Dietary restriction of two amino acids, tyrosine (Tyr) and phenylalanine (Phe), suppresses the malignant phenotype of B16-BL6 (BL6) murine melanoma. Our data indicate that Tyr and Phe restriction inhibits the ability of tumor cells to establish metastatic tumor foci. This proposal focuses on the mechanism responsible for this Tyr and Phe modulation and will test the hypothesis that it is multifactorial, encompassing decreased invasiveness, angiogenesis, and growth response.
The specific aims are to 1) examine invasion of BL6 murine melanoma and Tyr- and Phe-modulated BL6 variants through reconstituted basement membrane and selected matrix components and the response to chemotaxis, 2) examine differential proteolytic enzyme and protease inhibitor activities in media conditioned by BL6 and the modulated variants, 3) examine soluble angiogenic factors released into conditioned media by these tumor cells, and 4) determine if phenotypic modulation by Tyr and Phe restriction is associated with change in cell cycle characteristics, growth factors, or tumor suppressor gene expression. To confirm preliminary results indicating that Tyr and Phe limitation alters invasive characteristics of tumor cells, experiments are designed to specifically examine invasion of cells through substrates of extracellular matrix extracted from lung, Matrigel, collagen types I and IV, laminin, fibronectin, and elastin. To analyze differential release by our Tyr- and Phe-modulated variants of soluble proteolytic enzymes, conditioned media from cell cultures will be tested for ability to degrade collagen types I and IV, laminin, and elastin, in the presence and absence of protease inhibitors. These assays will be extended to determine release and activity in conditioned media of other soluble proteinases which are known to be involved in invasion, specifically cathepsins B and L, plasminogen activator, and chymotrypsin. The possibility of increased production and release of proteinase inhibitors will also be studied. Production and release of angiogenic factors such as fibroblast growth factor, transforming growth factor beta (TGF-beta), and angiogenin by BL6 and Tyr- and Phe-modulated BL6 variants will also be determined. Altered growth patterns and/or growth factor and tumor suppressor gene expression may contribute to the antimetastatic effects of Tyr and Phe restriction. We will investigate whether changes occur in the cell cycle and in expression of TGF-beta and the tumor suppressor genes, nm23 and TIMP (tissue inhibitors of metalloproteinases).
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