Potent antitumor activity of oncolytic adenovirus expressing C/EBPβ against hepatocellular carcinoma
Potent antitumor activity of oncolytic adenovirus expressing C/EBPβ against hepatocellular carcinoma
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表达 C/EBPβ 的溶瘤腺病毒对肝细胞癌的有效抗肿瘤活性
DOI：
10.1007/s10495-020-01595-4
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发表时间：
2020-02
期刊：
影响因子：
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通讯作者：
Hai-zhen Wang
中科院分区：
文献类型：
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作者：
Yan Zeng;Feng-di Li;Jiang-long Du;Yu-jia Xue;Jing Liu;Xin Cao;Hai-zhen Wang
肝细胞癌（HCC）是世界范围内第五大常见癌症和第二大常见癌症死亡原因。肝癌是一种发展迅速、治疗困难、疗效差、死亡率高的恶性肿瘤。因此，寻找有效的方法预防和治疗肝癌是当务之急。1998年，Freytag等人用携带CD/TK基因的复制型溶瘤腺病毒治疗癌症[1]。Zhang等在ONYX-015的基础上构建了溶瘤腺病毒载体ZD 55，其可在肿瘤细胞中特异性增殖和复制，但在正常细胞中不增殖。此外，它还含有一个用于插入外源基因的克隆位点。携带CD基因的溶瘤腺病毒ZD 55在体外和体内均显示出良好的抗癌作用[2]。引人注目的是，溶瘤病毒疗法促进肿瘤内T细胞浸润并改善抗PD-1免疫疗法[3]。C/EBPβ（CCAAT/enhancer-binding protein β）是C/EBP家族的6个成员之一，最初被命名为IL-6表达核因子（NF-IL 6）。作为一种多功能转录因子，C/EBPβ还可以调节细胞的增殖、分化和凋亡。过表达C/EBPβ可使HepG 2细胞阻滞于G1/S期。Sun等人证明，全身递送全长C/EBPβ/脂质体复合物可抑制裸鼠中人结肠癌的生长[4]。此外，C/EBPβ的3′ UTR具有肿瘤抑制活性[5]。在这项工作中构建了过表达全长C/EBPβ的溶瘤腺病毒（图1a）。Western blot检测C/EBPβ蛋白的过表达。结果显示，Ad-C/EBPβ和ZD 55-C/EBPβ感染后，均可检测到C/EBPβ蛋白的过表达（图1 B）。Western blot检测腺病毒E1 A和E1 B基因的蛋白表达。HEK 293中的野生型腺病毒（WT）表达E1 A和E1 B蛋白，而ZD 55-C/EBPβ病毒由于E1 B基因缺失而仅表达E1 A基因产物。Ad-C/EBPβ由于E1 A和E1 B55基因的缺失而不表达。在没有病毒处理的对照BEL 7404细胞中，如在没有病毒感染的情况下所预期的，没有检测到E1 A和E1 B基因的产物（图1c）。经病毒重组和鉴定后，比较了不同病毒感染正常NHLF-1细胞和恶性BEL 7404细胞的形态学特征。与ONYX-015病毒相比，ZD 55-C/EBPβ不仅具有良好的肿瘤选择性，而且对肿瘤细胞具有较强的杀伤作用（图1d）。用Ad-EGFP和ZD 55-EGFP病毒感染BEL 7404细胞后，检测报告基因EGFP的表达。ZD 55-EGFP病毒可表达绿色荧光，由于病毒能够复制，其荧光强度和持续时间均比Ad-EGFP强。比较不同肿瘤细胞（Bcap 37、A549、SW 620和HeLa）和正常细胞（NHLF-1）中EGFP的表达。ZD 55-EGFP病毒可在肿瘤细胞中特异性扩增和表达。在正常细胞中，EGFP的表达相对较低，因为病毒载体不复制（图1 e）。感染不同的细胞（BEL-7404、HeLa、HepG 2和NHLF-1
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the second most common cause of cancer mortality worldwide. HCC is known for its rapid development, difficult treatment, poor curative effect and high mortality. Therefore, it is urgent to find effective methods for the prevention and treatment of HCC. In 1998, Freytag et al. treated cancer with replicative oncolytic adenoviruses carrying the CD/TK gene [1]. Zhang et al. constructed the oncolytic adenoviral vector ZD55 on the basis of ONYX-015, which can proliferate and replicate specifically in tumor cells but does not proliferate in normal cells. In addition, it contains a cloning site for insertion of foreign genes. The oncolytic adenovirus ZD55 armed with the CD gene has shown good anticancer effects in vitro and in vivo [2]. Strikingly, oncolytic virotherapy promotes intratumoral T cell infiltration and improves anti-PD-1 immunotherapy [3]. C/EBPβ (CCAAT/enhancer-binding protein β) is one of six members of the C/EBP family, which was initially named nuclear factor for IL-6 expression (NF-IL6). As a multifunctional transcription factor, C/EBPβ can also regulate cell proliferation, differentiation and apoptosis. Overexpression of C/EBPβ can arrest HepG2 cells at the G1/S-phase boundary. Sun et al. demonstrated that systemic delivery of the full-length C/EBPβ/liposome complex suppressed the growth of human colon cancer in nude mice [4]. In addition, the 3′ UTR of C/EBPβ has tumor-suppressive activity [5]. An oncolytic adenovirus overexpressing full-length C/EBPβ was constructed in this work (Fig. 1 a). The overexpression of C/EBPβ protein was detected by Western blot. The results showed that overexpression of C/EBPβ protein could be detected after infection with Ad-C/EBPβ and ZD55-C/EBPβ (Fig. 1 b). The protein expression of the adenoviral E1A and E1B genes was identified by Western blot. The wild-type adenovirus (WT) in HEK293 expressed E1A and E1B proteins, while the ZD55-C/EBPβ virus only expressed E1A gene products because of the E1B gene deletion. Ad-C/EBPβ did not express either protein because of the deletion of the E1A and E1B55 genes. In the control BEL7404 cells without virus treatment, the products of the E1A and E1B genes were not detected as expected in the absence of virus infection (Fig. 1 c). After virus recombination and identification, we compared the morphological characteristics of normal NHLF-1 cells and malignant BEL7404 cells infected with different viruses. Compared with the ONYX-015 virus, ZD55-C/EBPβ not only had good tumor selectivity but also had a strong killing effect on tumor cells (Fig. 1 d). The expression of the reporter gene EGFP in BEL7404 cells was analyzed after infection with Ad-EGFP and ZD55-EGFP viruses. The ZD55-EGFP virus could express green fluorescence, which was stronger and longer lasting than that of Ad-EGFP because of the virus was able to replicate. Then, the expression of EGFP was compared between different tumor cells (Bcap37, A549, SW620 and HeLa) and normal cells (NHLF-1). The ZD55-EGFP virus could be specifically amplified and expressed in tumor cells. In normal cells, the expression of EGFP was relatively low because the viral vector did not replicate (Fig. 1 e). Different cells (BEL-7404, HeLa, HepG2, and NHLF-1) were infected