metallothionein의 RNA 간섭을 이용한 백금착제 항암제의 약물 내성 극복에 관한 연구

Abstract

Overexpression of metallothionein is one of the major issues for drug re-sistance in cancer cells. Although intracellular thiol-containing molecues such as metallothionein are induced in hypoxia due to its anti-oxidant effet, the overex-pression of metallothionein can induce resistance to platinum-based anticancer drug by its ability of detoxification in some cell lines. Hence, we hypothesized that knockdown of metallothionein by a short hairpin RNA (shRNA) delivery could help to restore the sensitivity to cisplatin for induction of apoptosis and cy-totoxicity. In this thesis, a new co-delivery system of cisplatin and shRNA to overcome resistance was designed and developed. The results showed that the 9-arginine-based reducible poly(oligo-D-arginine) (rPOA) used as a carrier could achieve efficient delivery of a short hairpin RNA against metallothionein (shMT) with low cytotoxicity and high transfection efficiency. Delivery of shRNA/rPOA polyplex targeting metallothionein resulted in down-regulation of metallothionein expression in B16F10 cancer cells. Down-regulation of metallothionein levels could lower the apoptosis threshold of cisplatin resistance compared to treatment with cisplatin alone or naked shMT plus cisplatin. Also, in the resistant cancer model, average tumor volume in the treated with both cisplatin and shMT/rPOA was significantly lower than those of treatment with cisplatin alone. Therefore, our study demonstrated that it is possible to use the rPOA to effectively deliver a shMT and knock down gene expression of metallothionein involved in cisplatin resistance. And it could improve drug sensitivity and therapeutic effect of cisplatin.|백금 착제 항암제는 고환암, 대장암, 난소암 등의 치료에 많이 쓰여지고 있는 약물 중 하나이지만, 심각한 부작용과 약물 효능 저하, 항암제 내성 발생 등의 문제점이 있다. 기존의 백금 착제 항암제에 대한 연구는 백금 착제 항암제의 구조 변화를 통한 부작용의 해결에 초점을 두고 진행이 되었다. 많은 개선을 보였음에도 불구하고, 근본적인 백금착제 항암제에 대한 내성 문제에 대해서는 해결하지 못했다. 많은 시스테인 기들을 가진 Metallothionein는 금속 및 산화 스트레스 물질과 결합하여 세포를 보호하는 역할을 하는 단백질로 알려져 있다. 하지만 암세포 내에서 과발현된 Metallothionein는 백금 착제 항암제와 결합을 하여 항암제의 약효를 떨어뜨리고 내성을 갖게 한다고 보고 되어지고 있다. 본 연구에서는 환원성 비바이러스 전달체를 이용하여 Metallothionein을 억제하는 shRNA를 투여하여 약물에 대한 내성을 억제하고, 동시에 백금 착제 항암제를 투여하여 복합치료 효과를 얻고자 하였다. MT shRNA 은 환원성 비바이러스 전달체인 양이온성 고분자 reducible poly(oligo D-arginine) (rPOA)와의 정전상호작용으로 결합하여 효과적으로 세포 도입을 하였다. 그리고 in vitro와 in vivo에서 MT shRNA와 백금 착제 항암제를 동시에 투여하는 복합 치료제에 의해 내성을 낮춤으로써, 항암제에 대한 민감도를 높여 암세포의 생존율을 낮춘 것을 확인하였다. 결론적으로, 약물 전달 시스템을 기반으로 한 저용량, 고효율의 복합 항암치료제가 항암제의 치료 효능를 높이고 부작용을 개선한 새로운 치료법으로 활용 가능함을 본 연구를 통해 검증하였다.; Overexpression of metallothionein is one of the major issues for drug re-sistance in cancer cells. Although intracellular thiol-containing molecues such as metallothionein are induced in hypoxia due to its anti-oxidant effet, the overex-pression of metallothionein can induce resistance to platinum-based anticancer drug by its ability of detoxification in some cell lines. Hence, we hypothesized that knockdown of metallothionein by a short hairpin RNA (shRNA) delivery could help to restore the sensitivity to cisplatin for induction of apoptosis and cy-totoxicity. In this thesis, a new co-delivery system of cisplatin and shRNA to overcome resistance was designed and developed. The results showed that the 9-arginine-based reducible poly(oligo-D-arginine) (rPOA) used as a carrier could achieve efficient delivery of a short hairpin RNA against metallothionein (shMT) with low cytotoxicity and high transfection efficiency. Delivery of shRNA/rPOA polyplex targeting metallothionein resulted in down-regulation of metallothionein expression in B16F10 cancer cells. Down-regulation of metallothionein levels could lower the apoptosis threshold of cisplatin resistance compared to treatment with cisplatin alone or naked shMT plus cisplatin. Also, in the resistant cancer model, average tumor volume in the treated with both cisplatin and shMT/rPOA was significantly lower than those of treatment with cisplatin alone. Therefore, our study demonstrated that it is possible to use the rPOA to effectively deliver a shMT and knock down gene expression of metallothionein involved in cisplatin resistance. And it could improve drug sensitivity and therapeutic effect of cisplatin.