PO-16 - ASK1 regulates tumor lung metastasis and platelet functions.

INTRODUCTION: Apoptosis signal-regulating kinase 1 (ASK1) is a MAP3K in the JNK and p38 MAPK pathways and responds to various stresses. Accumulating evidence indicates that ASK1 plays important roles in tumorigenesis by regulating apoptosis and inflammation. However, little is known about ASK1's roles in tumor metastasis. AIM: To investigate ASK1's roles in tumor metastasis. MATERIALS AND METHODS: We performed experimental lung metastasis model by intravenous injection of Lewis lung carcinoma cells constitutively expressing luciferase (3LL-Luc2 cells). As for the analysis of platelet functions, tail bleeding assay and ferric chloride-induced thrombosis model were utilized. RESULTS: We measured the transition of luciferase activity of the lung lysates up to 7 days as an indicator of lung metastasis. ASK1-/- mice showed markedly lower luciferase activity as early as 3 hours after injection compared to WT mice; hence ASK1 appears to be involved in the early stage of tumor lung metastasis, which is prior to the extravasation of tumor cells. Platelets aggregate and adhere to tumor cells in the early stage and are known to support hematogenous metastasis. ASK1-/- mice were normal in hematological parameters including platelet number, while analysis by western blot revealed that platelets of ASK1-/- mice exhibited markedly reduced phosphorylation of JNK and p38, both of which have been reported to regulate platelet functions such as platelet aggregation. We found that platelets of ASK1-/- mice were less responsive to specific aggregation agonists and that ASK1-/- mice showed bleeding tendency and defect in thrombosis. These phenotypes were also observed in megakaryocyte and platelet-specific ASK1 deficient mice. CONCLUSIONS: It is suggested that impaired platelet functions caused by ASK1 deficiency in platelets may attenuate tumor lung metastasis.

Postoperative chylothorax in congenital diaphragmatic hernia.

INTRODUCTION: The mechanisms responsible for postoperative chylothorax in Congenital Diaphragmatic Hernia (CDH) patients remain unclear. The aim of the present study was to examine the clinical features of CDH that may contribute to an association with postoperative chylothorax. MATERIAL AND METHODS: 198 neonates with CDH, in whom surgical repair of a diaphragmatic defect was performed between 1981 and 2008, were retrospectively studied. The patients were divided into 2 groups; patients with postoperative chylothorax (group I, n=11) and patients without postoperative chylothorax (group II, n=187). The clinical findings were compared between group I and group II to investigate potential predictive parameters for an association with chylothorax. Moreover, the clinical findings and treatments were evaluated in patients with chylothorax. RESULTS: 11 of the 198 infants (5.5%) developed a chylothorax. Although the incidence of a prenatal diagnosis was slightly higher in group I, no relationship with other clinical features was found which would indicate the severity of CDH or the occurrence of postoperative chylothorax. Treatment for chylothorax was drainage alone in 2 cases, total parenteral nutrition with drainage in 8 infants and additional intrathoracic OK-432 infusion in 1 patient. No patients required surgical intervention for chylothorax. No recurrences were observed in this patient series. CONCLUSIONS: It was concluded that postoperative chylothorax is not rare in infants after CDH repair. However, no statistically significant predictive parameters for chylothorax were identified, except for the presence of a prenatal diagnosis.

Efficacy and safety of torasemide in children with heart failure.

OBJECTIVE: To examine the efficacy and safety of torasemide in children with chronic heart failure (HF). METHODS: 102 children with chronic HF who had received oral torasemide were analysed. Of these, 62 (de novo group) were newly diagnosed as having HF and were given torasemide as a diuretic. The remaining 40 (replacement group) had been given furosemide for >3 months before the study, and furosemide was then replaced with torasemide. Clinical signs and symptoms of HF (assessed as the HF index), humoral factors and serum potassium concentrations before torasemide treatment were compared with those obtained 3-4 weeks after torasemide treatment. Patients were also monitored for adverse effects. RESULTS: In the de novo group, torasemide significantly improved the HF index with concomitant improvement in plasma brain natriuretic peptide concentration (median (interquartile range) 52 (51) vs 43 (49) pg/ml). In a randomly selected group of 25 de novo patients with ventricular septal defect, echocardiography showed that torasemide significantly improved left ventricular geometry and function. In the replacement group, brain natriuretic peptide concentrations were also significantly decreased from 50 (104) to 45 (71) pg/ml after substitution of torasemide, but the HF index showed only a tendency for improvement (p = 0.07). Torasemide also had a potassium-sparing effect (de novo group, no change in potassium concentration; replacement group, significant increase from 4.2 (0.5) to 4.3 (0.5) mEq/l), and caused a significant rise in serum aldosterone concentration, consistent with the anti-aldosterone effect of this drug. Serum concentrations of sodium and uric acid had not changed after torasemide treatment, and there were no serious adverse events that necessitated drug withdrawal. CONCLUSION: Torasemide can be safely used, and appears to be effective for treatment of HF in children. Future clinical trials are warranted to verify the present results.

EP2, a receptor for PGE2, regulates tumor angiogenesis through direct effects on endothelial cell motility and survival.

Prostaglandin E2 (PGE2), a major cyclooxygenase (COX) metabolite, plays important roles in tumor biology. We studied the role of EP2, a receptor for PGE2, in tumor angiogenesis using EP2 knockout mice. We found that deletion of the EP2 receptor impaired tumor angiogenesis and this finding was confirmed by an in vivo corneal angiogenesis model and an ex vivo aortic ring assay. To further characterize the cellular mechanisms of the EP2 receptor in angiogenesis, we isolated primary pulmonary endothelial cells (ECs) from wild-type (wt) and EP2-/- mice and observed that EP2-/- ECs exhibited defects in vascular branch formation when compared to wt ECs. In addition, EP2-/- ECs showed impaired cell motility on collagen-coated surface and they responded poorly to PGE2-induced cell migration compared to control cells. However, no difference in cell proliferation was observed between the EP2-/- and wt Ecs. In addition, EP2-/- ECs were more susceptible to apoptosis than wt cells under growth factor depletion conditions. Collectively, our data demonstrate that EP2 signaling in endothelium directly regulates tumor angiogenesis by contributing to cell survival and endothelial cell motility. Moreover, our finding suggests that EP2 is a major receptor in PGE2-mediated cell motility in ECs.

Moderate increase of mean daily temperature adversely affects fruit set of Lycopersicon esculentum by disrupting specific physiological processes in male reproductive development.

BACKGROUND AND AIMS: Global warming is gaining significance as a threat to natural and managed ecosystems since temperature is one of the major environmental factors affecting plant productivity. Hence, the effects of moderate temperature increase on the growth and development of the tomato plant (Lycopersicon esculentum) were investigated. METHODS: Plants were grown at 32/26 degrees C as a moderately elevated temperature stress (METS) treatment or at 28/22 degrees C (day/night temperatures) as a control with natural light conditions. Vegetative growth and reproductive development as well as sugar content and metabolism, proline content and translocation in the androecium were investigated. KEY RESULTS: METS did not cause a significant change in biomass, the number of flowers, or the number of pollen grains produced, but there was a significant decrease in the number of fruit set, pollen viability and the number of pollen grains released. Glucose and fructose contents in the androecium (i.e. all stamens from one flower) were generally higher in the control than METS, but sucrose was higher in METS. Coincidently, the mRNA transcript abundance of acid invertase in the androecium was decreased by METS. Proline contents in the androecium were almost the same in the control and METS, while the mRNA transcript level of proline transporter 1, which expresses specifically at the surface of microspores, was significantly decreased by METS. CONCLUSIONS: The research indicated that failure of tomato fruit set under a moderately increased temperature above optimal is due to the disruption of sugar metabolism and proline translocation during the narrow window of male reproductive development.

Gene and protein expression profiles of prostaglandin E2 receptor subtypes in the human corpus cavernosum.

Prostaglandin E1 leads to penile erection, mainly via prostaglandin E2 (EP) receptors. This study aimed to identify the expression profile of EP receptor genes in human corpus cavernosum. Using the quantitative real-time reverse transcription polymerase chain reaction, the mRNA levels of EP receptor subtypes were measured. In addition, expressions of EP receptor subtype proteins were determined by immunohistochemical method. Among the four subtypes, EP4 receptor mRNA expression was the highest, and EP2 receptor mRNA followed, whereas EP1 and EP3 receptor mRNAs were hardly observed. Expression level of EP4 receptor mRNA was significantly higher than that of EP2 receptor mRNA. Expression of both EP2 and EP4 receptor proteins were clearly detected in the cavernous smooth muscle. These results may suggest that EP4 receptor plays an important role among four EP receptor subtypes for relaxation of smooth muscle in the human corpus cavernosum.

Inhibition of osteoclast differentiation and bone resorption by cathepsin K antisense oligonucleotides.

We confirmed the expression of cathepsin K, the most abundant and specific cysteine protease found in osteoclasts, at the mRNA level in most of our cases of breast cancer, and even at the protein level in bone metastatic lesions. Therefore, we investigated the functions of cathepsin K in osteoclasts with special attention to bone metastasis from breast cancer. Mouse osteoclast-like cells (OCLs) were established by coculture of mouse bone marrow cells and osteoblastic cells. Rodent cathepsin K antisense (AS) or random control (CL) oligonucleotides were added on day 0, 3, or 6 of culture. Tartrate-resistant acid phosphatase staining confirmed the formation of OCLs after 9 d of incubation. AS treatment significantly reduced both the number of TRAP-positive cells and the percentage of multinuclear cells. For the pit-forming assay, after 9 d of incubation, mature OCLs were collected and incubated on ivory slices with AS or CL for 48 h. The antisense oligonucleotides also inhibited the bone-resorbing activity of OCLs. CL treatment did not affect either the number of TRAP-positive cells or pit formation. Cathepsin K may play important roles in bone resorption as well as in differentiation of osteoclasts. These findings indicate that the inhibition of this enzyme may prevent the development of bone metastasis from breast cancer.

Matrilysin stimulates DNA synthesis of cultured vascular endothelial cells and induces angiogenesis in vivo.

Matrilysin produced by human colon cancer cells may be involved in the progression and metastasis of cancer. In the present study, we investigated the association of matrilysin with angiogenesis. One microgram of recombinant matrilysin is confirmed to have increased [3H]-thymidine uptake in human umbilical vein endothelial cells. Then we used micro encapsulation and a mouse hemoglobin enzyme-linked immunosorbent assay system for in vivo quantitation of angiogenesis with BALB/c nu/nu athymic mice. Hundred micrograms of recombinant matrilysin induced angiogenesis to the same degree as 10 microg of basic fibroblast growth factor (bFGF). Angiogenesis was observed at the site implanted with human colon cancer WiDr cells in agarose micro beads. This was inhibited by subcutaneous injection of matrilysin-specific antisense oligonucleotide significantly by 53%. In conclusion, matrilysin may be associated with angiogenesis of human colon cancer through the direct proliferative action on endothelial cells.

Daily cocoa intake reduces the susceptibility of low-density lipoprotein to oxidation as demonstrated in healthy human volunteers.

Nine male volunteers were given 36 g of cocoa powder (containing 2610 mg of polyphenols) per day with sugar and 6 volunteers received an equivalent amount of sugar for 2 weeks. Conjugated diene production in LDL induced by 2-2' azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70) and copper ion were evaluated. The lag time was significantly prolonged at 1 and 2 weeks in V-70 and at 2 weeks in copper ion after cocoa powder consumption. The level of excretion of epicatechin in urine was significantly higher in the cocoa group than that in the control group. In conclusion, the antioxidants in cocoa powder might be absorbed and increase the resistance of human LDL to oxidation.

Magnetic resonance imaging in the evaluation of vaginal foreign bodies in a young girl.

A 7-year-old girl with foul-smelling, bloody vaginal discharge for more than 2 years was initially suspected of suffering from vaginal foreign bodies. Although plain radiography revealed no abnormal findings in the pelvis, magnetic resonance imaging (MRI) showed multiple low intensity objects and an intact vaginal wall. Four plastic toys were removed with forceps under general anesthesia. MRI is supposed to be the best technique for evaluating vaginal foreign bodies in young girls.

Prenatal findings in Brachmann-de Lange syndrome.

We present a case of Brachmann-de Lange syndrome, in which prenatal ultrasonographic evaluation demonstrated increased nuchal translucency, early onset of intrauterine growth retardation, and limb abnormalities in the first, second, and third trimester, respectively.

[Putaminal signal hypointensity on T2 weighted MR image in a patient with corticobasal degeneration].

In the course of a 74-year-old woman with corticobasal degeneration (CBD), bilateral putaminal signal hypointensity appeared on T2 weighted MR image. This right-handed woman noticed progressive difficulty in writing and using chopsticks during two years. Neurological examination in February 1998 showed apraxia, rigidity, akinesia and myoclonus, which were more marked in the right hand side. MRI revealed atrophy of the left frontotemporal lobes. These findings were compatible to CBD. Her symptoms progressed slowly. On re-admission due to aspiration pneumonia in December 1998, she showed difficulty in swallowing and walking in addition to the signs and symptoms described above. MRI revealed bilateral putaminal signal hypointensity on T2 weighted image. In the literature, totally 9 cases in 2 reports showed similar putaminal signal hypointensity. Although this finding is unusual in CBD, advanced degenerative process in the putamen might have induced this change due possibly to the accumulation of iron secondary to the nerve cell loss.

Role of prostaglandin E2 and leukotriene B4 in skin reaction induced by transdermal application of propranolol.

Dermal application of propranolol (PRL) induced formation of erythema and edema, and pseudoeosinophil infiltration in epidermis and dermis at the application site in guinea pigs. We investigated the production of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) at the application site of PRL and the role of these inflammatory chemical mediators in the occurrence of the skin reactions. PGE2 was found to be produced at the application site slightly after the accumulation of PRL released from the adhesive bandage in the patch test, and the amount of PGE2 increased continuously, with a peak value obtained at 24 h after application. The time-course changes resembled those of delta a* value, the index of erythema formation determined by colorimetric measurement, and edema formation. The production of PGE2 by dermal application of PRL was suppressed by local pretreatment with dexamethasone or indomethacin. However, no notable production of LTB4 was observed at the application site of PRL.

Reversal of intrapulmonary arteriovenous shunting detected by two-dimensional contrast-enhanced echocardiography after liver transplantation.

BACKGROUND: Intrapulmonary arteriovenous shunting (IPS), occasionally associated with advanced liver disease, may reverse after liver transplantation (LTx). Two-dimensional contrast-enhanced echocardiography, a convenient noninvasive study, has never been used to demonstrate disappearance of IPS after LTx. METHODS: For an 8-month-old girl undergoing living-related LTx, two-dimensional contrast-enhanced echocardiography was performed with the microbubble injection. The opacification of the microbubble in the left heart emerging within 3-6 beats after detection in the right heart was compared with that in the right heart. RESULTS: Microbubble opacification in the left heart was almost the same as that in the right heart (grade 3) shortly after LTx. However, the contrast in the left heart diminished (grade 1) as the respiratory condition improved and subsequently disappeared (grade 0). CONCLUSIONS: Two-dimensional contrast-enhanced echocardiography may be a feasible noninvasive method to evaluate the degree of IPS in the peritransplant period and observe disappearance of IPS after LTx.

Changes in clinicopathological findings after portal vein embolization.

BACKGROUND/AIMS: Portal vein embolization is becoming more common as a method of preventing hepatic failure after an extended hepatectomy but its mechanism is not well understood. This clinicopathological study focused on its mechanism. METHODOLOGY: Thirty patients who underwent extended hepatectomy after portal vein embolization were evaluated. Liver volume was measured before and after portal vein embolization, and histological studies were performed to examine morphological changes, morphometric parameters and apoptosis of hepatocytes. RESULTS: The mean volume of the non-embolized lobe grew significantly from 392 to 462 mL after portal vein embolization (P < 0.0001). The hypertrophy ratio of the non-embolized lobe (100 x volume change during portal vein embolization/volume before portal vein embolization, %) was correlated closely with the volume of the non-embolized lobe before portal vein embolization (r = -0.65, P < 0.0001). Histological study showed the embolized lobe hepatocytes to be atrophic, partly necrotic and apoptotic. In the non-embolized lobe, the mean hepatocyte volume was 8686 micron 3 (control: 6544 micron 3) and the mean hepatocyte count was 109 x 10(6)/mL (control: 122 x 10(6)/mL). CONCLUSIONS: The enlargement of the non-embolized lobe was caused by hypertrophy rather than hyperplasia suggesting hyperfunction. The resection of the atrophic embolized lobe, leaving the hypertrophic non-embolized lobe was thought to be less surgically stressful than hepatectomy without portal vein embolization.

Does pancreatico-biliary maljunction play a role in spontaneous perforation of the bile duct in children?

Spontaneous perforation of the bile duct (SPBD) is a rare disease in children. Pancreatico-biliary maljunction (PBM) has been postulated to contribute to its etiology. We have treated three children with SPBD over 30 years, two of whom had PBM. There was one boy and two girls aged 10 months to 2 years with symptoms of abdominal distension, vomiting, abdominal pain, jaundice, and acholic stools. The diagnosis of SPBD was made by paracentesis showing biliary ascites, and primary biliary and intra-abdominal drainage was performed in all cases. The site of perforation was at the connection of the common bile duct (CBD) with the cystic duct in all cases. In two cases reflux of contrast into the pancreatic duct was noted, the common channel was long (17 and 12 mm, respectively), and the bile amylase level in the CBD was abnormally high (50,000 and 67,000 IU/l, respectively). In the third patient there was no reflux of contrast into the pancreatic duct and the bile amylase and trypsin levels in the CBD and gallbladder were not measurable. Thus, SPBD in children may not be due solely to PBM, but may involve multiple mechanisms.

Inhibition of low-density lipoprotein oxidation by astaxanthin.

Marine animals produce astaxanthin which is a carotenoid and antioxidant. In this study we determined the in vitro and ex vivo effects of astaxanthin on LDL oxidation. The oxidation of LDL was measured in a 1 ml reaction system consisting of increasing concentrations of astaxanthin (12.5, 25.0, 50.0 microg/ml), 400 microM V-70 (2, 2'-azobis(4-methoxy-2, 4-dimethylvaleronitrile)), and LDL (70 microg/ml protein). Astaxanthin dose, dependently significantly prolonged the oxidation lag time (31.5, 45.4, 65.0 min) compared with the control (19.9 min). For the ex vivo study 24 volunteers (mean age 28.2 [SD 7.8] years) consumed astaxanthin at doses of 1.8, 3.6,14.4 and 21.6 mg per day for 14 days. No other changes were made in the diet. Fasting venous blood samples were taken at days 0, +14. LDL lag time was longer (5.0, 26.2, 42.3 and 30.7% respectively) compared with day 0 after consuming astaxanthin at doses of 1.8, 3.6,14.4 and 21.6 mg for 14 days compared with day 0, but there was no difference in oxidation of LDL between day 0 (lag time 59.9+/-7.2 min) and day 14 (57.2+/-6.0 min) in the control group. Our results provide evidence that consumption of marine animals producing astaxanthin inhibits LDL oxidation and possibly therefore contributes to the prevention of atherosclerosis.

Telomerase enzyme activity and RNA expression in adriamycin-resistant human breast carcinoma MCF-7 cells.

Telomerase activity has been reported in cancer cells after treatment with antineoplastic agents. Assessment of telomerase activity could be a valuable tool to measure the reduction of aggression caused by chemotherapy. This study was designed to investigate the significance of telomerase for chemotherapy with respect to Adriamycin (ADM)-resistance. MCF-7 and its ADM-resistant line (AdrR) were treated with ADM, 5-fluorouracil (5FU) or taxotere (TAXO). Telomerase activity and human telomerase RNA component (hTR) were quantitatively measured by the telomeric repeat amplification protocol assay and RT-PCR, respectively. Cell counting and MTT assay were also performed. In MCF-7, enzyme activity was significantly reduced by ADM and 5FU treatments. In AdrR, 5FU and TAXO reduced enzyme activity, while ADM significantly increased the activity. No significant changes in hTR were seen in these two cell lines after treatment with any of these drugs. When Bcl-2 expression was examined after drug treatments, ADM increased Bcl-2 expression in AdrR cells, while not changing it in MCF-7 cells. We conclude that an unusual reaction of telomerase activity in AdrR may explain, at least in part, one of the mechanisms of the malignant biological behavior related with the drug-resistance to ADM.