Mechanisms of radiosensitization by 2-methoxyestradiol in prostate cancer models

描述（由申请人提供）：使肿瘤细胞放射增敏且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可能有用。天然存在的雌激素代谢物 2-甲氧基雌二醇 (2-ME) 是增强前列腺肿瘤放射治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特性。该提案的主要目标是使用体外和体内前列腺癌模型评估 2-ME 的放射增敏特性，并研究 2-ME 与辐射之间相互作用的分子机制。具体目的是：1）研究2-ME是否可以在体外和体内使雄激素敏感和雄激素不敏感的前列腺癌模型放射增敏； 2) 研究2-ME抗血管生成在人内皮细胞和前列腺癌异种移植物放射增敏机制中的作用； 3) 确定 MAPK、CREB ​​和其他下游效应子在前列腺癌细胞和肿瘤中辐射与 2-ME 相互作用中的作用。由于目前对晚期前列腺癌的治疗受到疾病从雄激素依赖性状态进展到雄激素非依赖性状态的趋势的限制，因此将比较雄激素依赖性（LNCaP）和雄激素非依赖性（PC3、C4-2）细胞系的结果。克隆形成、细胞死亡、DNA 合成和其他测定将与裸鼠中的肿瘤异种移植（皮下和原位）研究一起进行。此外，将对小鼠正常盆腔组织进行组织学评估，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。将通过评估内皮细胞增殖（体外）和微血管密度（体内）来测量抗血管生成作用的参与。血管正常化和肿瘤氧合将被视为体内放射增敏的潜在机制。 MAPK、CREB、HIF-11 和 VEGF 的作用将使用传统技术（报告基因测定、EMSA 和蛋白质印迹）以及这些效应子的遗传调节进行研究。由于2-ME作为单一药物已显示出抗癌功效，并且在针对前列腺癌的II期临床试验中具有良好的耐受性，因此这些临床前研究将为2-ME作为前列腺癌放射治疗的辅助药物的可行性提供重要信息。这些研究的有希望的结果可能支持在人类前列腺癌患者中进行放射加 2-ME 的进一步临床试验。公共健康相关性：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，是增强前列腺肿瘤放射治疗的有希望的候选药物。由于 2-ME 作为单一药物显示出有限的抗癌活性，并且在人类前列腺癌 II 期临床试验中具有良好的耐受性，因此这些临床前研究将为 2-ME 与前列腺癌放射治疗联合的可行性提供重要信息。这些研究的有希望的结果可能支持在人类前列腺癌患者中进行放射加 2-ME 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.