Combined therapy using TS-suppressing vector and 5-FU

胸苷酸合成酶（TS）是5-FU抗肿瘤药物的靶分子。我们发现TS过表达与5-FU耐药性相关（中野等.例如，Br J Cancer 2006）。因此，为了开发针对TS过度表达癌症患者的治疗，我们一直在研究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，我们合成了几种siRNA。利用脂质体转染法将siRNA转染到TS高表达细胞A549和MAC 10中，确定了最有效的抗TS siRNA序列。在此基础上合成短发夹状RNA（short hairpin RNA，shRNA），将其插入带有人U6启动子的pBASi-hU 6 DNA质粒载体中，构建了TS抑制性shRNA质粒载体。利用COS-TPC方法构建了TS抑制性shRNA腺病毒载体（TSshRNA-Ad 5）。将TSshRNA-Ad 5转染到TS过表达的细胞株A549和MAC 10中，导致TS基因表达的90%以上的敲低。然后，我们浓缩并纯化TSshRNA-Ad 5。接下来，我们进行MTT测定以评估TSshRNA-Ad 5和5-FU在A549和MAC 10细胞中的抗肿瘤功效。因此，我们发现TSshRNA-Ad 5和5-FU联合治疗比单独使用TSshRNA-Ad 5或5-FU治疗具有更强的抗肿瘤作用。为了证实这些结果，我们正在使用5-FU耐药癌细胞（如NUGC-3/5-FU）进行额外的体外实验。此外，我们还使用荷瘤裸鼠进行了TS抑制载体和5-FU联合治疗的体内实验。此外，我们发现E2 F1过表达在非小细胞肺癌中诱导TS表达（Huang，et.艾尔。，Clin Cancer Res 2007）。

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).