Capillary zone electrophoresis-based cytotoxicity analysis of Caco-2 cells

A capillary zone electrophoresis-based method to evaluate the cytotoxicity of substances to Caco-2 cells was established. The estimation of the injected cell number (500-5000) and the minor effect of injection condition on cytotoxicity determination were investigated. Caco-2 cells the best model of the intestinal absorptive epithelium, were treated with substances and then stained with Trypan Blue and fixed with paraformaldehyde. The treated Caco-2 cells were detected simultaneously at 590 nm and 214 nm, and the absorbance ratio of the two wavelengths (R(590/214)) can reflect simultaneously the loss of cell membrane integrity and the degradation/leak of intracellular components and indicate the cytotoxicity of substances. The cytotoxicity of the four substances sodium sulfite (Na(2)SO(3)), methyl mercury (MeHg), paclitaxel (PTX), and cadmium chloride (CdCl(2)) were determined and compared. There was no obvious cytotoxicity caused by 20 μM Na(2)SO(3) for 24 h treatment, and the toxicity of the other three toxicants was sequenced as: CdCl(2) > MeHg > PTX. The results are in good agreement with the references and the conventional Trypan Blue exclusion counting assay.     

Continuous intact cell detection and viability determination by CE with dual‐wavelength detection

We introduce here a method for continuous intact cell detection and viability determination of individual trypan blue stained cells by CE with ultraviolet–visible dual‐wavelength detection. To avoid cell aggregation or damage during electrophoresis, cells after staining were fixed with 4% formaldehyde and were continuously introduced into the capillary by EOF. The absorbance of a cell at 590 nm was used to determine its viability. An absorbance of two milli‐absorbance unit at 590 nm was the clear cut‐off point for living and dead Hela cells in our experiments. Good viability correlation between the conventional trypan blue staining assay and our established CE method (correlation coefficient, R²=0.9623) was demonstrated by analysis of cell mixtures with varying proportions of living and dead cells. The CE method was also used to analyze the cytotoxicity of methylmercury, and the results were in good agreement with the trypan blue staining assay and 3‐(4,5‐dimethyl‐2‐thiazyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide methods. Compared with the 3‐(4,5‐dimethyl‐2‐thiazyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide method, our established CE method can be easily automated to report cell viability based on the state of individual cells. Tedious manual cell counting and human error due to investigator bias can be avoided by using this method.     

Catalytic determination of traces of silver(I) using the oxidation of Janus Green with peroxodisulfate.

A method for sensitive and selective determination of silver based on the catalytic effect of silver(I) ion on the oxidation of Janus Green by peroxodisulfate is described. o-Phenanthroline is used as an activator. The rate of the decrease in absorbance of Janus Green (at 615 nm) is proportional to the concentration of silver in the range of 0.3-4.0 ng mL(-1) and 4.0-500.0 ng mL(-1). The theoretical limit of detection was 0.25 ng mL(-1). The method is free from most interferences. The method was applied to the determination of silver in plants (the uptake of silver by plants), in photographic solutions, lake water and several synthetic samples.     

Design,Synthesis and Biological Evaluations of a Novel Series of Enediynes Constituted with DNA Cleavage,Alkylating and DNA Binding Agents via Varies Spacers

Among the three series of complicated combinatory enediynes, 3a?d were more active than 2a?c and 4a?b , in which 3a?b showed equally inhibitory activity against the growth of Hepa59T/VGH, KB and Hela with mean IC₅₀ values lower than 10 μg/mL. Compounds 2a and 3b exhibited the most potent inhibitory activity against the growth of Hela cells for 4.39 μg/mL. Derivative 3d displayed the greatest ability against the growth of Hepa59T/VGH cell lines for 6.95 μg/mL, while 3a showed more cytotoxic to KB cells than other analogs. Conjugates 2a‐c presented better responses than 3a‐d for the DNA cleavage assay of supercoiled ΦX174.     

Synthesis, Crystal Structure and Antitumor Activity of 4-tert-Butyl-N-（2-fluorophenyl）-5- （1H-1,2,4-triazol-1-yl）-thiazol-2-amine

The title compound has been synthesized by the reaction of 1-bromo-3,3-dime- thyl-1- (1H-1,2,4-triazol-1-yl)butan-2-one with 1-(2-fluorophenyl)thiourea, and its crystal structure was determined by single-crystal X-ray diffraction. The crystal belongs to the orthorhombic system, space group Pbca with a=15.2568(6), b=12.1533(5), c=16.7307(7) , Z=8, V=3102.2(2)3, Mr=317.39, Dc=1.359 g/cm3, S=1.05, μ=0.223 mm-1, F(000)=1328, the final R=0.034 and wR=0.097 for 2590 observed reflections (I>2σ(I)). X-ray crystal structure presents the intramolecular N-H…N hydrogen bond, which plays an important role in stabilizing the crystal structure. In addition, the preliminary biological test on the title compound shows good antitumor activity, with IC50 of 0.122 μmol/mL against the Hela cell line.     

Determination of 5-fluorouracil and 5-fluoro-2'-deoxyuridine-5'-monophosphate in pancreatic cancer cell line and other biological materials using capillary electrophoresis

Capillary zone electrophoresis (CZE) was used for the rapid determination of 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) in pancreatic cancer cell line (PANC-1), culture medium, plasma and pancreatic tissue. The assay is based upon protein precipitation with acetonitrile followed by a 9-min CZE analysis of the supernatant in an uncoated fused-silica capillary employing a borate buffer and on-column absorbance detection at 265 nm. Using 50 microl of sample, 5-FU levels between 4.12 and 132 microg/ml (31.7-1000 microM) were found to provide linear calibration graphs. Intra-day and inter-day RSD values evaluated from peak height ratios (n=5) were <7.6 and <8.8%, respectively. Corresponding RSD values of detection times (n=7) were <1 and <1.5%, respectively. The limits of detection for 5-FU and FdUMP were 1.72 and 5.29 microg/ml, respectively. As application, the accumulation of 5-FU by PANC-1 cells over a 4-h time period was investigated. Having a culture medium concentration of 100 microg/ml, the 5-FU cell content was estimated to become equal to that of the surrounding medium (i.e., 100 microg/ml or 3.61 fmol per cell with a volume of 4.7 pl) within that time period. The sensitivity of the assay was sufficient for the determination of 5-FU in all cell samples. FdUMP, however, could not be detected in these samples. Furthermore, the data obtained in uncoated capillaries are compared to those measured in a fused-silica capillary whose inner surface was coated with linear polyacrylamide (about 10-fold reduction of electroosmosis). The latter capillary format was found to be useless for simultaneous analysis of 5-FU and FdUMP in pancreatic cells but could be potentially useful for analysis of these compounds in plasma.     

Development and validation of a fast monoclonal based disequilibrium enzyme-linked immunosorbent assay for the detection of triphenylmethane dyes and their metabolites in fish

Malachite Green (MG), Crystal Violet (CV) and Brilliant Green (BG) are antibacterial, antifungal and antiparasitic agents that have been used for treatment and prevention of diseases in fish. These dyes are metabolized into reduced leuco forms (LMG, LCV, LBG) that can be present in fish muscles for a long period. Due to the carcinogenic properties they are banned for use in fish for human consumption in many countries including the European Union and the United States. HPLC and LC-MS techniques are generally used for the detection of these compounds and their metabolites in fish. This study presents the development of a fast enzyme-linked immunosorbent assay (ELISA) method as an alternative for screening purposes. A first monoclonal cell line producing antibodies to MG was generated using a hybridoma technique. The antibody had good cross-reactivates with related chromatic forms of triphenylmethane dyes such as CV, BG, Methyl Green, Methyl Violet and Victoria Blue R. The monoclonal antibody (mAb) was used to develop a fast (20 min) disequilibrium ELISA screening method for the detection of triphenylmethanes in fish. By introducing an oxidation step with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) during sample extraction the assay was also used to detect the presence of the reduced metabolites of triphenylmethanes. The detection capability of the assay was 1 ng g(-1) for MG, LMG, CV, LCV and BG which was below the minimum required performance limit (MRPL) for the detection method of total MG (sum of MG and LMG) set by the Commission Decision 2004/25/EC (2 ng g(-1)). The mean recoveries for fish samples spiked at 0.5 MRPL and MRPL levels with MG and LMG were between 74.9 and 117.0% and inter- and intra-assay coefficients of variation between 4.7 and 25.7%. The validated method allows the analysis of a batch of 20 samples in two to three hours. Additionally, this procedure is substantially faster than other ELISA methods developed for MG/LMG thus far. The stable and efficient monoclonal cell line obtained is an unlimited source of sensitive and specific antibody to MG and other triphenylmethanes.     

Azadirachta indica leaves mediated green synthesized copper oxide nanoparticles induce apoptosis through activation of TNF-α and caspases signaling pathway against cancer cells

Green nanotechnology elucidates highly prioritized anticancer activity. We synthesized Copper oxide nanoparticles (CuONPs) using leaves of Azadirachta indica (A. indica) plants and studied the molecular mechanism of cancer cell apoptosis. After their synthesis, with the help of expository tools like Fourier transform infrared spectroscopy (FT-IR), Transmission electron microscopy (TEM), Dynamic light scattering (DLS) and surface zeta potential we confirmed the successful synthesis of CuONPs. Here, crystalline structure of green synthesized CuONPs of 36?±?8?nm size and spherical shape was able to kill MCF-7 and Hela cells, estimated by MTT assay. Successful internalization of Cu⁺² ions inside the cell was estimated by the atomic absorption study. Cellular uptake of Cu⁺² ions inflicted significant Reactive Oxygen Species (ROS) generation inside the cancer cells, thereby leading to DNA fragmentation as observed by DAPI staining. In in vivo model, CuONPs reduced the breast tumor volume in Balb/C mice and increased the mean survival time through the alteration of pro-inflammatory cytokines level. In case of both in vivo and in vitro models, CuONPs altered the pro-inflammatory cytokine level and pro-apoptotic protein expressions. In future, green synthesized CuONPs might be beneficial for its application as an anticancer drug in in vivo (mice model) and in vitro, though further study is needed on its toxicity.     

A new kinetic spectrophotometric method for the determination of major metabolite of heroin in biological samples

This study describes a simple,rapid and selective catalytic kinetic spectrophotometric method for the determination of 6- monoacetylmorphine（6-MAM） as major metabolite of heroin in biological samples.The method is based upon the catalytic effect of 6-MAM on the oxidation of Janus Green by bromate in acid media.The reaction was followed spectrophotometrically by measuring the decrease in absorbance of Janus Green at 618 nm.The dependence of sensitivity on the reaction variables was studied.Under optimum conditions,two linear calibration curves over the range 0.1-1.0μg mL^-1 and 1.0-34.0μg mL^-1 of 6- MAM were obtained.The detection limit was 1.2×10²μg mL^-1 of 6-MAM.The relative standard deviations for six replicate determinations of 0.8 and 5.0μg mL^-1 of 6-MAM were 1.4 and 1.1%respectively.The effect of various species commonly associated with heroin in real samples was also investigated.The proposed method was successfully applied in human urine and serum samples with satisfactory results.     

Meso-substituted porphyrin photosensitizers with enhanced near-infrared absorption: Synthesis,characterization and biological evaluation for photodynamic therapy

Porphyrin derivatives are widely explored and used in photodynamic therapy, for their marvelous therapeutic properties. In order to fill in the gaps of insufficient photosensitizers with near-infrared absorption, three porphyrin molecules, 5,10,15,20-tetrakis(3,4-bis(2-(-2-(2-hydroxyethoxy)ethoxy)ethoxy)benzyl)zinc porphyrin(P1), 5,15-bis(3,4-bis(2-(-2-(2-hydroxyethoxy)ethoxy)ethoxy)benzyl)-10,20-bis(2-(2-(2-(4-ethynylphenoxy)ethoxy)ethoxy)ethanol)zincporphyrin(P2),5,15-bis(3,4-bis(2-(-2-(2-hydroxyethoxy)ethoxy)ethoxy)benzyl)-10,20-N,N-dibutyl-4-ethynylaniline zinc porphyrin(P3), were designed and synthesized targeting for more efficient cancer treatment. Excellent photophysical properties were illustrated by UV–vis absorption and emission spectra with enhanced absorbance between 650 and 750?nm and fluorescence emission within 600–800?nm. Besides, with high ¹O₂ quantum yield, especially P2 (0.89), all porphyrins were further evaluated in vitro by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay against Hela cells and exhibited negligible dark toxicity and robust phototoxicity. Fluorescence confocal laser microscopy confirmed cellular uptake and diffusion of these porphyrins, therefore demonstrated their potential and promising applications in photodynamic therapy.     

Antioxidant and anticancer activity of fresh corm extract from Romulea tempskyana (Iridaceae)

The antioxidant and anticancer properties of a medicinal plant, Romulea tempskyana (R. tempskyana) (Iridaceae) were investigated. The fresh corm water extract of R. tempskyana significantly increased cell viability against H(2)O(2) cytotoxicity(.) The extract also showed high inhibition of the lipid peroxidation activity (78.20%), reducing power (IC(50) 64.99?μg?mL(-1)) activity and hydroxyl (IC(50) 38.66?μg?mL(-1)), superoxide (IC(50) 25.99?μg?ml(-1)) and DPPH (IC(50) 19.88?μg?mL(-1)) radical scavenging activity. On the other hand, treatment of the cells with higher extract concentration showed the anticancer activity inducing cytotoxicity. The extract significantly affected Hepatoma G2 and H1299 cell proliferation (IC(50); 103.79 and 88.15?μg?mL(-1)). The amount of MDA (2-fold and 2.5-fold) and activities of several cellular antioxidant enzymes, including Se-GSPx (30%, 15%), non-Se-GSH-Px (11%, 16%) and GST (17%, 23%) increased in Hepatoma G2 and H1299 cells treated with IC(50) concentrations of extract, respectively. These findings suggest that R. tempskyana extract exhibits antioxidant and carcinogenesis-reducing potential.     

Photosensitizers containing the 1,8-naphthyridyl moiety and their use in dye-sensitized solar cells

Using a Friedl?nder condensation approach, we prepared a series of 2-(pyrid-2'-yl)-1,8-naphthyridines containing a carboethoxy group appended at the 4- and 4'-positions. Complexation of these ligands with Ru(II) and NaNCS led to the complexes [Ru(L)2(NCS)2], and subsequent hydrolysis of the ester groups afforded the carboxylic acid dyes 13b-15b. The more delocalized and electronegative nature of the 1,8-naphthyridyl moiety lowers the energy of the ligand pi*-level and extends the absorption envelope of these complexes well into the red. The system lacking a 4-carboxypyridine moiety shows poor absorbance in the blue region of the spectrum. Solar cells involving thin films of anatase TiO2 impregnated with these dyes were prepared, and their photovoltaic performance was evaluated. The incident photon-to-current efficiencies in the region beyond 625 nm were considerably greater than those of the prototype N3 dye.     

新型N-甲基吲哚类衍生物的设计、合成及抗肿瘤活性研究

本文以吲哚骨架为起点,设计合成了5个含吲哚骨架的新型有机小分子(4a-4e),通过NMR,IR和MS对目标化合物进行了结构表征.采用MTS法测试了目标化合物对SMMC-7721,Hela,MCF-7和HepG2癌细胞的体外抗肿瘤活性.结果表明,部分化合物选择性地作用于一种细胞,显示出较强的抑制肿瘤细胞增殖的活性,值得进一步研究.     

Synthesis and anti-tumour activities of sulphated polysaccharide obtained from Momordica charantia

A native polysaccharide (MCP2) was extracted and isolated from Momordica charantia. Four sulphated derivatives of MCP2 were prepared by chlorosulphonic acid method. The structures of the sulphated derivatives were characterised by FT-IR spectra. Depending on the reaction conditions, the sulphated derivatives showed different degree of substitution (DS) ranging from 0.56 to 1.10, and different weight-average molecular mass (Mw) ranging from 7.2 to 9.3?KDa. It implied the efficient substitution of hydroxyl groups in the polysaccharides by sulphated groups with degradation. The effects of the sulphated derivatives on inhibiting the growth of HepG2 cells and Hela cells in?vitro were compared with taking non-modified MCP2 as control. The sulphated derivatives inhibited the growth of HepG2 cells and Hela cells in?vitro significantly, which indicated that sulphated modification could enhance the anti-tumour activity of MCP2.     

Synthesis of Novel Amide Derivatives with In Vitro Antiproliferative and Cytotoxic Activity

Some amide derivatives are highly valuable products with antiproliferative and cytotoxic bioactivity. In this paper, 12 novel amide compounds were synthesized correspondingly by boc‐protection glycine, deprotection, and condensation reactions. The antiproliferative and cytotoxic activity of these compounds was also evaluated by human cervical cancer (Hela) and hepatoma carcinoma (SK‐Hep‐1) cancer cell lines. The assay revealed that eight compounds ( 5a–d , 6a–d ) exhibited activity against Hela cancer cells. Four of them ( 5a–d ) also showed activity against SK‐Hep‐1 cancer cells. © 2012 Wiley Periodicals, Inc. Heteroatom Chem 24:9–17, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/hc.21057     

Total synthesis and anticancer activity studies of the stereoisomers of asperphenamate and patriscabratine

<正>All stereoisomers of asperphenamate 1a and patriscabratine 2a were achieved with a high yield,and total synthesis of 2a is firstly described here.The absolute configuration of patriscabratine was determined as(S,S).The compounds 1a-d and 2a-d have been tested by MTT assay in T47D,MDA-MB231,HL60,Hela and SGC-7901 cell lines in vitro.Among them,the(R,S) stereoisomer shows the strongest anticancer effects,while the(S,R) shows the weakest one.     

Copper (II)-Surfactant Complex and Its Nano Analog as Potential Antitumor Agents

The in vitro anticancer activity of copper cetyl trimethyl ammonium bromide (Cu-CTAB)-loaded cyclodextrin nanoparticles on Ehrlich ascites carcinoma (EAC), colon cancer cells (HCT 116), liver cancer cells (HepG-2), breast cancer cells (MCF-7), and cervix cancer cells (Hela) was investigated using MTT due assay. Cyclodextrin nanoparticles loaded with Cu-CTAB exerted in vitro anticancer activity against the previous human cancer cell lines comparable to the activity of free non-entrapped in nanoparticles macro-particle Cu-CTAB. The nano analog was synthesized by physical loading using grinding with ball mill. The ratio between Cu-CTAB and cyclodextrin oligosaccharide was 1 Cu-CTAB: 3 cyclodextrin. The particle size of the nano derivative was determined using the transmitted electron microscope (TEM).     

In vitro antitumor activity of triterpenes from Ceriops tagal

The embryo of Ceriops tagal was extracted with 95% ethanol at room temperature, and four triterpenes (1-4) were separated from this extract. For the first time these triterpenes were the separated from this plant. Compounds (1-4) were tested in vitro for antitumor activity against three cell lines (human liver cancer cell (H-7402), human B-lymphoblastoid cell (Raji), and human cervical carcinoma cell (Hela)). Compounds 1 and 3 were effective to inhibit cell proliferation and growth of H-7402 and Hela, the IC(50) of them on H-7402 were 14.42 microg mL(-1) and 9.97 microg mL(-1), and the IC(50) of them on Hela were 11.84 microg mL(-1) and 11.32 microg mL(-1). All compounds 1-4 were not effective to inhibit cell proliferation and growth of Raji. The effects of compound 4 on inhibiting proliferation and growth of these three cancer cells was also not obvious.     

Effect of nucleic acid binding dyes on DNA extraction,amplification, and STR typing

We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond? Nucleic Acid Dye, GelGreen?, GelRed?, RedSafe?, SYBR^® Green I, and EvaGreen? were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR^® Green I; 99.6% for RedSafe?; 99.4% for EvaGreen?; 52.7% for Diamond? Dye; 50.6% for GelRed?, and; could not be determined for GelGreen?. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t‐test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreen^TM at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreen? (1X), although with reduced amplification products. RedSafe? (1X), Diamond? Dye (1X), and SYBR^® Green I (1X) all exhibited varying degrees of locus drop‐out with GelRed? generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection.     

Iodine-catalyzed three-component one-pot synthesis of novel 1,8-dioxo-decahydroacridines derivatives bearing benzene sulfonamide moiety

An efficient synthetic method for 1,8-dioxo-decahydroacridines derivatives bearing the biologically active sulfonamide moiety is described. Aromatic aldehyde reacted with 5,5-dimethyl-1,3-cyclohexanedione and sulfanilamide, with molecular iodine as catalyst, to give 1,8-dioxo-decahydroacridines derivatives in high to excellent yield. The structures of these compounds were established on the basis of elemental (C, H and N) and spectral analysis (¹H NMR, ¹³C NMR, MS and FTIR). All the compounds were tested for their cytotoxic activity in vitro against three human tumor cell lines: human mammary cancer cells (MCF-7), human cervical carcinoma cells (Hela), and human lung cancer cells (A549) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Most of them showed moderate to potent cytotoxic activity against the tested cell lines. Among them, the most active compound 4e exhibited more efficient activity (10.92 μM) against MCF-7 cells than cisplatin (11.06 μM).