Tomoya Kudo

Inhibition of Membrane-Type 1 Matrix Metalloproteinase at Cell-Matrix Adhesions

Membrane-type 1 matrix metalloproteinase (MT1-MMP) has been implicated in tumor invasion and metastasis. We previously reported that extracellular matrix degradation by MT1-MMP regulates cell migration via modulating sustained integrin-mediated signals. In this study, MT1-MMP-expressing cells were plated onto fibronectin-coated plates and monitored for cell-matrix adhesion formation and fibronectin degradation. The fibronectin was degraded and removed in line with the cell migration track. The migrating cells showed a polarized morphology and were in contact with the edge of fibronectin through the leading edge, in which cell-matrix adhesions are concentrated. Expression of MT1-MMP targeted to cell-matrix adhesions by fusing with the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK) promoted the initial fibronectin lysis at the cell periphery immediately after adhesion. These results suggest that fibronectin is degraded by MT1-MMP located at cell-matrix adhesions, which are concentrated at the leading edge of the migrating cells. To inhibit MT1-MMP at cell-matrix adhesion, the dominant negative form of MT1-MMP (MT1-Pex) was targeted to the cell-matrix adhesion by fusing with the FAT domain (MT1-Pex-FAT). MT1-Pex-FAT accumulated at cell-matrix adhesions and inhibited fibronectin degradation as well as FAK phosphorylation more effectively than parental MT1-Pex. MT1-Pex-FAT was also shown to suppress the invasion of tumor cells into three-dimensional collagen gel more strongly than MT1-Pex. These results suggest that MT1-MMP-mediated extracellular matrix lysis at cell-matrix adhesions induces the establishment of cell polarity, which facilitates cell-matrix adhesion turnover and subsequent cell migration. This model highlights the role of MT1-MMP at the leading edge of migrating cells.

Enterolosaponins A and B, novel triterpene bisdesmosides from Enterolobium contortisiliquum, and evaluation for their macrophage-oriented cytotoxic activity.

Two novel triterpene bisdesmosides, designated as enterolosaponin A (1) and B (2), were isolated from Enterolobium contortisiliquum. The chemical structures of 1 and 2 were determined by analysis of their extensive spectroscopic data, as well as hydrolysis followed by chromatographic study. Enterolosaponins have a 2-amino-2-deoxy-D-glucosyl unit (D-glucosamine) as one of the monosaccharides constituting their oligosaccharide moieties, which have been rarely found in natural product research. Enterolosaponin A (1) exhibited a highly selective cytotoxicity against BAC1.2F5 mouse macrophages, and it should be notable that the macrophage death caused by 1 was shown to be neither necrotic nor due to induction of apoptosis from morphology of the died cells, whose cytosol occurred in vacuolation.

Cathepsin G, a Neutrophil Protease, Induces Compact Cell-Cell Adhesion in MCF-7 Human Breast Cancer Cells

Cathepsin G is a serine protease secreted by activated neutrophils that play a role in the inflammatory response. Because neutrophils are known to be invading leukocytes in various tumors, their products may influence the characteristics of tumor cells such as the growth state, motility, and the adhesiveness between cells or the extracellular matrix. Here, we demonstrate that cathepsin G induces cell-cell adhesion of MCF-7 human breast cancer cells resulting from the contact inhibition of cell movement on fibronectin but not on type IV collagen. Cathepsin G subsequently induced cell condensation, a very compact cell colony, resulting due to the increased strength of E-cadherin-mediated cell-cell adhesion. Cathepsin G action is protease activity-dependent and was inhibited by the presence of serine protease inhibitors. Cathepsin G promotes E-cadherin/catenin complex formation and Rap1 activation in MCF-7 cells, which reportedly regulates E-cadherin-based cell-cell junctions. Cathepsin G also promotes E-cadherin/protein kinase D1 (PKD1) complex formation, and Go6976, the selective PKD1 inhibitor, suppressed the cathepsin G-induced cell condensation. Our findings provide the first evidence that cathepsin G regulates E-cadherin function, suggesting that cathepsin G has a novel modulatory role against tumor cell-cell adhesion.

Induction of multicellular 3-D spheroids of MCF-7 breast carcinoma cells by neutrophil-derived cathepsin G and elastase

In tumor metastasis, multicellular aggregates of tumor cells form and disseminate into the blood or lymph vessels from the tumor mass, following the formation of tumor cell emboli in distant vessels. However, the mechanism by which aggregates form in the tumor mass is unknown. Neutrophils often exist in tumors and are considered to affect tumor development. We observed that neutrophils had the capacity to induce the aggregation of MCF‐7 human breast carcinoma cells adhering to culture substrates. When MCF‐7 cells were cultured with rat inflammatory neutrophils, the soluble fraction of their lysate, and the conditioned medium of neutrophils stimulated with N‐formyl‐Met‐Leu‐Phe plus cytochalasin B, multicellular aggregates formed within 16 h, and tightly aggregated 3‐D spheroids formed when the cultures were prolonged. The spheroid‐inducing reaction was reversible and energy‐dependent. The MCF‐7 cells induced to aggregate by the neutrophil extract showed growth potential, although the growth rate of the cells was slightly reduced. The aggregation was dependent on E‐cadherin, because the spheroids dispersed into isolated cells on incubation with EGTA or anti‐E‐cadherin antibody following pipetting. The aggregation‐inducing activity in neutrophils was completely inhibited by soybean trypsin‐chymotrypsin inhibitor. Moreover, the commercially available human neutrophil elastase and cathepsin G induced the aggregation of MCF‐7 cells and formation of spheroids. The proteases secreted by infiltrated neutrophils in tumors are implicated in the dissemination of tumor aggregates from primary tumor sites. (Cancer Sci 2005; 96: 560 –570)

Synthesis and chiroptical properties of a π-conjugated polymer containing glucose-linked biphenyl units in the main chain capable of folding into a helical conformation

An optically active π-conjugated polymer (poly-9) containing glucose-linked biphenyl units in the main chain was synthesized through the copolymerization of 5,5′-diiodo-2,2′-bithiophene and a new diethynyl compound, whose molecular design has taken inspiration from naturally occurring ellagitannins. The chiroptical properties of poly-9 were investigated in solution and the solid state. The absorption and circular dichroism spectra of poly-9 were clearly changed depending on the solvents due to the conformational alteration within a single polymer chain. Because a corresponding model molecule and polymer did not show marked solvent-dependent spectral changes, the backbone conformation of poly-9 is considered to be capable of interconverting between random-coil and preferred-handed helical states in response to the exterior environment. We also found that poly-9 exhibited efficient circularly polarized luminescence with a green color, whose dissymmetry factor reached 1.9 × 10−2 when the backbone was folded into the helical conformation in the film state.

Chiral stationary phases consisting of π-conjugated polymers bearing glucose-linked biphenyl units: reversible switching of resolution abilities based on a coil-to-helix transition

A series of optically active π-conjugated polymers (poly-2–poly-5) consisting of alternating thieno[3,4-b]thiophene and glucose-linked biphenyl units were synthesized through copolymerizations by Sonogashira–Hagihara cross-coupling. Absorption and circular dichroism spectroscopy and theoretical calculations revealed that poly-2 undergoes a conformational transition between the random-coil and helix in both solution and the solid state in response to the external solvent environment (chloroform and acetonitrile). Coated-type chiral stationary phases (CSPs) for high-performance liquid chromatography were prepared from both the random-coil and helical poly-2, and the influence of the polymer backbone structure on the chiral recognition ability was evaluated. The two CSPs showed somewhat complementary resolution abilities and the kinds of resolved racemates were dependent on the backbone conformation. In addition, an immobilized-type CSP with universal solvent durability was also prepared through copper-catalyzed click cyclization between an alkyne-appended poly-2 analogue and an azide-functionalized silica gel. The resulting CSP displayed repeatable switching of the chiral recognition ability based on a coil-to-helix transition of the polymer backbone by alternate column treatment with common organic solvents, such as chloroform and acetonitrile.

Characterization of the growth-inhibitory and apoptosis-inducing activities of a triterpene saponin, securioside B against BAC1.2F5 macrophages.

BACKGROUND: Since the growth state of macrophages in local pathological sites is considered a factor that regulates the processes of many disease, such as tumors, inflammation, and atherosclerosis, the substances that regulate macrophage growth or survival may be useful for disease control. We previously reported that securiosides A and B, novel triterpene saponins, exerted macrophage-oriented cytotoxicity in the presence of a L-cell-conditioned medium containing macrophage colony-stimulating factor (M-CSF), while the compounds did not exhibit an effect on macrophages in the absence of the growth-stimulating factors. AIM: This study was undertaken to characterize the growth-inhibitory and the apoptosis-inducing activities of securioside B, focusing on the effects of the macrophage-growth factor(s), and to examine the implication of a mitochondria pathway in apoptosis induction. METHODS: The effect of securioside B on a murine macrophage cell line (BAC1.2F5) was examined by MTT assay and lactose dehydrogenase release assay in the presence of L-cell-conditioned medium, M-CSF, or granulocyte-macrophage CSF (GM-CSF). RESULT: Securioside B inhibited the growth of the cells stimulated by recombinant M-CSF or GM-CSF, but it scarcely induced cytolysis of the cells under the same conditions. Moreover, securioside B did not induce cell death when the compound only was added to the cells. On the other hand, the compound extensively induced apoptotic cell death in the presence of L-cell-conditioned medium, suggesting that apoptosis induction by securioside B requires the additional factor(s) present in L-cell-conditioned medium. Securioside B plus L-cell-conditioned medium induced the activation of caspase-3 and caspase-9, but not caspase-8. In addition, cytochrome c release from the mitochondria into the cytosol, and disrupted mitochondria membrane potential, was also observed in the apoptotic BAC1.2F5 cells. CONCLUSION: These data suggest that securioside B has growth-inhibitory activity against growth factor-stimulated macrophages, and that it induces apoptotic macrophage death through the activation of a mitochondrial pathway in the presence of L-cell-conditioned medium.