Hypoxia-regulated expression of attenuated diphtheria toxin a fused with hypoxia-inducible factor-1α oxygen-dependent degradation domain preferentially induces apoptosis of hypoxic cells in solid tumor
Hypoxia-regulated expression of attenuated diphtheria toxin a fused with hypoxia-inducible factor-1α oxygen-dependent degradation domain preferentially induces apoptosis of hypoxic cells in solid tumor
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DOI：
10.1158/0008-5472.can-05-0111
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发表时间：
2005-12-15
期刊：
影响因子：
11.2
通讯作者：
Takenaga, K
中科院分区：
文献类型：
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作者：
Koshikawa, N;Takenaga, K
实体瘤缺氧区域的肿瘤细胞对常规化疗和放疗具有抵抗力，因此成为癌症治疗的障碍。我们在这里报告了应用白喉毒素 A (DT-A) 的缺氧调节表达来杀死缺氧肿瘤细胞的可行性。构建表达载体，表达在血管内皮生长因子基因启动子控制下与缺氧诱导因子1α（HIF-1α）氧依赖性降解（ODD）结构域融合的DT-A，并在DT-A/ODD序列下游含有促红细胞生成素mRNA结合蛋白（ERBP）结合序列。体外泛素化测定表明，在含氧量正常的条件下，DT-A/ODD（而非 DT-A）以 von Hippel-Lindau 和氧依赖性方式被泛素化，其效率与 HIF-1 α 一样。 DT-A/ODD 表现出与 DT-A 相当的翻译抑制活性。 ERBP 结合序列可有效稳定各种细胞类型缺氧条件下的 mRNA。将表达 DT-A/ODD 的载体转染到高转移性 Lewis 肺癌 (3LL) 中，所有细胞均导致不依赖于缺氧的细胞凋亡诱导，这可能是由于其极端毒性。然而，表达减毒DT-A(W153F)/ODD或DT-A(H21A)/ODD的载体的转染导致缺氧依赖性细胞凋亡的诱导。编码DT-A(W153)/ODD的载体的脂质体基因转移在由对缺氧诱导的细胞凋亡具有较高抵抗力的所有变体细胞建立的实体瘤的缺氧区域中诱导细胞凋亡，但在含氧量正常的区域中不诱导细胞凋亡，并抑制由3LL-P29细胞建立的缺氧肿瘤的生长。这些结果表明，弱氧调节的 DT-A(W153F)/ODD 融合蛋白的表达可能用于杀死缺氧肿瘤细胞，同时最大限度地减少对含氧量正常的正常组织的损害。
Tumor cells in hypoxic areas of solid tumors are resistant to conventional chemotherapy and radiotherapy and thus are obstacles of cancer therapy. We report here the feasibility of applying hypoxia-regulated expression of diphtheria toxin A (DT-A) for killing hypoxic tumor cells. The expression vector was constructed to express DT-A fused with hypoxia-inducible factor-1 alpha (HIF-1 alpha) oxygen-dependent degradation (ODD) domain under the control of vascular endothelial growth factor gene promoter and contain erythropoietin mRNA-binding protein (ERBP)-binding sequence downstream of the DT-A/ODD sequence. In vitro ubiquitination assay showed that DT-A/ODD, but not DT-A, was ubiquitinated as efficient as HIF-1 alpha under normoxic conditions in a von Hippel-Lindau-and oxygen-dependent manner. DT-A/ODD exhibited a comparable translation inhibitory activity to DT-A. ERBP-binding sequence was effective in stabilizing mRNA under hypoxic conditions in various cell types. Transfection of the vector expressing DT-A/ODD into high-metastatic Lewis lung carcinoma (3LL) All cells resulted in induction of apoptosis independently of hypoxia, probably due to its extreme toxicity. However, transfection of the vector expressing attenuated DT-A(W153F)/ODD or DT-A(H21A)/ODD resulted in a hypoxia-dependent induction of apoptosis. Liposomal gene transfer of the vector encoding DT-A(W153)/ODD induced apoptosis in hypoxic, but not in normoxic, areas of solid tumors established by All variant cells with higher resistance to hypoxia-induced apoptosis and inhibited the growth of hypoxic tumors established by 3LL-P29 cells. These results suggest that hypoxia-regulated expression of attenuated DT-A(W153F)/ODD fusion protein is potentially of use for killing hypoxic tumor cells with minimizing the damage to normoxic normal tissues.