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Development of anti-cancer strategy to increase sensitivity of cancer cells to chemotherapy using siRNA combined with HVJ-E vector

利用siRNA联合HVJ-E载体开发提高癌细胞对化疗敏感性的抗癌策略

基本信息

批准号：
15300163
负责人：
KANEDA Yasufumi
金额：
$ 8.51万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Every cancer therapy appears to be transiently effective for cancer regression, but cancers gradually transform to be resistant to the therapy. Cancers also develop machineries to resist chemotherapy. Short interfering RNA (siRNA) has been evaluated as an attractive and effective tool for suppressing target protein by specifically digesting its mRNA. Suppression of the machineries using siRNA may enhance the sensitivity to chemotherapy in cancers when combined with effective delivery system. To enhance the anti-cancer effect of chemotherapy, we tested the ability of short interfering RNA(siRNA) to inhibit chemotherapy-resistant gene expression. We found that Rad51 was enhanced with cis-diamminedichloroplatinum (II) (CDDP ; cisplatin) in cancer cells. Overexpression of Rad51 caused cancer cells to become resistant to CDDP. When synthetic Rad 51 siRNA was delivered to HeLa cells using HVJ (hemagglutinating virus of Japan, Sendai virus) envelope vector, the delivery efficiency of siRNA was approximately 100%. No Rad51 transcripts were detected on day 2, and Rad51 protein completely disappeared for 4 days after siRNA transfer. The combination of Rad51 siRNA and CDDP caused increased DNA damage, which was detected by pulsed-field gel electrophoresis. When HeLa cells were incubated with 0.02 μg/ml CDDP for 3 hours after siRNA transfer, the number of colonies decreased to approximately 10% of that with scrambled siRNA. When Rad51 siRNA was delivered into tumors using HVJ envelope vector, the delivery efficiency was approximately 50%, and the Rad51 transcript level was reduced by approximately 60%. Rad51 siRNA combined with CDDP significantly inhibited the tumor growth when compared to siRNA or CDDP alone.Our results suggest that the combination of CDDP and Rad51 siRNA will be an effective anti-cancer protocol.

每一种癌症治疗似乎对癌症消退都是短暂有效的，但癌症会逐渐转变为对治疗产生抗性。癌症也会发展出抵抗化疗的机制。短干扰RNA（Short interfering RNA，siRNA）是通过特异性消化靶蛋白mRNA来抑制靶蛋白表达的有效工具。当与有效的递送系统结合时，使用siRNA抑制该机制可以增强癌症对化疗的敏感性。为了增强化疗的抗癌效果，我们测试了短干扰RNA（siRNA）抑制化疗耐药基因表达的能力。我们发现Rad 51在癌细胞中被顺式二氨二氯铂（CDDP ;顺铂）增强。Rad 51的过表达导致癌细胞对CDDP产生耐药性。当使用HVJ（日本血凝病毒，仙台病毒）包膜载体将合成的Rad 51 siRNA递送至HeLa细胞时，siRNA的递送效率约为100%。在第2天没有检测到Rad 51转录物，并且在siRNA转移后4天Rad 51蛋白完全消失。Rad 51 siRNA和CDDP的组合引起DNA损伤增加，这通过脉冲场凝胶电泳检测。当HeLa细胞在siRNA转移后与0.02 μg/ml CDDP孵育3小时时，集落数减少至使用乱序siRNA的集落数的约10%。当使用HVJ包膜载体将Rad 51 siRNA递送到肿瘤中时，递送效率约为50%，并且Rad 51转录物水平降低约60%。Rad 51 siRNA联合CDDP对肿瘤生长的抑制作用明显优于单独使用siRNA或CDDP，提示联合使用Rad 51 siRNA和CDDP可能是一种有效的抗肿瘤方案。