Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Deep-learning workflows of microscopic image analysis are sufficient for handling the contextual variations because they employ biological samples and have numerous tasks. The use of well-defined annotated images is important for the workflow. Cancer stem cells (CSCs) are identified by specific cell markers. These CSCs were extensively characterized by the stem cell (SC)-like gene expression and proliferation mechanisms for the development of tumors. In contrast, the morphological characterization remains elusive. This study aims to investigate the segmentation of CSCs in phase contrast imaging using conditional generative adversarial networks (CGAN). Artificial intelligence (AI) was trained using fluorescence images of the Nanog-Green fluorescence protein, the expression of which was maintained in CSCs, and the phase contrast images. The AI model segmented the CSC region in the phase contrast image of the CSC cultures and tumor model. By selecting images for training, several values for measuring segmentation quality increased. Moreover, nucleus fluorescence overlaid-phase contrast was effective for increasing the values. We show the possibility of mapping CSC morphology to the condition of undifferentiation using deep-learning CGAN workflows.

DOI： 10.3390/biom10060931

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FUJI TECHNOLOGY PRESS LTD.  

DOI： 10.20965/jaciii.2020.p0134

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Excess iron causes cancer and is thought to be related to carcinogenesis and cancer progression including stemness, but the details remain unclear. Here, we hypothesized that stemness in cancer is related to iron metabolism and that regulating iron metabolism in cancer stem cells (CSCs) may be a novel therapy. In this study, we used murine induced pluripotent stem cells that expressed specific stem cell genes such as Nanog, Oct3/4, Sox2, Klf4, and c-Myc, and two human cancer cell lines with similar stem cell gene expression. Deferasirox, an orally available iron chelator, suppressed expression of stemness markers and spherogenesis of cells with high stemness status in vitro. Combination therapy had a marked antitumor effect compared with deferasirox or cisplatin alone. Iron metabolism appears important for maintenance of stemness in CSCs. An iron chelator combined with chemotherapy may be a novel approach via suppressing stemness for CSC targeted therapy.

DOI： 10.3390/cancers11020177

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The surface reactivity of gold nanoparticles (AuNPs) is receiving attention as a radiosensitizer of cancer cells for radiation therapy and/or as a drug carrier to target cells. This study demonstrates the potential of DNA-AuNPs (prepared by mixing calf thymus DNA with HAuCl4 solution) as a radiosensitizer of human glioma cells that have cancer stem cell (CSC)-like properties, to reduce their survival. CSC-like U251MG-P1 cells and their parental glioblastoma U251MG cells are treated with a prepared DNA-AuNP colloid. The radiosensitivity of the resultant AuNP-associated cells are significantly enhanced. To reveal the mechanism by which survival is reduced, the generation of reactive oxygen species (ROS), apoptosis induction, or DNA damage in the cells is assayed using the fluorescent dye DCFDA, annexin V-FITC/PI, and foci formation of γ-H2AX, respectively. X-ray irradiation with administration of AuNPs overcomes the radioresistance of U251MG-P1 cells. It does not induce ROS generation or apoptosis in the cells but enhances the number of abnormal nuclei with abundant γ-H2AX foci, which is judged as cell death by mitotic catastrophe. The AuNP association with the cells effectively induces mitotic catastrophe in x-ray-irradiated CSC-like cells, implicating that DNA-AuNPs might be a promising tool to develop an efficient radiosensitizer against CSC.

DOI： 10.1088/1361-6528/aaedd5

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Language：English   Publishing type：Research paper (international conference proceedings)  

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Administration of bone marrow-derived mesenchymal stem cells (MSCs) is a possible treatment for graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation and other inflammatory conditions. To address the mechanism of immunosuppression by MSCs, in particular those derived from adipose tissue (AMSCs), AMSCs were isolated from three different mouse strains, and the suppressive capacity of the AMSCs thus obtained to suppress interferon (IFN)-γ generation in mixed lymphocyte reaction cultures serving as an in vitro model of GVHD were assessed. It was revealed that the AMSCs had a potent capacity to suppress IFN-γ production regardless of their strain of origin and that such suppression was not associated with production of interleukin-10. In addition, the results demonstrated that β2-microglobulin (β2m)-deficient AMSCs from β2m-/- mice were also potent suppressor cells, verifying the fact that the mechanism underlying the suppression by AMSCs is independent of major histocompatibility complex (MHC) class I expression or MHC compatibility. As AMSCs appear to have immunosuppressive properties, AMSCs may be a useful source of biological suppressor cells for the control of GVHD in humans.

DOI： 10.3892/etm.2018.6689

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Cancer cells often secrete extracellular vesicles (EVs) that carry heat shock proteins (HSPs) with roles in tumor progression. Oral squamous cell carcinoma (OSCC) belongs to head and neck cancers (HNC) whose lymph-node-metastases often lead to poor prognosis. We have examined the EV proteome of OSCC cells and found abundant secretion of HSP90-enriched EVs in lymph-node-metastatic OSCC cells. Double knockdown of HSP90α and HSP90β, using small interfering RNA significantly reduced the survival of the metastatic OSCC cells, although single knockdown of each HSP90 was ineffective. Elevated expression of these HSP90 family members was found to correlate with poor prognosis of HNC cases. Thus, elevated HSP90 levels in secreted vesicles are potential prognostic biomarkers and therapeutic targets in metastatic OSCC.

DOI： 10.1002/jcb.27039

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Previously, we have succeeded in converting induced pluripotent stem cells (iPSCs) into cancer stem cells (CSCs) by treating the iPSCs with conditioned medium of Lewis lung carcinoma (LLC) cells. The converted CSCs, named miPS-LLCcm cells, exhibited the self-renewal, differentiation potential, and potential to form malignant tumors with metastasis. In this study, we further characterized miPS-LLCcm cells both in vivo and in vitro. The tumors formed by subcutaneous injection showed the structures with pathophysiological features consisting of undifferentiated and malignant phenotypes generally found in adenocarcinoma. Metastasis in the lung was also observed as nodule structures. Excising from the tumors, primary cultured cells from the tumor and the nodule showed self-renewal, differentiation potential as well as tumor forming ability, which are the essential characters of CSCs. We then characterized the epigenetic regulation occurring in the CSCs. By comparing the DNA methylation level of CG rich regions, the differentially methylated regions (DMRs) were evaluated in all stages of CSCs when compared with the parental iPSCs. In DMRs, hypomethylation was found superior to hypermethylation in the miPS-LLCcm cells and its derivatives. The hypo- and hypermethylated genes were used to nominate KEGG pathways related with CSC. As a result, several categories were defined in the KEGG pathways from which most related with cancers, significant and high expression of components was PI3K-AKT signaling pathway. Simultaneously, the AKT activation was also confirmed in the CSCs. The PI3K-Akt signaling pathway should be an important pathway for the CSCs established by the treatment with conditioned medium of LLC cells.

DOI： 10.1016/j.tranon.2018.03.001

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We recently have established a successful xenograft model of human glioblastoma cells by enriching hyaluronic acid-dependent spheroid-forming populations termed U251MG-P1 cells from U251MG cells. Since U251MG-P1 cells have been confirmed to express CD44 along with principal stemness marker genes, OCT3/4, SOX2, KLF4 and Nanog, this CD44 expressing population appeared to majorly consist of undifferentiated cells. Evaluating the sensitivity to anti-cancer agents, we found U251MG-P1 cells were sensitive to doxorubicin with IC50 at 200 nM. Although doxorubicin has serious side-effects, establishment of an efficient therapy targeting undifferentiated glioblastoma cell population is necessary. We previously designed a chlorotoxin peptide fused to human IgG Fc region without hinge sequence (M-CTX-Fc), which exhibited a stronger growth inhibitory effect on the glioblastoma cell line A172 than an original chlorotoxin peptide. Combining these results together, we designed M-CTX-Fc conjugated liposomes encapsulating doxorubicin and used U251MG-P1 cells as the target model in this study. The liposome modified with M-CTX-Fc was designed with a diameter of approximately 100-150 nm and showed high encapsulation efficiency, adequate loading capacity of anticancer drug, enhanced antitumor effects demonstrating increasing uptake into the cells in vitro; M-CTX-Fc-L-Dox shows great promise in its ability to suppress tumor growth in vivo and it could serve as a template for targeted delivery of other therapeutics.

DOI： 10.3390/ijms19030659

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Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

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Adequate iron levels are essential for human health. However, iron overload can act as catalyst for the formation of free radicals, which may cause cancer. Cancer stem cells (CSCs), which maintain the hallmark stem cell characteristics of self-renewal and differentiation capacity, have been proposed as a driving force of tumorigenesis and metastases. In the present study, we investigated the role of iron in the proliferation and stemness of CSCs, using the miPS-LLCcm cell model. Although the anti-cancer agents fluorouracil and cisplatin suppressed the proliferation of miPS-LLCcm cells, these drugs did not alter the expression of stemness markers, including Nanog, SOX2, c-Myc, Oct3/4 and Klf4. In contrast, iron depletion by the iron chelators deferasirox and deferoxamine suppressed the proliferation of miPS-LLCcm cells and the expression of stemness markers. In an allograft model, deferasirox inhibited the growth of miPS-LLCcm implants, which was associated with decreased expression of Nanog and Sox2. Altogether, iron appears to be crucial for the proliferation and maintenance of stemness of CSCs, and iron depletion may be a novel therapeutic strategy to target CSCs.

DOI： 10.18632/oncotarget.21846

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Taxanes including paclitaxel and docetaxel are effective anticancer agents preferably sufficient for liposomal drug delivery. However, the encapsulation of these drugs with effective amounts into conventional liposomes is difficult due to their high hydrophobicity. Therefore, an effective encapsulation strategy for liposomal taxanes has been eagerly anticipated. In this study, the mixture of polyethoxylated castor oil (Cremophor EL) and ethanol containing phosphate buffered saline termed as CEP was employed as a solvent of the inner hydrophilic core of liposomes where taxanes should be incorporated. Docetaxel-, paclitaxel-, or 7-oxacetylglycosylated paclitaxel-encapsulating liposomes were successfully prepared with almost 100% of encapsulation efficiency and 29.9, 15.4, or 29.1 mol% of loading efficiency, respectively. We then applied the docetaxel-encapsulating liposomes for targeted drug delivery. Docetaxel-encapsulating liposomes were successfully developed HER2-targeted drug delivery by coupling HER2-specific binding peptide on liposome surface. The HER2-targeting liposomes exhibited HER2-specific internalization and enhanced anticancer activity in vitro. Therefore, we propose the sophisticated preparation of liposomal taxanes using CEP as a promising formulation for effective cancer therapies.

DOI： 10.3390/nano7100290

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Language：English   Publisher：(一社)日本癌学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Cancer-associated fibroblasts (CAFs) are one of the most prominent cell types in the stromal compartment of the tumor microenvironment. CAFs support multiple aspects of cancer progression, including tumor initiation, invasion, and metastasis. The heterogeneous nature of the stromal microenvironment is attributed to the multiple sources from which the cells in this compartment originate. The present study provides the first evidence that cancer stem cells (CSCs) are one of the key sources of CAFs in the tumor niche. We generated CSC-like cells by treating mouse induced pluripotent stem cells with conditioned medium from breast cancer cell lines. The resulting cell population expressed both CSC and pluripotency markers, and the sphere-forming CSC-like cells formed subcutaneous tumors in nude mice. Intriguingly, these CSC-like cells always formed heterogeneous populations surrounded by myofibroblast-like cells. Based on this observation, we hypothesized that CSCs could be the source of the CAFs that support tumor maintenance and survival. To address this hypothesis, we induced the differentiation of spheres and purified the myofibroblast-like cells. The resulting cells exhibited a CAF-like phenotype, suggesting that they had differentiated into the subpopulations of cells that support CSC self-renewal. These findings provide novel insights into the dynamic interplay between various microenvironmental factors and CAFs in the CSC niche.

DOI： 10.1038/s41598-017-07144-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

Lung cancer is one of the major causes of cancer-related mortality worldwide, and non-small-cell lung cancer is the most common form of lung cancer. Several studies had shown that thalidomide has potential for prevention and therapy of cancer. Therefore, the current study aimed to investigate the antitumor effects of two novel thalidomide analogs in human lung cancer A549 cells. The antiproliferative, antimigratory, and apoptotic effects in A549 cells induced by thalidomide analogs were examined. In addition, their effects on the expression of mRNAs encoding vascular endothelial growth factor165 (VEGF165) and matrix metalloproteinase-2 (MMP-2) were evaluated. Their influence on the tumor volume in nude mice was also determined. Results revealed that thalidomide analogs exhibited antiproliferative, antimigratory, and apoptotic activities with more pronounced effect than thalidomide drug. Furthermore, analogs 1 and 2 suppressed the expression levels of VEGF165 by 42% and 53.2% and those of MMP-2 by 45% and 52%, respectively. Thalidomide analogs 1 and 2 also reduced the tumor volume by 30.11% and 53.52%, respectively. Therefore, this study provides evidence that thalidomide analogs may serve as a new therapeutic option for treating lung cancer. (C) 2016 Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.biopha.2016.11.063

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Publisher：{OMICS} Publishing Group  

DOI： 10.4172/2157-7633.1000384

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HUMANA PRESS INC  

Transplantation of hematopoietic stem and progenitor cells (HSCs) i.e., self-renewing cells that retain multipotentiality, is now a widely performed therapy for many hematopoietic diseases. However, these cells are present in low number and are subject to replicative senescence after extraction; thus, the acquisition of sufficient numbers of cells for transplantation requires donors able to provide repetitive blood samples and/or methods of expanding cell numbers without disturbing cell multipotentiality. Previous studies have shown that HSCs maintain their multipotentiality and self-renewal activity if TCF3 transcription function is blocked under B cell differentiating conditions. Taking advantage of this finding to devise a new approach to HSC expansion in vitro, we constructed an episomal expression vector that specifically targets and transiently represses the TCF3 gene. This consisted of a vector encoding a transcription activator-like effector (TALE) fused to a Kruppel-associated box (KRAB) repressor. We showed that this TALE-KRAB vector repressed expression of an exogenous reporter gene in HEK293 and COS-7 cell lines and, more importantly, efficiently repressed endogenous TCF3 in a human B lymphoma cell line. These findings suggest that this vector can be used to maintain multipotentiality in HSC being subjected to a long-term expansion regimen prior to transplantation.

DOI： 10.1007/s12010-016-2187-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Libertas Academica Ltd.  

We performed gene expression microarray analysis coupled with spherical self-organizing map (sSOM) for artificially developed cancer stem cells (CSCs). The CSCs were developed from human induced pluripotent stem cells (hiPSCs) with the conditioned media of cancer cell lines, whereas the CSCs were induced from primary cell culture of human cancer tissues with defined factors (OCT3/4, SOX2, and KLF4). These cells commonly expressed human embryonic stem cell (hESC)/hiPSC-specific genes (POU5F1, SOX2, NANOG, LIN28, and SALL4) at a level equivalent to those of control hiPSC 201B7. The sSOM with unsupervised method demonstrated that the CSCs could be divided into three groups based on their culture conditions and original cancer tissues. Furthermore, with supervised method, sSOM nominated TMED9, RNASE1, NGFR, ST3GAL1, TNS4, BTG2, SLC16A3, CD177, CES1, GDF15, STMN2, FAM20A, NPPB, CD99, MYL7, PRSS23, AHNAK, and LOC152573 genes commonly upregulating among the CSCs compared to hiPSC, suggesting the gene signature of the CSCs.

DOI： 10.4137/CIN.S39839

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A plant-derived neocryptolepine core, the 5-Me-indolo[2,3-b]quinoline skeleton, was emblazoned with substituents at C11 and C2 and then tested against various cancer cell lines to find potent anticancer agents. In the in vitro antiproliferative activity assay against the breast cancer MDA-MB-453 cell line, the attachment of alkylamino substituents at C11 of the 5-Me-indolo[2,3-b]quinoline induced improved activities. Specifically, 11-(3-aminopropylamino) and 11-(4-aminobutylamino) derivatives indicated the highest activity and selectivity against MDA-MB-453 (IC = 0.3-0.5 μM) and also exhibited a higher cytotoxicity against the colon adenocarcinoma (WiDr) and ovarian cancer (SKOv3) cell lines. A synergistic effect by attachment of substituents at C2 was favorably observed with an electron-donating group, such as CH O, and unfavorably observed with an electron-withdrawing one, such as F and CF . Further modification of the terminal free amino group of the lariat attachment at C11 into the corresponding acylamides and 2,3-dihydrobenzo[e][1,3]thiazin-4-ones was not effective for the antiproliferative activity. The computer-assisted database analysis, COMPARE, suggested that 14e and 13b have a mode of action similar to actinomycin D and 13c has a mode of actions similar to vindesine sulfate or aclarubicin hydrochloride. However, the new compounds may have other unique mode of actions since the correlation coefficients (r) were in relatively low levels. 50 3 3

DOI： 10.1007/s00044-016-1508-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Docetaxel comprises one of the most effective anti-cancer drugs despite of serious side effects. Liposomes encapsulation is practically feasible to deliver the drug. However, due to the significant hydrophobicity, docetaxel will be integrated into the lipid bilayer resulting in poor encapsulation capacity. Here, we evaluated a remote loading strategy using a solubility gradient made between the two solvents for 7-glucosyloxyacetyldocetaxel, which has enhanced water solubility of docetaxel with a coupled glucose moiety. Therefore, 7-glucosyloxyacetyldocetaxel was more effectively encapsulated into liposomes with 71.0% of encapsulation efficiency than docetaxel. While 7-glucosyloxyacetyldocetaxel exhibited 90.9% of tubulin stabilisation activity of docetaxel, 7-glucosyloxyacetyldocetaxel encapsulated in liposomes significantly inhibited the growth of tumour in vivo with side effects less than unencapsulated drug. Collectively, the encapsulation of 7-glucosyloxyacetyldocetaxel into liposomes by remote loading under the solubility gradient is considered to be a promising application to prepare practical drug delivery system.

DOI： 10.3109/02652048.2016.1144815

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DOI： 10.4137/BIC.S33542

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：E-CENTURY PUBLISHING CORP  

Pancreatic ductal adenocarcinoma (PDAC) is the most representative form of pancreatic cancers. PDAC solid tumours are constituted of heterogeneous populations of cells including cancer stem cells (CSCs), differentiated cancer cells, desmoplastic stroma and immune cells. The identification and consequent isolation of pancreatic CSCs facilitated the generation of genetically engineered murine models. Nonetheless, the current models may not be representative for the spontaneous tumour occurrence. In the present study, we show the generation of a novel pancreatic iPSC-converted cancer stem cell lines (CSCcm) as a cutting-edge model for the study of PDAC. The CSCcm lines were achieved only by the influence of pancreatic cancer cell lines conditioned medium and were not subjected to any genetic manipulation. The xenografts tumours from CSCcm lines displayed histopathological features of ADM, PanIN and PDAC lesions. Further molecular characterization from RNA-sequencing analysis highlighted primary culture cell lines (1st CSCcm) as potential candidates to represent the pancreatic CSCs and indicated the establishment of the pancreatic cancer molecular pattern in their subsequent progenies 2nd CSCcm and 3rd CSCcm. In addition, preliminary RNA-seq SNPs analysis showed that the distinct CSCcm lines did not harbour single point mutations for the oncogene Kras codon 12 or 13. Therefore, PDAC-CSCcm model may provide new insights about the actual occurrence of the pancreatic cancer leading to develop different approaches to target CSCs and abrogate the progression of this fatidic disease.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：E-CENTURY PUBLISHING CORP  

To grow beyond a size of approximately 1-2 mm(3), tumor cells activate many processes to develop blood vasculature. Growing evidences indicate that the formation of the tumor vascular network is very complex, and is not restricted to angiogenesis. Cancer cell-derived tumor vasculatures have been recently described. Among them, endothelial differentiation of tumor cells have been directly related to cancer stem cells, which are cells within a tumor that possess the capacity to self-renew, and to exhibit multipotential heterogeneous lineages of cancer cells. Vasculogenic mimicry has been described to be formed by cancer cells expressing stemness markers. Thus, cancer stem cells have been proposed to contribute to vasculogenic mimicry, though its relation is yet to be clarified. Here, we analyzed the tumor vasculature by using a model of mouse cancer stem cells, miPS-LLCcm cells, which we have previously established from mouse induced pluripotent stem cells and we introduced the DsRed gene in miPS-LLCcm to trace them in vivo. Various features of vasculature were evaluated in ovo, in vitro, and in vivo. The tumors formed in allograft nude mice exhibited angiogenesis in chick chorioallantoic membrane assay. In those tumors, along with penetrated host endothelial vessels, we detected endothelial differentiation from cancer stem cells and formation of vasculogenic mimicry. The angiogenic factors such as VEGF-A and FGF2 were expressed predominantly in the cancer stem cells subpopulation of miPS-LLCcm cells. Our results suggested that cancer stem cells play key roles in not only the recruitment of host endothelial vessels into tumor, but also in maturation of endothelial linage of cancer stem cell's progenies. Furthermore, the undifferentiated subpopulation of the miPS-LLCcm participates directly in the vasculogenic mimicry formation. Collectively, we show that miPS-LLCcm cells have advantages to further study tumor vasculature and to develop novel targeting strategies in the future.

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Language：English   Publisher：日本癌学会  

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Language：Japanese   Publisher：Japan Society for Fuzzy Theory and Intelligent Informatics  

The significance degree of each component was calculated only by once SOM learning where the Spherical Self-Organizing-Map (SSOM) was used for the analysis. This method was applied to a great deal of gene data of the breast cancer. Then, the relation between each gene and the breast cancer was examined. Finally, the method succeeded in 22 genetic extraction to be connected with breast cancer carcinogenesis from 831 genes

DOI： 10.14864/fss.31.0_476

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Language：English   Publisher：INT INST ANTICANCER RESEARCH  

Conventional anticancer therapies such as chemotherapy are losing their sheen in the battle against cancer. Therefore, strategies for treatment of cancer need to be constantly modified to fulfill the growing demands of alternative therapies. Several viral and non-viral vectors have been exploited for anticancer gene therapy. But over the years bacteria have been proven to be an important candidate for successful evasion of cancer. They serve as invaluable source of tumor-specific anticancer genes, toxins, polysaccharides for synthesis of nanodrugs and gene-delivery vectors. The current review assesses the role of important bacterial groups in different spheres of anti-cancer research.

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DOI： 10.1158/1538-7445.AM2014-3016

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Language：English   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2014-3026

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DOI： 10.14800/ccm.236

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

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 7glucosyloxyacetylpaclitaxel in vivo. This data successfully demonstrates immunoliposomes as a promising candidate for the efficient delivery of paclitaxel glycoside.

DOI： 10.1371/journal.pone.0107976

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Inhibition of angiogenesis is currently perceived as a promising strategy in the treatment of cancer. The anti-angiogenicity of thalidomide has inspired a second wave of research on this teratogenic drug. The present study aimed to investigate the anti-proliferative and anti-angiogenic activities of two thalidomide dithiocarbamate analogs by studying their anti-proliferative effects on human umbilical vein endothelial cells (HUVECs) and MDA-MB-231 human breast cancer cell lines. Their action on the expression levels of IL-6, IL-8, TNF-alpha, VEGF(165), and MMP-2 was also assessed. Furthermore, their effect on angiogenesis was evaluated through wound healing, migration, tube formation, and nitric oxide (NO) assays. Results illustrated that the proliferation of HUVECs and MDA-MB-231 cells was not significantly affected by thalidomide at 625-100 mu M. Thalidomide failed to block angiogenesis at similar concentrations. By contrast, thalidomide dithiocarbamate analogs exhibited significant anti-proliferative action on HUVECs and MDA-MB-231 cells without causing cytotoxicity and also showed powerful anti-angiogenicity in wound healing, migration, tube formation, and NO assays. Thalidomide analogs 1 and 2 demonstrated more potent activity to suppress expression levels of IL-6, IL-8, TNF-alpha, VEGF(165), and MMP-2 than thalidomide. Analog 1 consistently, showed the highest potency and efficacy in all the assays. Taken together, our results support further development and evaluation of novel thalidomide analogs as antitumor and anti-angiogenic agents. (C) 2014 The Authors. Published by Elsevier B.V.

DOI： 10.1016/j.intimp2014.05.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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 CSC's properties. Therefore, miPS-LLCcm cells create their own in vitro niche to maintain themselves in the hierarchy of differentiating CSCs.

DOI： 10.1002/ijc.28648

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HINDAWI PUBLISHING CORPORATION  

Chlorotoxin (CTX) is a 36-amino acid peptide derived from Leiurus quinquestriatus (scorpion) venom, which inhibits low-conductance chloride channels in colonic epithelial cells. It has been reported that CTX also binds to matrix metalloproteinase-2 (MMP-2), membrane type-1 MMP, and tissue inhibitor of metalloproteinase-2, as well as CLC-3 chloride ion channels and other proteins. Pancreatic cancer cells require the activation of MMP-2 during invasion and migration. In this study, the fusion protein was generated by joining the CTX peptide to the amino terminus of the human IgG-Fc domain without a hinge domain, the monomeric form of chlorotoxin (M-CTX-Fc). The resulting fusion protein was then used to target pancreatic cancer cells (PANC-1) in vitro. M-CTX-Fc decreased MMP-2 release into the media of PANC-1 cells in a dose-dependent manner. M-CTX-Fc internalization into PANC-1 cells was observed. When the cells were treated with chlorpromazine (CPZ), the internalization of the fusion protein was reduced, implicating a clathrin-dependent internalization mechanism of M-CTX-Fc in PANC-1 cells. Furthermore, M-CTX-Fc clearly exhibited the inhibition of the migration depending on the concentration, but human IgG, as negative control of Fc, was not affected. The M-CTX-Fc may be an effective instrument for targeting pancreatic cancer.

DOI： 10.1155/2014/152659

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：E-CENTURY PUBLISHING CORP  

Induced pluripotent stem (iPS) cells may be a powerful tool in regenerative medicine, but their potential tumorigenicity is a significant challenge for the clinical use of iPS cells. Previously, we succeeded in converting miPS cells into cancer stem cells (CSCs) under the conditions of tumor microenvironment. Both stem cells and tumor cells are profoundly influenced by bi-directional communication with their respective microenvironment, which dictates cell fate determination and behavior. The microenvironment derived from iPS cells has not been well studied. In this paper, we have investigated the effects of secreted factors from Nanog-mouse iPS (miPS) cells on mouse Lewis lung cancer (LLC) cells that are found in the conditioned media. The results demonstrated that miPS cells secrete factors that can convert the epithelia phenotype of LLC cells to a mesenchymal phenotype, and that can promote tumorigenisity, migration and invasion. Furthermore, LLC cells that have been exposed to miPS conditioned medium became resistant to apoptosis. These various biological effects suggest that the miPS microenvironment contain factors that can promote an epithelial-mesenchymal transition (EMT) through an active Snail-MMP axis or by suppressing differentiation in LLC cells.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IVYSPRING INT PUBL  

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.

DOI： 10.7150/jca.8865

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Prior to gastrulation, the Wnt signaling pathway through stabilized beta-catenin enhances the differentiation of mouse ES cell into cardiomyocytes. We have recently shown that cardiomyocyte differentiation is enhanced by eosinophil cationic protein (ECP) through accelerated expression of marker genes of early cardiac differentiation. Furthermore, ECP enhanced the expression of Wnt3a in P19CL6 cells which were stimulated to differentiate into cardiomyocytes by DMSO. Following these findings, we evaluated in this study the potential of ECP to activate the Wnt/beta-catenin signaling pathway during cardiomyocyte differentiation. Analysis by real time qPCR revealed that ECP increased the expression of Frizzled genes such as Frizzled-1, -2, -4 and -10 in P19CL6 cells in the presence of DMSO. The increased expression of those Wnt receptors was found to inhibit the phosphorylation of beta-catenin resulting in the stabilization and translocation of beta-catenin into the nucleus of P19CL6 cells during the early stages of cardiomyocyte differentiation. When assessed for beta-catenin/TCF transcriptional activity with a TCF-luciferase (TOP/FOP) assay, ECP enhanced luciferase activity in P19CL6 cells during 48 h after transfection with TOP/FOP flash reporter in a stoichiometric manner. Collectively, this suggests that ECP can activate a canonical Wnt/beta-catenin signaling pathway by enhancing the stabilization of beta-catenin during cardiomyocyte differentiation.

DOI： 10.1007/s11033-012-2390-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IVYSPRING INT PUBL  

The oncogenic tyrosine kinase receptor ErbB2 is a prognostic factor and target for breast cancer therapeutics. In contrast with the other ErbB receptors, ErbB2 is hardly internalized by ligand induced mechanisms, indicating a prevalent surface expression. Elevated levels of ErbB2 in tumor cells are associated with its defective endocytosis and down regulation. Here we show that caveolin-1 expression in breast cancer derived SKBR-3 cells (SKBR-3/Cav-1) facilitates ligand induced ErbB2 endocytosis using an artificial peptide ligand EC-eGFP. Similarly, stimulation with humanized anti ErbB2 antibody Trastuzumab (Herceptin) was found to be internalized and co-localized with caveolin-1 in SKBR-3/Cav-1 cells. Internalized EC-eGFP and Trastuzumab in SKBR-3/Cav-1 cells were then delivered via caveolae to the caveolin-1 containing early endosomes. Consequently, attenuated Fc receptor mediated ADCC functions were observed when exposed to Trastuzumab and EC-Fc (EC-1 peptide conjugated to Fc part of human IgG). On the other hand, this caveolae dependent endocytic synergy was not observed in parental SKBR-3 cells. Therefore, caveolin-1 expression in breast cancer cells could be a predictive factor to estimate how cancer cells are likely to respond to Trastuzumab treatment.

DOI： 10.7150/jca.6470

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INFORMA HEALTHCARE  

We investigated the functional role of eosinophil cationic protein (ECP) in regulating cardiomyogenesis using mouse P19CL6 embryonic carcinoma cells. ECP was confirmed to accelerate the cardiomyocyte differentiation of P19CL6 cells by enhancing the rate and area size of beating of cardiomyocyte and by facilitating the expression of cardiomyocyte-specific genes, such as GATA4 and alpha-MHC. Since cardiomyocyte differentiation in vivo is considered to follow mesoderm induction, the induction of Brachyury, a marker of mesoderm, was assessed. Brachyury expression was found to be enhanced after the addition of ECP. This enhancement was due to the stimulation of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation by ECP. In this context, treatment with SU5402, an inhibitor of fibroblast growth factor (FGF) receptor 1, suppressed Brachyury expression, phosphorylation of ERK1/2, and cardiomyocyte differentiation induced by ECP. We concluded that ECP might induce mesoderm differentiation through FGF signaling pathway and enhance subsequent cardiomyocyte differentiation in concert with dimethyl sulfoxide in P19CL6 cells. ECP may be a novel factor for cardiomyocyte differentiation, which should be very useful to prepare adequate numbers of cardiomyocytes for therapeutic cell transplantation.

DOI： 10.3109/08977194.2012.709852

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

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, Yamanaka's 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.

DOI： 10.1371/journal.pone.0033544

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MAIK NAUKA/INTERPERIODICA/SPRINGER  

Green fluorescent protein (GFPuv) has been widely used as a reporter fused to individual targeting sequences. However, its state in liquid and its effect on other proteins are still unclear. The conformational polymorphisms of glutathione-S-transferase-green fluorescent protein (GST-GFPuv), GFPuv and GST were analyzed by native polyacrylamide gel, indicating that GST was in many different states while GFPuv and GST-GFPuv were only in four and two slightly different states. Four different circular dichroism spectra were obtained from the GFPuv polymorphisms. The single molecular behavior of GST-GFPuv and GFPuv was also characterized by MALDI-TOF MS. Thus, we demonstrated that: (1) there might be four different structural polymorphisms for the native GFPuv; (2) GFPuv could reduce its partner's polymorphism as a fusion protein. Although GFPuv had many merits as a reporter, its unreliability was found in the study. DOI: 10.1134/S0026893311060045

DOI： 10.1134/S0026893311060045

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DOI： 10.1134/S0026893311060045

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DOI： 10.1155/2012/975763

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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.

DOI： 10.1111/j.1582-4934.2011.01277.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The structural and spatial associations of Fe with O and C in the outer coat fibers of the Leptothrix ochracea sheath were shown to be substantially similar to the stalk fibers of Gallionella ferruginea, i.e., a central C core, probably of bacterial origin, and aquatic Fe interacting with O at the surface of the core.

DOI： 10.1128/AEM.06003-11

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Leptothrix species in aquatic environments produce uniquely shaped hollow microtubules composed of aquatic inorganic and bacterium-derived organic hybrids. Our group termed this biologically derived iron oxide as "biogenous iron oxide (BIOX)". The artificial synthesis of most industrial iron oxides requires massive energy and is costly while BIOX from natural environments is energy and cost effective. The BIOX microtubules could potentially be used as novel industrial functional resources for catalysts, adsorbents and pigments, among others if effective and efficient applications are developed. For these purposes, a reproducible system to regulate bacteria and their BIOX productivity must be established to supply a sufficient amount of BIOX upon industrial demand. However, the bacterial species and the mechanism of BIOX microtubule formation are currently poorly understood. In this study, a novel Leptothrix sp. strain designated OUMS1 was successfully isolated from ocherous deposits in groundwater by testing various culture media and conditions. Morphological and physiological characters and elemental composition were compared with those of the known strain L. cholodnii SP-6 and the differences between these two strains were shown. The successful isolation of OUMS1 led us to establish a basic system to accumulate biological knowledge of Leptothrix and to promote the understanding of the mechanism of microtubule formation. Additional geochemical studies of the OUMS1-related microstructures are expected provide an attractive approach to study the broad industrial application of bacteria-derived iron oxides.

DOI： 10.1007/s00284-011-9957-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC JOURNALS  

Although site-directed mutagenesis has been used in many fields, it still has low rate of success and high cost because of low-yield target products. A modified method for multi-site-directed mutagenesis was developed with shifted primer design and cold-start polymerase chain reaction (PCR). The developed method was successfully applied to hexapeptide gene synthesis and recombinant enterokinase gene modification in the plasmids pET41a and pET24b-EK. The efficiency was pronounced at a 1:10 molar ratio of 7-base mutant products to 705-bp fragment products as control. Even in a 10-base substitution mutagenic PCR, a 1:50 molar ratio of mutant products to 705-bp fragment products was reached. Meanwhile, the quality of mutants was proved through the transformation efficiency and sequencing. This method was beneficial to prepare high-quality multibase mutagenesis and also implied that large-scale multibase mutagenesis was feasible, efficient, economical, and productive.

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Language：English   Publisher：AMER SOC CELL BIOLOGY  

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Language：English   Publisher：AMER SOC CELL BIOLOGY  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2015-LB-144

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2015-4243

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DOI： 10.1007/978-3-319-21702-4

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2014-4461

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Language：English   Publisher：日本癌学会  

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DOI： 10.1002/9781118356203.ch6

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2013-LB-8

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Language：Japanese  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2012-5700

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2012-418

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Aluminum (Al) is the most abundant metal in the Earth's crust, but its trivalent ionic form is highly toxic to all organisms at low concentrations. How Al enters cells has not been elucidated in any organisms. Herein, we report a transporter, Nrat1 (Nramp aluminum transporter 1), specific for trivalent Al ion in rice. Nrat1 belongs to the Nramp (natural resistance-associated macrophage protein) family, but shares a low similarity with other Nramp members. When expressed in yeast, Nrat1 transports trivalent Al ion, but not other divalent ions, such as manganese, iron, and cadmium, or the Al-citrate complex. Nrat1 is localized at the plasma membranes of all cells of root tips except epidermal cells. Knockout of Nrat1 resulted in decreased Al uptake, increased Al binding to cell wall, and enhanced Al sensitivity, but did not affect the tolerance to other metals. Expression of Nrat1 is up-regulated by Al in the roots and regulated by a C2H2 zinc finger transcription factor (ART1). We therefore concluded that Nrat1 is a plasma membrane-localized transporter for trivalent Al, which is required for a prior step of final Al detoxification through sequestration of Al into vacuoles.

DOI： 10.1073/pnas.1004949107

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Language：Japanese  

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When inorganic phosphate, a common and essential element for organisms, was applied endogenously, a rejection reaction and superoxide generation were induced in pea tissues but phytoalexin production was not. Phosphate-induced superoxide generation was sensitive to cycloheximide (CHX) and salicylhydroxamic acid (SHAM), indicating that part of the generation was dependent upon the expression of peroxidase gene(s). Peroxidases (POXs) are well known not only to scavenge hydrogen peroxide with phenolics but also to generate superoxide via NADH oxidation in the presence of p-coumaric acid and manganese ion. We cloned five pea POX cDNAs that are predicted to be located outside of the cells. The accumulation of five POX mRNAs, NTPase mRNA, and phenylalanine ammonia-lyase mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction. The expression of the five POX genes was induced by a fungal elicitor. On the other hand, inorganic phosphate induced the accumulation of POX11, POX14, and POX21 mRNAs but not of POX13, POX29, and PsPAL1 mRNAs within 1-3∈h after treatment of pea seedlings. In view of these findings, we discuss inorganic phosphate as a signal transmitter inducing part of the plant defense responses. © The Phytopathological Society of Japan and Springer-Verlag 2006.

DOI： 10.1007/s10327-005-0261-9

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DOI： 10.1007/s10327-006-0283-y

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Applicant：国立大学法人 岡山大学, LSIPファンド運営合同会社

Application no：JP2014057572  Date applied：2014.3.19

Announcement no：WO2014-148562  Date announced：2014.9.25

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Applicant：国立大学法人 岡山大学

Application no：特願2014-042544  Date applied：2014.3.5

Announcement no：特開2014-207889  Date announced：2014.11.6

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Applicant：国立大学法人 岡山大学

Application no：特願2014-014778  Date applied：2014.1.29

Announcement no：特開2014-207883  Date announced：2014.11.6

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Applicant：国立大学法人 岡山大学

Application no：JP2013072178  Date applied：2013.8.20

Announcement no：WO2014-030641  Date announced：2014.2.27

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Applicant：国立大学法人 岡山大学

Application no：特願2013-082671  Date applied：2013.4.11

Announcement no：特開2014-204674  Date announced：2014.10.30

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Applicant：国立大学法人 岡山大学

Application no：特願2013-082671  Date applied：2013.4.11

Announcement no：特開2014-204674  Date announced：2014.10.30

Patent/Registration no：特許第6226545号  Date issued：2017.10.20

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Applicant：塩水港精糖株式会社, 濱田 博喜, 藤原 一郎, 国立大学法人 岡山大学

Application no：JP2013058242  Date applied：2013.3.22

Announcement no：WO2013-141346  Date announced：2013.9.26

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Applicant：塩水港精糖株式会社, 濱田 博喜, 藤原 一郎, 国立大学法人 岡山大学

Application no：特願2013-537718  Date applied：2013.3.22

Patent/Registration no：特許第5490326号  Date issued：2014.3.7

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