Masabumi Nagashima

The proton pump inhibitor inhibits cell growth and induces apoptosis in human hepatoblastoma

PurposeIn normal physiology, a vacuolar-type proton pump (V-ATPase) maintains an intracellular acid microenvironment in lysosome, endosome, and other endomembrane systems. Cancer cells overexpress V-ATPase compared with normal cells, and disturbances of the acid environment are thought to significantly impact the cancer cell infiltration and growth. Bafilomycin A1 (Baf-A1) is a specific inhibitor of the proton-pump inhibitor (PPI) V-ATPase. Neoplastic cells are reportedly more sensitive to Baf-A1 than normal cells, and the difference between the susceptibility to Baf-A1 in normal cells and that in cancer cells may become a target in the cancer therapy. With this in mind, we used cells of hepatoblastoma, the cancer type accounting for 80% of all childhood liver cancers, to investigate the effects of Baf-A1 as an inducer of cancer cell apoptosis and inhibitor of cancer cell reproductionMethods and resultsElectron microscopy showed significant morphological change of the hepatoblastoma cells of the Baf-A1-treated group compared with hepatoblastoma cells of the Baf-A1-free group. The rate of the apoptotic cell increased, and cell reproduction was inhibited. Moreover, the analysis of hepatoblastoma cells using the gene Chip gene expression analysis arrays showed that three of the 27 V-ATPase-related transcripts (ATP6V0D2, ATP6V1B1, and ATP6V0A1) were more weakly expressed in the Baf-A1-treated cells than in the Baf-A1-free cells. In normal human hepatic cells, on the other hand, the inhibition of cell growth of the Baf-A1-treated cells was negligible compared to that of the cells without Baf-A1 treatment. The result of apoptotic cell detection by morphological observations and flow cytometry revealed that Baf-A1 inhibits hepatoblastoma cellular reproduction by inducing apoptosis. On the other hand, the Baf-A1-conferred inhibition of cell growth was negligible in normal human hepatocytesConclusionThe V-ATPase inhibitor Baf-A1 has been proven to selectively inhibit the reproduction and induce the apoptosis of hepatoblastoma cells without adversely influencing normal hepatic cells. With these effects, V-ATPase inhibitors may hold promise as therapeutic agents for hepatoblastoma. Given that three V-ATPase-related genes (ATP6V0D2, ATP6V1B1, and ATP6V0A1) were more weakly expressed in the hepatoblastoma cells of the Baf-A1-treated group than in the Baf-A1-free cells, drug development targeting V-ATPase gene of hepatoblastomas is expected.

X-ray phase imaging: from synchrotron to hospital.

With the aim of clinical applications of X-ray phase imaging based on Talbot–Lau-type grating interferometry to joint diseases and breast cancer, machines employing a conventional X-ray generator have been developed and installed in hospitals. The machine operation especially for diagnosing rheumatoid arthritis is described, which relies on the fact that cartilage in finger joints can be depicted with a dose of several milligray. The palm of a volunteer observed with 19u2009s exposure (total scan time: 32u2009s) is reported with a depicted cartilage feature in joints. This machine is now dedicated for clinical research with patients.

Cadaveric and in vivo human joint imaging based on differential phase contrast by X-ray Talbot-Lau interferometry.

We developed an X-ray phase imaging system based on Talbot-Lau interferometry and studied its feasibility for clinical diagnoses of joint diseases. The system consists of three X-ray gratings, a conventional X-ray tube, an object holder, an X-ray image sensor, and a computer for image processing. The joints of human cadavers and healthy volunteers were imaged, and the results indicated sufficient sensitivity to cartilage, suggesting medical significance.

Suppression of proinflammatory cytokine production in macrophages by lansoprazole

Macrophages (MPs) produce increased levels of proinflammatory cytokines in Crohn’s disease; these cytokines are thought to play a central role in the occurrence of the disease. Biologics are currently available for anti-cytokine therapy, but treating intestinal inflammation through direct suppression of proinflammatory cytokine production could be more effective. P-ATPase inhibitors have been reported to be anti-inflammatory, and these inhibitors might suppress the production of MP proinflammatory cytokines. In this study, we examined the effect of two types of ATPase inhibitors on the expression patterns of typical proinflammatory cytokines. Peritoneal MPs from 6- to 8-week-old mice were cultured for 48xa0h in the presence of lansoprazole (P-ATPase inhibitor), bafilomycin A1 (V-ATPase inhibitor), or the control solvent dimethylsulfoxide. The MPs were then examined for cytokine expression by quantitative real-time polymerase chain reaction (PCR), and culture supernatants were examined for cytokine production with a multiplex assay in a suspension array system. The possible existence of P-ATPase mRNA in MPs was explored using reverse-transcriptase PCR. P-ATPase mRNA was not detected in MP cells. However, all examined proinflammatory cytokines decreased significantly in their mRNA and protein expression in the lansoprazole-treated group. Conversely, bafilomycin A1 increased the levels of these cytokines. Lansoprazole might be useful for the treatment of inflammatory bowel diseases (IBDs), including Crohn’s disease, as it suppresses the production of relevant MP proinflammatory cytokines. However, because P-ATPase was not detected in MPs, the mechanism is unclear and remains to be studied further in an IBD animal model.

Netrin-1 elevates the level and induces cluster formation of its receptor DCC at the surface of cortical axon shafts in an exocytosis-dependent manner.

During development, a diffusible axon guidance cue, netrin-1, plays a variety of important roles in the correct wiring of the nervous system by inducing axon outgrowth, attraction, repulsion and/or branching in various types of neurons. It has been reported that translocation of its receptor DCC (deleted in colorectal cancer) from an intracellular pool to the plasma membrane enhances outgrowth of rat spinal commissural axons in response to netrin-1 (Bouchard et al., 2004). To find out whether netrin-1 induces DCC translocation in cerebral cortical neurons, we examined changes in the level and distribution of DCC at the surface of hamster dissociated cortical axons in response to netrin-1. At the surface of cortical axon shafts, we observed netrin-1-evoked, exocytosis-dependent DCC clustering, which was accompanied by elevation of the DCC level. These changes in cell surface DCC occurred in axon shafts, but did not occur in growth cones. Taken together, these results indicate that cell surface DCC is modulated by netrin-1 through translocation of DCC to the plasma membrane via exocytosis in cerebral cortical neurons.

Effect of a dienogest for an experimental three-dimensional endometrial culture model for endometriosis.

The pathogenesis of endometriosis remains poorly understood at least in part because early stages of the disease process are difficult to investigate. Previous studies have proposed a three-dimensional fibrin matrix culture model to study human endometriosis. We examined the ultrastructural features of the endometriosis in this model and assessed the effect of a progestin on endometrial outgrowth and apoptosis in this culture system. Endometrial explants were placed in three-dimensional fibrin matrix culture and treated with and without various concentrations of the progestin dienogest. By the second week, endometrial gland-like formation was established in outgrowths both attached to and at a distance from the explants. These cells formed a combination of clumps and tubular monolayers surrounding a central cavity. Electron microscopy demonstrated that these cells are polarized with microvilli on the apical surface, desmosome-like structures, and basement membrane; features consistent with glandular epithelial cells. Outgrowth of endometrial stromal cells and glandular formation was impaired in response to dienogest in a dose-dependent manner. Our study shows that the human endometrial explants cultured in three-dimensional fibrin matrix establish outgrowths that ultrastructurally resemble ectopic endometrial implants. This model may provide insight into the cellular processes leading to endometriosis formation and enables screening of therapeutic compounds.

Development of the Talbot-Lau interferometry system available for clinical use

The prototype of the Talbot-Lau interferometer for clinical use was designed and applied to the preclinical examination. The human cadaveric hand and the mastectomy specimen were imaged. As a result, the images obtained by Talbot-Lau interferometry sensitively depicted the cartilages or the intraductal carcinoma. This result indicated that the Talbot-Lau interferometry would be a promising technology of the image diagnosis.

Application of X-ray grating interferometry for the imaging of joint structures

AbstractnConventional X-ray absorption contrast imaging does not depict soft tissues, such as cartilage, in sufficient detail. For visualization of the soft tissues, X-ray phase-contrast imaging is more sensitive than absorption-contrast imaging. The basic concept of the X-ray phase-contrast imaging used in this study is similar to that of differential interference contrast (Nomarski) microscopy. We applied Talbot–Lau X-ray interferometry to visualize the joint structures in the right hand and knee of a donated cadaver. This imaging system simultaneously produced three different types of images: an absorption image, a differential phase image, and a visibility image. The interface between the articular cartilage of the metacarpo-phalangeal joint and fluid or the bony cortex was clearly demonstrated on the differential phase image, whereas this interface was unclear on the absorption image. Within the knee joint, the surface of the articular cartilage was demonstrated both on the differential phase and visibility images; the medial collateral ligament and medial meniscus were also visualized successfully. These results are clinically significant for the diagnosis and therapeutic estimation of rheumatoid arthritis and related joint diseases. This feasibility study on the clinical application of this imaging tool was a collaborative effort of researchers in the fields of physics, radiology, and gross anatomy.

Shift in the function of netrin-1 from axon outgrowth to axon branching in developing cerebral cortical neurons

BackgroundNetrin-1, a multifunctional axon guidance cue, elicits axon outgrowth via one of its receptors deleted in colorectal cancer (DCC) in several types of neurons, including cerebral cortical neurons of embryonic mice. However, we and others have observed de novo formation of axon branches without axon outgrowth induced by netrin-1 in cortical culture of neonatal hamsters. These previous reports suggested the possibility that netrin-1 function might alter during development, which we here investigated using dissociated culture prepared from cerebral cortices of embryonic mice.ResultsImaging analysis revealed netrin-1-induced outgrowth in embryonic day (E) 14 axons and netrin-1-induced branching in E16 axons. Netrin-1-evoked filopodial protrusions, which sprouted on the shafts of E16 axons preceding branch formation, were visualized by a novel method called atmospheric scanning electron microscopy. Treatment with an anti-DCC function-blocking antibody affected both axon outgrowth and branching.ConclusionsMorphological analyses suggested a possibility of a shift in the function of netrin-1 in cortical axons during development, from promotion of outgrowth to promotion of branch formation starting with filopodial protrusion. Function-blocking experiments suggested that DCC may contribute not only to axon outgrowth but branching.

Netrin-1 regulates cell surface distribution of its receptor DCC in hamster primary cortical neurons

Deleted in colorectal cancer (DCC), a receptor for an axon guidance cue netrin-1, plays an important role in target innervation during development of central nervous system. In rat spinal commissural axons it is reported that intracellular vesicular pool of DCC exists and plasma membrane insertion of DCC occurs in response to netrin-1/protein kinase A. In addition, rapid depletion of cell surface DCC after netrin-1 treatment is reported in retinal growth cones of Xenopus laevis, which is dependent on endocytosis. In hamster cortical neurons netrin-1 is known to promote axon branching. In order to clarify whether translocation of DCC to, and/or endocytic removal of DCC from the plasma membrane occur by netrin-1 stimulation, we examined the changes in the level and distribution of DCC at the cell surface of hamster cortical neurons, employing total internal reflection fluorescence microscopy as well as cell-surface labeling techniques. A change in the distribution of cell surface DCC was observed in response to netrin-1 within 5 min.