Shun-ichi Wada

Lanostane-type triterpenoids from the sclerotia of Inonotus obliquus possessing anti-tumor promoting activity.

Two new lanostane-type triterpenoids, 1 and 2 besides two known lanostane-type triterpenoids, 3 and 4 were isolated from the sclerotia of Inonotus obliquus. Their structures were determined to be lanosta-8,23E-diene-3beta,22R,25-triol (1) and lanosta-7:9(11),23E-triene-3beta,22R,25-triol (2) by spectral data. These compounds were tested for their anti-tumor-promoting activity using a short-term in vitro assay for EBV-EA activation induced by TPA. Compounds 1, 2 and 4 were stronger than the positive control, oleanolic acid. The most abundant compound 4 was investigated for the inhibitory effect in a two-stage carcinogenesis test on mouse skin using DMBA as an initiator and TPA as a promoter. Compound 4 was found to exhibit the potent anti-tumor promoting activity in the in vivo carcinogenesis test.

Cellular uptake of Aib-containing amphipathic helix peptide.

Cell-penetrating peptides (CPPs) are useful tools for the delivery of hydrophilic bioactive molecules, such as peptides, proteins, and oligonucleotides, across the cell membrane. To realize the delivery of therapeutic macromolecules by CPPs, the CPPs are required to show resistance to protease and no cytotoxicity. In order to produce potent non-toxic and protease-resistant CPPs with high cellular uptake, we designed an amphipathic helix peptide using α-aminoisobutyric acid (Aib, U) and named it MAP(Aib). In the MAP(Aib) molecule, five Aib residues are aligned on the hydrophobic face of the helix and five lysine (K) residues are aligned on the hydrophilic face. MAP(Aib) showed potent resistance to trypsin and pronase compared with MAP, an amphipathic helix peptide formed by usual amino acids. Fluorescein-labeled MAP(Aib) efficiently traversed the A549 cell membrane, diffusing into the cytoplasm and slightly into the nucleus without exerting any cytotoxicity. In contrast, MAP was poorly taken up by the cell. These results indicate that the incorporation of Aib residues into CPPs markedly improves cellular uptake and MAP(Aib) may be a useful tool for the delivery of hydrophilic macromolecules.

Bisphenol A 3,4-quinone induces the conversion of xanthine dehydrogenase into oxidase in vitro.

In the present study, we assessed the influence of bisphenol A (BPA) and bisphenol A 3,4-quinone (BPAQ) on the conversion of xanthine dehydrogenase (XD) into xanthine oxidase (XO) in the rat liver in vitro. BPA up to 100 micromol/L did not affect the XO and XD activities in the partially purified cytosolic fraction from rat liver, whereas BPAQ (2-10 micromol/L) dose-dependently enhanced the XO activity concomitant with a decrease in the XD activity, implying that BPAQ, but not BPA, can convert XD into the reactive oxygen species (ROS) producing the form XO. Furthermore, it was found that BPAQ could increase the generation of ROS and oxidize the guanine moiety of deoxyguanosine in the DNA of primary rat hepatocyte cultures. These results suggest that BPAQ has the potential to convert XD into XO in the liver, which in turn may lead to ROS generation and oxidative DNA damage in this region.

Anti-tumor-initiating effects of spiro-biflavonoids from Abies sachalinensis.

Cancer chemoprevention, the prevention of cancer by ingestion of chemical agents that reduce the risk of carcinogenesis, is one of the potent ways to reduce morbidity and mortality. We have been searching for cancer chemopreventive agents from the leaves and barks of coniferous trees that have been treated as waste in the forestry industry. We have previously reported the isolation of spiro‐biflavonoids, named as abiesinols, and a neolignan from the MeOH extract of the bark of Abies sachalinensis. These compounds were tested for their inhibitory effects on the activation of (±)‐(E)‐methyl‐2‐[(E)‐hydroxyimino]‐5‐nitro‐6‐methoxyhex‐3‐enamide (NOR 1), a nitric oxide (NO) donor, as a primary screening test for anti‐tumor initiators. All compounds tested exhibited potent inhibitory effects on NOR 1 activation. Furthermore, abiesinol A, bearing a spiro‐biflavonoid skeleton, showed remarkable anti‐tumor‐initiating activity in the in vivo two‐stage mouse skin carcinogenesis test using peroxynitrite (ONOO−; PN) as the initiator and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) as the promoter.

Effect of Ala replacement with Aib in amphipathic cell-penetrating peptide on oligonucleotide delivery into cells.

A number of cell-penetrating peptides (CPPs) have been characterized and their usefulness as delivery tools has been clarified. As one of the CPPs, model amphipathic peptide (MAP) was developed by integrating both hydrophobic and hydrophilic amino acids in its sequence. In our previous work, we designed MAP(Aib) by replacing five alanine (Ala) residues on the hydrophobic face of the helix in the MAP sequence with α-aminoisobutyric acid (Aib) residues, and the replacement resulted in higher helix propensity, stronger resistance to protease, and higher cell membrane permeability than MAP. As a next step, we examined the efficiency of oligonucleotide (ODN) delivery into cells by MAP(Aib) in comparison with that by MAP. The electrostatically formed MAP(Aib)/ODN complex was more easily taken up by cells than the MAP/ODN complex, and the ODN delivery by MAP(Aib) was via an endocytic pathway. We demonstrated that the incorporation of Aib residues into CPPs enhances the delivery of hydrophilic molecules, such as ODN, into cells.

In vitro inhibitory effects of DNA topoisomerase II by fernane-type triterpenoids isolated from a Euphorbia genus

Several kinds of naturally occurring fernane-type triterpenoids isolated from a Euphorbia genus were tested on the inhibitory effects of DNA Topoisomerases I (Topo I) and II (Topo II) activities. A-ring cleaved 3,4-seco-8 beta H-ferna-4(23),9(11)-dien-3-oic acid and its 3-hydroxyl derivative were found to be selective inhibitors of Topo II activity without the stabilization of a DNA/Topo II cleavable complex.

Anti-tumor-initiating effects of phenolic compounds isolated from the bark of Picea jezoensis var. jezoensis.

We have previously reported the isolation of nine phenolic compounds including three new flavonostilbenes, jezonocinols A, B, and C, from the MeOH extract of the bark of Picea jezoensis var. jezoensis. Further investigation of the MeOH extract led to the isolation of three new stilbene-type compounds and one new 1,4-benzodioxane-type compound, together with seven known phenolic compounds. These compounds were tested for their inhibitory effects on the activation of (+/-)-(E)-methyl-2-[(E)-hydroxy-imino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitric oxide (NO) donor, as a primary screening test for anti-tumor initiators. All compounds tested exhibited potent inhibitory effects on NOR 1 activation. Furthermore, jezonocinol B, the most potent inhibitor of NOR 1 activation, showed remarkable anti-tumor-initiating activity in the in vivo two-stage mouse skin carcinogenesis test using peroxynitrite (ONOO(-); PN) as the initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as the promoter.

Cancer Chemopreventive Activity of Oleanane‐Type Triterpenoids from the Stem Bark of Betula ermanii

In search for cancer chemopreventive agents from natural sources, three oleanane‐ and four known lupane‐type triterpenoids, and sitosterol from the stem bark of Betula ermanii were tested for their inhibitory effects on Epstein–Barr virus early antigen (EBV‐EA) activation induced by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Among them, 3β‐acetoxy‐12α‐hydroxyoleanan‐13β,28‐olide (1) and 3β‐acetoxy‐11α,12α‐epoxyoleanan‐13β,28‐olide (2) were investigated for the inhibitory effect in a two‐stage carcinogenesis test on mouse skin using 7,12‐dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter. 3β‐Acetoxy‐11α,12α‐epoxyoleanan‐13β,28‐olide (2) was found to exhibit the potent antitumor promoting activity in the in vivo carcinogenesis test.

Translocation of an Aib-containing peptide through cell membranes.

The biophysical characteristics and channel-forming activity of peptaibols inserted into artificial membranes have been studied over the last 30 years. However, to our knowledge, no studies have addressed directly their behavior in living cells. In this work, a novel strategy has been employed to precisely assess the living cell membrane-penetrating activity of a fluorescein-labeled Aib (alpha-aminoisobutyric acid)-containing peptide derived from a peptaibol, trichorovin-XIIa (TV-XIIa). We have demonstrated for the first time that the peptide containing an unusual amino acid residue, Aib, is taken up by cells via a non endocytic pathway. The replacement of Aib in the TV-XIIa sequence with Ala inhibits the cellular uptake.

Design of cyclic RGD-conjugated Aib-containing amphipathic helical peptides for targeted delivery of small interfering RNA.

To achieve the targeted delivery of siRNA, five conjugates of Aib-containing amphipathic helical peptides with mono-, di-, and trivalent cRGDfC [cyclo(-Arg-Gly-Asp-d-Phe-Cys-)], which is known to bind to αVβ3 integrin, at several positions of the amphipathic helical peptide were designed and synthesized. Among the five conjugates, the monovalent cRGDfC conjugating at position 20 of the amino acid sequence of the helical peptide through the formation of a disulfide bond (PI) and the divalent cRGDfC conjugating at positions 2 and 14 of the amino acid sequence of the helical peptide through the formation of disulfide bonds (PIII) significantly enhanced the delivery of fluorescence-labeled siRNA into A549 cells as the peptide/siRNA complex formed by electrostatic interaction. The cellular uptake of the PI/siRNA complex was mediated by both endocytic and non-endocytic pathways, whereas that of the PIII/siRNA complex was enabled by endocytosis. Furthermore, the cellular uptake of the PI/siRNA complex might involve specific interactions of the RGD group with the αVβ3 integrin receptor. Next, the RNAi effect of the peptide/siRNA complex on luciferase expression in A549-Luc cells was examined. Luciferase expression was significantly decreased in the presence of the complex at the concentration of 1.0μM PI/10nM siRNA. In contrast, the PIII/siRNA complex did not show the RNAi effect under the same conditions. However, extending the incubation time led to the suppression of the luciferase expression in the presence of the PIII/siRNA complex. Considering that the cellular uptake of the PIII/siRNA complex is mediated by the endocytic pathway, the release of siRNA from the endosome into the cytosol might require a long time. We present herein a useful and unique tool for the delivery of siRNA.