Expression of matrix metalloproteinase 2 (MMP-2), membrane-type 1 MMP and tissue inhibitor of metalloproteinase 2 and activation of proMMP-2 in pancreatic duct adenocarcinomas in hamsters treated with N-nitrosobis(2-oxopropyl)amine
Katsumichi Iki1,
Masahiro Tsutsumi,
Akira Kido,
Hiroyuki Sakitani,
Makoto Takahama,
Masatoshi Yoshimoto,
Masaaki Motoyama2,
Kunihiko Tatsumi2,
Tsukasa Tsunoda1 and
Yoichi Konishi3
Department of Oncological Pathology, Cancer Center, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-0813,
1 Department of Gastroenterological Surgery, Kawasaki Medical University, 577 Matsusima, Kurashiki, Okayama 701-0192 and
2 Fuji Memorial Research Institute, Otsuka Pharmaceutical Co., Shiga, 1-11-1, Karasaki, Otsu, Shiga 520-01, Japan
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Abstract
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In order to assess the significance of changes in metalloproteinase activity in pancreatic carcinogenesis, the expression of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9, respectively), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2, and membrane-type 1 MMP (MT1-MMP) and MT2-MMP in ductal lesions in a rapid-production model for pancreatic duct carcinomas (PCs) in hamsters initiated with N-nitrosobis(2-oxopropyl)amine (BOP) and in subcutaneous transplantable tumors of hamster pancreatic duct carcinoma (HPDs) was investigated. Northern analysis revealed MMP-2, MMP-9, TIMP-2 and MT1-MMP mRNAs to be overexpressed in PCs. Immunohistochemically, elevated levels of MMP-2 were apparent in early duct epithelial hyperplasias and staining increased from atypical hyperplasias to carcinomas. Gelatin zymography demonstrated clear activation of proMMP-2 but not proMMP-9 in both of primary and HPD tumors, the MT1-MMP mRNA level and proMMP-2 activation being significantly correlated (r = 0.893, P < 0.001). In our rapid production model, 0.1 and 0.2% OPB-3206, an inhibitor of MMPs, given in the diet after two cycles of augmentation pressures for 48 days decreased the incidence and number of carcinomas. Gelatin zymography demonstrated that OPB-3206 inhibited activation of proMMP-2 in pancreatic cancer tissues. These results indicate that overexpression of MMP-2, TIMP-2 and MT1-MMP, and cell surface activation of proMMP-2 by MT1-MMP, are involved in the development of PCs, and that MMP-2 expression at the protein level appears in the early phase of pancreatic duct carcinogenesis. OPB-3206 may be a candidate chemopreventive agent for pancreatic ductal adenocarcinomas.
Abbreviations: BOP, N-nitrosobis(2-oxopropyl)amine; GAPDH, glyceraldehyde-3-phosphatase dehydrogenase; HPDs, subcutaneous transplantable tumors of hamster pancreatic duct carcinoma; MMP, matrix metalloproteinase; MT-MMP, membrane-type MMP; PC, pancreatic duct adenocarcinoma; SSC, standard saline citrate; TIMP, tissue inhibitor of metalloproteinase.
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Introduction
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The matrix metalloproteinases (MMPs) are a family of so far 20 identified proteolytic enzymes that degrade substances within the extracellular matrix (1,2). Since destruction of basement membrane and extracellular matrix is essential for cancer invasion and metastasis to occur, the involvement of MMPs in neoplasia has recently been attracting attention. Among MMPs, the gelatinases, MMP-2 and MMP-9, are particularly important since they digest type collagen, the main component of basement membranes (3,4), and are known to contribute to the invasion and metastasis of various human malignancies, for example in the lung (5,6), stomach (7), breast (810) and pancreas (11,12). The proteolytic activity of MMPs depends on the balance between the levels of activated enzymes and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs) (11). Recently, it was found that epithelial cell membrane-bound MMPs, membrane-type MMPs (MT-MMPs), play crucial roles in the activation of MMPs. For instance, recent in vitro studies have demonstrated that free TIMP-2 first combines with MT-MMP, which exists on the cell surface, and with the free latent form of MMP-2, then with the TIMP-2/MT-MMP complex to create a subsequent complex of TIMP-2/MT-MMP/MMP-2, which finally activates the latent form of MMP-2 into the activated form (3,8,1315).
The pancreatic duct adenocarcinoma (PC) is the fifth leading cause of cancer death in both the US and Japan (16,17). Due to its silent clinical course, at the time of diagnosis the vast majority of pancreatic cancer cases are incurable with a very poor prognosis. In order to control the disease, it is indispensible to detect the tumor as early as possible and, in turn, to prevent its subsequent progression. An experimental model suitable for investigation of human PC development has been established in hamsters using the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) and related compounds (1820). To facilitate studies of the underlying mechanisms, we have developed a rapid-production model of PCs (2123), incorporating the principle of selection by resistance to cytotoxicity demonstrated earlier for liver carcinogenesis in rats (24,25). This model has not only provided a major stimulus to understanding factors for tumor induction but also serves as a bio-assay for identification of risk factors and appropriate chemopreventive or chemotherapeutic agents (26). We have also established transplantable adenocarcinoma lines from resultant cancers (27,28).
With regard to expression and activation of MMPs, it has been reported that the active form of MMP-2 is detectable in human primary pancreatic cancers and metastatic lesions in the liver (12). An imbalance in expression of MMPs and TIMPs has also been described (11). However, despite many reports of MMP action in vitro, there are few findings about the role of MMPs in carcinogenesis in vivo with experimental models especially for pancreatic carcinogenesis. Assuming the context in the last paragraph, in the present experiment, we studied expression and activation of MMPs and TIMPs during pancreatic duct carcinogenesis in order to obtain information useful for understanding the mechanisms of its progression stage using the above-mentioned rapid-production model. In addition, the effects of OPB-3206 (3S-[4-(N-hydroxyamino)-2R-isobutylsuccinyl]amino-1-methoxy-3,4-dihydrocarbostyril), an inhibitor of MMPs (29), on development of ductal lesions in our rapid production model were assessed.
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Materials and methods
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Animals and treatments
Female Syrian golden hamsters (Japan SLC, Shizuoka, Japan), 7 weeks old and weighing ~100 g each at the commencement were used. They were housed three to a plastic cage in an air-conditioned room at 24 and 60% humidity, with a daily 12 h alternating cycle of light and dark. Pancreatic ductal lesions were induced by the rapid-production model protocol shown in Figure 1
. The details of the procedures have been described previously (1524). Briefly, 70 mg BOP per kg body wt (Nacalai Tesque, Kyoto, Japan) were injected s.c. for initiation. Twelve days thereafter, the hamsters were subjected to the first cycle of augmentation pressure which consisted of four daily i.p. injections of 500 mg DL-ethionine per kg body wt (Nacalai Tesque) while being maintained on a choline-deficient diet (Dyets, Bethlehem, PA) followed by a i.p. injection of 800 mg L-methionine per kg body wt (Nacalai Tesque). The animals were then returned to the basal diet (Oriental MF; Oriental Yeast Co., Tokyo, Japan) and given 20 mg BOP per kg body wt. Augmentation pressure was repeated and all hamsters were killed under ether anesthesia 98 days after the beginning of the experiment. Each pancreas was carefully removed and macroscopically detectable whitish-yellow tumors were excised. A portion of each tumor was immediately frozen in liquid nitrogen for total RNA and protein extractions. Remaining portion were fixed in 10% buffered formalin and embedded in paraffin for histological and immunohistochemical analyses. Transplantable tumors, the hamster pancreatic duct carcinomas (HPDs), used in the present experiment were generations at 140 to 145 and were excised and stored at 80°C for mRNA and protein extraction. The doubling time of transplanted tumors was ~45 days. Hamsters without any treatments were used as controls.

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Fig. 1. Experimental protocol of the rapid production model for PCs in hamsters. Large thick arrow, 70 mg/kg BOP s.c.; medium thick arrow, 20 mg/kg BOP s.c.; hollow arrow, 500 mg/kg DL-ethionine i.p.; thin arrow, 800 mg/kg L-methionine i.p. Detailed procedures were described in Materials and methods.
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cDNA probes for northern blotting
cDNA probes for MMP-2, MMP-9, TIMP-1 and TIMP-2 were obtained by reverse transcriptase-PCR amplification. Briefly, total RNA was prepared from normal rat skin and bone tissues, and cDNA was synthesized using Moloney-murine leukemia virus reverse transcriptase (Gibco BRL, Gaithersburg, MD) and 20mer oligodeoxythymidylic acid primers. Adequate synthesized oligonucleotide pairs were prepared for the PCR amplification with the primer sequences: MMP-2, 5'-TGATCTCCAGTGCCCTC-3' and 5'-TGGCGAACACAGCACTC-3'; MMP-9, 5'-AGCGAGACACTAAAGGCCAT-3' and 5'-TGCAGGACAAATAGGAGCGT-3'; TIMP-1, 5'-CCAGAAATCATCGAGACCACCT-3' and 5'-GGCAGGCAAAGTGATCGCTCT-3'; and TIMP-2, 5'-TGACAAAGAGGACAGAAAGT-3' and 5'-TATTCCTTCTTTCCTCCAAC-3'. PCR amplification was performed in 200 µl of reaction mixture under the following conditions: MMP-2, 40 cycles at 94°C for 45 s, 60°C for 1 min and 72°C for 1 min; MMP-9, 40 cycles at 94°C for 45 s, 63°C for 1 min and 72°C for 1 min; TIMP-1, 30 cycles at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min; TIMP-2, 35 cycles at 94°C for 45 s, 58°C for 1 min and 72°C for 1 min. DNA fragments amplified by PCR were electrophoresed in 1% agarose gels and then extracted using a Wizard PCR Preps kit (Promega, Madison, WI), their sequences being determined with an ABI PRISM Genetic Analyzer (Perkin Elmer, Foster City, CA). The lengths of the products were: MMP-2, 0.41 kb; MMP-9, 0.47 kb; TIMP-1, 0.42 kb and TIMP-2, 0.44 kb. cDNA probes for MT1-MMP and MT2-MMP were generous gifts from Dr Hiroshi Sato (Department of Molecular Oncology, Cancer Research Institute, Kanazawa University, Japan) (7,8).
Northern blot analysis for MMPs, TIMPs and MT-MMPs
Total RNA was isolated from frozen tissues using the lithium chlorideurea method (30). Aliquots of 20 µg of total RNA were electrophoresed in 1% agarose/formaldehyde gels, capillary blotted in 20x standard saline citrate (SSC) onto Hybond N nylon membranes (Amersham, Tokyo, Japan) and fixed by baking at 80°C. Radiolabeled DNA probes were prepared with [
-32P]dCTP using a Random Primed DNA Labeling kit (Pharmacia). Nylon membranes were pretreated with a mixture of 50% formides, 5x SSC, 0.1 M phosphate buffer (pH 7.4), 5x Denhardt's solution, 0.1% SDS, 5 mM EDTA and 100 µg/ml salmon testis DNA, for 4 h at 37°C. Then northern hybridization was performed for 24 h in the same buffer with the labeled probes at 37°C. Nylon membranes were washed in 2x SSC and 0.1% SDS at room temperature and then twice in 2x SSC and 0.1% SDS at 55°C for 20 min. Densitometric quantification of the blots was performed with a BAS1000 image analyzer (Fuji Photo Film, Tokyo, Japan). The blots were then reprobed for glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) to confirm loading and transfer of similar amounts of RNA for each sample. The GAPDH levels were also used to normalize the densitometric quantification.
Immunohistochemistry and western blot analysis of MMP-2 protein
Tissue sections were dewaxed and dehydrated through ethanol; endogenous peroxidase was blocked with hydrogen peroxide and non-specific protein binding was reduced using goat serum. A mouse monoclonal antibody to human MMP-2 was generated using synthetic peptides with the amino acid sequence of VTPRDKPMGPLLVATF, corresponding to the C-terminal sequence (Fuji Chemical Industries, Takaoka, Shizuoka, Japan) according to the protocol recommended by the manufacturer. Binding in tissue sections was immunohistochemically demonstrated using an LSAB kit (DAKO, Glostrup, Denmark). Expression levels were classified into four categories using the following criteria: negative, not stained; slightly positive, <20% stained; positive, 2050% stained; and strongly positive, >50% stained.
For western blotting, 10 mg portions of frozen tissues were homogenized in 500 µl of a lysis buffer containing 1% Triton X-100, 50 mM Tris and 300 mM NaCl. The lysates were incubated on ice for 15 min with occasional vortexing, and insoluble material was removed by centrifugation. The resultant supernates were subjected to 10% SDSPAGE. After the electrophoresis, the samples were electroblotted onto polyvinylidene difluoride transfer membranes (Millipore, Bedford, MA). After 1 h of incubation in a blocking solution of 5% dried milk in TBST buffer (10 mM Tris-base pH 8.0, 150 mM NaCl, 0.1% Tween-20), membranes were incubated for 1 h at room temperature with the mouse monoclonal anti-human MMP-2 antibody (1 µg/ml). After washing in TBST buffer, membranes were incubated with peroxidase-conjugated anti-mouse immunoglobulin G (Vector Laboratories, Burlingame, CA), and the blots were visualized by the enhanced chemiluminescence (ECL) detection method using an ECL kit (Amersham).
SDSPAGE zymography of gelatinase activity for MMPs
Gelatin zymography to reveal gelatinase activity was performed according to the method of Nakajima et al. (31). Standardized aliquots of tissue homogenate, prepared as described above for western blotting, were subjected to electrophoresis in 10% SDSPAGE gels polymerized with 0.1% gelatin. Gels were washed three times each for 30 min in Triton X-100 to remove SDS. And then incubated for 16 h at 37°C in 50 mM TrisHCl buffer, pH 7.5, containing 200 mM NaCl and 10 mM CaCl2. Another set of gels were incubated under identical conditions in the presence of 20 mM EDTA to confirm that proteins exerting gelatinase activity were MMPs. Gels were fixed and stained with 0.5% Coomassie blue R-250 in a mixture of 30% methanol and 10% acetic acid for 15 min at room temperature, and destained with the same solution without Coomassie blue. They were dried for 2 days at room temperature. Ratios for proMMP-2 activation were estimated by computer-assisted densitometric scanning of 58 and 62 kDa proteolytic bands, corresponding to active and latent species of MMP-2, respectively (32), using BAS 1000.
Effects of OPB-3206 on development of pancreatic duct lesions in the rapid production model
Figure 2
shows the chemical structure of OPB-3206. The compound was kindly supplied by Otsuka Pharmaceutical (Ohtsu, Japan). A total of 46 hamsters were divided into three groups. Group 1 received basal diet (Oriental MF) without OPB-3206, and groups 2 and 3 received 0.1 and 0.2% OPB-3206 in the diet for 48 days after two cycles of augmentation pressures as shown in Figure 1
. The diets containing OPB-3206 were prepared once a week and stored in a cold room. All hamsters were killed under ether anesthesia 98 days after the beginning of the experiment. The pancreas was flattened on a filter paper to avoid shrinkage by fixation, divided into the splenic, gastric, and duodenal lobes, and fixed in 10% buffered formalin. After fixation, horizontal slices were made through the three lobes and the tissues were routinely processed for hematoxylineosin staining. Portions of macroscopic tumors were frozen in liquid nitrogen for zymography. The diagnostic criteria for ductal lesions, hyperplasia, atypical hyperplasia, carcinoma in situ and invasive carcinoma have been described previously (33).
Statistics
Statistical analyses were performed using the
2 test and the two-tailed MannWhitney U-test. Statistical significance was concluded when P-values were <0.05.
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Results
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Expression of MMP-2, MMP-9, TIMP-2 and MT1-MMP mRNAs
Northern analysis for MMP-2, MMP-9, TIMP-1, TIMP-2, MT1-MMP and MT2-MMP was performed with total RNAs extracted from primary PCs, HPDs, and normal pancreas tissues. Figure 3
shows the representative blots. A single mRNA transcript was evident for each enzyme in all of the examined samples with the exceptions of three transcripts noted for TIMP-2, with sizes of 1.0, 3.5 and 4.9 kb, and none for MT2-MMP. Results of densitometric analysis are summarized in Figure 4
. The mRNA levels of MMP-2, MMP-9, TIMP-2 and MT1-MMP were all significantly increased in both primary PCs and HPDs as compared with the normal pancreas case. In contrast, no alterations in TIMP-1 mRNA levels was evident.

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Fig. 3. Representative results of northern blot analysis for mRNA expression of MMP-2, MMP-9, TIMP-1, TIMP-2, MT1-MMP and MT2-MMP in primary PCs (lanes 14), HPDs (lanes 58) and normal pancreas tissues (lanes 912).
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Fig. 4. The results of densitometric analysis of mRNA levels of MMP-2, MMP-9, TIMP-1, TIMP-2, MT1-MMP and MT2-MMP from Figure 3 in normal pancreas tissues (NP), PCs and HPDs standardized to the GAPDH mRNA levels (arbitary units). Bars, mean; *, with significant difference; NS, without significant difference.
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Immunohistochemistry and western blot analysis for MMP-2 protein
Immunohistochemical staining of MMP-2 revealed normal pancreatic acinar cells and islet cells to be negative to slightly positive, while ductal cells were totally negative (Figure 5A
). Increase was detected in the cytoplasm of cells in hyperplasias (Figure 5B
), atypical hyperplasias (Figure 5C
) and primary PCs (Figure 5D
). Gradual increase in the incidence and extent of staining was observed with progression to malignancy (Table I
). To confirm the immunohistochemical results, standardized aliquots with appropriate buffer of tissue homogenates were subjected to western blot analysis. The latent form of MMP-2 was identified in the pancreatic adenocarcinomas and the reaction of the human MMP-2 antibody with MMP-2 protein in hamster pancreatic adenocarcinomas was confirmed.

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Fig. 5. Immunohistochemistry for MMP-2 protein in primary pancreatic duct lesions arising in the rapid production model (2123). (A) normal pancreatic duct; (B) hyperplasia; (C) atypical hyperplasia; (D) invasive carcinoma.
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Table I. Immunohistochemical findings for MMP-2 protein expression in pancreatic ductal lesions induced by BOP in our rapid production model
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SDSPAGE zymography for gelatinase activity of MMP-2
The results of SDSPAGE zymography for gelatinase activity are shown in Figure 6
. Bands of 92, 62 and 58 kDa were attributed to proMMP-9, proMMP-2 and MMP-2 which were abolished by EDTA. The incidence of proMMP-9 expression detected as 92 kDa was 10/16 (63%) in primary adenocarcinomas, 10/10 (100%) in HPDs and 0% in normal tissues. The active form of MMP-9 was not detected either in primary adenocarcinomas or HPDs. Normal tissues were also negative. The incidence of proMMP-2 detected as 62 kDa was 16/16 (100%) adenocarcinomas, 10/10 (100%) in HPDs and 9/9 (100%) in normal tissues. The active form of MMP-2 detected as 58 kDa was found in 12/16 (75%) primary adenocarcinomas and 10/10 (100%) HPDs, whereas no activity was apparent in normal tissues. The results for proMMP-2 activation measured by densitometry and expressed as MMP-2/proMMP-2 plus MMP-2 are shown in Figure 7
. The ratios in primary adenocarcinomas and HPDs were 0.46 ± 0.15 and 0.60 ± 0.30, respectively, being significantly higher than that in normal pancreatic tissues (0.18 ± 0.04) (Figure 7
, left panel). Furthermore, proMMP-2 activation correlated linearly with the MT1-MMP mRNA level (Figure 7
, right panel).

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Fig. 6. Representative SDSPAGE zymography findings for gelatinase activity of MMP-2 in primary PCs, PC + EDTA, HPDs and normal pancreas tissue (NP).
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Fig. 7. Ratios of proMMP-2 activation (activated to latent forms) and the correlation with MT1-MMP mRNA levels (right panel) in normal pancreas tissues (NP), primary PCs and HPDs. Bars, mean; *, significant difference (left panel).
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Effects of OPB-3206 on BOP-initiated pancreatic duct carcinogenesis in the rapid production model
There were no significant differences in terms of final body weights, mean pancreas weights, average diet intake or average water intake among the groups. All pancreatic lesions were of duct epithelial cell origin, and no acinar or islet cell lesions were observed. Data for incidences and mean numbers of ductal lesions are summarized in Table II
. There were no significant differences in terms of the incidences of hyperplasia, atypical hyperplasia and carcinoma in situ among the groups, while the incidence of invasive carcinoma in group 2 was significantly less than that of group 1. The mean numbers of invasive carcinoma and of total lesions were significantly decreased in group 2 and also that of invasive carcinomas in group 3, as they compared with group 1. Gelatin zymography demonstrated that activation of proMMP-2 was decreased in PCs of hamsters treated with 0.1 and 0.2% OPB-3206, respectively (Figure 8
).
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Table II. Effects of OPB-3206 on the development of pancreatic ductal lesions in the rapid production model for pancreatic adenocarcinomas
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Fig. 8. Representative data for proMMP-2 activation in PCs in hamsters with or without OPB-3206 treatments.
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Discussion
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The present investigation clearly demonstrated that MMP-2, MMP-9, TIMP-2 and MT1-MMP are upregulated at the mRNA level in PCs and that MMP-2 protein is immunohistochemically detectable in ductal lesions during pancreatic carcinogenesis in hamsters. It is generally accepted that MT1-MMP is not only an extracellular matrix-degrading proteinase, but also an activator and receptor for MMP-2. The latent form of the MT1-MMP is activated by proteolysis, either in the Golgi by furin family proteinase or on the cell surface by a proteinase such as a plasmin.
The activated MT1-MMP then binds the inhibitor TIMP-2 by its N-terminal inhibitory domain. The C-terminal domain of the bound TIMP-2 then acts as a receptor for binding the C-terminal hemopexin domain of proMMP-2. A second molecule of MT1-MMP cleaves and activates the bound proMMP-2 to give active MMP-2 that can remain membrane-bound or be released (3). In the present experiment, the expression of MMP-2, TIMP-2 and MT1-MMP detected by northern blot analysis and activation of proMMP-2 observed by gelatin zymography, together with the correlation between MT1-MMP expression and proMMP-2 activation, suggest that cell surface activation of proMMP-2 by MT1-MMP occurs in PCs. Furthermore, the immunohistochemical finding of increased MMP-2 protein in duct epithelial hyperplasias, indicates that this might be a relatively early event during the development of carcinomas in the present model (26). Detailed studies of the time schedule of such activation are now required.
OPB-3206 is a novel agent, designed to be administered orally, which inhibits MMP activity by binding reversibly to the zinc binding region of the enzymes (29). Inhibition in vitro is effected at the following IC50 values; MMP-1, 7x107; MMP-2, 5x106; MMP-9, 5x107 (information obtained from Otsuka Pharmaceutical). In the present experiment, OPB-3206 specifically inhibited the expression of MMP-2, as assessed by gelatin zymography, and also reduced both the incidences and numbers of invasive carcinomas. Thus, direct evidence was obtained of an involvement of MMP-2 in cancer invasion by PCs.
Carcinogenesis is now generally considered to feature multiple steps in which genetic alterations accumulate (34). A number of lesions at the DNA level are known to be characteristic of human pancreatic carcinomas, but their sequential appearance cannot be determined in man. Hamster pancreatic cancers closely resemble their human counterparts in terms of histology, biology and genetic alterations. Previously, we reported that k-ras mutation is an early event, which can be detected in hyperplasias but increases in incidences with progression to carcinomas (35,36), whereas p53 mutations normally occur first in frank malignancies (37). Shortened telomere and increased telomerase activity can be detected in primary adenocarcinomas, HPDs and cultured cell lines (38). The present findings of enhanced expression of MMP-2, TIMP-2 and MT1-MMP at the mRNA level, and MMP-2 at the protein level, along with proMMP-2 activation, suggest that alterations in metalloproteinase function are important for pancreatic adenocarcinoma development and progression and may be treated in attempts to control this deadly disease.
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Acknowledgments
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We express our gratitude to Ms Kazue Ichida, Rie Maeda, Hiroko Masuda and Michiko Iki for expert technical assistance. This work was supported by a Grant-in Aid (to Y.K.) from the Ministry of Education, Science, Sports and Culture and by Grant-in-Aid (to Y.K.) from the Ministry of Health and Welfare for the Comprehensive 10 Year Strategy for Cancer Control, Japan.
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Notes
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3 To whom correspondence should be adressed Email: ykonishi{at}nmu-gw.cc.naramed-u.ac.jp 
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Received December 3, 1998;
revised March 12, 1999;
accepted March 25, 1999.