Takayuki Kudoh

Domino Double Michael−Claisen Cyclizations: A Powerful General Tool for Introducing Quaternary Stereocenters at C(4) of Cyclohexane-1,3-diones and Total Synthesis of Diverse Families of Sterically Congested Alkaloids

Reactions of substituted acetone derivatives with acrylic acid esters (>200 mol %) in the presence of t-BuOK (200 mol %) in t-BuOH-THF (1:1 by volume) turned out to proceed as a cascade process consisting of the first Michael addition, the second Michael addition, and the last Claisen reaction to afford 4,4-disubstituted cyclohexane-1,3-diones. Only more substituted enolates play the role of a Michael donor in this cascade process, and therefore the ketone took up two alkoxycarbonylethyl groups on the same carbon bearing more substituents. Such intermediates were followed by intramolecular Claisen reactions leading to cyclohexane-1,3-diones bearing quaternary stereogenic centers at C(4), which bears an alkoxycarbonylethyl group and the substituent of the starting acetone derivatives. Thus-obtained 4,4-disubstituted cyclohexane-1,3-diones were successfully employed for total syntheses of intricate alkaloids of biological interest such as (+)-aspidospermidine, (+/-)-galanthamine, (+/-)-lycoramine, and (+/-)-mesembrine, all featuring quaternary stereogenic centers. DFT calculations provided us with clear-cut explanations for the observed chemoselectivity of the cascade process involving ketone-based enolates under thermodynamically controlled conditions.

A model of cancer stem cells derived from mouse induced pluripotent stem cells

Cancer stem cells (CSCs) are capable of continuous proliferation and self-renewal and are proposed to play significant roles in oncogenesis, tumor growth, metastasis and cancer recurrence. CSCs are considered derived from normal stem cells affected by the tumor microenvironment although the mechanism of development is not clear yet. In 2007, Yamanakas group succeeded in generating Nanog mouse induced pluripotent stem (miPS) cells, in which green fluorescent protein (GFP) has been inserted into the 5′-untranslated region of the Nanog gene. Usually, iPS cells, just like embryonic stem cells, are considered to be induced into progenitor cells, which differentiate into various normal phenotypes depending on the normal niche. We hypothesized that CSCs could be derived from Nanog miPS cells in the conditioned culture medium of cancer cell lines, which is a mimic of carcinoma microenvironment. As a result, the Nanog miPS cells treated with the conditioned medium of mouse Lewis lung carcinoma acquired characteristics of CSCs, in that they formed spheroids expressing GFP in suspension culture, and had a high tumorigenicity in Balb/c nude mice exhibiting angiogenesis in vivo. In addition, these iPS-derived CSCs had a capacity of self-renewal and expressed the marker genes, Nanog, Rex1, Eras, Esg1 and Cripto, associated with stem cell properties and an undifferentiated state. Thus we concluded that a model of CSCs was originally developed from miPS cells and proposed the conditioned culture medium of cancer cell lines might perform as niche for producing CSCs. The model of CSCs and the procedure of their establishment will help study the genetic alterations and the secreted factors in the tumor microenvironment which convert miPS cells to CSCs. Furthermore, the identification of potentially bona fide markers of CSCs, which will help the development of novel anti-cancer therapies, might be possible though the CSC model.

Novel and simple loading procedure of cisplatin into liposomes and targeting tumor endothelial cells

Although intravenous administration of high levels of cisplatin (CDDP) are limited due to its severe side effects, efficient delivery of CDDP directly to the tumor should improve the therapeutic response while potentially by-passing significant side effects. High loading of CDDP into liposomes is one technique that could be used as a potential drug delivery system. Since cis-diamminedinitratoplatinum (CDDP3) is highly soluble in water and converts to CDDP in the presence of chloride ions, we encapsulated CDDP3 into liposomes in the absence of chloride ions and supplemented chloride ions to prepare CDDP-encapsulated liposomes (CDDP-Lip) resulting in a significantly improved loading efficiency of CDDP. We further conjugated the CDDP-Lip with Sialyl Lewis(X) (CDDP-SLX-Lip) because we previously demonstrated Sialyl Lewis(X) enhanced efficient accumulation of liposomes into tumors in vivo. CDDP-SLX-Lip treated mice showed a survival rate of 75% at 14 days even if a lethal level of CDDP was injected into mice. Loss of body weight was negligible and no histological abnormality was found in a variety of normal tissues. Accumulation of CDDP-SLX-Lip was about 6 times more than that of CDDP-Lip or CDDP. As the result, there was better antitumor activity of CDDP-SLX-Lip than that of CDDP-Lip with significantly less toxic effects in normal tissues.

Development of 111In-Labeled Liposomes for Vulnerable Atherosclerotic Plaque Imaging

Macrophage infiltration is a common characteristic feature of atherosclerotic-vulnerable plaques. Macrophages recognize phosphatidylserine (PS) exposed on the surface of apoptotic cells, which triggers the engulfment of the apoptotic cells by macrophages through phagocytosis. In this study, we prepared radiolabeled PS liposomes for detection of vulnerable plaques. Methods: PS liposomes were prepared by lipid film hydration. Phosphatidylcholine (PC) liposomes were prepared as controls. Liposomes (100 or 200 nm) were generated by an extruder to produce PS100, PS200, PC100, and PC200 liposomes. These were then radiolabeled by encapsulating 111In-nitrilotriacetic acid using an active-loading method. 111In liposomes were incubated with cultured macrophages for 2 h, and the uptake level was measured. For biodistribution studies, the 111In liposomes were injected intravenously into ddY mice. In addition, the 111In liposomes were injected into apolipoprotein E–deficient (apoE−/−) mice, and the aortas were harvested for autoradiography and oil red O staining. For SPECT imaging, 111In liposomes were injected intravenously into Watanabe heritable hyperlipidemic rabbits and scanned 48 h after injection. Results: The radiochemical yields were greater than 95% for all the prepared 111In liposomes. The level of in vitro uptake by macrophages was 60.5, 14.7, 32.0, and 14.4 percentage injected dose per milligram of protein for 111In-PS100, 111In-PC100, 111In-PS200, and 111In-PC200, respectively. In biodistribution studies, high spleen uptake was seen with PC liposomes. Liver uptake was high for all liposomes but was lowest with 111In-PS200. The blood half-lives were 3.2, 22.0, 3.6, and 7.4 min for 111In-PS100, 111In-PC100, 111In-PS200, and 111In-PC200, respectively. The distribution of 111In-labeled PS liposomes into atherosclerotic regions determined by autoradiography was well matched with the results of oil red O staining in apoE−/− mice. The target-to-nontarget ratios were 2.62, 2.23, 3.27, and 2.51 for 111In-PS100, 111In-PC100, 111In-PS200, and 111In-PC200, respectively. The aorta was successfully visualized by SPECT at 48 h after 111In-labeled PS liposome injection; however, high liver uptake was also observed. Discussion: From the in vitro uptake study, it has been demonstrated that macrophage targeting was accomplished by PS modification. Also, an atherosclerotic region was successfully detected by 111In-PS200 in apoE−/− mice and Watanabe heritable hyperlipidemic rabbits in vivo. Liposome modification to obtain slower blood clearance and lower liver uptake would be required to improve the SPECT images.

Cancer stem cells maintain a hierarchy of differentiation by creating their niche

The self‐renewal and differentiation properties of cancer stem cells (CSCs) are regulated and maintained by the CSC niche. However, the mechanism of this maintenance, especially the maintenance contributed by differentiated cancer cells, remains to be fully elucidated. Recently, we have established a model of CSCs, miPS‐LLCcm, from mouse induced pluripotent stem cells (miPSCs). In vitro cultured miPS‐LLCcm cells were autonomously balanced with stem‐like cells and differentiated cells including vascular endothelial cells. Under these conditions, the CSC properties appeared to be stable in the presence of the factor(s) secreted by the differentiated cells. The factor(s) activated Notch signaling and promoted self‐renewal of CSCs. In addition, the secreted factor(s) appeared to regulate the differentiation lineage of CSCs. Our results indicate that the differentiated progenies of CSCs containing vascular endothelium play important roles for regulating the CSCs properties. Therefore, miPS‐LLCcm cells create their own in vitro niche to maintain themselves in the hierarchy of differentiating CSCs.

Efficient Drug Delivery of Paclitaxel Glycoside: A Novel Solubility Gradient Encapsulation into Liposomes Coupled with Immunoliposomes Preparation

Although the encapsulation of paclitaxel into liposomes has been extensively studied, its significant hydrophobic and uncharged character has generated substantial difficulties concerning its efficient encapsulation into the inner water core of liposomes. We found that a more hydrophilic paclitaxel molecule, 7-glucosyloxyacetylpaclitaxel, retained tubulin polymerization stabilization activity. The hydrophilic nature of 7-glucosyloxyacetylpaclitaxel allowed its efficient encapsulation into the inner water core of liposomes, which was successfully accomplished using a remote loading method with a solubility gradient between 40% ethylene glycol and Cremophor EL/ethanol in PBS. Trastuzumab was then conjugated onto the surface of liposomes as immunoliposomes to selectively target human epidermal growth factor receptor-2 (HER2)-overexpressing cancer cells. In vitro cytotoxicity assays revealed that the immunoliposomes enhanced the toxicity of 7-glucosyloxyacetylpaclitaxel in HER2-overexpressing cancer cells and showed more rapid suppression of cell growth. The immunoliposomes strongly inhibited the tumor growth of HT-29 cells xenografted in nude mice. Notably, mice survived when treated with the immunoliposomes formulation, even when administered at a lethal dose of 7-glucosyloxyacetylpaclitaxel in vivo. This data successfully demonstrates immunoliposomes as a promising candidate for the efficient delivery of paclitaxel glycoside.

Enhanced internalization of ErbB2 in SK‐BR‐3 cells with multivalent forms of an artificial ligand

Targeting and down‐regulation of ErbB2, a member of EGF receptor family, is regarded as one of the key aspect for cancer treatment because it is often overexpressed in breast and ovarian cancer cells. Although natural ligands for ErbB2 have not been found, unlike other ErbB receptors, EC‐1, a 20‐amino acid circular peptide, has been shown to bind to ErbB2 as an artificial ligand. Previously we showed EC‐1 peptide did not induce the internalization of ErbB2 in SK‐BR‐3 cells. In this report, we designed divalent and multivalent forms of EC‐1 peptide with the Fc portion of the human IgG and bionanocapsule modified with ZZ‐tag on its surface to improve the interaction with ErbB2. These forms showed higher affinity to ErbB2 than that of EC‐1 monomer. Furthermore, prominent endosomal accumulation of ErbB2 occurred in SK‐BR‐3 cells when stimulated with EC‐Fc ligand multivalently displayed on the surface of the bionanocapsule, whereas SK‐BR‐3 cells as themselves displayed stringent mechanism against ErbB2 internalization without stimulation. The multivalent form of EC‐1 peptide appeared to internalize ErbB2 more efficiently than divalent form did. This internalization was unaffected by the inhibition of clathrin association, but inhibited when the cholesterol was depleted which explained either caveolar or GPI‐AP‐early endocytic compartment (GEEC) pathway. Because of the lack of caveolin‐1 expression, caveolar machinery may be lost in SK‐BR‐3 cell line. Therefore, it is suggested that the multivalent form of EC‐1 induces the internalization of ErbB2 through the GEEC pathway.

Chlorotoxin Fused to IgG-Fc Inhibits Glioblastoma Cell Motility via Receptor-Mediated Endocytosis.

Chlorotoxin is a 36-amino acid peptide derived from Leiurus quinquestriatus (scorpion) venom, which has been shown to inhibit low-conductance chloride channels in colonic epithelial cells. Chlorotoxin also binds to matrix metalloproteinase-2 and other proteins on glioma cell surfaces. Glioma cells are considered to require the activation of matrix metalloproteinase-2 during invasion and migration. In this study, for targeting glioma, we designed two types of recombinant chlorotoxin fused to human IgG-Fcs with/without a hinge region. Chlorotoxin fused to IgG-Fcs was designed as a dimer of 60 kDa with a hinge region and a monomer of 30 kDa without a hinge region. The monomeric and dimeric forms of chlorotoxin inhibited cell proliferation at 300 nM and induced internalization in human glioma A172 cells. The monomer had a greater inhibitory effect than the dimer; therefore, monomeric chlorotoxin fused to IgG-Fc was multivalently displayed on the surface of bionanocapsules to develop a drug delivery system that targeted matrix metalloproteinase-2. The target-dependent internalization of bionanocapsules in A172 cells was observed when chlorotoxin was displayed on the bionanocapsules. This study indicates that chlorotoxin fused to IgG-Fcs could be useful for the active targeting of glioblastoma cells.

Characterization of Cancer Stem-Like Cells Derived from Mouse Induced Pluripotent Stem Cells Transformed by Tumor-Derived Extracellular Vesicles

Several studies have shown that cancer niche can perform an active role in the regulation of tumor cell maintenance and progression through extracellular vesicles-based intercellular communication. However, it has not been reported whether this vesicle-mediated communication affects the malignant transformation of normal stem cells/progenitors. We have previously reported that the conditioned medium derived from the mouse Lewis Lung Carcinoma (LLC) cell line can convert mouse induced pluripotent stem cells (miPSCs) into cancer stem cells (CSCs), indicating that normal stem cells when placed in an aberrant microenvironment can give rise to functionally active CSCs. Here, we focused on the contribution of tumor-derived extracellular vesicles (tEVs) that are secreted from LLC cells to induce the transformation of miPSCs into CSCs. We isolated tEVs from the conditioned medium of LLC cells, and then the differentiating miPSCs were exposed to tEVs for 4 weeks. The resultant tEV treated cells (miPS-LLCev) expressed Nanog and Oct3/4 proteins comparable to miPSCs. The frequency of sphere formation of the miPS-LLCev cells in suspension culture indicated that the self-renewal capacity of the miPS-LLCev cells was significant. When the miPS-LLCev cells were subcutaneously transplanted into Balb/c nude mice, malignant liposarcomas with extensive angiogenesis developed. miPS-LLCevPT and miPS-LLCevDT, the cells established from primary site and disseminated liposarcomas, respectively, showed their capacities to self-renew and differentiate into adipocytes and endothelial cells. Moreover, we confirmed the secondary liposarcoma development when these cells were transplanted. Taken together, these results indicate that miPS-LLCev cells possess CSC properties. Thus, our current study provides the first evidence that tEVs have the potential to induce CSC properties in normal tissue stem cells/progenitors.

Visible-Light-Derived Photocatalyst Based on TiO2−δNδ with a Tubular Structure

We succeeded in achieving visible-light responsiveness on a tubular TiO(2) sample through the treatment of a tubular TiO(2) that has a large surface area with an aqueous solution of ammonia or triethylamine at room temperature and subsequent calcination at 623 K, which produced a nitrided tubular TiO(2) sample. It was found that the ease of nitridation is dependent on the surface states; washing the tubular TiO(2) sample with an aqueous acidic solution is very effective and indispensable. This treatment causes the appearance of acidic sites on the tubular TiO(2), which was proved by the following experiments: NH(3) temperature-programmed desorption and two types of organic reactions exploiting the acid properties. The prepared samples, TiO(2-δ)N(δ), efficiently absorb light in the visible region, and they exhibit a prominent feature for the decomposition of methylene blue in an aqueous solution at 300 K under irradiation with visible light, indicating the achievement of visible-light responsiveness on the tubular TiO(2) sample. This type of tubular TiO(2-δ)N(δ) sample has merit in the sense that it has a large surface area and a characteristic high transparency for enabling photocatalytic reactions because it has a tubular structure and is composed of thin walls.