Site-directed Chemotherapy for Breast Cancer using Novel Angiogenesis Inhibitor

描述（由申请人提供）：最近有证据表明甲状腺激素也可能在癌变中起作用，该领域的开创性研究表明，甲状腺激素如3,5,3'-三碘- l -甲状腺原氨酸（T3）和l -甲状腺素（T4）可以刺激癌细胞增殖，从而发挥生长因子的作用。我们已经在人皮肤微血管内皮细胞实验和鸡毛囊尿囊膜（CAM）和血管生成的Matrigel模型中证明了T3和T4都具有强大的促血管生成作用。综上所述，这些发现表明甲状腺激素和/或相关信号通路的拮抗剂可能是治疗癌症的有力靶点。我们实验室最近的研究表明，四碘甲状腺乙酸（tetraiodothyroacetic acid, tetrac）是一种脱氨甲状腺激素类似物，能够阻止T4在细胞膜上与1vB3结合，对细胞增殖和血管生成具有深远的抑制作用。发现四聚氰胺抑制癌细胞增殖促使我们研究它是否也可能刺激细胞对压力的反应，从而逆转耐药性的发展。我们的研究结果表明，tetrac对耐药性有影响，并暗示至少有两种途径在细胞对化疗的反应中起关键作用。在这个探索性的应用中，我们将研究四氯甲烷作为治疗癌症的治疗工具的潜在用途，特别是它在抑制化疗耐药性发展方面的可能作用。目的是确定抗血管生成和抗增殖作用是否在质膜而不是在基因组水平启动。为此，我们制备了PLGA纳米颗粒，其表面与四氯乙烯共价连接。初步数据表明，在与癌细胞孵育后，颗粒优先定位于质膜。假设：利用四氯乙烯共价连接纳米颗粒（TclNPs）将四氯乙烯优先靶向质膜，将为研究这种激素拮抗剂的质膜受体介导的作用提供一种独特的方法。在本研究的背景下，我们将研究其抗血管生成和抗增殖的作用。由于全身使用甲状腺药物可能对多种生理途径产生潜在影响，因此选择性地将Tetrac靶向质膜上的受体可能足以显著抑制侵袭性肿瘤的增殖，而且副作用更少。使用生物可降解的TclNPs装载化疗药物紫杉醇，也可用于在肿瘤部位控制药物的释放，因此，有望产生更好的抗肿瘤活性，降低紫杉醇毒性。具体目标：1；实验将评估TclNPs在体外MCF7细胞和内皮细胞中的功能活性。荧光显微镜的初步数据显示，负载荧光素的TclNPs优先定位于乳腺癌MCF7细胞的质膜。进一步的研究将确定TclNPs的结合特性，以及它们对增殖和细胞迁移的影响。TclNPs的作用也将在与肿瘤细胞耐药发展或逆转相关的途径上进行测试。将TclNPs的效果与这些体系中游离四氢碳的效果进行比较。2. 在对裸鼠进行纳米靶向治疗之前，将在鸡绒毛膜尿囊膜（CAM）肿瘤植入模型的卵子中进行初步实验，以确定TclNPs的最佳配方。CAM模型允许对卵子的生物活性进行预筛选，同时限制使用更有知觉和昂贵的小鼠物种。在CAM模型中显示出最佳抗肿瘤和抗血管生成活性的纳米颗粒配方将在实验方法中描述的原位乳腺癌模型中进行测试。雌性胸腺小鼠将耐药MCF7人类乳腺癌细胞原位植入第四乳腺。我们将评估靶向纳米颗粒TclNPs在减少肿瘤生长和肿瘤血管生成方面的有效性。我们将评估这些配方在限制与全身给药相关的潜在毒性方面的功效，并确定TclNPs与紫杉醇或不含紫杉醇是否能限制耐药性的发展，正如我们初步的体外研究所表明的那样。在这些研究中评估的新型纳米颗粒系统结合了靶向和抗肿瘤活性的特性，并且由于Tetrac，紫杉醇和PLGA纳米颗粒系统都被批准用于患者，因此在可预见的未来可能会有潜在的临床应用。

DESCRIPTION (provided by applicant): Recent evidence has been provided that thyroid hormones may also play a role in carcinogenesis, and pioneering investigations in this area have demonstrated that the thyroid hormones such as 3,5,3'-triiodo-L-thyronine (T3) and L-Thyroxine (T4) can stimulate cancer cell proliferation and thus act as growth factors. We have shown that both T3 and T4 exert potent pro-angiogenic effects in the human dermal microvascular endothelial cell assays and in the chick chorioallantoic membrane (CAM) and Matrigel model of angiogenesis. Taken together, these findings suggest that antagonist for thyroid hormones and/or related signaling pathways may represent compelling targets to treat cancer. Recent studies from our laboratory have shown that tetraiodothyroacetic acid (tetrac), a deaminated thyroid hormone analog, capable of preventing the binding of T4 to 1vB3 at the cell membrane exerted profound inhibitory effects on cellular proliferation and angiogenesis. The finding that tetrac inhibits cancer cell proliferation prompted us to investigate whether it may also stimulate cellular response to stress and thus reverse the development of drug resistance. Our results demonstrate that tetrac impacts on drug resistance and implicate at least two pathways that play key roles in cellular response to chemotherapy. In this Exploratory Application we will investigate the potential use of tetrac as a therapeutic tool for the treatment of cancer, particularly with regards to its possible role in suppressing the development of resistance to chemotherapy. The goal is to determine whether the anti-angiogenic and anti-proliferative actions of tetrac are initiated at the plasma membrane and not at the genomic level. To this end, we have generated PLGA nanoparticles covalently - linked to Tetrac on their surfaces. Preliminary data have shown that upon incubation with cancer cells, particles are preferentially localized to the plasma membrane. Hypothesis: Preferential targeting of Tetrac to the plasma membrane using Tetrac covalently-linked to nanoparticles (TclNPs) will provide a unique approach to study plasma membrane receptor-mediated actions of this hormone antagonist. In the context of the proposed study, we will investigate its anti- angiogenic and anti-proliferative actions. Because thyroid agents used systemically could potentially impact on multiple physiological pathways, the selective targeting of Tetrac to receptor(s) at the plasma membrane may be sufficient to significantly inhibit proliferation of aggressive tumors with fewer side effects. The use of biodegradable TclNPs loaded with a chemotherapeutic agent Paclitaxel can also be used for a controlled release of this drug at the tumor site and thus, is expected to result in better anti-tumor activity with reduced Paclitaxel toxicity. Specific Aims: 1. Experiments will be performed to evaluate the functional activities of TclNPs in vitro in MCF7 cells and in endothelial cells. Preliminary data using fluorescence microscopy showed that fluorescein-loaded TclNPs localize preferentially at the plasma membrane of the breast cancer MCF7 cells. Further studies will be performed to determine binding characteristics of TclNPs, and their effects on proliferation and cell migration. The effects of TclNPs also will be tested on pathways associated with the development or reversal of drug resistance in tumor cells. The effect of TclNPs will be compared to those of free Tetrac in these systems. 2. Pilot experiments to determine the optimum formulations of TclNPs will be performed in ova in the chick chorioallantoic membrane (CAM) tumor implant model of tumor growth and angiogenesis prior to performance of nanoparticle-targeted treatments of nude mice. The CAM model permits in ova pre-screening for bioactivity while limiting the use of more sentient and costly murine species. Nanoparticle formulations that show optimum anti-tumor and anti-angiogenesis activity in the CAM model will be tested in the orthotopic breast cancer model described in Experimental Methods. 3. Female athymic mice will have drug-resistant MCF7 human breast cancer cells implanted orthotopically into the fourth mammary gland. We will evaluate the effectiveness of targeted nanoparticles TclNPs, in reducing tumor growth and tumor angiogenesis. We will evaluate the efficacy of these formulations in limiting potential toxicities associated with systemic Tetrac administration and determine whether TclNPs, with or without Paclitaxel act to limit the development of drug resistance, as suggested by our preliminary in vitro studies. The novel nanoparticle system to be evaluated in these studies combines the properties of targeting and anti-tumor activities, and because Tetrac, Paclitaxel and PLGA nanoparticle systems are all approved for use in patients, could find potential clinical application in the foreseeable future. PUBLIC HEALTH RELEVANCE: Cancer cells have the unique ability to develop resistance to chemotherapeutic drugs, and so research on ways to reverse this phenomenon would have significant value in the treatment of cancer patients. This project will use a combination of two drugs, one of which impairs the cancer cell's ability to develop drug resistance, in a mouse model of breast cancer. A novel technology, the use of nanoparticles to encapsulate the test drugs, and direct them to tumors will be tested to determine whether these nanoparticles can improve the delivery of drugs and minimize the associated toxicities.