Introduction

Metastasis is one of the most important events in cancer pathogenesis, responsible for the most part of the human mortality through cancer.
The development of sofisticated biochemical, immunological and genetic techniques that can be applied to spontaneous primary tumors and their metastases was an important stage in research and led to the detailed analysis of the tumoral progression, matastasis and therapy of the cancerous disease and its metastases. At present, it is well known that metastasis is dependent on both host’s characteristics and tumor’s characteristics. 
For studying the experimental tumor metastases, it is certainly useful to have animal tumor models that mimic well the metastatic disease in humans. Some of these medels can have a limited usefulness and answer to some specific questions regarding the metastatic process, while others can be useful in studying a large area of problems related to cancer biology and metastases therapy.
Walker carcinosarcoma 256 is a tumor that appears spontaneously in the mammary glands of the rat, discovered by Walker. Initially, the tumor was diagnosticated as adenocarcinoma, in which structure there is also a sarcomatoid cell type population. Walker carcinosarcoma 256 is supported by subcutaneous grafts in Wistar rats, it has a metastatic potential, and the invasion of locoregional lymph nodes appears in the terminal phase of the tumoral growth, when tumor necrosis phenomena are accentuated. It is a standard tumor used in lymph node metastasis models, in the preclinical screening of the antitumor substances, and also in different experimental models of chemotherapy and radiation therapy.
The goal of our study was to elucidate the activity of cathepsin D, correlated with evolution and metastasis parameters. 

Materials and method

Biological samples 
The study was conducted on 60 normal, healthy Wistar albino rats, with a 200 g medium weight, who were inoculated with 1,000.000 tumoral cells/ml, in the right side. 
Cell viability was assessed using 0.1% Trypan Blue in TFS. Rats were sacrificed at 13, 15, 18, 21 and 25 days from grafts, when the primary tumor was evaluated and biologic tissue was sampled for obtaining the tumor cytosol. The local tumor was measured using two diameters, and the tumor volume was calculated using the following formula:  
V = a x b² x 0.52 (a = the large diameter of the tumor; b = the small diameter of the tumor; 0.52 = thickness of the tegument).
Obtaining the cytosol
The homogenization of the tissue for isolating the cytosolic fraction was made using 10 mM Tris-HCl buffer, pH=7.4. The homogenate was centrifuged at 5000xg, for 10 minutes, and the supernatant was ultracentrifuged at 100000xg for one hour.
Determinations
The cytosolic protein concentration was determined by two methods, Lowry and Bradford, with bovine serum albumin (BSA) as standard. The method used for the biochemical determination of the cathepsin D was similar to the method described by Capony. The reaction took place in Acetate Buffer 1M pH 3.5, with 4% hemoglobin as substrate, and it was stopped with 2 ml of 3% trichloroacetic acid. 
A standard curve was made, for whose plotting we used pure cathepsin D, obtained from Sigma company. For control, 20 µl of pepsatin A were added to the reaction, resulting a final concentration of 10-6 M. A second control sample was realized, adding the tumor extract followed by the trichloroacetic acid, which stopped the reaction.

Results and discussion

Walker carcinosarcoma 256 had a good growth, the tumor became palpable at 8-14 days after the inocula­tion, and the micrometastases were present in the liver. 
At 15-21 days after the inoculation we could notice hepatic macrometastases. In the final phases, they covered the entire liver surface and after 7 days the rat’s mortality from our study was almost 100%.
The evolution and metastatic process of the Walker carcinosarcoma 256 were assessed by measuring the tumor volume and by the quantitative evaluation of the hepatic tumors. 
Table 1 presents the evaluation of hepatic metastases, in the evolution course of Walker carcinosarcoma 256, in Wistar rats subcutaneously grafted with 1x106 living cells. 
The results presented in Table 1 are means obtained from two lots grafted at different moments, in which the rats presented quite similar evolutions.
The tumor volume was significantly increased (p<0.05) in the 18th day versus the 13th day, and the metastasis parameters were significantly different (p<0.05) from those from the 13th day and even from those from the 15th day.
The 15th day can be considered the moment of the obvious appearance of liver metastases in our experimental conditions.
From the primary tumor sampled in days 13, 15, 18, 21 and 25 it was prepared the tumor cytosol in which there were dosed the cytosolic protein and the cathepsin D. The activity of cathepsin D was expressed in pmoli/mg cytosolic protein. 
The results presented in Table 2 indicate the modification of this degradative enzyme in the primary tumor, subcutaneously grafted, in the process of enzyme formation.
The activity of cathepsin in the primary tumor in the 13th day doesn’t differ significantly from normal. A significant increase of the enzyme activity was noticed in the 15th day, compared to the activity measured at 13 days after inoculation. The activity measured in days 18, 21 and 25 doesn’t differ significantly from the one measured in the 15th day. The differences in the cathepsin D activity, in the primary tumor, in days 15, 18, 21 and 25 are elevated, with statistical significance. 
Cathepsin D activity is 2.12 times higher in the 25th day compared to the 13th day. The presence in the tumoral tissue of an enhanced enzymatic activity of cathepsin D, in a metastasizing tumor such as Walker carcinosarcoma 256, is in agreement with the presumed role of cathepsin D in the metastasis process.

Conclusions

It was noticed a positive correlation between the growth time of lysosomal enzyme, cathepsin D, in Walker carcinosarcoma 256, and the appearance of micro- and macrometastases in liver. 
The mechanisms responsible for the intratumoral lysosomal enzyme growth are unidentified, but they might be related to macrophage infiltration and other tumor-host interactions, which can facilitate the tumor cell disemination. Our results suggest that the inhibition of this enzyme may open the way for new therapeutic methods for controlling the malignant disease.  atepsina Dcarcinosarcom Walker 256proteaze