While the ultimate goal of this investigation continues to be the characterization of chemopreventive strategies to reduce the genotoxic damage to normal tissues by ionizing radiation during the treatment of potentially curable neoplastic disease, the focus of this application is directed to the investigation of the inhibitory effects of thiols on the process of spontaneous metastasis development. This study will utilize the SA-NH sarcoma that is capable of being grown in C3H mice as a model of spontaneous metastasis formation. SA-NH cell lines are also available for growth under in vitro conditions. The thiols chosen for study are amifostine, N-acetylcysteine (NAC), and captopril because each is currently in clinical use and each has been observed to have an inhibitory effect on metastases development in rodent tumor models. It is anticipated that if any or all of these thiols are found effective in inhibiting metastases formation in mice, their use as anti-metastatic agents could rapidly be translated to clinical protocols for cancer treatment. This study will focus only on thiol related properties that can affect certain well characterized steps in the metastatic process. Three hypotheses will be tested. First, because thiols are sulfhydryl doners they can stimulate the intracellular production of angiostatin, an inhibitor of angiogenesis, from plasminogen. Second, by virtue of their ability to chelate zinc, thiols can inhibit zinc binding to the zinc requiring matrix metalloproteinases (MMPs). In this manner MMP activities required for tumor cell invasion into normal tissues are inhibited. And third, thiols can enhance gene expression and enzyme activity of MnSOD in tumor cells which in turn leads to a reduced metastatic phenotype. Techniques to be used include Northern blot analysis to assess MnSOD gene expression; Western blot analysis to assess angiostatin production; zymogram analysis to measure MMP activities; a spontaneous metastases assay involving the assessment of pulmonary metastases formed following the surgical removal of the primary tumor; and an artificial metastasis assay involving the assessment of pulmonary tumors formed following the injection of viable tumor cells treated under in vitro conditions and then injected into the lateral tail veins of recipient animals.
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