Mechanisms of radiosensitization by 2-methoxyestradiol in prostate cancer models

说明书(申请人提供)：对肿瘤细胞进行放射增敏而不会产生不可接受的正常组织毒性的辅助剂在前列腺癌的放射治疗中具有潜在的应用价值。自然产生的雌激素代谢物2-甲氧基雌二醇(2-ME)是提高前列腺癌放射治疗效果的一个有前景的候选药物。2-ME干扰微管功能，具有抗肿瘤和抗血管生成作用。该方案的主要目的是利用体外和体内前列腺癌模型评价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。其具体目的是：1)研究2-ME在体内外对雄激素敏感和雄激素不敏感的前列腺癌模型的放射增敏作用；2)研究2-ME对人内皮细胞和前列腺癌移植瘤放射增敏机制中的抗血管生成作用；3)确定MAPK、CREB和其他下游效应分子在放射与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发展到雄激素非依赖状态的趋势的限制，因此将雄激素依赖(LNCaP)和雄激素非依赖(PC3，C4-2)细胞株的结果进行比较。克隆形成、细胞死亡、DNA合成和其他分析将与裸鼠肿瘤异种移植(皮下和原位)研究一起进行。此外，还将对小鼠正常的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来检测微管与MAPK之间的相互作用，以测试2-ME是否破坏已知的MAPK与2-微管蛋白之间的结合。通过评估血管内皮细胞增殖(体外)和微血管密度(体内)来衡量抗血管生成效应的参与程度。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。将使用传统技术(报告基因分析、EMSA和Western blotting)以及这些效应器的遗传调控来研究MAPK、CREB、HIF-11和VEGF的作用。由于2-ME作为一种单一药物已经显示出抗癌效果，并且在前列腺癌的II期临床试验中耐受性良好，这些临床前研究将为2-ME作为前列腺癌放射治疗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会支持进一步的放射加2-ME在人类前列腺癌患者中的临床试验。公共卫生相关性：2-甲氧基雌二醇(2-ME)是一种自然产生的雌激素衍生物，根据我们最近的动物模型初步数据，它是一种很有前途的前列腺癌放射治疗候选药物。由于2-ME作为一种单一药物显示出有限的抗癌活性，并且在人类前列腺癌的II期临床试验中耐受性良好，这些临床前研究将提供有关2-ME与前列腺癌放射治疗相结合的可行性的重要信息。这些研究的有希望的结果可能会支持进一步的放射加2-ME在人类前列腺癌患者中的临床试验。

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.