Browsing by Author "Ishida, Tatsuhiro"

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    Agitation during lipoplex formation harmonizes the interaction of siRNA to cationic liposomes.
    (2012) Barichello, José Mario; Kizuki, Shinji; Tagami, Tatsuaki; Soares, Luiz Alberto Lira; Ishida, Tatsuhiro; Kikuchi, Hiroshi; Kiwada, Hiroshi
    We recently demonstrated that agitation during lipoplex formation (vorLTsiR) improves the gene knockdown effect of siRNA because the resultant decrease in lipoplex size leads to an enhanced uptake by cells. In furthering this line of research, the present study was focused on the interaction of siRNA to cationic liposomes during lipoplex preparation. A fluorescence resonance energy transfer (FRET) study indicated that the application of agitation in the presence of siRNA effectively reorganized positively charged lipids (DC-6-14 and DOPE) in an order that effectively promoted further electrostatic interaction between the negatively charged phosphate backbone of siRNA and the positively charged lipids in the cationic liposome membrane. A circular dichroism (CD) study indicated that the agitation did not bring about a change in the A-form helix of siRNA, therefore the interactions between the lateral anionic groups of siRNA – responsible for the characteristic bands of the A-form helix – and cationic liposomes were effectively promoted. Factorial design coupled with response surface methodology was used to statistically analyze the influence of vortex speed and time and siRNA dose on the in vitro gene knockdown effects of siRNA-lipoplex that were spontaneously formulated (spoLTsiR) along with that formulated under agitation (vorLTsiR). The analysis indicated that vortex speed plays the most important role in enhancing the gene knockdown effect of siRNA among the three variables, although all three are important. It was concluded that the high energy transmitted by applying agitation during lipoplex formation harmonized the interaction of siRNA to positively charged lipids (DC-6-14 and DOPE) in cationic liposomes, resulting in a superior gene knockdown efficacy of vorLTsiR compared to spoLTsiR. Our study suggests that the preparation procedure is one of the critical factors in producing the enhanced gene knockdown effect of siRNA.
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    Argonaute2 is a potential target for siRNA-based cancer therapy for HT1080 human fibrosarcoma.
    (2001) Tagami, Tatsuaki; Suzuki, Takuya; Hirose, Kiyomi; Barichello, José Mario; Yamazaki, Naoshi; Asai, Tomohiro; Oku, Naoto; Ishida, Tatsuhiro; Kiwada, Hiroshi
    Small interfering RNAs (siRNAs) are small RNA molecules that have a potent, sequence-specific gene silencing effect and therefore show promise for therapeutic use as molecular-targeted drugs for the treatment of various genetic diseases, including cancer. The aim of the present study was to evaluate whether Argonaute2 (Ago2) is a therapeutically effective target for siRNA-based cancer therapy. Ago2 is the key protein in mammalian RNAi and is also known as the only member of the Ago family that mediates the microRNA (miRNA)-dependent cleavage of targeted mRNAs. It is assumed that these unique properties of the Ago2 protein can play a central role in the regulation of the miRNA pathway and subsequent translational inhibition of miRNA-targeted mRNAs, including cell survival and cancer progression. To assess its therapeutic effect, siRNA against Ago2 (Ago2-siRNA) was transfected into HT1080 human fibrosarcoma cells, which are malignant cancer cells. Ago2 gene silencing resulted in the inhibition of cell growth and the induction of apoptosis and G0/G1 arrest in the cell cycle. In addition, Ago2 knockdown induced morphological changes and actin stress fiber formation in the cells. The results of a microarray study showed that Ago2 suppression stimulated several crucial genes related to apoptosis, the cell cycle, immune response, cell adhesion, metabolism, etc. Repeated intratumoral injection of Ago2-siRNA/cationic liposome complex induced tumor growth suppression in an HT1080 xenograft model. These results suggest that the suppression of the Ago2 gene may be useful for the inhibition of cancer progression and that Ago2 may be a desirable target for siRNA-based cancer therapy.