Anti-cancer effect of R3V6 peptide-mediated delivery of an anti-microRNA-21 antisense-oligodeoxynucleotide in a glioblastoma animal model.

MicroRNA-21 (miR-21) expression in glioblastoma inhibits the expression of pro-apoptotic genes, thereby promoting tumor growth. A previous study showed that the amphiphilic R3V6 peptide is an efficient carrier of the anti-miR-21 antisense oligodeoxynucleotide (antisense-ODN) into cells in vitro. In the current study, in vivo delivery of antisense-ODN using the R3V6 peptide was evaluated in a glioblastoma animal model. In vitro transfection showed that the R3V6 peptide delivered antisense-ODN more efficiently than polyethylenimine (25 kDa, PEI25k) in C6 glioblastoma cells. For in vivo evaluation, antisense-ODN/R3V6 complex was injected intratumorally into a C6 glioblastoma xenograft animal model. Tumor growth was suppressed by the injection of the antisense-ODN/R3V6 complex, compared with the antisense-ODN/PEI25k and scrambled-antisense-ODN (scr-antisense-ODN)/R3V6 complexes. Real-time RT-PCR showed that miR-21 levels were reduced most efficiently by the antisense-ODNR3V6 complex in tumors. Due to inhibition of miR-21, expression of the programed cell death 4 (PDCD4) gene was promoted in tumors, resulting in the induction of apoptosis of tumor cells. These results suggest that delivery of antisense-ODN using R3V6 peptides may be useful for the development of antisense-ODN therapy for glioblastoma.

Peptide micelle-mediated delivery of tissue-specific suicide gene and combined therapy with avastin in a glioblastoma model.

Bevacizumab (Avastin) is an angiogenesis inhibitor used as a treatment for various cancers. In this study, the combination therapy of Avastin and glioblastoma-specific thymidine kinase gene [pEpo-NI2-SV-herpes simplex virus thymidine kinase(HSVtk)] was evaluated in a glioblastoma animal model. The R7L10 peptide was used as a gene carrier of pEpo-NI2-SV-HSVtk. Gel retardation assays confirmed that R7L10 formed stable complexes with pEpo-NI2-SV-HSVtk. R7L10 protected DNA from nuclease digestion. R7L10 had lower transfection efficiency than polyethylenimine (PEI; 25 kDa). However, the in vitro and in vivo toxicity assays showed that R7L10 had lower cytotoxicity than PEI, suggesting that R7L10 is safer than PEI. For the combination therapy, Avastin was injected intravenously and the pEpo-NI2-SV-HSVtk/R7L10 complexes were injected intratumorally in the glioblastoma animal model. Tumor growth was most effectively inhibited by the combination therapy of Avastin and the gene. The immunostaining results confirmed that the HSVtk genes were expressed in the groups with the pEpo-NI2-SV-HSVtk/R7L10 complex. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed a higher level of apoptotic cells in the combination group than the pEpo-NI2-SV-HSVtk/R7L10 complex or Avastin group. In conclusion, the combination of Avastin and the glioblastoma-specific HSVtk gene has a higher antitumor effect than single therapy of Avastin or HSVtk after intratumoral administration in glioblastoma animal model.

Delivery of anti-microRNA-21 antisense-oligodeoxynucleotide using amphiphilic peptides for glioblastoma gene therapy.

Inhibition of microRNA-21 (miR-21) has been shown to promote apoptosis of cancer cells and to reduce tumor size in glioblastoma. However, efficient carriers for antisense-oligodeoxynucleotide (antisense-ODN) against miR-21 have not yet been developed. In this study, the R3V6 peptide (R3V6) was evaluated as a carrier of antisense-ODN. In a gel retardation assay, R3V6 formed a complex with an antisense-ODN. The serum stability assay showed that R3V6 protected it from nucleases more efficiently than polyethylenimine (PEI; 25 kDa, PEI25k). A Renilla luciferase gene with a 3'-untranslated region (3'-UTR) recognizable by miR-21 (psiCHECK2-miR-21-UTR) was constructed for the antisense-ODN assay. psiCHECK2-miR-21-UTR expressed less Renilla luciferase in the cells with a higher level of miR-21 due to the effect of miR-21. In an in vitro transfection assay, the R3V6 peptide delivered anti-miR-21 antisense-ODN into cells more efficiently than PEI (25 kDa, PEI25k) and lipofectamine. As a result, antisense-ODN/R3V6 complex inhibited miR-21 and increased Renilla luciferase expression more efficiently than antisense-ODN/PEI25k or antisense-ODN/Lipofectamine complexes in both C6 and A172 glioblastoma cells. Furthermore, the antisense-ODN/R3V6 complexes reduced the level of miR-21 and induced apoptosis of glioblastoma cells. These results suggest that the R3V6 peptide may be a useful carrier of antisense-ODN for glioblastoma gene therapy.

Combined delivery of BCNU and VEGF siRNA using amphiphilic peptides for glioblastoma.

Combined delivery of chemical drug and therapeutic gene has been introduced as an efficient method for the treatment of cancers such as glioblastoma. In this study, bis-chloroethylnitrosourea (BCNU) and vascular endothelial growth factor (VEGF) small interfering RNA (VEGF-siRNA) were co-delivered into C6 glioblastoma cells using a non-toxic peptide-based carrier. The R3V6 peptides, which are composed of 3-arginine and 6-valine, formed self-assembled micelles in aqueous solution. BCNU, a hydrophobic anti-cancer drug, was loaded into the hydrophobic core of the micelles, forming BCNU-loaded R3V6 micelles (R3V6-BCNU). In gel retardation assay, R3V6-BCNU formed a stable complex with siRNA. In vitro transfection assay showed that the VEGF-siRNA/R3V6-BCNU complex had the highest transfection efficiency into C6 cells at a 1:20 weight ratio (VEGF-siRNA:R3V6-BCNU). In addition, the VEGF-siRNA/R3V6-BCNU complexes had higher delivery efficiency than lipofectamine or naked siRNA. VEGF expressions were remarkably decreased by transfection of the VEGF-siRNA/R3V6 or VEGF-siRNA/R3V6-BCNU complexes. Furthermore, R3V6-BCNU delivered BCNU more efficiently into the cells than BCNU only. Therefore, R3V6 delivered both VEGF-siRNA and BCNU efficiently into the glioblastoma cells. The results suggest that R3V6-BCNU may be useful for combined delivery of siRNA and chemical drug into cancer cells.

Combined delivery of HMGB-1 box A peptide and S1PLyase siRNA in animal models of acute lung injury.

The combinational therapy with S1Plyase siRNA (siS1PLyase) and recombinant high mobility group box-1 box A peptide (HMGB1A) may have a synergistic effect for the treatment of acute lung injury (ALI). For efficient delivery, the R3V6 peptides were used as a carrier. The ternary complex of siS1PLyase, HMGB1A, and R3V6 was produced by charge interaction. The siS1PLyase/HMGB1A/R3V6 ternary complex delivered siRNA into the LA-4 lung epithelial cells more efficiently than polyethylenimine (PEI) and Lipofectamine. Furthermore, the ternary complex was non-toxic. The siS1PLyase/HMGB1A/R3V6 complex reduced the levels of IL-6 and TNF-α more efficiently than HMGB1A only or siS1PLyase/R3V6 complex in the LPS activated macrophage cells. In vivo administration of the siS1PLyase/HMGB1A/R3V6 complex reduced the S1PLyase level efficiently in an LPS-induced ALI model. Furthermore, the complex reduced the inflammatory response and apoptosis in the ALI model. In conclusion, siS1PLyase and HMGB1A have a synergistic therapeutic effect for the treatment of ALI. Furthermore, R3V6 is an efficient carrier for combined delivery of siS1PLyase and HMGB1A.

Post-translational regulation of gene expression using the ATF4 oxygen-dependent degradation domain for hypoxia-specific gene therapy.

Solid tumors have hypoxic regions in their cores, due to low blood supply levels. Therefore, hypoxia-specific gene regulation systems have been developed for tumor-specific gene therapy. In this study, the oxygen-dependent degradation (ODD) domain on activating transcription factor-4 (ATF4) was evaluated for post-translational regulation of proteins. The ATF4 ODD cDNA was amplified by RT-PCR, and a luciferase plasmid containing the ATF4 ODD domain, pSV-Luc-ATF4-ODD, was constructed. Luciferase expression was induced under hypoxia by the ATF4 ODD domain in transfection assays into N2A neuroblastoma cells, C6 glioblastoma cells, and U87 glioblastoma cells. In the transfection assay with pSV-Luc-ATF4-ODD, RT-PCR results showed that the mRNA level did not change under hypoxia. This suggests that the induction of luciferase under hypoxia was mediated by post-translational regulation. A plasmid expressing thymidine kinase from herpes simplex virus (HSV-tk), pSV-HSVtk-ATF4-ODD, was constructed with the ATF4 ODD cDNA. The transfection assay with pSV-TK-ATF4-ODD showed that the ATF4 ODD domain induced HSV-tk expression under hypoxia and facilitated the death of C6 cells in the presence of ganciclovir (GCV). Furthermore, pSV-HSVtk-ATF4-ODD induced caspase-3 activity in the hypoxic cells. In conclusion, the ATF4 ODD may be useful for hypoxia-specific gene therapy by post-translational regulation of gene expression.

The box A domain of high mobility group box-1 protein as an efficient siRNA carrier with anti-inflammatory effects.

High mobility group box-1 (HMGB-1) is a DNA binding nuclear protein and pro-inflammatory cytokine. The box A domain of HMGB-1 (rHMGB-1A) exerts an anti-inflammatory effect, inhibiting wild-type HMGB-1 (wtHMGB-1). In this study, HMGB-1A was evaluated as an siRNA carrier with anti-inflammatory effects. HMGB-1A was expressed and purified by consecutive nickel chelate chromatography, cationic exchange chromatography, and polymixin B chromatography. Purified rHMGB-1A demonstrated an anti-inflammatory effect, reducing tumor necrosis factor-α (TNF-α) in wtHMGB-1 or lipopolysaccharide (LPS) activated macrophages. In gel retardation assay, rHMGB-1A formed a stable complex with siRNA at or above a 1:2 weight ratio (siRNA:rHMGB-1A). A heparin competition assay showed that an siRNA/rHMGB-1A complex released siRNA more easily than an siRNA/polyethylenimine (PEI, 25 kDa) complex. Luciferase siRNA/rHMGB-1A reduced firefly luciferase expression at a similar level as luciferase siRNA/PEI complex. Furthermore, TNF-α siRNA/rHMGB-1A synergistically reduced TNF-α expression in LPS activated macrophages. Therefore, rHMGB-1A may be useful as an siRNA carrier with anti-inflammatory effects in siRNA therapy for various inflammatory diseases.