Combined therapy using TS-suppressing vector and 5-FU

胸苷酸合酶(TS)是5-FU类抗肿瘤药物的靶向分子。我们发现TS过表达与5-FU耐药有关(Nakano，et.等人，BR J癌症2006)。因此，为了开发针对TS高表达肿瘤患者的治疗方法，我们一直在研究TS抑制载体与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。通过脂质体转染法将siRNA导入TS过表达的细胞，如A549和Mac10，我们确定了最有效的针对TS的siRNA序列。在此基础上合成短发夹状RNA后，将其插入带有人U6启动子的pBAsi-HU6DNA载体中，构建了抑制TS的shRNA载体。然后，利用COS-TPC方法构建了TS抑制shRNA腺病毒载体(TSshRNA-Ad5)。将TSshRNA-Ad5导入TS高表达细胞系A549和Mac10，可使TS基因表达下调90%以上。然后，我们对TSshRNA-Ad5进行了浓缩和纯化。接下来，我们用四甲基偶氮唑盐比色法检测TSshRNA-Ad5和5-FU对A549和Mac10细胞的抗肿瘤作用。因此，我们发现TSshRNA-Ad5和5-FU联合治疗比单纯TSshRNA-Ad5或5-FU具有更强的抗肿瘤作用。为了证实这些结果，我们正在进行额外的体外实验，使用5-FU耐药的癌细胞，如NUGC-3/5-FU。此外，我们还利用荷瘤裸鼠进行了TS抑制载体与5-FU联合治疗的体内实验。此外，我们还发现在非小细胞肺癌中E2F1的过表达可以诱导TS的表达。等人，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).