Mechanisms of radiosensitization by 2-methoxyestradiol in prostate cancer models

描述（由申请人提供）：使肿瘤细胞放射敏感而没有不可接受的正常组织毒性的佐剂在前列腺癌的放射治疗中有潜在的用途。天然雌激素代谢物2-甲氧基雌二醇（2-ME）是增强前列腺肿瘤放疗的一个有希望的候选者。2-ME干扰微管功能，具有抗肿瘤和抗血管生成的特性。本研究的主要目的是通过体外和体内前列腺癌模型来评估2-ME的放射增敏特性，并研究2-ME与辐射相互作用的分子机制。具体目的是：1)研究2-ME对雄激素敏感型和雄激素不敏感型前列腺癌模型的放射增敏作用；2)研究抗血管生成参与2- me对人内皮细胞和前列腺癌异种移植物放射增敏的机制；3)确定MAPK、CREB等下游效应物在前列腺癌细胞和肿瘤中辐射与2-ME相互作用中的作用。由于目前晚期前列腺癌的治疗受到疾病从雄激素依赖状态进展到雄激素独立状态的趋势的限制，因此将比较雄激素依赖（LNCaP）和雄激素独立（PC3, C4-2）细胞系的结果。克隆性、细胞死亡、DNA合成和其他检测将与裸鼠肿瘤异种移植（皮下和原位）研究一起进行。此外，小鼠正常盆腔组织将进行组织学评估，以评估正常组织的影响。为了解决2-ME作用的初始事件，将通过免疫荧光显微镜和其他定量技术检查微管和MAPK之间的相互作用，以测试2-ME是否破坏MAPK和2-微管蛋白之间的已知结合。参与抗血管生成效应将通过评估内皮细胞增殖（体外）和微血管密度（体内）来测量。血管正常化和肿瘤氧合将被认为是体内放射致敏的潜在机制。MAPK、CREB、HIF-11和VEGF的作用将使用传统技术（报告基因测定、EMSA和Western blotting）以及这些效应物的遗传调节来研究。由于2-ME作为单一药物在前列腺癌的II期临床试验中显示出抗癌效果，并且耐受性良好，这些临床前研究将为2-ME作为前列腺癌放疗辅助剂的可行性提供重要信息。这些研究的令人鼓舞的结果可能支持放射加2-ME在人类前列腺癌患者中的进一步临床试验。公共卫生相关性：根据我们最近在动物模型中的初步数据，2-甲氧基雌二醇（2-ME）是一种天然存在的雌激素衍生物，是一种有希望增强前列腺肿瘤放疗的候选者。由于2-ME作为单一药物在人类前列腺癌的II期临床试验中显示出有限的抗癌活性，并且耐受性良好，这些临床前研究将提供关于2-ME与前列腺癌放疗联合可行性的重要信息。这些研究的令人鼓舞的结果可能支持放射加2-ME在人类前列腺癌患者中的进一步临床试验。

DESCRIPTION (provided by applicant): Adjuvant agents that radiosensitize tumor cells without unacceptable normal tissue toxicity are potentially useful in the radiation therapy of prostate cancer. A promising candidate for enhancement of prostate tumor radiotherapy is the naturally occurring estrogen metabolite, 2-methoxyestradiol (2-ME). 2-ME interferes with microtubule function and has both anti-tumor and anti-angiogenic properties. The broad objectives of this proposal are to evaluate the radiosensitizing properties of 2-ME using both in vitro and in vivo prostate cancer models, and to study the molecular mechanisms of interaction between 2-ME and radiation. The specific aims are: 1) to investigate whether 2-ME can radiosensitize androgen-sensitive and androgen-insensitive prostate cancer models in vitro and in vivo; 2) to study the involvement of anti-angiogenesis in the mechanism of radiosensitization by 2-ME in human endothelial cells and prostate cancer xenografts; and 3) to determine the roles of MAPK, CREB, and other downstream effectors in the interaction between radiation and 2-ME in prostate cancer cells and tumors. Since current therapy for advanced prostate cancer is limited by the tendency of the disease to progress from an androgen-dependent state to an androgen-independent state, results from both androgen-dependent (LNCaP) and androgen-independent (PC3, C4-2) cell lines will be compared. Clonogenic, cell death, DNA synthesis, and other assays will be performed along with tumor xenograft (subcutaneous and orthotopic) studies in nude mice. In addition, mouse normal pelvic tissue will be histologically evaluated to assess normal tissue effects. To address the initial events of 2-ME action, the interaction between microtubules and MAPK will be examined by immunofluorescence microscopy and other quantitative techniques to test whether 2-ME disrupts the known binding between MAPK and 2-tubulin. Involvement of anti-angiogenic effects will be measured by evaluating endothelial cell proliferation (in vitro) and microvessel density (in vivo). Vascular normalization and tumor oxygenation will be addressed as potential mechanisms of radiosensitization in vivo. The roles of MAPK, CREB, HIF-11, and VEGF will be studied using traditional techniques (reporter gene assays, EMSA, and Western blotting), along with genetic modulation of these effectors. Since 2-ME as a single agent has shown anti-cancer efficacy and was well-tolerated in Phase II clinical trials against prostate cancer, these pre-clinical studies will provide important information regarding the feasibility of 2-ME as an adjuvant agent in the radiotherapy of prostate cancer. Promising results from these studies may support further clinical trials of radiation plus 2-ME in human prostate cancer patients. PUBLIC HEALTH RELEVANCE: 2-methoxyestradiol (2-ME), a naturally occurring derivative of estrogen, is a promising candidate for enhancement of prostate tumor radiotherapy, based upon our recent preliminary data in animal models. Since 2-ME as a single agent has shown limited anti-cancer activity and was well-tolerated in Phase II clinical trials against prostate cancer in humans, these pre-clinical studies will provide important information regarding the feasibility of 2-ME in combination with the radiotherapy of prostate cancer. Promising results from these studies may support further clinical trials of radiation plus 2-ME in human prostate cancer patients.