Eribulin mesilate suppresses experimental metastasis of breast cancer cells by reversing phenotype from epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET) states.

BACKGROUND: Eribulin mesilate (eribulin), a non-taxane microtubule dynamics inhibitor, has shown trends towards greater overall survival (OS) compared with progression-free survival in late-stage metastatic breast cancer patients in the clinic. This finding suggests that eribulin may have additional, previously unrecognised antitumour mechanisms beyond its established antimitotic activity. To investigate this possibility, eribulin's effects on the balance between epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) in human breast cancer cells were investigated. METHODS: Triple negative breast cancer (TNBC) cells, which are oestrogen receptor (ER-)/progesterone receptor (PR-)/human epithelial growth receptor 2 (HER2-) and have a mesenchymal phenotype, were treated with eribulin for 7 days, followed by measurement of EMT-related gene and protein expression changes in the surviving cells by quantitative real-time PCR (qPCR) and immunoblot, respectively. In addition, proliferation, migration, and invasion assays were also conducted in eribulin-treated cells. To investigate the effects of eribulin on TGF-ß/Smad signalling, the phosphorylation status of Smad proteins was analysed. In vivo, the EMT/MET status of TNBC xenografts in mice treated with eribulin was examined by qPCR, immunoblot, and immunohistochemical analysis. Finally, an experimental lung metastasis model was utilised to gauge the metastatic activity of eribulin-treated TNBC in the in vivo setting. RESULTS: Treatment of TNBC cells with eribulin in vitro led to morphological changes consistent with transition from a mesenchymal to an epithelial phenotype. Expression analyses of EMT markers showed that eribulin treatment led to decreased expression of several mesenchymal marker genes, together with increased expression of several epithelial markers. In the TGF-ß induced EMT model, eribulin treatment reversed EMT, coincident with inhibition of Smad2 and Smad3 phosphorylation. Consistent with these changes, TNBC cells treated with eribulin for 7 days showed decreased capacity for in vitro migration and invasiveness. In in vivo xenograft models, eribulin treatment reversed EMT and induced MET as assessed by qPCR, immunoblot, and immunohistochemical analyses of epithelial and mesenchymal marker proteins. Finally, surviving TNBC cells pretreated in vitro with eribulin for 7 days led to decreased numbers of lung metastasis when assessed in an in vivo experimental metastasis model. CONCLUSIONS: Eribulin exerted significant effects on EMT/MET-related pathway components in human breast cancer cells in vitro and in vivo, consistent with a phenotypic switch from mesenchymal to epithelial states, and corresponding to observed decreases in migration and invasiveness in vitro as well as experimental metastasis in vivo. These preclinical findings may provide a plausible scientific basis for clinical observations of prolonged OS by suppression of further spread of metastasis in breast cancer patients treated with eribulin.

Formation and evolution of carbonaceous asteroid Ryugu: Direct evidence from returned samples.

Samples of the carbonaceous asteroid Ryugu were brought to Earth by the Hayabusa2 spacecraft. We analyzed 17 Ryugu samples measuring 1 to 8 millimeters. Carbon dioxide-bearing water inclusions are present within a pyrrhotite crystal, indicating that Ryugu's parent asteroid formed in the outer Solar System. The samples contain low abundances of materials that formed at high temperatures, such as chondrules and calcium- and aluminum-rich inclusions. The samples are rich in phyllosilicates and carbonates, which formed through aqueous alteration reactions at low temperature, high pH, and water/rock ratios of <1 (by mass). Less altered fragments contain olivine, pyroxene, amorphous silicates, calcite, and phosphide. Numerical simulations, based on the mineralogical and physical properties of the samples, indicate that Ryugu's parent body formed ~2 million years after the beginning of Solar System formation.

Energy metabolism and mitochondrial defects in X-linked Charcot-Marie-Tooth (CMTX6) iPSC-derived motor neurons with the p.R158H PDK3 mutation.

Charcot-Marie-Tooth (CMT) is a group of inherited diseases clinically and genetically heterogenous, characterised by length dependent degeneration of axons of the peripheral nervous system. A missense mutation (p.R158H) in the pyruvate dehydrogenase kinase 3 gene (PDK3) has been identified as the genetic cause for an X-linked form of CMT (CMTX6) in two unrelated families. PDK3 is one of four PDK isoenzymes that regulate the activity of the pyruvate dehydrogenase complex (PDC). The balance between kinases (PDKs) and phosphatases (PDPs) determines the extend of oxidative decarboxylation of pyruvate to generate acetyl CoA, critically linking glycolysis and the energy producing Krebs cycle. We had shown the p.R158H mutation causes hyperactivity of PDK3 and CMTX6 fibroblasts show hyperphosphorylation of PDC, leading to reduced PDC activity and ATP production. In this manuscript we have generated induced pluripotent stem cells (iPSCs) by re-programming CMTX6 fibroblasts (iPSCCMTX6). We also have engineered an isogenic control (iPSCisogenic) and demonstrated that genetic correction of the p.R158H mutation reverses the CMTX6 phenotype. Patient-derived motor neurons (MNCMTX6) show increased phosphorylation of the PDC, energy metabolism defects and mitochondrial abnormalities, including reduced velocity of trafficking mitochondria in the affected axons. Treatment of the MNCMTX6 with a PDK inhibitor reverses PDC hyperphosphorylation and the associated functional deficits founds in the patient motor neurons, demonstrating that the MNCMTX6 and MNisogenic motor neurons provide an excellent neuronal system for compound screening approaches to identify drugs for the treatment of CMTX6.

Induced alpha helix in the VP16 activation domain upon binding to a human TAF.

Activation domains are functional modules that enable sequence-specific DNA binding proteins to stimulate transcription. The structural basis for the function of activation domains is poorly understood. A combination of nuclear magnetic resonance (NMR) and biochemical experiments revealed that the minimal acidic activation domain of the herpes simplex virus VP16 protein undergoes an induced transition from random coil to alpha helix upon binding to its target protein, hTAFII31 (a human TFIID TATA box-binding protein-associated factor). Identification of the two hydrophobic residues that make nonpolar contacts suggests a general recognition motif of acidic activation domains for hTAFII31.

Development of a medical-text parsing algorithm based on character adjacent probability distribution for Japanese radiology reports.

OBJECTIVES: The objectives of this study were to investigate the transitional probability distribution of medical term boundaries between characters and to develop a parsing algorithm specifically for medical texts. METHODS: Medical terms in Japanese computed tomography (CT) reports were identified using the ChaSen morphological analysis system. MeSH-based medical terms (51,385 entries), obtained from the metathesaurus in the Unified Medical Language System (UMLS, 2005AA), were added as a medical dictionary for ChaSen. A radiographer corrected the set of results containing 300 parsed CT reports. In addition, two radiologists checked the medical term parsing of 200 CT sentences. RESULTS: We obtained modified inter-annotator agreement scores for the text corrected by the radiologists. We retrieved the transitional probability as the conditional probability of a uni-gram, bi-gram, and tri-gram. The highest transitional probability P(Ci | Ci- 2(*)Ci- 1) was 1.00. For an example of anatomical location, the term "pulmonary hilum" was parsed as a tri-gram. CONCLUSIONS: Retrieval of transitional probability will improve the accuracy of parsing compound medical terms.

Thermal and impact histories of 25143 Itokawa recorded in Hayabusa particles.

Understanding the origin and evolution of near-Earth asteroids (NEAs) is an issue of scientific interest and practical importance because NEAs are potentially hazardous to the Earth. However, when and how NEAs formed and their evolutionary history remain enigmas. Here, we report the U-Pb systematics of Itokawa particles for the first time. Ion microprobe analyses of seven phosphate grains from a single particle provide an isochron age of 4.64 ± 0.18 billion years (1σ). This ancient phosphate age is thought to represent the thermal metamorphism of Itokawa's parent body, which is identical to that of typical LL chondrites. In addition, the incorporation of other particles suggests that a significant shock event might have occurred 1.51 ± 0.85 billion years ago (1σ), which is significantly different from the shock ages of 4.2 billion years of the majority of shocked LL chondrites and similar to that of the Chelyabinsk meteorite. Combining these data with recent Ar-Ar studies on particles from a different landing site, we conclude that a globally intense impact, possibly a catastrophic event, occurred ca. 1.4 Ga ago. This conclusion enables us to establish constraints on the timescale of asteroid disruption frequency, the validity of the crater chronology and the mean lifetime of small NEAs.

Human LBP-32/MGR is a repressor of the P450scc in human choriocarcinoma cell line JEG-3.

Steroid hormones regulate a wide range of physiologic functions in humans. The cholesterol side-chain cleavage enzyme P450scc regulates the initial step of biosynthesis of all steroid hormones. We investigated the expression of P450scc by studying a potential regulator of P450scc, LBP-32/MGR. Using a Northern blot, we found that LBP-32/MGR mRNA was expressed mainly in the human placenta. Using radiation hybrid mapping, we identified LBP-32/MGR on human chromosome 2p25. Recombinant LBP-32/MGR protein bound preferentially to a DNA fragment from the promoter of P450scc in vitro and exhibited clear nuclear localization in transfected cells. Luciferase reporter gene assays showed that LBP-32/MGR specifically repressed transcriptional activation of the human P450scc promoter. Because placental P450scc expression is essential for pregnancy and steroid biosynthesis, the placental expression and transcriptional repressor activity of LBP-32/MGR in JEG-3 cells suggest it has a role as a transcriptional modulator of steroid biosynthesis.

Hydrophobic DNA binding of esperamicin requires conformational distortion of the host DNA.

Evidence is presented that the sequence-specific DNA binding of esperamicin is accompanied by structural distortion of the host DNA that depends on hydrophobic interactions. In the first part of this paper, we describe the effects of conformational freedom of DNA on the DNA-cutting efficiency of esperamicin. If conformational distortion of DNA is significantly required for binding of the drug, then the drug should possess a lower binding/cleaving efficiency for conformationally more restricted DNAs. Our results on model DNAs reveal a substantial decrease in the DNA-cleaving efficiency of esperamicin as the conformational freedom of DNA decrease. In the second part we demonstrate that esperamicin binds to DNA with considerably higher affinity in solutions containing organic solvents. This observation indicates that the required distortion of DNA depends on hydrophobic interactions. Molecular origins of the sequence-specific binding are also discussed on the basis of all available data.

Predictive value of serum immunosuppressive acidic protein for lung metastasis after amputation of murine osteosarcoma of the lower limb.

A spontaneously occurring murine osteosarcoma cell line (POS-1) was inoculated into the footpads of 88 mice. In 59 mice, the tumor-bearing leg was amputated at 1, 3, 5, or 7 weeks after inoculation and the mice sacrificed at 9 weeks. Lung metastasis was observed in 82.6-100% of the mice undergoing leg amputation at 5 or 7 weeks after inoculation. There were no lung metastasis in the mice amputated at 1 or 3 weeks. The serum immunosuppressive acidic protein (IAP) concentration showed a significant increase at 5 or 7 weeks after inoculation (P<0.005 and P<0.0005, respectively). When amputation was done 5 weeks after inoculation, the serum IAP concentration increased further to 5.9 times the pre-amputation level 1 week after the amputation. The IAP concentration increased only 1.3-fold in the control group without inoculation. Thus, a high serum IAP concentration before amputation and a further increase after amputation were indicators of lung metastasis in this mouse model of osteosarcoma.

Acceleration of lung metastasis by up-regulation of CD44 expression in osteosarcoma-derived cell transplanted mice.

The effect of CD44-phenotypic expression on metastasis to the lung was studied using a spontaneous murine osteosarcoma-derived cell line, POS-1, stimulated with lipopolysaccharide (LPS). POS-1 cells were inoculated into the hind paws of 20 C3H/HeJ mice and produced a visible mass in all mice in 5 weeks, and these transplanted tumors resulted in lung metastasis in all mice. The number of metastatic foci in the lungs was 12.0+/-2.1 (mean+/-SD) with LPS-stimulated cells, which was significantly higher than that of unstimulated cells (5.8+/-1.4; N=10 for each; P<0.05). Hyaluronate (HA), a ligand of CD44, inhibited a number of lung metastases in a dose-dependent manner (0.5% HA, 3.0+/-1.1; 0.005% HA, 5.1+/-1.5; without HA, 8.6+/-1.7; N=10 for each; P<0.05, each group with HA versus the group without HA). Adhesion assay by coculturing POS-1 cells and lung microvascular endothelial cells on culture plate showed that the adhesion was significantly lower in HA treated POS-1 than those without HA (1.18+/-0.12 and 2.74+/-0.17, respectively, P<0.05). These results suggest that lung metastasis was accelerated by up-regulation of CD44.

SB209670, a potent endothelin receptor antagonist, prevents or delays axonal degeneration after spinal cord injury.

We developed a rat spinal cord transection injury model and investigated whether endogenous endothelin takes part in axonal degeneration after injury, by using a potent nonselective endothelin receptor antagonist, SB209670. Light microscopic analysis showed that axonal degeneration of the spinal cord was clearly observed one week after injury, supported by immunohistochemical study with anti-neurofilament antibody. Electron microscopic observation showed enlargement and shrinking of spinal axons in the injured sites one week after injury. Application of SB209670 to the lesion sites markedly inhibited axonal damage after injury. These results suggest that endogenous endothelin plays a role in axonal degeneration after spinal cord injury and that SB209670 prevents or delays the axonal degeneration after CNS damage.

Endogenous endothelin-1 initiates astrocytic growth after spinal cord injury.

We developed a rat spinal cord injury model and investigated whether endogenous endothelin (ET)-1 plays a role in astrocytic growth after injury. Immunohistochemical study showed that the number of immature astrocytes (ACs) exhibiting strong reactivity to the monoclonal antibody, RC1, markedly increased 24 h after the injury. Injection of a potent nonselective ET receptor antagonist, SB209670, into the lesion sites significantly inhibited the appearance of RC1-positive cells 24 h after the injury. In conjunction with this result, the increase in immunostaining density of 5-bromo-2'-deoxyuridine in the spinal cord 24 h after the injury was inhibited by the injection of SB209670. The tissue content of ET-1-LI was significantly increased 12 and 24 h after the injury. These results suggest that endogenous ET-1 is involved in astrocytic growth after spinal cord injury.

Development of Dunn osteosarcoma inoculated into subcutaneous air pouch in mice.

Dunn osteosarcoma cell growth was observed in C3H/Hej mouse subcutaneous air pouch. Inoculation with Dunn osteosarcoma cell suspension was performed by injection into air pouches of 70 mice on Day 7 after the initial injection of air. The developmental pattern of the tumor cell colonies was classified histologically into five stages. In Stage 0 (stage of no tumor cells), there were no tumor cells. In Stage 1 (focal stage), the colonies were limited to the lining-cell layers. In Stage 2, (segmental stage) the colonies protruded into the cavity of the pouch. In Stage 3 (annular stage), the total surface of the inner wall of the pouch was occupied by tumor cells. In Stage 4 (occlusive stage), the cavity of the pouch was fully occupied by tumor cells. The tumor was observed to develop to stage 1 in all mice 7 days after inoculation. The colonies were all found to be settled initially in the deeper layer of lining cells. Thus, the mouse air-pouch model is useful for direct observation of osteosarcoma cell growth.

Luxury perfusion phenomenon in acute herpes simplex virus encephalitis.

In a patient with acute herpes simplex virus (HSV) encephalitis, positron emission tomography (PET) demonstrated increased cerebral blood flow in the affected temporal lobe accompanied by reduction in the cerebral oxygen extraction fraction and the cerebral metabolic rate of oxygen, i.e., luxury perfusion. Follow-up PET studies showed reduction in cerebral perfusion until it was more closely coupled with oxygen metabolism after the resolution of the acute inflammation. These findings support previous single photon emission computed tomographic data and provide a pathophysiological background for the occurrence of hyperperfusion in HSV encephalitis. This is an interesting example of the luxury perfusion phenomenon occurring in a disease other than cerebral ischemia.

Assessing the bilateral geometrical differences of the tibia--are they the same?

Contralateral bones are often used in many medical applications but it is assumed that their bilateral differences are insignificant. Previous studies used a limited number of distance measurements in quantifying the corresponding differences; therefore, little is known about their bilateral 3D surface asymmetries. The aim of the study is to develop a comprehensive method to quantify geometrical asymmetries between the left and right tibia in order to provide first results on whether the contralateral tibia can be used as an equivalent reference. In this study, 3D bone models were reconstructed from CT scans of seven tibiae pairs, and 34 variables consisting of 2D and 3D measurements were measured from various anatomical regions. All 2D measurements, and lateral plateau and distal subchondral bone surface measurements showed insignificant differences (p>0.05), but the rest of the surfaces showed significant differences (p<0.05). Our results suggest that the contralateral tibia can be used as a reference especially in surgical applications such as articular reconstructions since the bilateral differences in the subchondral bone surfaces were less than 0.3mm. The method can also be potentially transferable to other relevant studies that require the accurate quantification of bone bilateral asymmetries.

Assessment of four methodologies (microparticle enzyme immunoassay, chemiluminescent enzyme immunoassay, affinity column-mediated immunoassay, and flow injection assay-tandem mass spectrometry) for measuring tacrolimus blood concentration in Japanese liver transplant recipients.

Therapeutic drug monitoring (TDM) and subsequent dosage adjustment for individual patients in the treatment with tacrolimus are required after liver transplantation to prevent rejection and over-immunosuppression, which leads to severe infection and adverse reactions including nephrotoxicity. The purpose of this study was to evaluate the analytical performance among commercially available immunoassay methods, which were microparticle enzyme immunoassay (MEIA), chemiluminescent enzyme immunoassay (CLIA), and affinity column-mediated immunoassay (ACMIA), compared with an assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, the flow injection assay (FIA-MS/MS) was also evaluated to determine whether it could be available as a new method of analysis in tacrolimus therapy. The blood tacrolimus concentrations in samples from liver transplant recipients (n = 102) were measured using MEIA, CLIA, ACMIA, and LC-MS/MS. Additional blood samples from liver transplant recipients (n = 54) were analyzed using both FIA-MS/MS and LC-MS/MS. Because the assay performance and characteristics of MEIA, CLIA, ACMIA, and FIA-MS/MS are relatively different, the measured data should be carefully considered depending on the methodology.

A new X-ray fluorescence spectroscopy for extraterrestrial materials using a muon beam.

The recent development of the intense pulsed muon source at J-PARC MUSE, Japan Proton Accelerator Research Complex/MUon Science Establishment (10(6) s(-1) for a momentum of 60 MeV/c), enabled us to pioneer a new frontier in analytical sciences. Here, we report a non-destructive elemental analysis using µ(-) capture. Controlling muon momentum from 32.5 to 57.5 MeV/c, we successfully demonstrate a depth-profile analysis of light elements (B, C, N, and O) from several mm-thick layered materials and non-destructive bulk analyses of meteorites containing organic materials. Muon beam analysis, enabling a bulk analysis of light to heavy elements without severe radioactivation, is a unique analytical method complementary to other non-destructive analyses. Furthermore, this technology can be used as a powerful tool to identify the content and distribution of organic components in future asteroidal return samples.

Neutron activation analysis of a particle returned from asteroid Itokawa.

A single grain (~3 micrograms) returned by the Hayabusa spacecraft was analyzed by neutron activation analysis. This grain is mainly composed of olivine with minor amounts of plagioclase, troilite, and metal. Our results establish that the Itokawa sample has similar chemical characteristics (iron/scandium and nickel/cobalt ratios) to chondrites, confirming that this grain is extraterrestrial in origin and has primitive chemical compositions. Estimated iridium/nickel and iridium/cobalt ratios for metal in the Itokawa samples are about five times lower than CI carbonaceous chondrite values. A similar depletion of iridium was observed in chondrule metals of ordinary chondrites. These metals must have condensed from the nebular where refractory siderophile elements already condensed and were segregated.