Lu Qin, Yu De-Hong, Fang Chao, Liu Fang, Ye Xun, Zhao Yi, Qin Jie, Guo Xiao-Kui, Liang Min, Hu Fang, Chen Hong-Zhuan
Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Cancer Biol Ther. 2009 Jun;8(12):1125-32. doi: 10.4161/cbt.8.12.8445. Epub 2009 Jun 17.
Virotherapy employing conditionally replicative adenovirus (CRAd) represents a novel targeted strategy for the hepatocellular carcinoma (HCC) treatment. In this study, we explored the potential influence of E3 region, which encodes several TRAIL-inhibiting proteins (E3-6.7K, E3-10.4K/14.5K and E3-14.7K), on CRAd mediated cytotoxicity to HCC cells. Two E1B-55 kDa-deleted CRAds containing E3 region (Ad.DeltaE1B) or no E3 region (Ad.DeltaE1B.DeltaE3) were fabricated. Ad.DeltaE1B.DeltaE3 exhibited higher cytocidal potency than Ad.DeltaE1B in all tested HCC cells (Hep3B, BEL-7404, BEL-7402, HuH7, PLC/PRF/5 and HepG2), suggesting that Ad.DeltaE1B.DeltaE3 mediated cytotoxicity was partly attributed to the absence of E3 region encoding TRAIL-inhibiting proteins. In representative Hep3B cells, Ad.DeltaE1B.DeltaE3 led to more drop of mitochondrial membrane potential (MMP) and much lower ATP level than Ad.DeltaE1B. Moreover, Ad.DeltaE1B.DeltaE3 induced early apoptotic cells and the late apoptotic/necrotic cells for three and four times more than those infected by Ad.DeltaE1B. The cytotoxicity to all TRAIL endogenously expressing HCC cells and MMP drop of Hep3B cells induced by Ad.DeltaE1B.DeltaE3 but not Ad.DeltaE1B could be significantly inhibited by z-vad-fmk, a pan caspase inhibitor, suggesting that the endogenous TRAIL-mediated apoptotic pathway may be implicated in the cytocidal potency of Ad.DeltaE1B.DeltaE3 on HCC cells although other unknown mechanisms may be also involved. Our findings provided the first evidence that CRAd without E3 region might be a smart choice for the virotherapy of HCC.
使用条件性复制腺病毒（CRAd）的病毒疗法是一种用于肝细胞癌（HCC）治疗的新型靶向策略。在本研究中，我们探讨了编码几种TRAIL抑制蛋白（E3-6.7K、E3-10.4K/14.5K和E3-14.7K）的E3区域对CRAd介导的对HCC细胞的细胞毒性的潜在影响。构建了两种缺失E1B-55 kDa且含有E3区域（Ad.DeltaE1B）或不含有E3区域（Ad.DeltaE1B.DeltaE3）的CRAd。在所有测试的HCC细胞（Hep3B、BEL-7404、BEL-7402、HuH7、PLC/PRF/5和HepG2）中，Ad.DeltaE1B.DeltaE3表现出比Ad.DeltaE1B更高的杀细胞效力，这表明Ad.DeltaE1B.DeltaE3介导的细胞毒性部分归因于缺失编码TRAIL抑制蛋白的E3区域。在代表性的Hep3B细胞中，Ad.DeltaE1B.DeltaE3导致线粒体膜电位（MMP）下降更多，ATP水平比Ad.DeltaE1B低得多。此外，Ad.DeltaE1B.DeltaE3诱导的早期凋亡细胞和晚期凋亡/坏死细胞比Ad.DeltaE1B感染的细胞多三到四倍。一种泛半胱天冬酶抑制剂z-vad-fmk可显著抑制Ad.DeltaE1B.DeltaE3而非Ad.DeltaE1B对所有内源性表达TRAIL的HCC细胞的细胞毒性以及对Hep3B细胞的MMP下降，这表明内源性TRAIL介导的凋亡途径可能与Ad.DeltaE1B.DeltaE3对HCC细胞的杀细胞效力有关，尽管可能还涉及其他未知机制。我们的研究结果首次证明，不含E3区域的CRAd可能是HCC病毒疗法的明智选择。
