Introduction
Metalloproteinas play a significant role both in physiological and pathological process in the human body, acting as mediators in remodelling and degrada-tion of extracellular space components. Much attention has been paid to their involvement in neoplasia, mech-anisms of invasion and formation of metastas. An increa in the level of metalloproteinas initiates spread of cancer cells, thus causing tumor invasion and formation of distant metastas [14]. It has been shown that MMP-8 is creted by squamous epithelial carci-noma cells, MMP-7 is produced by cancer cells of the stomach, breast and lungs, while MMP-2, MMP-9 and MMP-14 by epithelial cells of various cancers [10]. Bramhall et al.[2] performed comparative analysis of the expression of metalloproteinas (MMP-2, MMP-3, MMP-7 and MMP-11) in pancreatic ductal carcino-ma and healthy pancreas (intended for transplantation) using Northern blot method and in situ hybridization Their results clearly indicated an increa in the expression of metalloproteinas in cancer specimens as compared to healthy tissues.
Matrix metalloproteina 9 (MMP-9) is produced by neoplastic cells as well as by host cells that form the connective tissue stroma indispensable for tumor growth. In neoplastic invasion, MMP-9, involved in degradation and remodelling of the extracellular matrix, and in breakdown of the bament membrane of vesls and ducts, helps overcome the physical barriers and contributes to the formation
of distant metastas. It also promotes tumor neovascularization. Numerous studies have been conducted on its role in the devolopment of carcinomas of the breast, the bladder, the pancreas, the colon, the prostate or hypophysial adenomas [4]. A rela-tionship has been described between MMP-9 activity and tumor metastasis formation and patients' survival. For instance, it has been demonstrated in patients with advanced breast cancer that the higher the MMP-9 activity at the time of tumor diagnosis, the shorter over-all survival [18]. Similarly, MMP-9 expression has been found to be an unfavorable prognostic marker in non-small cell lung cancer [17].
Therefore, the current study objective was to asss the expression of MMP-9 in pancreatic ductal cancer, and to analy the correlation of MMP-9 with chon
FOLIA HISTOCHEMICA
ET CYTOBIOLOGICA
V ol. 45, No. 1, 2007
对齐命令pp. 37-40
Expression of matrix metalloproteina 9 in pancreatic ductal carcinoma is associated with tumor me
tastasis formation
Anna Pryczynicz1, Katarzyna Guziñska-Ustymowicz1, Violetta Dymicka-Piekarska2, Jolanta Czy¿ewska2and Andrzej Kemona1
1Department of General Pathomorphology and
2Department of Clinical Laboratory Diagnostics, Medical University of Bia³ystok, Bia³ystok, Poland
Abstract:The objective of the current study was to asss the expression of matrix metalloproteina 9 (MMP-9) in pan-creatic ductal carcinoma and to examine its correlation with chon clinico-anatomical parameters. The study group con-sisted of 36 patients with pancreatic ductal carcinoma. Tumors were stained using immunohistochemical method (NCL-MMP-9, Novocastra). No correlation was found between tumor MMP-9 expression and age, gender or grade of histologi-cal malignancy. However, statistical analysis revealed a relationship between tumor MMP-9 expression and histological type (adenocarcinoma mucinosum) of pancreatic carcinoma. The expression was strongly correlated with lymph node involvement and occurrence of distant metastas (p<0.00001). The results indicate a correlation between the expression of MMP-9 in pancreatic ductal carcinoma and wor prognosis (shown by lymph node involvement and distant metastas). Key words:Pancr
eatic carcinoma - Matrix metalloproteina-9 (MMP-9) - Immunohistochemistry
独龙牛Correspondence: K. Guziñska-Ustymowicz, Dept. of General Pa-
thomorphology, Medical University of Bia³ystok, Waszyngtona 13,
15-269 Bia³ystok, Poland; e-mail: pl
anatomo-clinical parameters: age and gender, histological type of cancer, grade of histological malignancy, lymph node involvement and formation of distant metastas.
Materials and methods
Material.The study involved a group of 36 pancreatic ductal car-cinoma patients (8 men, 28 women; of them 17 aged <60 and 19aged 60 or above) surgically treated at the Department of Gas-troenterological Surgery, Medical University of Bia³ystok, in the years 1999-2004. Hematoxylin-eosin -stained ction were exam-ined according to the TNM classification. Each tumor was c-tioned parallel to the longest axis and at least one total oblong-c-tion (2-3 mm thick) was obtained. It was then divided into small blocks, 1-1.5 cm in diameter, to obtain 4-8 ctions consisting of tumor and adjacent macroscopically unchanged tissues.
Immunohistochemistry.Immunohistochemical investigations were performed using monoclonal antibody (Novocastra/NCL-
MMP-9/clone 2C3) directed against human matrix metallopro-teina 9. Slides of 4 μm-thick rial ctions of the primary tumor were prepared from each patient. A standard avidin-biotin immunoperoxida method (Novostain Super ABC Kit Universal)was ud for the detection of MMP-9. Briefly, the slides were dewaxed using xylene, transferred to alcohol, placed in citric acid buffer (pH=6.0) and heated in a microwave oven (700 W) for 10min to expo antigens. Endogenous peroxida activity was blocked by incubating the ctions in 3% hydrogen peroxide in methanol for 10 min. The slides were then washed 3 times in phos-phate-buffered saline (PBS) and incubated in normal hor rum for 15 min to reduce nonspecific antibody binding. After washing with PBS, the slides were incubated overnight at room temperature with monoclonal antibody (Novocastra/NCL-MMP9/clone 2C3;dilution 1:40, Biokom, Poland) was ud. Nonspecific mou IgG was ud as a negative control. The reaction products were visual-ized with diaminobenzidine DAB (DAKO S3000, Dako, Poland). Evaluation of samples.Cytoplasmic immunostaining and poor stromal immunostaining (Fig. 1 and 2) was obrved. Expression
38
A. Pryczynicz
et al.
Fig. 1.Expression of MMP-9 in cancer cells (magn. × 40).
Fig. 2.Cytoplasmic expression of MMP-9(magn. × 40).
pop设计was mi-quantitatively assd in neoplastic cells of the primary tumor and was defined as follows:
•negative - indicating lack of reaction to the prence of MMP-9 or reaction in <30% of cells,
•positive - reaction to MMP-9 visible in >30% of cells.
The percentage of MMP-9 positive cells was calculated in 500 cancer cells in each preparation, at a magnification of 400 x. The results were subjected to statistical analysis using exact Fisher test and χ2test. The value p<0.05 was accepted as the level of significance. Results and discussion
In recent years, literature reports have described the relationship of the degradation of the extracellular matrix, particularly of the bament membrane, as the basic mechanism facilitating invasion and formation of metastas by cancer cells. The extracellular matrix, which consists of vari
怀孕能喝碳酸饮料吗ous types of collagens, laminin, fibronectin, elastin and proteoglycans, is degraded by proteinas, such as metalloproteinas. The expres-sion of metalloproteinas is variously regulated and correlates with invasiveness and formation of metas-tas in carcinomas of the thyroid, the prostate, the ovaries, the stomach and the lungs [16].
We analyzed the expression of matrix metallopro-teina-9 (MMP-9) in correlation with chon anato-mo-clinical parameters (Table 1). No statistically sig-nificant relationship was found with age and gender of the studied patients.
The studied group of tumors consisted of adenocar-cinoma-type and adenocarcinoma mucinosum neo-plasms. The MMP-9 expression strongly correlated with the prence of mucus, previously shown to facil-itate cancer spread. Therefore, adenocarcinoma muci-nosum is more aggressive than ordinary adenocarcino-ma and more difficult to treat. However, no literature data are available on the relationship between MMP-9 expression and creting or non-creting histological type of carcinoma. Gress et al.[5] have noticed that matrix metalloproteina 9 can be associated with the process that leads to strong desmoplastic proliferation of the connective tissue stroma compo-nents obrved in pancreatic carcinomas.
The role of matrix metalloproteina 9 has been the subject of many current studies revealing that MMP-9 expression in tumor correlates with shorter survival time. Tumor size, lymph node involvement or distant metastas significantly correlate with shorter survival [7]. Moreover, MMP-9 has been found to contribute to the aggressive behaviour of pancreatic carcinoma and thus lies behind the poor prognosis [6].
In our study, the expression of matrix metallopro-teina 9 was obrved in all patients with lymph node involvement. Migration of cancer cells via the lymphat-ic system plays an esntial role in tumor invasion and in the formation of metastas in lymph nodes. Degra-dation of extracellular matrix by metalloproteinas is the necessary prerequisite for cell migration. Migration of cancer cells can be enhanced through MMP-9 over-expression and is reduced through overexpression of TIMP(tissue inhibitor of metalloproteina) or applica-tion of MMP-9 inhibitors [1,3]. Yamamoto et al.[19] have confirmed that both cancer cells and stromal cells are the major source of matrix metalloproteina 9 in pancreatic ductal carcinoma tissues (among 70 cas, 93% of carcinoma cells and 60% of stroma cells were MMP-9 positive). However, MMP-9 expression was insignificantly correlated with such pathological factors as tumor size, lymph node involvement, formation of distant metastas or tumor stage. Nevertheless, each tumor shows different expression of metalloproteina
9 and it is likely that MMP-9 differently affects invasive-ness and formation of metastas of the respective tumors.According to Maatt et al.[11], matrix metallo-proteina 9 does not have a special role in pancreatic ductal carcinoma. The authors analyzed the expression
39
MMP-9 in pancreatic ductal carcinoma
Table 1.Expression of MMP-9 in pancreatic ductal adenocarcino-
ma in association with chon anatomoclinical parameters
of MMP-9 in 8 cas of pancreatic tumor. The reaction was pronounced only in tumor epithelial cells, but it was negative in stromal cells in most cas (only sporadic fibroblasts and endothelial cells were positive). Zucker et al.[20] obrved MMP-9 expression only in 19 out of 45 (43%) pancreatic ductal carcinomas but not in healthy pancreas specimens (p=0.0009). Six of 19 (32%) pan-creatic tumors showed either moderate or strong expres-sion. No expression of MMP-9 was found in adjacent tis-sues, stromal cells of the cancer or in the normal pan-creas. No correlation was obrved between MMP-9 expression with histological differentiation of the cancer, tumor size, lymph node involvement or survival time.
We found MMP-9 expression in all patients with dis-tant metastas. Numerous reports em to confirm that matrix metalloproteina 9 has a significant effect on the formation of metastas of pancreatic ductal carci-noma. Matsuyama et al. [12] have proved that pancreat-ic ductal carcinomas with metastas show much high-er MMP-9 expression than metastasis-free carcinomas. Similarly, Nagakawa et al.[13], who analyzed 32 cas of pancreatic ductal carcinoma, found distant metastas in 31 of them and demonstrated a major role of matrix metalloproteina 9 in cancer cell infiltration of blood vesls, which is a risk factor of tumor spread. Angio-genesis, i.e. neovascularizat
ion, also plays an outstand-ing role in the formation of distant metastas. It has been shown that tumor without additional blood vesls can reach 1-2 mm in diameter and needs nutrients sup-plied by blood vesls to grow and proliferate. Angio-genesis is strictly regulated by the system of stimulators and inhibitors, starting with the relea of proteas that decompo the bament membrane and the extracellu-lar matrix. Metalloproteina 9 helps cancer cells pass through the extracellular matrix barrier and participates in the modulation of signals that affect cell transforma-tion, growth factors, angiogenesis and apoptosis [8,9,15]. The results of our study indicate a correlation between the expression of MMP-9 in pancreatic ductal carcinoma and wor prognosis (shown by lymph node involvement and distant metastas).
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Received: August 29, 2006
Accepted after revision: October 23, 2006
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