Induction of differentiation of human leukemia cells by various combinations of cytokines and low-molecular-weight inducers

To explore agents for differentiation therapy of leukemias, various combinations of cytokines and low-molecular-weight inducers were examined for differentiation-inducing activity toward three kinds of human leukemia-derived cell lines. The strongest differentiation inducing activity on promyelocytic HL60 cells and histiocytic U937 cells was obtained by combining recombinant tumor necrosis factor (rTNF), interferon-gamma (IFN-gamma), retinoic acid (RA), and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). For myeloblastic ML1 cells, the combination of rTNF, IFN-gamma, and RA had the strongest differentiation-inducing activity.     

Reduction of MTT by flavonoids in the absence of cells

The tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) is used to determine cell viability in assays of cell proliferation and cytotoxicity. MTT is reduced in metabolically active cells to yield an insoluble purple formazan product. However, in this study, using a colorimetric method with MTT, we demonstrated that luteolin and quercetin (both are flavonoids) can reduce MTT in the absence of living cells. We also examined effects of some experimental conditions on the reaction, such as concentrations of flavonoids, incubation time, the structure and the existing form in solvents of flavonoids. The maximal absorbance values, which were 0.355 (luteolin) and 0.491 (quercetin), were observed when the concentrations of flavonoids were 200 microg/ml. Incubating MTT with flavonoids for 8 h, the absorbance values got the maximum, which were 0.320 (luteolin) and 0.398 (quercetin). The ability of flavonoids reducing MTT in RPMI-1640 with 10% fetal calf serum was higher than that in anhydrous ethanol. And the ability of quercetin reducing MTT was higher than that of luteolin both in RPMI-1640 with 10% fetal calf serum and anhydrous ethanol. Based on the results, this undescribed reaction can significantly influence the results of experiments using the MTT assay to measure the effects of flavonoids on cell growth.     

A topographically optimized substrate with well-ordered lattice micropatterns for enhancing the osteogenic differentiation of murine mesenchymal stem cells

Although recently a growing number of reports demonstrate that topography or geometry of the substrate also plays an important role in the fate of the stem cells, most of these studies are usually completed by a few distinct patterns such as simple lines, posts, etc. As a result, there is a lack of quantitative analysis of the relationship between topographical variation and the differentiation of stem cells. Here, the effectiveness of topography variation is studied systematically in several microengineered substrates on osteogenic differentiation. It is found that the effectiveness of the osteogenic differentiation has a peak around 3 μm in the interval length of micropatterns.     

Sedimentation field flow fractionation to study human erythroleukemia cell megakaryocytic differentiation after short period diosgenin induction

Anti-cancer differentiation therapy could be one strategy to stop cancer cell proliferation. We propose a new sedimentation field flow fractionation (SdFFF) cell separation application in the field of cancer research. It concerns the study of megakaryocytic differentiation processes after a short exposure to an inducting agent (diosgenin). Washout process and early dual SdFFF separation--removing the influence of diosgenin and decreasing the influence of undifferentiated cells--resulted in the preparation of an enriched population to study the mechanism and kinetics of megakaryocytic differentiation. A short exposure to diosgenin was able to induce complete differentiation leading to maximal maturation which ended naturally after 192h incubation without the influence of a secondary effect of diosgenin. The study of isolated undifferentiated cells also showed that no resistance to diosgenin was observed. This result suggested different sensitivities to differentiation induction, and SdFFF cell separation would be of great interest to explore this phenomena.     

高效液相色谱法测定茶饮料中的黄酮类化合物

建立了茶饮料中六种黄酮类化合物的高效液相色谱检测方法。流动相采用梯度洗脱。结果表明芦丁、漆黄素、木犀草素、芹菜素在0.025~20 mg·L-1范围内呈现良好线性关系,桑色素、槲皮素在0.050~20 mg·L-1范围内呈现良好线性关系。加标回收率在84.5%~107.5%。该方法精密度良好(六种黄酮类化合物的相对标准偏差均不大于3.2%),检测限为6.2~34μg·L-1。该方法简便,灵敏、重现性好,可用于同时分析茶饮料中的黄酮化合物。     

Induction of apoptosis in human promyelocytic leukemia HL60 cells by panaxynol and panaxydol

Panaxynol and panaxydol are naturally occurring polyacetylenes, isolated from the lipophilic fractions of Panax notoginseng, that exert anti-proliferative effects against malignant cells. However, to the best of our knowledge, no study concerning the inhibitory effects of the two polyacetylenes on cell growth of human promyelocytic leukemia cells has been reported. In this paper, we examined the antiproliferation and proapoptotic effects of panaxynol and panaxydol on HL60 cells and investigated their mechanism of action. Cell growth inhibition of panaxynol and panaxydol were determined by trypan blue dye exclusion assays. Apoptosis of cells was revealed by morphological observation, analysis for nuclear DNA distribution and by annexin V-FITC/ PI staining using flow cytometry. It was found that panaxynol and panaxydol markedly inhibited proliferation of HL60 cells in a time- and dose-dependent manner via an apoptotic pathway. In concern with these ?ndings, Western blot analysis showed proteolytic activation of PKCδ, caspase-3 activation and cleavage of poly (ADP [adenosine diphosphate]-ribose) polymerase in HL60 cells treated by panaxynol and panaxydol. In conclusion, panaxynol and panaxydol have profound effects on growth and apoptosis of HL60 cells, suggesting those substances are worthy of further exploration as potential anti-cancer agents.     

Maturation of murine bone marrow-derived dendritic cells induced by Radix Glycyrrhizae polysaccharide

Radix Glycyrrhizae polysaccharide (GP), the most important component of Radix Glycyrrhizae, has been reported to have many immunopharmacological activities. However, the mechanism by which GP affects dendritic cells (DCs) has not been elucidated. In this study, we investigated the effect of GP on murine bone marrow-derived DCs and the potential pathway through which GP exerts this effect. Mononuclear cells (MNCs) were isolated from murine bone marrow and induced to become DCs by culturing with GM-CSF and IL-4. Six days later, DCs were divided into three groups: control group, GP group and LPS group. After 48 h of treatment, phenotypic figures and antigen uptake ability were determined by FACS analysis. The proliferation of DC-stimulated allogenic CD3+ T cells was detected by WST-1. IL-12 p70 and IFN-γ, which are secreted by DCs and CD3+ T cells respectively, were quantified by ELISA. Additionally, IL-12 p40 mRNA expression was determined by real-time PCR. Alterations in TLR4-related signaling pathways were examined by performing an antibody neutralization experiment. Treatment of DCs with GP resulted in the enhanced expression of the cell surface molecules CD80, CD86 and MHC I-A/I-E. GP also increased the production of IL-12 p70 by DCs in a time-dependent manner. The endocytosis of FITC-dextran by DCs was suppressed by GP administration. Furthermore, GP-treated DCs enhanced both the proliferation and IFN-γ secretion of allogenic CD3+ T cells. Finally, the effects of GP on DCs were partially reduced by using inhibitors of TLR4, NF-κB, p38 MAPK or JNK. In conclusion, GP can induce the maturation of DCs, and does so, in part, by regulating a TLR4-related signaling pathway.     

H1 histone and nucleosome repeat length alterations associated with the in vitro differentiation of murine embryonal carcinoma cells to extra-embryonic endoderm

The histone compositions and average distance between nucleosomes have been determined for F9.22 and PSA1 murine embryonal carcinoma cell lines, for primary extra-embryonic endoderm derived from the in vitro differentiation of PSA1 embryonal carcinoma cells, and for two long-term extra-embryonic endodermal cell lines. A change in the relative proportions of two forms of the H1 histones (H1A and H1B) was found to correlate with the extra-embryonic endodermal differentiated phenotype. The embryonal carcinoma cells had a ratio of H1A/H1B of 1.49 or greater. In contrast, extra-embryonic endoderm from either cell lines or freshly isolated from differentiating embryonal carcinoma cell cultures had a ratio of H1A/H1B of less than 0.9. Partial peptide mapping of gel purified H1A and H1B suggest the two proteins differ in primary structure. The nucleosome repeat length of the embryonal carcinoma cell lines was 196 bp of DNA. Primary extra-embryonic endoderm was found to have a value of 205 bp, but the long-term extra-embryonic endodermal cell lines had an average nucleosome repeat length of 187 bp. Since both freshly isolated primary endoderm and the long-term endodermal cell lines express differentiated functions (basement membrane glycoproteins and plasminogen activator activity), there appears to be no simple correlation between the nucleosome repeat length and the expression of these differentiated functions.     

Induction of apoptosis in human leukemia cells by 3-deazaadenosine is mediated by caspase-3-like activity

3-Deazaadenosine (DZA), one of the potent inhibitors of S-adenosylhomocysteine hydrolase, is known to possess several biological properties including an induction of apoptosis. To evaluate a possibility that DZA may be utilized for the treatment of human leukemia, we studied molecular events of cell death induced by DZA in human leukemia HL-60 and U-937 cells. DZA induced a specific cleavage of poly ADP-ribose polymerase (PARP) and an activation of the cysteine protease caspase-3/CPP32 which is known to cleave PARP. DZA-mediated nuclear DNA-fragmentation was completely blocked in the presence of a universal inhibitor of caspases (z-VAD-fmk) or the specific inhibitor of caspase-3 (z-DEVD-fmk) unlike of cycloheximide (CHX). DNA fragmentation was preceded by the lowering of c-myc mRNA in the DZA treated cells. In addition, DZA-induced apoptosis was blocked by pretreatment with adenosine transporter inhibitors such as nitrobenzylthioinosine (NBTI) and dipyridamole (DPD). Taken together, these results demonstrate that DZA-induced apoptosis initiated through an active transport of DZA into human leukemia cells, is dependent on the caspase-3-like activity without de novo synthesis of proteins and possibly involves c-myc down-regulation.     

Induction of multinucleated cells caused by UVA exposure in different stages of the cell cycle

Fibroblasts of the line 3T3 from swiss albino mice were exposed to ultraviolet A (UVA) irradiation. The cells were synchronized by treatment with nocodazole and mitotic shake-off, and then exposed to UVA irradiation in different stages of the cell cycle. Their photosensitivity varied through the cell cycle, being greatest in the G2 phase. UVA irradiation was found to induce the formation of multinucleated cells. Cells in the G1 phase were found to be most prone to multinucleation 15 min after UVA irradiation, while cells exposed to UVA irradiation in S and G2 phases contained the largest fractions of multinucleated cells 24 h after treatment. The present results indicate that multinucleated cells are formed by fusion of two or more cells shortly after UVA irradiation of early G1 cells, while impairment of cytokinesis is a possible explanation for the delayed formation of multinucleated cells after irradiation in S and G2.     

Identification of flavonoids by TLC scanning analysis

Summary A TLC-densitometric method for flavonoid analysis is described. Results are comparable to those obtained with classical methods. As the isolation step can be avoided results can be observed with 100 ng samples.     

Strategies for differentiation of isobaric flavonoids using liquid chromatography coupled to electrospray ionization mass spectrometry

Flavonoids are a class of secondary plant metabolites existing in great variety in nature. Due to this variety, identification can be difficult, especially as overlapping compounds in both chromatographic separations and mass spectrometric detection are common. Methods for distinguishing isobaric flavonoids using MS² and MS³ have been developed. Chromatographic separation of various plant extracts was done with RP‐HPLC and detected with positive ESI‐MS operated in information‐dependent acquisition (IDA) mode. Two methods for the determination of flavonoid identity and substitution pattern, both featuring IDA criteria, were used together with the HPLC equipment. A third method where the collision energy was ramped utilized direct infusion. With the developed strategies, it is possible to differentiate between many isobaric flavonoids. Various classes of flavonoids were found in all of the plant extracts, in the red onion extract 45 components were detected and for 29 of them the aglycone was characterized, while the substituents were tentatively identified for 31 of them. For the strawberry extract, those numbers were 66, 30 and 60, and for the cherry extract 99, 56 and 71. The great variety of flavonoids, several of them isobaric, found in each of the extracts highlights the need for reliable methods for flavonoid characterization. Methods capable of differentiating between most of the isobars analyzed have been developed. Copyright © 2014 John Wiley & Sons, Ltd.     

Study of antioxidant properties of flavonoids by voltammetry

Flavonoids are a large group of natural phenolic compounds contained in high concentrations in vegetables, fruits, etc. Antioxidant and redox properties of some flavonoids such as catechin, quercetin, dihydroquercetin, and rutin were investigated in this work. Optimal concentration and time of action of flavonoids were obtained. To determine the more effective range of antioxidant activity, mathematical models and the response surfaces of investigated flavonoids were determined using methods of experiment design. Oxidation potentials of the compounds were also obtained, E?=?0.3?÷?0.4?V. Moreover, the antioxidant activity of flavonoids depends on the redox properties and the structure of the flavonoids. The antioxidant activity of flavonoids, which is correlated to reversible potentials for this compound is good. Finally, the use of these substances as antioxidants with therapeutic effects has been recommended in human diet.     

Analysis of isoflavones and flavonoids in human urine by UHPLC

A rapid, ultra high-performance liquid chromatographic (UHPLC) method has been developed and validated for simultaneous identification and analysis of the isoflavones genistein, daidzein, glycitin, puerarin, and biochanin A, and the flavonoids (±)-catechin, (−)-epicatechin, rutin, hesperidin, neohesperidin, quercitrin, and hesperetin in human urine. Urine samples were incubated with β-glucuronidase/sulfatase. UHPLC was performed with a Hypersil Gold (50 × 2.1 mm, 1.9 μm) analytical column. Elution was with a gradient prepared from aqueous trifluoroacetic acid (0.05%) and acetonitrile. UV detection was performed at 254 and 280 nm. The calibration curves were indicative of good linearity (r ² ≥ 0.9992) in the range of interest for each analyte. LODs ranged between 15.4 and 107.0 ng mL⁻¹ and 3.9 and 20.4 ng mL⁻¹ for flavonoids and isoflavones, respectively. Intra-day and inter-day precision (C.V., %) was less than 3.9% and 3.8%, respectively, and accuracy was between 0.03% and 5.0%. Recovery was 70.35–96.58%. The method is very rapid, simple, and reliable, and suitable for pharmacokinetic analysis. It can be routinely used for simultaneous determination of these five isoflavones and seven flavonoids in human urine. The method can also be applied to studies after administration of pharmaceutical preparations containing isoflavones and flavonoids to humans.     

Determination of flavonoids in citrus juices by micro-HPLC-ESI/MS

Flavonoids are a large class of naturally occurring aromatic secondary plant metabolites. They constitute one of the most characteristic classes found in nature and more than 4000 flavonoids have been identified and divided into several subclasses. Flavonoids have several effects on human health, mainly related to their antioxidant activity. As a result of the increased interest in the biologically active compounds in food, many research studies have investigated the detection and quantification of antioxidants. Citrus plants are of great interest since they accumulate large amounts of flavonoids and fruit and juices of these species are consumed in large quantities. Despite the extensive research performed on Citrus flavonoids, many compounds still remain unidentified. In this work, flavonoids of five citrus juices: lemon, grapefruit, bergamot, orange, and mandarin were analyzed by microHPLC coupled on-line with an MS detector equipped with an ESI source operating in negative mode. The use of microcolumn HPLC greatly enhanced detection performance. In total, 20 flavonoids of two different subclasses (flavanone and flavone glycosides) were identified with the help of retention data and MS spectral information.     

Simultaneous determination of seven flavonoids in Potentilla multifida by HPLC

A reversed-phase high-performance liquid chromatographic method is described for the simultaneous determination of seven flavonoids in Potentilla multifida: hyperin, quercetin-3-O-beta-D-glucopyranoside, luteolin-7-O-beta-D-glucuronide, apigenin-7-O-beta-D-glucuronide, quercetin, tribuloside, and apigenin. The method involves the use of a Hypersil octadecylsilyl silica (ODS) analytical column (125A, 5 microm, 4.6 x 250 mm) at 25 degrees C with the mixture of acetonitrile and aqueous H(3)PO(4) as the mobile phase and detection at 254 nm. The recovery of the method is 95.4-104.8%, and linearity (r > 0.9998) is obtained for all the flavonoids. The results indicate that the flavonoid content of P. multifida varied significantly from locality to locality.