0.03
0.03
3.51
3.18
2.70
2.81
2.82
6
6
6
6
6
0.26
0.22
0.06f
0.09
0.04
464
327
174
287
332
6
6
6
6
6
38f
32
12
23f
34f
169 6 19
119 6 11
94 6 4
137 6 15
115 6 10
163
219
240
290
230
6
6
6
6
6
43
19
9
35
8
Cc
12
17
22
27
32
weeks
weeks
weeks
weeks
weeks
0.11 6 0.01
0.18 6 0.05
0.1
0.13 6 0.03
0.26 6 0.03
3.55
2.83
2.47
2.69
2.64
6
6
6
6
6
0.24
0.11
0.05
0.10
0.06
620
372
207
415
478
6
6
6
6
6
63
66
40
47
19
182
135
86
144
169
6
6
6
6
6
13
11
10
20
19
234
222
219
254
217
6
6
6
6
6
43
19
11
17
29
GCd
12
17
22
27
32
weeks
weeks
weeks
weeks
weeks
0.1
0.1
0.1
0.1
0.1
3.40
3.60
3.60
3.62
3.65
6
6
6
6
6
0.20
0.05
0.07
0.05
0.05
51
47
53
40
45
6
6
6
6
6
4
7
5
5
6
42
38
40
35
40
6
6
6
6
6
5
6
5
6
7
182
188
199
195
195
6
6
6
6
6
9
7
8
5
3
SCe
12
17
22
27
32
weeks
weeks
weeks
weeks
weeks
0.1
0.1
0.1
0.1
0.1
3.50
3.60
3.65
3.60
3.60
6
6
6
6
6
0.06
0.05
0.06
0.05
0.05
44
47
48
44
42
6
6
6
6
6
5
4
5
5
4
38
40
38
37
38
6
6
6
6
6
6
4
4
7
5
189
183
182
189
202
6
6
6
6
6
4
4
3
6
7
aMean 6 SEM.
bGlycyrrhizin plus
6
6
6
6
NDEA.
cSaline plus NDEA.
dGlycyrrhizin only.
eSaline only.
fP , 0.05, compared
60
with the C group of the corresponding week.
Inhibitions of hepatocellular carcinomas
Fig. 2. Changes in body weights in the four groups. *P , 0.05, compared
with the GL group; **P , 0.05, compared with the SC group.
Results
Body weight and liver weight
The changes in body weight are shown in Figure 2. Body
weights increased with time except that the body weight in
the C group decreased after 22 weeks. Body weights of the
GC group were greater than those of the GL group throughout
the experiment (P , 0.05, each). Body weights of the C group
were lower than the SC group at 12, 17, 27 and 32 weeks
(P , 0.05, each). Mice in the GL group had similar body
weights to the C group throughout the experiment. Liver
weights of the GL group at 12 and 32 weeks were higher than
those of the C group at the corresponding weeks (data
not shown). Although liver weight/body weight (LW/BW)
gradually decreased with time in the SC and GC groups, it
gradually increased in the GL and C groups (data not shown).
At 32 weeks, LW/BW in the GL group was significantly higher
than in the GC group (P , 0.05, data not shown) and the C
group was also higher than the SC group (P , 0.05; data
not shown).
Liver function tests
Serum bilirubin levels in the GL and C groups at 32 weeks
were higher than those at 12 weeks (P , 0.05, respectively;
Table I). However, no difference was observed between the
GL and C groups. Serum albumin levels gradually decreased
in the GL and C groups. However, the albumin decrease in
the GL group was smaller than that in the C group. Serum
albumin in the GL group was higher than in the C group
throughout the experiment. At 22 weeks, serum albumin in
the GL group was significantly higher than that in the C group
(P , 0.05). Serum AST in the GL group was lower than in
the C group throughout the experiment. Levels of serum AST
at 12, 27 and 32 weeks in the GL group were significantly
lower than those in the C group (P , 0.05, respectively).
Changes in serum ALT were similar to those of AST. Serum
ALT was slightly lower in the GL group, compared with the
C group at 32 weeks. Levels of AFP were ,1 ng/ml in all
samples (data not shown). There were no changes in serum
bilirubin, albumin, AST, ALT, ALP or AFP levels between
the GC and SC groups throughout the experiment (Table I).
Tumor incidence
No tumors were observed until 17 weeks in the GL and C
groups. At 22 weeks in the C group, a tumor which was a
histologically hyperplastic nodule developed. After 27 weeks,
Fig. 3. Tumor incidence in mice. Numbers of tumors (A) and HCC (B) are
shown.
adenoma developed in the C group. The numbers of tumors
in the C group at 22, 27 and 32 weeks were 0.11 6 0.11,
0.44 6 0.34 and 1.64 6 0.36/liver, respectively. However, the
number of tumors in the GL group at 32 weeks was 0.71 6
0.22/liver, which was significantly lower than in the C group
(P , 0.05; Figure 3A). HCC first developed in the C group
at 20 weeks. The numbers of HCC in the GL and C groups
at 32 weeks were 0.29 6 0.13 and 0.82 6 0.18 /liver,
respectively. There was a significant difference between the
two groups (P , 0.05; Figure 3B). Histological examination
at 32 weeks showed that 18 tumors in the C group were
composed of nine HCCs and nine adenomas, and that 10
tumors in the GL group were composed of four HCCs and six
adenomas (data not shown). Macroscopic examination of
HCCs in the C group at 32 weeks is shown in Figure 4A. The
color of HCCs was whitish and the tumor edges were sharp.
The tumors were clearly discriminated from the surrounding
tissues (Figure 4B). Cystic dilatation of bile ducts and mild
fatty changes were features observed in non-cancerous tissues
in this experimental model. Microscopic examination revealed
that HCC had a trabecular structure, which is a typical feature
of hepatocellular carcinoma (Figure 4C) (20). Although three
mice in the C group at 32 weeks exhibited lung metastasis
(Figure 4D), no mice in the GL group had lung metastasis.
Therefore, the results suggest that this experimental model is
61
G.Shiota et al.
Fig. 4. Gross and microscopic appearances of HCC. (A) HCCs (arrows) are whitish and their margins are clear. (B) HCC (T) and surrounding tissue (N) are
shown. Cystic dilatation of bile ducts and mild fatty change are characteristic features of non-cancerous tissues (magnification 340). (C) Trabecular
appearance of HCC tissue (magnification 3200). (D) Lung metastasis in mice from the C group at 32 weeks (magnification 340).
useful to assess anti-cancerous drugs. Table II shows the
incidence of HCC in the four groups at 32 weeks. The
incidence rate of HCC in the GL group was 3/14 (21%), which
was significantly ,7/11 (64%) of the C group (P , 0.05). No
tumors were observed in the GC and SC groups throughout
the experiment. These data suggest that GL decreased the
occurrence of HCC in mice treated with NDEA.
Discussion
HCC is one of the most common cancers worldwide (1).
Although many risk factors have been reported (3), viral
infection is an important factor for HCC (2,5,6). In the general
population of Japan, the carrier rates of HBV and HCV are
1.3 and 1.5%, respectively. However, HBV and HCV carriers
account for 20 and 70% of HCC in Japan (6), and the incidence
of HCC is ~3-fold higher in chronic hepatitis C than in chronic
hepatitis B (5). These data suggest that HCC develops at a
fairly high rate in chronic viral liver diseases, especially in
HCV carriers. Therefore, chemoprevention of HCC may be as
important as therapies for HCC.
IFN has been reported to be effective for reducing and
eliminating HCV RNA (24). IFN has been therapeutically
useful for patients with chronic hepatitis C with viral clearance
rates of ,40–50% (25,26). Furthermore, IFN-α (6 mU 3 days
a week for 12–24 weeks) significantly decreased the incidence
of HCC in cirrhotic patients (27), and responders to IFN
62
Table II. Incidence of hepatocellular carcinoma in mice at 32 weeks
Groups
No. of mice
No. of mice that
developed HCC
Percentage
GLa
Cb
GCc
SCd
14
11
3
3
3
7
0
0
21e
64
0
0
aGlycyrrhizin
plus NDEA.
bSaline plus NDEA.
cGlycyrrhizin only.
dSaline only.
eP , 0.05, compared
with C group.
therapy exhibited a lower cumulative incidence of HCC (28).
Thus, IFN therapy decreases viral replication, resulting in a
lower incidence of HCC. In addition, IFN may activate the
immune system, or directly reduce cell proliferation which
leads to the development of HCC or precancerous cells.
This is the first experimental animal study in which GL was
used as a chemopreventive agent of HCC. However, the
inhibitory mechanism of HCC by GL remains unclear. It has
been recently reported that long-term treatment with GL for
chronic hepatitis C was effective in lowering the incidence of
HCC (29). The 10 year rates of cumulative HCC incidence in
patients treated with and without GL were 7 and 12%, and 15
Inhibitions of hepatocellular carcinomas
year rates were 12 and 25%, respectively. The relative risk of
HCC in patients treated without GL was 2.49-fold higher than
patients treated with GL (P , 0.05, GL-treated versus control
group). The authors concluded that the anti-cancerous effect
of GL may be associated with its anti-inflammatory effect.
Nitrosoamine elevation has been reported in viral hepatitis
(17,18). Therefore, GL may decrease nitrosoamine levels
in liver tissues. Furthermore, in our preliminary study, the
expression rate of proliferating cell nuclear antigen (PCNA)
in hepatocytes of the GL group at 7 weeks was 3-fold higher
than that in the C group (data not shown). In addition, the
apoptotic rate of hepatocytes in the GL group at 12 weeks
was similar to that in the C group. However, it increased to
0.41 6 0.09% in the GL group at 32 weeks, which was 2-fold
higher than that in the C group at the corresponding week
(data not shown). Therefore, modulation of cell proliferation
and apoptosis by GL may be associated with inhibition of
HCC in NDEA-treated mice. The inhibitory effect of GL on
hepatocarcinogenesis requires further investigation to clarify
the mechanism of these findings.
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Received May 5, 1998; revised August 18, 1998; accepted September 23, 1998
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