Salmonella typhimurium VNP-20009 (VNP) is a non-pathogenic attenuated strain, which, as a facultative anaerobe, preferentially accumulates in hypoxic regions of solid tumors. Here, VNP was utilized as a delivery vehicle of the anti-tumor protein Lipidated azurin, Laz, which is produced by the meningitis-causing bacterium Neisseria meningitides. In brain cancer cells, Laz has been demonstrated to induce apoptosis through an interaction with the tumor suppressor protein p53. In this study, the laz gene, including its signal sequence, was cloned downstream of a hypoxia inducible promoter (HIP-1), before being electroporated into VNP. Successful ectopic expression and export of the Laz protein by VNP under hypoxic conditions were confirmed by Western blot analysis of the cell-free culture medium. Effective expression of Laz by VNP was investigated in two glioblastoma cell lines: LN-229 and U-373, with the latter line carrying a mutated version of p53; as well as in the breast cancer line MCF-7. Cytotoxicity of the VNP-Laz was assessed by determining the fluorescence of the apoptotic marker caspases 3/7. Compared to the purified Laz, VNP-Laz, significantly induced apoptosis in MCF-7, LN-229 and, to a much lower extent in U-373 cells, suggesting a p53-linked mechanism. Our results might represent a new approach of targeted gene delivery and suggest a potential application in brain tumor therapy.

鼠伤寒沙门氏菌VNP-20009 (VNP) 是一种非致病性减毒菌株，作为兼性厌氧菌，优先在实体瘤的缺氧区域积聚。在这里，VNP 被用作抗肿瘤蛋白脂化天青蛋白 Laz 的递送载体，Laz 是由引起脑膜炎的细菌脑膜炎奈瑟菌产生的。在脑癌细胞中，Laz 已被证明可以通过与肿瘤抑制蛋白 p53 相互作用来诱导细胞凋亡。在这项研究中， laz基因（包括其信号序列）在电穿孔至 VNP 之前被克隆到缺氧诱导启动子 (HIP-1) 的下游。无细胞培养基的蛋白质印迹分析证实了 VNP 在缺氧条件下成功异位表达和输出 Laz 蛋白。在两种胶质母细胞瘤细胞系 LN-229 和 U-373 中研究了 VNP 有效表达 Laz，后者携带 p53 的突变版本；以及乳腺癌系列 MCF-7。通过测定凋亡标记物半胱天冬酶 3/7 的荧光来评估 VNP-Laz 的细胞毒性。与纯化的 Laz 相比，VNP-Laz 显着诱导 MCF-7、LN-229 细胞凋亡，并且在 U-373 细胞中诱导凋亡的程度低得多，这表明 p53 相关机制。我们的结果可能代表了一种靶向基因递送的新方法，并表明其在脑肿瘤治疗中的潜在应用。