The roles of proteolytic enzymes in the development of tumour-induced bone disease in breast and prostate cancer.

Tumour-induced bone disease is a common clinical feature of advanced breast and prostate cancer and is associated with considerable morbidity for the affected patients. Our understanding of the molecular mechanisms underlying the development of bone metastases is incomplete, but proteolytic enzymes are implicated in a number of processes involved in both bone metastasis and in normal bone turnover, including matrix degradation, cell migration, angiogenesis, tumour promotion and growth factor activation. Malignant as well as non-malignant cells in the primary and secondary sites express these enzymes, the activity of which may be regulated by soluble factors, cell- or matrix-associated components, as well as a number of cell signalling pathways. A number of secreted and cell surface-associated proteolytic enzymes are implicated in tumour-induced bone disease, including the matrix metalloproteinases, lysosomal cysteine proteinases and plasminogen activators. This review will introduce the role of proteolytic enzymes in normal bone turnover and give an overview of the studies in which their involvement and regulation in the development of bone metastases in breast and prostate cancer has been described. The results from trials involving protease inhibitors in clinical development will also be briefly discussed.

A potential role for TGFbeta in the regulation of uveal melanoma adhesive interactions with the hepatic endothelium.

PURPOSE: TGFbeta has been shown to have a regulatory effect on uveal melanoma invasion, but it is not known which processes are specifically influenced. The purpose of this study was to analyze the effect of TGFbeta stimulation on the adhesive interactions of uveal melanomas with the extracellular matrix (ECM) and endothelium and, in addition, its effect on the secretion of collagenases. METHODS: Invasive and a noninvasive uveal melanoma cell lines, supported by short-term primary uveal melanoma cultures, were used to assess the effect of TGFbeta on ECM and endothelial adhesion and degradation of the ECM. Changes in cell adhesion molecule expression were assessed by flow cytometry, and conditioned media were analyzed by gelatin zymography. Assays of adhesion to ECM substrates and endothelial cells were also performed. RESULTS: Treatment with TGFbeta increased low basal levels of adhesion molecule and latent MMP-2 expression, as well as adhesion to hepatic endothelial cells by the noninvasive cell line. Conversely, TGFbeta reduced adhesion to laminin and a laminin-binding integrin by invasive cells but had no effect on their adhesion to the endothelium. CONCLUSIONS: In this preliminary study, TGFbeta was found to upregulate levels of MMP-2, reduce adhesion to laminin, and downregulate expression of laminin-binding integrins. Specifically, TGFbeta was found to increase adhesion of noninvasive uveal melanoma cells to the hepatic, but not the dermal, endothelium and may therefore contribute to the preferential targeting of the liver by uveal melanomas.

Combined effects of zoledronic acid and doxorubicin on breast cancer cell invasion in vitro.

The bisphosphonate zoledronic acid and the cytotoxic drug doxorubicin induce synergistic levels of apoptosis in breast cancer cells. As zoledronic acid and doxorubicin have been shown to reduce cell invasion and migration, we have investigated if these drugs also act synergistically on breast cancer invasion in vitro. MCF7 cells were treated with 0.05 microM doxorubicin/4 h followed by 1 or 10 microM zoledronic acid/24 h (or the reverse sequence). To study invasion, MCF7 cells were either grown on Transwell membranes coated with Matrigel or in a 24-well plate. Cells were treated sequentially using the above drug combinations, prior to starting the invasion assays for 48 h. Cell growth and death were also assessed under the same conditions. We found that invasion of MCF7 cells treated with zoledronic acid and doxorubicin was significantly reduced when compared with control, but the effect was dependent on drug sequence. At 1 microM, zoledronic acid significantly reduced invasion only if cells were pre-treated with doxorubicin, but cell growth was unaffected. For 10 microM zoledronic acid, invasion was reduced when administered before or after the doxorubicin, but this dose of zoledronic acid caused a significant reduction in MCF7 growth. Apoptosis was not induced by any of the drug doses and combinations. We conclude that pre-treatment with 0.05 microM doxorubicin followed by 1 microM zoledronic acid reduces invasion when cells were grown on Matrigel. For 10 microM zoledronic acid, pre- or post-doxorubicin also reduces invasion, but for this combination inhibition of cell growth may contribute to the reduction in invasion observed.

Preclinical evidence for the effect of bisphosphonates and cytotoxic drugs on tumor cell invasion.

Bisphosphonates (BPs) are stable pyrophosphate analogs currently used in the treatment of patients with metastatic bone disease, known to affect bone resorption by reducing osteoclast activity. Use of these drugs in adjuvant therapy is currently under investigation following reports of an effect of BPs on tumor cell apoptosis in preclinical models. Recent evidence has suggested that BPs might also affect tumor cell invasion in vitro, and the component processes of adhesion, migration and degradation, through mechanisms including inhibition of prenylation of intracellular small GTPases such as Ras and Rho. The effects potentially may be enhanced through co-administration with chemotherapy agents, as both synergistic and additive effects have been described in vitro. This review discusses the preclinical evidence for the potential use of BPs and cytotoxic drugs for inhibiting tumor cell invasion, a key process in cancer progression.

Stimulation and inhibition of uveal melanoma invasion by HGF, GRO, IL-1alpha and TGF-beta.

PURPOSE: To investigate potential factors involved in uveal melanoma migration and invasion in vitro. METHODS: Using a microchemotaxis chamber, the effects were studied of a range of stimulators and inhibitors on a series of 10 primary uveal melanomas and 2 uveal melanoma cell lines, by assessing invasion through an 8- micro m pore membrane, precoated with an extracellular matrix solution. In addition, invasion in response to the effect of cells and conditioned media derived from the liver and other tissues was studied for one uveal melanoma culture, by using double-chambered wells, and invasion was assessed through an 8- micro m pore membrane, precoated with synthetic extracellular matrix. In all instances, invading cells were counted under x400 magnification on the lower surface of the membrane. Levels of invasion were correlated with histopathologic markers of prognosis. RESULTS: Conditioned media and cells derived from other tissues, including the liver, increased cellular invasion of the uveal melanoma cell line studied. For specific regulators, maximum stimulation of invasion was induced by hepatic growth factor (HGF), growth-related oncogene (GRO), and macrophage inflammatory protein (MIP)-1beta, whereas significant inhibition was induced by IL-1alpha, TGF-beta1, and TGF-beta2. CONCLUSIONS: The primary site of metastasis in patients with uveal melanoma is the liver. For the degree of site specificity commonly seen, regulators involved in the process may be expressed at the secondary sites, promoting adhesion, migration, invasion, and proliferation of tumor cells. HGF, GRO, MIP-1beta, IL-1alpha, TGF-beta1, and TGF-beta2 may play a significant role in regulating invasion of uveal melanoma cells.

An in vitro assay to assess uveal melanoma invasion across endothelial and basement membrane barriers.

PURPOSE: To develop a modified in vitro invasion assay to assess uveal melanoma invasion across endothelial and basement membrane barriers. METHODS: Using permeable cell culture supports, endothelial cells were grown to confluence on an 8-microM pore polycarbonate membrane precoated with an artificial basement membrane. Primary uveal melanomas were grown as short-term cultures at 37 degrees C and 5% CO2 and invaded through the endothelial cell layer and basement membrane. Invading cells were counted under x400 magnification on the lower surface of the membrane. Levels of invasion were correlated with histopathologic markers of prognosis. The relative invasion of individual tumors was established by comparison of invasion through both endothelial and basement membrane barriers with invasion through basement membrane components alone. RESULTS: A series of 13 primary tumors were studied using the modified invasion assay. Tumors varied in their propensity to permeate both barriers. In all cases the endothelial cell layer reduced invasion, but the effect varied between tumors. CONCLUSIONS: Some tumors were more adept at overcoming the additional endothelial cell layer, whereas invasion of others was severely inhibited. Tumor invasion through the transendothelial model was found to correlate more closely with clinical characteristics associated with invasion, than was invasion through basement membrane components alone. The transendothelial model may represent a more realistic model for the in vitro study of invasion of uveal melanoma cells, providing a useful in vitro system for the investigation of cellular interactions during the invasion process.