EDITORIAL

Ligand-mediated drug delivery systems have enormous potential for improving the efficacy of cancer treatment. In particular, Arg-Gly-Asp peptides are promising ligand molecules for targeting α_vβ₃/α_vβ₅ integrins, which are overexpressed in angiogenic sites and tumors, such as intractable human glioblastoma (U87MG). We here achieved highly efficient drug delivery to U87MG tumors by using a platinum anticancer drug-incorporating polymeric micelle (PM) with cyclic Arg-Gly-Asp (cRGD) ligand molecules. Intravital confocal laser scanning microscopy revealed that the cRGD-linked polymeric micelles (cRGD/m) accumulated rapidly and had high permeability from vessels into the tumor parenchyma compared with the PM having nontargeted ligand, “cyclic-Arg-Ala-Asp” (cRAD). As both cRGD/m- and cRAD-linked polymeric micelles have similar characteristics, including their size, surface charge, and the amount of incorporated drugs, it is likely that the selective and accelerated accumulation of cRGD/m into tumors occurred <i>via</i> an active internalization pathway, possibly transcytosis, thereby producing significant antitumor effects in an orthotopic mouse model of U87MG human glioblastoma

Confirmation of the screening system of the shRNA library by using the shRNA directed against human Ago2 (pcPURU6i-Ago2).

<p>(A) General schematic diagram of the screening procedure. (B) Schematic diagram of the optimization for the screening condition using Ago2 as a positive control. (C) The inhibitory effects of the shRNA directed against human Ago2 (pcPURU6i-Ago2) on RNAi. The second transfection was performed at 72 h, 80 h and 88 h after the first transfection, and the luciferase activity was determined at 80 h, 88 h and 96 h after the first transfection. Data was shown as mean and S.D. (n = 3). *<i>P</i><0.05.</p

Phylogenetic tree showing homologs of DDX3 in selected species, formatted with TreeView (Page, 1996) using the ClustalW output.

<p>Accession numbers of sequences used in the calculation includes: HuDDX3, human DDX3; NP_076829, HuDDX4; human DDX4, NP_077726; HuDDX18, human DDX18, NP_006764; HuDDX23, human DDX23, NP_004809; HuGU2, human GU2, NP_076950; DmBel, <i>D. melanogaster</i> Bel, NP_536783; CeMut-14, <i>C. elegans</i> MUT-14, NP_504490; CeGLH-1, <i>C. elegans</i> GLH-1, NP_491963; CeGLH-2, <i>C. elegans</i> GLH-2, NP_491876; CeGLH-3, <i>C. elegans</i> GLH-3, NP_491681; CeGLH-4, <i>C. elegans</i> GLH-4, NP_491207; CeY38A10A.6, <i>C. elegans</i> CeY38A10A.6, NP_504574; ScDbp1p, <i>Saccharomyces cerevisiae</i> Dbp1p, NP_015206; and AraNM_126777, <i>Arabidopsis thaliana</i> putative helicase, NP_178818.G.</p

Screening of the shRNA library for identifying essential factors involved in RNAi pathway.

<p>(A) The first screening scheme and results of the shRNA library directed against human helicases and RNAi-related genes. The screening was performed in duplicate. (B) Results of control experiments for inhibitory effects on RNAi. Luciferase data before correction (upper panel), results of control experiments (middle panel), and data after correction (bottom panel) are shown. Clones #1, #2 and #46 are empty shRNA vector (negative control), and clone #24 is Ago2 (positive control). (C) Multi-target analysis using five shRNA vectors directed against various sites in DDX3 mRNA. #, clone that showed reduced cell proliferation. Data was shown as mean and S.D. (n = 3). *<i>P</i><0.05.</p

Effect of a dominant-negative mutant of DDX3 on RNAi. HeLa S3 cells were transfected with 250 ng of the DDX3-expression vector or DDX3 mutant expression vector, 10 ng of the shRNA vector directed against firefly luciferase, 25 ng of the firefly luciferase expression vector, and 2.5 ng of the <i>Renilla</i> luciferase-expression vector.

<p>Cells were harvested 24 h after transfection and assayed for luciferase activity. Data was shown as mean and S.D. (n = 3). *<i>P</i><0.05.</p

Colocalization of DDX3 with Ago2 in HeLa S3 cells.

<p>(A) Subcellular localization of Flag-tagged DDX3 (Red) and HA-tagged Ago2 (Green) in HeLa S3 cells, as determined by anti-Flag and anti-HA immunofluorescence microscopy. (B) Flag-tagged DDX3 is colocalized with the P-body marker DCP1a in P-body granules.</p

Suppression of the expression of DDX3 reduces the effects of RNAi on expression of genes for GFP and DsRed.

<p>HeLa S3 cells were transfected with an shRNA vector against firefly luciferase (A–F) or an shRNA vector against DDX3 (G–L) and selected by exposure to puromycin. Then, the cells were cotransfected with an shRNA vector directed against GFP or DsRed, a GFP-expression vector and a DsRed-expression vector. Finally, 48 h after the second transfection, the expression of GFP and DsRed was monitored by fluorescence microscopy.</p