Motoyuki Shimonaka

Differential genotoxicity of chemical properties and particle size of rare metal and metal oxide nanoparticles

Nanoparticles of rare metal compounds are used in various products. However, their carcinogenicity and genotoxicity have not been sufficiently evaluated. The tumor‐initiating and ‐promoting potentials of four rare metals, indium oxide (In2O3), dysprosium oxide (Dy2O3), tungsten oxide (WO3) and molybdenum (Mo), with a well‐defined particle diameter were evaluated. The mutagenicity of these rare metals was investigated by Ames test using five bacteria strains, and transformability of these rare metals was investigated by cell‐transformation assay using v‐Ha‐ras‐transfected BALB/c 3T3 cells (Bhas 42 cells). Nano‐sized Dy2O3 showed strong mutagenesis in all five bacteria strains tested with and without metabolic activation, while micro‐sized particles showed weak mutagenesis in two bacterial strains. Dy2O3 induced transformation colonies of Bhas 42 cell dose‐dependently, although there was no difference in the number of transformed foci between nano‐sized and micro‐sized particles. Nano‐sized In2O3 and WO3 showed positive mutagenic response in TA1537 and TA98, respectively, whereas the micro‐sized metal oxide particles showed no mutagenesis in the test bacterial strains. Both nano‐sized and micro‐sized In2O3 showed similar levels of transformability. However, nano‐sized and micro‐sized WO3 did not show any transformability. Both nano‐sized and micro‐sized Mo particles showed neither mutagenesis nor transformability. These results suggest that mutagenicity of rare metals depends on their particle size, although transformability depends on their chemical components but not on their particle size. Copyright

Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmanns thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-β-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbβ3-myosin complex is formed as a primary axis to promote platelet contraction.

Myosin light chain kinase expression induced via tumor necrosis factor receptor 2 signaling in the epithelial cells regulates the development of colitis-associated carcinogenesis.

It has been suggested that prolonged inflammatory bowel diseases (IBD) may lead to colitis-associated carcinogenesis (CAC). We previously observed that the NF-κB activation in colonic epithelial cells is associated with increased tumor necrosis factor receptor 2 (TNFR2) expression in CAC development. However, the mechanism by which epithelial NF-κB activation leading to CAC is still unclear. Myosin light chain kinase (MLCK) has been reported to be responsible for the epithelial permeability associated with TNF signaling. Therefore we focused on the role of MLCK expression via TNFR2 signaling on CAC development. Pro-tumorigenic cytokines such as IL-1β, IL-6 and MIP-2 production as well as INF-γ and TNF production at the lamina propria were increased in the setting of colitis, and further in tumor tissues in associations with up-regulated TNFR2 and MLCK expressions in the epithelial cells of a CAC model. The up-regulated MLCK expression was observed in TNF-stimulated colonic epithelial cells in a dose-dependent fashion in association with up-regulation of TNFR2. Silencing TNFR2, but not TNFR1, resulted in restoration of epithelial tight junction (TJ) associated with decreased MLCK expression. Antibody-mediated blockade of TNF signaling also resulted in restoration of TJ in association with suppressed MLCK expression, and interestingly, similar results were observed with suppressing TNFR2 and MLCK expressions by inhibiting MLCK in the epithelial cells. Silencing of MLCK also resulted in suppressed TNFR2, but not TNFR1, expression, suggesting that the restored TJ leads to reduced TNFR2 signaling. Such suppression of MLCK as well as blockade of TNFR2 signaling resulted in restored TJ, decreased pro-tumorigenic cytokines and reduced CAC development. These results suggest that MLCK may be a potential target for the prevention of IBD-associated tumor development.

Impact of plasminogen on an in vitro wound healing model based on a perfusion cell culture system

Vascular reorganization in wound healing is a complex process, which involves coagulation, endothelial cell proliferation and migration, basement membrane regeneration, and fibrinolysis. During this healing process, the hemostatic system and the angiogenic system are intimately interconnected. To elucidate the contribution of plasminogen in the process of wound healing, we have established a perfusion cell culture system. Using this novel cell culture system, we found that addition of plasminogen in the perfusion medium allowed the “scratch-wounded” endothelial cells to recover completely, while mini-plasminogen only affected the migration but not the proliferation of the endothelial cells. In the process of recovery with the addition of plasminogen, significant plasmin activity could only be detected when the growth of the endothelial cells have almost reached confluence. This finding indicates that wound healing is triggered and promoted during the absence of the proteolytic activity of plasmin. In addition, we could not detect any matrix metalloproteinase activity in the perfusion culture medium throughout the whole culture period. However, we did found that the circulating medium collected from the perfusion system at the early phase of the healing process has stimulatory activity on the growth of endothelial cells, but the proliferative activity decreased back to the basal level when the cells reached confluence. Thus, by using the perfusion cell culture system, we found that proliferation of endothelial cells is regulated by plasminogen and the wound healing process is controlled by a temporal interaction between the endothelial cells and plasminogen.

Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration

Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides.

Reduced sulfation of chondroitin sulfate in thyroglobulin derived from human papillary thyroid carcinomas

The presence of a chondroitin sulfate (CS) chain on human thyroglobulin (Tg) distinguishes it from Tg of other species; the role played by this chain in normal thyroid function is unclear. In the present study, we determined the structure of the CS oligosaccharides in human thyroid‐derived Tg. Q‐Sepharose anion exchange column chromatography of thyroid extracts indicated that the negative charge of human Tg was primarily due to the presence of the CS chain. Interestingly, the Tg of papillary carcinomas was less negatively charged, suggesting that its CS side chain was less sulfated. Structural analysis of the CS in Tg revealed that its most abundant disaccharide is the ΔDi‐0S unit (50.2 ± 18.3%), which is not sulfated. The ΔDi‐0S, ΔDi‐6S (31.7 ± 13.7%) and ΔDi‐diSD (12.8 ± 4.3%) units comprise more than 90% of the disaccharides in normal Tg. However, the ΔDi‐6S (0.0–21.2%) and ΔDi‐diSD (0.0–7.7%) units were significantly reduced in Tg extracted from papillary thyroid carcinomas, whereas ΔDi‐0S (86.0 ± 21.3%) was increased. These results suggest that the Tg in papillary carcinomas has a less sulfated CS side chain and, by virtue of that fact, is less negatively charged. What role this change in carcinoma cells has in their transformation and spread remains to be determined. (Cancer Sci 2007; 98: 1577–1581)

Tamoxifen inhibits the proliferation of non‑melanoma skin cancer cells by increasing intracellular calcium concentration

Tamoxifen is an estrogen receptor (ER) antagonist used as first-line chemotherapy in breast cancer. Recent studies suggest that tamoxifen may be effective not only for ER‑positive but also for ER‑negative cancer cases. The aim of the present study was to investigate the antiproliferative effect of tamoxifen against human non‑melanoma skin cancer cells. Tamoxifen inhibited the proliferation of the skin squamous cell carcinoma (SCC) cell lines A431, DJM‑1 and HSC‑1. A431 cells did not express ER‑α or -β, suggesting that tamoxifen may exert antiproliferative effects on skin SCC cells via a non‑ER‑mediated pathway. Tamoxifen increased the intracellular calcium concentration of skin SCC cells, and this increase in intracellular calcium concentration by calcium ionophore A23187 suppressed the proliferation of skin SCC cells. These data indicate that tamoxifen inhibited the proliferation of human skin SCC cells via increasing intracellular calcium concentration. Voltage-gated calcium channels and non‑selective cation channels are involved in the increase in intracellular calcium concentration induced by tamoxifen. The broad-spectrum protein kinase C (PKC) inhibitor phloretin significantly attenuated the antiproliferative effect of tamoxifen on skin SCC cells. From these data, it may be concluded that tamoxifen inhibits the proliferation of skin SCC cells by induction of extracellular calcium influx via calcium channels in the plasma membrane and by subsequent activation of PKC.

Anti‑proliferative effect of ridaifen‑B on hepatoma cells

Ridaifens (RIDs), a novel series of tamoxifen derivatives, exhibit a potent growth-inhibitory effect against numerous tumor cells regardless of the expression of estrogen receptors, and are thus promising candidates as novel anti-tumor drugs. RID-B is a first generation RIDs, and inhibits the proliferation of several tumor cell lines. However, the potentially growth inhibitory effect of RID-B against hepatoma cells, and the detailed mechanism underlying RID-B-mediated tumor cell death remain to be elucidated. The purpose of the current study was to evaluate the anti-proliferative effect of RID-B against hepatoma cells. The anti-proliferative effect of RID-B against human hepatoma Huh-7 cells was investigated by cell proliferation assay using WST-1 reagent, and caspase-3 activity was evaluated by using specific fluorescent substrate. In addition, DNA fragmentation in Huh-7 cells induced by RID-B was estimated by terminal deoxynucleotidyl transferase dUTP nick-end labelling assay, and binding of RID-B to double-stranded DNA was confirmed by mass spectrometry. RID-B (0.5, 1 and 2 µM) inhibited the growth of Huh-7 cells, seemingly dose-dependently, but did not inhibit the growth of normal primary rat hepatocytes in the same concentration range. Furthermore, the caspase-3 activity of Huh-7 cells was increased by RID-B (0.5 and 5 µM), and the anti-proliferative effect of RID-B (1 µM) on Huh-7 cells was partially suppressed by the addition of the caspase inhibitor, Z-VAD-FMK. Additionally, RID-B (10 µM) directly bound to double-stranded DNA, and the addition of DNA suppressed RID-B-mediated cell growth inhibition and DNA fragmentation in Huh-7 cells. From these data, it may be concluded that RID-B inhibited cell growth and induced apoptosis via activating caspase-3 and binding to DNA directly, leading to DNA fragmentation in hepatoma cells.

SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation

Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.