|
I-Shyan
Sheen, Liver Reserch Unit, Chang Gung Memorial Hospital, Taipei,
Taiwan, China
Kuo-Shyang Jeng, Department of Surgery, Mackay Memorial
Hospital, Taipei, Taiwan, China
Wen-Juei Jeng, National Yang-Ming University Medical College,
Taipei, Taiwan, China
Chi-Juei Jeng, National Taiwan University Medical College,
Taipei, Taiwan, China
Yi-Ching Wang, Shu-Ling Gu, Shin-Yun Tseng, Chien-Ming Chu,
Chia-Hui Lin, Department of Medical Research, Mackay Memorial
Hospital, Taipei, Taiwan, China
Kuo-Ming Chang, Department of Pathology, Mackay Memorial
Hospital, Taipei, Taiwan
, China
Supported by Grants From The New Century Health Care
Promotion Foundation, Taiwan, China and Professor Wen-Pin Lien
Correspondence to: Kuo-Shyang Jeng, M.D., F.A.C.S., Department of
Surgery, Mackay Memorial Hospital, No.92, Sec2, Chung-san North
Road, Taipei, Taiwan, China. issheen.jks@msa.hinet.net
Telephone: +886-2-5433535
Fax: +886-2-7065704
Received: 2004-06-08
Accepted: 2004-07-27
Abstract
AIM: To investigate the effect and possible mechanisms of
antiangiogenesis therapy for HCC in rats.
METHODS: Adult male LEW/SsN rats were divided into 3 groups, 25
animals each. Group A was the control group. Groups B and C were
given diethylnitrosamine, 5 mg/kg/d. In addition, group C rats
received an intraperitoneal injection of fumagillin, 30 mg/(kg.d).
Five animals in each group were killed at 6th, 12th, 18th, 20th and
24th wk to evaluate the development of HCC and metastasis. Weight of
the rats, liver tumors, and number of organs involved by HCC were
measured at each stage. We compared methionine aminopeptidase-2
(MetAP-2) mRNA, Bcl-2 mRNA, telomerase mRNA, and telomerase activity
at 24th wk in the liver tissue of group A rats and tumor tissue of
HCC from group B and C rats.
RESULTS: No HCC developed in group A, but tumors were present in
group B and C rats by the 18th wk. At wk 20 and 24, the median liver
weight in group B was 0.64 g (range: 0.58-0.70 g) and 0.79 g (range:
0.70-0.90 g) (P = 0.04), and that in group C was 0.37 g
(range: 0.35-0.42 g) and 0.39 g (range: 0.35-0.47 g) (P =
0.67). The liver weight in group C rats was significantly lower than
that in group B rats (P = 0.009). At the same time, the
median metastasis score (number of organ systems involved) was 3
(range2-3) in group B, and 1 (range 1-2) in group C, a significant
difference between the groups (P = 0.007, 0.004). The levels
of MetAP-2 mRNA were significantly higher in groups B and C than in
group A (P = 0.025), and significantly higher in group C than
in group B (P = 0.047). The level of Bcl-2 mRNA was
significantly higher in group B than in group A (P = 0.024),
but lower in group C than in group B, although not significantly (P
= 0.072). Telomerase mRNA was significantly higher in group B
than in group A (P = 0.025), but significantly lower in group
C than in group B (P = 0.016). The same inter-group
relationship was also true for telomerase activity (P = 0.025
and 0.046).
CONCLUSION: Fumagillin effectively inhibits both liver tumor growth
and metastasis in rats in vivo. A possible mechanism is fumagillin-induced
inhibition of MetAP-2, which plays an essential role in endothelial
cell proliferation. Inhibition of MetAP-2 also results in inhibition
of Bcl-2 and telomerase activity.
ã 2005
The WJG Press and Elsevier Inc. All rights reserved.
Key
words: Hepatocellular carcinoma; Antiangiogenesis therapy;
Fumagillin; MetAP-2
Sheen IS, Jeng KS,
Jeng WJ, Jeng CJ, Wang YC, Gu SL, Tseng SY, Chu CM, Lin CH, Chang
KM. Fumagillin treatment of hepatocellular carcinoma in rats: An in
vivo study of antiangiogenesis. World J Gastroenterol
2005; 11(6): 771-777
http://www.wjgnet.com/1007-9327/11/771.asp
INTRODUCTION
Hepatocellular
carcinoma (HCC), a leading cause of death in Taiwan and many Asian
countries, is difficult to treat because of early progression and
metastasis. It is well known that angiogenesis is essential for the
survival, growth, and metastasis of tumor cells[1-5].
Angiogenesis, formation of new blood vessels from the existing
vascular bed, is a complex multistep process. There is extracellular
matrix remodeling and binding of angiogenic factors to specific
endothelial cell (EC) receptors, which results in EC proliferation,
invasion of basement membrane, migration, differentiation, and
formation of new capillary tubes. Their anastomoses develop a
vascular network. There is much interest in inhibiting angiogenesis
as a treatment strategy[6-9].
Fumagillin
and its derivatives, such as TNP 470, are well-known antiangiogenic
agents[9-18]. There are few published studies, however,
of the in vivo effects of these agents on experimentally induced HCC
in an animal model. The aim of this study was to evaluate the
therapeutic effect and possible mechanisms of antiangiogenesis in a
rat model of HCC.
MATERIALS AND METHODS
Animals
Pathogen-free adult male LEW/SsN rats at the age of 8 wk
were purchased from the National Science Council, Taiwan. They were
fed standard diet chow pellets and water ad libitum. The study was
begun when the rats were 12 wk old, and their median body weight
(BW) was 372.5 g (range: 350-394 g). All experiments were performed
according to standard guidelines for animal experiments and approved
by the Animal Ethics Committees of Mackay Memorial Hospital.
Treatment of rats
The 75 rats were divided into 3 groups, 25 each. Group A
rats were used as controls, receiving food only and no medication.
To induce hepatocarcinogenesis, groups B and C rats were given
diethylnitrosamine (DEN) (C4H10N2O)
(Sigma Chemical, St. Louis, MO. USA) in water at a dose of 5 mg/(kg.d).
Group C rats received, in addition to DEN, intraperitoneal
injections of fumagillin (C26H34O7)
(Sigma Chemical, St. Louis, MO, USA) 0.3 mg/(kg.d) beginning at the
18th wk of DEN induction.
Gross and histologic examination
Five rats in each group were sacrificed at 6th, 12th, 18th,
20th and 24th wk to evaluate the development of liver tumors and
their changes. We measured the body weight, whole liver weight, and
the number of involved organ systems of each rat. Liver and HCC
specimens were examined by pathologists. The tumor weight was
estimated by subtracting the liver weight of group A rats from that
of group B or C rats. In examining for metastasis, we gave a score
of 1 for HCC limited to the liver, 2 for extrahepatic extension or
metastasis within the peritoneal cavity, and 3 if there were both
intraperitoneal and lung metastases.
Molecular mechanisms
To investigate the molecular mechanisms of HCC inhibition by
fumagillin, we detected methionine aminopeptidase 2 (MetAP-2) mRNA,
Bcl-2 mRNA, and telomerase mRNA and telomerase activity from samples
of the resected livers of group A and the resected tumors of groups
B and C, in the 24th wk of treatment. GAPDH mRNA was used as a
control.
Extraction of RNA
We homogenized resected tissue completely in 1mL of RNA-BeeTM
(Tel-Test, Protech Technology Enterprises Co., Ltd, Friendswood,
TX), added 0.2 mL chloroform, and shook vigorously for 15-30 s. We
stored the samples on ice for 5 min and then centrifuged at 12 000 g
for 15 min. We transferred the supernatant to a new 1.5 mL Eppendorf
tube and precipitated the solution with 0.5 mL of isopropanol for 5
min at 4 °C.We centrifuged the tube at 12 000 g for 5 min at 4 °C before removing the supernatant and washed the RNA pellet with
1 mL of isopropanol, shook to dislodge the pellet from the side of
the tube. We centrifuged the pellet again at 12 000 g for 5 min at 4
°C, removed the supernatant, and washed the RNA pellet once with
75% ethanol, shook to dislodge the pellet from the side of the tube.
We suspended the pellet in at least 1 mL of 75% ethanol and
centrifuged it at 7 500 g for 5 min at 4 °C before carefully removing the ethanol. The RNA was to air
dried and then dissolved in DEPC-H2O (50-100 mL)
and stored at -80 °C.
Reverse transcription
We heated the RNA sample at 55 °C for 10 min, chilled it on ice, and then added the following
reagents: 4 mL
5 XRT buffer containing Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L
MgCl2, and 10 mmol/L DTT (dithiothreitol); 3 mL
10 mmol/L dNTP (deoxyribonucleoside triphosphate); 1.6 mL
Oligo-d(T)18 and 0.4 mL
random hexamers (N)6 (1 ug/mL);
0.5 mL
RNase inhibitor (40 units/mL);
3 mL
25 mmol/L MnCl2; 6 mL
RNA in DEPC-H2O; and 0.5 mL
DEPC-H2O. We incubated the mixture at 70 °C for 2 min and then chilled it to 23 °C to anneal the primer to the RNA. We added 1 mL
of M-MLV RTase (Moloney murine leukemia virus reverse transcriptase,
200 units/mL,
Promega) and incubated it for 10 min at 23 °C followed by 60 min at 40 °C. We then heated it at 94 °C for 5 min, chilled it on ice, and stored the cDNA at -20 °C.
By PCR amplification of MetAP-2, Bcl-2, telomerase, and GAPDH
cDNA
First-strand cDNA synthesis was carried out using 2 mg
of total RNA purified from 50 mg tissue. Reverse transcription was
performed in a 20 mL
final volume containing 2 mg
of random hexamer (Gene Teks Bioscience Inc., Taipei), and 1.5 mmol/L
each of dATP, dCTP, dGTP, and dTTP. Each reaction mix was incubated
for 8 min at 23 °C with 20 U of rRNasin (RNase inhibitor; Promega, Madison, WI)
followed by incubation with 200 U of Moloney murine leukemia virus
reverse transcriptase (Gibco-BRL, Paisley, UK) for 60 min at 40 °C followed by 5 min at 94 °C. PCR was performed in a final volume of 50 mL,
by using 2 mL
of cDNA solution in a mix containing 0.4 mmol/L deoxynucleotide
triphosphates, 40 pmol of both sense and antisense oligonucleotide
primers according to the MetAP-2, Bcl-2 and telomerase type to be
detected, 2.5 mmol/L MgCl2, 2.5 U of Taq DNA polymerase (Promega)
and 5 mL
of 10 min Taq DNA polymerase reaction buffer (500 mmol/L KCl, 100
mmol/L Tris-HCl [pH9.0], 1% Triton-X-100). PCR primer sequences of
the sense and antisense oligonucleotides for MetAP-2, Bcl-2 and
telomerase, as well as the direction, size and reaction conditions
are shown in Table 1. For example, the MetAP2 phosphorothioate
anti-sense oligonucleotide (5’-AGTATTT ACTTTCTCCCAAG-3’) and its
relative scrambled sequence (S’-CTTGG GAGAAAGTAAATACT- 3’) were
synthesized by Sigma-Genosys Ltd, Woodlands, TX, USA. The anti-sense
start position on the MetAP2 mRNA coding region was 1 284. This
region corresponds to the large helical domain insertion on the
surface of the type 2 isozyme. GAPDH was used as a control, with the
quantities of the other mRNA products reported as a fraction of
their intensity compared to GAPDH mRNA. To eliminate any possibility
of genomic DNA contamination, PCR amplification reaction was carried
out on each sample for RNA extraction. As another internal
contamination control, PCR amplification was also carried out on a
sample of reaction mixture in the absence of cDNA.
Telomeric repeat amplification protocol (TRAP) assay
Either
an unamplified conventional standard or a polymerase chain
reaction-ELISA-based assay (Roche Molecular Biochemicals, Foster
City, CA) was used to measure telomerase activity. Cell equivalents
(1×103
to 5×103)
were used to visualize the DNA ladder according to the standard
protocol. For polymerase chain reaction-ELISA, 2×103
cell equivalents were used. The polymerase chain reaction-ELISA
protocol was provided by the assay kit manufacturer (Roche Molecular
Biochemicals). Each set of TRAP assays included control reaction
tubes without any extract or with RNase A (200 mg/mL)-treated
extracts. To quantify the levels of telomerase activity, the average
densitometric optical density of the first six TRAP bands after a
primer band was reported as a ratio of the internal TRAP assay
standard band.
Quantification
of telomerase activity
After the TRAP reaction, hybridization and ELISA, the level
of telomerase activity in a given sample was determined by comparing
the signal from the sample to the signal obtained using a control
template (TS8; solutions 4 or 5). The control templates provided
with the TeloTAGGG telomerase PCR ELISAplus are ready-to-use
solutions containing TS8 at a concentration of 0.001 mol/mL and 0.1
mol/mL. The control templates used were identical to a telomerase
elongation product with 8 telomeric repeats. However, because
amplification of the TRAP products and the internal standard (IS)
are competitive, the signal of the internal control might be near
background level when analyzing samples with very high telomerase
activity.
Relative
telomerase activities (RTA) within different samples were obtained
using the following formula:
| RTA
= |
(AS-ASO)/AS,IS |
×
100 |
| (ATS8-ATS8,0)/ATS8,IS |
AS:
absorbance of sample; AS,0: absorbance of heat- or RNase-treated
sample; AS,IS: absorbance of internal standard (IS) of
the sample; ATS8: absorbance of control template (TS8), ATS8,0:
absorbance of lysis buffer; ATS8,IS: absorbance of the
internal standard of the control template.
Statistical analysis
A statistical software package (SPSS for Windows, version
8.0, Chicago, IL) was used, with Student’s t test for continuous
variables and x2 or Fisher’s exact test for categorical
variables. Non-parametric data were analyzed with Mann-Whitney test
or Kruskal-Wallis test. Significance was accepted at P<0.05.
RESULTS
Hepatocarcinogenesis
No tumor was found in the liver of group A rats at any time
point in the study. All group B and C rats developed diffuse
neoplasms in all lobes of the liver by the end of 18th wk. However,
after fumagillin treatment, hepatic tumors in group C rats at wk 20
and 24 had necrosis and hemorrhage, no change was seen in group B.
Both group B and C rats had a slight but insignificant increase in
weight from wk 20 to 24; the difference in the changes between the
two groups was also not significant (P>0.05, Table 2).
Table1 Sequence of sense and antisense primers in
reverse transcription-polymerase chain reaction (RT-PCR) analysis
for MetAP-2, Bcl-2 and telomerase mRNA expression
| Name |
Sequence |
Direction |
Expected
product size (bp) |
PCR
conditions for pair of primers |
| MetAP-2-S |
TGG
CGG GCG TGGAAG AGG |
Sense |
282 |
1
cycles: 94 °C,
7 min 50 cycles: 94 °C,
40 s; 54 °C,
40; 72 °C,
1 min |
| MetAP-2-AS |
GCA
CCA TCA CCATCA CCA TCT CC |
Antisense |
282 |
1
cycle: 72 °C,
10 min; 4 °C
overnight |
| Bcl-2-S |
AGA
TGA AGA CTCCGC GCC CCT CAG G |
Sense |
566 |
1
cycles: 94 °C,
7 min 50 cycles: 94 °C,
40 s; 54 °C,
40; 72 °C,
1 min |
| Bcl-2-AS |
CCA
GGT ATG CACCCA GAG TGA TG |
Antisense |
566 |
1
cycles: 72 °C,
1 min; 4 °C
overnight |
| Telomerase-S |
GAC
ATG GAG AACAAG CTG TTT GC |
Sense |
185 |
1
cycles: 94 °C,
7 min 50 cycles: 94 °C,
40 s; 54 °C,
40; 72 °C,
1 min |
| Telomerase-AS |
ACA
GGG AAG TTCACC ACT GTC |
Antisense |
185 |
1
cycle: 72 °C,
10 min; 4 °C
overnight |
| GAPDH-S |
ACC
ACA GTC CATGCC ATC AC |
Sense |
485 |
1
cycles: 94 °C,
7 min 50 cycles: 94 °C,
40 s; 54 °C,
40; 72 °C,
1 min |
| GAPDH-AS |
TCC
ACC ACC CTGTTG CTG TA |
Antisense |
485 |
1
cycle: 72 °C,
10 min; 4 °C
overnight |
Table
2 Body
weight of rats at wk 20 and 24
|
Rats
(n = 10) |
Body
weight (g) |
| 20th
wk |
24th
wk |
| Group
B |
DEN
only |
398 |
400 |
|
|
[386-409] |
[384-411] |
| Group
C |
DEN
+ Fumagillin |
397 |
398 |
|
|
[380-410] |
[382-414] |
| P
value |
|
NS |
NS |
Weight:
median [range]; P value: non-parametric test (Mann-Whitney U test)
Group A (control): 372 [350-380] g at wk 20 and 380 [351-394] g at
wk 24.
The
median tumor weights in group B at wk 20 and 24 were 0.64 g (range
0.58-0.70 g) and 0.79 g (0.70-0.90 g) respectively, a significant
increase. Those in group C were 0.37 g (range 0.35-0.42 g) and 0.39
g (0.35-0.47 g). The tumor weight in group C rats was significantly
lower than that in group B at wk 20 and 24 (P = 0.009, P =0.009,
Table 3), suggesting that fumagillin inhibited the tumors.
Metastasis
Group B rats had a median metastasis score of 3 (range 2-3)
at wk 20 and 24. Group C rats had a median score of 1 (range 1-2) at
wk 20 and 24. The difference between the two groups was
statistically significant (P = 0.007, P = 0.004, Table
3).
Table 3 Inhibitory
effect of fumagillin on hepatic tumor growth and metastasis in LEW
rats at wk 20 and 24
| Treatment
groups |
Liver
tumor (B/C-A) (g, median) |
No.
of HCC-involved organs1 |
| 20th
wk |
24th
wk |
20th
wk |
24th
wk |
| Group
B
DEN only |
0.641 |
0.791 |
3 |
3 |
| (n
= 5) |
[0.58-0.70] |
[0.70-0.90] |
[2-3] |
[3-3] |
| Group
C DEN + Fumagillin |
0.37 |
0.39 |
1 |
1 |
| (n
= 5) |
[0.35-0.42] |
[0.35-0.47] |
[1-2] |
[1-2] |
| P
value |
0.009 |
0.009 |
0.007 |
0.004 |
Weight:
median [range] of 5 rats in each step; P value: non-parametric test
(Mann-Whitney U test); 1organs involved: 1 (liver only),
or 2 to 3 (lung or/and peritoneum, in addition to liver). Liver
tumor (B/C-A): B rat liver tumor weight =
liver weight of group B rat minus that of group A rat; C rat
liver tumor weight = liver weight of group C rat minus that of group A rat.
Microscopic
findings
All rats had evidence of HCC after 18 wk of treatment with
DEN. However, after administration of fumagillin for 2 wk, tumors in
group C rats had dilated bile ducts and sinusoids, and karyorrhectic
changes of endothelial cells lining the sinusoids. After 6 wk of
fumagillin treatment, multiple areas with varying degrees of
necrosis and hemorrhage were found in tumors of the group C rats.
Some cancer cells had membrane blebbing, cytoplasmic vacuolization,
and mitochondrial body formation, and there were neutrophil and
histolytic infiltration. These changes were not present in HCC of
group B rats at wk 20 and 24.
Assays for MetAP-2, GAPDH, Bcl-2, and telomerase mRNA
The results of quantitative RT-PCR analysis are shown in
Table 4. The median intensity of MetAP-2 mRNA (compared with GAPDH)
in liver tissue of group A and HCC tissue of groups B and C at the
24th wk was 0.34 (range 0.32-0.36), 0.50 (0.33-0.70) and 0.58 (range
0.40-0.73) respectively. The groups all differed significantly from
one another. Comparable results for Bcl-2 mRNA were 0 in group A
(range 0-0.19), 0.45 in group B (range 0.22-0.63) and 0.38 in group
C (range 0-0.32). Differences among the 3 groups and between groups
A and C as well as between groups B and C were not significant.
However, the difference between groups A and B was significant (P
= 0.024). The median value of telomerase mRNA was 0.30 in group
A (range 0.29-0.32), 0.43 in group B (range 0.35-0.47), and 0.34 in
group C (range 0.29-0.37). The value for group B was significantly
higher than that for group A or C (P = 0.025, 0.016), while
the value did not differ significantly in groups A and C (P =
0.655).
Telomerase activity
The median telomerase activity of HCC tissue at wk 24 in the
3 groups was 47.6% (range 46-71%), 225.0% (187-310%), and 203.5%
(94-292%) respectively. Telomerase activity in group A was
significantly lower than that in group B (P = 0.025) or C (P
= 0.025). The activity
was significantly higher in group B than that in group C (P =
0.046, Table 4).
Table
4 Comparison of
MetAP-2 mRNA, Bcl-2 mRNA, telomerase mRNA and telomerase activity
among the 3 groups of rats at wk 24
| Parameters |
Group |
P
value |
| A
(n = 3) |
B
(n = 5) |
C
(n = 5) |
A
vs B |
A
vs C |
B
vs C |
| MetAP-2
mRNA |
0.34 |
0.50 |
0.58 |
0.025 |
0.025 |
0.047 |
| (0.32-0.36) |
(0.33-0.70) |
(0.40-0.73) |
|
|
|
|
| Bcl-2
mRNA |
0 |
0.45 |
0.38 |
0.024 |
0.608 |
0.072 |
| (0-0.19) |
(0.22-0.63) |
(0-0.42) |
|
|
|
|
| Telomerase
mRNA |
0.30 |
0.43 |
0.34 |
0.025 |
0.655 |
0.016 |
|
(0.29-0.32) |
(0.35-0.47) |
(0.29-0.37) |
|
|
|
| Telomerase |
47.6 |
225.0 |
203.5 |
0.025 |
0.025 |
0.046 |
| Activity
(%) |
(46-71) |
(187-310) |
(94-292) |
|
|
|
Non-parametric
test: Mann-Whitney and Kruskal-Wallis tests.
DISCUSSION
Our
study has confirmed the inhibitory effects of fumagillin on HCC and
allowed us to develop a model describing its effects both at tissue
and cellular level and at molecular level. The advantage of our
investigation is that it was an in vivo rather in vitro study or
one, which used subcutaneously implanted tumors. Evaluating HCC
progression and inhibition in situ in the liver can increase our
understanding of the disease.
Tumor
weight in the group B animals treated with DEN increased
significantly from wk 20 to wk 24, confirming its effect on
progression of HCC. However, in group C rats being given both DEN
and fumagillin, the tumor weight was significantly lower than that
in group B rats at wk 20 and 24. In addition, the fumagillin-treated
rats had a lower metastasis score than those treated with DEN alone.
Fumagillin
has been reported to cause weight loss[18,19], although
Kin et al.[20] found that liver weight as a
function of body weight is actually higher in rats treated with
fumagillin derivative TNP-470[20]. In our fumagillin-treated
rats, insignificant weight loss was only at wk 20 and 24. We think
that this is most likely due to the low dose we used. In addition,
the tumor weight was determined by comparing whole liver weight with
that of controls. Thus, fumagillin-induced body weight loss cannot
explain the lower tumor weight in the fumagillin-treated rats.
It
is proposed that the mechanism by which fumagillin inhibits HCC is
by inhibiting angiogenesis, specifically by blocking EC
proliferation. By inducing apoptosis of ECs, vascularization is
disrupted, leading to infarction of HCC.
It
is well documented that fumagillin agents directly inhibit
proliferation and migration of ECs in vitro and in vivo in various
tumor models, such as in tumors implanted subcutaneously in mice[21].
Folkman[22] has stated that the goal of antiangiogenic
therapy is to maximize apoptosis of ECs in tumor vascular beds. Fox et
al.[5] pointed out that this is a particularly
attractive approach, as ECs are directly accessible through the
blood and because they are ‘normal’ cells and therefore unlikely
to become resistant to treatment. Similarly, according to Prox et
al.[23], fumagillin derivatives do not directly
inhibit proliferation of pancreatic cancer cells, but they inhibit
EC proliferation, increasing apoptosis of tumor cells by reducing
microvessel density.
At
24 wk, our fumagillin-treated rats had massive hemorrhage and
necrosis in the liver tumors, a finding not seen in the rats treated
with DEN alone. Damage to the vessels supplying the tumor could
certainly account for these changes, as ischemia can result in both
apoptosis and necrosis of cancer cells. There is a critical
difference between these two causes of cell death. Necrosis occurs
when the cell suffers a major insult. Damage is generally so severe
that the cell loses its ability to maintain membrane integrity,
rapid swelling results in bursting of the membrane and release of
its contents. This sets up a chain reaction, as toxic enzymes
released from the dead cells attack surrounding cells. A wave of
necrosis radiates out from the initial site of damage.
There
is as yet no evidence that fumagillin can kill HCC cells. However,
Catalano et al.[24] noted that fumagillin might
induce apoptosis by early mitochondrial damage in malignant
mesothelioma cells. Yoshida et al.[25] reported
that fumagillin-like agents inhibit both the growth and migration of
human hepatoma and vascular ECs in vitro and may suppress in vivo
growth of hepatoma, associated with a reduction in the
microvasculature and macrophage counts. The speculation that
fumagillin inhibits not only ECs but also cancer cells to some
degree warrants more studies.
Methionine
aminopeptidases (MetAPs) are enzymes involved in the removal of
N-terminal methionine from peptides and proteins. The molecular
target of fumagillin is MetAP-2, which appears to be important in EC
growth[26]. This enzyme’s effects appear to include
protein co-translational or posttranslational processing and
myristoylation, as well as regulation of protein stability[27].
Sin et al.[28] demonstrated that fumagillin
selectively inhibits MetAP-2 protein in vivo by covalently binding
to it and blocking its aminopeptidase activity. This would disrupt
post-translational modification with failure of myristoylation,
contributing to EC cytostasis, with arrest in the late G1 phase.
If
MetAP-2 plays a comparable role in tumor cells, that would further
support the hypothesis that fumagillin directly inhibits tumor
cells. Studies have shown that in vitro exposure of human
microvascular endothelial cells (HMVECs) to 1 nmol/L fumagillin for
24 h results in a two- to six-fold increase in MetAP2 protein in all
cell types[29]. Up-regulation of MetAP-2 gene thus seems
to be a common phenomenon in cells treated with fumagillin. It is
hypothesized that the loss of MetAP-2 catalytic function in cells
exposed to fumagillin leads to up-regulation of the gene. This
response might itself contribute to cytostatic inhibition of ECs,
possibly via an excessive increase in the ribosomal regulatory (p67)
function of increased MetAP-2 protein in its free form or bound to
fumagillin.
DEN
treatment significantly increased rat liver MetAP-2 mRNA over that
in untreated rats in our study. We attribute this to rapid cell
growth, and hence increased expression of MetAP-2 mRNA, during
carcinogenesis. Fumagillin-treated rats had an even higher MetAP-2
mRNA level than those treated with DEN alone, a difference that
achieved statistical significance (P = 0.047). With
fumagillin inhibition, MetAP-2 mRNA in HCC probably first decreased
and then increased to compensate for loss of MetAP-2 catalytic
activity. However, it did not quite achieve a two-fold increase over
the level in the control rats. This might be due to the fact that we
used a relatively low dose of fumagillin.
MetAP2
also affects two key regulators of proliferation and programmed cell
death, namely Bcl-2 and telomerase. Inhibition of MetAP-2 in
mesothelioma cells reduces both mRNA and protein expression of the
anti-apoptosis gene bcl-2 as well as telomerase activity. This
suggests a major role for MetAP2 in proliferative and apoptosis
pathways[24,30,31]. However, the mechanism by which
MetAP2 regulates bcl-2 expression remains unknown. MetAP2 is not a
transcription factor; therefore, it is unlikely that it directly
regulates bcl-2 gene expression. Instead, by its posttranslational
processing effects, MetAP2 may alter the function of bcl-2
transcription factors.
The
bcl-2 gene encodes a 26-kDa protein that protects cells against
apoptosis in a variety of experimental systems. Bcl-2 maintains
mitochondrial integrity by regulating the opening of the transition
pore, thus preventing release into the cytosol of caspase
activators. Furthermore, Bcl-2 protein prevents apoptosis by
inhibiting lipid peroxidation of the cell membrane. It may therefore
be important in protecting ECs against apoptosis as they are engaged
in forming new vessels in tumors. It may also potentiate their
differentiation into functional blood vessels. Second, Bcl-2 might
potentiate the ability of ECs to differentiate. Some authors have
reported that MetAP2 inhibition causes a time-dependent
down-regulation of the bcl-2 gene, whereas it does not alter
expression of the pro-apoptotic gene, bax. Another mechanism for
downregulation of bcl-2 expression, at least in selected systems, is
by an increase in p53 expression induced by fumagillin. In our
study, Bcl-2 mRNA was significantly higher in DEN-treated rats than
in controls. It was somewhat lower in the fumagillin-treated rats
than in group B, but the difference was not statistically
significant. It is possible this was because of our small sample
number. Or, it is possible that down-regulation of bcl-2 does not
play a significant role in this particular model of tumor
inhibition.
In
addition to deregulation of apoptosis, it is increasingly clear that
oncogenesis is driven by the activation of telomerase, a
ribonucleoprotein complex that adds telomeric repeats (hexanucleotide
5’-TTAGGG-3’) to the ends of replicating chromosomes. Telomerase
is thought to be responsible for cell immortality, primarily by
protecting chromosomes from rearrangement. Telomerase activity is
not detected in normal liver, but it has been detected in the vast
majority of human cancer cells. This has raised the possibility that
telomerase may be an important target for therapy aimed at
controlling cell growth. Kishimoto et al.[31]
emphasized the critical step of telomerase activation in
hepatocarcinogenesis and tumor progression. Takahashi et al.[32]
reported that telomerase reactivation during hepatocarcinogenesis
might be regulated only by hTERT, whereas increased telomerase
activity in tumor progression might be regulated by both hTERT
(reverse transcript) and hTERT (RNA component). Shimada et al.[33]
maintained that the higher the telomerase activity in HCC, the
higher the malignant potential.
The
relationship between Bcl-2 and telomerase activity remains
controversial. Recent studies have shown an association between
telomerase activation and Bcl-2 deregulation in a wide range of
human carcinoma cells. Elkak et al.[34] found that
telomerase activity is higher in the Bcl-2-expressing cases of
colorectal cancer than in Bcl-2-non-expressing cases, suggesting
that Bcl-2 expression may be related to telomerase activity in
colorectal carcinoma. Iida et al.[30] and Elkak et
al.[34] hypothesized that telomerase reactivation in
human breast cancer is associated with increased immunohistochemical
expression of Bcl-2. Mandal et al.[35] reported
that the stable overexpression of Bcl-2 in human cancer cells with
low Bcl-2 expression is accompanied with increased levels of
telomerase activity. In low-grade tumors, Bcl-2 is inversely
correlated with telomerase activity. Ohmura et al.[36]
suggested that the biological role of the Bcl-2 protein is altered
by the degree of tumor aggressiveness, so that it works with
telomerase against genetic instability. HCC is an aggressive
malignancy, and we propose Bcl-2 and telomerase work together in
this tumor. It is possible that MetAP2 acts as upstream of Bcl-2,
while the Bcl-2 site of action is likely to be upstream of that of
telomerase and caspases.
We
quantified both telomerase mRNA and telomerase activity to ensure
the accuracy of our results. DEN-treated rats had significantly
higher values for both at 24th wk compared to control and fumagillin-treated
rats. Telomerase mRNA in fumagillin-treated rats did not differ
significantly from that in controls, although the telomerase
activity remained significantly higher in group C than in group A.
There are three possible explanations for this. First, as suggested
by Kenmochi et al.[37] and Ohta et al.[38],
differentiation may worsen as the tumor progresses; there may be
localized spread of the tumor, including intrahepatic metastasis or
portal vein thrombosis. Second, as the treated tumor necroses,
regeneration of hepatocytes may increase telomerase activity. Third,
it has been reported that telomerase may be expressed in
lymphocytes. Lymphocytic infiltration may occur during tumor
necrosis, so that the total telomerase activity we measured included
a proportion generated by lymphocytes, thus overestimating that
contributed by the tumor.
Our
study demonstrated the ability of fumagillin to inhibit both
progression of HCC in the liver itself and systemic metastasis in
vivo in DEN-treated rats. We also examined three molecular targets
of fumagillin in HCC. We found that HCC tissue in fumagillin-treated
rats had a compensatory elevation of MetAP-2 mRNA after an initial
decrease, with associated decreases in Bcl-2 mRNA, telomerase mRNA,
and telomerase activity. These results may be attributed mainly to
inhibition of ECs by fumagillin. A possible mechanism is that
fumagillin-induced inhibition of MetAP-2 plays an essential role in
EC proliferation. Inhibition of MetAP-2 also results in inhibition
of Bcl-2 and telomerase activity.
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Edited
by
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