Assessment of protease activity in endothelial cells and its role in tumour angiogenesis and spread

Thakur, Sapna (2006) Assessment of protease activity in endothelial cells and its role in tumour angiogenesis and spread. (PhD thesis), Kingston University, .


Angiogenesis is essential for the growth of a tumour, as it provides tumour cells with nutrients and oxygen for their survival. As the tumour expands, neovascularisation is facilitated by the release of enzymes called proteases, which degrade extracellular matrix and facilitate the metastatic spread of cancer. TNF[alpha] and IL-1[beta] are potent cytokines that share the ability to stimulate angiogenesis, hence their possible significance in metastasis has been a focus of intense research. TNF[alpha] and IL-1[beta] have been shown to regulate the activity of proteases such as MMP's and serine protease. The aim of this study was to investigate the effect of TNF[alpha] and IL-1[beta] on cathepsin B and DPP IV activity and their protein levels in HRT 18, HT 29 cells and HUVEC's. Further experiments were conducted to assess the viability of the cells upon treatment with the cytokines. In addition the potency of inhibitors Mu-Phe-Hph-FMK, for cathepsin B enzyme, and Gly-Pro-Gly-Gly, for DPP IV enzyme were assayed in the three cell lines studied. Addition of TNF[alpha] and IL-1[beta] resulted in the reduction of intracellular cathepsin B and DPP IV activity and an increase in its extracellular activity in HRT 18 and HT 29 cells, suggesting that the cytokines induced the release of the enzymes or may have inactivated intracellular enzyme while activating the latent extracellular enzyme. However, in HUVECs, both the cytokines led to an increase in intracellular as well as extracellular cathepsin B activity, possibly by activating the latent form of enzyme present within and outside the cell. With respect to DPP IV, there was an increased intracellular and extracellular activity with TNF[alpha], but with IL-1[beta], an increase in intracellular activity and a decrease in extracellular activity were observed, suggesting involvement of a different mechanism for the exopeptidase enzyme in HUVECs. The two Cytokines had a cytotoxic/cytostatic effect on all three cell lines, with prominent reduction in cell viability of HUVECs. In conclusion the varied response of intracellular and extracellular activity and protein levels of cathepsin B and DPP IV, in different cell lines suggests that TNF[alpha] and IL-1[beta] may act as important modulators of proteases in the process of angiogenesis in cancer and normal endothelial cells. Thus understanding the pleiotropic nature of these cytokines will further broaden the knowledge of involvement of these cytokines in cancer progression/cancer regression.

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