Ryuichiro Kimura

In this study, we analyzed the size-dependent bioactivities of fucoidan by comparing the cytotoxic effects of native fucoidan and fucoidan lipid nanoparticles on osteosarcoma in vitro and in vivo. In vitro experiments indicated that nanoparticle fucoidan induced apoptosis of an osteosarcoma cell line more efficiently than native fucoidan. The more potent effects of nanoparticle fucoidan, relative to native fucoidan, were confirmed in vivo using a xenograft osteosarcoma model. Caco-2 cell transport studies showed that permeation of nanoparticle fucoidan was higher than native fucoidan. The higher bioactivity and superior bioavailability of nanoparticle fucoidan could potentially be utilized to develop novel therapies for osteosarcoma.

Background: Crassocephalum crepidioides, a plant distributed in Okinawa Islands, is known in folk medicine; however, its anticancer activity has not been investigated. The aim of this study was to determine thein vitroandin vivoantitumor activities ofC. crepidioideson murine Sarcoma 180 (S-180) and related molecular mechanisms.Methods: The antitumor effect ofC. crepidioideswas evaluated in S-180-cell-bearing mice. Cell growth was assessed using a colorimetric assay. Nitrite and nitrate levels were measured by colorimetry. The expression levels of inducible NO synthase (iNOS) in murine RAW264.7 macrophages was assessed by reverse transcriptase-polymerase chain reaction. Activation of iNOS promoter was detected by reporter gene. Activation of nuclear factor-κB (NF-κB) was evaluated by electrophoretic mobility shift assay. The role of NF-κB signaling was analyzed using inhibitors of NF-κB and dominant-negative mutants, and Western blot analysis.Results: C. crepidioidesextract delayed tumor growth in S-180-bearing mice. However, it did not inhibit S-180 cell growthin vitro. Supernatant of culturedC. crepidioides-stimulated RAW264.7 macrophages was cytotoxic to S-180 cells. This cytotoxicity was associated with nitric oxide (NO) production. NF-κB signaling pathway was crucial for the transcriptional activation of iNOS gene. Isochlorogenic acid, a component ofC. crepidioides, induced NF-κB activation and iNOS expression.Conclusions: The results highlight the oncolytic and immunopotentiation properties ofC. crepidioidesmediated through NF-κB-induced release of NO from macrophages.

Background: DICER is an RNase III family endoribonuclease that processes precursor microRNAs (pre-miRNAs) and long double-stranded RNAs, generating microRNA (miRNA) duplexes and short interfering RNA duplexes with 20~23 nucleotides (nts) in length. The typical form of pre-miRNA processed by the Drosha protein is a hairpin RNA with 2-nt 3' overhangs. On the other hand, production of mature miRNA from an endogenous hairpin RNA with 5' overhangs has also been reported, although the mechanism for this process is unknown.Results: In this study, we show that human recombinant DICER protein (rDICER) processes a hairpin RNA with 5' overhangsin vitroand generates an intermediate duplex with a 29 nt-5' strand and a 23 nt-3' strand, which was eventually cleaved into a canonical miRNA duplex via a two-step cleavage. The previously identified endogenous pre-miRNA with 5' overhangs, pre-mmu-mir-1982 RNA, is also determined to be a substrate of rDICER through the same two-step cleavage.Conclusions: The two-step cleavage of a hairpin RNA with 5' overhangs shows that DICER releases double-stranded RNAs after the first cleavage and binds them again in the inverse direction for a second cleavage. These findings have implications for how DICER may be able to interact with or process differing precursor structures.