Effect of Blonanserin on the Proliferation and Migration of Glioblastoma Cells.

Glioblastoma is a highly malignant and invasive brain tumor, and there is an urgent need to establish a treatment option that prevents its growth and metastasis. Blonanserin is an antipsychotic drug widely used in the treatment of schizophrenia. It has recently been reported to inhibit the growth of breast cancer cells. In this study, we investigated the effect of blonanserin on the proliferation and migration of glioblastoma cells. The anti-proliferative activity of blonanserin was evaluated in terms of cell viability, competition, and cell death pathways in glioblastoma. Cell viability studies showed that blonanserin had growth inhibitory ability regardless of the malignancy of glioblastoma cells, but at concentrations close to its IC50, it only had a slight cell death-inducing effect. Blonanserin showed growth inhibitory activity without D2 antagonism following an independent competition analysis using blonanserin and D2 antagonists. When the anti-migration activity of U251 cells was measured, blonanserin was found to attenuate cell migration. Furthermore, treatment with blonanserin at concentrations close to its IC50 value inhibited extensive filament actin formation. In conclusion, blonanserin inhibited the proliferation and migration of glioblastoma cells independent of D2 antagonism. The present study shows that blonanserin may serve as a seed compound for the discovery of new glioblastoma therapeutics to prevent the growth and metastasis of glioblastoma.

CYP4F2 gene polymorphism as a contributor to warfarin maintenance dose in Japanese subjects.

WHAT IS KNOWN AND OBJECTIVE: Polymorphisms in the gene encoding CYP4F2 may partly explain the variability in warfarin maintenance dose by altering the metabolism of vitamin K. To determine the genetic factors that cause large inter-patient variability in warfarin efficacy, we investigated the relationship between serum warfarin concentration and CYP4F2 V433M (1347C>T, rs2108622) polymorphism in Japanese subjects. METHODS: Gene variations in VKORC1, CYP2C9 and CYP4F2 were analysed in 126 Japanese patients treated with warfarin. The daily dosage of warfarin, concentration of S- and R-warfarin in plasma, and prothrombin time international normalized ratio (PT-INR) was used as the pharmacokinetic and pharmacodynamic indices. RESULTS AND DISCUSSION: The maintenance dose of warfarin was larger in the CYP4F2 1347 CT genotype group (3·59±1·80 mg/day, P=0·027) than in the CYP4F2 CC genotype group (2·88±1·00 mg/day). CYP4F2 1347C>T polymorphism significantly affected serum R-warfarin concentration when the VKORC1-1639 genotypes are AG and GG. WHAT IS NEW AND CONCLUSION: Although a significant inter-patient difference in warfarin maintenance dose was observed between the CYP4F2 CC and CT genotypes, serum S-warfarin concentration was not significantly different between them. An effect of CYP4F2 V433M polymorphism on warfarin maintenance dose was observed but was relatively small when compared to the effects of CYP2C9 and VKOR polymorphism.

Subtype-specific translocation of the delta subtype of protein kinase C and its activation by tyrosine phosphorylation induced by ceramide in HeLa cells.

We investigated the functional roles of ceramide, an intracellular lipid mediator, in cell signaling pathways by monitoring the intracellular movement of protein kinase C (PKC) subtypes fused to green fluorescent protein (GFP) in HeLa living cells. C(2)-ceramide but not C(2)-dihydroceramide induced translocation of delta PKC-GFP to the Golgi complex, while alpha PKC- and zeta PKC-GFP did not respond to ceramide. The Golgi-associated delta PKC-GFP induced by ceramide was further translocated to the plasma membrane by phorbol ester treatment. Ceramide itself accumulated to the Golgi complex where delta PKC was translocated by ceramide. Gamma interferon also induced the delta PKC-specific translocation from the cytoplasm to the Golgi complex via the activation of Janus kinase and Mg(2+)-dependent neutral sphingomyelinase. Photobleaching studies showed that ceramide does not evoke tight binding of delta PKC-GFP to the Golgi complex but induces the continuous association and dissociation of delta PKC with the Golgi complex. Ceramide inhibited the kinase activity of delta PKC-GFP in the presence of phosphatidylserine and diolein in vitro, while the kinase activity of delta PKC-GFP immunoprecipitated from ceramide-treated cells was increased. The immunoprecipitated delta PKC-GFP was tyrosine phosphorylated after ceramide treatment. Tyrosine kinase inhibitor abolished the ceramide-induced activation and tyrosine phosphorylation of delta PKC-GFP. These results suggested that gamma interferon stimulation followed by ceramide generation through Mg(2+)-dependent sphingomyelinase induced delta PKC-specific translocation to the Golgi complex and that translocation results in delta PKC activation through tyrosine phosphorylation of the enzyme.

Three distinct mechanisms for translocation and activation of the delta subspecies of protein kinase C.

We expressed delta subspecies of protein kinase C (delta-PKC) fused with green fluorescent protein (GFP) in CHO-K1 cells and observed the movement of this fusion protein in living cells after three different stimulations. The delta-PKC-GFP fusion protein had enzymological characteristics very similar to those of the native delta-PKC and was present throughout the cytoplasm in CHO-K1 cells. ATP at 1 mM caused a transient translocation of delta-PKC-GFP to the plasma membrane approximately 30 s after the stimulation and a sequent retranslocation to the cytoplasm within 3 min. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 microM), induced a slower translocation of delta-PKC-GFP, and the translocation was unidirectional. Concomitantly, the kinase activity of delta-PKC-GFP was increased by these two stimulations, when the kinase activity of the immunoprecipitated delta-PKC-GFP was measured in vitro in the absence of PKC activators such as phosphatidylserine and diacylglycerol. Hydrogen peroxide (H2O2; 5 mM) failed to translocate delta-PKC-GFP but increased its kinase activity more than threefold. delta-PKC-GFP was strongly tyrosine phosphorylated when treated with H2O2 but was tyrosine phosphorylated not at all by ATP stimulation and only slightly by TPA treatment. Both TPA and ATP induced the translocation of delta-PKC-GFP even after treatment with H2O2. Simultaneous treatment with TPA and H2O2 further activated delta-PKC-GFP up to more than fivefold. TPA treatment of cells overexpressing delta-PKC-GFP led to an increase in the number of cells in G2/M phase and of dikaryons, while stimulation with H2O2 increased the number of cells in S phase and induced no significant change in cell morphology. These results indicate that at least three different mechanisms are involved in the translocation and activation of delta-PKC.

1-BP inhibits NF-kappaB activity and Bcl-xL expression in astrocytes in vitro and reduces Bcl-xL expression in the brains of rats in vivo.

1-Bromopropane (1-BP) has been widely used as a substitute for chlorofluorocarbon that destroys the ozone layer. Although the central neurotoxicity of 1-BP has been recently reported, a molecular mechanism is not clear. In particular, the effects on cells in brain have not been fully analyzed. Here, we studied the effects of 1-BP on the activation of transcription factors involved in anti-apoptotic function or cell survival in astrocytes. Astrocytoma cell lines, U251, U373 and VM, or murine primary astrocytes were used for in vitro assay. DNA binding activities of NF-kappaB in these cells induced by interleukin (IL)-1 or LPS were inhibited by 1-BP. Consequently, the treatment of U251 cells with 1-BP resulted in suppression of NF-kappaB reporter activity. Furthermore, 1-BP blocked IkappaBalpha degradation, which is important for NF-kappaB activation. In addition, the level of Bcl-xL mRNA, which is known as an anti-apoptotic gene, were reduced in U251 treated with 1-BP or in the brain from rat exposed to 1-BP (400 ppm, 12 weeks). These results suggest that subchronic inhalation exposure to 1-BP vapor may affect the Bcl-xL expression in astrocytes.

Case study of volatile organic compounds in indoor air of a house before and after repair where sick building syndrome occurred.

A housewife in her late thirties, mother of two children, had an indefinite complaint about the indoor air quality of her house. Inspectors from a public health center treating the housewife's complaint quantified formaldehyde (FA) in high concentration exceeding Japanese national guideline of FA in some rooms of the house. We also determined FA and total volatile organic compounds (TVOCs) in higher concentrations more than the national guidelines. Remodeling of the house was performed to improve the air quality as follows. Vinyl wallpaper was exchanged to plant made paper, plywood made doors were exchanged to pure wood made doors, plywood stairs were covered with plant cork and so on. After remodeling the house, we measured the concentrations of FA and TVOCs again. The concentrations of the chemicals in the indoor air decreased which approve effectiveness of the remodeling. Moreover complaints of the housewife lessened. This also proved the effectiveness of the remodeling. Four years after the inspection, we visited the house again and found that the concentration of FA in the house was still lower than that of national guideline. The housewife was evaluated in a good healthy condition by her answers to our questions related to indoor air quality, daily life, physical condition, and so on.

Subjective symptoms of medical students exposed to formaldehyde during a gross anatomy dissection course.

The purpose of this study is to investigate the safety of and to try to find the best plan to cope with exposure to FA for students during a gross anatomy dissection course. The FA exposure level and subjective symptoms was estimated. The relationship between exposure to FA and subjective symptoms of irritation were discussed for times; before, in the beginning period, in the middle period, and upon completion of the Anatomy Dissection Course. The geometric means of FA concentration were 32.7 micrograms/m3 (before), 891.3 micrograms/m3 (beginning), 763.3 micrograms/m3 (middle), and 238.9 micrograms/m3 (completion), respectively. Among them, FA-related symptoms were observed in 61.1 percent; 28.0 percent fell strong stress during the course, and 27.4 percent complained that their normal life situation was affected. Our results indicate that such subjective symptoms during the anatomy dissection course were related to the period spent in the anatomy dissection room. Our study suggests that shortening the time of each anatomy dissection practical class and reduction of the number of cadaver tables could help to reduce symptoms.

Immunochemical characterization and toxicological significance of P-450HFLb purified from human fetal livers.

Immunochemical properties of P-450HFLb purified from human fetal livers were investigated. P-450HFLb cross-reacted with antibodies to rat P-4501A1 but not with antibodies to CYP2A6, CYP2C9, CYP3A7 (P-450HFLa) and rat CYP2B1. In addition, P-450HFLb also cross-reacted with both monospecific antibodies to rat CYP1A1 and CYP1A2. However, P-450HFLb was shown to be an immunochemically distinct form of cytochrome P-450 from P-450PA (human CYP1A2). Immunoblot analysis of human fetal livers with the antibodies to P-450HFLb showed that P-450HFLb was expressed in all fetal livers studied although there appeared to be individual differences in the amounts of P-450HFLb expressed in fetal livers. The formation of mutagens from IQ (but not from AFB1) in fetal liver homogenates was inhibited by the antibodies to P-450HFLb in a dose dependent manner. These results suggest that P-450HFLb may be a form of human cytochrome P-450 classified into CYP1 gene family, and that the cytochrome P-450 is, in part, responsible for the mutagenic activation of IQ in human fetal livers as well as CYP3A7 (P-450HFLa).

Differential catalytic properties in metabolism of endogenous and exogenous substrates among CYP3A enzymes expressed in COS-7 cells.

The catalytic properties of CYP3A7 in the metabolism of endogenous and exogenous substrates were compared with those of CYP3A4 and CYP3A5 using COS-7 expressing enzymes. The highest activities of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone 3-sulfate (DHEA-S) 16alpha-hydroxylase were observed in COS-7 cells expressing CYP3A7. In contrast, the activity of testosterone 6beta-hydroxylase of CYP3A7 expressed in COS-7 cells was much less than that of CYP3A4 expressed in COS-7 cells. The rate of carbamazepine 10, 11-epoxidation was the greatest in COS-7 cells expressing CYP3A4, followed by CYP3A5 and CYP3A7. On the other hand, the formation of reductive metabolite of zonisamide was the highest in COS-7 cells expressing CYP3A4, followed by CYP3A7 and CYP3A5. Furthermore, the addition of triazolam resulted in a decrease in 6beta-hydroxylation catalyzed by CYP3A7, but not by CYP3A4, whereas the pretreatment of microsomes with triacetyloleandomycin (TAO) resulted in a decrease in the reaction catalyzed by CYP3A4, but not by CYP3A7. Together with these results, it was suggested that CYP3A7 exerts differential catalytic properties not only in metabolism of endogenous substrates but also in drug metabolism compared to CYP3A4 and CYP3A5.

Saccharomyces cerevisiae cultured under aerobic and anaerobic conditions: air-level oxygen stress and protection against stress.

Cells of Saccharomyces cerevisiae were grown aerobically and anaerobically, and levels of the protective compounds, cysteine and glutathione, and activities of defensive enzymes, catalase and superoxide dismutase, against an oxygen stress were determined and compared in both cells. Aerobiosis increased both the compounds and enzyme activities. The elevated synthesis of glutathione could be associated with the increased levels of cysteine which in its turn was found to be controlled by the oxygen-dependent activation of cystathionine beta-synthase.

Diabetes and cardiovascular disease in a prospective population survey in Japan: The Hisayama Study.

To elucidate the effect of glucose intolerance on cardiovascular disease in the current Japanese population, we performed a 75-g oral glucose tolerance test in 2,427 Hisayama residents aged 40-79 years in 1988, who were free from a previous history of stroke or myocardial infarction, and followed them prospectively for 5 years. The prevalence of diabetes (NIDDM) among men was 13% and that of impaired glucose tolerance (IGT) was 20%; the corresponding values for women were 9 and 19%, respectively. The age- and sex-adjusted incidence of cerebral infarction (6.5 per 1,000 person-years, P < 0.01) and coronary heart disease (5.0 per 1,000 person-years, P < 0.05) was significantly higher in subjects with NIDDM than in those with normal glucose tolerance (1.9 and 1.6 per 1,000 person-years, respectively). In addition, subjects with IGT and NIDDM had a higher risk of cardiovascular disease including stroke and coronary heart disease than did those with normal glucose tolerance after adjustment for age and sex, namely the relative risk for IGT was 1.9 (95% CI 1.2-3.2), and the relative risk for NIDDM was 3.0 (95% CI 1.8-5.2). These associations remained significant even after controlling for six other risk factors including hypertension in multivariate analysis. Our data suggest that NIDDM is a significant risk factor for both cerebral infarction and coronary heart disease and also that IGT itself is a risk factor for cardiovascular disease in the general Japanese population today.

Glucocorticoids potentiate the action of atrial natriuretic polypeptide in adrenalectomized rats.

Adrenal insufficiency in human and rat is associated with an impairment of the diuretic response to water load, and only glucorticoids (GCs) restore this deficit. Our observation that GCs potentiate atrial natriuretic polypeptide (ANP)-stimulated cGMP production in cultured renal cells prompted us to examine the possibility that GCs may restore the diuretic response through the potentiation of ANP action. Initially, changes in urine volume and ANP levels were studied in adrenalectomized (Adx) and sham-operated intact rats after an oral water load of 5 ml/100 g BW. Urine volume after water load was 4.5 +/- 0.5 ml/30 min in the intact rats, whereas it was 0.8 +/- 0.2 ml/30 min in the Adx rats. In the intact rats, a significant increase in plasma ANP level was observed 30 min after the water load, whereas no increase was observed in Adx rats. This defective ANP response may be involved in the impairment of the diuretic response in Adx rats. Indeed, pretreatment of Adx rats with dexamethasone (Dex, 20 micrograms/100 g BW) increased plasma ANP levels even before water load and improved diuretic response. Subsequently, effect of iv administration of human or rat ANP at a pharmacological dose (2.5 micrograms/100 g BW) on urine volume, osmolarity, and urinary excretion of cGMP, and sodium was studied in Adx rats that received an oral water load 30 min before ANP. Dex treatment was achieved by per os administration 3 h before the ANP injection. In Adx rats, the urine volume after ANP administration was 1.2 +/- 0.1 ml/30 min, and pretreatment with Dex markedly increased the urine volume to 6.3 +/- 0.4 ml/30 min. Dex also increased ANP-induced osmolar and sodium excretion by 2.6- and 2.9-fold, respectively. Although urinary excretion of cGMP was increased in Adx rats by ANP administration, a further significant increase was observed by the pretreatment with Dex. Injection of (Bu)2cGMP to Adx rats pretreated with Dex resulted in a significant increase in urine volume and osmolar and sodium excretion. However, no significant increase in urine volume was observed in Adx rats not pretreated with Dex. The present study suggests that GCs restore the diuretic response to acute water load not only by increasing the secretion of ANP but also by potentiating ANP-stimulated cGMP production. Furthermore, GCs may augment ANP action at one or more steps other than cGMP formation because administration of (Bu)2cGMP to Adx rats did not correct the diuretic response to water load.

Inhibition of growth hormone (GH) secretion by a mutant GH-I gene product in neuroendocrine cells containing secretory granules: an implication for isolated GH deficiency inherited in an autosomal dominant manner.

Isolated GH deficiency (IGHD) type II is a disease inherited in an autosomal dominant manner. Although point mutations at the donor splice site of intron 3 of the GH-I gene have been identified in patients, the mechanism of how such mutations result in severe GH deficiency is unclear. Recently, we identified two mutations in Japanese patients with IGHD type II, G to A substitutions at the first (mutA) and fifth (mutE) nucleotides of intron 3. Messenger ribonucleic acids skipping exon 3 were transcribed from both mutant GH-I genes. We studied in this report the synthesis and secretion of GH encoded by the mutant GH-I genes and tested whether inhibition of wild-type GH secretion by mutant products could be demonstrated in cultured cell lines. A metabolic labeling study in COS-1 cells revealed that a mutant GH with a reduced molecular mass was synthesized from the mutant messenger ribonucleic acid and retained in the cells for at least 6 h. On the other hand, the wild-type GH was rapidly secreted into the medium. Coexpression of mutant and wild-type GH did not result in any inhibition of wild-type GH secretion in COS-1 or HepG2 cells. However, coexpression of mutant GH resulted in significant inhibition of wild-type GH secretion in somatotroph-derived MtT/S cells as well as in adrenocorticotroph-derived AtT-20 cells, without affecting cell viability. We conclude that the dominant negative effect of mutant GH on the secretion of wild-type GH is at least in part responsible for the pathogenesis of IGHD type II. Our results also suggest that neuroendocrine cell type-specific mechanisms, including intracellular storage of the secretory proteins, are involved in the inhibition.

Hyperinsulinemia and left ventricular geometry in a work-site population in Japan.

The present study was designed to test whether hyperglycemia or hyperinsulinemia influences left ventricular mass and geometry. An echocardiogram and 75-g oral glucose tolerance test were performed in 210 normotensive and 180 mildly to moderately hypertensive male workers in a bus company who were free from cardiac diseases and were not taking medication for hypertension and diabetes mellitus. When we divided subjects into four groups according to the left ventricular geometric pattern using left ventricular mass index of 110 g/m2 and relative wall thickness (ratio of 2 x posterior wall thickness to end-diastolic left ventricular diameter) of 0.44, body mass index and systolic blood pressure were higher in those with concentric hypertrophy and eccentric hypertrophy. In addition, hemoglobin A(Ic) level and the sum of fasting and 2-hour postload serum glucose levels were higher in subjects with concentric hypertrophy. In subjects without diabetes mellitus (n=336), 2-hour postload serum insulin level and the sum of fasting and 2-hour postload serum insulin levels tended to be higher in those with concentric hypertrophy and concentric remodeling. In multiple regression analysis, the sum of glucose levels (or hemoglobin A(Ic) level) in all subjects and the sum of insulin (or 2-hour postload insulin) levels in subjects without diabetes mellitus significantly correlated with relative wall thickness, independent of age, systolic blood pressure, and body mass index. Neither glucose nor insulin levels correlated with left ventricular mass index. Our results suggest that hyperglycemia and hyperinsulinemia may promote concentric changes in the left ventricle in normotensive and mildly to moderately hypertensive men.

Overexpression of glutathione-S-transferase A1 in benign adrenocortical adenomas from patients with Cushing's syndrome.

Benign adrenocortical adenoma is a major primary cause of Cushing's syndrome. Although numerous studies have been performed, the molecular mechanism of adrenocortical adenoma is yet to be elucidated. In this study we endeavored to identify genes differentially regulated in adrenocortical adenoma by suppression PCR-based complementary DNA (cDNA) subtractive hybridization. The cDNA population in atrophied nontumorous adrenal gland adjacent to the adenoma was subtracted from that in the adenoma. Then adenoma-specific cDNAs were amplified by PCR. We cloned several cDNAs that are selectively up-regulated in the adenoma, one of which was identified to encode glutathione-S-transferase A1 (GSTA1). Northern blot analysis revealed that GSTA1 messenger ribonucleic acid was abundantly expressed in the adenoma compared with that in the adjacent atrophied nontumorous gland. Western blot analysis and immunohistochemistry showed high expression of GSTA1 also at the protein level. In concordance with this finding, GST activity was significantly higher in the adenoma than in the adjacent atrophied nontumorous gland. To clarify the role of GSTA1 in adrenocortical cells, GST activity in the H295R human adrenocortical cell line was inhibited by ethacrynic acid. Inhibition of GSTs interfered with proliferation of the cells. We, therefore, hypothesize that overexpression of GSTA1 in adrenocortical adenomas might be involved in the growth of tumor cells. We also speculate that this overexpression might be an adaptive response to excess cortisol production.

The human homolog of Diminuto/Dwarf1 gene (hDiminuto): a novel ACTH-responsive gene overexpressed in benign cortisol-producing adrenocortical adenomas.

To elucidate the molecular mechanism of the pathogenesis of benign functioning adrenocortical adenomas causing Cushing's syndrome, we employed suppression PCR-based cDNA subtractive hybridization to identify novel genes that are differentially expressed in the adenoma. In this report we describe the adenoma-specific overexpression of the human homolog of the Diminuto/Dwarf1 (hDiminuto) gene. Northern blot analysis revealed that hDiminuto mRNA was overexpressed in the adenoma tissue of 14 patients with Cushing's syndrome in comparison to the adjacent nontumorous adrenal gland. In situ hybridization using hDiminuto cRNA probe showed its abundant expression in the tumor cells, whereas the nontumorous cells showed a low level of expression. As the atrophic adjacent gland may not represent the normal architecture, we examined the expression pattern of hDiminuto mRNA in normal human adrenal cortex. In situ hybridization revealed that it was expressed in all layers of the normal adrenal cortex. In situ apoptosis detection by the TUNEL method revealed that a low level of hDiminuto expression in the atrophic, adjacent gland was associated with numerous TUNEL-positive cells in all layers of cortex. In contrast almost no apoptotic cell was detected in the tumor or in the normal adrenal cortex where hDiminuto expression was abundant. These results are compatible with a recent report that hDiminuto acts as an antiapoptotic factor in neurons. The expression of hDiminuto in the normal adrenal cortex was most abundant in the zona fasciculata, suggesting its possible regulation by ACTH/cAMP. Indeed, forskolin treatment of H295R human adrenocortical cells resulted in a significant induction of the mRNA in a time- and dose-dependent manner. To further demonstrate the physiological regulation, an in vivo experiment was carried out in dexamethasone-treated rats. ACTH administration to these rats increased the mRNA expression. These results led us to speculate that the overexpression of hDiminuto in the adenoma could be due to the abundant expression of ACTH receptor, as we previously described. Diminuto is involved in steroid synthesis and cell elongation in plants. We, therefore, hypothesize that hDiminuto might be involved in the molecular events of adrenocortical tumorigenesis by facilitating steroid synthesis and cell growth.