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- The novel thymidylate synthase inhibitor trifluorothymidine (TFT) and TRAIL synergistically eradicate non-small cell lung cancer cells. [JOURNAL ARTICLE]
- Cancer Chemother Pharmacol 2014 Apr 18.
TRAIL, a tumor selective anticancer agent, may be used for the treatment of non-small cell lung cancer (NSCLC). However, TRAIL resistance is frequently encountered. Here, the combined use of TRAIL with trifluorothymidine (TFT), a thymidylate synthase inhibitor, was examined for sensitizing NSCLC cells to TRAIL.Interactions between TRAIL and TFT were studied in NSCLC cells using growth inhibition and apoptosis assays. Western blotting and flow cytometry were used to investigate underlying mechanisms.The combined treatment of TFT and TRAIL showed synergistic cytotoxicity in A549, H292, H322 and H460 cells. For synergistic activity, the sequence of administration was important; TFT treatment followed by TRAIL exposure did not show sensitization. Combined TFT and TRAIL treatment for 24 h followed by 48 h of TFT alone was synergistic in all cell lines, with combination index values below 0.9. The treatments affected cell cycle progression, with TRAIL inducing a G1 arrest and TFT, a G2/M arrest. TFT activated Chk2 and reduced Cdc25c levels known to cause G2/M arrest. TRAIL-induced caspase-dependent apoptosis was enhanced by TFT, whereas TFT alone mainly induced caspase-independent death. TFT increased the expression of p53 and p21/WAF1, and p53 was involved in the increase of TRAIL-R2 surface expression. TFT also caused downregulation of cFLIP and XIAP and increased Bax expression.TFT enhances TRAIL-induced apoptosis in NSCLC cells by sensitizing the apoptotic machinery at different levels in the TRAIL pathway. Our findings suggest a possible therapeutic benefit of the combined use of TFT and TRAIL in NSCLC.
- Development of hollow/porous floating beads of metoprolol for pulsatile drug delivery. [JOURNAL ARTICLE]
- Eur J Drug Metab Pharmacokinet 2014 Apr 18.
The purpose of this work was to develop hollow calcium pectinate beads for floating pulsatile release of metoprolol tartrate intended for chronopharmacotherapy. Floating pulsatile concept was applied to increase the gastric residence of the dosage form having lag phase followed by a burst release. To overcome limitations of various approaches for imparting buoyancy, hollow/porous beads were prepared by simple process of acid-base reaction during ionotropic cross-linking using low methoxy pectin, xanthan gum, sodium carboxy methyl cellulose, guar gum, locust bean, gellan gum and calcium chloride as a cross-linking agent. Based on the preliminary studies optimized polymers were selected for formulation design with different polymers ratio concentrations. The obtained floating beads were studied for entrapment efficiency, buoyancy study, swelling index, surface morphology, in vitro release, stability studies and in vivo floating study. The floating beads obtained were porous, float up to 12-24 h. The radiological studies (X-rays) pointed out the capability of the system to release drug in lower parts of GIT after a programmed lag time for hypertension. The floating beads provided expected two-phase release pattern with initial lag time during floating in acidic medium followed by rapid pulse release in phosphate buffer. From the accelerated stability studies, it was observed that the formulations are quite stable. All formulations followed first-order release kinetics by diffusion mechanism. This approach suggested the use of hollow calcium pectinate microparticles as promising floating pulsatile drug delivery system for site- and time-specific release of drugs acting as per chronotherapy of diseases.
- Formation of elongated fascicle-inspired 3D tissues consisting of high-density, aligned cells using sacrificial outer molding. [JOURNAL ARTICLE]
- Lab Chip 2014 Apr 17.
The majority of muscles, nerves, and tendons are composed of fiber-like fascicle morphology. Each fascicle has a) elongated cells highly aligned with the length of the construct, b) a high volumetric cell density, and c) a high length-to-width ratio with a diameter small enough to facilitate perfusion. Fiber-like fascicles are important building blocks for forming tissues of various sizes and cross-sectional shapes, yet no effective technology is currently available for producing long and thin fascicle-like constructs with aligned, high-density cells. Here we present a method for molding cell-laden hydrogels that generate cylindrical tissue structures that are ~100 μm in diameter with an extremely high length to diameter ratio (>100 : 1). Using this method we have successfully created skeletal muscle tissue with a high volumetric density (~50%) and perfect cell alignment along the axis. A new molding technique, sacrificial outer molding, allows us to i) create a long and thin cylindrical cavity of the desired size in a sacrificial mold that is solid at a low temperature, ii) release gelling agents from the sacrificial mold material after the cell-laden hydrogel is injected into fiber cavities, iii) generate a uniform axial tension between anchor points at both ends that promotes cell alignment and maturation, and iv) perfuse the tissue effectively by exposing it to media after melting the sacrificial outer mold at 37 °C. The effects of key parameters and conditions, including initial cavity diameter, axial tension, and concentrations of the hydrogel and gelling agent upon tissue compaction, volumetric cell density, and cell alignment are presented.
- Construction of expression vectors for metabolic engineering of the vanillin-producing actinomycete Amycolatopsis sp. ATCC 39116. [JOURNAL ARTICLE]
- Appl Microbiol Biotechnol 2014 Apr 18.
Amycolatopsis sp. ATCC 39116 is able to synthesize the important flavoring agent vanillin from cheap natural substrates. The bacterium is therefore of great interest for the industry and used for the fermentative production of vanillin. In order to improve the production of natural vanillin with Amycolatopsis sp. ATCC 39116, the strain has been genetically engineered to optimize the metabolic flux towards the desired product. Extensive metabolic engineering was hitherto hampered, due to the lack of genetic tools like functional promoters and expression vectors. In this study, we report the establishment of a plasmid-based gene expression system for Amycolatopsis sp. ATCC 39116 that allows a further manipulation of the genotype. Four new Escherichia coli-Amycolatopsis shuttle vectors harboring different promoter elements were constructed, and the functionality of these regulatory elements was proven by the expression of the reporter gene gusA, encoding a β-glucuronidase. Glucuronidase activity was detected in all plasmid-harboring strains, and remarkable differences in the expression strength of the reporter gene depending on the used promoter were observed. The new expression vectors will promote the further genetic engineering of Amycolatopsis sp. ATCC 39116 to get insight into the metabolic network and to improve the strain for a more efficient industrial use.
- Anthocyans as tertiary chemopreventive agents in bladder cancer: anti-oxidant mechanisms and interaction with mitomycin C. [JOURNAL ARTICLE]
- Mutagenesis 2014 Apr 17.
Bladder cancer is associated with high rates of recurrence making tertiary chemoprevention an attractive intervention strategy. Anthocyanins have been shown to possess chemopreventive properties and are detectable in urine after oral ingestion, with higher concentrations achievable via intravesical administration alongside current chemotherapeutic regimens. Yet their apparent ability to protect against certain DNA damage may in turn interfere with cancer treatments. Our aim was therefore to determine the potential of anthocyanins as chemopreventive agents in bladder cancer, their mode of action and effects, both alone and in combination with mitomycin C (MMC). In this study we showed that mirtoselect, a standardised mixture of anthocyanins, possesses significant anti-proliferative activity, causing growth inhibition and apoptosis in bladder cancer cell lines. The anti-oxidative potential of mirtoselect was examined and revealed significantly fewer H2O2-induced DNA strand breaks, as well as oxidised DNA bases in pre-treated cells. In contrast, endogenous levels of oxidised DNA bases were unaltered. Investigations into the possible protective mechanisms associated with these anti-oxidant properties revealed that mirtoselect chelates metal ions. In mirtoselect/MMC combination studies, no adverse effects on measures of DNA damage were observed compared to treatment with MMC alone and there was evidence of enhanced cell death. Consistent with this, significantly more DNA crosslinks were formed in cells treated with the combination. These results show that mirtoselect exerts effects consistent with chemopreventive properties in bladder cancer cell lines and most importantly does so without adversely affecting the effects of drugs used in current treatment regimens. We also provide evidence that mirtoselect's anti-oxidative mechanism of action is via metal ion chelation. Overall these results suggest that mirtoselect could be an effective chemopreventive agent in bladder cancer and provides the necessary pre-clinical data for future in vivo animal studies and clinical trials.
- Pharmacological and Structure-Activity Relationship Evaluation of 4-aryl-1-Diphenylacetyl(thio)semicarbazides. [Journal Article]
- Molecules 2014; 19(4):4745-59.
This article describes the synthesis of six 4-aryl-(thio)semicarbazides (series a and b) linked with diphenylacetyl moiety along with their pharmacological evaluation on the central nervous system in mice and computational studies, including conformational analysis and electrostatic properties. All thiosemicarbazides (series b) were found to exhibit strong antinociceptive activity in the behavioural model. Among them, compound 1-diphenylacetyl-4-(4-methylphenyl)thiosemicarbazide 1b was found to be the most potent analgesic agent, whose activity is connected with the opioid system. For compounds from series a significant anti-serotonergic effect, especially for compound 1-diphenylacetyl-4-(4-methoxyphenyl)semicarbazide 2b was observed. The computational studies strongly support the obtained results.
- Protective Effects of Kaempferol on Lipopolysaccharide-Induced Mastitis in Mice. [JOURNAL ARTICLE]
- Inflammation 2014 Apr 18.
Kaempferol isolated from the root of Zingiberaceae plants galangal and other Chinese herbal medicines have been reported to have anti-inflammatory properties. However, the anti-inflammatory effects of kaempferol on lipopolysaccharide (LPS)-induced mastitis are unknown and their underlying molecular mechanisms remain to be explored. The aim of this study was to evaluate the effects of kaempferol on LPS-induced mouse mastitis. The mouse model of mastitis was induced by injection of LPS through the duct of mammary gland. Kaempferol was injected 1 h before and 12 h after induction of LPS intraperitoneally. The present results showed that kaempferol markedly reduced infiltration of neutrophilic granulocyte, activation of myeloperoxidase (MPO), expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in a dose-dependent manner, which were increased in LPS-induced mouse mastitis. Furthermore, kaempferol suppressed the phosphorylation of nuclear factor-κB (NF-κB) p65 subunit and the degradation of its inhibitor IκBα. All results suggest that anti-inflammatory effects of kaempferol against the LPS-induced mastitis possibly through inhibition of the NF-κB signaling pathway. Kaempferol may be a potential therapeutic agent for mastitis.
- Hypoxia enhances the antiglioma cytotoxicity of b10, a glycosylated derivative of betulinic Acid. [Journal Article]
- PLoS One 2014; 9(4):e94921.
B10 is a glycosylated derivative of betulinic acid with promising activity against glioma cells. Lysosomal cell death pathways appear to be essential for its cytotoxicity. We investigated the influence of hypoxia, nutrient deprivation and current standard therapies on B10 cytotoxicity. The human glioma cell lines LN-308 and LNT-229 were exposed to B10 alone or together with irradiation, temozolomide, nutrient deprivation or hypoxia. Cell growth and viability were evaluated by crystal violet staining, clonogenicity assays, propidium iodide uptake and LDH release assays. Cell death was examined using an inhibitor of lysosomal acidification (bafilomycin A1), a cathepsin inhibitor (CA074-Me) and a short-hairpin RNA targeting cathepsin B. Hypoxia substantially enhanced B10-induced cell death. This effect was sensitive to bafilomycin A1 and thus dependent on hypoxia-induced lysosomal acidification. Cathepsin B appeared to mediate cell death because either the inhibitor CA074-Me or cathepsin B gene silencing rescued glioma cells from B10 toxicity under hypoxia. B10 is a novel antitumor agent with substantially enhanced cytotoxicity under hypoxia conferred by increased lysosomal cell death pathway activation. Given the importance of hypoxia for therapy resistance, malignant progression, and as a result of antiangiogenic therapies, B10 might be a promising strategy for hypoxic tumors like malignant glioma.
- Transcriptomes and shRNA Suppressors in a TP53 Allele-specific Model of Early-onset Colon Cancer in African Americans. [JOURNAL ARTICLE]
- Mol Cancer Res 2014 Apr 17.
African Americans are disproportionately affected by early-onset, high-grade malignancies. A fraction of this cancer health disparity can be explained by genetic differences between individuals of African or European descent. Here the wild-type Pro/Pro genotype at the TP53Pro72Arg (P72R) polymorphism (SNP:rs1042522) is more frequent in African Americans with cancer than in African Americans without cancer (51% vs 37%), and is associated with a significant increase in the rates of cancer diagnosis in African Americans. To test the hypothesis that p53 allele-specific gene expression may contribute to African American cancer disparities, p53 hemizygous knockout variants were generated and characterized in the RKO colon carcinoma cell line, which is wild-type for p53 and heterozygous at theTP53Pro72Arg locus. Transcriptome profiling, using RNAseq, in response to the DNA-damaging agent etoposide revealed a large number of p53-regulated transcripts, but also a subset of transcripts that were TP53Pro72Arg allele specific. In addition, a shRNA-library suppressor screen for p53 allele-specific escape from p53-induced arrest was performed. Several novel RNAi suppressors of p53 were identified, one of which, PRDM1beta (BLIMP-1), was confirmed to be an Arg-specific transcript. PRDM1beta silences target genes by recruiting H3K9 trimethyl (H3K9me3) repressive chromatin marks, and is necessary for stem cell differentiation. These results reveal a novel model for African American cancer disparity, in which the TP53 codon 72 allele influences lifetime cancer risk by driving damaged cells to differentiation through an epigenetic mechanism involving gene silencing. Implications: TP53 P72R polymorphism significantly contributes to increased African American cancer disparity.
- A Draft Genome of the Honey Bee Trypanosomatid Parasite Crithidia mellificae. [Journal Article]
- PLoS One 2014; 9(4):e95057.
Since 2006, honey bee colonies in North America and Europe have experienced increased annual mortality. These losses correlate with increased pathogen incidence and abundance, though no single etiologic agent has been identified. Crithidia mellificae is a unicellular eukaryotic honey bee parasite that has been associated with colony losses in the USA and Belgium. C. mellificae is a member of the family Trypanosomatidae, which primarily includes other insect-infecting species (e.g., the bumble bee pathogen Crithidia bombi), as well as species that infect both invertebrate and vertebrate hosts including human pathogens (e.g.,Trypanosoma cruzi, T. brucei, and Leishmania spp.). To better characterize C. mellificae, we sequenced the genome and transcriptome of strain SF, which was isolated and cultured in 2010. The 32 megabase draft genome, presented herein, shares a high degree of conservation with the related species Leishmania major. We estimate that C. mellificae encodes over 8,300 genes, the majority of which are orthologs of genes encoded by L. major and other Leishmania or Trypanosoma species. Genes unique to C. mellificae, including those of possible bacterial origin, were annotated based on function and include genes putatively involved in carbohydrate metabolism. This draft genome will facilitate additional investigations of the impact of C. mellificae infection on honey bee health and provide insight into the evolution of this unique family.