The State Key Laboratory of Pharmaceutical Biotechnology and School of Stomatology, Affiliated Stomatological Hospital, Nanjing University, Nanjing, P R China.
Mol Cell Proteomics. 2011 Jun;10(6):M111.009399. doi: 10.1074/mcp.M111.009399. Epub 2011 Apr 7.
Solid tumors often contain hypoxic and necrotic areas that can be targeted by attenuated Salmonella typhimurium VNP20009 (VNP). We sought to develop a hypoxia- inducible promoter system based on the tumor-specific delivered strain VNP to confine expression of therapeutic gene specifically or selectively within the tumor microenvironment. A hypoxia-inducible promoter - adhE promoter was screened from the hypoxia-regulated endogenous proteins of Salmonella through two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight/time-of-flight MS-based proteomics approaches. The efficiency and specificity of the selected adhE promoter were validated first in both bacteria and animal tumor models. The adhE promoter could specifically drive GFP gene expression under hypoxia, but not under normoxia. Furthermore, luciferase reporter expression controlled by the system was also confined to the tumors. Finally, we investigated the anticancer efficacy of VNP delivering human endostatin controlled by our adhE promoter system in both murine melanoma and Lewis lung carcinoma models. Our results demonstrated that by the dual effects of tumoricidal and anti-angiogenic activities, the recombinant Salmonella strain could generate enhanced antitumor effects compared with those of unarmed VNP treatment or untreated control. The recombinant VNP could retard tumor growth significantly and extend survival of tumor-bearing mice by inducing more apoptosis and more severe necrosis as well as inhibiting blood vessel density within tumors. Therefore, VNP carrying the endostatin gene under our tumor-targeted expression system holds promise for the treatment of solid tumors.
实体瘤常含有缺氧和坏死区域，这些区域可以被减毒鼠伤寒沙门氏菌 VNP20009（VNP）靶向。我们试图开发一种基于肿瘤特异性传递菌株 VNP 的缺氧诱导启动子系统，将治疗基因的表达特异性或选择性限制在肿瘤微环境中。通过二维凝胶电泳和基质辅助激光解吸电离飞行时间/飞行时间 MS 基于蛋白质组学的方法，从沙门氏菌中缺氧调节的内源性蛋白筛选出缺氧诱导启动子-adhE 启动子。首先在细菌和动物肿瘤模型中验证了所选 adhE 启动子的效率和特异性。adhE 启动子可在缺氧下特异性驱动 GFP 基因表达，但在常氧下不能。此外，受该系统控制的荧光素酶报告基因表达也局限于肿瘤。最后，我们研究了受我们的 adhE 启动子系统控制的人血管内皮抑制素表达的 VNP 在小鼠黑色素瘤和 Lewis 肺癌模型中的抗癌功效。我们的结果表明，通过杀瘤和抗血管生成活性的双重作用，与无武装 VNP 治疗或未治疗对照相比，重组沙门氏菌菌株可产生增强的抗肿瘤作用。重组 VNP 通过诱导更多的细胞凋亡和更严重的坏死以及抑制肿瘤内血管密度，显著延缓肿瘤生长并延长荷瘤小鼠的存活时间。因此，携带内皮抑素基因的 VNP 在我们的肿瘤靶向表达系统下有望用于治疗实体瘤。
