Sequential MMP-2 Alteration in DMBA-Induced Hamster Cheek Pouch Carcinogenesis.
Y-J Chen * and M-Y Chou
*Dep. of Oral & Maxillofacial Surgery, Chung Shan Medical University, Taichung, Taiwan

Abstract
Objective: The matrix metalloproteinases (MMPs) are produced by the cancer cells or through induction of surrounding stromal cells. The ability of MMP-2 to degrade basement membrane type IV collagen appears especially important in cancer progression. However, the sequential alteration of MMP-2 in oral carcinogenesis has not yet been well demonstrated. The aim of this study was to investigate the sequential MMP-2 alteration in DMBA-induced hamster cheek pouch model. Methods: Thirty-five out-bred, young (7 weeks old), male Syrian golden hamsters were randomly divided into 5 experimental groups (week 4, 6, 8, 10, and 12 DMBA treated groups; each with 5 animals) and two control groups (6 animals each). The pouches of each experimental group were bilaterally painted with a 0.5% DMBA solution three times a week. Each animal of one control group was similarly treated with mineral oil only, while the other control group remained untreated throughout the experiment. All pouches were studied both grossly and histologically. Tissues from each group were used to evaluate MMP-2 activity by gelatin zymography. Results: The main gelatinase secreted by hamster cheek pouch migrated at 72 kDa and represented MMP-2. The values of MMP-2 activity were found increased in a time-dependent manner (p<0.05). From the AlphaImager 2000 densitometer, the amount of MMP-2 was about 1.4 fold on week 4 (hyperkeratosis), 1.6 fold on week 6 (dysplasia), 2.0 fold on week 10 (leukoplakia), and 3.7 fold on week 12 (carcinoma) compared with control, respectively. Conclusion: These results demonstrate that the up-regulation of MMP-2 expression in DMBA-induced oral carcinogenesis, suggesting that MMP-2 may play an important role in the pathogenesis of oral cancer.

Introduction
Degradation of the extracellular matrix and basement membrane is an essential component of several steps in tumor invasion, angiogenesis, and metastasis (Liotta and Stetler-Stevenson 1991; Goldberg and Eisen 1991; Hart et al. 1989). Different classes of enzymes have been implicated in this process, including members of the zinc-dependent proteinase family known collectively as matrix metalloproteinases (MMPs) (Matrisian 1990). The ability of tumor cells to produce these metalloproteinases has been implicated in the process of tumor progression. The MMPs, in particular the type IV collagen specific collagenases, MMP-2 and MMP-9, participate in the degradation of ECM components including the basement membrane, which separates epithelia from stroma. The type IV collagen specific collagenases, encoded by two distinct genes, are secreted in latent form, which require activational cleavage yielding the 62 and 82 kDa active enzymes, respectively. In vitro observations have independently shown that MMP-2 and MMP-9 production and type IV collagen content correlate with metastatic potential. In addition, increased MMP-2 and MMP-9 expression as well as loss of basement membrane type IV collagen have also been reported in many human solid tumors. In head and neck squamous cell carcinoma (SCC), immunohistochemistry indicated that MMP-2 was mainly expressed in cancer cells. Because MMP-2 degrades type IV collagen, the level of MMP-2 in carcinomas may be a useful indicator of the degree of invasion and metastasis. (Ryo Kawata et al. 2002). Using zymography, Sutinen et al. (1998) indicate that the studied MMPs are clearly up-regulated during invasion in oral SCC. However, there was also a clear, although weak, up-regulation of the expression of the MMPs in some of the lichen planus and dysplastic lesions. An appropriate model system for basic research in carcinogenesis is a prerequisite to understand the processes in the pathogenesis and subsequently, prevention and treatment of cancer. Hamster buccal pouch mucosa, following experimental carcinogenesis using 7,12-dimethyl-benz(a)-anthracene (DMBA), is one of the most widely accepted experimental models of oral carcinogenesis (Gimenez-Conti and Slaga 1993).
In spite of anatomical and histological differences between pouch mucosa and human buccal tissue, carcinogenesis protocols induce premalignant changes and carcinomas that are similar to the development of premalignancy and malignancy in human oral cancer. Hyperkeratosis, dysplasia, and exophytic and endophytic SCCs are usually observed in the cheek pouch model. The expression of MMP-2 in oral SCC had reported in several present studies. Cross-section studies at several stages in oral carcinogenesis had discussed. However, the sequential alteration of MMP-2 in oral carcinogenesis has not yet been well demonstrated. The aim of this study was to investigate the sequential MMP-2 alteration in DMBA-induced hamster cheek pouch model.

Material and method
Thirty-five out-bred, young (7 weeks old), male Syrian golden hamsters were randomly divided into 5 experimental groups (week 4, 6, 8, 10, and 12 DMBA treated groups; each with 5 animals) and two control groups (6 animals each). The pouches of each experimental group were bilaterally painted with a 0.5% DMBA solution three times a week. Each animal of one control group was similarly treated with mineral oil only, while the other control group remained untreated throughout the experiment. All pouches were studied both grossly and histologically. Tissues from each group were used to evaluate MMP-2 activity by gelatin zymography. Zymography was performed according to a previously published method. In brief, cell culture media were concentrated using a concentration device (Centrikon, Millipore, Illkrich, France). Samples containing 16 µg protein were electrophoresed on 8% non-reducing SDS-PAGE gels containing 0.1% gelatin as a substrate. Gels were washed two times in 2.5% TritonX ­100 for 30 min. Gels were pre-incubated in reaction buffer (40 mM Tris-HCl pH 8.0, 10 mM CaCl2, 0.01% NaN3) for 30 min and subsequently incubated in reaction buffer for 12 h at 37°C. Gelatinolytic activity was visualized as clear areas in a Coomassie Blue R-250 stained gel. The gelatin cleavage rate was analyzed from the photographed gels with a densitometer (AlphaImager 2000, Alpha Immotech, San Leonardo, CA, USA).

Result                                                                                                                                                                                                                                                               Fig 1A. Normal hamster cheek pouch B. Numerous tumors (12weeks of DMBA applications)  Fig 2A. Hyperkeratosis after 4 weeks of DMBA applications (H&E 200×)  B. The expression of MMP-2 in hyperkeratosis (IHC 200×). Fig 3A. Dysplasia after 6 weeks of DMBA applications (H&E 200×). B. The expression of MMP-2 in dysplasia. (IHC 200×). Fig 4A. Leukoplakia with dysplasia after 10 weeks of DMBA applications (H&E 200×). B. The expression of MMP-2 in leukoplakia with dysplasia (IHC 200×). Fig 5A. Carcinoma induced by application of DMBA after 12 weeks (H&E 200×). B. The expression of MMP-2 in carcinoma. The immunoreactivity to MMP-2 was expressed in a cancer nest (IHC 200×).                                               Gelatin zymography
Fig 6. Gelatin zymogram of MMPs secreted by hamster cheek pouch. Gelatinolytic activity and molecular weight positions of MMP-2 and MMP-9 activity are indicated. The optical density values of MMP-2 were calculated from their gelatinolytic activity by using the AlphaImager 2000 densitometer (p<0.05).

Conclusion
Increased gelatinase expression loss of basement membrane type IV collagen has been reported in many human solid tumors. However, the sequential alteration of MMP-2 in oral carcinogenesis has not yet been well demonstrated. In this study, we use DMBA-induced hamster cheek pouch model to investigate the sequential MMP-2 alteration. These results demonstrate that the up-regulation of MMP-2 expression in DMBA-induced oral carcinogenesis, suggesting that MMP-2 may play an important role in the pathogenesis of oral cancer. And the hamster cheek pouch is an appropriate model system for basic research in carcinogenesis and a prerequisite to understand the processes in the pathogenesis and subsequently, prevention and treatment of cancer.

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