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1.
Biomed Chromatogr ; 28(12): 1624-32, 2014 Dec.
Article in English | MEDLINE | ID: mdl-24782399

ABSTRACT

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of Grayanotoxin I (GTX I) and Grayanotoxin III (GTX III) in rat whole blood. Grayanotoxins (GTXs) and clindamycin as internal standard (IS) were extracted from rat blood via solid-phase extraction using PEP solid-phase extraction cartridges. Chromatographic separation of the analytes was achieved on a Kinetex C18 (100 × 2.1 mm, 2.6 µm) reversed-phase column using a gradient elution with the mobile phase of 1% acetic acid in water and methanol at a flow rate of 0.2 mL/min. Electrospray ionization mass spectrometry was operated in the positive ion mode with multiple reaction monitoring. The calibration curves obtained were linear over the concentration range of 1-100 ng/mL with a lower limit of quantification of 1 ng/mL for GTXs. The relative standard deviation of intra-day and inter-day precision was below 6.8% and accuracy ranged from 94.8 to 106.6%. The analytes were stable in the stability studies. The validated method was successfully applied to the quantification and toxicokinetic study of GTXs in rats for the first time after oral administration of 11.52 mg/kg mad honey and 0.35 mg/kg GTX III, respectively.


Subject(s)
Chromatography, Reverse-Phase/methods , Diterpenes/blood , Diterpenes/pharmacokinetics , Tandem Mass Spectrometry/methods , Administration, Oral , Animals , Diterpenes/administration & dosage , Diterpenes/chemistry , Linear Models , Male , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and Specificity , Solid Phase Extraction/methods , Spectrometry, Mass, Electrospray Ionization/methods , Toxicokinetics
2.
Carcinogenesis ; 33(12): 2520-8, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22907530

ABSTRACT

To evaluate the significance of C-C chemokine receptor type 5 (CCR5) in lung tumor development, we compared carcinogen-induced tumor growth in CCR5 knockout (CCR5(-/-)) mice and wild-type (CCR5(+/+)) mice. CCR5(-/-) mice showed reduced urethane (1g/kg)-induced tumor incidence when compared with those of CCR5(+/+) mice. We investigated the activation of nuclear factor-kappaB/STAT3 since these are implicated transcription factors in the regulation of genes involving tumor growth. Significant inhibition of DNA-binding activity of nuclear factor-kappaB and STAT3, and the translocation of p50 and p65 into the nucleus and the phosphorylation of IĸB were found in the lungs of CCR5(-/-) mice compared with the lungs of CCR5(+/+) mice. Expression of apoptotic protein such as cleaved caspase-3, cleaved PARP and Bax was elevated, whereas the expression levels of survival protein such as Bcl-2 and cIAP1 was decreased in the lungs of CCR5(-/-) mice. Interestingly, we found that the level of monocyte chemoattractant protein-1 (MCP-1), a tumor growth-promoting cytokine, was significantly reduced in the lung tumor tissue and blood of CCR5(-/-) mice compared with the level in CCR5(+/+) mice. In addition, CCR5 small interfering RNA (siRNA) and inhibitor of MCP-1 blocked lung cancer cell growth, which was abolished by the addition of MCP-1 protein in cultured lung cancer cells. Moreover, inactivation of CD8(+) cytotoxic T cell and dendritic cells was significantly increased in the blood, lung tumors and spleens of CCR5(-/-) mice compared with that of CCR5(+/+) mice. Therefore, these results showed that CCR5 deficiency suppressed lung tumor development through the inhibition of nuclear factor-kappaB/STAT3 pathways and the downregulation of MCP-1 in the carcinogen-induced lung tumor model.


Subject(s)
Chemokine CCL2/antagonists & inhibitors , Lung Neoplasms/prevention & control , NF-kappa B/antagonists & inhibitors , Receptors, CCR5/physiology , Animals , Apoptosis , CCR5 Receptor Antagonists , CD8-Positive T-Lymphocytes/physiology , Dendritic Cells/physiology , Disease Models, Animal , Humans , Lung Neoplasms/chemically induced , Lung Neoplasms/pathology , Mice , Mice, Inbred C57BL , NF-kappa B/physiology , STAT3 Transcription Factor/physiology , Urethane/toxicity
3.
Chirality ; 24(12): 1037-46, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22847739

ABSTRACT

Enantiomeric separations of N-phthaloyl (N-PHT), N-tetrachlorophthaloyl (N-TCPHT), and N-naphthaloyl (N-NPHT) α-amino acids and their esters were examined on several kinds of polysaccharide-derived chiral stationary phases (CSPs). Resolution capability of CSPs was greater Chiralcel OF than the others for N-PHT and N-NPHT α-amino acids and their esters. In N-TCPHT α-amino acids and their esters, good enantioselectivities showed Chiralcel OG for N-TCPHT α-amino acids, Chiralpak AD for N-TCPHT α-amino acid methyl esters, and Chiralcel OD for N-TCPHT α-amino acid ethyl esters, respectively. From the results of liquid chromatography and computational chemistry, it is concluded that l-form is preferred and more retained with electrostatic interaction in case of interaction between N-PHT α-amino acid derivatives and Chiralcel OF, N-TCPHT α-amino acid derivatives and Chiralcel OD, and N-NPHT α-amino acid derivatives and Chiracel OF. On the other hand, d-form is preferred and more retained with van der Waals interaction in case of interaction between N-TCPHT α-amino acid ester derivatives and Chiralcel OG and Chiralpak AD.


Subject(s)
Amino Acids/chemistry , Amino Acids/isolation & purification , Chromatography, Liquid/methods , Molecular Dynamics Simulation , Polysaccharides/chemistry , Esters , Molecular Conformation , Stereoisomerism
4.
Biomed Chromatogr ; 26(4): 429-33, 2012 Apr.
Article in English | MEDLINE | ID: mdl-21830227

ABSTRACT

A simple LC-MS/MS method has been developed and validated for the quantification of endogenous myo- and chiro-inositol in human urine. myo- and chiro-Inositol were completely resolved from other carbohydrates and there were no interference peaks in human urine. The correlation coefficient (n = 3) was greater than 0.9991 over the range 0.05-25.0 µg/mL with the weighted (1/C²) least square method. Precision (%RSD) and accuracy (%RE) were 0-10.0% and 0-6.0% for the intra-day assay (n = 5) and 0-14.3% and 0-10.0% for the inter-day assay (n = 5). myo- and chiro-Inositol have been shown to be stable in human urine stored at room temperature and for three freeze-thaw cycles.


Subject(s)
Inositol/urine , Tandem Mass Spectrometry/methods , Biomarkers/urine , Chromatography, Liquid/methods , Humans , Insulin Resistance , Sensitivity and Specificity
5.
Can J Physiol Pharmacol ; 89(3): 232-7, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21423297

ABSTRACT

A germacranolide sesquiterpene lactone, 2α,5-epoxy-5,10-dihydroxy-6α-angeloyloxy-9ß-(3-methylbutyloxy)-germacran-8α,12-olide (EDAG), isolated from Carpesium triste var. manshuricum, showed inhibitory activity in the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) mRNA and protein in LPS-activated macrophage cells. Molecular analysis reveals that these suppressive effects are correlated with the inhibition of NF-κB activation by EDAG. Immunoblotting showed that EDAG suppressed the LPS-induced degradation of I-κBα and decreased nuclear translocation of p65. Furthermore, EDAG showed reduced phosphorylation of ERK1/2 and p38 MAPK, whereas activation of JNK was not changed. These data suggest, at least in part, that EDAG utilizes the signal cascades of ERK1/2, p38 MAPK, and NF-κB for the suppression of iNOS gene expression.


Subject(s)
Asteraceae , Down-Regulation/drug effects , NF-kappa B/antagonists & inhibitors , Nitric Oxide Synthase Type II/antagonists & inhibitors , Sesquiterpenes, Germacrane/pharmacology , Animals , Cell Line , Down-Regulation/physiology , Mice , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/metabolism , Sesquiterpenes, Germacrane/isolation & purification
6.
Biochem Biophys Res Commun ; 391(3): 1400-4, 2010 Jan 15.
Article in English | MEDLINE | ID: mdl-20026303

ABSTRACT

Carabrol, isolated from Carpesium macrocephalum, showed anti-inflammatory potential in LPS-induced RAW 264.7 murine macrophages. In present study, carabrol demonstrated the inhibitory activity on pro-inflammatory cytokines such as IL-1beta, IL-6 and TNF-alpha. In addition, mRNA and protein levels of iNOS and COX-2 were reduced by carabrol. Molecular analysis revealed that these suppressive effects were correlated with the inactivation of p38 and JNK via inhibition of NF-kappaB activation. Immunoblotting showed that carabrol suppressed LPS-induced degradation of I-kappaBalpha and decreased nuclear translocation of p65. Taken together, these results suggest that carabrol can be a modulator of pro-inflammatory signal transduction pathway in RAW 264.7 cells.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Asteraceae/chemistry , Cyclooxygenase 2/drug effects , Nitric Oxide Synthase Type II/antagonists & inhibitors , Sesquiterpenes/chemistry , Active Transport, Cell Nucleus/drug effects , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Cell Line , Cyclooxygenase 2/biosynthesis , I-kappa B Kinase/metabolism , Interleukin-1beta/antagonists & inhibitors , Interleukin-6/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , Lipopolysaccharides/pharmacology , Mice , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/biosynthesis , Phosphorylation/drug effects , Sesquiterpenes/pharmacology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolism
7.
Behav Pharmacol ; 21(5-6): 572-5, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20700049

ABSTRACT

This experiment was designed to know whether (-)-epigallocatethin-3-O-gallate (EGCG) counteracts caffeine-induced hyperactivity, and its potential mechanisms in mice. EGCG inhibited methamphetamine-induced, cocaine-induced and caffeine-induced horizontal hyperlocomotion and rearing activity. EGCG also inhibited hyperlocomotion and rearing activity induced by apomorphine, a D1/D2-like agonist. Moreover, EGCG inhibited climbing behavior, a typical stereotyped behavior induced by stimulation of dopamine receptors through the activation of those receptors by apomorphine. From this experiment, we suggest that EGCG inhibits hyperactivity induced by psychostimulants including caffeine, in part by modulating dopaminergic transmission, and these inhibitory effects of EGCG counteract the stimulant actions of caffeine in green tea.


Subject(s)
Caffeine/toxicity , Catechin/analogs & derivatives , Central Nervous System Stimulants/toxicity , Hyperkinesis/prevention & control , Animals , Catechin/pharmacology , Hyperkinesis/chemically induced , Male , Mice , Mice, Inbred ICR , Receptors, Dopamine D1/drug effects , Receptors, Dopamine D1/metabolism , Receptors, Dopamine D2/drug effects , Receptors, Dopamine D2/metabolism , Stereotyped Behavior/drug effects , Tea/chemistry
8.
Electrophoresis ; 30(10): 1661-9, 2009 May.
Article in English | MEDLINE | ID: mdl-19343727

ABSTRACT

A robust and convenient sheathless CE/ESI-MS interface realized with an ionophore membrane-packed electro-conduction channel is described. Sheathless interfaces that may provide higher sensitivity for MS detection than sheath flow-supported interfaces generally show instability and short lifetimes due to their imperfection in making an electrical contact with the emitter tip. In this work, we designed a sheathless interface based on an ionophore membrane-packed electro-conduction channel. At the joining point of the CE capillary and the emitter capillary, the conduction channel was implemented toward the exterior of the interface body, where a platinum wire electrode was placed. The conduction channel transferred the electric field from the external Pt electrode to the joining point, but prevented the effluent of CE from leaking. The interface body was designed to have receptacles for standard capillary tubing with finger-tight fittings, which allowed easy replacement of capillary tubing. Stable electrospray was observed for an extended time period without any signs of bubbling or damage to the emitter tip. No significant increment of dead-volume at the interface was observed for well-aligned capillaries. Sensitive and stable CE-MS detection of the model compound of creatinine and uric acid was demonstrated.


Subject(s)
Electrophoresis, Capillary/methods , Spectrometry, Mass, Electrospray Ionization/methods , Electric Conductivity , Equipment Design/methods , Ionophores/chemistry
9.
Int J Oncol ; 34(5): 1221-30, 2009 May.
Article in English | MEDLINE | ID: mdl-19360335

ABSTRACT

Berberine has anti-tumor properties in some cancer cells including prostate cancer, but the exact mechanisms and in vivo effects are unclear. We investigated anti-cancer activity of berberine in vitro and in vivo, and possible mechanisms in prostate cancer cells. Berberine treatment inhibited cell cancer growth in a concentration (0-50 microM) and time- (0-48 h) dependent manner without any growth inhibition in normal human prostate epithelial PWR-1E cells. However, the p53 expressing LNCaP cells were more susceptible against berberine than the p53 lacking PC-3 cells. The cell arrest in G0/G1 phase, apoptotic cell death and the expression of apoptotic cell death proteins Bax and caspase-3 was much higher in berberine-treated LNCaP cells than those in PC-3 cells. Exploration of p53 siRNA or pifithrin-alpha, a p53 inhibitor to the LNCaP cells, suppressed berberine-induced cell death and expression of apoptosis-related proteins. In xenograft in vivo studies, berberine reduced tumor weights and volumes accompanied with apoptotic cell death and increased expression of apoptotic cell death proteins, however, the extent of inhibitory effect was more significant in LNCaP cell-bearing mice. Therefore, these results indicated that berberine inhibited p53-dependent prostate cancer cell death.


Subject(s)
Apoptosis/drug effects , Berberine/pharmacology , Cell Proliferation , Prostatic Neoplasms/pathology , Tumor Suppressor Protein p53/physiology , Animals , Cell Cycle/drug effects , Cell Cycle/genetics , Cells, Cultured , Down-Regulation/drug effects , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Models, Biological , Neoplasm Proteins/metabolism , Prostatic Neoplasms/metabolism , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor Assays
10.
J Pharmacol Sci ; 111(2): 124-36, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19834284

ABSTRACT

Nuclear transcription factor-kappaB (NF-kappaB) is constitutively activated in prostate and colon cancers and is related with the resistance of cancer cells against chemotherapeutics. Previously, we found that obovatol, an active compound isolated from Magnolia obovata, inhibited cancer cell growth through inhibition of NF-kappaB activity. We investigated here whether obovatol could sensitize cancer cells against docetaxel through inhibition of NF-kappaB activity in prostate cancer (LNCaP and PC-3) and colon cancer (SW620 and HCT116) cells. The combination treatment with each drug at one half the respective IC(50) dose (5 microM obovatol + 5 nM docetaxel) was more effective and significant (60%-70%) in the inhibition of cancer cell growth than single treatment by each drug (20%-40%); inhibition was exerted through a significant increase of apoptosis induction (60%-80%) by the combination treatment compared to the single treatment (10%-30%). Correlating well with the synergistic inhibition (combination indices are less than 1 in all cell types), the combination significantly inhibited NF-kappaB activities as well as expression of NF-kappaB target apoptotic cell death proteins, but decreased anti-apoptotic cell death proteins. Similar combination effects of obovatol with other chemotherapeutic agents (paclitaxel, cisplatin, and doxorubicin) on the inhibition of cell growth and NF-kappaB activity were also found. These results indicate that obovatol augments cell growth inhibition by chemotherapeutics through inactivation of NF-kappaB and suggest that obovatol may have therapeutic advantages in the combination treatment with other chemotherapeutics. [Supplementary Figure: available only at http://dx.doi.org/10.1254/jphs.09048FP].


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Colonic Neoplasms/metabolism , NF-kappa B/antagonists & inhibitors , Prostatic Neoplasms/metabolism , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/chemistry , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Proliferation/drug effects , Cell Survival/drug effects , Colonic Neoplasms/pathology , Docetaxel , Drug Synergism , Fluorescent Dyes/metabolism , HCT116 Cells , Humans , In Situ Nick-End Labeling , Indoles/metabolism , Inhibitory Concentration 50 , Magnolia/chemistry , Male , Molecular Structure , NF-kappa B/metabolism , Phenyl Ethers/chemistry , Phenyl Ethers/pharmacology , Plant Leaves/chemistry , Prostatic Neoplasms/pathology , Taxoids/administration & dosage , Taxoids/chemistry , Taxoids/pharmacology
11.
Arch Pharm Res ; 32(5): 755-65, 2009 May.
Article in English | MEDLINE | ID: mdl-19471891

ABSTRACT

Ginsenoside Rg3, the main constituent isolated from Panax ginseng, has been of interest for use as a cancer preventive or therapeutic agent. We investigated here whether Rg3 can inhibit the activity of NF-kappaB, a key transcriptional factor constitutively activated in colon cancer that confers cancer cell resistance to chemotherapeutic agents. To investigate whether RG3 can suppress activation of NF-kappaB, and thus inhibit cancer cell growth, we examined the susceptibility of colon cancer cells (SW620 and HCT116) to treatment with Rg3 (25, 50, 75, 100 microM) and RG3-induced activation of NF-kappaB. RG3 dose-dependently inhibited cancer cell growth through induction of apoptosis and decreased NF-kappaB activity. In a further study of compounds in colon cancer, we used half of the IC(50) dose, values in combined treatments of Rg3 (50 microM) with conventional agents - docetaxel (5 nM), paclitaxel (10 nM) cisplatin (10 microM) and doxorubicin (2 microM). Compared to treatment with Rg3 or chemotherapy alone, combined treatment was more effective (i.e., there were synergistic effects) in the inhibition of cancer cell growth and induction of apoptosis and these effects were accompanied by significant inhibition of NF-kappaB activity. NF-kappaB target gene expression of apoptotic cell death proteins (Bax, caspase-3, caspase-9) was significantly enhanced, but the expression of anti-apoptotic genes and cell proliferation marker genes (Bcl-2, inhibitor of apoptosis protein (IAP-1) and X chromosome IAP (XIAP), Cox-2, c-Fos, c-Jun and cyclin D1) was significantly inhibited by the combined treatment compared to Rg3 or docetaxel alone. These results indicate that ginsenoside Rg3 inhibits NF-kappaB, and enhances the susceptibility of colon cancer cells to docetaxel and other chemotherapeutics. Thus, ginsenoside Rg3 could be useful as an anti-cancer or adjuvant anti-cancer agent.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Colonic Neoplasms/metabolism , Ginsenosides/pharmacology , NF-kappa B/antagonists & inhibitors , Taxoids/pharmacology , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , Cell Cycle Proteins/metabolism , Cell Proliferation/drug effects , Cisplatin/pharmacology , Colonic Neoplasms/pathology , Docetaxel , Dose-Response Relationship, Drug , Doxorubicin/pharmacology , HCT116 Cells , Humans , Inhibitory Concentration 50 , NF-kappa B/metabolism , Paclitaxel/pharmacology
12.
Eur J Pharmacol ; 582(1-3): 17-25, 2008 Mar 17.
Article in English | MEDLINE | ID: mdl-18241858

ABSTRACT

Biphenolic components in Magnolia obovata including magnolol and honokiol have shown several pharmacological activities such as anti-tumor, anti-oxidant and anti-inflammatory effects. Previously in cultured macrophage Raw264.7 cells and fibroblast, we found that obovatol, an active compound isolated from M. obovata inhibited NF-kappaB activity which has been known to be a significant transcriptional factor to control of cancer cell growth. We investigated here whether obovatol could inhibit NF-kappaB activity, and thereby inhibit cancer cell growth in prostate (LNCaP and PC-3) and colon cancer (SW620 and HCT116) cells. Treatment of obovatol (10, 15, 20, 25 microM) inhibits cancer cell growth in the absence or the presence of tumor necrosis factor-alpha (TNF-alpha , 10 ng/ml) and tetradecanoyl phorbol acetate (TPA 10 or 50 nM) in a concentration-dependent manner through induction of apoptotic cell death. Cytotoxic activity was not observed in normal cells with up to 50 muM obovatol. It was also found that obovatol inhibited TNF-alpha and TPA-induced transcriptional and DNA binding activities of NF-kappaB. In further study, obovatol decreased translocation p65 and p50 into nucleus via decrease of phosphorylation of IkappaB. Correlated well with the induction of apoptosis, obovatol increased the expression of the apoptotic genes; Bax, caspase-3, caspase-9, whereas inhibited expression of anti-apoptotic genes; Bcl-2, inhibitor of apoptosis protein (IAP-1) and X chromosome IAP (XIAP) as well as the cell proliferation marker genes; Cox-2, c-Fos, c-Jun and cyclin D1. These results suggest that obovatol inhibits prostate and colon cancer cell growth via induction of apoptotic cell death, and that inhibition of NF-kappaB may be a significant as its action mechanism.


Subject(s)
Apoptosis , Biphenyl Compounds/pharmacology , Colonic Neoplasms/pathology , NF-kappa B/antagonists & inhibitors , Phenyl Ethers/pharmacology , Prostatic Neoplasms/pathology , Caspases/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Colonic Neoplasms/metabolism , Dose-Response Relationship, Drug , Humans , Male , NF-kappa B/metabolism , Phosphorylation , Prostatic Neoplasms/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Transcription, Genetic , Tumor Necrosis Factor-alpha/pharmacology
13.
Anticancer Res ; 28(6A): 3777-84, 2008.
Article in English | MEDLINE | ID: mdl-19189664

ABSTRACT

Berberine, an alkaloid, has anti-tumor properties in some cancer cells, but action mechanisms are not clear yet. We here investigated the anticancer activity of berberine and possible mechanisms in human neuroblastoma SK-N-SH and SK-N-MC cells. The p53-expressing cells, SK-N-SH (IC50=37 microM) were more susceptible to berberine than the p53-deficient cells, SK-N-MC (IC50 > or =100 microM) without cytotoxic effect on the cortical neuronal cells. Berberine caused cell cycle arrest in G0/G1 phase and apoptotic cell death, and these effects were much greater in SK-N-SH cells than those in SK-N-MC cells. Berberine much greatly decreased G0/G1 phase-associated cyclin and cyclin-dependent kinase (cyclin D1, cyclin E, Cdk2, and Cdk4) expression, and increased apoptotic gene expression and activation of caspase-3 in SK-N-SH cells. Exploration of p53 siRNA or pifithrin-alpha (PFT-alpha), a p53 inhibitor, in the SK-N-SH cells resulted in increase of IC50 values for cell viability, and decreased apoptotic cell death, expression of p53 and activation of caspase-3. Therefore, these results showed that berberine causes p53-dependent apoptotic death of neuroblastoma cells, and suggested that berberine may be useful as an anticancer agent for neuroblastoma.


Subject(s)
Apoptosis/drug effects , Berberine/pharmacology , Neuroblastoma/drug therapy , Tumor Suppressor Protein p53/metabolism , Animals , Apoptosis/physiology , Cell Growth Processes/drug effects , Cell Growth Processes/physiology , Cell Line, Tumor , Cell Nucleus/metabolism , Female , G1 Phase/drug effects , Humans , Neuroblastoma/metabolism , Neuroblastoma/pathology , Pregnancy , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Rats , Rats, Sprague-Dawley , Resting Phase, Cell Cycle/drug effects , bcl-2-Associated X Protein/biosynthesis
14.
Arch Pharm Res ; 31(10): 1347-53, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18958427

ABSTRACT

A 1:1 mixture of acriflavine (ACF; CAS 8063-24-9) and guanosine is under evaluation in preclinical studies as a possible antitumor agent. Guanosine is known to potentiate the anti-cancer activity of ACF. We therefore investigated the pharmacokinetics of guanosine following administration of the ACF/guanosine mixture in rats. Rats were given guanosine (1 or 5 mg/kg) or ACF/guanosine (2 or 10 mg/kg) by i.v. bolus; or guanosine (3 or 15 mg/kg) or ACF/guanosine (6 or 30 mg/kg) by i.m. injection. We found that guanosine was rapidly cleared from the blood and transferred to tissues after i.m. administration of ACF/guanosine. The mean plasma half-lives (t(1/2)) at the alpha and beta phases were 0.091 and 6.86 h, or 0.09 and 7.51 h at a dose of 1 or 5 mg/kg guanosine, respectively. ACF had no effect on the plasma disappearance of guanosine following either i.v. bolus or i.m. administration of the combination mixture. Moreover, the ACF combination with guanosine did not significantly alter the values of MRT, V(dss), and CL(t) of guanosine. Guanosine exhibited linear pharmacokinetics over the dose range from 1 to 5 mg/kg for i.v. doses and 3 to 15 mg/kg for i.m. doses. The bioavailability of guanosine after i.m. administration was 84% for 3 mg/kg dose and 88% for 15 mg/kg dose. ACF had no effects on biliary and urinary excretion of guanosine after i.m. administration. The cumulative amount of guanosine in urine after i.m. administration was about 5-fold larger than that in bile, indicating that guanosine is mostly excreted into the urine. Guanosine was widely distributed in all tissues examined in this study, but was most highly concentrated in the kidney after i.m. administration, followed by slow excretion to bile or urine. ACF had no effect on the tissue distribution of guanosine following i.m. administration. These characterizations of the pharmacokinetics of guanosine after administration of the ACF/guanosine combination will be useful in providing preclinical and clinical bases for the potential application of this combination to the treatment of cancer.


Subject(s)
Acriflavine , Antineoplastic Agents , Guanosine/pharmacokinetics , Animals , Area Under Curve , Drug Combinations , Guanosine/administration & dosage , Indicators and Reagents , Injections, Intramuscular , Injections, Intravenous , Male , Rats , Rats, Sprague-Dawley , Tissue Distribution
15.
Mol Cancer Ther ; 6(2): 675-83, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17308063

ABSTRACT

We investigated whether the snake venom toxin (SVT) from Vipera lebetina turanica inhibits cell growth of human prostate cancer cells by inducing apoptosis and also studied possible signaling pathways involved in this cell death. SVT inhibited growth of PC-3 and DU145 cells, androgen-independent prostate cancer cells, but not LNCaP cells, a human androgen-dependent prostate cancer cell. Cells were arrested in the G(2)-M phase by SVT with a concomitant decrease in the expression of the G(2)-M phase regulatory protein cyclin B1 and were also arrested in the G(1)-S phase with decreasing expression of cyclin-dependent kinase 4, cyclin D1 and cyclin E. In addition to the growth-inhibitory effect, SVT increased the induction of apoptotic cell death. Untreated PC-3 cells show high DNA binding activity of nuclear factor kappaB (NF-kappaB), an antiapoptotic transcriptional factor, but this was inhibited by SVT and accompanied by a significant inhibition of p50 translocation into the nucleus, as well as phosphorylation of inhibitory kappaB. Consistent with the induction of apoptosis and inhibition of NF-kappaB, this toxin increased the expression of proapoptotic proteins such as p53, Bax, caspase-3, and caspase-9, but down-regulated antiapoptotic protein Bcl-2. However, SVT did not show an inhibitory effect on cell growth and caspase-3 activity in cells carrying mutant p50 and inhibitory kappaB kinase plasmids. Confocal microscopy analysis showed that SVT is taken up into the nucleus of the cells. These findings suggest that a nanogram concentration range of SVT from V. lebetina turanica could inhibit hormone-refractory human prostate cancer cell growth, and the effect may be related to NF-kappaB signal-mediated induction of apoptosis.


Subject(s)
Apoptosis/drug effects , NF-kappa B/metabolism , Neoplasms, Hormone-Dependent/drug therapy , Prostatic Neoplasms/drug therapy , Snake Venoms/pharmacology , Toxins, Biological/pharmacology , Viperidae , Animals , Caspases/metabolism , Cell Cycle/drug effects , Cell Line, Tumor/drug effects , Cell Nucleus , Cyclins/metabolism , Humans , Male , NF-kappa B/genetics , Phosphorylation/drug effects , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein Transport
16.
Mol Cancer Ther ; 6(10): 2786-97, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17938270

ABSTRACT

Molecular mechanisms underlying epothilone-induced apoptotic cell death were investigated in SW620 human colon cancer cells. Treatment with epothilone B and D at different concentrations (1-100 nmol/L) dose-dependently inhibited cell growth and caused cell cycle arrest at G2-M, which was followed by apoptosis. Consistent with this induction of apoptotic cell death, epothilone B and D enhanced the constitutional activation of nuclear factor-kappaB (NF-kappaB) via IkappaB degradation through IkappaB kinase (IKKalpha and IKKbeta) activation, and this resulted in p50 and p65 translocation to the nucleus. Moreover, cells treated with sodium salicylic acid, an IKK inhibitor, or transiently transfected with mutant IKKalpha and beta did not show epothilone-induced cell growth inhibition or p50 translocation, although p65 was still translocated to the nucleus. Treatment with epothilone B and D also enhanced beta-tubulin polymerization and the formation of p50/beta-tubulin complex. However, beta-tubulin polymerization was not inhibited in the cells treated by sodium salicylic acid or transiently transfected with mutant IKKalpha and beta. Moreover, epothilone B and D increased the expressions of NF-kappaB-dependent apoptotic cell death regulatory genes, i.e., Bax, p53, and the active form of caspase-3, but reduced Bcl-2 expression, and these actions were partially reversed by salicylic acid. In addition, caspase-3 inhibitor reduced epothilone B-induced cell death and NF-kappaB activation. These findings suggest that the activation of NF-kappaB/IKK signals plays an important role in the epothilone-induced apoptotic cell death of SW620 colon cancer cells in a tubulin polymerization-independent manner.


Subject(s)
Apoptosis/drug effects , Colonic Neoplasms/drug therapy , Epothilones/pharmacology , I-kappa B Kinase/metabolism , NF-kappa B/metabolism , Tubulin/metabolism , Apoptosis/physiology , Blotting, Western , Caspase 3/metabolism , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Electrophoretic Mobility Shift Assay , Flow Cytometry , Fluorescent Antibody Technique , Humans , In Situ Nick-End Labeling , Luciferases/metabolism , Microtubules/drug effects , Microtubules/metabolism , Protein Transport , Proto-Oncogene Proteins c-bcl-2/metabolism , Salicylic Acid/pharmacology , Signal Transduction , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism , bcl-2-Associated X Protein/metabolism
17.
Arch Pharm Res ; 30(4): 502-6, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17489368

ABSTRACT

The chicken embryo has been well used in studies of the developmental process, and during development sphingosine and sphingosine 1-phosphate (So1P) are considered critical mediators of cell death and survival. In this study, we compared the sphingolipid contents of chicken embryos during the early embryonic development period from day 3 to day 6. HPLC analyses of sphingosine and So1P in chicken embryos revealed that sphingosine levels were greatly reduced on day 4 whereas So1P levels were not significantly changed. Sphingosine kinase (Sphk) activities, which require sphingosine as substrate to produce So1P, were also greatly reduced on day 4. Collectively, we found sphingosine levels and Sphk activities, but not So1P levels are changed in early stage of chicken embryos development.


Subject(s)
Embryonic Development , Lysophospholipids/analysis , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Sphingosine/analogs & derivatives , Animals , Chick Embryo , Lysophospholipids/physiology , Sphingosine/analysis , Sphingosine/physiology
18.
Arch Pharm Res ; 30(3): 372-80, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17424946

ABSTRACT

Preclinical studies are currently underway to examine the potential antitumor effects of a 1:1 mixture of acriflavine (ACF; CAS 8063-24-9) and guanosine. Guanosine potentiates the anticancer activity of some compounds. However, the effects of guanosine on the pharmacokinetics of ACF in mammals are unknown. Therefore, this study investigated the effects of guanosine on the pharmacokinetics of ACF after administering a 1:1 mixture of ACF and guanosine in rats. The rats were given either 10 mg/kg of the mixture or 5 mg/kg ACF via an intravenous bolus injection; or 30 mg/kg of the mixture or 15 mg/kg ACF intramuscularly. An HPLC-based method, which was validated in this laboratory, was used to analyze the levels of trypaflavine (TRF) and proflavine (PRF) in the plasma, bile, urine, and tissue homogenates. It was found that TRF and PRF were rapidly cleared from the blood and transferred to the tissues after the i.v. bolus or i.m. injection of the combination mixture. Both TRF and PRF were found to be most highly concentrated in the kidneys after the i.v. bolus or i.m. injection, followed by slow excretion to the bile or urine. Guanosine had no effect on the plasma disappearance of TRF or PRF after the i.v. bolus injection. However, guanosine led to a prolongation of the plasma levels of PRF after the i.m. administration of the combination mixture, resulting in a 2 fold increase in the bioavailability (BA) of PRF The concentrations of TRF and PRF in all the tissues examined were similar in the groups given the mixture and ACF. However, guanosine led to a prolongation of the biliary and urinary excretions of both TRF and PRF after the i.v. bolus (1.25 fold) or i.m. (1.5-2.4 folds) injection. These prolonged effects of guanosine on the plasma disappearance or urinary excretion of TRF and PRF might be one reason for the enhanced antitumor effects of ACF. However, more study will be needed to further examine this potential mechanism.


Subject(s)
Acriflavine/administration & dosage , Acriflavine/pharmacokinetics , Antineoplastic Agents/administration & dosage , Guanosine/administration & dosage , Guanosine/pharmacology , Animals , Bile/metabolism , Chromatography, High Pressure Liquid , Drug Combinations , Male , Rats , Rats, Sprague-Dawley , Tissue Distribution
19.
Arch Pharm Res ; 30(5): 556-60, 2007 May.
Article in English | MEDLINE | ID: mdl-17615673

ABSTRACT

Four known germacranolide sesquiterpene lactones, 2alpha,5-epoxy-5,10-dihydroxy-6alpha-angeloyloxy-9beta-isobutyloxy-germacran-8alpha,12-olide (1), 2alpha,5-epoxy-5,10-dihydroxy-6alpha,9beta-diangeloyloxy-germacran-8alpha,12-olide (2, divaricin B), 2alpha,5-epoxy-5,10-dihydroxy-6alpha-angeloyloxy-9beta-(2-methylbutyloxy)-germacran-8alpha,12-olide (3) and 2alpha,5-epoxy-5,10-dihydroxy-6alpha-angeloyloxy-9beta-(3-methylbutyloxy)-germacran-8alpha,12-olide (4), were isolated from the chloroform-soluble fraction of the whole plants of Carpesium triste var. manshuricum. Their chemical structures were determined using spectroscopic methods, including 2D-NMR. All the isolates showed significant cytotoxicities (ED50 value: 4.3-16.8 microM) against five human tumor cell lines; A549, SK-OV-3, SK-MEL-2, XF498 and HCT15.


Subject(s)
Antineoplastic Agents, Phytogenic/isolation & purification , Asteraceae/chemistry , Lactones/isolation & purification , Sesquiterpenes, Germacrane/isolation & purification , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Cell Line, Tumor , Humans , Lactones/chemistry , Lactones/pharmacology , Magnetic Resonance Spectroscopy , Sesquiterpenes, Germacrane/chemistry , Sesquiterpenes, Germacrane/pharmacology
20.
Arch Pharm Res ; 30(10): 1350-7, 2007 Oct.
Article in English | MEDLINE | ID: mdl-18038915

ABSTRACT

An immunoglobulin (IgG) preparation with micro-amount of histamine fixed on the active protein fraction has been used to increase the resistance to allergic reactions. However, excessive histamine may cause hypo- or hypertension, headache, or anaphylactic shock and so the histamine content of the drug is strictly controlled by a regulation: 0.15 microg of histamine dihydrochloride is allowed for 12 mg of immunoglobulin. In this study, a liquid chromatographic method to determine micro-amount of histamine in the pharmaceutical was developed and validated. This method include a sample cleanup by a solid phase extraction (SPE) using a polystyrene-divinyl benzene (PS-DVB) polymeric sorbent and high-performance liquid chromatography after precolumn fluorescent labeling of the histamine with o-phthaldialdehyde. The drug sample was loaded to the SPE cartridge after adjusting to pH 9.5. After successive washings of the cartridge with water and 30% aqueous methanol, histamine was then eluted with 100 mM sodium acetate (pH 9.5)-methanol (20:80, v/v). An aliquot from the eluate was labeled with o-phthaldialdehyde-mercaptoethanol (OPA-ME) for fluorescence detection at the excitation maximum of 340 nm and emission maximum of 450 nm. HPLC analysis was performed on a phenyl-hexyl column with an acetonitrile-phosphate buffer (pH 6.8; 50 microM) (35:65, v/v) as the mobile phase. The retention times of histamine and 3-methylhistamine (IS) were approximately 7.2 and 9.5 min, respectively. The quantitation range was between 0.01-0.2 mg/mL of histamine showing good linearity (r=0.9996). This analytical method would provide a potential mean for the strict control of histamine content in the pharmaceutical product.


Subject(s)
Anti-Allergic Agents/chemistry , Chromatography, High Pressure Liquid/methods , Histamine/analysis , Immunoglobulin G/chemistry , Solid Phase Extraction , Acetonitriles/chemistry , Anti-Allergic Agents/standards , Buffers , Calibration , Chromatography, High Pressure Liquid/standards , Hydrogen-Ion Concentration , Indicators and Reagents/chemistry , Ion Exchange Resins/chemistry , Mercaptoethanol/chemistry , Methanol/chemistry , Methylhistamines/analysis , Polystyrenes/chemistry , Quality Control , Reproducibility of Results , Sodium Acetate/chemistry , Solvents/chemistry , Spectrometry, Fluorescence , o-Phthalaldehyde/chemistry
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