Vitamin K3 derivative induces apoptotic cell death in neuroblastoma via downregulation of MYCN expression.

Neuroblastoma is a pediatric malignant tumor arising from the sympathetic nervous system. The patients with high-risk neuroblastomas frequently exhibit amplification and high expression of the MYCN gene, resulting in worse clinical outcomes. Vitamin K3 (VK3) is a synthetic VK-like compound that has been known to have antitumor activity against various types of cancers. In the present study, we have asked whether VK3 and its derivative, VK3-OH, could have the antitumor activity against neuroblastoma-derived cells. Based on our results, VK3-OH strongly inhibited cell proliferation and induced apoptotic cell death compared to VK3. Treatment of MYCN-driven neuroblastoma cells with VK3-OH potentiated tumor suppressor p53 accompanied by downregulation of anti-apoptotic Bcl-2 and Mcl-1. Interestingly, VK3-OH also suppressed the MYCN at mRNA and protein levels. Furthermore, we found downregulation of LIN28B following VK3-OH treatment in MYCN-amplified and overexpressed neuroblastoma cells. Collectively, our current findings strongly suggest that VK3-OH provides a potential therapeutic strategy for patients with MYCN-driven neuroblastomas.

Skin Permeation of Testosterone from Viscoelastic Lecithin Reverse Wormlike Micellar Solution.

We evaluated testosterone-containing lecithin reverse wormlike micelles (reverse worms) composed of a polar substance/lecithin/isopropyl myristate for transdermal application. Water, D-ribose, or tetraglycerol were used as the polar substance and were key ingredients for forming the reverse worms. Using the reverse worms, 1 wt% of testosterone could be stably solubilized. When using D-ribose as polar substance, the maximum zero-shear viscosity of the reverse worms solution was higher than that of systems using water or tetraglycerol as the polar substance. The mechanism of skin permeation of testosterone from reverse worms solution was elucidated using skin permeation experiments with hairless mouse skin. When the structure of the reverse worms transitioned to lamellar liquid crystals at the skin/formulation interface, testosterone became supersaturated in the formulations. The structural transition occurred in systems using water or D-ribose as the polar substance, increasing the flux of testosterone. The flux of testosterone from reverse worms solution thus depends on the type of polar substance used.

Glycosidic inhibitors of melanogenesis from leaves of Passiflora edulis.

A new flavonoid glycoside, chrysin 6-C-ß-rutinoside (chrysin α-L-rhamnopyranosyl-(1→6)-C-ß-glucopyranoside; 2), and two new triterpene glycosides, (31R)-31-O-methylpassiflorine (7) and (31S)-31-O-methylpassiflorine (8), along with 14 known glycosides, including three flavonoid glycosides, 1, 3, and 4, six triterpene glycosides, 5, 6, and 9-12, three cyano glycosides, 13-15, and two other glycosides, 16 and 17, were isolated from a MeOH extract of the leaves of Passiflora edulis (passion flower; Passifloraceae). The structures of new compounds were elucidated on the basis of extensive spectroscopic analysis and comparison with literature data. Upon evaluation of compounds 1-17 against the melanogenesis in the B16 melanoma cells induced with α-melanocyte-stimulating hormone (α-MSH), three compounds, isoorientin (1), 2, and (6S,9R)-roseoside (17), exhibited inhibitory effects with 37.3-47.2% reduction of melanin content with no, or almost no, toxicity to the cells (90.8-100.2% cell viability) at 100 µM. Western blot analysis showed that compound 2 reduced the protein levels of MITF, TRP-1, and tyrosinase, in a concentration-dependent manner while exerted almost no influence on the level of TRP-2, suggesting that this compound inhibits melanogenesis on the α-MSH-stimulated B16 melanoma cells by, at least in part, inhibiting the expression of MITF, followed by decreasing the expression of TRP-1 and tyrosinase. In addition, compounds 1-17 were evaluated for their inhibitory effects against the EpsteinBarr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells.

A new reverse worm-like micellar system from a lecithin, multivalent carboxylic acid and oil mixture.

We developed new lecithin organogels composed of reverse worm-like micelles with lecithin/multivalent carboxylic acid/oil systems, and discussed their phase behavior and rheological properties. The most important findings in this study are the following. From a screening test of many carboxylic acids for gelation, it was found that the number and position of the carboxyl groups of the multivalent carboxylic acids are the determinants for the formation of reverse worm-like micelles, and appropriate carboxylic acids such as citric acid and 1,2,3-propanetricarboxylic acid can change the lecithin/oil solution into a gel. Furthermore, upon addition of these carboxylic acids, the zero-shear viscosity of solutions increased monotonically until phase separation or cloudiness occurred. For example, when citric acid was used, the maximum zero-shear viscosity of the solution was 70,000,000 times larger than that of n-decane. From studies on the scaling of rheological parameters, it was found that further addition of multivalent carboxylic acids not only induced the formation of linear reverse worm-like micelles but also brought about their branching.

Antiviral activity of diarylheptanoid stereoisomers against respiratory syncytial virus in vitro and in vivo.

We previously showed that (5S)-5-hydroxy-7-(4-hydroxyphenyl)-1-phenylhept-3-one (AO-0011) and (5S)-5-methoxy-1,7-diphenylhept-3-one (AO-0016) isolated from Alpinia officinarum exhibited stronger anti-influenza virus activity and anti-respiratory syncytial virus (RSV) activity, respectively, than the other isolated diarylheptanoids. In this study, we synthesized an enantiomer (AO-0503) and racemate (AO-0504) of AO-0011 and an enantiomer (AO-0514) of AO-0016. The anti-RSV activities of the three stereoisomers (AO-0503, AO-0504, and AO-0514) and AO-0011 were examined in vitro and in vivo to evaluate the stereoisomeric effect on anti-RSV activity. In a plaque reduction assay using human epidermoid carcinoma cells, all four diarylheptanoids significantly exhibited anti-RSV activity, and AO-0514 and AO-0016 exhibited stronger anti-RSV activity than AO-0503, AO-0504, and AO-0011. In a murine RSV infection model, all four diarylheptanoids with anti-RSV activity in vitro were also significantly effective in reducing virus titers in the lungs of RSV-infected mice. In the histopathological analysis of RSV-infected lungs, the oral administration of even AO-0514, which showed the lowest reduction of virus titers in the lungs, was significantly effective in reducing the infiltration of lymphocytes and in reducing the interferon-γ level, which is a marker of severity of pneumonia due to RSV infection, in bronchoalveolar lavage fluids prepared from RSV-infected mice. Although the stereoisomeric effects of diarylheptanoids on anti-RSV activity varied moderately, all four diarylheptanoids examined were suggested to ameliorate pneumonia and have a potential anti-RSV activity in vivo. They are possibly mother compounds for the development of an anti-RSV drug in the future.

Vitamin K3 analogs induce selective tumor cytotoxicity in neuroblastoma.

We investigated the cytotoxicity of eight vitamin K3 (VK3) analogs against neuroblastoma cell lines (IMR-32, LA-N-1, NB-39, and SK-N-SH) and normal cell lines (human umbilical vein endothelial cells (HUVEC) and human dermal fibroblasts (HDF)) using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. 2-[(2-Methoxy)ethylthio]-3-methyl-1,4-naphthoquinone (VK3-OCH(3)) showed especially potent cytotoxic activities against neuroblastoma cells compared with normal cells. In a Hoechst 33342 staining experiment, apoptotic morphologies characterized by cell shrinkage, nuclear condensation, and nuclear fragmentation were observed in IMR-32 and LA-N-1 cells after 48 h of treatment with 10(-5) M of VK3-OCH(3). To clarify the molecular mechanisms of apoptosis induced by VK3-OCH(3), we examined the expression of apoptosis related proteins using a Proteome Profiler Array and western blotting. Heme oxygenase (HO)-1 was remarkably increased by VK3-OCH(3) compared with the control (173% in IMR-32 and 170% in LA-N-1 at 24 h). Moreover, caveolin-1 was induced by VK3-OCH(3) at 48 h. In addition, VK3-OCH(3) arrested the cell cycle at the G2/M phase in IMR-32 cells. These results suggest that VK3-OCH(3) exhibited a selective antitumor activity via HO-1-related mechanisms.

Cucurbitane triterpenoids from the leaves of Momordica charantia, and their cancer chemopreventive effects and cytotoxicities.

Seventeen cucurbitane-type triterpenoids, 1-17, including six new compounds, (23E)-3ß,25-dihydroxy-7ß-methoxycucurbita-5,23-dien-19-al (1), (23S*)-3ß-hydroxy-7ß,23-dimethoxycucurbita-5,24-dien-19-al (6), (23R*)-23-O-methylmomordicine IV (7), (25ξ)-26-hydroxymomordicoside L (8), 25-oxo-27-normomordicoside L (9), and 25-O-methylkaravilagenin D (12), were isolated from a MeOH extract of the leaves of Japanese Momordica charantia. The structures of new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. Compounds 1-17 were examined for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, a known primary screening test for inhibitors of tumor promotion. Four compounds, 1, (23E)-3ß,7ß-dihydroxy-25-methoxycucurbita-5,23-dien-19-al (2), karavilagenin D (11), and 12, showed potent inhibitory effects on EBV-EA induction with IC(50) values in the range of 242-264 mol ratio/32 pmol TPA. In addition, compounds 1 and 11 exhibited inhibitory effects on skin-tumor promotion in an in vivo two-stage mouse skin carcinogenesis test based on 7,12-dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as a promoter. Furthermore, upon evaluation of the cytotoxic activities of compounds 1-17 against human cancer cell lines, compounds 2, 5-7, 9, and 14 showed potent activities against HL60 cell line, and compound 2 against SK-BR-3 cell line.

Electrochemical detection of sugar-related compounds using boron-doped diamond electrodes.

Electrochemical detection of sugar-related compounds was conducted using a boron-doped diamond (BDD) electrode as a detector for flow-injection analysis (FIA). Sugar-related compounds oxidize at high applied potentials, for which the BDD electrode is suitable for electrochemical measurements. Conditions for an FIA system with a BDD detector were optimized, and the following detection limits were achieved for sugar-related compounds: monosaccharides, 25-100 pmol; sugar alcohols, 10 pmol; and oligosaccharides, 10 pmol. The detection limit for monosaccharide D-glucose (Glu) was 105 pmol (S/N = 3). A linear range was acquired from the detection limit to 50 nmol, and the relative standard deviation was 0.65% (20 nmol, n = 6). A high-performance liquid chromatography (HPLC) column was added to the system between the sample injector and the detector and detection limits to the picomole level were achieved, which is the same for the HPLC system and the FIA system. The electrochemical oxidation reaction of Glu was examined using cyclic voltammetry with the BDD detector. The reaction proved to be irreversible, and proceeded according to the following two-step mechanism: (1) application of a high potential (2.00 V vs. Ag/AgCl) to the electrode causes water to electrolyze on the electrode surface with the simultaneous generation of a hydroxyl radical on the surface, and (2) the hydroxyl radical indirectly oxidizes Glu. Thus, Glu can be detected by an increase in the oxidation current caused by reactions with hydroxy radicals.

Stereochemically controlled asymmetric 1,2-reduction of enones mediated by a chiral sulfoxide moiety and a lanthanum(III) ion.

Enantiomerically pure (Z)-ß-sulfinyl allylic alcohols of either handedness can be readily prepared from (Z)-ß-sulfinyl enones using NaBH(4) or DIBAL reductants in the presence of LaCl(3) as a chelating agent. A chiral sulfoxide auxiliary induces the remote 1,2-asymmetric reduction (1,4-induction) to afford various chiral allylic alcohols in high yields with excellent stereoselectivities (up to 100% de).

In vitro and in vivo anti-influenza virus activity of diarylheptanoids isolated from Alpinia officinarum.

BACKGROUND: Diarylheptanoids (AO-0002 [7-(4''-hydroxy-3''-methoxyphenyl)-1-phenyl-4E-hepten-3-one] and AO-0011 [(5S)-5-hydroxy-7-(4''-hydroxyphenyl)-1-phenyl-3-heptanone]) isolated from Alpinia officinarum have been reported to exhibit anti-influenza virus activity in vitro. Hence, efficacies against influenza virus infection and the mode of antiviral action were evaluated in vivo and in vitro, respectively. METHODS: In a murine influenza virus infection model, diarylheptanoids were orally administered three times daily to mice infected with influenza A/PR/8/34 virus for 6 days after infection. AO-0002 was examined for its antiviral activity against the wild types of influenza viruses A/PR/8/34 (H1N1), oseltamivir-resistant A/PR/8/34 (H1N1), A/Bangkok/93/03 (H1N1), A/Ishikawa/7/82 (H3N2), A/Fukushima/13/43 (H3N2), B/Singapore/222/79 and B/Fukushima/15/93 in plaque reduction or yield reduction assays. The mode of anti-influenza virus action was assessed by a virus adsorption assay, immunofluorescence assay of viral antigens, and inhibition of viral messenger RNA synthesis using real-time reverse transcriptase PCR. RESULTS: AO-0002 at 100 mg/kg was significantly effective in reducing the body weight loss and prolonging survival times of infected mice without toxicity, but AO-0011 was not. AO-0002 at 30 and 100 mg/kg significantly reduced virus titres in bronchoalveolar lavage fluids of the lungs on days 3 and 6 after infection. AO-0002 exhibited anti-influenza virus activity against all viruses used, including the oseltamivir-resistant strain in vitro. The compound had no effect on virus adsorption or invasion into cells, but dose-dependently suppressed the expression of viral messenger RNA and antigens. CONCLUSIONS: AO-0002 was suggested to have a different anti-influenza virus action to that of oseltamivir and was verified to show anti-influenza activity in vitro and in vivo.

Asymmetric synthesis of gamma-hydroxy alpha-enones by 1,8-diazabicyclo[5.4.0]undec-7-ene-catalyzed stereoselective rearrangement of chiral alpha-sulfinyl enones.

The asymmetric rearrangement of optically active alpha-sulfinyl enone 1 induced by catalytic DBU and triphenylphosphine gave optically active gamma-hydroxy alpha-enone derivatives (up to 99% ee) in good yield following treatment with aqueous hydrogen peroxide.

Chirality in dynamic supramolecular nanotubes induced by a chiral solvent.

Amplification of chirality has been reported in polymeric systems. It has also been shown that related effects can occur in polymer-like dynamic supramolecular aggregates, if a subtle balance between noncovalent interactions allows the coupling between a chiral information and a cooperative aggregation process. In this context, we report a strong majority-rules effect in the formation of chiral dynamic nanotubes from chiral bisurea monomers. Furthermore, similar helical nanotubes (with the same circular dichroism signature) can be obtained from racemic monomers in a chiral solvent. Competition experiments reveal the relative strength of the helical bias induced by the chiral monomer or by the chiral solvent. The nanotube handedness is imposed by the monomer chirality, whatever the solvent chirality. However, the chirality of the solvent has a significant effect on the degree of chiral induction.

Effect of cell differentiation for neuroblastoma by vitamin k analogs.

BACKGROUND: Lack of receptor tyrosine kinase (TrkA), a high-affinity nerve growth factor (NGF) receptor, is closely associated with the malignant progression of neuroblastoma (NB) and its prognosis. Vitamin K3 (VK3) analogs inhibit the activity of protein tyrosine phosphatases (PTPases), which causes hydrolysis of the phosphate groups bound to the tyrosine residues on tyrosine kinase, resulting in sustained tyrosine phosphorylation. METHODS: In order to reverse this abnormal NGF/TrkA signal transduction in NB cells, we synthesized new VK3 analogs and examined their activity against NB cells. RESULTS: VK3 analogs increased or maintained the expression level of c-fos mRNA in the NB cells, which express the downstream genes of NGF/TrkA signal transduction. Moreover, the expression level of GAP-43 mRNA, which is a marker of neurite outgrowth and neuronal differentiation, was increased and morphological differentiation was also observed. VK3 analogs (especially COOH analog) continued to express c-fos and GAP-43 mRNAs and induced differentiation of NB cells after stimulation of NGF by strong inhibition of PTPase without affecting TrkA autophosphorylation. CONCLUSIONS: Vitamin K3 analogs may have potential as clinical therapeutic agents for NB.

Inhibitory effect of the rhizomes of Alpinia officinarum on TPA-induced inflammation and tumor promotion in two-stage carcinogenesis in mouse skin.

The methanol extract of galangal (the rhizomes of Alpinia officinarum L.) exhibited remarkable antitumor-promoting activity on an in vivo two-stage carcinogenesis test of mice using 7,12-dimethylbenz[a]anthracene as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. Seven diarylheptanoids (1-7) were isolated and identified from the active fraction of the methanol extracts of the galangal. These compounds, 1-7, were evaluated for their inhibitory effects on TPA-induced inflammation (1 microg/ear) in mice. These compounds (1-7) tested showed marked anti-inflammatory effects, with a 50% inhibitory dose of 0.8-2.7 micromol/ear.

Cucurbitane-type triterpenoids from the fruit of Momordica charantia.

The structures of three new cucurbitane-type triterpenoids isolated from the methanol extract of the fruit of Japanese Momordica charantia were established as (19R,23E)-5beta,19-epoxy-19-methoxycucurbita-6,23,25-trien-3beta-ol (1), (23E)-3beta-hydroxy-7beta-methoxycucurbita-5,23,25-trien-19-al (2), and (23E)-3beta-hydroxy-7beta,25-dimethoxycucurbita-5,23-dien-19-al (3) on the basis of spectroscopic methods. These compounds were accompanied by the known (19R,23E)-5beta,19-epoxy-19,25-dimethoxycucurbita-6,23-dien-3beta-ol (4) and (19R,23E)-5beta,19-epoxy-19-methoxycucurbita-6,23-diene-3beta,25-diol (5). This is the first report of the isolation of tetracyclic triterpenoids possessing a delta23,25-conjugated diene system, viz., 1 and 2, from a natural source.

Significance of survivin mRNA expression in prognosis of neuroblastoma.

Neuroblastoma (NB) is the most common malignant solid tumor in childhood, and among all childhood malignancies is second in prevalence only to leukemia. In NB we need to both make an accurate diagnosis and rapidly analyze the expression of genetic prognostic factors such as MYCN, H-ras, and trkA. Moreover, it has recently become important to analyze the expression of survivin mRNA, a member of the inhibitor of apoptosis protein family. Expression of the survivin gene is related to tumorigenesis and inhibition of apoptosis in some malignant tumors. We investigated its expression by reverse transcription-polymerase chain reaction (RT-PCR) in NB cell lines (SK-N-SH, NB-39, and IMR-32), two normal blood cell samples, and 13 clinical NB tumor samples. All three NB cell lines had high levels of mRNA expression for this gene, but normal blood cells had no expression. We detected expression of survivin mRNA in 7 of the 13 NB tumor samples (54%). Two NB patients were in stage I disease, 6 in stage II, and 5 in stage IV(A). Quantitative analysis by RT-PCR revealed that the ratio between survivin mRNA and human glyceraldehyde-3-phosphate dehydrogenase (h-GAPDH) mRNA was very low in stages I and II (0-0.017). In contrast, in advanced NBs (stage IV(A)) the ratio was much higher (0-0.050). The prognoses of the three patients in the advanced stage who had high ratios of expression were poor. A high level of expression of survivin mRNA indicates a high grade of malignancy, high likelihood of recurrence, and poor prognosis.

Usefulness of tyrosine hydroxylase mRNA for diagnosis and detection of minimal residual disease in neuroblastoma.

Neuroblastoma (NB) is the most common malignant solid tumor in childhood and, among all childhood malignancies, is second only to leukemia. NB originates before birth in the neural crest, which develops into the adrenal medullae and sympathetic ganglia. In the adrenal medulla, tyrosine hydroxylase (TH) is the first enzyme in the pathway of catecholamine synthesis. We used reverse transcription polymerase chain reaction (RT-PCR) to examine the expression of TH mRNA in NB and Ewing's sarcoma cell lines, small round cell tumors (SRCTs) containing NB, and other clinical tumor samples (osteosarcoma, osteochondroma, and Wilms' tumor). In total, we analyzed 33 clinical tumor samples. TH mRNA was expressed in all three NB cell lines examined, but not in two ES cell lines or in a breast cancer cell line. We detected TH mRNA in 23 of 25 NB tumor samples (92%), but in none of the SRCTs or other clinical tumor samples. This RT-PCR technique showed a sensitivity for TH mRNA of one NB cell per 10(5) negative cells. Based on these results, the detection of TH mRNA is very useful both as a tumor marker for NB and for detecting minimal residual disease. Therefore, we can use this method to detect tumor cell contamination before hematopoietic stem cell transplantation.

Sunpollenol and five other rearranged 3,4-seco-tirucallane-type triterpenoids from sunflower pollen and their inhibitory effects on Epstein-Barr virus activation.

Six new rearranged 3,4-seco-tirucallane-type triterpenoids (1-6) have been isolated from the diethyl ether extract of the pollen grains of sunflower (Helianthus annuus). These compounds were evaluated with respect to their inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) in Raji cells. All of the compounds tested showed potent inhibitory effects on EBV-EA activation (97-100% inhibition at 1 x 10(3) mol ratio/TPA).

Diagnostic significance and clinical applications of chimeric genes in Ewing's sarcoma.

Ewing's sarcoma (ES) is one of the most malignant bone and soft tissue tumors in childhood. Morphologically, ES belongs to the small round cell tumors (SRCT). ES, peripheral primitive neuroectodermal tumor (PNET), and Askin's tumor are classified as ES family tumors (ESFT) because they share a common chromosomal translocation. The EWS-FLI1 chimeric gene is generated by t (11; 22). Other reciprocal translocations resulting in formation of chimeric genes between EWS and ETS family genes (ERG, ETV1, E1AF, and FEV) are t (21; 22), t (7; 22), t (17; 22), and t (2; 22), respectively. Although it is generally difficult to distinguish ES from SRCT, we could easily and quickly distinguish ES from other SRCT by using reverse transcription polymerase chain reaction (RT-PCR). We looked for specific chimeric genes in 23 tumor samples, including three ES clinical samples. We detected five chimeric genes in the three ES samples. Three chimeric genes, all EWS-FLI1, were detected in one ES sample. Different chimeric genes, EWS-ERG and EWS-ETV1, were detected in the other two ES samples. Moreover, because we could not detect specific chimeric genes in samples from non-ESFT, it may be possible to use this technique to diagnose ESFT and to detect tumor cell contamination before hematopoietic stem cell transplantation.