뇌종양의 신생혈관억제를 위한 RAGE binding peptide가 결합된 유전자 전달체 개발

Abstract

뇌종양, 직장암, 유방암과 같은 다양한 종류의 종양에서 Receptor for advanced glycation endproduct (RAGE)의 과발현이 발견되어왔다. RAGE와 S100 단백질들, high mobility group box1과 같은 그 리간드와의 결합은 침투,전이, 신생혈관생성의 종양 세포 성장에 관여한다. 이러한 RAGE/리간드 간의 상호작용을 막는 것은 MAP kinase, NF-kB를 통한 RAGE의 세포 신호를 억제한다. 이 연구에서는 RAGE 결합 단백질인 RAGE binding peptide (RBP)를 polyethyleneimmine (PEI)에 합성하여 RAGE를 타겟으로 하는 유전자 전달체를 개발하였다. RBP가 결합된 PEI (PEI-RBP)를 생산하기 위하여, PEI와 RBP는 SPDP 가교제를 이용하여 합성되었다. PEI-RBP는 pDNA와 1:2 이상의 질량비에서 (pDNA:Carrier) 나노 크기의 양전하의 복합체를 형성했다. Rat의 뇌종양 C6 세포상에서 PEI-RBP의 최적화된 유전자 전달 효율은 질량비 1:5였으며, PEI보다 높은 유전자 전달효율과 낮은 독성을 나타내었다. RBP 경쟁 실험을 통해 RBP 존재 하에 낮아진 유전자 전달 효율을 보였으며, RBP가 적용된 C6 세포 표면의 RAGE가 감소되었다. 이러한 결과는 RBP가 RAGE와의 결합 후 RAGE를 통한 새포 내 유입을 유도함을 시사한다. 또한, RAGE/리간드 상호작용의 산물인 세포성장인자 VEGF의 분비량이 RBP와 pDNA/PEI-RBP의 처리시 감소되었음을 VEGF ELISA를 통해 밝혀내었다. 뇌종양 동물 모델에서 조직학적, 형광 관찰평가를 통해 RBP와 PEI-RBP의 항암효과를 세포사멸정도와 RAGE와 VEGF, α-SMA의 감소 정도를 통해 입증하였다. 따라서 개발된 RBP 결합 양이온성 유전자 전달체는 그 자체로의 뇌종양 치료 효과와 더불어 치료 유전자의 전달을 통해 RAGE를 타겟으로 하는 유전자 전달체로서 매우 유용할 것이다.|In various types of tumors, including brain, colorectal, breast cancers, overexpressed Receptor for advanced glycation endproduct (RAGE) has been detected. Interactions between RAGE and its ligand, such as S100 proteins, High mobility group box 1 (HMGB1), involved in tumor cell growth including cell invasion, migration, angiogenesis. Blockade of RAGE/ligand interactions can inhibit RAGE mediated cell signaling pathway such as MAP kinase and NF-kB activation. In this study, RAGE binding peptide (RBP) was developed to block the RAGE interactions with ligands, and conjugated to polyethyleneimmine (PEI, 25kDa) for RAGE targeted gene delivery carrier. For the production of RBP labeled PEI (PEI-RBP), PEI and RBP was crosslinked by SPDP crosslinking reagent. The PEI-RBP formed nano-sized cationic complexes with pDNA at and above 1:2 weight ratio (pDNA:Carrier). The optimized transfection efficiency of PEI-RBP against C6 rat glial cell was 1:5 weight ratio and showed higher gene delivery efficiency and lower cytotoxicity than PEI. In RBP competition assay, the transfection efficiency was reduced in presence of free RBP. In addition, the amounts of RAGE can bind with ligand in cell surface was decreased in RBP transfected C6 cells. These observations suggest that the RBP induced RAGE mediate cellular uptake followed by RAGE/RBP interactions. the VEGF ELISA data revealed that the secretion of VEGF, one of productions from RAGE/ligands tumor cell growth signaling pathway, decreased in free RBP and pDNA/PEI-RBP complexes, respectively. Histological and fluorescence evaluation in vivo rat glioblastoma model showed the anti-tumor effect of free RBP and PEI-RBP, confirming the increased apoptosis positive cells, the blockade of RAGE, decreased VEGF and α-Smooth muscle actin (α-SMA) secretion. Accordingly, developed RBP labeled cationic carrier, PEI-RBP may be useful not only for the treatment of glioblastoma itself also for delivery of therapeutic gene as a RAGE targeted gene carrier.; In various types of tumors, including brain, colorectal, breast cancers, overexpressed Receptor for advanced glycation endproduct (RAGE) has been detected. Interactions between RAGE and its ligand, such as S100 proteins, High mobility group box 1 (HMGB1), involved in tumor cell growth including cell invasion, migration, angiogenesis. Blockade of RAGE/ligand interactions can inhibit RAGE mediated cell signaling pathway such as MAP kinase and NF-kB activation. In this study, RAGE binding peptide (RBP) was developed to block the RAGE interactions with ligands, and conjugated to polyethyleneimmine (PEI, 25kDa) for RAGE targeted gene delivery carrier. For the production of RBP labeled PEI (PEI-RBP), PEI and RBP was crosslinked by SPDP crosslinking reagent. The PEI-RBP formed nano-sized cationic complexes with pDNA at and above 1:2 weight ratio (pDNA:Carrier). The optimized transfection efficiency of PEI-RBP against C6 rat glial cell was 1:5 weight ratio and showed higher gene delivery efficiency and lower cytotoxicity than PEI. In RBP competition assay, the transfection efficiency was reduced in presence of free RBP. In addition, the amounts of RAGE can bind with ligand in cell surface was decreased in RBP transfected C6 cells. These observations suggest that the RBP induced RAGE mediate cellular uptake followed by RAGE/RBP interactions. the VEGF ELISA data revealed that the secretion of VEGF, one of productions from RAGE/ligands tumor cell growth signaling pathway, decreased in free RBP and pDNA/PEI-RBP complexes, respectively. Histological and fluorescence evaluation in vivo rat glioblastoma model showed the anti-tumor effect of free RBP and PEI-RBP, confirming the increased apoptosis positive cells, the blockade of RAGE, decreased VEGF and α-Smooth muscle actin (α-SMA) secretion. Accordingly, developed RBP labeled cationic carrier, PEI-RBP may be useful not only for the treatment of glioblastoma itself also for delivery of therapeutic gene as a RAGE targeted gene carrier.