ORIGINAL PAPER

Metalloproteinases 2 and -9 activity during promotionand progression stages of rat liver carcinogenesis

Kelly Silva Furtado Æ Paulo Wagner Pires Æ Luis Antonio Justulin Jr. ÆMaria Aparecida Marchesan Rodrigues Æ Sergio Luis Felisbino ÆLuis Fernando Barbisan

Received: 26 September 2008 / Accepted: 17 November 2008

� Springer Science+Business Media B.V. 2008

Abstract Activity of metalloproteinases 2 and 9 (MMP-2

and 9) during promotion and progression of rat liver car-

cinogenesis was investigated in a modified resistant

hepatocyte model. Development of preneoplastic liver

lesions positive for glutathione S-transferase 7-7-(GST-P 7-

7-positive PNL) and tumors besides hepatocytes positive

for proliferating cell nuclear antigen (PCNA) were quan-

tified and compared to MMP-2 and-9 activity using gelatin

zymography. Marked increases in GST-P 7-7-positive PNL

development, PCNA labeling indices, MMP-2 (pro, inter-

mediate and active forms) and pro-MMP-9 activity were

observed after proliferative stimulus induced by 2-ace-

tylaminofluorene (2-AAF) exposure cycles. After 2-AAF

withdrawal, increase in MMP-2 activity was detected only

in neoplastic mixed lesions, whereas active MMP-9 was

increased in both PLN and neoplastic tissues. Our findings

suggest that MMP-2 may be associated with proliferative

events induced by 2-AAF rather than with selective growth

of PNL and that MMP-9 could be associated with pro-

gression of PNL and neoplastic mixed lesions.

Keywords Rat liver carcinogenesis � Resistant

hepatocyte model � Metalloproteinases � Preneoplastic

and neoplastic lesions

Introduction

Rat liver carcinogenesis is a multistep and complex process

that includes initiation, promotion and progression stages

(Pitot 2001). Foci of altered hepatocytes and hyperplastic

nodules have been described as putative preneoplastic

lesions (PNL) in various experimental models of rat liver

carcinogenesis and it is argued that they are capable of

progressing to hepatocellular adenomas and carcinomas

(Bannasch and Zerban 1992; Hasegawa and Ito 1994).

Recently, different types of PNL with similar morpholog-

ical and biochemical changes in the hepatocellular

phenotype were identified in human chronic liver diseases

associated with hepatocellular carcinoma (Su and Ban-

nasch 2003; Libbrecht et al. 2005).

The resistant hepatocyte (RH) model is one of the most

classical for the study of the different steps of rat liver

carcinogenesis (Farber and Sarma 1987). In this model,

diethylnitrosamine (DEN)-initiating agent induces single

cells and small foci of putative initiated hepatocytes that

are promoted with 2-acetylaminofluorene (2-AAF), asso-

ciated with a 70% partial hepatectomy (PH) (Farber and

Sarma 1987). 2-AAF is a mito-inhibitor to normal hepa-

tocytes but does not inhibit a subpopulation of the

glutathione S-transferase (GST-P) 7-7-positive hepato-

cytes. Partial hepatectomy acts as a mitotic stimulus

resulting in activation of hepatic stem cells (oval cells) and

a rapid growth of the initiated cells subpopulation into

larger GST-P 7-7-positive lesions. Some lesions eventually

progress into grossly visible nodules and, over months,

K. S. Furtado � M. A. M. Rodrigues

School of Medicine, Department of Pathology, UNESP Sao

Paulo State University, Botucatu, SP 18618-000, Brazil

P. W. Pires

Department of Cell Biology, Institute of Cell Biology,

UNICAMP, Campinas, SP 13083-950, Brazil

K. S. Furtado � L. A. Justulin Jr. � S. L. Felisbino �L. F. Barbisan (&)

Department of Morphology, Institute of Biosciences, UNESP

Sao Paulo State University, Botucatu, SP 18618-000, Brazil

e-mail: barbisan@ibb.unesp.br

123

J Mol Hist

DOI 10.1007/s10735-008-9206-x

tumors arise (Sell and Dunsford 1989; Tiwawech et al.

1991).

The liver extracellular matrix (ECM) is not only a

passive structural support, since it plays key roles in pro-

viding a structural framework and maintaining the

differentiated phenotype and normal function of hepato-

cytes, sinusoidal, endothelial and stellate cells (Martinez-

Hernandez and Amenta 1993). ECM turnover is a vital step

in tissue remodeling that accompanies both physiological

and pathological processes, including aging, fibrosis and

tumor development (Brew et al. 2000; Visse and Nagase

2003; Chakraborti et al. 2003; Vihinen et al. 2005; Ra and

Parks 2007). Matrix metalloproteinases (MMPs) are a

family of zinc-dependent endopeptidases that degrade most

ECM components in both normal and pathophysiological

processes, and their activity is tightly regulated by the

tissue inhibitors of metalloproteinases (TIMPs) (Brew et al.

2000; Visse and Nagase 2003; Chakraborti et al. 2003;

Vihinen et al. 2005; Ra and Parks 2007). MMPs are syn-

thesized as inactive pro-forms which require subsequent

cleavage in order to become active (Visse and Nagase

2003; Ra and Parks 2007). Based on their substrate spec-

ificity and structural similarity, MMPs are often classified

into six subgroups: collagenases, gelatinases, stromelysins,

matrilysin, membrane-type MMPs and other MMPs (Vi-

hinen et al. 2005; Ra and Parks 2007). Perfect regulation of

ECM degradation is essential for tissue homeostasis

maintenance, and an imbalance between TIMPs and MMPs

rates may participate in tumor progression and metastasis

in several tissues and organs (Brew et al. 2000; Chakraborti

et al. 2003).

A small number of studies investigated the potential role

of MMPs in experimental liver carcinogenesis. Wood and

Archer (2001) examined the role of gelatinases during the

early PNL growth in both Fischer 344 (susceptible) and

Copenhagen—Cop (resistant) rats. These authors reported

a higher increase of hepatic MMP-2 and -9 activity in

Fischer 344 rats than in Cop rats, nevertheless no inhibition

on liver regeneration or early GST-P 7-7-positive PNL

growth after PH was observed when the Fischer 344 rats

were treated with batimastat, a MMP inhibitor. Thus, the

increase of hepatic gelatinases activity could be non

essential for the early PNL expansion in DEN-initiated and

2AAF-treated rats. Gao et al. (2002) described an increase

in MMP-2 mRNA and in pro and active MMP-2 and pro-

MMP-9 activity during HCC development in rats initiated

with DEN.

Therefore, the present study was designed to evaluate

the activity and in situ immunohistochemistry expression

of metalloproteinases 2 and 9 at the promotion/progression

stages of liver carcinogenesis in male Wistar rats submitted

to a modified RH model. In addition, MMP-2 and -9

findings were compared to hepatic cell proliferation, p21ras

and cyclooxygenase-2 (COX-2) protein immunoreactivity,

PNL development and tumor phenotypes.

Materials and methods

Animals and treatments

Four-week-old male Wistar rats were obtained from Multi-

disciplinary Center for Biological Investigation (CEMIB,

UNICAMP Campinas-SP, Brazil) and housed in polypro-

pylene cages (five animals/cage) covered with metallic grids

in a room maintained at 22 ± 2�C, 55 ± 10% humidity and

with a 12-h light-dark cycle. They were fed commercial

Purina chow (LABINA, Paulınia, SP, Brazil) and water ad

libitum during a 2-week acclimatization period. Then, the

animals were randomly allocated into four experimental

groups (Fig. 1): non-treated group (G1), a single dose of

200 mg/kg b.w. of diethylnitrosamine (DEN, Sigma-

Aldrich Co., St. Louis Mo, USA)-initiated group (G2),

DEN-initiated plus 0.02% 2-acetylaminofluorene (2-AAF,

Sigma-Aldrich Co., St. Louis Mo, USA, 1 cycle of 3 weeks

and 3 cycles of 2 weeks)-promoted group (G3) and 2-AAF-

treated group (G4). All animals were subjected to 70%

partial hepatectomy (PH) at week 3 and sacrificed at weeks

8, 20 or 35. Body weight and food/water intake were mea-

sured twice a week during the entire experimental period.

In this study, as Wistar non-isogenic strain is more

resistant than Fischer 344 isogenic strain to liver carcino-

genesis (Asamoto et al. 1989; Pascale et al. 2002), we

introduced three additional cycles of treatment with 2-AAF

to increase the development of hepatic preneoplastic lesions

and the tumor burden in male Wistar rats. Besides DEN plus

2-AAF-treated group (main group), we also introduced all

control groups including non-treated and DEN or 2-AAF-

treated groups. The protocols used were consistent with the

Ethical Principles for Animal Research adopted by the

Brazilian College of Animal Experimentation (COBEA).

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methods’’)

J Mol Hist

123

Tissue processing, histology and immunohistochemical

procedures

Immediately before necropsy, blood samples were collected

and the serum levels of alanine aminotransferase (ALT) and

albumin were measured spectrometrically to monitor

hepatocellular injury and function (Ramaiah 2007). At

necropsy, the liver was removed and weighted, and repre-

sentative samples were either fixed in 4% phosphate-

buffered formalin during 24 h for paraffin embedding or

frozen in liquid nitrogen for gelatin zymography. The par-

affin blocks were cut into 5-lm-thick sections and stained

with hematoxylin–eosin (HE) for histological analysis,

Gomori’s silver impregnation for reticulin fibers evaluation

and for immunohistochemical analysis of glutathione S-

transferase P form (GST-P 7-7), proliferating cell nuclear

antigen (PCNA) and metalloproteinases 2 (MMP-2) or 9

(MMP-9), p21ras and cyclooxygenase-2 (COX-2) markers.

In HE staining, PNL and neoplastic lesions (hepatocel-

lular adenoma or carcinoma) were classified according to

previous published criteria (Bannasch and Zerban 1992).

PCNA, GST-P 7-7, MMP-2, MMP-9, p21ras and COX-2

expression were immunohistochemically detected using the

avidin–biotin–peroxidase method. Briefly, deparaffinized

5-lm-thick serial liver sections on poly-L-lysine coated

slides were treated with 3% H2O2 in phosphate-buffered

saline for 15 min, nonfat milk for 60 min, anti-PCNA

(Dako Corporation, Carpinterie, CA, USA, clone PC10,

1:200 dilution), anti-GST-P 7-7 (Medical and Biological

Laboratories Co., Tokyo, Japan, clone 311, 1:1000 dilu-

tion), anti-MMP-2 and MMP-9 (Santa Cruz Biotechnology,

California, USA, clones C-19 and C-20, respectively, 1:200

dilution), p21ras (Santa Cruz, clone SC 32, 1:25 dilution)

or COX- 2 (Cayman Chemical Co, MI, EUA, 1:200 dilu-

tion) antibodies, biotinylated horse anti-mouse IgG, anti-

goat or anti-rabbit IgG antibodies (Dako Corporation,

Carpinterie, CA, USA, 1:200 dilution) for 60 min, and

avidin–biotin–peroxidase solution (Vector Laboratories

Inc, CA, USA, 1:1:50 dilution). Antigen retrieval for

MMP-2 and 9, p21ras and COX-2 was performed using

0.01 M citrate buffer (pH 6.0) boiling in microwave oven

(1,300 W) twice for 5 min each. Chromogen color was

accomplished with 3,30-diaminobenzidine tetrahydrochro-

ride (DAB, Sigma–Aldrich Co., St. Louis MO, USA) as the

substrate to demonstrate the sites of peroxidase binding.

The slides were counterstained with Harris’s hematoxylin.

Gelatin zymography for MMP-2 and MMP-9

All hepatic samples stored in liquid nitrogen for gelatin

zymography analysis were histologically checked for the

presence of preneoplastic and/or neoplastic lesions. Samples

from liver tumors did not contain surrounding parenchyma,

since they were easily identified macroscopically and iso-

lated. Thus, we compared liver samples containing liver

tumors or an extensive development of PNL (G3 and G4

groups), or few PNL lesions (G2 group) or unaltered liver

parenchyma (G1 group).

Frozen liver samples (*300 mg) were mechanically

homogenized in 50 mM Tris buffer pH 7.5 plus 1% triton-

X 100 and CaCl2 10 mM with a Polytron for 30 s at 4�C,

centrifuged and protein extracted on supernatant was

quantified by Bradford method. Aliquots (30 lg protein)

from liver extracts (non-altered, containing PNL or neo-

plastic lesions) were subjected to electrophoresis in gelatin

containing polyacrylamide (8% acrylamide) gels in the

presence of SDS under non-reducing conditions (Pires

et al. 2008). The gelatin substrate was present at 0.1% final

concentration in the gel. The gels (0.75 mm thick) were

electrophoresed for 2 h at 100 V, 4�C, in a Bio-Rad

MiniProtean II system (Bio-Rad Laboratories, Inc., Rich-

mond, CA, USA). Following electrophoresis, the gels were

washed by gentle shaking at room temperature with 2.5%

Triton X-100 (two changes) for 1 h. The gels were incu-

bated overnight (16 h) in 50 mM Tris–HCl (pH 8.4)

containing 5 mM CaCl2 and 1 lM ZnCl2 at 37�C. After

incubation, the gels were stained by Coomassie Brilliant

Blue R-250. Areas of proteolysis appeared as clear zones

against a blue background. Molecular mass determinations

were made with reference to prestained protein standards

(Bio-Rad Laboratories) co-electrophoresed into the gels.

The integrated optical density (IOD) spectrophotometry

was determinated in an Image Master VDS version 3.0

(Pharmacia Biotechnology, Piscataway, NJ, USA). The

values of IOD were analyzed statistically and plotted in

histograms.

GST-P positive PNL morphometry and PCNA labeling

analysis

PNL was detected by GST-P expression in liver sections

(3). GST-P 7-7-positive PNL were measured using a Nikon

photomicroscope (Microphot-FXA, Tokyo, Japan) con-

nected to a KS-300 apparatus (Kontron Elektronic,

Munich, Germany). Data from morphometric analysis of

putative preneoplastic liver lesions positive for GST-P 7-7

are frequently presented as number per liver area (cm2) or

per volume (cm3). Both two- and three-dimensionally

expressed quantitative results for GST-P 7-7 positive PNL

were found to be adequate to demonstrate the modifying

potential of test chemicals on chemically induced hepato-

carcinogenesis in rodents (Imaida et al. 1989). As the

immunohistochemical detection of GST-P 7-7-positive

PNL was performed in various fragments of all liver lobes

reminiscent after partial hepatectomy, our results were

presented as number per cm2, mean size (mm2) and

J Mol Hist

123

percentage of liver area occupied by these lesions (% liver

area). (Pinheiro et al. 2003; Pires et al. 2008).

PCNA labeling indices (PCNA LI %) for each animal

were estimated by counting the number of immunohisto-

chemically labeled hepatocytes nuclei (S-Phase) per total

number of counted hepatocytes (*2,000) in normal or

bearing PNL parenchyma (Pinheiro et al. 2003; Pires et al.

2008). PCNA LI% was also measured in neoplastic lesions

diagnosed at week 35.

Statistical analysis

Statistical analysis was performed using the Jandel Sigma

Stat Software (Jandel Corporation, San Rafael, CA, USA).

The body weight, body weight-gain, absolute and relative

liver, PCNA-labeling, GST-P 7-7-positive PNL and IOD

data and tumor multiplicity were analyzed by ANOVA test

or Kruskal–Wallis test. The incidence of different types of

PNL and neoplastic lesions was examined using v2 test or

the Fischer exact test. Data were considered statistically

significant when P \ 0.05.

Results

General findings

Body weight from animals of non-treated group (G1) and

DEN-treated group (G2) progressively increased during all

experimental periods. However, there was a marked

reduction in food intake (data not shown) and body-weight

gain in DEN plus 2-AAF-treated group (G3) and 2-AAF-

treated group (G4) during the cycles of 2-AAF treatment

(P \ 0.001, Fig. 2). A significant reduction in final body

weight was observed in the DEN plus 2-AAF-treated group

(G3) in all periods analyzed (weeks 8, 20 and 35)

(P \ 0.001, Table 1). The reduced food intake and body

weight gain observed in the 2-AAF-treated groups (G3 and

G4) is due to severe hepatoxicity induced by 2-AAF

treatment (Bitsch et al. 2000).

The relative liver weights from 2-AAF-treated groups

(G3 and G4) were significantly higher than the non-treated

group (G1) and DEN-treated group (G2) mainly at weeks

20 and 35 (P \ 0.001, Table 1). The alanine aminotrans-

ferase (ALT) and albumin serum levels did not differ

among the experimental groups at weeks 8, 20 and 35

(Table 1).

Preneoplastic and neoplastic lesions

The main hepatic lesions observed in carcinogen-treated

animals were PNL characterized by altered foci of hepato-

cytes and hyperplastic nodules with clear cell and/or

eosinophilic or basophilic cell phenotypes. Neoplastic

lesions included adenomas, hepatocarcinomas (HCC), cho-

langiomas, cholangiocarcinomas and mixed neoplastic

lesions like hepatocholangiocellular adenomas and carci-

nomas (Table 2). Benign and malignant lesions were

identified at weeks 20 and 35 predominantly in the DEN plus

2-AAF-treated group (G3) (Table 2). Some animals from

DEN-treated groups (G2 and G3) and/or 2AAF-treated

groups (G3 and G4) developed kidney adenomas and car-

cinomas or sebaceous and squamous cell carcinomas in the

Zymbal’s gland, respectively. These chemically induced

neoplasms are demonstrative of the organotropic activity of

DEN and 2-AAF on non-liver targets (Verna et al. 1996 a, b).

A rapid development of GST-P 7-7-positive PNL (i.e.,

mainly in mean size and percentage of occupied liver area)

and neoplastic lesions (i.e., mixed lesions, adenomas, HCC

and cholangioma and cholangiocarcinoma) was observed in

this modified RH model (Table 3). 2-AAF is a genotoxic

hepatocarcinogen with both initiating (weak) and promoting

(strong) activities on rat liver carcinogenesis (Tiwawech

et al. 1991, Verna et al. 1996b). Specially at week 20, the

promoting potential of this hepatocarcinogen was well

characterized in the 2-AAF-treated group with an increase of

6.75 times the liver area occupied by PNL when compared to

week 8 (Table 3). In 2-AAF-treated animals, the prolifera-

tive stimulus induced by partial hepatectomy resulted in oval

cell hyperplasia and an expansive growth of initiated cells

into larger GST-P 7-7-positive PNL in the absence of

hepatic inflammation, necrosis and fibrosis. However, there

was a reduction in the expansive growth of GST-P 7-7-

positive PNL after 2-AAF withdrawal (Table 3), probably

as a consequence of a decrease in cell proliferation and

remodeling (Tiwawech et al. 1991). In contrast, a modest

development of GST-P 7-7-positive lesions and rare GST-P

7-7-positive PNL were observed in the liver from DEN-

treated group (G2) and non-treated group (G1), respectively,

0

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300

400

500

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(G1) Control

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(G3) DEN+2-AAF

(G4) 2-AAF

Weeks

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Fig. 2 Effects of treatments on body weight. A significant reduction

in body weight was observed in groups treated with DEN plus 2-AAF.

(G3 \ G1 = G2 = G4, P \ 0.001) DEN, diethylnitrosamine

(200 mg/kg, b.w., i.p.); 2-AAF, 2-acethylaminofluorene (0.02% in

diet)

J Mol Hist

123

mainly at weeks 20 and 35 (Table 3). The finding of few

GST-P 7-7-positive PNL in the non-treated group (G1) has

been observed in senile rats (Hasegawa and Ito 1994).

The distribution of reticular fibers in the non-affected

liver parenchyma was restricted to Disse’s spaces, adjacent

to centrolobular veins and in the portal space (data not

shown). An increase in both thickness and number of

reticular fibers was observed at the compressive border that

embraces some neoplastic lesions like adenomas, HCC and

mixed neoplastic lesions, especially in the DEN plus

2-AAF-treated group (G3) (Fig. 3a). Absence of reticular

fibers within HCC was a peculiar finding, whereas in mixed

Table 1 Final body and relative liver weights and biochemical data in the experimental groups at weeks 8, 20 and 35 (values are mean ± SD)

Group/treatmenta Number of rats Body weight (g) Relative liver weight (g) ALT (U/l) Albumin (g/dl)

Week 8

(G1) Control 5 386.0 ± 33.6 3.0 ± 0.2 46.0 ± 6.2 3.0 ± 0.10

(G2) DEN 5 362.4 ± 33.6 3.1 ± 0.2 45.2 ± 8.6 3.1 ± 0.15

(G3) DEN ? 2-AAF 5 267.1 ± 55.7* 5.2 ± 0.6* 74.5 ± 14.6* 3.0 ± 0.20

(G4) 2-AAF 5 332.6 ± 29.9 3.4 ± 0.5 52.0 ± 16.3 2.9 ± 0.15

Week 20

(G1) Control 5 474.8 ± 70.9 2.8 ± 0.3 35.1 ± 11.2 3.2 ± 0.3

(G2) DEN 5 462.2 ± 42.4 2.8 ± 0.2 37.0 ± 9.2 2.9 ± 0.2

(G3) DEN ? 2-AAF 5 355.7 ± 33.2* 5.7 ± 0.7* 46.2 ± 7.5* 3.2 ± 0.2

(G4) 2-AAF 5 377.76 ± 28.7* 4.1 ± 0.4* 39.2 ± 3.3 2.9 ± 0.4

Week 35

(G1) Control 10 511.0 ± 41.3 3.2 ± 0.3 57.9 ± 10.7 3.0 ± 0.2

(G2) DEN 10 491.8 ± 47.9 3.0 ± 0.3 54.0 ± 10.5 3.1 ± 0.25

(G3) DEN ? 2-AAF 10 442.6 ± 34.8* 6.1 ± 1.4* 63.9 ± 12.1 3.1 ± 0.4

(G4) 2-AAF 14 518.8 ± 38.4 3.7 ± 0.3* 55.6 ± 15.9 3.2 ± 0.4

a DEN diethylnitrosamine (200 mg/kg b.w., i.p.); 2-AAF 2-acethylaminofluorene (0.02% in basal diet).* Different from G1 and G2 groups,

P \ 0.05

Table 2 Incidence of preneoplastic and neoplastic liver lesions in the experimental groups at weeks 8, 20 and 35

Group/treatmenta PNLb Neoplastic lesionsc

CC/CE CB Adenoma HCC Cholang Cholangioc Mixed

Week 8

G1 (control) 0 0 0 0 0 0 0

G2 (DEN) 5 (100%)* 0 0 0 0 0 0

G3 (DEN ? 2-AAF) 5 (100%)* 0 0 0 0 0 0

G4 (2-AAF) 5 (100%)* 0 0 0 0 0 0

Week 20

G1 (control) 2 (40%) 0 0 0 0 0 0

G2 (DEN) 4 (80%) 1 (20%) 0 0 0 0 0

G3 (DEN ? 2-AAF) 5 (100%) 3 (60%) 3 (60%) 0 0 1 (20%) 1 (20%)

G4 (2-AAF) 5 (100%) 0 2 (40%) 0 0 0 0

Week 35

G1 (control) 4 (40%) 1 (10%) 0 0 0 0 0

G2 (DEN) 10 (100%)* 8 (80%)* 0 0 0 0 0

G3 (DEN ? 2-AAF) 9 (90%)* 2 (20%) 7 (70%)* 4 (40%)* 1 (10%) 0 5 (50%)*

G4 (2-AAF) 14 (100%)* 1 (7%) 2 (14%) 1 (7, 1%) 1 (7, 1%) 0 0

a DEN diethylnitrosamine (200 mg/kg b.w., i.p.); 2-AAF 2-acethylaminofluorene (0.02% in diet); bPNL prenoplastic lesions with follows

phenothypes: CC/CE clear cells/eosinophilic cells or CB basophilic cells; cHCC hepatocellular carcinoma; Cholang cholangiona; Cholangioccholangiocarcinoma; Mixed hepatocholangioma or hepatocholangiocarcinoma;* Different from G1 and G2 groups, P \ 0.05

J Mol Hist

123

neoplastic lesions an increase in thickness and number of

reticular fibers was observed in areas associated with

cholangiocellular lesions (Fig. 3d).

MMP-2 and-9, p21ras and COX-2 immunoreactivity

and gelatinases

The immunolabeling for MMP-2 and -9, both pro and

active forms, performed at week 35 showed that these

enzymes were expressed in non-parenchymal cells, in

hepatocytes from PNL and adenomas (data not shown), and

hepatocholangiocellular adenomas and carcinomas

(Fig. 3b, c, e, f). All HCC analyzed at week 35 did not

expressed MMP-2 and -9. However, the immunoreactivity

for these MMPs was observed in the surrounding cells at

the tumor margin (Fig. 3b, C). At week 35, co-expression

of p21ras, COX-2 and MMP-9 was observed in most

neoplastic lesions from G3 and G4 groups. However, in

cholangiocellular areas of mixed lesions and in cholan-

giomas and cholangiocarcinomas, the MMP-9

immunoreactivity in bile ductal neoplastic cells was not

associated to p21ras and COX-2 co-expression (Fig. 4).

Data regarding metalloproteinases 2 and 9 (MMP-2 and

-9) activity in all experimental periods are shown in

Figs. 5, 6 and 7. A significant increase in the activity of pro

(72 kDa), intermediate (64 kDa) and active forms (59 kDa)

of MMP-2 and pro-MMP-9 (92 kDa) was observed in both

2-AAF-treated groups (G3 and G4) at weeks 8 and 20

(0.002 \ P \ 0.05) but not at week 35, after a 15-week

period of 2-AAF withdrawal. However, in neoplastic

lesions (i.e., predominantly hepatocholangiocellular ade-

nomas and carcinomas) these enzymatic activities

remained high after the 2-AAF withdrawal. The active

form (81 kDa) of MMP-9 was detected only at week 35,

especially in the liver parenchyma from 2-AAF-treated

groups (G3 and G4) and in mixed neoplastic lesions from

DEN plus 2-AAF-treated group (G3).

PCNA analysis

The rates of hepatic cell proliferation were significantly

higher in liver parenchyma from 2-AAF-treated groups (G3

and G4) than in the respective control groups (G1 and G2)

in all experimental periods (0.05 \ P \ 0.001, Figs. 5c, 6c

and 7c). A significant reduction in PCNA LI% was

observed in the liver parenchyma from 2-AAF-treated

groups (G3 and G4) at week 35, after a 15-week period of

2-AAF withdrawal. In contrast, the neoplastic lesions from

DEN plus 2-AAF-treated group (G3) maintained high

PCNA labeling indices at week 35 (Fig. 7c).

Discussion

The present study showed the dynamic changes in

metalloproteinases -2 and -9 activity during the promotion

and progression stages of chemically induced liver carci-

nogenesis in male Wistar rats submitted to a modified

resistant hepatocyte (RH) model.

Table 3 Analysis of GST-P 7-7-positive lesions in liver of the different experimental groups at weeks 8, 20 and 35 (values are mean ± SD)

Group/treatmenta Number of rats GST-P 7-7-positive datab

Number/cm2 Mean size (mm2) % Liver area

Week 8

G1 (Control) 05 0 0 0

G2 (DEN) 05 7.87 ± 4.04* 0.05 ± 0.01* 0.53 ± 0.33*

G3 (DEN ? 2-AAF) 05 34.71 ± 15.99*,** 1.74 ± 0.76*,** 62.69 ± 4.19*,**

G4 (2-AAF) 05 5.38 ± 2.41*,**,*** 0.86 ± 0.43*,**,*** 3.96 ± 1.05*,**,***

Week 20

G1 (Control) 05 0.66 ± 1.23 0.01 ± 0.01 0.02 ± 0.04

G2 (DEN) 05 24.73 ± 5.41* 0.02 ± 0.003* 1.11 ± 0.74*

G3 (DEN ? 2-AAF) 05 35.01 ± 5.52*,** 0.44 ± 0.08*,** 69.27 ± 9.59*,**

G4 (2-AAF) 05 27.31 ± 7.11*,*** 0.28 ± 0.16*,** 26.99 ± 16.22*,**,***

Week 35

G1 (Control) 10 1.83 ± 1.61 0.01 ± 0.01 0.08 ± 0.07

G2 (DEN) 10 25.98 ± 4.81* 0.03 ± 0.01 2.07 ± 0.56*

G3 (DEN ? 2-AAF) 10 20.11 ± 9.79* 0.51 ± 0.10*,** 55.20 ± 11.62*,**

G4 (2-AAF) 14 32.36 ± 5.95*,*** 0.17 ± 0.08*,**,*** 20.64 ± 8.95*,**,***

a DEN diethylnitrosamine (200 mg/kg b.w., i.p.); 2-AAF 0.02% 2-acethylaminofluorene in diet; bGST-P-positive lesions PNL and neoplastic

lesions (adenoma, hepatocellular carcinoma and mixed lesions); *,**,*** Different from G1, G2 or G3, respectively, 0.005 \ P \ 0.001

J Mol Hist

123

Various studies have shown that metalloproteinases and

their inhibitors are essential for liver regeneration after

partial hepatectomy or carbon tetrachloride (CCl4)-medi-

ated injury in rats, since these zinc-dependent proteases are

involved in the hepatic extracellular matrix remodelation

after surgical ablation, damage or fibrosis (Kim et al. 2000;

Kinnitel et al. 2000; Watanabe et al. 2000; Xu et al. 2004).

In hepatic fibrosis models, MMP-2 and-9 are produced

mainly by the activated hepatic stellate cells (HSCs), in

response to extracellular matrix changes induced by many

hepatotoxic agents, including ethanol and CCl4 (Kinnitel

et al. 2000; Watanabe et al. 2000; Xu et al. 2004). How-

ever, there are few studies investigating the role of MMPs

during different stages of rat chemically induced liver

carcinogenesis.

Wood and Archer (2001) showed that treatment with

batimastat, a MMP inhibitor, did not alter liver regenera-

tion or early PNL development in male Fischer 344 rats

sacrificed early after partial hepatectomy in a modified RH

model, suggesting that the increase in MMP-2 and -9

activity is not crucial for growth of normal or altered liver

parenchyma. In the present study we have observed a

significant increase in the gelatinolytic activity of all forms

of MMP-2 (72, 64 and 59 kDa) and pro-MMP-9 (92 kDa)

in the liver parenchyma with extensive development of

GST-P 7-7-positive PNL from 2-AAF-treated groups (G3

and G4), when compared to the respective control groups

(G1 and G2) at weeks 8 and 20. This initial increase of

MMP-2 and-9 activity in the liver parenchyma bearing

PNL may be associated to the ECM turnover due to the

hepatomegaly induced by 2-AAF exposure (Bitsch et al.

2000). In fact, a significant increase in the absolute and

relative liver weights and in cell proliferation rates was

observed after each cycle of 2-AAF-treatment. Therefore,

the increase in MMP-2 (72, 64 and 59 kDa) and pro-MMP-

9 (92 kDa) activity during this early stage of rat

Fig. 3 Neoplastic liver lesions

from DEN plus 2-AAF-treated

group (G3): a gomori staining

for reticular fibers, bimmunohistochemical staining

for MMP-2 and c MMP-9 at

HCC border. d gomori staining

for reticular fibers, eimmunohistochemical staining

for MMP-2 and f MMP-9 in a

hepatocholangioma. DEN,

diethylnitrosamine (200 mg/kg,

b.w., i.p.) and 2-AAF, 2-

acethylaminofluorene (0.02% in

diet)

J Mol Hist

123

hepatocarcinogenesis may be due to the global expansion

of hepatic cell compartment induced by 2-AAF-treatment

rather than the selective growth of PNL, as previously

described (Wood and Archer 2001; Pires et al. 2008).

At the end of experiment, the activity of pro, intermediate

and active forms of MMP-2 and pro-MMP-9 in samples

from non-neoplastic liver parenchyma was similar among

the different groups treated or non-treated with carcinogens.

Therefore, the differences in MMP-2 and pro-MMP-9

activity at this stage of lesion development did not directly

reflect the differences in the percentage of liver occupied by

PNL (*0.08, 2, 55 and 20% for G1, G2, G3 and G4 groups,

respectively). Thus, the differences of MMP-2 and pro-

MMP-9 activity observed may be related to differences in

cell proliferation rates observed after immediate (weeks 8

and 20) or prolonged period after 2-AAF withdrawal.

Interestingly, a high gelatinolytic activity of these MMPs

was observed in samples of hepatocholangiomas and he-

patocholangiocarcinomas from DEN plus 2-AAF-treated

(G3) group. Furthermore, MMP-2 is known to be constitu-

tively expressed in all tissues due to a different gene

promoter region (Ra and Parks 2007). Thus an increased

MMP-2 activity could be explained by the increase in cell

number within hepatocholangiocellular adenomas and car-

cinomas (Gianelli et al. 2003; Foda and Zucker 2001). Also,

the normal and PNL-bearing liver samples from all experi-

mental groups presented a remarkable MMP-9 activity

(81 kDa) but with a higher activity in both preneoplastic and

neoplastic lesions from DEN plus 2-AAF-treated group

(G3). As the expression of MMP-9 has been implicated in

the invasion and metastasis steps of the carcinogenic pro-

cess, the higher activation of MMP-9 observed in the liver

parenchyma containing PNL from 2-AAF-treated (G3 and

G4) groups or in mixed lesions from DEN plus 2-AAF-

treated (G3) group may from be probably related to the

progression of the carcinogenic cascade, which demands a

higher ECM degradation for the progression of these neo-

plastic lesions (Foda and Zucker 2001). In fact, the

malignant phenotype and invasiveness ability of H-Ras-

transformed rat liver epithelial cells and of conexin-26-

tranfected HepG2 cells is mediated by up-regulation of

MMP-9 activity (Yano and Yamasaki 2001; Lee et al. 2006).

An increase of p21ras and COX-2 expression has been

previously described during progression of rat liver carci-

nogenesis (Gulbis et al. 1993; Denda et al. 2002). Lee et al.

(2006) have shown that WB-F344 rat liver epithelial cells

transfected with v-H-Ras (H-ras WB cells) presented an

increase in MMP-9 activity, but not MMP-2, and in COX-2

expression. In H-ras WB cells, the MMP-9 and COX-2 up-

regulation occurred through the activation of ERK1/2 and

NF-kB pathways taking to invasive and migrative pheno-

types. Our findings showed that co-expression of p21ras,

COX-2 and MMP-9 depends on the morphological pattern

of the malignant lesions induced in this model.

In humans, HCC is commonly found surrounded by a

fibrous capsule, and requires the degradation of this barrier

Fig. 4 Immunohistochemical

staining for COX-2, p21ras and

MMP-9 in a cholangioma from

DEN plus 2-AAF-treated group

(G3): a HE staining, b COX-2

expression, c p21ras expression

and d MMP-9 expression in a

nodule near to a cholangioma

negative for MMP-9

J Mol Hist

123

to grow (Kaneyoshi et al. 2001). On the other hand, the

well differentiated HCC induced in the RH model adopted

here does not involve the formation of a fibrous capsule as

in human HCC. Hence, the role of MMP-2 and-9, in

association with other MMPs, at this stage of rat liver

carcinogenesis, could be important to degrade the reticular

fibers at the tumor margin. The increase in expression and

activity of MMP-2 and -9 at the lesion border indicate that

these enzymes are produced by the surrounding normal

cells, confirming that tumor–host interaction may have a

central role at the progression stage of carcinogenesis

(Caudroy et al. 2002).

In conclusion, the findings of the present study suggest

that: (1) the early increase in MMP-2 and pro-MMP-9

activity observed in the liver parenchyma from 2-AAF-

treated rats may be associated to the global proliferative

stimulus induced by this carcinogen; (2) the mixed neo-

plastic, cholangiocellular lesions and HCC have different

(G1) (G2) (G3) (G4)

- 92

- 72- 64- 59

Kda)lortnoc

%(yrte

moti sneD

(G2) (G3) (G4)

0

0.5

1.0

1.5

2.0

Pro

MMP-2

Intermediate

MMP-2

Active

MMP-2

Pro

MMP-9

MMPs

‡*

2.0

4.0

6.0

8.0

10.0

12.0

%IL

AN

CP

Group

2.0

4.0

6.0

8.0

10.0

(G1) (G2) (G4)(G3)0

‡* ‡*†*

‡* ‡* ‡*‡*

‡** ‡**

A

B

C

Fig. 5 a Representative gelatin zymography from liver samples after

8 weeks of experiment. The clear bands of 92, 72, 64 and 59 kDa

corresponds to the pro-MMP-9, pro-MMP-2, intermediate-MMP-2

and active-MMP-2, respectively. b Densitometric analysis of gelat-

olytic bands for MMP-2 and MMP-9. The values are expressed as

average of relative IOD, normalized to G1 group values. c PCNA

labeling indices (LI%). Groups: non-treated (G1), DEN-treated (G2)

DEN plus 2-AAF-treated (G3) and 2-AAF-treated (G4): DEN,

diethylnitrosamine (200 mg/kg. b.w., i.p.) and 2-AAF, 2-acethylami-

nofluorene (0.02% in diet). �,� Different from G1 group or G1 and

G2 groups, respectively. * P 0.05 \ P \ 0.09 and ** P \ 0.001

(G1) (G2) (G3) (G4

- 92

- 72- 64

- 59

Kda

2.0

4.0

6.0

8.0

2.0

4.0

6.0

8.0

% IL

AN

CP

Group(G1) (G2) (G4)(G3)

0

ActiveMMP-2

0

0.5

1.0

1.5

2.0

2.5

3.0

(G2) (G3) (G4)

ProMMP-2

IntermediateMMP-2

ProMMP- 9

)l ortnoc%(

yr temotisne

DMMPs

‡*‡*

‡*

‡*‡* ‡*

‡*‡*

‡*‡*

A

B

C

Fig. 6 a Representative gelatin zymography from liver samples after

20 weeks of experiment. The clear bands of 92, 72, 64 and 59 kDa

corresponds to the pro-MMP-9, pro-MMP-2, intermediate-MMP-2

and active-MMP-2, respectively. b Densitometric analysis of gelat-

olytic bands for MMP-2 and MMP-9. The values are expressed as

average of relative IOD, normalized to the G1 group values. c PCNA

labeling indices (LI%). Groups: non-treated (G1), DEN-treated (G2)

DEN plus 2-AAF-treated (G3) and 2-AAF-treated (G4): DEN,

diethylnitrosamine (200 mg/kg. b.w., i.p.) and 2-AAF, 2-acethylami-

nofluorene (0.02% in diet). � Different from G1 and G2 groups.

* P \ 0.001

J Mol Hist

123

patterns of immunoreactivity for MMP-2 and 9, p21ras and

COX-2 and 3) the activation of pro-MMP-9 is the better

marker associated to the progression of GST-P- positive

PNL. As RAS and COX-2 are key genes associated to PNL

progression to HCC, further studies on the role of these

pathways should be carried out in this model.

Acknowledgments This work was supported by CAPES, FAPESP

and TOXICAM. Kelly S. Furtado was recipient of a fellowship from

CAPES.

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-92

-64- 59

-72

-81

(G1) (G2) (G3) (G4) Kda (T1) (T2) (T3)

- 92

- 64- 59

- 72

- 81

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yrtemotisne

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