Cathepsin B and cathepsin D expression in the progression of colorectal adenoma to carcinoma

Abstract

Lysosomal proteinases, cathepsin B (CB) and cathepsin D (CD) have been implicated in the progression of several human tumors. In the present study, the antigen levels of CB and CD, and their immunohistochemical staining were compared in paired colorectal tumors (n=64) and background colon tissue of the same patients with clinicopathological staging. The antigen levels, were found to be significantly higher in cancer tissue (mean 35.79 ng/mg protein for CB and 3.97 ng/mg protein for CD) than in corresponding normal mucosa (24.62 ng/mg protein for CB and 2.69 ng/mg protein for CD). CB antigen levels were positively correlated with differentiation grade and Duke's stage (P<0.001 and P=0.041, respectively), but not correlated with nodal status. CD antigen levels were not correlated with the previous parameters. Staining intensity for both antigens increased from adenoma to adenocarcinoma. The degree of staining for CB and CD was associated with differentiation grade (P=0.004 and 0.001, respectively), Dukes' stage (P=0.002 and 0.001, respectively) and lymph node involvement (P=0.002 and P<0.001, respectively).

Introduction

Tumor invasion and metastasis is a multi-step process that involves, at various stages, the penetration of host ECM by cancer cells. The ability of tumor cells to invade tissues and metastasize is thought to involve an increased expression of proteinases and/or a decrease in the levels of proteinase inhibitors [1]. Proteinases may facilitate metastasis in a number of different ways including detachment of individual cells from the primary tumor, invasion of surrounding tissues to allow contact with vascular channels, degradation of the basement membrane during both intravasation and extravasation, and invasion of tissues during formation of secondary tumor sites. Several classes of proteinases have been implicated in this process including matrix metalloproteinases, cathepsins B and l (cysteine proteinases), cathepsin D (aspartic proteinase), and plasminogen activators (serine proteinases) [2], [3], [4], [5], [6], [7], [8]. It is possible that human tumors may use combinations of these enzymes working synergistically to facilitate invasion, or alternatively, one specific proteinase may play a dominant role in tissue invasion for a given cancer.

In the western world, colon carcinoma is a major cause of death, mainly due to local invasion and distal metastasis. Defining the role of proteinases in the progression of colon cancer can be important for the better understanding of processes involved in invasion and metastasis, development of prognostic markers and design of selective inhibitors of the proteases intended for therapy [9], [10].

Cathepsin B (CB) is the major representative of the cysteine proteinases and is present in lysosomes of all types of cells under normal conditions. It is involved in lysosomal turnover of proteins and basement membrane-specific components [11]. The enzyme exhibits broad substrate specificity at both acid and neutral pH [11] and functions both as an exopeptidase and as an endopeptidase [12]. The enzyme is synthesized as a precursor, which is enzymatically inactive [11]. Many human cell lines have been proved to exhibit qualitative and quantitative differences in the regulation of CB expression [13]. CB is also engaged in proteolytic cascades to activate other proteinases, which in turn, mediate ECM degradation [14]. In vivo inhibition of CB activity delays growth of primary tumors [15]. The results of the role of CB in colorectal cancer progression are contradictory. This is probably due to the different techniques employed to assess its involvement in the disease process. Campo et al. [4] showed that expression of CB in human colorectal carcinomas correlates with tumor progression and shortened patient survival. On the other hand, CB mRNA levels and activity were determined in homogenates of colorectal carcinomas by Murnane et al. and Iacobuzio-Donahue et al. [16], [17] who found the highest levels of mRNA and activity in early stages of tumor progression. Recently, Hazen et al. [5] found an inverse relationship between differentiation grade and CB activity.

CD is an aspartyl proteinase present within the lysosome of most mammalian cells and functions in the normal degradation of intracellular and endocytosed proteins. It is initially synthesized as a 52 kDa precursor protein. This form is enzymatically inactive and, following a proteolytic process, is cleaved into an active two-chain form. In breast cancer, the 52 kDa proenzyme is abnormally hypersecreted and this overexpression is believed to increase the metastatic potential of the tumor by the digestion of extracellular matrix [18]. High level of CD is strongly associated with poor prognosis in patients with primary breast cancer [19], [20], [21]. In colon cancer the expression of CD has been evaluated by several methods [6], [22], [23], [24].

Colon carcinoma is a frequent neoplasm in which, local extension and metastases are determinants of prognosis of the disease and are most likely regulated, among other factors, by increased proteolytic activity of tumor cells. Although the expression of each cathepsin in colon cancer has been examined by several methods, the concomitant expression of both cathepsins has not been examined until now, at least using immunolocalization and ELISA methods at the same time. Our aim was to analyse the expression of CB and CD in a series of primary human colon carcinomas and to compare this expression to that of adenomas and normal colon mucosa in order to establish their possible concomitant presence and its putative role in colon cancer progression.