RUNX1-Survivin Axis Is a Novel Therapeutic Target for Malignant Rhabdoid Tumors.

Malignant rhabdoid tumor (MRT) is a highly aggressive pediatric malignancy with no effective therapy. Therefore, it is necessary to identify a target for the development of novel molecule-targeting therapeutic agents. In this study, we report the importance of the runt-related transcription factor 1 (RUNX1) and RUNX1-Baculoviral IAP (inhibitor of apoptosis) Repeat-Containing 5 (BIRC5/survivin) axis in the proliferation of MRT cells, as it can be used as an ideal target for anti-tumor strategies. The mechanism of this reaction can be explained by the interaction of RUNX1 with the RUNX1-binding DNA sequence located in the survivin promoter and its positive regulation. Specific knockdown of RUNX1 led to decreased expression of survivin, which subsequently suppressed the proliferation of MRT cells in vitro and in vivo. We also found that our novel RUNX inhibitor, Chb-M, which switches off RUNX1 using alkylating agent-conjugated pyrrole-imidazole polyamides designed to specifically bind to consensus RUNX-binding sequences (5'-TGTGGT-3'), inhibited survivin expression in vivo. Taken together, we identified a novel interaction between RUNX1 and survivin in MRT. Therefore the negative regulation of RUNX1 activity may be a novel strategy for MRT treatment.

A RUNX-targeted gene switch-off approach modulates the BIRC5/PIF1-p21 pathway and reduces glioblastoma growth in mice.

Glioblastoma is the most common adult brain tumour, representing a high degree of malignancy. Transcription factors such as RUNX1 are believed to be involved in the malignancy of glioblastoma. RUNX1 functions as an oncogene or tumour suppressor gene with diverse target genes. Details of the effects of RUNX1 on the acquisition of malignancy in glioblastoma remain unclear. Here, we show that RUNX1 downregulates p21 by enhancing expressions of BIRC5 and PIF1, conferring anti-apoptotic properties on glioblastoma. A gene switch-off therapy using alkylating agent-conjugated pyrrole-imidazole polyamides, designed to fit the RUNX1 DNA groove, decreased expression levels of BIRC5 and PIF1 and induced apoptosis and cell cycle arrest via p21. The RUNX1-BIRC5/PIF1-p21 pathway appears to reflect refractory characteristics of glioblastoma and thus holds promise as a therapeutic target. RUNX gene switch-off therapy may represent a novel treatment for glioblastoma.

RUNX1 transactivates BCR-ABL1 expression in Philadelphia chromosome positive acute lymphoblastic leukemia.

The emergence of tyrosine kinase inhibitors as part of a front-line treatment has greatly improved the clinical outcome of the patients with Ph+ acute lymphoblastic leukemia (ALL). However, a portion of them still become refractory to the therapy mainly through acquiring mutations in the BCR-ABL1 gene, necessitating a novel strategy to treat tyrosine kinase inhibitor (TKI)-resistant Ph+ ALL cases. In this report, we show evidence that RUNX1 transcription factor stringently controls the expression of BCR-ABL1, which can strategically be targeted by our novel RUNX inhibitor, Chb-M'. Through a series of in vitro experiments, we identified that RUNX1 binds to the promoter of BCR and directly transactivates BCR-ABL1 expression in Ph+ ALL cell lines. These cells showed significantly reduced expression of BCR-ABL1 with suppressed proliferation upon RUNX1 knockdown. Moreover, treatment with Chb-M' consistently downregulated the expression of BCR-ABL1 in these cells and this drug was highly effective even in an imatinib-resistant Ph+ ALL cell line. In good agreement with these findings, forced expression of BCR-ABL1 in these cells conferred relative resistance to Chb-M'. In addition, in vivo experiments with the Ph+ ALL patient-derived xenograft cells showed similar results. In summary, targeting RUNX1 therapeutically in Ph+ ALL cells may lead to overcoming TKI resistance through the transcriptional regulation of BCR-ABL1. Chb-M' could be a novel drug for patients with TKI-resistant refractory Ph+ ALL.

RUNX inhibitor suppresses graft-versus-host disease through targeting RUNX-NFATC2 axis.

Patients with refractory graft-versus-host disease (GVHD) have a dismal prognosis. Therefore, novel therapeutic targets are still needed to be identified. Runt-related transcriptional factor (RUNX) family transcription factors are essential transcription factors that mediate the essential roles in effector T cells. However, whether RUNX targeting can suppress, and GVHD is yet unknown. Here, we showed that RUNX family members have a redundant role in directly transactivating NFATC2 expression in T cells. We also found that our novel RUNX inhibitor, Chb-M', which is the inhibitor that switches off the entire RUNX family by alkylating agent-conjugated pyrrole-imidazole (PI) polyamides, inhibited T-cell receptor mediated T cell proliferation and allogenic T cell response. These were designed to specifically bind to consensus RUNX-binding sequences (TGTGGT). Chb-M' also suppressed the expression of NFATC2 and pro-inflammatory cytokine genes in vitro. Using xenogeneic GVHD model, mice injected by Chb-M' showed almost no sign of GVHD. Especially, the CD4 T cell was decreased and GVHD-associated cytokines including tissue necrosis factor-α and granulocyte-macrophage colony-stimulating factor were reduced in the peripheral blood of Chb-M' injected mice. Taken together, our data demonstrates that RUNX family transcriptionally upregulates NFATC2 in T cells, and RUNX-NFATC2 axis can be a novel therapeutic target against GVHD.

Suppression of malignant rhabdoid tumors through Chb-M'-mediated RUNX1 inhibition.

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive pediatric malignancy primarily affecting infants and young children. Intensive multimodal therapies currently given to MRT patients are not sufficiently potent to control this highly malignant tumor. Therefore, additive or alternative therapy for these patients with a poor prognosis is necessary. We herein demonstrated that the inhibition of runt-related transcription factor 1 (RUNX1) by novel alkylating conjugated pyrrole-imidazole (PI) polyamides, which specifically recognize and bind to RUNX-binding DNA sequences, was highly effective in the treatment of rhabdoid tumor cell lines in vitro as well as in an in vivo mouse model. Therefore, suppression of RUNX1 activity may be a novel strategy for MRT therapy.

[Simultaneous Bilateral Pneumothorax due to the Communication of Both Thoracic Cavity].

We report a case of a simultaneous bilateral pneumothorax (buffalo chest). A 75-year-old man who had undergone resection of an esophageal carcinoma had difficulty in breathing and lost consciousness. He was transported to our hospital and diagnosed as a simultaneous bilateral pneumothorax. He underwent bilateral chest drainages, and was hospitalized. Because of the continued air leak, an operation was performed. First, thoracoscopic bullectomy was performed from the left side. Changing the position, the water poured in the left thoracic cavity to test for air leaks flowed out to the right drain in large quantities;thus, a communication between both sides of the thoracic cavity became clear, although we could not find a pleural defect between the thoracic cavities.

[Surgical Case of Lung Cancer with Anomalous Right Pulmonary Vein;Report of a Case].

We report a case of an anomalous pulmonary vein with right V8. A 67-year-old man was found to have an abnormal shadow on chest X-ray film. He had a diagnosis of clinical stage IB lung adenocarcinoma. Preoperative three-dimensional computed tomographic angiography (3D-CT angiography) showed middle lobe pulmonary vein directly flow into the left atrium and the right V8 flow into the middle lobe pulmonary vein. Based on these findings, right lower lobectomy was safely and successfully performed. A complete understanding of the anatomic features of the pulmonary vascular branches prior to surgery is essential for safe and accurate pulmonary resection.

Fetal androgen signaling defects affect pancreatic ß-cell mass and function, leading to glucose intolerance in high-fat diet-fed male rats.

We previously demonstrated that androgen signaling expands pancreatic ß-cell mass in the sexual maturation period (Am J Physiol Endocrinol Metab 314: E274-E286, 2018). The aim of this study was to elucidate whether fetal androgen signaling plays important roles in ß-cell mass development and ß-cell function in adulthood, defects of which are associated with type 2 diabetes mellitus. In the pancreas of male fetuses, androgen receptor (AR) was strongly expressed in the cytoplasm and at the cell membrane of Nkx6.1-positive ß-cell precursor cells but was markedly reduced in insulin-positive ß-cells. Administration of the anti-androgen flutamide to pregnant dams during late gestation reduced ß-cell mass and Ki67-positive proliferating ß-cells at birth in a male-specific manner without affecting body weight. The decrease of ß-cell mass in flutamide-exposed male rats was not recovered when rats were fed a standard diet, whereas it was fully recovered when rats were fed a high-fat diet (HFD), at 6 and 12 wk of age. Flutamide exposure in utero led to the development of glucose intolerance in male rats due to a decrease in insulin secretion when fed HFD but not standard diet. Insulin sensitivity did not differ between the two groups irrespective of diet. These results indicated that the action of fetal androgen contributed to ß-cell mass expansion in a sex-specific manner at birth and to the development of glucose intolerance by decreasing the secretion of insulin in HFD-fed male rats. Our data demonstrated the involvement of fetal androgen signaling in hypothesized sex differences in the developmental origins of health and disease by affecting pancreatic ß-cell function.

Androgen signaling expands ß-cell mass in male rats and ß-cell androgen receptor is degraded under high-glucose conditions.

A deficient pancreatic ß-cell mass increases the risk of type 2 diabetes mellitus. Here, we investigated the effects of testosterone on the development of pancreatic ß-cell mass in male rats. The ß-cell mass of male rats castrated at 6 wk of age was reduced to ~30% of that of control rats at 16 wk of age, and castration caused glucose intolerance. Loss of ß-cell mass occurred because of decreases in islet density per pancreas and ß-cell cluster size. Castration was negatively associated with the number of Ki-67-positive ß-cells and positively associated with the number of TUNEL-positive ß-cells. These ß-cell changes could be prevented by testosterone treatment. In contrast, castration did not affect ß-cell mass in male mice. Androgen receptor (AR) localized differently in mouse and rat ß-cells. Testosterone enhanced the viability of INS-1 and INS-1 #6, which expresses high levels of AR, in rat ß-cell lines. siRNA-mediated AR knockdown or AR antagonism with hydroxyflutamide attenuated this enhancement. Moreover, testosterone did not stimulate INS-1 ß-cell viability under high d-glucose conditions. In INS-1 ß-cells, d-glucose dose dependently (5.5-22.2 mM) downregulated AR protein levels both in the presence and absence of testosterone. The intracellular calcium chelator (BAPTA-AM) could prevent this decrease in AR expression. AR levels were also reduced by a calcium ionophore (A23187), but not by insulin, in the absence of the proteasome inhibitor MG132. Our results indicate that testosterone regulates ß-cell mass, at least in part, by AR activation in the ß-cells of male rats and that the ß-cell AR is degraded under hyperglycemic conditions.

Androgen receptor silences thioredoxin-interacting protein and competitively inhibits glucocorticoid receptor-mediated apoptosis in pancreatic ß-Cells.

Androgen receptor (AR) is known to bind to the same cis-element that glucocorticoid receptor (GR) binds to. However, the effects of androgen signaling on glucocorticoid signaling have not yet been elucidated. Here, we investigated the effects of testosterone on dexamethasone (DEX, a synthetic glucocorticoid)-induced apoptosis of pancreatic ß-cells, which might be involved in the pathogenesis of type 2 diabetes mellitus in males. We used INS-1 #6 cells, which were isolated from the INS-1 pancreatic ß-cell line and which express high levels of AR. Testosterone and dihydrotestosterone inhibited apoptosis induced by DEX in INS-1 #6 cells. AR knockdown and the AR antagonist hydroxyflutamide each diminished the anti-apoptotic effects of testosterone. AR was localized in the nucleus of both INS-1 #6 cells and pancreatic ß-cells of male rats. Induction of thioredoxin-interacting protein (TXNIP) is known to cause pro-apoptotic effects in ß-cells. Testosterone suppressed the DEX-induced increase of TXNIP at the transcriptional level. A Chromatin immunoprecipitation assays showed that both AR and GR competitively bound to the TXNIP promoter in ligand-dependent manners. Recombinant DNA-binding domain of AR bound to the same cis-element of the TXNIP promoter that GR binds to. Our results show that AR and GR competitively bind to the same cis-element of TXNIP promoter as a silencer and enhancer, respectively. These results indicate that androgen signaling functionally competes with glucocorticoid signaling in pancreatic ß-cell apoptosis.

Fabrication and Characterization of Single Phase α-Alumina Membranes with Tunable Pore Diameters.

Nanoporous and single phase α-alumina membranes with pore diameters tunable over a wide range of approximately 60-350 nm were successfully fabricated by optimizing the conditions for anodizing, subsequent detachment, and heat treatment. The pore diameter increased and the cell diameter shrunk upon crystallization to α-alumina by approximately 20% and 3%, respectively, in accordance with the 23% volume shrinkage resulting from the change in density associated with the transformation from the amorphous state to α-alumina. Nevertheless, flat α-alumina membranes, each with a diameter of 25 mm and a thickness of 50 µm, were obtained without thermal deformation. The α-alumina membranes exhibited high chemical resistance in various concentrated acidic and alkaline solutions as well as when exposed to high temperature steam under pressure. The Young's modulus and hardness of the single phase α-alumina membranes formed by heat treatment at 1250 °C were notably decreased compared to the corresponding amorphous membranes, presumably because of the nodular crystallite structure of the cell walls and the substantial increase in porosity. Furthermore, when used for filtration, the α-alumina membrane exhibited a level of flux higher than that of the commercial ceramic membrane.

Flight distance and avoidance score in thoroughbred foals and yearlings and the relationship with handling frequency in the young ages.

Flight and avoidance reactions from human were examined using 168 postweaning Thoroughbred foals in 22 breeding farms. Further 114 yearlings of 168 foals were tested in the following summer. The foal handlings by the stabler were asked in questionnaire. The relationship between the behavioural reactions and the foal handling frequencies was analyzed. The flight reaction was estimated as the distance from the animal to a stranger when the animal began to flight away from his approach. The avoidance scores were set up from (1) (not resistant) to (5) (touch rejection) from human touching. In the stabler questionnaire, handling frequencies of "body brushing", "rectal temperature measurement", "hoof cleaning", and "stall cleaning" in the early nursing period were asked. The handling frequencies were scored from (1) (not done) to (5) (every day). In the preliminary test, a measurement reliability of the flight distance and the avoidance score was confirmed. The mean flight distances were 0.56 m and 0.27 m in the postweaning foals and the yearlings, respectively. Touch-avoidance scores of the highest frequency were (3) and (2) in the postweaning foals and the yearlings, respectively. As the results of Spearman's rank-correlation analysis, "body brushing" showed highly negative relationships with "flight distance" (ρ=-0.31, P<0.001) and "avoidance score" (ρ=-0.37, P<0.001) in the postweaning foals. In the yearlings, "hoof cleaning" also showed significantly negative relationships with these behavioural indices (ρ=-0.24, P<0.01; ρ=-0.22, P<0.01, respectively).

Assessment of emotional reaction induced by visual stimulation based on cross-correlation between pulse wave transmission time and heart rate in the Mayer wave-band.

Mayer wave (0.1Hz fluctuation) included in heart rate variability and blood pressure variability appears apparently in the resting state. On the other hand, it can be predicted that a strong emotional reaction may affect the relationship between these variabilities. This prediction suggests that the human emotional reaction can be quantified by the maximum correlation coefficient rho/sub max/ between heart rate and blood pressure whose frequency components are limited to the Mayer wave-band. However, the conventional method of obtaining rho/sub max/ needs a bulky and expensive device for measuring continuous blood pressure. In this study, a smaller and cheaper device for measuring pulse wave transmission time (PTT) has been developed. This work has shown that the PTT can give rho/sub max/ instead of blood pressure and that rho/sub max/ obtained by the PTT may significantly reflect the emotional reaction on the basis of an experiment using nine healthy subjects with nine self-produced devices in which pictures were presented to the subjects to induce their emotional reactions.