Inhibition of heat shock protein 90 exerts an antitumour effect in angiosarcoma: involvement of the vascular endothelial growth factor signalling pathway.

BACKGROUND: Angiosarcoma is a rare malignant neoplasm derived from endothelial cells, and because advanced angiosarcoma is resistant to standard chemotherapy its prognosis is poor. Therefore, new therapies are urgently needed. Heat shock protein (HSP)90 has been identified as a molecular chaperone that regulates various cancer-related proteins. Numerous clinical trials are currently testing the effectiveness of HSP90 inhibitors in various types of malignancies. OBJECTIVES: To investigate the role of HSP90 in the pathogenesis of angiosarcoma and whether the inhibition of HSP90 may have antitumour activity. METHODS: The expression of HSP90 protein in angiosarcoma was examined using immunohistochemistry and immunoblotting. The effects of HSP90 inhibition were proven using proliferation, migration and invasion assay in angiosarcoma cells. The mechanism of antitumour effect by HSP90 inhibition was investigated by the transfection of small interfering RNA (siRNA). RESULTS: The levels of HSP90 protein expression in cultured angiosarcoma cell lines were markedly increased compared with those in normal tissue cell lines. Immunohistochemical analyses revealed that the expression of HSP90 protein was strongly detected in angiosarcoma tissues compared with that in normal dermal vessels or senile angioma tissues. Ganetespib, an HSP90 inhibitor, with or without taxanes, inhibited the proliferation of angiosarcoma cells via apoptosis in a dose-dependent manner. HSP90 siRNA suppressed the proliferation, migration and invasion of angiosarcoma cells. Knock-down of HSP90 did not suppress vascular endothelial growth factor receptor 2 directly, but selectively suppressed several downstream targets of vascular endothelial growth factor signalling in angiosarcoma cells. CONCLUSIONS: HSP90 could be a novel therapeutic target for angiosarcoma.

Experimental validation of a novel compact focusing scheme for future energy-frontier linear lepton colliders.

A novel scheme for the focusing of high-energy leptons in future linear colliders was proposed in 2001 [P. Raimondi and A. Seryi, Phys. Rev. Lett. 86, 3779 (2001)]. This scheme has many advantageous properties over previously studied focusing schemes, including being significantly shorter for a given energy and having a significantly better energy bandwidth. Experimental results from the ATF2 accelerator at KEK are presented that validate the operating principle of such a scheme by demonstrating the demagnification of a 1.3 GeV electron beam down to below 65 nm in height using an energy-scaled version of the compact focusing optics designed for the ILC collider.

Systematic evaluation of magnetic sensitivities of anisotropic magnetoresistive sensors at liquid helium temperature for superconducting cavities.

Trapped magnetic flux in bulk superconductors reduces the quality factor Q in superconducting radio-frequency (SRF) cavities. However, the mechanisms underlying flux trapping and radio-frequency loss are not well understood. Detailed observation of the magnetic distributions is important for understanding such phenomena. Magnetic field mapping is useful for observing the magnetic field distribution around SRF cavities. Measuring the change in the magnetic field around the cavity elucidates the flux trapping behavior. Anisotropic magnetoresistive (AMR) sensors are inexpensive and small devices that can detect magnetic flux density. The magnetic sensitivities of AMR sensors need to be evaluated at liquid helium temperature for the magnetic field mapping of SRF cavities. In this study, a test stand was constructed to calibrate the magnetic sensitivities of AMR sensors in liquid helium, and 110 AMR sensors were tested using this stand. The magnetic sensitivities were evaluated systematically. A solenoid coil was used to control the uniform external magnetic field and to measure the magnetic sensitivity at low temperatures. All AMR sensors exhibited suitable sensitivities to the magnetic field around the SRF cavity. The variation in these sensitivities in all AMR sensors was ∼1%. The AMR sensors were found to have sufficient sensitivity for mapping the magnetic field around the exterior surface of the SRF cavity.

Urinary neutral glycosphingolipid analysis of patients with Fabry's disease; rapid isocratic elution from high-performance liquid chromatography as per-o-benzoyl derivatives.

Neutral glycosphingolipids from urinary sediments of six patients with Fabry's disease and 11 members of the family of one propositus were analyzed using high-performance liquid chromatography. Per-o-benzoyl derivatives of GlcCer, LacCer, GbOse3Cer and GbOse4Cer were clearly resolved by a solvent mixture of hexane/dioxane/isopropanol (75:25:1, v/v) on a normal-phase silica column. Using our isocratic solvent system, the analysis was completed within 15 min. The smallest amount of glycolipid that could be detected by HPLC was 50 pmol and a linear response was shown at 230 nm up to 400 pmol. The calculated peak area of GbOse4Cer was higher than those of GlcCer, LacCer and GbOse3Cer. The molar ratios of GbOse3Cer to monohexosyl ceramide (CMH) in the urinary sediments were: Fabry hemizygotes, 36.33 +/- 25.54 (n = 6); heterozygotes, 0.94 +/- 0.50 (n = 4); and controls, 0.11 +/- 0.06 (n = 5). The molar ratio CDH/CMH was also higher in patients (8.42 +/- 6.23) than in controls (0.77 +/- 0.23). The female H was a rare example of a carrier with typical clinical manifestations. From the urinary sediment analysis, females E, G and J were suspected to be Fabry heterozygotes, although no clinical signs were observed at the time of examination.

Evaluation of combined therapy with chemoembolization and ethanol injection for advanced hepatocellular carcinoma.

To evaluate the therapeutic efficacy of transcatheter arterial chemoembolization (TACE) combined with percutaneous ethanol injection therapy (PEIT) for advanced hepatocellular carcinoma, we studied the effectiveness of TACE therapy combined with PEIT (50 cases) and TACE alone (50 cases). In both groups, patients had multiple lesions, or a single lesion with a diameter exceeding 2 cm or with vascular invasion (stages II, III, and IV in the tumor staging classification of the Liver Cancer Study Group of Japan). The clinical features in the two groups were comparable. The cumulative survival rates with TACE-PEIT were 95.0% for 1 year, 72.5% for 2 years, and 50.0% for 3 years, whereas the rates with TACE alone were 92.5% for 1 year, 57.5% for 2 years, and 20.0% for 3 years. The survival rate in the TACE-PEIT group was significantly higher than that in the TACE alone group. Moreover, the survival rate of patients with stage II or III disease in the TACE-PEIT group was significantly better than that in the TACE alone group, and the survival rate of patients with Child's classification B or C in the TACE-PEIT group was significantly higher than that in the TACE alone group. Multivariate analysis using Cox's proportional hazard regression model showed that the most significant prognostic factors in the TACE-PEIT group were tumor embolus in the portal vein and the number of tumors. These results suggest the effectiveness of combining TACE and PEIT for the treatment of advanced hepatocellular carcinoma.

Prospective and randomized trial of lipiodol-transcatheter arterial chemoembolization for treatment of hepatocellular carcinoma: a comparison of epirubicin and doxorubicin (second cooperative study). The Cooperative Study Group for Liver Cancer Treatment of Japan.

A randomized, controlled clinical trial was conducted to compare the use of epirubicin (EPI) and doxorubicin (DOX) in Lipiodol (Laboratoire Guerbet, Roissy-Charles-de-Gaulle Cedex, France)-transcatheter arterial chemoembolization as a treatment of hepatocellular carcinoma. One hundred ninety-two hospitals participated, and 415 patients were enrolled in the study during the period between October 1989 and December 1990. The patients were randomly allocated to group A (EPI) or group B (DOX) by a centralized telephone registration. The actual doses of EPI and DOX were 72 mg/body and 48 mg/body, respectively. The 1-, 2-, and 3-year survival rates were, respectively, 69%, 44%, and 33% for group A and 73%, 54%, and 37% for group B. There were no statistically significant differences (P = .2296, log-rank test). When each group of patients was classified retrospectively into high-risk and low-risk subgroups based on the severity index calculated by the Cox regression model from the significant prognostic factors (the pretreatment tumor size, the pretreatment serum alpha-fetoprotein level, tumor encroachment, and Child's classification), the survival curve of the low-risk DOX subgroup was significantly superior to that of the low-risk EPI subgroup (P = .0182). However, there was no significant difference between the high-risk subgroups (P = .4606). The change in the serum alpha-fetoprotein level, the extent of Lipiodol accumulation in the tumor, and the extent of tumor reduction after the treatment did not show any significant differences between the groups. The white blood cell count in group B showed a tendency to decrease slightly more than in group A at 3 weeks after Lipiodol-transcatheter arterial chemoembolization. In conclusion, there was no statistically significant difference between the survival curves of the EPI and DOX groups in Lipiodol-transcatheter arterial embolization treatment of hepatocellular carcinoma.

Multiple, unique, and common p53 mutations in a thorotrast recipient with four primary cancers.

Four primary cancers found at autopsy of a patient who received the thorium-based contrast agent Thorotrast 50 years ago and who was healthy up until a few months before his death from liver failure were analyzed for p53 mutations. The data suggest that the chronic alpha-irradiation may be a large causative factor. Multiple mutations were found in all the cancer tissues: two foci of a cholangiocellular carcinoma, a tubular adenocarcinoma of the stomach, a squamous cell carcinoma of the lung, and an adenocarcinoma of Vater's ampulla. The total number of point mutations detected were 13. Moreover, homozygous aberrations were detected in a large area of normal small intestine and noncancer liver tissues suggesting that nontumor cells which harbored p53 abnormalities gained a survival advantage and clonally expanded.

Corticotropin-releasing factor but not urocortin is involved in adrenalectomy-induced adrenocorticotropin release.

Urocortin, a new corticotropin-releasing factor (CRF)-related peptide, has been reported to have the ability to bind to CRF receptors and to stimulate adrenocorticotropin (ACTH) secretion from the rat anterior pituitary in vivo and in vitro. In this study, we examined the effect of intravenous administration of urocortin-antiserum to investigate the role of endogenous urocortin on ACTH secretion from rat anterior pituitary after adrenalectomy. Male Sprague-Dawley rats, which were maintained in a conscious and undisturbed condition, were administered non-immunized rabbit serum (NRS), CRF-antiserum or urocortin-antiserum at a volume of 1 ml/kg b.w. 15 min before the injection of secretagogues. Synthetic rat urocortin (2 microg/kg B.W.) increased plasma ACTH concentrations by about sixfold the basal concentration. The pretreatment with urocortin-antiserum but not CRF-antiserum abolished the urocortin-induced increase in plasma ACTH concentrations. In adrenalectomized rats, plasma ACTH concentrations were markedly increased at basal conditions, and rapidly reduced after the administration of CRF-antiserum. By contrast, administration of urocortin-antiserum did not alter ACTH secretion induced by adrenalectomy. Our results suggest that endogenous urocortin is unlikely to be involved in ACTH release in adrenalectomized rats.

Local expression of IFN-gamma mRNA in skin lesions of patients with dermatophytosis.

Delayed type hypersensitivity (DTH) response to dermatophyte antigens is one of the host defense mechanisms in dermatophytosis. Skin lesions of dermatophytosis were examined for the cytokine mRNAs expression using the reverse transcriptase/polymerase chain reaction (RT-PCR) method. Interferon-gamma (IFN-gamma) mRNA was expressed in all of the dermatophytosis lesions. IFN-gamma plays an important role in the effector phase of the DTH reaction. Therefore, these findings indicate that DTH response is elicited in the skin lesions with dermatophytosis.

Treatment of murine angiosarcoma with etoposide, TNP-470 and prednisolone.

To develop effective therapies for angiosarcoma, we investigated the anti-tumor effects of etoposide (ETO), TNP-470 and prednisolone (PSL) using an established murine angiosarcoma cell line (ISOS-1). We examined the direct anti-tumor and anti-angiogenic effects of these drugs on ISOS-1 cells and normal murine microvascular endothelial cells (mECs) in vitro. Cell growth of ISOS-1 was inhibited significantly by ETO, moderately by TNP-470, and not at all by PSL (IC(50): 0.25 microg/ml, 10 microg/ml, >8000 microg/ml, respectively). One the other hand, cell growth of mECs was inhibited significantly by TNP-470, slightly by PSL, and negligibly by ETO (IC(50): 0.85 ng/ml, 0.7 microg/ml, 10 microg/ml, respectively). In an in vivo assay, tumor growth of ISOS-1 was significantly inhibited by more than 2.5 mg/kg of ETO dose-dependently, and by more than 30 mg/kg of TNP-470, and 100 mg/kg of PSL individually. Combination treatments of ETO+TNP-470 and TNP-470+PSL showed synergistic enhancement of inhibition (% control inhibition: ETO vs. TNP-470 vs. ETO+TNP-470: 55 versus 55 vs. 16%) (% control inhibition: TNP-470 vs. PSL vs. TNP-470+PSL: 41 vs. 86 vs. 21%). ETO+PSL combination treatment, however, failed to show significant enhancement of anti-tumor effects. In conclusion, our results indicated that TNP-470 may be a very effective drug for angiosarcoma treatment, especially in combination with ETO or PSL. We eagerly anticipate the use of TNP-470 in clinical treatment of angiosarcoma.

Establishment of a new murine-phenotypic angiosarcoma cell line (ISOS-1).

A cell line, designated ISOS-1, was established from a tumor formed by transplantation of a human angiosarcoma into mice with severe combined immunodeficiency (SCID). The cells showed endothelial properties, based on the uptake of Dil-Ac-LDL and binding of UEA-I/GSA-I lectins, but were negative for CD11b and Pan Cytokeratin. However, the cells lost differentiated characteristics such as expression of von Willebrand factor, contact inhibition growth and tube formation activity. These findings indicate that ISOS-1 is a poorly-differentiated endothelial cell line. At the 81st passage, all of the cells were positive for H-2Dd in various intensity, but not HLA-ABC. The metaphase chromosomes consistently showed a characteristic mouse, but not human, telocentric form. Furthermore, this cell line produced fatal tumor growth in SCID mice and also in BALB/c mice. These results suggest that ISOS-1 is a murine-phenotypic angiosarcoma cell line.

Evaluation of recombinant interleukin-2 immunotherapy for human hemangiosarcoma in a SCID mice model (WB-SCID).

We treated the patients with cutaneous hemangiosarcoma with recombinant interleukin-2 (rIL-2) immunotherapy that showed clear therapeutic effects. This immunotherapy is popular for the treatment of hemangiosarcoma in Japan. The purpose of this study is to clarify the clinical effects in an animal experiment. After establishing a SCID mouse model of human hemangiosarcoma WB-SCID, we used this model to investigate anti-tumor effects of rIL-2 and LAK cells. We demonstrated that hemangiosarcoma cells are LAK-sensitive, and LAK cells induced by rIL-2 suppress the growth of hemangiosarcoma. Our results may assure the clinical effects of rIL-2 immunotherapy on hemangiosarcoma.

Effect of urocortin and its interaction with adrenocorticotropin (ACTH) secretagogues on ACTH release.

We examined the effect of urocortin (Ucn) on the adrenocorticotropin (ACTH) release from cultured rat anterior pituitary cells and AtT 20 cells. Synthetic rat (r)Ucn was not soluble in 0.1 N HCl but soluble in alkaline solvents with diminished corticotropin-releasing activity. rUcn dissolved in 0.1 M sodium phosphate buffer as a stock solution maintained its bioactivity and had the equal corticotropin-releasing activity with rat/human corticotropin-releasing factor (r/hCRF). rUcn stimulated the adrenocorticotropin release via CRF-receptors accompanied by the additive effect with r/hCRF, the synergistic effect with arginine vasopressin and the dose-dependent inhibition of a potent CRF-receptor antagonist.

Urocortin in human placenta and maternal plasma.

Plasma immunoreactive (IR-) urocortin (Ucn) and corticotropin-releasing factor (CRF) levels in pregnant women were measured by their specific radioimmunoassays after extraction. Although plasma IR-CRF levels were increased in pregnant women as compared to men and non-pregnant women, there was no difference of plasma IR-Ucn levels among groups. Ucn mRNA was detected in cytotrophoblasts and syncytiotrophoblasts by in situ hybridization. A reverse-phase high-performance liquid chromatography (HPLC) showed the major peak of IR-Ucn in placenta and plasma that had similar chromatographic mobility to synthetic Ucn1-40. These data suggest that Ucn is produced and processed into the same form of synthetic Ucn in placenta, but not secreted into maternal blood.

Analysis of prognostic factors in patients with hepatocellular carcinoma treated by transcatheter arterial embolization.

We studied 240 cases of unresected hepatocellular carcinoma (HCC) using Cox's proportional hazard model to elucidate which factors would be closely related with the survival period after treatment by transcatheter arterial embolization (TAE) in the presence or absence of iodized oil. The results were as follows. The cumulative survival values obtained after TAE were 67.5% for 1 year, 32.0% for 2 years, and 20.5% for 3 years. The most significant prognostic factor was the degree of extension of tumor embolus in the portal vein or its branch. The tumor extension and the tumor type were also important factors. Age, sex, and AFP, HBsAg, and HCV Ab values were not useful as prognostic factors. This study provides a rational background for the selection of treatment for HCC. Furthermore, knowledge of the prognostic factors is useful for the management of patients, particularly in maintaining their good quality of life.

Effects of transcatheter arterial chemoembolization with oral chemotherapy on hepatic neoplasms.

An investigation was carried out into the effects of lipiodol-transcatheter arterial chemoembolization (L-TACE) therapy on hepatocellular carcinoma (HCC) and metastatic liver cancer, as well as the effects of oral 5-fluorouracil administration after L-TACE. For L-TACE, lipiodol mixed with adriamycin (doxorubicin) was injected through a catheter inserted into the tumor feeding artery and this was followed by embolization with a gelatin sponge. Twenty national hospitals throughout Japan participated in this multicenter co-operative open trial. A total of 102 patients became the subjects of study, including 75 HCC patients, 12 metastatic liver cancer patients treated with L-TACE, and 15 HCC patients who had hepatectomy after L-TACE. In 22% of the HCC patients and in 42% of the metastatic liver cancer patients, the tumor size was reduced by more than 50% after L-TACE. 73% of the 63 HCC patients showed a more than 50% reduction of the levels of serum alpha-fetoprotein. Although the survival rates of the HCC patients who had a hepatic resection were better than those who had not, there was no statistically significant difference between the survival rates of the HCC patients and those of the metastatic liver cancer patients treated with L-TACE. The survival rates of the HCC patients after L-TACE did not change as a result of oral 5-fluorouracil administration. It was therefore concluded that L-TACE is an effective way of treating both HCC patients and metastatic liver cancer patients, and that repeated L-TACE should be considered for some patients whose serum levels of alpha-fetoprotein rose again after L-TACE. Further follow-up studies will be needed to discover the effects of oral chemotherapy after L-TACE.

The effect of ketamine isomers on both mice behavioral responses and c-Fos expression in the posterior cingulate and retrosplenial cortices.

Ketamine, a non-competitive NMDA receptor antagonist, is a racemic mixture. S(+) ketamine is presumed to be more potent as an anesthetic than R(-) ketamine, and causes less postanesthetic stimulation of locomotor activity than R(-) ketamine in animals at equihypnotic doses. In the present study, we investigated the effect of S(+), R(-), and racemic ketamines on mice behavioral responses and c-Fos expression in the posterior cingulate and retrosplenial cortices (PC/RS), which are suggested to be the brain regions responsible for NMDA-receptor-antagonist-induced psychotomimetic activity. Ataxia and head weaving and c-Fos expression in the PC/RS were significantly more induced by both S(+) and racemic ketamines than by R(-) ketamine at the same dose. S(+) ketamine induced significantly more potent ataxia than racemic ketamine at the same dose. Ketamine-induced c-Fos expression in the PC/RS correlated well with the intensity of behavioral responses. These results imply that R(-) ketamine is weaker than both S(+) and racemic ketamines in a psychotomimetic effect. Also, S(+) ketamine is more potent than racemic ketamine in a psychotomimetic effect and possibly in an anesthetic effect. They also indicate that PC/RS is at least one of the specific brain regions responsible for ketamine-induced behavioral responses in animals and a psychotomimetic activity in humans.

Endoscopic follow-up study of development of gastric antral vascular ectasia associated with liver cirrhosis.

Gastric antral vascular ectasia is an important cause of chronic gastrointestinal blood loss. However, its development and progression have not yet been clarified. We investigated its early lesions and progression by reviewing endoscopic films of five patients with gastric antral vascular ectasia followed for liver cirrhosis. In all patients, early findings were prepyloric red spots. In two patients, anemia due to gastrointestinal bleeding was already observed when vascular lesions were confined to the distal antrum. In the other three patients, anemia was observed 1-2 years after they showed a diagnostic pattern of gastric antral vascular ectasia. The vascular lesions gradually thickened and extended throughout the antrum, with the complete picture shown in 1.5-5 years. The pattern of distribution was classified into three types: diffuse spotty, diffuse confluent, and striped. These types could be predicted before the complete formation. Gastric antral vascular ectasia associated with liver cirrhosis started as prepyloric red spots and extended to the proximal antrum in various ways and varying time courses of less than 5 years; this entity may cause hemorrhage even in the early stage.

Thrombotic thrombocytopenic purpura developed suddenly during interferon treatment for chronic hepatitis C.

A 57-year-old man had abnormal hepatic function identified in April 1994. In October 1994, chronic hepatitis C was diagnosed. Based on the findings of a liver biopsy, administration of recombinant interferon (rIFN)-alpha2b was begun. In the 16th week of treatment, the patient experienced headache and fever and developed a markedly decreased, platelet count and hemolytic anemia. He was admitted on May 19, 1995 and thrombotic thrombocytopenic purpura (TTP) was diagnosed. He died on the 3rd hospital day. The causes of TTP have yet to be elucidated, but in this patient the occurrence of TTP appeared to be related to the IFN treatment for chronic hepatitis C.

Distribution and concentration of urocortin, and effect of adrenalectomy on its content in rat hypothalamus.

We developed a specific and sensitive radioimmunoassay (RIA) for rat urocortin (rUcn) and investigated the tissue distribution and concentration of immunoreactive (IR-)Ucn in rats. Antiserum was obtained by immunizing rabbits with synthetic rUcn21-35 coupled with bovine thyroglobulin. 125I-[Tyr]18-rUcn19-37 was used as the tracer. The RIA detected synthetic rUcn1-40 as low as 0.4 fmol/tube, and did not cross-react with other corticotropin-releasing factor-related peptides. IR-Ucn was widely distributed in central nervous system, endocrine organs, and digestive system. Its concentration was highest in pituitary (11.0 +/- 1.36 pmol/g.w.w., mean +/- SEM, n=4). Reverse-phase HPLC revealed that hypothalamic IR-Ucn had similar chromatographic mobility to synthetic rUcn1-40. However, bilateral adrenalectomy did not influence the hypothalamic IR-Ucn content. Our results suggest that Ucn may play important roles in various tissues in normal rats, but not behave as a hypothalamic hypophysiotropic factor in mediating adrenocorticotropin secretion in adrenalectomized rats.