腺病毒载体表达针对 Wnt2B 的短发夹 RNA 对 Wnt2B 过表达肿瘤具有有效的抗肿瘤活性。

Adenoviral vector expressing short hairpin RNA targeting Wnt2B has an effective antitumour activity against Wnt2B2-overexpressing tumours.

机构信息

Department of General Thoracic Surgery, Breast and Endocrinological Surgery, Faculty of Medicine, Kagawa University, Miki-cho, Kita-gun, Kagawa, Japan.

出版信息

Eur J Cancer. 2012 May;48(8):1208-18. doi: 10.1016/j.ejca.2011.05.003. Epub 2011 Jun 7.

DOI:10.1016/j.ejca.2011.05.003

PMID:21646015

Abstract

BACKGROUND

The Wnt family encodes multi-functional signalling glycoproteins regulating various normal and pathological processes including tumourigenesis. Wnt2B overexpression is thought to affect tumour progression through the activation of the canonical Wnt pathway.

METHOD

Experimental studies were conducted using a Wnt2B-inhibiting vector to establish gene therapy against Wnt2B2-overexpressing tumours. A replication-deficient recombinant adenoviral vector expressing short hairpin RNA targeting Wnt2B (Ad-shWnt2B) was constructed. Three Wnt2B2-overexpressing human tumour cells, including A549 cells, Hela cells and PANC1 cells, were used. Thereafter, cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Next, a human tumour xenograft model in nude mice was prepared by subcutaneously implanting tumours derived from A549 cells. Ad-shWnt2B was administered via intratumoural injection every 4days.

RESULTS

First, immunohistochemical studies revealed that high levels of Wnt2B expression appeared in proliferative normal tissues and many human tumour tissues. Furthermore, the Wnt2B2 gene expression was associated with c-Myc and survivin expressions in human lung cancer. Transduction with Ad-shWnt2B effectively downregulated the Wnt2B2 expression in all the three Wnt2B2-overexpressing tumour cells (p<0.0001). The transduction with Ad-shWnt2B significantly reduced the percentage of viable cells in all the Wnt2B2-overexpressing tumour cells (p<0.005). In addition, transduction with Ad-shWnt2B significantly downregulated c-Myc and survivin in A549 cells (p<0.005). Furthermore, the treatment with Ad-shWnt2B exerted a significant antitumour effect against the Wnt2B2-overexpressing A549 xenografts by inducing apoptosis (p<0.01).

CONCLUSIONS

Cancer gene therapy using an adenoviral vector expressing short hairpin RNA (shRNA) against Wnt2B was, therefore, found to have a strong antitumour effect against Wnt2B2-overexpressing tumours.

摘要

背景

Wnt 家族编码多功能信号糖蛋白，调节包括肿瘤发生在内的各种正常和病理过程。Wnt2B 的过表达被认为通过激活经典 Wnt 途径影响肿瘤进展。

方法

使用抑制 Wnt2B 的载体建立针对 Wnt2B 过表达肿瘤的基因治疗，进行实验研究。构建表达针对 Wnt2B 的短发夹 RNA 的复制缺陷重组腺病毒载体（Ad-shWnt2B）。使用三种 Wnt2B2 过表达的人肿瘤细胞，包括 A549 细胞、Hela 细胞和 PANC1 细胞。然后，使用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐（MTT）测定法评估细胞活力。接下来，通过皮下植入源自 A549 细胞的肿瘤，在裸鼠中制备人肿瘤异种移植模型。每隔 4 天通过肿瘤内注射给予 Ad-shWnt2B。

结果

首先，免疫组织化学研究表明，Wnt2B 高表达出现在增殖性正常组织和许多人类肿瘤组织中。此外，Wnt2B2 基因表达与人类肺癌中的 c-Myc 和 survivin 表达相关。Ad-shWnt2B 的转导可有效下调三种 Wnt2B2 过表达肿瘤细胞中的 Wnt2B2 表达（p<0.0001）。Ad-shWnt2B 的转导可显著降低所有 Wnt2B2 过表达肿瘤细胞中存活细胞的百分比（p<0.005）。此外，Ad-shWnt2B 的转导可显著下调 A549 细胞中的 c-Myc 和 survivin（p<0.005）。此外，Ad-shWnt2B 的治疗对 Wnt2B2 过表达的 A549 异种移植物具有显著的抗肿瘤作用，通过诱导细胞凋亡（p<0.01）。