Anti-tumor activities of chondroitinase AC and chondroitinase B: inhibition of angiogenesis, proliferation and invasion
Introduction
The growth of tumors requires tumor cell proliferation as well as the formation of new blood vessels. Metastasis of the primary tumor and growth of secondary tumors involve a complex sequence of events, which has been referred to as the “metastatic cascade” (Sneath and Mangham, 1998). While many growth factors, enzymes and extracellular matrix components are involved in these events, works by other investigators have suggested that chondroitin sulfate proteoglycans may play a key role in tumor growth and metastasis.
In examining the functions of chondroitin sulfates in metastasis, two different approaches have been used: inhibiting the formation of chondroitin sulfate proteoglycans with β-d-xyloside (ρ-nitrophenyl-β-d-xylopyranoside), and enzymatically removing chondroitin sulfates from cells with chondroitinase ABC. Both methods have similar results and effectively decrease tumor cell invasion and endothelial migration and adhesion Henke et al., 1996, Faassen et al., 1992, Faassen et al., 1993.
Taken together, studies utilizing inhibitors or enzymes, which degrade chondroitin sulfates, indicate that chondroitin sulfates play important roles in the processes of tumor growth and metastasis Iida et al., 1996, Sneath and Mangham, 1998. However, it is not clear from these prior studies which chondroitin sulfate proteoglycans were involved. Chondroitin sulfate proteoglycans exist in three major forms: chondroitin sulfate A, chondroitin sulfate B (dermatan sulfate) and chondroitin sulfate C. Recent work has demonstrated that the different forms of chondroitin sulfates have different functions. Dermatan sulfate is important in controlling fibroblast proliferation, and chondroitin sulfates A and C regulate integrin-mediated cell adhesion Denholm et al., 2000, Moyano et al., 1999.
The purpose of the present study was to determine the relative importance of the different forms of chondroitin sulfate in cellular activities related to metastasis. To accomplish this, two specific glycosaminoglycan lyases were utilized: chondroitinase AC (substrates are chondroitin sulfates A and C) and chondroitinase B (substrate is chondroitin sulfate B or dermatan sulfate). The effects of these two glycosaminoglycan lyases on several tumor and endothelial cell functions were compared, and the mechanisms underlying the inhibition of these activities were examined.
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Materials
Human melanoma (SK-MEL-2) and calf pulmonary artery endothelial cells were from ATCC, Manassas, VA. Dulbecco's minimal essential medium, Fischer's medium, phosphate buffered saline (PBS) and fetal bovine serum were from Gibco, Grand Island, NY. Dermatan sulfate, chondroitin sulfates A and C, and heparan sulfate were purchased form Celsus Laboratories, Cincinnati, OH. Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were purchased from Peprotech, Rocky Hill,
Angiogenesis and endothelial cell proliferation
Capillary endothelial cells were treated with 0.1–10 IU/ml of chondroitinase AC or chondroitinase B, and angiogenesis, as measured by the formation of capillary-like structures, was examined. Endothelial cells treated with chondroitinase AC contained fewer capillary-like structures than untreated controls did Fig. 1, Fig. 2. Angiogenesis was inhibited by 46% and 72%, following treatment with 1.0 and 10 IU/ml of chondroitinase AC, respectively. In comparison, β-d-xyloside, which blocks the
Discussion
Comparison of the relative effects of chondroitinase AC with those of chondroitinase B, indicated that both chondroitin and dermatan sulfates are involved in regulating a number of cellular activities linked to metastasis. At the start of these experiments, it had been thought that the use of the two specific chondroitinase enzymes would reveal a clearly predominant role for either chondroitin or dermatan sulfates in each of these various activities. It would appear that chondroitin sulfates A
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