A phase I/II trial of AT9283, a selective inhibitor of aurora kinase in children with relapsed or refractory acute leukemia: challenges to run early phase clinical trials for children with leukemia.

Aurora kinases regulate mitosis and are commonly overexpressed in leukemia. This phase I/IIa study of AT9283, a multikinase inhibitor, was designed to identify maximal tolerated doses, safety, pharmacokinetics, and pharmacodynamic activity in children with relapsed/refractory acute leukemia. The trial suffered from poor recruitment and terminated early, therefore failing to identify its primary endpoints. AT9283 caused tolerable toxicity, but failed to show clinical responses. Future trials should be based on robust preclinical data that provide an indication of which patients may benefit from the experimental agent, and recruitment should be improved through international collaborations and early combination with established treatment strategies.

Novel reuterin-related compounds suppress odour by periodontopathic bacteria.

OBJECTIVE: Halitosis is caused by volatile sulphur compounds including methyl mercaptan (CH3 SH) in the oral cavity and is a serious problem that limits interpersonal social communication. The aim of study was to evaluate the effects of reuterin-related compounds (RRCs) on halitosis-related periodontopathic bacteria in vitro. MATERIALS AND METHODS: RRC-01, RRC-02 and RRC-03 (32 and 64 µg ml-1 ) in culture media containing Fusobacterium nucleatum JCM8523 and Porphyromonas gingivalis ATCC33277 were used. The effects of RRCs on CH3 SH production and detectable odour by F. nucleatum and P. gingivalis were examined by CH3 SH production assay and organoleptic test, respectively. The number of bacterial cells was also measured using an ATP assay. In P. gingivalis treated with RRCs, the expression of mgl gene, which is responsible for CH3 SH production, was examined by qRT-PCR. RESULTS: CH3 SH production and the score of detectable odour from F. nucleatum and P. gingivalis culture media containing RRCs were significantly lower than that without RRCs (P < 0.05). The expression of mgl gene in P. gingivalis was significantly downregulated by RRC-01 (P < 0.01), but not by RRC-02 or RRC-03. CONCLUSIONS: RRCs are potent oral care products for preventing halitosis via reducing CH3 SH production.

Induction of non-apoptotic cell death by sodium 5,6-benzylidene-L-ascorbate in a human salivary gland tumor cell line.

We investigated whether sodium 5,6-benzylidene-L-ascorbate (SBA) induces apoptotic cell death in a human salivary gland tumor cell line HSG, using two different cytochemical methods. Millimolar concentrations of SBA dose-dependently reduced the viable cell number of HSG cells, accompanied by the detachment of dying cells from the culture plates. The nuclei of the dying cells were not stained with TUNEL reagent, indicating the lack of DNA nicks or fragments. On the other hand, the nuclei of epigallocatechin gallate (EGCG)-treated cells (positive control) were TUNEL-positive, demonstrating the production of DNA nicks or fragments. Furthermore, the cytoplasms of SBA-treated cells were not stained with M30 monoclonal antibody, which reacts with the degradation products of cytokeratin 18 by the activated caspases, in contrast to those of EGCG-treated cells. These results suggest that SBA induces non-apoptotic cell death, possibly necrosis, in HSG cells.

Cytotoxic activity of polyprenylalcohols and vitamin K2 derivatives.

Cytotoxic activity of 9 polyprenylalcohols and 6 vitamin K2 derivatives (MK-1 to MK-6) with various lengths of prenyl units was investigated. Among these compounds, geranylgeraniol with 4 prenyl units, and MK-2 with 2 prenyl units, showed the highest cytotoxic activity against human oral tumor cell lines (HSC-2, HSG), without induction of internucleosomal DNA fragmentation. Higher molecular weight compounds showed selective cytotoxicity against tumor cell lines than normal human gingival fibroblasts HGF. ESR spectroscopy showed that all polyprenylalcohols did not produce radical, nor scavenged O2- generated by hypoxanthine and xanthine oxidase reaction, and only slightly enhanced the radical intensity of sodium ascorbate. Vitamin K2 derivatives scavenged O2- more efficiently, but did not produce radical (except MK-3) and only slightly modified the ascorbate radical intensity. Cytotoxic activity of these compounds might be affected by the molecular weight, hydrophobicity, van der Waals area and stabilization of hydration of the molecule.

Use of the monoclonal antibody M30 for detecting HSG cell apoptosis.

An immunocytochemical method using a monoclonal antibody (MoAb), M30, which reacts with the product resulting from the cleavage of cytokeratin 18 by activated caspase, was applied to detect the apoptosis of human salivary gland tumor (HSG) cells induced by epigallocatechin gallate (EGCG), gallic acid (GA) and sodium ascorbate (SA). EGCG, GA and SA dose-dependently induced HSG cell death. Immunoreactive products were significantly observed in the cytoplasm of HSG cells after treatment with all these compounds. The reactions occurred with lower concentrations of these agents and after shorter treatment times, in comparison with DNA fragmentation detected by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. These results suggest that immunocytochemical staining with the MoAb M30 may be useful for detecting the apoptosis-inducing activities of various chemical compounds.

Cytotoxic activity of low molecular weight polyphenols against human oral tumor cell lines.

A total of 150 chemically-defined natural and synthetic polyphenols (flavonoids, dibenzoylmethanes, dihydrostilbenes, dihydrophenanthrenes and 3-phenylchromen-4-ones), with molecular weights ranging from 224 to 824, were investigated for cytotoxic activity against normal, tumor and human immunodeficiency virus (HIV)-infected cells. They showed higher cytotoxic activity against human oral squamous cell carcinoma HSC-2 and salivary gland tumor HSG cell lines than against normal human gingival fibroblasts HGF. Many of the active compounds had a hydrophilic group (hydroxyl group) in the vicinity of a hydrophobic group (prenyl, phenyl, methylcyclohexene or methylbenzene moiety), similar to isoprenoid-substituted flavones. Substitution of hydrophobic group (prenyl or geranyl group) did not significantly change the cytotoxic activity of flavanones, isoflavans, chalcones or 5-hydroxy-3-phenoxychromen-4-ones. However, the prenylation(s) of an isoflavone and a 2-arylbenzofuran significantly enhanced the cytotoxic activity. Agarose gel electrophoresis showed that active components induced internucleosomal DNA fragmentation in human promyelocytic leukemic HL-60 cells, but not in HSC-2 cells. Most of the polyphenols failed to reduce the cytophathic effect of HIV infection in MT-4 cells.

Induction of apoptosis by flavones, flavonols (3-hydroxyflavones) and isoprenoid-substituted flavonoids in human oral tumor cell lines.

Various flavones, flavonols (3-hydroxyflavones) and isoprenoid-substituted flavones (flavonols) were investigated for their cytotoxic activity. Most of these compounds were more cytotoxic against human oral squamous cell carcinoma and salivary gland tumor cell lines than human gingival fibroblasts. The cytotoxic activity of flavonoids was generally higher than that of tannin-related compounds. Flavonoids induced apoptotic cell death characterized by DNA fragmentation (as identified by TUNEL method) and activation of caspase(s) (as identified by degradation products of cytokeratin 18 with M30 monoclonal antibody). ESR spectroscopy revealed that higher concentrations of flavonoids produced radicals under alkaline conditions. However, not all of them enhanced the radical intensity of sodium ascorbate, suggesting that the redox potential of flavonoids differs considerably from samples to samples. Catalase failed to eliminate the cytotoxic activity of flavonoids, reducing the possibility of the involvement of hydrogen peroxide for the cytotoxicity induction by them.

Induction of apoptosis by cooperative action of vitamins C and E.

Millimolar concentrations of sodium ascorbate (vitamin C) induced apoptotic cell death in human promyelocytic leukemic HL-60 cells. The apoptotic cells displayed a smaller cell volume, disappearance of cell surface microvilli, appearance of cytoplasmic vacuoles, chromatin condensation, nuclear fragmentation and production of apoptotic bodies. The apoptosis-inducing activity of sodium ascorbate was significantly enhanced by noncytotoxic concentrations of CuCl2, but was almost completely eliminated by FeCl3. CuCl2 transiently stimulated the hydrogen peroxide (H2O2) production by sodium ascorbate, whereas FeCl3 slightly reduced the H2O2 production. alpha-Tocopherol (vitamin E) slightly enhanced the radical and H2O2 productions, and apoptosis induction by sodium ascorbate. The effect of alpha-tocopherol seems to be rather specific for ascorbic acid, since alpha-tocopherol did not significantly affect the cytotoxic activity of CuCl2, FeCl3 nor gallic acid. The present study demonstrated the cooperative action of vitamins C and E.

Role of hydrogen peroxide for cell death induction by sodium 5,6-benzylidene-L-ascorbate.

The role of hydrogen peroxide in the induction of cell death in human promyelocytic leukemic HL-60 cells by sodium 5,6-benzylidene-L-ascorbate (SBA) and its degradation product, ascorbic acid, was investigated. Millimolar concentrations of these compounds induced cell death, characterized by cell shrinkage, nuclear and internucleosomal DNA fragmentation, disappearance of microvilli and condensation of chromatin near the nuclear membrane. Catalase significantly reduced the cytotoxic activity of these compounds, whereas superoxide dismutase, nitric oxide (NO) generator, NO scavenger and NO synthase inhibitor were inactive, suggesting the possible role of H2O2. Determination of H2O2 with the peroxyoxalate chemiluminescence demonstrated that sodium ascorbate and SBA produced H2O2 in amounts necessary for cell death induction.

Induction of apoptosis by dopamine in human oral tumor cell lines.

Dopamine dose-dependently reduced the viable cell number of both human salivary gland tumor HSG and oral squamous cell carcinoma HSC-2, HSC-4, and NA cells. CoCl2 significantly reduced both the cytotoxic activity and radical intensity of dopamine (determined by ESR spectroscopy). Dopamine produced DNA fragments (demonstrated by TUNEL method) and induced degradation of cytokeratin by activated caspase in HSG cells (detected by an immunocytochemical method, using a specific M30 monoclonal antibody). FACS analysis demonstrated that dopamine induced DNA fragmentation, a biochemical hallmark of apoptosis, in human promyelocytic leukemia HL-60 cells. The addition of catalase did not prevent the apoptosis-inducing activity of dopamine, reducing the possibility of the involvement of H2O2 for dopamine-induced apoptosis. Dopamine transiently induced p38 mitogen-activated protein kinase (MAP kinase) phosphorylation. However, an inhibitor of p38 MAP kinase phosphorylation, SB203680, failed to inhibit the dopamine-induced apoptosis. These data suggest that p38 phosphorylation at an early stage may not be a causative event for apoptosis.

Radical modulation activity of lignins from a mangrove plant, Ceriops decandra (Griff.) Ding Hou.

The radical modulation activity of hot water and alkaline extracts from leaf of Ceriops decandra, a mangrove plant, was investigated using ESR spectroscopy. IR and NMR analyses demonstrate that the leaf extracts have a lignin-like polyphenolic structure. All these extracts produced radical(s) under alkaline conditions. The radical intensity of sodium ascorbate was slightly reduced at lower concentrations of the extracts, but it was synergistically enhanced at higher concentrations. All the extracts effectively scavenged superoxide anion, produced by hypoxanthine-xanthine oxidase reaction. Pretreatment of mice with the extracts significantly protected them from the lethal infection by E. coli. Similar activity was found in lignins from pine seed shell of Pinus parviflora Sieb. et Zucc. These data further support the medicinal efficacy of plant extracts.

Improvement of bond strength in metal-ceramic systems using a gold intermediate layer.

The mechanism of bonding between metal and ceramic in systems using the functionally graded method with pure gold and gold mixture as a primer was examined. Four types of samples, porcelain, porcelain-gold, porcelain-metal and porcelain-gold-metal were prepared. The gold intermediate layer was fired at 1000 degrees C. For porcelain and metal, low-fusing opaque, body porcelain and palladium alloy were used. The intermediate layer was composed of three layers; pure gold, gold-palladium and gold-porcelain layer. During the bending test of each sample, the porcelain peeled away from the porcelain-metal system, while porcelain with the gold intermediate layer remained on the metal surface even after maximal loading. The bond strength of the porcelain-gold-metal system was much higher than that of the porcelain-metal system, and the toughness of the former was much greater than that of the latter. Laser microscopy and scanning electron microscopy (SEM) showed a smooth interface between the intermediate layer and the metal which suggested proper chemical bonding, and no gap was observed. At the interface between the porcelain and the gold intermediate alloy, a good mechanical anchor lock was observed. Electron probe microanalysis (EPMA) showed a clear distribution of each element (e.g. Si, Au and Pd) in the porcelain, gold intermediate layer and metal frame.

Cross-sectional echocardiographic study on the mitral valve prolapse associated with secundum atrial septal defect: pre- and post-operative comparison.

Cross-sectional echocardiography was performed on 77 patients with secundum atrial septal defect (ASD). Mitral valve prolapse (MVP) was recognized in 41 out of the 77 (53.2%); anterior MVP was found in 36 patients, three cases revealed posterior MVP, and two were diagnosed as having both anterior and posterior MVP. Pre- and post-operative study was carried out on 53 cases; anterior MVP decreased in incidence from 28 patients to 17 following closure of ASD, whereas posterior MVP remained unchanged, as did both anterior and posterior MVP. The left ventricular distortion ratio (LVDR) was measured in the short axis view in 45 cases. After the repair of ASD, the LVDR decreased both in end-systole and end-diastole (p less than 0.01), that is, the left ventricle became more circular in configuration. Pre- and post-operatively, MVP patients revealed a significantly greater LVDR than those without it in end-systole, and post-operatively in end-diastole as well (p less than 0.01). Prior to operation, the short axis area of the left ventricle (LVSAA) was statistically larger in patients with MVP than without it in end-diastole (p less than 0.05). However, there existed no relationship between the presence of MVP and the pulmonic to systemic flow ratio, right ventricular dimension index, right ventricular systolic pressure and size of ASD at the time of operation.

Clinical study on the right-sided Austin Flint murmur using intracardiac phonocardiography.

Right heart catheterization was carried out on 14 patients with pulmonic regurgitation using intracardiac phonocardiography. All the patients showed pulmonic regurgitant murmur in the right ventricular outflow tract. In addition, seven out of the 14 patients showed mid-diastolic and presystolic murmurs maximally in the outflow tract of the right ventricle. Furthermore, inspiration increased the loudness of these diastolic murmurs in four patients. These findings were compatible with those of right-sided Austin Flint murmur due to functional tricuspid stenosis in pulmonic incompetence. Ten out of the 14 patients had pulmonary hypertension and all the subjects with a rightsided Austin Flint murmur showed elevated pulmonary arterial pressure. Thus, pulmonic regurgitation with pulmonary hypertension is thought to be closely related to the right-sided Austin Flint murmur and the turbulence resulting from antegrade flow across a closing tricuspid valve may be responsible for the genesis of the murmur.

Clinical study on the abnormal flow patterns in Ebstein's anomaly using pulsed Doppler echocardiography.

Pulsed Doppler echocardiography (PDE) was performed in 10 patients with Ebstein's anomaly and 10 cases of tricuspid regurgitation secondary to mitral stenosis. Distal atrialized right ventricle (ATRV): In all patients with Ebstein's anomaly, tricuspid regurgitation flow was recognized by PDE. In this lesion with moderate tricuspid regurgitation, a widely dispersed dot pattern was recorded during systole However, in the cases with severe tricuspid regurgitation a relatively smooth dot pattern was recognized. In the case with marked delay in pressure rise in the right ventricle, PDE showed a bimodal regurgitant flow pattern. The interval between the onset of QRS and that of tricuspid regurgitant flow with right ventricular pressure rise was measured. The interval corrected for heart rate ranged from 0.10 to 0.35 with an average of 0.19 +/- 0.08 sec. In the subjects with secondary tricuspid regurgitation, it ranged from 0.07 to 0.11 sec. This interval was significantly prolonged in Ebstein's anomaly as compared to that in secondary tricuspid regurgitation (p less than 0.001). Proximal ATRV: Tricuspid regurgitant flow was detected in 6 to 10 patients with Ebstein's anomaly. The disturbed flow was less apparent in the proximal ATRV than in the distal ATRV.

Clinical investigation of aortic insufficiency by means of pulsed Doppler echocardiography.

Pulsed Doppler echocardiography (PDE) was performed on 41 patients with aortic insufficiency (AI), isolated or associated with other cardiac diseases, using an ATL 500A pulsed Doppler system. The diagnosis was confirmed by angiocardiography in 25 cases. The purpose of the present study was to analyze the disturbed flow due to AI, to investigate the sensitivity of PDE to this lesion, and to compare with the angiographic severity of AI (Sellers). The transducer was placed on the left sternal border and the flow pattern was recorded at the aortic valve orifice and the proximal and distal left ventricular outflow tract (LVOT), using a strip chart recorded at a paper speed of 100 mm/sec. The specific feature of AI was a widely dispersed dot pattern which began at the aortic valve closure and extended to late diastole. The severity of AI was graded by supravalvular aortogram in 25 patients. In grades I and II, the abnormal dot pattern due to AI was mostly detected at the aortic valve orifice and the distal LVOT, but it was rather difficult to detect the disturbed flow at the proximal LVOT. In contrast, in grades III and IV, the disturbed flow was recorded at all the sampling sites; with severe aortic regurgitation, it was detected at a wider range in the left ventricular cavity. In grades I and II, the abnormal dot dispersion at the proximal LVOT was not so large at its onset in diastole but it tended to increase after the mitral valve opening, whereas in the majority of patients of grades III and IV, a large dot dispersion was recognized from its onset to late diastole. The typical flow pattern due to AI was detected at the LVOT in 38 out of 41 patients (92.7%). Moreover, it was detected in all the patients with angiographically proved AI except for 1 case of grade I (96.0%).

Effect of maternal alcohol consumption on the fetal thyroid in the rat.

To investigate the effect of maternal alcohol consumption on the development of the fetal thyroid gland, Sprague-Dawley rats were given 20% ethanol for 4 weeks prior to mating and 30% ethanol throughout gestation. Pair-fed controls received an isocaloric amount of corn starch and chow, with water ad libitum, and ad libitum controls received rat chow and water. On Days 17, 18, 19, and 20 of gestation, the fetuses were weighed and the fetal thyroids were removed for histometric observation. On Days 19 and 20, the fetal thyroids of alcohol-exposed fetuses weighed significantly less than those of the two control groups, but more than the control thyroids 1 day earlier. Maternal alcohol consumption caused a significant decrease in both the follicular cell height and the follicle diameter of the fetal thyroid on all days examined. In the alcohol group on Days 19 and 20 of gestation, the cell height was less than, and the follicle diameter was approximately equal to those in the two controls 2 days earlier. These results indicate that, as a consequence of maternal alcohol consumption, growth of the fetal thyroid gland is retarded, and there are indications of fetal hypothyroidism, as seen from the histometric data. This latter is suggestive of a retarded thyrotropic activity of the fetal pituitary gland.

Studies on cytotoxicity of nickel ions using C3H10T1/2 fibroblast cells.

Cytotoxicity of Ni ions were examined using C3H mouse derived 10T1/2 fibroblast cells. It became evident that Ni ions had dose-dependent cytotoxicity. The cell number incubated in DME medium containing 0.04 mM/L Ni ion for 6 days was reduced to half that in control DME medium without Ni. The cell totally disappeared in DME medium containing 2 mM/L Ni ion. The dissolution of Ni ions from Ni-containing metallic restorations must be lower than these concentration levels so that oral tissues might not be damaged. No neoplastic transformation was found on all cells examined.

Studies on cytotoxic effect of nickel ions on three cultured fibroblasts.

Cytotoxicity of Ni ions on three fibroblasts such as L929, Balb/3T3 clone A31 and MC3T3-E1 were examined by cell count (CC) and Neutral Red assay (NR). Three cells were incubated for 6 days in 1 ml DME medium containing Ni ions which ranged from 0 to 2 mM/l. The results clarified that Ni ions had dose-dependent cytotoxicity. L929 possessed the largest TC50 values (the amount of Ni ion that caused 50% cell death or 50% cell viability) of 0.12 mM/l (CC) and 0.32 mM/l (NR), and Balb/3T3 clone A31 had the least values of 0.05 mM/l (CC) and 0.09 mM/l (NR), whilst MC3T3-E1 had the intermediate values of 0.08 mM/l (CC) and 0.15 mM/l (NR). The dissolution of Ni ions from Ni-containing metallic restorations must be lower than these concentration levels so that body tissues might not be severely damaged.

Cytotoxic activity of hydrolyzable tannins against human oral tumor cell lines--a possible mechanism.

Hydrolyzable tannins showed higher cytotoxic activity against human oral squamous cell carcinoma and salivary gland tumor cell lines than against normal human gingival fibroblasts, whereas gallic acid, a component unit of tannins, showed much weaker selective cytotoxicity. The cytotoxic activity of dimeric compounds was generally higher than that of monomeric compounds. Macrocyclic ellagitannin oligomers, such as oenothein B, woodfordin C and woodfordin D showed the greatest cytotoxic activity, and their activity (per given number of molecules) was one order higher than those of gallic acid and epigallocatechin gallate, a major component of green tea. These compounds induced apoptotic cell death characterized by DNA fragmentation (as demonstrated by the TUNEL method) and cleavage of cytokeratin 18 by activated caspase(s) (as demonstrated by M30 monoclonal antibody). ESR spectroscopy revealed that these macrocyclic compounds at higher concentrations produced their own radicals and significantly enhanced the radical intensity of sodium ascorbate, possibly by their prooxidant actions. Catalase failed to eliminate their apoptosis-inducing activity, reducing the possibility of the involvement of hydrogen peroxide production in the extracellular fraction. These observations suggested that the antitumor activity of macrocyclic ellagitannin oligomers reported previously might be explained by their apoptosis-inducing activity.