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Antisense down-regulation of 5-FU related enhances cytotoxicity of 5-FU in human lung cancer

5-FU相关的反义下调增强了5-FU在人肺癌中的细胞毒性

基本信息

批准号：
13671237
负责人：
KONDO Kazuya
金额：
$ 2.3万
依托单位：
THE University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Thymidylate synthase (TS), the key enzyme in de novo synthesis of thymidine, is an important target for antitumor chemothrapy of 5-FU. We used antisense oligodeoxynucleotide (ODN) to down-regulate the expression of TS. We made 5 antisense ODNs which is complementary to TS Mrna, and one random ODN. Transfection of ODNs was performed using HVJ-liposome. After incubating human lung cancer cell lines (Ma10 and Ma25) and antisense ODNs with HVJ-liposome, the TS expression of cell lines was examined by RT-PCR method and TS activity was measured. TS expression was repressed from 50% to 20% by incubaating cell lines and antisense ODNs and TS expression. It was impossible that antisense ODNs of TS inhibited the expression of TS. Chu e al. reported that the TS protein itself binds to the T Mrna both at the translational start site and in the coding region, inhibiting translational processing of the message.Next year, we examined whether CDDP (anticancer drug) conjugated by liposome (liposome-CDDP) enhance anticancer effect. The IC50 of CDDP was 40ug/ML in human lung cancer cell line Ma44-3 in vitro. Meanwhile, the IC50 of liposome-CDDP was 10ug/ML for 48 hr incubation. And it was 1.5ug/ML for 96 hr incubation. When thecubation time becomes longer, the IC50 of liposome-CDDP was lower. The use of Rhodamine-labeled liposome-CDDP revealed that the lipsome-CDDP attached the cytoplasm of cancer cells in vitro. Rhodamine-labeled liposome-CDDP revealed that the liposome-CDDP attached the cytoplasm of cancer cells in vitro. Rhodamine-labeled liposome-CDDP was administrared in SCID mice using a nebulizer (through airway). Rhodamine-labeled liposome was observed in the lung of the mice. These results demonstrate that the administration of liposome-CDDP through airway may become a therapy for lung cancer.

胸苷酸合酶（TS）是胸苷从头合成的关键酶，是5-FU抗肿瘤化疗的重要靶点。我们使用反义寡脱氧核苷酸（ODN）来下调 TS 的表达。我们制作了 5 个与 TS Mrna 互补的反义 ODN，以及 1 个随机 ODN。使用 HVJ 脂质体进行 ODN 转染。将人肺癌细胞系（Ma10和Ma25）与反义ODN与HVJ-脂质体一起孵育后，通过RT-PCR方法检测细胞系的TS表达并测量TS活性。通过孵育细胞系以及反义 ODN 和 TS 表达，TS 表达被抑制从 50% 至 20%。 TS的反义ODN不可能抑制TS的表达。楚等人。报道称，TS蛋白本身在翻译起始位点和编码区均与T Mrna结合，抑制信息的翻译加工。明年，我们检查了脂质体缀合的CDDP（抗癌药物）（脂质体-CDDP）是否增强抗癌效果。 CDDP在体外人肺癌细胞系Ma44-3中的IC50为40ug/ML。同时，脂质体-CDDP孵育48小时的IC50为10ug/ML。 96 小时孵育的浓度为 1.5ug/ML。孵育时间越长，脂质体-CDDP的IC50越低。使用罗丹明标记的脂质体-CDDP表明脂质体-CDDP在体外附着于癌细胞的细胞质。罗丹明标记的脂质体-CDDP显示，脂质体-CDDP在体外附着于癌细胞的细胞质。使用喷雾器（通过气道）对 SCID 小鼠施用罗丹明标记的脂质体-CDDP。在小鼠肺部观察到罗丹明标记的脂质体。这些结果表明，通过气道施用脂质体-CDDP可能成为肺癌的治疗方法。