EXPERIMENTAL RADIOTHERAPY--CARCINOGENESIS, AND PROTECTOR

虽然本研究的最终目标仍然是表征化学预防策略，以减少在治疗可能可治愈的肿瘤疾病过程中电离辐射对正常组织的遗传毒性损伤，但本申请的重点是研究硫醇对自发转移发展过程的抑制作用。 本研究将利用能够在C3 H小鼠中生长的SA-NH肉瘤作为自发转移形成的模型。 SA-NH细胞系也可用于在体外条件下生长。 选择用于研究的硫醇是氨磷汀、N-乙酰半胱氨酸（NAC）和卡托普利，因为每一种目前都在临床上使用，并且已经观察到每一种对啮齿动物肿瘤模型中的转移发展具有抑制作用。 预计如果发现这些硫醇中的任何一种或全部有效抑制小鼠中的转移形成，则它们作为抗转移剂的用途可以迅速转化为癌症治疗的临床方案。 本研究将仅关注可能影响转移过程中某些充分表征步骤的硫醇相关性质。 将检验三个假设。 首先，因为硫醇是巯基供体，所以它们可以刺激来自纤溶酶原的血管生成抑制剂血管生成抑制素的细胞内产生。 第二，凭借其螯合锌的能力，硫醇可以抑制锌与需要锌的基质金属蛋白酶（MMP）的结合。 以这种方式，肿瘤细胞侵入正常组织所需的MMP活性被抑制。 第三，巯基可以增强肿瘤细胞中MnSOD的基因表达和酶活性，这反过来又导致转移表型减少。 使用的技术包括评估MnSOD基因表达的北方印迹分析;评估血管抑素产生的Western印迹分析;测量MMP活性的酶谱分析;涉及评估手术切除原发肿瘤后形成的肺转移的自发转移测定;以及一种人工转移测定，其涉及评估在注射在100 μ L下处理的活肿瘤细胞后形成的肺肿瘤。体外条件下，然后注射到受体动物的侧尾静脉中。

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.