The growing of solid tumors highly depends on angiogenesis. In this new vessel formation process the integrins, expressed on endothelial cells are important mediators. Inhibition of function of these integrins is currently one of the directions in cancer research. Moreover, integrins expressed on cancer cells are involved in migration of these cells during metastasis. In the proposed research plan, we will investigate ct 1131 integrin (VLA-1), which is recognized as a specific collagen IV receptor. We will focus our research on two inhibitors of VLA-1, obtustatin and viperisrastatin. Both of them belong to disintegrin family, and are low molecular mass inhibitors (4.2 kDa) of VLA-1. The activity of these disintegrins has been localized within their integrin-binding loop as an active sequence KTS. Obtustatin showed potent angiostatic activity in the chicken CAM model in vivo, and in tube EC formation assay in vitro. Moreover, obtustatin inhibited Lewis lung cancer development in syngeneic mouse model. The activity of KTS-disintegrins will be further tested on growing tumor induced by mouse melanoma B 16 and M-3 cell lines in the syngeneic mouse, and human melanoma cell lines, MV3, HS.939T, A-375, C32, and A2058 in nude mice. The participation VLA-1 in metastasis of these cell lines to the lung will be investigated in vivo using syngeneic and nude mice. To explain the mechanism of the angiostatic effect of KTS containing disintegrins, series of experiments will be performed to investigate their effect on microvascular EC. The preliminary data showed that obtustatin induced apoptosis in these cells and inhibited their proliferation. Based on the previous reports indicating involvement of VLA-1 in signal transduction pathway dependent on MAPK, the effect of KTS-disintegrins on activation of this pathway will be evaluated. Moreover, radial migration assay in collagen gel will be proposed. That in vitro assay imitates movement of EC during early stage of neovascularization after dissolution of the basement membrane. The effect of both disintegrins in EC motility in this model will be tested. Obtustatin and its naturally occurring analog viperisrastatin may be models for designing of synthetic or recombinant compounds, which may be useful in cancer therapy. In summary, the work with KTS containing disintegrins may lead to the better understanding of the involvement of VLA-1 in cancer progression and metastasis, as well as contribute to an understanding of the role of VLA-1 in angiogenesis.
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