Combined therapy using TS-suppressing vector and 5-FU

使用 TS 抑制载体和 5-FU 的联合治疗

• 批准号: 18591438
• 负责人: UENO Masaki
• 金额: $ 2.57万
• 依托单位: Kagawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591438/
• 关键词: gene therapy; thymidylate synthase; 5-FU; adenoviral vector; RNA interference; chemotherapy

Thymidylate synthase (TS) is a target molecule of 5-FU-derived anti-tumor drugs. We found a TS overexpression to be associated with the 5-FU resistance (Nakano, et. al., Br J Cancer 2006). Therefore, in order to develop the treatment against patients with TS-overexpressing cancers, we have been studying on the combined therapy with a TS-suppressing vector and 5-FU. First, to determine the effective siRNA sequence against TS, we synthesized several siRNAs. With the transfection of the siRNA into TS-overexpressing cells, such as A549 and MAC10, using the lipofection method, we determined the most effective siRNA sequence against TS. After the synthesize of shRNA (short hairpin RNA) based on this siRNA, we constructed a TS-suppressing shRNA plasmid vector by inserting the shRNA into the pBAsi-hU6 DNA plasmid vector with the human U6 promoter. Then, using the COS-TPC method, we constructed a TS-suppressing shRNA adenoviral vector (TSshRNA-Ad5). The transfection of TSshRNA-Ad5 into TS-overexpressing.lls, A549 and MAC10, resulted in more than 90% of knockdown of the TS gene expression. Then, we concentrated and purified the TSshRNA-Ad5. Next, we performed the MTT assay to evaluate the anti-tumor efficacy of the TSshRNA-Ad5 and 5-FU in A549 and MAC10 cells. Consequently, we found that the combined therapy with both TSshRNA-Ad5 and 5-FU had a stronger anti-tumor effect than the simple treatment of TSshRNA-Ad5 or 5-FU did. In order to confirm these results, we are performing the additional in vitro experiments using 5-FU-resistant cancer cells, such as NUGC-3/5-FU. In addition, we are also performing the in vivo experiment of the combined therapy with the TS-suppressing vector and 5-FU using the tumor-bearing nude mice. Furthermore, we found an E2F1 overexpression to induce the TS expression in non-small cell lung cancer (Huang, et. Al., Clin Cancer Res 2007).

胸苷酸合酶（TS）是5-FU衍生抗肿瘤药物的靶分子。我们发现TS过表达与5-FU耐药性有关（Nakano等，Br J Cancer 2006）。因此，为了开发针对过表达TS的癌症患者的治疗，我们一直在研究与TS抑制载体和5-FU的联合治疗。首先，为了确定针对TS的有效siRNA序列，我们合成了几个siRNA。通过将siRNA转染到过表达TS的细胞中，例如A549和Mac10，使用LiPofection方法，我们确定了针对TS的最有效的siRNA序列。基于此siRNA合成shRNA（短发夹RNA）之后，我们通过将shRNA插入人类U6启动子中，构建了pbasi-Hu6 DNA质粒载体中的shRNA质粒载体。然后，使用COS-TPC方法，我们构建了一个抑制TS的shRNA腺病毒载体（TSSHRNA-AD5）。将tsshrna-ad5转染到ts-Overexpressing.lls，A549和Mac10中导致TS基因表达的敲低超过90％。然后，我们集中并纯化了Tsshrna-Ad5。接下来，我们进行了MTT分析，以评估A549和MAC10细胞中TsshRNA-AD5和5-FU的抗肿瘤功效。因此，我们发现与TSSHRNA-AD5和5-FU的联合疗法相比，抗肿瘤效应比TSSHRNA-AD5或5-FU的简单治疗具有更强的抗肿瘤作用。为了确认这些结果，我们使用5-FU耐药性癌细胞（例如NUGC-3/5-FU）进行了其他体外实验。此外，我们还使用含有肿瘤的裸小鼠进行了与TS抑制载体和5-FU联合治疗的体内实验。此外，我们发现了E2F1的过表达，可在非小细胞肺癌中诱导TS表达（Huang等，等，Clin Cancer Res 2007）。

项目成果

MRP-1/CD9 gene transduction regulates the actin cytoskeleton through the downregulation of WAVE2

• DOI: 10.1038/sj.onc.1209654
• 发表时间: 2006-10-19
• 期刊: ONCOGENE
• 影响因子: 8
• 作者: Huang, C-L;Ueno, M.;Miyake, M.
• 通讯作者: Miyake, M.

The expression of osteopontin is increased in vessels with blood-brain bather impairment

血脑屏障受损的血管中骨桥蛋白的表达增加

• 发表时间: 2008
• 期刊: Neuropathol Appl Neurobiol 34
• 作者: Iwanaga;Y;Ueno;M;et. al.
• 通讯作者: et. al.

The expression of osteopontin is increased in vessels with blood-brain barrier impairment

• DOI: 10.1111/j.1365-2990.2007.00877.x
• 发表时间: 2008-04-01
• 期刊: NEUROPATHOLOGY AND APPLIED NEUROBIOLOGY
• 影响因子: 5
• 作者: Iwanaga, Y.;Ueno, M.;Sakamoto, H.
• 通讯作者: Sakamoto, H.

Two pathways of apoptosis are simultaneously induced in the embroyonal brains of neural cell-specific HIF-lalpha-deficient mice

在神经细胞特异性 HIF-lα 缺陷小鼠的胚胎脑中同时诱导两种凋亡途径

• 发表时间: 2006
• 期刊: Histochemistry and Cell Biology 125
• 作者: Masaki Ueno;et al.
• 通讯作者: et al.

The HAUSP gene plays an important role in non-small cell lung carcinogenesis through p53-dependent pathways

• DOI: 10.1002/path.1931
• 发表时间: 2006-04-01
• 期刊: JOURNAL OF PATHOLOGY
• 影响因子: 7.3
• 作者: Masuya, D;Huang, C;Sumitomo, S
• 通讯作者: Sumitomo, S