Inhibition of Bcl-2 and Head & Neck Tumor Angiogenesis

描述（由申请人提供）：肿瘤细胞依赖血管生成生存、增殖和转移。头颈部肿瘤血管化程度高，侵袭性强，常发生局部转移。因此，破坏肿瘤血管网络可能有利于口腔癌患者的治疗。已知血管内皮生长因子（vascular endothelial growth factor， VEGF）是肿瘤血管生成的强诱导剂，VEGF通过上调抗凋亡的Bcl-2的表达来提高内皮细胞的存活。用抗体（如阿瓦斯汀）或其受体之一的抑制剂（如PTK787）抑制VEGF信号传导可导致肿瘤血管的选择性消融和肿瘤生长的抑制。这些结果表明，VEGF/Bcl-2通路对维持肿瘤血管系统至关重要。基于结构的30个数据库搜索导致开发了一种新的Bcl-2小分子抑制剂（TW-37）。TW-37体外诱导肿瘤细胞凋亡，体内抑制前列腺肿瘤（PC-3）的生长。最近的实验表明，TW-37也能诱导体外新生血管内皮细胞凋亡，但不能诱导人真皮成纤维细胞或正常前列腺细胞凋亡。然而，TW-37阻断Bcl-2的功能是否足以破坏肿瘤血管并抑制头颈部肿瘤生长尚不清楚。本研究的长期目标是了解治疗性抑制Bcl-2对肿瘤血管生成和肿瘤生长的影响。本研究的目的是评估TW-37对血管生成的影响，以及对口腔肿瘤微血管密度和生长的影响。我们计划通过研究tw -37诱导内皮细胞凋亡过程的机制及其对体外毛细血管发芽的影响来实现这些目标。利用SCID小鼠血管生成模型评价TW-37对免疫缺陷小鼠血管生成的影响。此外，我们将在SCID小鼠中植入经荧光素酶转导的口腔肿瘤细胞，通过体内生物发光分析TW-37对肿瘤生长、侵袭和转移的影响。内皮细胞凋亡对TW-37抗肿瘤作用的影响将通过在SCID小鼠中产生血管化的内皮细胞，这些内皮细胞稳定地以显性负Caspase-9（抵抗TW-37诱导的凋亡）转导，或在Bax小鼠中植入小鼠口腔肿瘤细胞来评估。本研究成果将进一步加深我们对Bcl-2在肿瘤新生血管内皮细胞中的作用的认识，并可能为Bcl-2作为分子靶点进一步开发治疗高度血管化肿瘤的药物提供支持。这项工作可能表明，Bcl-2的小分子抑制剂代表了一类新的药物，可诱导肿瘤细胞凋亡和抗血管生成，这是两种不同的，可能是协同的抗肿瘤作用。

DESCRIPTION (provided by applicant): Tumor cells depend on angiogenesis to survive, proliferate, and metastasize. Head & neck tumors are highly vascularized, invasive, and frequently develop local metastasis. Therefore, disruption of the tumor vascular network might be beneficial for treatment of patients with oral cancer. It is known that vascular endothelial growth factor (VEGF) is a strong inducer of tumor angiogenesis, and that VEGF enhances endothelial cell survival by upregulating the expression of the anti-apoptotic Bcl-2. Inhibition of VEGF signaling with an antibody (e.g. Avastin), or with an inhibitor of one its receptors (e.g. PTK787) results in selective ablation of tumor blood vessels and inhibition of tumor growth. These results demonstrate that the VEGF/Bcl-2 pathway is critical for the maintenance of tumor vasculature. Structure based 30-database searching led to the development of a novel small molecule inhibitor of Bcl-2 (TW-37). TW-37 induces apoptosis of tumor cells in vitro, and, inhibits growth of prostate tumors (PC-3) in vivo. Recent experiments demonstrated that TW-37 also induces apoptosis of neovascular endothelial cells in vitro, but not human dermal fibroblasts or normal prostate cells. However, it is not known if blockade of Bcl-2's function with TW-37 is sufficient to disrupt tumor blood vessels and to inhibit head & neck tumor growth. The broad long-term goals of this research are to understand the effect of therapeutic inhibition of Bcl-2 on tumor angiogenesis and tumor growth. The objectives of this application are to evaluate the effect of TW-37 on angiogenesis, and to evaluate its effect on the microvessel density and growth of oral tumors. We plan to accomplish these objectives by studying the mechanisms involved in the process of TW-37-induced endothelial cell apoptosis, and its effects on capillary sprouting in vitro. The SCID Mouse Model of Human Angiogenesis will be used to evaluate the effect of TW-37 in human blood vessels developed in immunodeficient mice. In addition, we will implant oral tumor cells transduced with Luciferase in SCID mice to analyze the effect of TW-37 on tumor growth, invasion, and metastasis by in vivo bioluminescence. The impact of endothelial cell apoptosis on TW-37's anti-tumor effect will be evaluated by generating tumors vascularized with endothelial cells stably transduced with dominant negative Caspase-9 (resistant to TW-37-induced apoptosis) in SCID mice, or by implanting murine oral tumor cells in Bax mice. The knowledge generated here will enhance our understanding about the role of Bcl-2 in neovascular endothelial cells of tumors, and may provide support for Bcl-2 as a molecular target for further development of drugs for treatment of highly vascularized tumors. This work may demonstrate that small molecule inhibitors of Bcl-2 represent a novel class of drugs that induce tumor cell apoptosis and are anti-angiogenic, two distinct and perhaps synergistic anti-tumor effects.