Functional and Molecular Characters of Cancer Stem Cells Through Development to Establishment.

Cancer stem cells (CSCs) are small subpopulation sharing similar properties like normal stem such as self-renewal and differentiation potential to direct tumor growth. Last few years, scientists considered CSCs as the cause of phenotypic heterogeneity in diverse cancer types. Also, CSCs contribute to cancer metastasis and recurrence. The cellular and molecular regulators influence on the CSCs' phenotype changing their behaviors in different stages of cancer progression. CSC markers play significance roles in cancer diagnosis and characterization. We delineate the cross-talks between CSCs and the tumor microenvironment that supports their intrinsic properties including survival, stemness, quiescence and their cellular and molecular adaptation. An insight into the markers of CSCs specific to organs is described.

Cripto-1 as a Potential Target of Cancer Stem Cells for Immunotherapy.

The immune system has been found to be suppressed in cancer patients. Cancer cells are extremely resistant to chemotherapeutic drugs, conventional immunotherapy, or cancer antigen vaccine therapy. Cancer immunotherapy, which is mainly based on immune checkpoint inhibitors, such as those for PD-1, PD-L1, and CTLA4, is an effective treatment method. However, no immunotherapeutic target has been found that retains validity in the face of tumor diversity. The transforming growth factor (TGF)-ß cytokine family possesses broad biological activity and is involved in the induction and/or transdifferentiation of helper T cells, which are important in immunotherapy. Nodal is a member of the TGF-ß family playing important roles in tissue stem cells and cancer stem cells (CSCs), interacting with the co-receptor Cripto-1, as well as with Activin type IB (Alk4) and Activin typeIIreceptors, and maintaining stemness and Notch and Wnt/ß-catenin signaling in CSCs. In recent years, it has been reported that Cripto-1 could be a potential therapeutic target in CSCs. Here, we review the accumulated literature on the molecular mechanisms by which Cripto-1 functions in CSCs and discuss the potential of Cripto-1 as an immunotherapeutic target in CSCs.

A Novel Artificially Humanized Anti-Cripto-1 Antibody Suppressing Cancer Cell Growth.

Cripto-1 is a member of the EGF-CFC/FRL1/Cryptic family and is involved in embryonic development and carcinogenesis. We designed a novel anti-Cripto-1 artificial antibody and assessed the recognition to the antigen and the potential to suppress the growth of cancer stem cells. First, single chain antibody clones were isolated by bio-panning with the affinity to recombinant Cripto-1 protein from our original phage-display library. Then, the variable regions of heavy chain VH and light chain VL in each clone were fused to constant regions of heavy chain CH and light chain CL regions respectively. These fused genes were expressed in ExpiCHO-S cells to produce artificial humanized antibodies against Cripto-1. After evaluation of the expression levels, one clone was selected and the anti-Cripto-1 antibody was produced and purified. The purified antibody showed affinity to recombinant Cripto-1 at 1.1 pmol and immunoreactivity to cancer tissues and cell lines. The antibody was available to detect the immunoreactivity in tissue microarrays of malignant tumors as well as in Cripto-1 overexpressing cells. Simultaneously, the antibody exhibited the potential to suppress the growth of human colon cancer derived GEO cells overexpressing Cripto-1 with IC50 at approximately 110 nM. The artificially humanized antibody is proposed to be a good candidate to target cancer cells overexpressing Cripto-1.

Isolation and characterization of cancer stem cells derived from human glioblastoma.

Cancer stem cell (CSC) is considered as a cause of cancer recurrence and metastasis. Simultaneously CSCs are responsible for the heterogeneous population in tumor tissues due to their differentiation potential. However, the characterizations of CSCs are still not enough and cancer stem cell lines widely available is desired to be established for the advancement of cancer research. In this study, we tried to isolate and characterize stem like cells from human glioblastoma cell line U-251MG cells. U-251MG P1 cells, which was previously condensed in the presence of hyaluronic acid as CD44 positive population were subjected to single cell isolation procedure. Although 5 clones were isolated, only one clone exhibited high expression of CD44, Nanog, OCT3/4 and SOX2, and named U-251MGSC1. The sphere forming ability of U-251MGSC1 cell was significantly higher than the parental U-251MG cells. Tumorigenicity of U-251MG-SC1 cells were higher than that of U-251MG cells. U-251MGSC1 cells exhibited higher expression of CD44, SOX2, Nestin and A2B5 than U-251MG cells in vitro and in vivo. The expression of GFAP and NF-M was enhanced when the cells were treated with the conditioned medium of U-251MG cells indicating the potential of differentiation. Sphere forming ability was more efficient than that of U-251MG cells and was enhanced in the presence of hyaluronic acid, which enhanced the cell growth as well. U-251MGSC1 cells exhibited rapid growth tumor in nude mice and efficient metastatic ability in transmembrane assay when compared with U-251MG cells. As the result, we concluded U-251MGSC1 cell was a glioblastoma CSC line derived from the parental U-251MG cells. U-251MGSC1 cells will be a good tool to develop effective therapeutic agents against CSCs and to elucidate the properties of glioma derived CSCs and the mechanism of tumor development in brain.

Diagnostic ability of limited volume cone beam computed tomography with small voxel size in identifying the superior and inferior walls of the mandibular canal.

BACKGROUND: The aim of this study was to evaluate the visibility of the superior and inferior walls of the mandibular canal separately using limited volume cone beam computed tomography (CBCT) with small voxel size. METHODS: CBCT cross-sectional images of 86 patients obtained by 3D Accuitomo FPD and reconstructed with a voxel size of 0.08 mm were used for the evaluation. A 30-mm range of the mandible just distal to the mental foramen was divided into three equal areas (areas 1, 2, and 3, from anterior to posterior). Each area contained 10 cross-sectional images. Two observers evaluated the visibility of the superior and inferior walls of the mandibular canal on each of the cross-sectional images in these three areas. The visibility ratio in each area was determined as the number of cross-sectional images with a visible wall divided by 10. RESULTS: In all areas, the visibility ratio of the superior wall was significantly lower than that of the inferior wall. As for variance among the three areas, the ratio was highest in the most posterior area (area 3) and tended to decrease gradually towards the mental foramen for both walls. Cases in which more than two thirds of the superior wall could be identified (visibility ratio of 0.7 or more) in areas 1, 2, and 3 were 44, 62, and 66%, respectively. CONCLUSIONS: The superior wall was significantly more poorly visualized than the inferior wall in all areas examined. The visibility of the superior wall on CBCT images was limited even when a limited volume device with small voxel size was used.

A high-fat diet increases the incidence of mammary cancer inc-Ha-ras proto-oncogene transgenic rats.

Mammary cancer is the most common type of cancer and the fifth most common cause of cancer-related deaths among Japanese women. The recent sharp increase in the number of women diagnosed with mammary cancer per year is thought to be associated with increased fat intake resulting from changes in the dietary habits of contemporary Japanese citizens. In this study, human c-Ha-ras proto-oncogene transgenic (Hras128) rats, which are highly susceptible to mammary carcinogens, were fed high- or low-fat diets to examine the relationship between fat consumption and the development of mammary cancer. Female 7-week-old Hras128 rats and wild-type littermates were administered benzo[a]pyrene. A week later, the animals were randomly assigned to high-fat or low-fat diet groups (45% or 10% of calories from fat, respectively). After 12 weeks, the rats were sacrificed and autopsied, and mammary tumors were excised and processed for microscopic observation. Mammary tumors were found in 11 of the 12 animals in the high-fat diet group and in 5 of the 12 animals in the low-fat diet group, and the numbers of mammary gland tumors per animal in these groups were 1.7 and 0.7, respectively. Notably, the observed differences in incidence and multiplicity of mammary tumors between the two groups were statistically significant. These results suggest a positive relationship between the incidence of breast cancer and high fat intake.

Eosinophil Cationic Protein Shows Survival Effect on H9c2 Cardiac Myoblast Cells with Enhanced Phosphorylation of ERK and Akt/GSK-3ß under Oxidative Stress.

Eosinophil cationic protein (ECP) is well known as a cationic protein contained in the basic granules of activated eosinophils. Recent studies have reported that ECP exhibits novel activities on various types of cells, including rat neonatal cardiomyocytes. Here we evaluated the effects of ECP on rat cardiac myoblast H9c2 cells. Our results showed that ECP enhanced the survival of the cells, in part by promoting the ERK and Akt/GSK-3ß signaling pathways. ECP attenuated the cytotoxic effects of H2O2 on H9c2 cells as well as the production of reactive oxygen species, the number of apoptotic cells and caspase 3/7 activity in the cells. In conclusion, ECP activated the ERK and Akt/GSK-3ß pathways, resulting in anti-oxidative effects on H9c2 cells that attenuated apoptosis.

Focal adhesion-localization of START-GAP1/DLC1 is essential for cell motility and morphology.

There is a class of GTPase activating proteins for the Rho family GTPases (RhoGAPs) that contain the steroidogenic acute regulatory protein (STAR)-related lipid transfer (START) domain. In mammals three genes encode such proteins and they are designated START-GAP1-3 or deleted in liver cancer 1-3 (DLC1-3). In this study, we examined the intracellular localization and roles of START-GAP1/DLC1 in cell motility. Immunofluorescence microscopic analysis of NRK cells and HeLa cells revealed that START-GAP1 was localized in focal adhesions. Amino acid residues 265-459 of START-GAP1 were found to be necessary for focal adhesion targeting and we name the region "the focal adhesion-targeting (FAT) domain." It was previously known that ectopic expression of START-GAP1 induced cell rounding. We demonstrated that the FAT domain of START-GAP1 was partially required for this morphological change. Furthermore, expression of this domain in HeLa cells resulted in dissociation of endogenous START-GAP1 from focal adhesions as a dominant negative modulator, reducing cell migration and spreading. Taken together, START-GAP1 is targeted to focal adhesions via the FAT domain and regulates actin rearrangement through down-regulation of active RhoA and Cdc42. Its absence from focal adhesions could, therefore, cause abnormal cell motility and spreading.