Mechanisms of Acquired Drug Resistance to the HDAC6 Selective Inhibitor Ricolinostat Reveals Rational Drug-Drug Combination with Ibrutinib.

PubMed

Amengual, Jennifer E; Prabhu, Sathyen A; Lombardo, Maximilian; Zullo, Kelly; Johannet, Paul M; Gonzalez, Yulissa; Scotto, Luigi; Serrano, Xavier Jirau; Wei, Ying; Duong, Jimmy; Nandakumar, Renu; Cremers, Serge; Verma, Akanksha; Elemento, Olivier; O'Connor, Owen A

2017-06-15

Purpose: Pan-class I/II histone deacetylase (HDAC) inhibitors are effective treatments for select lymphomas. Isoform-selective HDAC inhibitors are emerging as potentially more targeted agents. ACY-1215 (ricolinostat) is a first-in-class selective HDAC6 inhibitor. To better understand the discrete function of HDAC6 and its role in lymphoma, we developed a lymphoma cell line resistant to ACY-1215. Experimental Design: The diffuse large B-cell lymphoma cell line OCI-Ly10 was exposed to increasing concentrations of ACY-1215 over an extended period of time, leading to the development of a resistant cell line. Gene expression profiling (GEP) was performed to investigate differentially expressed genes. Combination studies of ACY-1215 and ibrutinib were performed in cell lines, primary human lymphoma tissue, and a xenograft mouse model. Results: Systematic incremental increases in drug exposure led to the development of distinct resistant cell lines with IC 50 values 10- to 20-fold greater than that for parental lines. GEP revealed upregulation of MAPK10, HELIOS, HDAC9, and FYN, as well as downregulation of SH3BP5 and LCK. Gene-set enrichment analysis (GSEA) revealed modulation of the BTK pathway. Ibrutinib was found to be synergistic with ACY-1215 in cell lines as well as in 3 primary patient samples of lymphoma. In vivo confirmation of antitumor synergy was demonstrated with a xenograft of DLBCL. Conclusions: The development of this ACY-1215-resistant cell line has provided valuable insights into the mechanistic role of HDAC6 in lymphoma and offered a novel method to identify rational synergistic drug combinations. Translation of these findings to the clinic is underway. Clin Cancer Res; 23(12); 3084-96. ©2016 AACR . ©2016 American Association for Cancer Research.

Cytotoxic, Antiproliferative and Apoptotic Effects of Perillyl Alcohol and Its Biotransformation Metabolite on A549 and HepG2 Cancer Cell Lines.

PubMed

Oturanel, Ceren E; Kıran, İsmail; Özşen, Özge; Çiftçi, Gülşen A; Atlı, Özlem

2017-01-01

A monoterpene, perillyl alcohol, has attracted attention in medicinal chemistry since it exhibited chemo-preventive and therapeutic properties against a variety of cancers. In the present work, it was aimed to obtain derivatives of perillyl alcohol through microbial biotransformation and investigate their anticancer activities against A549 and HepG2 cancer cell lines. Biotransformation studies were carried out in a α-medium for 7 days at 25oC. XTT assay was performed to investigate the anticancer activities of perillyl alcohol and its biotransformation metabolite, dehydroperillic acid, against A549 and HepG2 cell lines and their selectivity using healthy cell line, NIH/3T3. Cell proliferation ELISA, BRDU (colorimetric) assay was used for measurement of proliferation in replicative cells in which DNA synthesis occurs. Flow cytometric analyses were also carried out for measuring apoptotic cell percentages, caspase 3 activation and mitochondrial membrane potential. Biotransformation of perillyl alcohol with Fusarium culmorum yielded dehydroperillic acid in a yield of 20.4 %. In in vitro anticancer studies, perillyl alcohol was found to exert cytotoxicity against HepG2 cell line with an IC50 value of 409.2 μg/mL. However, this effect was not found to be selective because of its higher IC50 (250 μg/mL) value against NIH/3T3 cell line. On the other hand, dehydroperillic acid was found to be effective and also selective against A549 cell line with an IC50 value of 125 μg/mL and a selectivity index (SI) value of 400. Apoptosis inducing effects of dehydroperillic acid was better in A549 cell line. Dehydroperillic acid may be a good candidate for therapy of lung adenocarcinoma and may show this anticancer activity by inducing apoptosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies.

PubMed

Noh, Soo Min; Shin, Seunghyeon; Lee, Gyun Min

2018-03-29

To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1 and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation between the specific mAb productivity and these three gene copies (R 2  ≤ 0.012). Taken together, GS-mediated gene amplification does not occur in a single round of selection at a MSX concentration up to 50 μM. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX.

O-naphthoquinone isolated from Capraria biflora L. induces selective cytotoxicity in tumor cell lines.

PubMed

de S Wisintainer, G G N; Scola, G; Moura, S; Lemos, T L G; Pessoa, C; de Moraes, M O; Souza, L G S; Roesch-Ely, M; Henriques, J A P

2015-12-21

Biflorin is an o-naphthoquinone isolated from the roots of the plant Capraria biflora L. (Scrophulariaceae). In this study, the cytotoxic effects of biflorin were verified, and late apoptosis was detected in various cancer cell lines by in situ analysis. The cytotoxicity was further evaluated exclusively for 48 h of treatment in different tumor and non-tumor cell lines (Hep-2, HeLa, HT-29, A-375, and A-549, and HEK-293, respectively). The results indicated that biflorin induced selective cytotoxicity in tumor cells. HeLa cells were more susceptible to biflorin, followed by HT-29, A-549, A-375, and Hep-2 at all concentrations (range 5-50 μg/mL), and the highest half-maximal inhibitory concentration IC50 (56.01 ± 1.17 μg/mL) was observed in HEK-293 cells. Late apoptotic/necrotic events, observed by in situ immunostaining with Annexin V, varied with each cell line; an increase in late apoptotic events was observed corresponding to the increase in biflorin dosage. Hep-2 cells showed a greater percentage of late apoptotic events among the tumor cell lines when treated with higher concentrations of biflorin (69.63 ± 2.28%). The non-tumor HEK-293 line showed greater resistance to late apoptotic events, as well as a lower level of cytotoxicity (77.69 ± 6.68%) than the tested tumor lines. The data presented indicate that biflorin showed an important, possibly selective, cytotoxicity against tumor cell lines, thereby revealing a promising novel substance with potential anticancer activity for tumor therapy.

Relationship between Salt Tolerance and Resistance to Polyethylene Glycol-Induced Water Stress in Cultured Citrus Cells 1

PubMed Central

Ben-Hayyim, Gozal

1987-01-01

Salt-tolerant selected cells of Shamouti orange (Citrus sinensis) and Sour orange (Citrus aurantium) grew considerably better than nonselected cells at any NaCl concentration tested up to 200 millimolar. Also, the growth response of each treatment was identical in the two species. However, the performance of cells of the two species under osmotic stress induced by polyethylene glycol (PEG), which is presumably a nonabsorbed osmoticum, was significantly different. The nonselected Shamouti cell lines were significantly more sensitive to osmotic stress than the selected cells. The salt adapted Shamouti cells were apparently also adapted to osmotic stress induced by PEG. In Sour orange, however, the selected lines had no advantage over the nonselected line in response to osmotic stress induced by PEG. This response was also similar quantitatively to the response of the selected salt-tolerant Shamouti cell line. It seems that the tolerance to salt in Shamouti, a partial salt excluder, involves an osmotic adaptation, whereas in Sour orange, a salt accumulator, such an adaptation apparently does not occur. PEG-induced osmotic stress causes an increase in the percent dry weight of salt-sensitive and salt-tolerant cells of both species. No such increase was found under salt stress. The size of control and stressed cells is not significantly different. PMID:16665715

Rapid high-throughput characterisation, classification and selection of recombinant mammalian cell line phenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling.

PubMed

Povey, Jane F; O'Malley, Christopher J; Root, Tracy; Martin, Elaine B; Montague, Gary A; Feary, Marc; Trim, Carol; Lang, Dietmar A; Alldread, Richard; Racher, Andrew J; Smales, C Mark

2014-08-20

Despite many advances in the generation of high producing recombinant mammalian cell lines over the last few decades, cell line selection and development is often slowed by the inability to predict a cell line's phenotypic characteristics (e.g. growth or recombinant protein productivity) at larger scale (large volume bioreactors) using data from early cell line construction at small culture scale. Here we describe the development of an intact cell MALDI-ToF mass spectrometry fingerprinting method for mammalian cells early in the cell line construction process whereby the resulting mass spectrometry data are used to predict the phenotype of mammalian cell lines at larger culture scale using a Partial Least Squares Discriminant Analysis (PLS-DA) model. Using MALDI-ToF mass spectrometry, a library of mass spectrometry fingerprints was generated for individual cell lines at the 96 deep well plate stage of cell line development. The growth and productivity of these cell lines were evaluated in a 10L bioreactor model of Lonza's large-scale (up to 20,000L) fed-batch cell culture processes. Using the mass spectrometry information at the 96 deep well plate stage and phenotype information at the 10L bioreactor scale a PLS-DA model was developed to predict the productivity of unknown cell lines at the 10L scale based upon their MALDI-ToF fingerprint at the 96 deep well plate scale. This approach provides the basis for the very early prediction of cell lines' performance in cGMP manufacturing-scale bioreactors and the foundation for methods and models for predicting other mammalian cell phenotypes from rapid, intact-cell mass spectrometry based measurements. Copyright © 2014 Elsevier B.V. All rights reserved.

Loss in MCL-1 function sensitizes non-Hodgkin's lymphoma cell lines to the BCL-2-selective inhibitor venetoclax (ABT-199)

PubMed Central

Phillips, D C; Xiao, Y; Lam, L T; Litvinovich, E; Roberts-Rapp, L; Souers, A J; Leverson, J D

2015-01-01

As a population, non-Hodgkin's lymphoma (NHL) cell lines positive for the t(14;18) translocation and/or possessing elevated BCL2 copy number (CN; BCL2High) are exquisitely sensitive to navitoclax or the B-cell lymphoma protein-2 (BCL-2)-selective inhibitor venetoclax. Despite this, some BCL2High cell lines remain resistant to either agent. Here we show that the MCL-1-specific inhibitor A-1210477 sensitizes these cell lines to navitoclax. Chemical segregation of this synergy with the BCL-2-selective inhibitor venetoclax or BCL-XL-selective inhibitor A-1155463 indicated that MCL-1 and BCL-2 are the two key anti-apoptotic targets for sensitization. Similarly, the CDK inhibitor flavopiridol downregulated MCL-1 expression and synergized with venetoclax in BCL2High NHL cell lines to a similar extent as A-1210477. A-1210477 also synergized with navitoclax in the majority of BCL2Low NHL cell lines. However, chemical segregation with venetoclax or A-1155463 revealed that synergy was driven by BCL-XL inhibition in this population. Collectively these data emphasize that BCL2 status is predictive of venetoclax potency in NHL not only as a single agent, but also in the adjuvant setting with anti-tumorigenic agents that inhibit MCL-1 function. These studies also potentially identify a patient population (BCL2Low) that could benefit from BCL-XL (navitoclax)-driven combination therapy. PMID:26565405

Loss in MCL-1 function sensitizes non-Hodgkin's lymphoma cell lines to the BCL-2-selective inhibitor venetoclax (ABT-199).

PubMed

Phillips, D C; Xiao, Y; Lam, L T; Litvinovich, E; Roberts-Rapp, L; Souers, A J; Leverson, J D

2015-11-13

As a population, non-Hodgkin's lymphoma (NHL) cell lines positive for the t(14;18) translocation and/or possessing elevated BCL2 copy number (CN; BCL2(High)) are exquisitely sensitive to navitoclax or the B-cell lymphoma protein-2 (BCL-2)-selective inhibitor venetoclax. Despite this, some BCL2(High) cell lines remain resistant to either agent. Here we show that the MCL-1-specific inhibitor A-1210477 sensitizes these cell lines to navitoclax. Chemical segregation of this synergy with the BCL-2-selective inhibitor venetoclax or BCL-XL-selective inhibitor A-1155463 indicated that MCL-1 and BCL-2 are the two key anti-apoptotic targets for sensitization. Similarly, the CDK inhibitor flavopiridol downregulated MCL-1 expression and synergized with venetoclax in BCL2(High) NHL cell lines to a similar extent as A-1210477. A-1210477 also synergized with navitoclax in the majority of BCL2(Low) NHL cell lines. However, chemical segregation with venetoclax or A-1155463 revealed that synergy was driven by BCL-XL inhibition in this population. Collectively these data emphasize that BCL2 status is predictive of venetoclax potency in NHL not only as a single agent, but also in the adjuvant setting with anti-tumorigenic agents that inhibit MCL-1 function. These studies also potentially identify a patient population (BCL2(Low)) that could benefit from BCL-XL (navitoclax)-driven combination therapy.

Isolation of parafluorophenylalanine-resistant mutants from HeLa cell cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yim, L.K.; Stuart, W.D.

This report describes a method to isolate temperature-conditional phenylalanine transport mutants from the transformed human cell line HeLa. Using ultraviolet light as a mutagenic agent and DL-parafluorophenylalanine (PFPA), a poisonous analogue of L-phenylalanine, as a selective agent, mutagenized cells were selected for survival in the presence of PFPA at a temperature of 39 degrees C. Survivors of the mutagenesis and selection procedures were removed from the culture dishes by trypsin and cloned at a temperature of 35 degrees C. Seven of these lines isolated demonstrated continued resistance to PFPA at 39 degrees C. These lines were tested for uptake ofmore » L-phenylalanine at an external concentration of 100 microM and for continued resistance to PFPA at two concentrations. Cells were tested at 35 and at 39 degrees C. The data were compared to those obtained for the parental HeLa cell line under identical conditions. The seven mutant cell lines demonstrated varying resistances to PFPA and varying levels of accumulation of L-phenylalanine when tested at 35 and 39 degrees C. Three mutant lines were additionally tested for L-phenylalanine tRNA charging levels and for transport of L-arginine. The lines had parental cell levels of tRNA charging and L-arginine transport which suggest that the induced genetic defect affects a specific L-phenylalanine transport system.« less

An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

PubMed

Muller, Ryan Y; Hammond, Ming C; Rio, Donald C; Lee, Yeon J

2015-12-01

The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

Novel tag-and-exchange (RMCE) strategies generate master cell clones with predictable and stable transgene expression properties.

PubMed

Qiao, Junhua; Oumard, André; Wegloehner, Wolfgang; Bode, Juergen

2009-07-24

Site-specific recombinases have revolutionized the systematic generation of transgenic cell lines and embryonic stem cells/animals and will ultimately also reveal their potential in the genetic modification of induced pluripotent stem cells. Introduced in 1994, our Flp recombinase-mediated cassette exchange strategy permits the exchange of a target cassette for a cassette with the gene of interest, introduced as a part of an exchange vector. The process is "clean" in the sense that it does not co-introduce prokaryotic vector parts; neither does it leave behind a selection marker. Stringent selection principles provide master cell lines permitting subsequent recombinase-mediated cassette exchange cycles in the absence of a drug selection and with a considerable efficiency (approximately 10%). Exemplified by Chinese hamster ovary cells, the strategy proves to be successful even for cell lines with an unstable genotype.

Selective cytotoxicity of transformed cells but not normal cells by a sialoglycopeptide growth regulator in the presence of tumor necrosis factor

NASA Technical Reports Server (NTRS)

Woods, K. M.; Fattaey, H.; Johnson, T. C.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

The tumor necrosis factor-alpha (TNF)-resistant, SV40-transformed, murine fibroblast cell lines, F5b and F5m, became sensitive to TNF-mediated cytolysis after treatment with a biologically active 18 kDa peptide fragment (SGP) derived from a 66-kDa parental cell surface sialoglycoprotein. Neither TNF nor the SGP alone exhibited cytotoxicity to the two SV40-transformed cell lines. However, Balb/c 3T3 cells, incubated with SGP alone or with SGP and TNF, were not killed. Therefore, SGP can selectively sensitize cells for TNF alpha-mediated cytotoxicity. This selective sensitization may be due to the previously documented ability of the SGP to selectively mediate cell cycle arrest.

Augmenting Trastuzumab Therapy Against Breast Cancer Through Selective Activation of NK Cells

DTIC Science & Technology

2013-10-01

selection and assessed for purity (>90% purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Breast cancer cell lines...at a ratio of 1:1. After 24 hours, NK cells were isolated by negative selection and assessed for purity (>90% purity as defined by CD3-CD56+ flow ... cytometry ) and activation (>50% expression of CD137). Chromium-labeled breast cancer cell lines including MCF7 (A), BT474M1 (B), HER18 (C), and SKBR3

Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations.

PubMed

Lin, Yao-Cheng; Boone, Morgane; Meuris, Leander; Lemmens, Irma; Van Roy, Nadine; Soete, Arne; Reumers, Joke; Moisse, Matthieu; Plaisance, Stéphane; Drmanac, Radoje; Chen, Jason; Speleman, Frank; Lambrechts, Diether; Van de Peer, Yves; Tavernier, Jan; Callewaert, Nico

2014-09-03

The HEK293 human cell lineage is widely used in cell biology and biotechnology. Here we use whole-genome resequencing of six 293 cell lines to study the dynamics of this aneuploid genome in response to the manipulations used to generate common 293 cell derivatives, such as transformation and stable clone generation (293T); suspension growth adaptation (293S); and cytotoxic lectin selection (293SG). Remarkably, we observe that copy number alteration detection could identify the genomic region that enabled cell survival under selective conditions (i.c. ricin selection). Furthermore, we present methods to detect human/vector genome breakpoints and a user-friendly visualization tool for the 293 genome data. We also establish that the genome structure composition is in steady state for most of these cell lines when standard cell culturing conditions are used. This resource enables novel and more informed studies with 293 cells, and we will distribute the sequenced cell lines to this effect.

Magnetic field design for selecting and aligning immunomagnetic labeled cells.

PubMed

Tibbe, Arjan G J; de Grooth, Bart G; Greve, Jan; Dolan, Gerald J; Rao, Chandra; Terstappen, Leon W M M

2002-03-01

Recently we introduced the CellTracks cell analysis system, in which samples are prepared based on a combination of immunomagnetic selection, separation, and alignment of cells along ferromagnetic lines. Here we describe the underlying magnetic principles and considerations made in the magnetic field design to achieve the best possible cell selection and alignment of magnetically labeled cells. Materials and Methods Computer simulations, in combination with experimental data, were used to optimize the design of the magnets and Ni lines to obtain the optimal magnetic configuration. A homogeneous cell distribution on the upper surface of the sample chamber was obtained with a magnet where the pole faces were tilted towards each other. The spatial distribution of magnetically aligned objects in between the Ni lines was dependent on the ratio of the diameter of the aligned object and the line spacing, which was tested with magnetically and fluorescently labeled 6 microm polystyrene beads. The best result was obtained when the line spacing was equal to or smaller than the diameter of the aligned object. The magnetic gradient of the designed permanent magnet extracts magnetically labeled cells from any cell suspension to a desired plane, providing a homogeneous cell distribution. In addition, it magnetizes ferro-magnetic Ni lines in this plane whose additional local gradient adds to the gradient of the permanent magnet. The resultant gradient aligns the magnetically labeled cells first brought to this plane. This combination makes it possible, in a single step, to extract and align cells on a surface from any cell suspension. Copyright 2002 Wiley-Liss, Inc.

Generating mammalian stable cell lines by electroporation.

PubMed

A Longo, Patti; Kavran, Jennifer M; Kim, Min-Sung; Leahy, Daniel J

2013-01-01

Expression of functional, recombinant mammalian proteins often requires expression in mammalian cells (see Single Cell Cloning of a Stable Mammalian Cell Line). If the expressed protein needs to be made frequently, it can be best to generate a stable cell line instead of performing repeated transient transfections into mammalian cells. Here, we describe a method to generate stable cell lines via electroporation followed by selection steps. This protocol will be limited to the CHO dhfr-Urlaub et al. (1983) and LEC1 cell lines, which in our experience perform the best with this method. Copyright © 2013 Elsevier Inc. All rights reserved.

A Double Selection Approach to Achieve Specific Expression of Toxin Genes for Ovarian Cancer Gene Therapy

DTIC Science & Technology

2007-11-01

cells ( gray fill), cells preincubated with PBS and infected with virus (solid line), and cells preincubated with recombinant knob protein and incubated...U118-hCAR-tailless cells (b). Gray line¼ cells alone, solid line¼ cells+Ad5Luc1-CK1, dashed line¼ cells+Ad5Luc1. Figure 5 Ad5Luc1-CK1CAR-independent...line, whereas U118MG-hCAR-tailless stably expresses the extracellular domain of human CAR. Cells were infected with Ad5Luc1 ( gray bar) and Ad5-r1 (black

RNA deep sequencing as a tool for selection of cell lines for systematic subcellular localization of all human proteins.

PubMed

Danielsson, Frida; Wiking, Mikaela; Mahdessian, Diana; Skogs, Marie; Ait Blal, Hammou; Hjelmare, Martin; Stadler, Charlotte; Uhlén, Mathias; Lundberg, Emma

2013-01-04

One of the major challenges of a chromosome-centric proteome project is to explore in a systematic manner the potential proteins identified from the chromosomal genome sequence, but not yet characterized on a protein level. Here, we describe the use of RNA deep sequencing to screen human cell lines for RNA profiles and to use this information to select cell lines suitable for characterization of the corresponding gene product. In this manner, the subcellular localization of proteins can be analyzed systematically using antibody-based confocal microscopy. We demonstrate the usefulness of selecting cell lines with high expression levels of RNA transcripts to increase the likelihood of high quality immunofluorescence staining and subsequent successful subcellular localization of the corresponding protein. The results show a path to combine transcriptomics with affinity proteomics to characterize the proteins in a gene- or chromosome-centric manner.

Gene transfer preferentially selects MHC class I positive tumour cells and enhances tumour immunogenicity.

PubMed

Hacker, Ulrich T; Schildhauer, Ines; Barroso, Margarita Céspedes; Kofler, David M; Gerner, Franz M; Mysliwietz, Josef; Buening, Hildegard; Hallek, Michael; King, Susan B S

2006-05-01

The modulated expression of MHC class I on tumour tissue is well documented. Although the effect of MHC class I expression on the tumorigenicity and immunogenicity of MHC class I negative tumour cell lines has been rigorously studied, less is known about the validity of gene transfer and selection in cell lines with a mixed MHC class I phenotype. To address this issue we identified a C26 cell subline that consists of distinct populations of MHC class I (H-2D/K) positive and negative cells. Transient transfection experiments using liposome-based transfer showed a lower transgene expression in MHC class I negative cells. In addition, MHC class I negative cells were more sensitive to antibiotic selection. This led to the generation of fully MHC class I positive cell lines. In contrast to C26 cells, all transfectants were rejected in vivo and induced protection against the parental tumour cells in rechallenge experiments. Tumour cell specificity of the immune response was demonstrated in in vitro cytokine secretion and cytotoxicity assays. Transfectants expressing CD40 ligand and hygromycin phosphotransferase were not more immunogenic than cells expressing hygromycin resistance alone. We suggest that the MHC class I positive phenotype of the C26 transfectants had a bearing on their immunogenicity, because selected MHC class I positive cells were more immunogenic than parental C26 cells and could induce specific anti-tumour immune responses. These data demonstrate that the generation of tumour cell transfectants can lead to the selection of subpopulations that show an altered phenotype compared to the parental cell line and display altered immunogenicity independent of selection marker genes or other immune modulatory genes. Our results show the importance of monitoring gene transfer in the whole tumour cell population, especially for the evaluation of in vivo therapies targeted to heterogeneous tumour cell populations.

Fibroblast Growth Factor Receptors Are Components of Autocrine Signaling Networks in Head and Neck Squamous Cell Carcinoma Cells

PubMed Central

Marshall, Marianne E.; Hinz, Trista K.; Kono, Scott A.; Singleton, Katherine R.; Bichon, Brady; Ware, Kathryn E.; Marek, Lindsay; Frederick, Barbara A.; Raben, David; Heasley, Lynn E.

2011-01-01

Purpose We previously reported that a fibroblast growth factor (FGF) receptor (FGFR) signaling pathway drives growth of lung cancer cell lines of squamous and large cell histologies. Herein, we explored FGFR dependency in cell lines derived from the tobacco-related malignancy, head and neck squamous cell carcinoma (HNSCC). Experimental Design FGF and FGFR mRNA and protein expression was assessed in nine HNSCC cell lines. Dependence on secreted FGF2 for cell growth was tested with FP-1039, an FGFR1-Fc fusion protein. FGFR and EGFR-dependence was defined by sensitivity to multiple inhibitors selective for FGFRs or EGFR. Results FGF2 was expressed in eight of the nine HNSCC cell lines examined. Also, FGFR2 and FGFR3 were frequently expressed while only two lines expressed FGFR1. FP-1039 inhibited growth of HNSCC cell lines expressing FGF2, identifying FGF2 as an autocrine growth factor. FGFR inhibitors selectively reduced in vitro growth and ERK signaling in three HNSCC cell lines while three distinct lines exhibited responsiveness to both EGFR and FGFR inhibitors. Combinations of these drugs yielded additive growth inhibition. Finally, three cell lines were highly sensitive to EGFR TKIs with no contribution from FGFR pathways. Conclusions FGFR signaling was dominant or co-dominant with EGFR in six HNSCC lines while three lines exhibited little or no role for FGFRs and were highly EGFR-dependent. Thus, the HNSCC cell lines can be divided into subsets defined by sensitivity to EGFR and FGFR-specific TKIs. FGFR inhibitors may represent novel therapeutics to deploy alone or in combination with EGFR inhibitors in HNSCC. PMID:21673064

Careful Selection of Reference Genes Is Required for Reliable Performance of RT-qPCR in Human Normal and Cancer Cell Lines

PubMed Central

Jacob, Francis; Guertler, Rea; Naim, Stephanie; Nixdorf, Sheri; Fedier, André; Hacker, Neville F.; Heinzelmann-Schwarz, Viola

2013-01-01

Reverse Transcription - quantitative Polymerase Chain Reaction (RT-qPCR) is a standard technique in most laboratories. The selection of reference genes is essential for data normalization and the selection of suitable reference genes remains critical. Our aim was to 1) review the literature since implementation of the MIQE guidelines in order to identify the degree of acceptance; 2) compare various algorithms in their expression stability; 3) identify a set of suitable and most reliable reference genes for a variety of human cancer cell lines. A PubMed database review was performed and publications since 2009 were selected. Twelve putative reference genes were profiled in normal and various cancer cell lines (n = 25) using 2-step RT-qPCR. Investigated reference genes were ranked according to their expression stability by five algorithms (geNorm, Normfinder, BestKeeper, comparative ΔCt, and RefFinder). Our review revealed 37 publications, with two thirds patient samples and one third cell lines. qPCR efficiency was given in 68.4% of all publications, but only 28.9% of all studies provided RNA/cDNA amount and standard curves. GeNorm and Normfinder algorithms were used in 60.5% in combination. In our selection of 25 cancer cell lines, we identified HSPCB, RRN18S, and RPS13 as the most stable expressed reference genes. In the subset of ovarian cancer cell lines, the reference genes were PPIA, RPS13 and SDHA, clearly demonstrating the necessity to select genes depending on the research focus. Moreover, a cohort of at least three suitable reference genes needs to be established in advance to the experiments, according to the guidelines. For establishing a set of reference genes for gene normalization we recommend the use of ideally three reference genes selected by at least three stability algorithms. The unfortunate lack of compliance to the MIQE guidelines reflects that these need to be further established in the research community. PMID:23554992

Selective Targeting of Cancer Stem Cells by 2-Aminodihydroquinoline Analogs.

PubMed

Park, Heejoo; Yu, Yeongji; Kim, Hyejin; Lee, Eun; Lee, Hani; Jeon, Raok; Kim, Woo-Young

2017-04-01

Many aminodihydroquinoline compounds have been studied to determine their cytotoxicity to cancer cells. However, anti-cancer stem cells (CSCs) activity of aminodihydroquinoline has not been tested in spite that CSC is believed to do an important roles in chemotherapy resistance and recurrence. The CSC selective targeting activities of 10 recently synthesized 2-aminodihydroquinoline analogs were examined on CSCs and bulk culture of a glioblastoma cell line. A diethylaminopropyl substituted aminodihydroquinoline, 5h, showed a strong anti-CSC effect and general cytotoxicity. However, a benzyl substituted aminodihydroquinoline, 5i, displayed the most effective anti-CSC effect, with no or small significant cytotoxic effect in bulk culture conditions. While 5h temporarily enhanced CSC marker-positive cells and eventually suppressed the CSC population, which is similar to other cytotoxic anticancer reagents reported, 5i selectively eliminated CSC marker-positive cells based on fluorescence activated cell sorter (FACS) analysis. 5h also temporarily activated some genes associated with signaling required for CSC, while 5i selectively suppressed these genes supporting that the differential effects are resulted from different molecular responses. In addition, the selective CSC effect is also found against a colon cancer cell line. Collectively, we suggest that these two novel aminodihydroquinoline compounds possess novel anti-CSC effects in colon and brain tumor derived cell lines probably through independent pathways.

Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

PubMed

Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

2016-01-01

The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling.

Cinnamides as selective small-molecule inhibitors of a cellular model of breast cancer stem cells.

PubMed

Germain, Andrew R; Carmody, Leigh C; Nag, Partha P; Morgan, Barbara; Verplank, Lynn; Fernandez, Cristina; Donckele, Etienne; Feng, Yuxiong; Perez, Jose R; Dandapani, Sivaraman; Palmer, Michelle; Lander, Eric S; Gupta, Piyush B; Schreiber, Stuart L; Munoz, Benito

2013-03-15

A high-throughput screen (HTS) was conducted against stably propagated cancer stem cell (CSC)-enriched populations using a library of 300,718 compounds from the National Institutes of Health (NIH) Molecular Libraries Small Molecule Repository (MLSMR). A cinnamide analog displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control cell line (HMLE_sh_eGFP). Herein, we report structure-activity relationships of this class of cinnamides for selective lethality towards CSC-enriched populations. Copyright © 2013. Published by Elsevier Ltd.

Myricetin inhibits proliferation of cisplatin-resistant cancer cells through a p53-dependent apoptotic pathway

PubMed Central

HUANG, HAIZHI; CHEN, ALLEN Y.; YE, XINGQIAN; LI, BINGYUN; ROJANASAKUL, YON; RANKIN, GARY O.; CHEN, YI CHARLIE

2015-01-01

Cisplatin is a commonly used drug for cancer treatment by crosslinking DNA, leading to apoptosis of cancer cells, resistance to cisplatin treatment often occurs, leading to relapse. Therefore, there is a need for the development of more effective treatment strategies that can overcome chemoresistance. Myricetin is a flavonoid from fruits and vegetables, showing anticancer activity in various cancer cells. In this study, we found myricetin exhibited greater cytotoxicity than cisplatin in two cisplatin-resistant ovarian cancer cell lines, OVCAR-3 and A2780/CP70, and it was less cytotoxic to the normal ovarian cell line IOSE-364. Myricetin selectively induced apoptosis in both cisplatin-resistant cancer cell lines, but did not induce apoptosis in the normal ovarian cell line. It induced both Bcl-2 family-dependent intrinsic and DR5 dependent extrinsic apoptosis in OVCAR-3 cells. P53, a multifunctional tumor suppressor, regulated apoptosis in OVCAR-3 cells through a Bcl-2 family protein-dependent pathway. Myricetin did not induce cell cycle arrest in either ovarian cancer cell line. Because of its potency and selectivity against cisplatin-resistant cancer cells, myricetin could potentially be used to overcome cancer chemoresistance against platinum-based therapy. PMID:26315556

Establishment and characterization of Macaca fascicularis lymphoblastoid cell lines.

PubMed

Manning, C H; Heise, E R

1992-01-01

A panel of cynomolgus macaque lymphoblastoid cell lines (LCL) was established by transforming peripheral blood mononuclear cells (PBMC) with Herpesvirus papio (HVP), and selected lines were examined by flow cytometry. Results indicate that HVP-transformed macaque LCL are phenotypically heterogeneous and resemble human Epstein-Barr virus (EBV)-transformed LCL in the abundant expression of major histocompatibility complex (MHC) class I and class II molecules. At least some lines are of B cell origin.

Selective tumor cell targeting by the disaccharide moiety of bleomycin.

PubMed

Yu, Zhiqiang; Schmaltz, Ryan M; Bozeman, Trevor C; Paul, Rakesh; Rishel, Michael J; Tsosie, Krystal S; Hecht, Sidney M

2013-02-27

In a recent study, the well-documented tumor targeting properties of the antitumor agent bleomycin (BLM) were studied in cell culture using microbubbles that had been derivatized with multiple copies of BLM. It was shown that BLM selectively targeted MCF-7 human breast carcinoma cells but not the "normal" breast cell line MCF-10A. Furthermore, it was found that the BLM analogue deglycobleomycin, which lacks the disaccharide moiety of BLM, did not target either cell line, indicating that the BLM disaccharide moiety is necessary for tumor selectivity. Not resolved in the earlier study were the issues of whether the BLM disaccharide moiety alone is sufficient for tumor cell targeting and the possible cellular uptake of the disaccharide. In the present study, we conjugated BLM, deglycoBLM, and BLM disaccharide to the cyanine dye Cy5**. It was found that the BLM and BLM disaccharide conjugates, but not the deglycoBLM conjugate, bound selectively to MCF-7 cells and were internalized. The same was also true for the prostate cancer cell line DU-145 (but not for normal PZ-HPV-7 prostate cells) and for the pancreatic cancer cell line BxPC-3 (but not for normal SVR A221a pancreas cells). The targeting efficiency of the disaccharide was only slightly less than that of BLM in MCF-7 and DU-145 cells and comparable to that of BLM in BxPC-3 cells. These results establish that the BLM disaccharide is both necessary and sufficient for tumor cell targeting, a finding with obvious implications for the design of novel tumor imaging and therapeutic agents.

Responses to the Selective Bruton's Tyrosine Kinase (BTK) Inhibitor Tirabrutinib (ONO/GS-4059) in Diffuse Large B-cell Lymphoma Cell Lines.

PubMed

Kozaki, Ryohei; Vogler, Meike; Walter, Harriet S; Jayne, Sandrine; Dinsdale, David; Siebert, Reiner; Dyer, Martin J S; Yoshizawa, Toshio

2018-04-23

Bruton's tyrosine kinase (BTK) is a key regulator of the B-cell receptor signaling pathway, and aberrant B-cell receptor (BCR) signaling has been implicated in the survival of malignant B-cells. However, responses of the diffuse large B-cell lymphoma (DLBCL) to inhibitors of BTK (BTKi) are infrequent, highlighting the need to identify mechanisms of resistance to BTKi as well as predictive biomarkers. We investigated the response to the selective BTKi, tirabrutinib, in a panel of 64 hematopoietic cell lines. Notably, only six cell lines were found to be sensitive. Although activated B-cell type DLBCL cells were most sensitive amongst all cell types studied, sensitivity to BTKi did not correlate with the presence of activating mutations in the BCR pathway. To improve efficacy of tirabrutinib, we investigated combination strategies with 43 drugs inhibiting 34 targets in six DLBCL cell lines. Based on the results, an activated B-cell-like (ABC)-DLBCL cell line, TMD8, was the most sensitive cell line to those combinations, as well as tirabrutinib monotherapy. Furthermore, tirabrutinib in combination with idelalisib, palbociclib, or trametinib was more effective in TMD8 with acquired resistance to tirabrutinib than in the parental cells. These targeted agents might be usefully combined with tirabrutinib in the treatment of ABC-DLBCL.

Responses to the Selective Bruton’s Tyrosine Kinase (BTK) Inhibitor Tirabrutinib (ONO/GS-4059) in Diffuse Large B-cell Lymphoma Cell Lines

PubMed Central

Vogler, Meike; Jayne, Sandrine; Dinsdale, David; Siebert, Reiner

2018-01-01

Bruton’s tyrosine kinase (BTK) is a key regulator of the B-cell receptor signaling pathway, and aberrant B-cell receptor (BCR) signaling has been implicated in the survival of malignant B-cells. However, responses of the diffuse large B-cell lymphoma (DLBCL) to inhibitors of BTK (BTKi) are infrequent, highlighting the need to identify mechanisms of resistance to BTKi as well as predictive biomarkers. We investigated the response to the selective BTKi, tirabrutinib, in a panel of 64 hematopoietic cell lines. Notably, only six cell lines were found to be sensitive. Although activated B-cell type DLBCL cells were most sensitive amongst all cell types studied, sensitivity to BTKi did not correlate with the presence of activating mutations in the BCR pathway. To improve efficacy of tirabrutinib, we investigated combination strategies with 43 drugs inhibiting 34 targets in six DLBCL cell lines. Based on the results, an activated B-cell-like (ABC)-DLBCL cell line, TMD8, was the most sensitive cell line to those combinations, as well as tirabrutinib monotherapy. Furthermore, tirabrutinib in combination with idelalisib, palbociclib, or trametinib was more effective in TMD8 with acquired resistance to tirabrutinib than in the parental cells. These targeted agents might be usefully combined with tirabrutinib in the treatment of ABC-DLBCL. PMID:29690649

Analysis of renal cancer cell lines from two major resources enables genomics-guided cell line selection

NASA Astrophysics Data System (ADS)

Sinha, Rileen; Winer, Andrew G.; Chevinsky, Michael; Jakubowski, Christopher; Chen, Ying-Bei; Dong, Yiyu; Tickoo, Satish K.; Reuter, Victor E.; Russo, Paul; Coleman, Jonathan A.; Sander, Chris; Hsieh, James J.; Hakimi, A. Ari

2017-05-01

The utility of cancer cell lines is affected by the similarity to endogenous tumour cells. Here we compare genomic data from 65 kidney-derived cell lines from the Cancer Cell Line Encyclopedia and the COSMIC Cell Lines Project to three renal cancer subtypes from The Cancer Genome Atlas: clear cell renal cell carcinoma (ccRCC, also known as kidney renal clear cell carcinoma), papillary (pRCC, also known as kidney papillary) and chromophobe (chRCC, also known as kidney chromophobe) renal cell carcinoma. Clustering copy number alterations shows that most cell lines resemble ccRCC, a few (including some often used as models of ccRCC) resemble pRCC, and none resemble chRCC. Human ccRCC tumours clustering with cell lines display clinical and genomic features of more aggressive disease, suggesting that cell lines best represent aggressive tumours. We stratify mutations and copy number alterations for important kidney cancer genes by the consistency between databases, and classify cell lines into established gene expression-based indolent and aggressive subtypes. Our results could aid investigators in analysing appropriate renal cancer cell lines.

A chemical screen in diverse breast cancer cell lines reveals genetic enhancers and suppressors of sensitivity to PI3K isotype-selective inhibition

PubMed Central

Torbett, Neil E; Luna, Antonio; Knight, Zachary A.; Houk, Andrew; Moasser, Mark; Weiss, William; Shokat, Kevan M.; Stokoe, David

2011-01-01

Synopsis The Phosphoinositide-3-kinase (PI3K) pathway regulates cell proliferation, survival and migration and is consequently of great interest for targeted cancer therapy. Using a panel of small molecule PI3K isoform-selective inhibitors in a diverse set of breast cancer cell lines, we demonstrate that the biochemical and biological responses were highly variable and dependent on the genetic alterations present. p110α inhibitors were generally effective in inhibiting the phosphorylation of Akt and S6, two downstream components of PI3K signaling, in most cell lines examined. In contrast, 110β selective inhibitors only reduced Akt phosphorylation in PTEN mutant cell lines, and was associated with a lesser decrease in S6 phosphorylation. PI3K inhibitors reduced cell viability by causing a cell cycle arrest in the G1 phase of the cell cycle, with multi-targeted inhibitors causing the most potent effects. Cells expressing mutant Ras were resistant to the cell cycle effects of PI3K inhibition, which could be reversed using inhibitors of Ras signaling pathways. Taken together our data indicates that these compounds, alone or in suitable combinations, may be useful as breast cancer therapeutics, when used in appropriate genetic contexts. PMID:18498248

Gemcitabine: Selective cytotoxicity, induction of inflammation and effects on urothelial function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Farr, Stefanie E; Chess-Williams, Russ; McDermott,

Intravesical gemcitabine has recently been introduced for the treatment of superficial bladder cancer and has a favourable efficacy and toxicity profile in comparison to mitomycin c (MMC), the most commonly used chemotherapeutic agent. The aim of this study was to assess the cytotoxic potency of gemcitabine in comparison to MMC in urothelial cell lines derived from non-malignant (UROtsa) and malignant (RT4 and T24) tissues to assess selectivity. Cells were treated with gemcitabine or mitomycin C at concentrations up to the clinical doses for 1 or 2 h respectively (clinical duration). Treatment combined with hyperthermia was also examined. Cell viability, ROSmore » formation, urothelial function (ATP, acetylcholine and PGE2 release) and secretion of inflammatory cytokines were assessed. Gemcitabine displayed a high cytotoxic selectivity for the two malignant cell lines (RT4, T24) compared to the non-malignant urothelial cells (UROtsa, proliferative and non-proliferative). In contrast, the cytotoxic effects of MMC were non-selective with equivalent potency in each of the cell lines. The cytotoxic effect of gemcitabine in the malignant cell lines was associated with an elevation in free radical formation and was significantly decreased in the presence of an equilibrative nucleoside transporter inhibitor. Transient changes in urothelial ATP and PGE{sub 2} release were observed, with significant increase in release of interleukin-6, interleukin-8 and interleukin-1β from urothelial cells treated with gemcitabine. The selectivity of gemcitabine for malignant urothelial cells may account for the less frequent adverse urological effects with comparison to other commonly used chemotherapeutic agents. - Highlights: • Intravesical gemcitabine has recently been introduced to treat bladder cancer. • Gemcitabine is selectively toxic for malignant urothelial cells. • Urothelial ATP, PGE{sub 2} and inflammatory cytokines were altered by gemcitabine. • Selectivity of gemcitabine may account for less frequent urological side effects.« less

Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines.

PubMed Central

Cheng, J; Haas, M

1990-01-01

Human T-cell leukemia and T-cell acute lymphoblastic leukemia cell lines were studied for alterations in the p53 tumor suppressor gene. Southern blot analysis of 10 leukemic T-cell lines revealed no gross genomic deletions or rearrangements. Reverse transcription-polymerase chain reaction analysis of p53 mRNA indicated that all 10 lines produced p53 mRNA of normal size. By direct sequencing of polymerase chain reaction-amplified cDNA, we detected 11 missense and nonsense point mutations in 5 of the 10 leukemic T-cell lines studied. The mutations are primarily located in the evolutionarily highly conserved regions of the p53 gene. One of the five cell lines in which a mutation was detected possesses a homozygous point mutation in both p53 alleles, while the other four cell lines harbor from two to four different point mutations. An allelic study of two of the lines (CEM, A3/Kawa) shows that the two missense mutations found in each line are located on separate alleles, thus both alleles of the p53 gene may have been functionally inactivated by two different point mutations. Since cultured leukemic T-cell lines represent a late, fully tumorigenic stage of leukemic T cells, mutation of both (or more) alleles of the p53 gene may reflect the selection of cells possessing an increasingly tumorigenic phenotype, whether the selection took place in vivo or in vitro. Previously, we have shown that the HSB-2 T-cell acute lymphoblastic leukemia cell line had lost both alleles of the retinoblastoma tumor suppressor gene. Taken together, our data show that at least 6 of 10 leukemic T-cell lines examined may have lost the normal function of a known tumor suppressor gene, suggesting that this class of genes serves a critical role in the generation of fully tumorigenic leukemic T cells. Images PMID:2144611

Beating the odds: The poisson distribution of all input cells during limiting dilution grossly underestimates whether a cell line is clonally-derived or not.

PubMed

Zhou, Yizhou; Shaw, David; Lam, Cynthia; Tsukuda, Joni; Yim, Mandy; Tang, Danming; Louie, Salina; Laird, Michael W; Snedecor, Brad; Misaghi, Shahram

2017-09-23

Establishing that a cell line was derived from a single cell progenitor and defined as clonally-derived for the production of clinical and commercial therapeutic protein drugs has been the subject of increased emphasis in cell line development (CLD). Several regulatory agencies have expressed that the prospective probability of clonality for CHO cell lines is assumed to follow the Poisson distribution based on the input cell count. The probability of obtaining monoclonal progenitors based on the Poisson distribution of all cells suggests that one round of limiting dilution may not be sufficient to assure the resulting cell lines are clonally-derived. We experimentally analyzed clonal derivatives originating from single cell cloning (SCC) via one round of limiting dilution, following our standard legacy cell line development practice. Two cell populations with stably integrated DNA spacers were mixed and subjected to SCC via limiting dilution. Cells were cultured in the presence of selection agent, screened, and ranked based on product titer. Post-SCC, the growing cell lines were screened by PCR analysis for the presence of identifying spacers. We observed that the percentage of nonclonal populations was below 9%, which is considerably lower than the determined probability based on the Poisson distribution of all cells. These results were further confirmed using fluorescence imaging of clonal derivatives originating from SCC via limiting dilution of mixed cell populations expressing GFP or RFP. Our results demonstrate that in the presence of selection agent, the Poisson distribution of all cells clearly underestimates the probability of obtaining clonally-derived cell lines. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

Development of a recombinant human ovarian (BG1) cell line containing estrogen receptor α and β for improved detection of estrogenic/antiestrogenic chemicals.

PubMed

Brennan, Jennifer C; Bassal, Arzoo; He, Guochun; Denison, Michael S

2016-01-01

Estrogenic endocrine-disrupting chemicals are found in environmental and biological samples, commercial and consumer products, food, and numerous other sources. Given their ubiquitous nature and potential for adverse effects, a critical need exists for rapidly detecting these chemicals. The authors developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the US Environmental Protection Agency (USEPA) and Organisation for Economic Co-operation and Development (OECD) as a bioanalytical method to detect estrogen receptor (ER) agonists/antagonists. Unfortunately, these cells appear to contain only 1 of the 2 known ER isoforms, ERα but not ERβ, and the differential ligand selectivity of these ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. To improve the estrogen screening bioassay, BG1Luc4E2 cells were stably transfected with an ERβ expression plasmid and positive clones identified using ERβ-selective ligands (genistein and Br-ERβ-041). A highly responsive clone (BG1LucERβc9) was identified that exhibited greater sensitivity and responsiveness to ERβ-selective ligands than BG1Luc4E2 cells, and quantitative reverse-transcription polymerase chain reaction confirmed the presence of ERβ expression in these cells. Screening of pesticides and industrial chemicals identified chemicals that preferentially stimulated ERβ-dependent reporter gene expression. Together, these results not only demonstrate the utility of this dual-ER recombinant cell line for detecting a broader range of estrogenic chemicals than the current BG1Luc4E2 cell line, but screening with both cell lines allows identification of ERα- and ERβ-selective chemicals. © 2015 SETAC.

Development of a recombinant human ovarian (BG1) cell line containing estrogen receptor α and β for improved detection of estrogenic/antiestrogenic chemicals

PubMed Central

Brennan, Jennifer C.; Bassal, Arzoo; He, Guochun; Denison, Michael S.

2016-01-01

Estrogenic endocrine disrupting chemicals are found in environmental and biological samples, commercial and consumer products, food, and numerous other sources. Given their ubiquitous nature and potential for adverse effects, there is a critical need for rapidly detecting these chemicals. We developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the USEPA and OECD as a bioanalytical method to detect estrogen receptor (ER) agonists/antagonists. Unfortunately, these cells appear to contain only one of the two known ER isoforms, ERα but not ERβ, and the differential ligand selectivity of these ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. To improve the estrogen screening bioassay, BG1Luc4E2 cells were stably transfected with an ERβ expression plasmid and positive clones identified using ERβ-selective ligands (genistein and Br-ERβ-041). A highly responsive clone (BG1LucERβc9) was identified that exhibited greater sensitivity and responsiveness to ERβ-selective ligands than BG1Luc4E2 cells and qRT-PCR confirmed the presence of ERβ expression in these cells. Screening of pesticides and industrial chemicals identified chemicals that preferentially stimulated ERβ-dependent reporter gene expression. Together, these results not only demonstrate the utility of this dual ER recombinant cell line for detecting a broader range of estrogenic chemicals than the current BG1Luc4E2 cell line, but screening with both cell lines allows identification of ERα and ERβ-selective chemicals. PMID:26139245

Discovery of tumor-specific irreversible inhibitors of stearoyl CoA desaturase | Office of Cancer Genomics

Cancer.gov

A hallmark of targeted cancer therapies is selective toxicity among cancer cell lines. We evaluated results from a viability screen of over 200,000 small molecules to identify two chemical series, oxalamides and benzothiazoles, that were selectively toxic at low nanomolar concentrations to the same 4 of 12 human lung cancer cell lines. Sensitive cell lines expressed cytochrome P450 (CYP) 4F11, which metabolized the compounds into irreversible inhibitors of stearoyl CoA desaturase (SCD). SCD is recognized as a promising biological target in cancer and metabolic disease.

Identification of a selective small molecule inhibitor of breast cancer stem cells.

PubMed

Germain, Andrew R; Carmody, Leigh C; Morgan, Barbara; Fernandez, Cristina; Forbeck, Erin; Lewis, Timothy A; Nag, Partha P; Ting, Amal; VerPlank, Lynn; Feng, Yuxiong; Perez, Jose R; Dandapani, Sivaraman; Palmer, Michelle; Lander, Eric S; Gupta, Piyush B; Schreiber, Stuart L; Munoz, Benito

2012-05-15

A high-throughput screen (HTS) with the National Institute of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) compound collection identified a class of acyl hydrazones to be selectively lethal to breast cancer stem cell (CSC) enriched populations. Medicinal chemistry efforts were undertaken to optimize potency and selectivity of this class of compounds. The optimized compound was declared as a probe (ML239) with the NIH Molecular Libraries Program and displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control line (HMLE_sh_GFP). Copyright © 2012 Elsevier Ltd. All rights reserved.

Defining new criteria for selection of cell-based intestinal models using publicly available databases

PubMed Central

2012-01-01

Background The criteria for choosing relevant cell lines among a vast panel of available intestinal-derived lines exhibiting a wide range of functional properties are still ill-defined. The objective of this study was, therefore, to establish objective criteria for choosing relevant cell lines to assess their appropriateness as tumor models as well as for drug absorption studies. Results We made use of publicly available expression signatures and cell based functional assays to delineate differences between various intestinal colon carcinoma cell lines and normal intestinal epithelium. We have compared a panel of intestinal cell lines with patient-derived normal and tumor epithelium and classified them according to traits relating to oncogenic pathway activity, epithelial-mesenchymal transition (EMT) and stemness, migratory properties, proliferative activity, transporter expression profiles and chemosensitivity. For example, SW480 represent an EMT-high, migratory phenotype and scored highest in terms of signatures associated to worse overall survival and higher risk of recurrence based on patient derived databases. On the other hand, differentiated HT29 and T84 cells showed gene expression patterns closest to tumor bulk derived cells. Regarding drug absorption, we confirmed that differentiated Caco-2 cells are the model of choice for active uptake studies in the small intestine. Regarding chemosensitivity we were unable to confirm a recently proposed association of chemo-resistance with EMT traits. However, a novel signature was identified through mining of NCI60 GI50 values that allowed to rank the panel of intestinal cell lines according to their drug responsiveness to commonly used chemotherapeutics. Conclusions This study presents a straightforward strategy to exploit publicly available gene expression data to guide the choice of cell-based models. While this approach does not overcome the major limitations of such models, introducing a rank order of selected features may allow selecting model cell lines that are more adapted and pertinent to the addressed biological question. PMID:22726358

Ontological representation, integration, and analysis of LINCS cell line cells and their cellular responses.

PubMed

Ong, Edison; Xie, Jiangan; Ni, Zhaohui; Liu, Qingping; Sarntivijai, Sirarat; Lin, Yu; Cooper, Daniel; Terryn, Raymond; Stathias, Vasileios; Chung, Caty; Schürer, Stephan; He, Yongqun

2017-12-21

Aiming to understand cellular responses to different perturbations, the NIH Common Fund Library of Integrated Network-based Cellular Signatures (LINCS) program involves many institutes and laboratories working on over a thousand cell lines. The community-based Cell Line Ontology (CLO) is selected as the default ontology for LINCS cell line representation and integration. CLO has consistently represented all 1097 LINCS cell lines and included information extracted from the LINCS Data Portal and ChEMBL. Using MCF 10A cell line cells as an example, we demonstrated how to ontologically model LINCS cellular signatures such as their non-tumorigenic epithelial cell type, three-dimensional growth, latrunculin-A-induced actin depolymerization and apoptosis, and cell line transfection. A CLO subset view of LINCS cell lines, named LINCS-CLOview, was generated to support systematic LINCS cell line analysis and queries. In summary, LINCS cell lines are currently associated with 43 cell types, 131 tissues and organs, and 121 cancer types. The LINCS-CLO view information can be queried using SPARQL scripts. CLO was used to support ontological representation, integration, and analysis of over a thousand LINCS cell line cells and their cellular responses.

Multifunctionalized iron oxide nanoparticles for selective drug delivery to CD44-positive cancer cells

NASA Astrophysics Data System (ADS)

Aires, Antonio; Ocampo, Sandra M.; Simões, Bruno M.; Josefa Rodríguez, María; Cadenas, Jael F.; Couleaud, Pierre; Spence, Katherine; Latorre, Alfonso; Miranda, Rodolfo; Somoza, Álvaro; Clarke, Robert B.; Carrascosa, José L.; Cortajarena, Aitziber L.

2016-02-01

Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro.

Integrated QSAR study for inhibitors of hedgehog signal pathway against multiple cell lines:a collaborative filtering method

PubMed Central

2012-01-01

Background The Hedgehog Signaling Pathway is one of signaling pathways that are very important to embryonic development. The participation of inhibitors in the Hedgehog Signal Pathway can control cell growth and death, and searching novel inhibitors to the functioning of the pathway are in a great demand. As the matter of fact, effective inhibitors could provide efficient therapies for a wide range of malignancies, and targeting such pathway in cells represents a promising new paradigm for cell growth and death control. Current research mainly focuses on the syntheses of the inhibitors of cyclopamine derivatives, which bind specifically to the Smo protein, and can be used for cancer therapy. While quantitatively structure-activity relationship (QSAR) studies have been performed for these compounds among different cell lines, none of them have achieved acceptable results in the prediction of activity values of new compounds. In this study, we proposed a novel collaborative QSAR model for inhibitors of the Hedgehog Signaling Pathway by integration the information from multiple cell lines. Such a model is expected to substantially improve the QSAR ability from single cell lines, and provide useful clues in developing clinically effective inhibitors and modifications of parent lead compounds for target on the Hedgehog Signaling Pathway. Results In this study, we have presented: (1) a collaborative QSAR model, which is used to integrate information among multiple cell lines to boost the QSAR results, rather than only a single cell line QSAR modeling. Our experiments have shown that the performance of our model is significantly better than single cell line QSAR methods; and (2) an efficient feature selection strategy under such collaborative environment, which can derive the commonly important features related to the entire given cell lines, while simultaneously showing their specific contributions to a specific cell-line. Based on feature selection results, we have proposed several possible chemical modifications to improve the inhibitor affinity towards multiple targets in the Hedgehog Signaling Pathway. Conclusions Our model with the feature selection strategy presented here is efficient, robust, and flexible, and can be easily extended to model large-scale multiple cell line/QSAR data. The data and scripts for collaborative QSAR modeling are available in the Additional file 1. PMID:22849868

Integrated QSAR study for inhibitors of Hedgehog Signal Pathway against multiple cell lines:a collaborative filtering method.

PubMed

Gao, Jun; Che, Dongsheng; Zheng, Vincent W; Zhu, Ruixin; Liu, Qi

2012-07-31

The Hedgehog Signaling Pathway is one of signaling pathways that are very important to embryonic development. The participation of inhibitors in the Hedgehog Signal Pathway can control cell growth and death, and searching novel inhibitors to the functioning of the pathway are in a great demand. As the matter of fact, effective inhibitors could provide efficient therapies for a wide range of malignancies, and targeting such pathway in cells represents a promising new paradigm for cell growth and death control. Current research mainly focuses on the syntheses of the inhibitors of cyclopamine derivatives, which bind specifically to the Smo protein, and can be used for cancer therapy. While quantitatively structure-activity relationship (QSAR) studies have been performed for these compounds among different cell lines, none of them have achieved acceptable results in the prediction of activity values of new compounds. In this study, we proposed a novel collaborative QSAR model for inhibitors of the Hedgehog Signaling Pathway by integration the information from multiple cell lines. Such a model is expected to substantially improve the QSAR ability from single cell lines, and provide useful clues in developing clinically effective inhibitors and modifications of parent lead compounds for target on the Hedgehog Signaling Pathway. In this study, we have presented: (1) a collaborative QSAR model, which is used to integrate information among multiple cell lines to boost the QSAR results, rather than only a single cell line QSAR modeling. Our experiments have shown that the performance of our model is significantly better than single cell line QSAR methods; and (2) an efficient feature selection strategy under such collaborative environment, which can derive the commonly important features related to the entire given cell lines, while simultaneously showing their specific contributions to a specific cell-line. Based on feature selection results, we have proposed several possible chemical modifications to improve the inhibitor affinity towards multiple targets in the Hedgehog Signaling Pathway. Our model with the feature selection strategy presented here is efficient, robust, and flexible, and can be easily extended to model large-scale multiple cell line/QSAR data. The data and scripts for collaborative QSAR modeling are available in the Additional file 1.

Effects of 1,3,5-triphenyl-4,5-dihydro-1H-pyrazole derivatives on cell-cycle and apoptosis in human acute leukemia cell lines.

PubMed

Santos Bubniak, Lorena Dos; Gaspar, Pâmela Cristina; de Moraes, Ana Carolina Rabello; Bigolin, Alisson; de Souza, Rubia Karine; Buzzi, Fátima Campos; Corrêa, Rogério; Filho, Valdir Cechinel; Bretanha, Lizandra Czermainski; Micke, Gustavo Amadeu; Nunes, Ricardo José; Santos-Silva, Maria Cláudia

2017-05-01

Pyrazoline is an important 5-membered nitrogen heterocycle that has been extensively researched. Ten derivatives were synthesized and tested for antileukemic effects on 2 human acute leukemia cell lines, K562 and Jurkat. The most cytotoxic of these derivatives, compound 21, was chosen for investigation of cytotoxicity mechanisms. The results obtained with selectivity calculations revealed that compound 21 is more selective for acute leukemia (K562 and Jurkat cell lines) than for other tumor cell lines. Moreover, compound 21 was not cytotoxic to normal cell lines, indicating a potential use in clinical tests. Compound 21 caused a significant cell cycle arrest in the S-phase in Jurkat cells and increased the proportion of cells in the sub G0/G1 phase in both cell lines. Cells treated with compound 21 demonstrated morphological changes characteristic of apoptosis in the EB/AO assay, confirmed by externalization of phosphatidylserine by the annexin V - fluorescein isothiocyanate method and by DNA fragmentation. An investigation of cytotoxicity mechanisms suggests the involvement of an intrinsic apoptosis pathway due to mitochondrial damage and an increase in the ratio of mitochondrial Bax/Bcl2. Pyrazoline 21 obeyed Lipinski's "rule of five" for drug-likeness. Based on these preliminary results, the antileukemic activity of compound 21 makes it a potential anticancer agent.

B cell signatures of BCWD-resistant and susceptible lines of rainbow trout: a shift towards more EBF-expressing progenitors and fewer mature B cells in resistant animals.

PubMed

Zwollo, Patty; Ray, Jocelyn C; Sestito, Michael; Kiernan, Elizabeth; Wiens, Gregory D; Kaattari, Steve; StJacques, Brittany; Epp, Lidia

2015-01-01

Bacterial cold water disease (BCWD) is a chronic disease of rainbow trout, and is caused by the Gram-negative bacterium Flavobacterium psychrophilum (Fp), a common aquaculture pathogen. The National Center for Cool and Cold Water Aquaculture has bred two genetic lines of rainbow trout: a line of Fp-resistant trout (ARS-Fp-R or R-line trout) and a line of susceptible trout (ARS-Fp-S, or S-line). Little is known about how phenotypic selection alters immune response parameters or how such changes relate to genetic disease resistance. Herein, we quantify interindividual variation in the distribution and abundance of B cell populations (B cell signatures) and examine differences between genetic lines of naive animals. There are limited trout-specific cell surface markers currently available to resolve B cell subpopulations and thus we developed an alternative approach based on detection of differentially expressed transcription factors and intracellular cytokines. B cell signatures were compared between R-line and S-line trout by flow cytometry using antibodies against transcription factors early B cell factor-1 (EBF1) and paired domain box protein Pax5, the pro-inflammatory cytokine IL-1β, and the immunoglobulin heavy chain mu. R-line trout had higher percentages of EBF(+) B myeloid/ progenitor and pre-B cells in PBL, anterior and posterior kidney tissues compared to S-line trout. The opposite pattern was detected in more mature B cell populations: R-line trout had lower percentages of both IgM(+) mature B cells and IgM-secreting cells in anterior kidney and PBL compared to S-line trout. In vitro LPS-activation studies of PBL and spleen cell cultures revealed no significant induction differences between R-line and S-line trout. Together, our findings suggest that selective resistance to BCWD may be associated with shifts in naive animal developmental lineage commitment that result in decreased B lymphopoiesis and increased myelopoiesis in BCWD resistant trout relative to susceptible trout. Copyright © 2014 Elsevier Ltd. All rights reserved.

In vitro cytotoxicity and differential cellular sensitivity of derivatives of diamino acids. II. N1-methyl, N1-allyl, N1-(2-chloroethyl) and N1-propargyl nitrosoureas.

PubMed

Dulude, H; Salvador, R; Gallant, G

1995-01-01

The in vitro cytotoxicity and differential cellular sensitivity of a series of new N1-methyl, N1-allyl, N1-2-chloroethyl and N1-propargyl nitrosourea derivatives of diamino acids were determined in the National Cancer Institute's primary antitumor drug screen. The compounds tested showed an in vitro anticancer activity similar to commercialized nitrosoureas such as CCNU, BCNU, MeCCNU, chlorozotocin, streptozotocin and PCNU. The alkylating moiety of the nitrosoureas seems to play a role in the general selectivity of our compounds. The N1-methyl and N1-2-chloroethyl nitrosourea derivatives are more selective for central nervous system cell lines, the N1-allyl nitrosourea derivatives are more selective for lung cancer cell lines and the N1-propargyl nitrosoureas are more selective for leukemia cell lines.

RNA-seq reveals distinctive RNA profiles of small extracellular vesicles from different human liver cancer cell lines.

PubMed

Berardocco, Martina; Radeghieri, Annalisa; Busatto, Sara; Gallorini, Marialucia; Raggi, Chiara; Gissi, Clarissa; D'Agnano, Igea; Bergese, Paolo; Felsani, Armando; Berardi, Anna C

2017-10-10

Liver cancer (LC) is one of the most common cancers and represents the third highest cause of cancer-related deaths worldwide. Extracellular vesicle (EVs) cargoes, which are selectively enriched in RNA, offer great promise for the diagnosis, prognosis and treatment of LC. Our study analyzed the RNA cargoes of EVs derived from 4 liver-cancer cell lines: HuH7, Hep3B, HepG2 (hepato-cellular carcinoma) and HuH6 (hepatoblastoma), generating two different sets of sequencing libraries for each. One library was size-selected for small RNAs and the other targeted the whole transcriptome. Here are reported genome wide data of the expression level of coding and non-coding transcripts, microRNAs, isomiRs and snoRNAs providing the first comprehensive overview of the extracellular-vesicle RNA cargo released from LC cell lines. The EV-RNA expression profiles of the four liver cancer cell lines share a similar background, but cell-specific features clearly emerge showing the marked heterogeneity of the EV-cargo among the individual cell lines, evident both for the coding and non-coding RNA species.

Effects of space flight exposure on cell growth, tumorigenicity and gene expression in cancer cells

NASA Astrophysics Data System (ADS)

Yang, Cheng; Li, Yuehui; Zhang, Zhijie; Luo, Chen; Tong, Yongqing; Zhou, Guohua; Xie, Pingli; Hu, Jinyue; Li, Guancheng

2008-12-01

It is well recognized that harsh outer space environment, consisting of microgravity and radiation, poses significant health risks for human cells. To investigate potential effects of the space environment exposure on cancer cells we examined the biological changes in Caski cells carried by the "Shen Zhou IV" spaceship. After exposure for 7 days in spaceflight, 1440 survival subclonal cell lines were established and 4 cell lines were screened. 44F10 and 17E3 were selected because of their increased cell proliferation and tumorigenesis, while 48A9 and 31F2 had slower cytological events. Experiments with cell proliferation assay, flow cytometry, soft agar assay, tumorigenesis assay and DNA microarray analysis have shown that selected cell lines presented multiple biological changes in cell morphology, cell growth, tumorigenicity and gene expression. These results suggest that space environment exposure can make significant biological impact on cancer cells and provide an entry point to find the immunological target of tumorigenesis.

Warburg and Crabtree Effects in Premalignant Barrett's Esophagus Cell Lines with Active Mitochondria

PubMed Central

Suchorolski, Martin T.; Paulson, Thomas G.; Sanchez, Carissa A.; Hockenbery, David; Reid, Brian J.

2013-01-01

Background Increased glycolysis is a hallmark of cancer metabolism, yet relatively little is known about this phenotype at premalignant stages of progression. Periodic ischemia occurs in the premalignant condition Barrett's esophagus (BE) due to tissue damage from chronic acid-bile reflux and may select for early adaptations to hypoxia, including upregulation of glycolysis. Methodology/Principal Findings We compared rates of glycolysis and oxidative phosphorylation in four cell lines derived from patients with BE (CP-A, CP-B, CP-C and CP-D) in response to metabolic inhibitors and changes in glucose concentration. We report that cell lines derived from patients with more advanced genetically unstable BE have up to two-fold higher glycolysis compared to a cell line derived from a patient with early genetically stable BE; however, all cell lines preserve active mitochondria. In response to the glycolytic inhibitor 2-deoxyglucose, the most glycolytic cell lines (CP-C and CP-D) had the greatest suppression of extra-cellular acidification, but were able to compensate with upregulation of oxidative phosphorylation. In addition, these cell lines showed the lowest compensatory increases in glycolysis in response to mitochondrial uncoupling by 2,4-dinitrophenol. Finally, these cell lines also upregulated their oxidative phosphorylation in response to glucose via the Crabtree effect, and demonstrate a greater range of modulation of oxygen consumption. Conclusions/Significance Our findings suggest that cells from premalignant Barrett's esophagus tissue may adapt to an ever-changing selective microenvironment through changes in energy metabolic pathways typically associated with cancer cells. PMID:23460817

Genetic transformation of tobacco NT1 cells with Agrobacterium tumefaciens.

PubMed

Mayo, Kristin J; Gonzales, Barbara J; Mason, Hugh S

2006-01-01

This protocol is used to produce stably transformed tobacco (Nicotiana tabacum) NT1 cell lines, using Agrobacterium tumefaciens-mediated DNA delivery of a binary vector containing a gene encoding hepatitis B surface antigen and a gene encoding the kanamycin selection marker. The NT1 cultures, at the appropriate stage of growth, are inoculated with A. tumefaciens containing the binary vector. A 3-day cocultivation period follows, after which the cultures are rinsed and placed on solid selective medium. Transformed colonies ('calli') appear in approximately 4 weeks; they are subcultured until adequate material is obtained for analysis of antigen production. 'Elite' lines are selected based on antigen expression and growth characteristics. The time required for the procedure from preparation of the plant cell materials to callus development is approximately 5 weeks. Growth of selected calli to sufficient quantities for antigen screening may require 4-6 weeks beyond the initial selection. Creation of the plasmid constructs, transformation of the A. tumefaciens line, and ELISA and Bradford assays to assess protein production require additional time.

Selection of Metastatic Breast Cancer Cells Based on Adaptability of Their Metabolic State

PubMed Central

Singh, Balraj; Tai, Karen; Madan, Simran; Raythatha, Milan R.; Cady, Amanda M.; Braunlin, Megan; Irving, LaTashia R.; Bajaj, Ankur; Lucci, Anthony

2012-01-01

A small subpopulation of highly adaptable breast cancer cells within a vastly heterogeneous population drives cancer metastasis. Here we describe a function-based strategy for selecting rare cancer cells that are highly adaptable and drive malignancy. Although cancer cells are dependent on certain nutrients, e.g., glucose and glutamine, we hypothesized that the adaptable cancer cells that drive malignancy must possess an adaptable metabolic state and that such cells could be identified using a robust selection strategy. As expected, more than 99.99% of cells died upon glutamine withdrawal from the aggressive breast cancer cell line SUM149. The rare cells that survived and proliferated without glutamine were highly adaptable, as judged by additional robust adaptability assays involving prolonged cell culture without glucose or serum. We were successful in isolating rare metabolically plastic glutamine-independent (Gln-ind) variants from several aggressive breast cancer cell lines that we tested. The Gln-ind cells overexpressed cyclooxygenase-2, an indicator of tumor aggressiveness, and they were able to adjust their glutaminase level to suit glutamine availability. The Gln-ind cells were anchorage-independent, resistant to chemotherapeutic drugs doxorubicin and paclitaxel, and resistant to a high concentration of a COX-2 inhibitor celecoxib. The number of cells being able to adapt to non-availability of glutamine increased upon prior selection of cells for resistance to chemotherapy drugs or resistance to celecoxib, further supporting a linkage between cellular adaptability and therapeutic resistance. Gln-ind cells showed indications of oxidative stress, and they produced cadherin11 and vimentin, indicators of mesenchymal phenotype. Gln-ind cells were more tumorigenic and more metastatic in nude mice than the parental cell line as judged by incidence and time of occurrence. As we decreased the number of cancer cells in xenografts, lung metastasis and then primary tumor growth was impaired in mice injected with parental cell line, but not in mice injected with Gln-ind cells. PMID:22570721

University of Texas Southwestern Medical Center: NSCLC Cell Lines with Loss of SMARCA4 Expression are Hypersensitive to Inhibitors of Aurora Kinase A | Office of Cancer Genomics

Cancer.gov

A genome-wide siRNA screen was employed to identify genes that were selectively toxic for a non-small cell lung cancer (NSCLC) cell line that lacked expression of SMARCA4, but were not toxic in non-cancerous immortalized human bronchial epithelial cells lacking SMARCA4 expression. Among the selectively toxic genes were several mapping to the molecular machinery regulating activity of Aurora kinase A on the mitotic spindle.

University of Texas Southwestern Medical Center (UTSW): NSCLC Cell Lines with Loss of SMARCA4 Expression are Hypersensitive to Inhibitors of Aurora Kinase A | Office of Cancer Genomics

Cancer.gov

A genome-wide siRNA screen was employed to identify genes that were selectively toxic for a non-small cell lung cancer (NSCLC) cell line that lacked expression of SMARCA4, but were not toxic in non-cancerous immortalized human bronchial epithelial cells lacking SMARCA4 expression. Among the selectively toxic genes were several mapping to the molecular machinery regulating activity of Aurora kinase A on the mitotic spindle.

In vitro evaluation of human hybrid cell lines generated by fusion of B-lymphoblastoid cells and ex vivo tumour cells as candidate vaccines for haematological malignancies.

PubMed

Mohamed, Yehia S; Dunnion, Debbie; Teobald, Iryna; Walewska, Renata; Browning, Michael J

2012-10-12

Fusions of dendritic cells (DCs) and tumour cells have been shown to induce protective immunity to tumour challenge in animal models, and to represent a promising approach to cancer immunotherapy. The broader clinical application of this approach, however, is potentially constrained by the lack of replicative capacity and limited standardisation of fusion cell preparations. We show here that fusion of ex vivo tumour cells isolated from patients with a range of haematological malignancies with the human B-lymphoblastoid cell line (LCL), HMy2, followed by chemical selection of the hybridomas, generated stable, self-replicating human hybrid cell lines that grew continuously in tissue culture, and survived freeze/thawing cycles. The hybrid cell lines expressed HLA class I and class II molecules, and the major T-cell costimulatory molecules, CD80 and CD86. All but two of 14 hybrid cell lines generated expressed tumour-associated antigens that were not expressed by HMy2 cells, and were therefore derived from the parent tumour cells. The hybrid cell lines stimulated allogeneic T-cell proliferative responses and interferon-gamma release in vitro to a considerably greater degree than their respective parent tumour cells. The enhanced T-cell stimulation was inhibited by CTLA4-Ig fusion protein, and by blocking antibodies to MHC class I and class II molecules. Finally, all of five LCL/tumour hybrid cell lines tested induced tumour antigen-specific cytotoxic T-cell responses in vitro in PBL from healthy, HLA-A2+ individuals, as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity. These data show that stable hybrid cell lines, with enhanced immunostimulatory properties and potential for therapeutic vaccination, can be generated by in vitro fusion and chemical selection of B-LCL and ex vivo haematological tumour cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Plasma stable, pH-sensitive fusogenic polymer-modified liposomes: A promising carrier for mitoxantrone.

PubMed

Ghanbarzadeh, Saeed; Arami, Sanam; Pourmoazzen, Zhaleh; Ghasemian-Yadegari, Javad; Khorrami, Arash

2014-07-01

pH-sensitive liposomes are designed to undergo acid-triggered destabilization. In the present study, we prepared polymer-modified, plasma stable, pH-sensitive fusogenic mitoxantrone liposomes to increase efficacy and selectivity on cancer cell lines. Conventional liposomes were prepared using cholesterol and dipalmitoyl-sn-glycero-3-phosphatidylethanolamine. Dioleoylphosphatidylethanolamine and a cholesteryl derivative, poly(monomethylitaconate)-co-poly(N,N-dimethylaminoethyl methacrylate) (PMMI-co-PDMAEMA), were used for the preparation of pH-sensitive fusogenic liposomes. Using polyethylene glycol (PEG)-poly(monomethylitaconate)-CholC6 (PEG-PMMI-CholC6) copolymers instead of cholesterol introduced pH-sensitive and plasma stability properties simultaneously in prepared liposomes. All formulations were prepared by thin film hydration method and subsequently, pH-sensitivity and stability in human serum were evaluated. The ability of pH-sensitive fusogenic liposomes to enhance the mitoxantrone cytotoxicity and selectivity in cancerous cell lines was assessed in vitro compared to normal cell line using human breast cancer cell line (MCF-7), human prostate cancer cell line (PC-3), and human umbilical vein endothelial cells line. Results revealed that both PMMI-co-PDMAEMA and PEG-PMMI-CholC6-based formulations showed pH-sensitive property and were found to rapidly release mitoxantrone under mildly acidic conditions. Nevertheless, only the PEG-PMMI-CholC6-based liposomes preserved pH-sensitivity after incubation in plasma. Mitoxantrone loaded-pH-sensitive fusogenic liposomes exhibited a higher cytotoxicity than the control conventional liposomes on MCF-7 and PC-3 cell lines. On the contrary, both pH-sensitive fusogenic liposomes showed lower cytotoxic effect on human umbilical vein endothelial cell line. Plasma stable, pH-sensitive fusogenic liposomes are promising carriers for enhancing the efficiency and selectivity, besides reduction of the side effects of anticancer agents. © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Reduction of ammonia and lactate through the coupling of glutamine synthetase selection and downregulation of lactate dehydrogenase-A in CHO cells.

PubMed

Noh, Soo Min; Park, Jin Hyoung; Lim, Myung Sin; Kim, Jong Won; Lee, Gyun Min

2017-02-01

Chinese hamster ovary (CHO) cell cultivation for production of therapeutic proteins is accompanied by production of metabolic wastes, mostly ammonia and lactate. To reduce ammonia production, the glutamine synthetase (GS) system was used to develop therapeutic monoclonal antibody (mAb)-producing CHO cells (SM-0.025). Additionally, the lactate dehydrogenase-A (LDH-A) was downregulated with shRNA to reduce lactate production in SM-0.025. The resulting mAb-producing cell lines (#2, #46, and #52) produced less ammonia than the host cell line during the exponential phase due to GS protein overexpression. LDH-A downregulation in SM-0.025 not only reduced lactate production but also further reduced ammonia production. Among the three LDH-A-downregulated clones, clone #2 had the highest mAb production along with significantly reduced specific lactate and ammonia production rates compared to those in SM-0.025. Waste reduction increased the galactosylation level of N-glycosylation, which improved mAb quality. LDH-A downregulation was also successfully applied to the host cell lines (CHO K1 and GS knockout CHO-K1). However, LDH-A downregulated host cells could not survive the pool-selection process wherein glutamine was excluded and methionine sulfoximine was added to the media. Taken together, LDH-A downregulation in the mAb-producing cell line generated with the GS system successfully reduced both ammonia and lactate levels, improving mAb galactosylation. However, LDH-A downregulation could not be applied to host cell lines because it hampered the selection process of the GS system.

Estrogen Regulates Bone Turnover by Targeting RANKL Expression in Bone Lining Cells.

PubMed

Streicher, Carmen; Heyny, Alexandra; Andrukhova, Olena; Haigl, Barbara; Slavic, Svetlana; Schüler, Christiane; Kollmann, Karoline; Kantner, Ingrid; Sexl, Veronika; Kleiter, Miriam; Hofbauer, Lorenz C; Kostenuik, Paul J; Erben, Reinhold G

2017-07-25

Estrogen is critical for skeletal homeostasis and regulates bone remodeling, in part, by modulating the expression of receptor activator of NF-κB ligand (RANKL), an essential cytokine for bone resorption by osteoclasts. RANKL can be produced by a variety of hematopoietic (e.g. T and B-cell) and mesenchymal (osteoblast lineage, chondrocyte) cell types. The cellular mechanisms by which estrogen acts on bone are still a matter of controversy. By using murine reconstitution models that allow for selective deletion of estrogen receptor-alpha (ERα) or selective inhibition of RANKL in hematopoietic vs. mesenchymal cells, in conjunction with in situ expression profiling in bone cells, we identified bone lining cells as important gatekeepers of estrogen-controlled bone resorption. Our data indicate that the increase in bone resorption observed in states of estrogen deficiency in mice is mainly caused by lack of ERα-mediated suppression of RANKL expression in bone lining cells.

Glutamine synthetase gene knockout-human embryonic kidney 293E cells for stable production of monoclonal antibodies.

PubMed

Yu, Da Young; Lee, Sang Yoon; Lee, Gyun Min

2018-05-01

Previously, it was inferred that a high glutamine synthetase (GS) activity in human embryonic kidney (HEK) 293E cells results in elevated resistance to methionine sulfoximine (MSX) and consequently hampers GS-mediated gene amplification and selection by MSX. To overcome this MSX resistance in HEK293E cells, a GS-knockout HEK293E cell line was generated using the CRISPR/Cas9 system to target the endogenous human GS gene. The GS-knockout in the HEK293E cell line (RK8) was confirmed by Western blot analysis of GS and by observation of glutamine-dependent growth. Unlike the wild type HEK293E cells, the RK8 cells were successfully used as host cells to generate a recombinant HEK293E cell line (rHEK293E) producing a monoclonal antibody (mAb). When the RK8 cells were transfected with the GS expression vector containing the mAb gene, rHEK293E cells producing the mAb could be selected in the absence as well as in the presence of MSX. The gene copies and mRNA expression levels of the mAb in rHEK293E cells were also quantified using qRT-PCR. Taken together, the GS-knockout HEK293E cell line can be used as host cells to generate stable rHEK293E cells producing a mAb through GS-mediated gene selection in the absence as well as in the presence of MSX. © 2018 Wiley Periodicals, Inc.

Exploiting persistent infection for selection of bovine herpesvirus 4 recombinants.

PubMed

Donofrio, G; Martignani, E; Cavirani, S; Flammini, C F

2005-09-01

Bovine herpesvirus 4 (BoHV-4) is a gamma-herpesvirus with no clear disease association, and due to its biological characteristics, has been suggested as a gene delivery vector. It was demonstrated previously that recombinant BoHV-4 carrying a neomycin-resistance gene was able to infect a human rhabdomyosarcoma cell line (RD-4), resulting in no detectable cytopathic effect (CPE) and allowing selection of G418-resistant persistently-infected cells containing circular episomal viral DNA [Donofrio, G., Cavirani, S., van Santen, V.L., 2000a. Establishment of a cell line persistently infected with recombinant BoHV-4. J. Gen. Virol. 81, 1807-1814.]. Those cells produce infectious virus and infection is predominantly non-permissive and non-cytopathic. Starting from these results, the ability of RD-4 cells to sustain persistent infection was combined with positive selection activity conferred by the neomycin-expression cassette insert, as an easier way to select recombinants of BoHV-4 following homologous recombination in permissive cells. A tool for selecting BoHV-4 recombinants was developed by drug positive selection.

Cell specific aptamer-photosensitizer conjugates as a molecular tool in photodynamic therapy

PubMed Central

Mallikaratchy, Prabodhika; Tang, Zhiwen

2010-01-01

This paper describes the application of a molecular construct of a photosensitizer and an aptamer for photo-therapeutically targeting tumor cells. The key step in increasing selectivity in chemotherapeutic drugs is to create effective molecular platforms that could target cancer cells but not normal cells. Recently, we have developed a strategy via cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to obtain cell specific aptamers using intact viable cells as targets to select aptamers that can recognize cell membrane proteins with high selectivity and excellent affinity. We have identified an aptamer TD05 that only recognizes Ramos cells, a Burkitt’s lymphoma cell line. Here, the high specificity of aptamers in target cell binding and an efficient phototherapy reagent, Ce6, are molecularly engineered to construct a highly selective Aptamer-photosensitizer conjugates (APS) to effectively destroy target cancer cells. Introduction of the APS conjugates followed by irradiation of light selectively destroyed target Ramos cells but not acute lymphoblastic leukemia and myeloid leukemia cell lines. This study demonstrates that the use of cancer specific aptamers conjugated to a photosensitizer will enhance the selectivity of photodynamic therapy. Coupled with the advantages of the cell-SELEX in generating multiple effective aptamers for diseased cell recognition, we will be able to develop highly efficient photosensitizer based therapeutical reagents for clinical applications. PMID:18058891

Novel cell-based odorant sensor elements based on insect odorant receptors.

PubMed

Mitsuno, Hidefumi; Sakurai, Takeshi; Namiki, Shigehiro; Mitsuhashi, Hiroyuki; Kanzaki, Ryohei

2015-03-15

Development of cell-based odorant sensor elements combined not only high degree of sensitivity and selectivity but also long-term stability is crucial for their practical applications. Here we report the development of a novel cell-based odorant sensor element that sensitively and selectively detects odorants and displays increased fluorescent intensities over a long period of time. Our odorant sensor elements, based on Sf21 cell lines expressing insect odorant receptors, are sensitive to the level of several tens of parts per billion in solution, can selectively distinguish between different types of odorants based on the odorant selectivity intrinsic to the expressed receptors, and have response times of approximately 13s. Specifically, with the use of Sf21 cells and insect odorant receptors, we demonstrated that the established cell lines stably expressing insect odorant receptors are able to detect odorants with consistent responsiveness for at least 2 months, thus exceeding the short life-span normally associated with cell-based sensors. We also demonstrated the development of a compact odorant sensor chip by integrating the established insect cell lines into a microfluidic chip. The methodology we established in this study, in conjunction with the large repertoire of insect odorant receptors, will aid in the development of practical cell-based odorant sensors for various applications, including food administration and health management. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of Cell-SELEX Technology and Its Application in Cancer Diagnosis and Therapy.

PubMed

Chen, Man; Yu, Yuanyuan; Jiang, Feng; Zhou, Junwei; Li, Yongshu; Liang, Chao; Dang, Lei; Lu, Aiping; Zhang, Ge

2016-12-10

SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed.

Development of Cell-SELEX Technology and Its Application in Cancer Diagnosis and Therapy

PubMed Central

Chen, Man; Yu, Yuanyuan; Jiang, Feng; Zhou, Junwei; Li, Yongshu; Liang, Chao; Dang, Lei; Lu, Aiping; Zhang, Ge

2016-01-01

SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed. PMID:27973403

Introduction of new genetic markers on human chromosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Satoh, Hitoshi; Barrett, J.C.; Oshimura, Mitsuo

1991-03-01

The purpose of this study was to use DNA transfection and microcell chromosome transfer techniques to engineer a human chromosome containing multiple biochemical markers for which selectable growth conditions exist. The starting chromosome was a t(X;3)(3pter{yields}3p12::Xq26{yields}Xpter) chromosome from a reciprocal translocation in the normal human fibroblast cell line GM0439. This chromosome was transferred to a HPRT (hypoxanthine phosphoribosyltransferase)-deficient mouse A9 cell line by microcell fusion and selected under growth conditions for the HPRT gene on the human t(X;3) chromosome. A resultant HAT-resistant cell line (A9(GM0439)-1) contained a single human t(X;3) chromosome. These results demonstrate that microcell chromosome transfer can bemore » used to select chromosomes containing multiple markers.« less

Quantitative structure-cytotoxicity relationship of phenylpropanoid amides.

PubMed

Shimada, Chiyako; Uesawa, Yoshihiro; Ishihara, Mariko; Kagaya, Hajime; Kanamoto, Taisei; Terakubo, Shigemi; Nakashima, Hideki; Takao, Koichi; Saito, Takayuki; Sugita, Yoshiaki; Sakagami, Hiroshi

2014-07-01

A total of 12 phenylpropanoid amides were subjected to quantitative structure-activity relationship (QSAR) analysis, based on their cytotoxicity, tumor selectivity and anti-HIV activity, in order to investigate on their biological activities. Cytotoxicity against four human oral squamous cell carcinoma (OSCC) cell lines and three human oral normal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Tumor selectivity was evaluated by the ratio of the mean CC50 (50% cytotoxic concentration) against normal oral cells to that against OSCC cell lines. Anti-HIV activity was evaluated by the ratio of CC50 to EC50 (50% cytoprotective concentration from HIV infection). Physicochemical, structural, and quantum-chemical parameters were calculated based on the conformations optimized by the LowModeMD method followed by density functional theory (DFT) method. Twelve phenylpropanoid amides showed moderate cytotoxicity against both normal and OSCC cell lines. N-Caffeoyl derivatives coupled with vanillylamine and tyramine exhibited relatively higher tumor selectivity. Cytotoxicity against normal cells was correlated with descriptors related to electrostatic interaction such as polar surface area and chemical hardness, whereas cytotoxicity against tumor cells correlated with free energy, surface area and ellipticity. The tumor-selective cytotoxicity correlated with molecular size (surface area) and electrostatic interaction (the maximum electrostatic potential). The molecular size, shape and ability for electrostatic interaction are useful parameters for estimating the tumor selectivity of phenylpropanoid amides. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

The Broad Institute: Screening for Dependencies in Cancer Cell Lines Using Small Molecules | Office of Cancer Genomics

Cancer.gov

Using cancer cell-line profiling, we established an ongoing resource to identify, as comprehensively as possible, the drug-targetable dependencies that specific genomic alterations impart on human cancers. We measured the sensitivity of hundreds of genetically characterized cancer cell lines to hundreds of small-molecule probes and drugs that have highly selective interactions with their targets, and that collectively modulate many distinct nodes in cancer cell circuitry.

Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

PubMed

Perez-Cunningham, Jessica; Boyer, Scott W; Landon, Mark; Forsberg, E Camilla

2016-08-01

Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells and platelets. This new "Vav-GFP" mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells (HSPCs). With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells and persists in donor-derived progeny after transplantation of HSPCs. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. By crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in hematopoietic stem cells (HSCs). These data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Picking Cell Lines for High-Throughput Transcriptomic Toxicity ...

EPA Pesticide Factsheets

High throughput, whole genome transcriptomic profiling is a promising approach to comprehensively evaluate chemicals for potential biological effects. To be useful for in vitro toxicity screening, gene expression must be quantified in a set of representative cell types that captures the diversity of potential responses across chemicals. The ideal dataset to select these cell types would consist of hundreds of cell types treated with thousands of chemicals, but does not yet exist. However, basal gene expression data may be useful as a surrogate for representing the relevant biological space necessary for cell type selection. The goal of this study was to identify a small (< 20) number of cell types that capture a large, quantifiable fraction of basal gene expression diversity. Three publicly available collections of Affymetrix U133+2.0 cellular gene expression data were used: 1) 59 cell lines from the NCI60 set; 2) 303 primary cell types from the Mabbott et al (2013) expression atlas; and 3) 1036 cell lines from the Cancer Cell Line Encyclopedia. The data were RMA normalized, log-transformed, and the probe sets mapped to HUGO gene identifiers. The results showed that <20 cell lines capture only a small fraction of the total diversity in basal gene expression when evaluated using either the entire set of 20960 HUGO genes or a subset of druggable genes likely to be chemical targets. The fraction of the total gene expression variation explained was consistent when

SAHA-induced TRAIL-sensitisation of Multiple Myeloma cells is enhanced in 3D cell culture.

PubMed

Arhoma, A; Chantry, A D; Haywood-Small, S L; Cross, N A

2017-11-15

Multiple Myeloma (MM) is currently incurable despite many novel therapies. Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is a potential anti-tumour agent although effects as a single agent are limited. In this study, we investigated whether the Histone Deacetylase (HDAC) inhibitor SAHA can enhance TRAIL-induced apoptosis and target TRAIL resistance in both suspension culture, and 3D cell culture as a model of disseminated MM lesions that form in bone. The effects of SAHA and/or TRAIL in 6 Multiple Myeloma cell lines were assessed in both suspension cultures and in an Alginate-based 3D cell culture model. The effect of SAHA and/or TRAIL was assessed on apoptosis by assessment of nuclear morphology using Hoechst 33342/Propidium Iodide staining. Viable cell number was assessed by CellTiter-Glo luminescence assay, Caspase-8 and -9 activities were measured by Caspase-Glo™ assay kit. TRAIL-resistant cells were generated by culture of RPMI 8226 and NCI-H929 by acute exposure to TRAIL followed by selection of TRAIL-resistant cells. TRAIL significantly induced apoptosis in a dose-dependent manner in OPM-2, RPMI 8226, NCI-H929, U266, JJN-3 MM cell lines and ADC-1 plasma cell leukaemia cells. SAHA amplified TRAIL responses in all lines except OPM-2, and enhanced TRAIL responses were both via Caspase-8 and -9. SAHA treatment induced growth inhibition that further increased in the combination treatment with TRAIL in MM cells. The co-treatment of TRAIL and SAHA reduced viable cell numbers all cell lines. TRAIL responses were further potentiated by SAHA in 3D cell culture in NCI-H929, RPMI 8226 and U266 at lower TRAIL + SAHA doses than in suspension culture. However TRAIL responses in cells that had been selected for TRAIL resistance were not further enhanced by SAHA treatment. SAHA is a potent sensitizer of TRAIL responses in both TRAIL sensitive and resistant cell lines, in both suspension and 3D culture, however SAHA did not sensitise TRAIL-sensitive cell populations that had been selected for TRAIL-resistance from initially TRAIL-sensitive populations. SAHA may increase TRAIL sensitivity in insensitive cells, but not in cells that have specifically been selected for acquired TRAIL-resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

RNA-seq reveals distinctive RNA profiles of small extracellular vesicles from different human liver cancer cell lines

PubMed Central

Berardocco, Martina; Radeghieri, Annalisa; Busatto, Sara; Gallorini, Marialucia; Raggi, Chiara; Gissi, Clarissa; D’Agnano, Igea; Bergese, Paolo; Felsani, Armando; Berardi, Anna C.

2017-01-01

Liver cancer (LC) is one of the most common cancers and represents the third highest cause of cancer-related deaths worldwide. Extracellular vesicle (EVs) cargoes, which are selectively enriched in RNA, offer great promise for the diagnosis, prognosis and treatment of LC. Our study analyzed the RNA cargoes of EVs derived from 4 liver-cancer cell lines: HuH7, Hep3B, HepG2 (hepato-cellular carcinoma) and HuH6 (hepatoblastoma), generating two different sets of sequencing libraries for each. One library was size-selected for small RNAs and the other targeted the whole transcriptome. Here are reported genome wide data of the expression level of coding and non-coding transcripts, microRNAs, isomiRs and snoRNAs providing the first comprehensive overview of the extracellular-vesicle RNA cargo released from LC cell lines. The EV-RNA expression profiles of the four liver cancer cell lines share a similar background, but cell-specific features clearly emerge showing the marked heterogeneity of the EV-cargo among the individual cell lines, evident both for the coding and non-coding RNA species. PMID:29137313

Selective antiproliferative and apoptotic effects of flavonoids purified from Rhus verniciflua Stokes on normal versus transformed hepatic cell lines.

PubMed

Son, Young-Ok; Lee, Kyung-Yeol; Lee, Jeong-Chae; Jang, Hyon-Seok; Kim, Jong-Ghee; Jeon, Young-Mi; Jang, Yong-Suk

2005-01-15

Considerable attention is being concentrated on dietary flavonoids in developing novel cancer-preventive approaches due to their potential ability to induce selective apoptosis of cancer cells. In this study, we prepared a flavonoid-containing fraction from a crude acetone extract of Rhus verniciflua Stokes (RVS), traditionally used as a food additive and as an herbal medicine, and named RVS chloroform-methanol fraction (RCMF). We evaluated the effects of RCMF on proliferation and apoptosis using mouse embryonic primary hepatic cells (MPHC), embryonic normal hepatic cell line (BNL CL.2), and its SV40-mediated transformed cell line (BNL SV A.8). We also investigated the effects of RCMF on the antioxidant defense system in those cells. This study demonstrated that RCMF exhibited a selective growth inhibition and apoptosis induction on transformed cells. BNL SV A.8 cells were more sensitive to RCMF-mediated cytotoxicity than were MPHC or BNL CL.2. RCMF-mediated reduction of MnSOD activity and glutathione (GSH) content in BNL SV A.8 cells is thought to be associated with RCMF-induced apoptosis. Our findings suggest that RCMF is an agent which may be capable of inducing growth inhibition and apoptosis of hepatic tumor cells.

Application of a haematopoetic progenitor cell-targeted adeno-associated viral (AAV) vector established by selection of an AAV random peptide library on a leukaemia cell line

PubMed Central

Stiefelhagen, Marius; Sellner, Leopold; Kleinschmidt, Jürgen A; Jauch, Anna; Laufs, Stephanie; Wenz, Frederik; Zeller, W Jens; Fruehauf, Stefan; Veldwijk, Marlon R

2008-01-01

Background For many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low. Advancements in vector development now allows the generation of target cell-selected AAV capsid mutants. Methods To determine its suitability, the method was applied on a chronic myelogenous leukaemia (CML) cell line (K562) to obtain a CML-targeted vector and the resulting vectors tested on leukaemia, non-leukaemia, primary human CML and CD34+ peripheral blood progenitor cells (PBPC); standard AAV2 and a random capsid mutant vector served as controls. Results Transduction of CML (BV173, EM3, K562 and Lama84) and AML (HL60 and KG1a) cell lines with the capsid mutants resulted in an up to 36-fold increase in CML transduction efficiency (K562: 2-fold, 60% ± 2% green fluorescent protein (GFP)+ cells; BV173: 9-fold, 37% ± 2% GFP+ cells; Lama84: 36-fold, 29% ± 2% GFP+ cells) compared to controls. For AML (KG1a, HL60) and one CML cell line (EM3), no significant transduction (<1% GFP+ cells) was observed for any vector. Although the capsid mutant clone was established on a cell line, proof-of-principle experiments using primary human cells were performed. For CML (3.2-fold, mutant: 1.75% ± 0.45% GFP+ cells, p = 0.03) and PBPC (3.5-fold, mutant: 4.21% ± 3.40% GFP+ cells) a moderate increase in gene transfer of the capsid mutant compared to control vectors was observed. Conclusion Using an AAV random peptide library on a CML cell line, we were able to generate a capsid mutant, which transduced CML cell lines and primary human haematopoietic progenitor cells with higher efficiency than standard recombinant AAV vectors. PMID:18789140

The potential of achiral sponge-derived and synthetic bromoindoles as selective cytotoxins against PANC-1 tumor cells.

PubMed

Lorig-Roach, Nicholas; Hamkins-Indik, Frances; Johnson, Tyler A; Tenney, Karen; Valeriote, Frederick A; Crews, Phillip

2018-01-11

Our quest to isolate and characterize natural products with in vitro solid tumor selectivity is driven by access to repositories of Indo-Pacific sponge extracts. In this project an extract of a species of Haplosclerida sponge obtained from the US NCI Natural Products Repository displayed, by in vitro disk diffusion assay (DDA) and IC 50 determinations, selective cytotoxicity with modest potency to a human pancreatic cancer cell line (PANC-1) relative to the human lymphoblast leukemia cell line (CCRF-CEM). Two brominated indoles, the known 6-bromo conicamin ( 1 ) and the new derivative, 6-Br-8-keto-conicamin A ( 2 ), were identified and 2 (IC 50 1.5 μM for the natural product vs 4.1 μM for the synthetic material) was determined to be responsible for the cytotoxic activity of the extract against the PANC-1 tumor cell line. The new natural product and ten additional analogs were prepared for further SAR testing.

Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

PubMed Central

Carson, Cheryl; Raman, Pichai; Tullai, Jennifer; Xu, Lei; Henault, Martin; Thomas, Emily; Yeola, Sarita; Lao, Jianmin; McPate, Mark; Verkuyl, J. Martin; Marsh, George; Sarber, Jason; Amaral, Adam; Bailey, Scott; Lubicka, Danuta; Pham, Helen; Miranda, Nicolette; Ding, Jian; Tang, Hai-Ming; Ju, Haisong; Tranter, Pamela; Ji, Nan; Krastel, Philipp; Jain, Rishi K.; Schumacher, Andrew M.; Loureiro, Joseph J.; George, Elizabeth; Berellini, Giuliano; Ross, Nathan T.; Bushell, Simon M.; Erdemli, Gül; Solomon, Jonathan M.

2015-01-01

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels. PMID:26098886

The Role of the MHV Receptor and Related Glycoproteins in Murine Hepatitis Virus Infection of Murine Cell Lines

DTIC Science & Technology

1995-04-13

rhodamine-coupled goat anti -mouse antibody . A rare , fused Cl 1 D giant cell was selected to show (Al, while extensive fusion was common throughout the...mouse anti - MHV-AS9 antiserum. To quantify the lev el of susceptibility of cells to MHV infection , ten randomly selected fields for each sample...named CealO) was discovered and found to be co-expressed with MHVR in the CI 1 D and F40 lines of mouse fibroblasts. A monoclonal anti - MHVR

Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35.

PubMed

Zhao, Menglin; Wang, Jiaxian; Luo, Manyu; Luo, Han; Zhao, Meiqi; Han, Lei; Zhang, Mengxiao; Yang, Hui; Xie, Yueqing; Jiang, Hua; Feng, Lei; Lu, Huili; Zhu, Jianwei

2018-07-01

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines.

Aspects of Salt Tolerance in a NaCl-Selected Stable Cell Line of Citrus sinensis.

PubMed

Ben-Hayyim, G; Kochba, J

1983-07-01

A NaCl-tolerant cell line which was selected from ovular callus of ;Shamouti' orange (Citrus sinensis L. Osbeck) proved to be a true cell line variant. This conclusion is based on the following observations. (a) Cells which have been removed from the selection pressure for at least four passages retain the same NaCl tolerance as do cells which are kept constantly on 0.2 molar NaCl. (b) Na(+) and Cl(-) uptake are considerably lower in salt-tolerant cells (R-10) than in salt-sensitive cells (L-5) at a given external NaCl concentration. (c) Growth of salt-tolerant cells is markedly suppressed upon replacement of NaCl by KCl, whereas the growth of salt-sensitive cells is only slightly affected. Accumulation of K(+) and Cl(-) accompanies the inhibition of growth. Experiments carried out with sodium and potassium sulfate suggest that the toxic effect is due to the accumulated Cl(-). (d) Removal of Ca(2+) from the growth medium severely inhibits the growth of salt-tolerant cells in the presence of NaCl, while it has a minor effect on growth of salt-sensitive cells in the presence of NaCl. (e) Electron micrographs show that the salt-tolerant cells have very big vacuoles when exposed to salt, while the size of the vacuoles of the salt-sensitive cells does not change.

Aspects of Salt Tolerance in a NaCl-Selected Stable Cell Line of Citrus sinensis1

PubMed Central

Ben-Hayyim, Gozal; Kochba, Joshua

1983-01-01

A NaCl-tolerant cell line which was selected from ovular callus of `Shamouti' orange (Citrus sinensis L. Osbeck) proved to be a true cell line variant. This conclusion is based on the following observations. (a) Cells which have been removed from the selection pressure for at least four passages retain the same NaCl tolerance as do cells which are kept constantly on 0.2 molar NaCl. (b) Na+ and Cl− uptake are considerably lower in salt-tolerant cells (R-10) than in salt-sensitive cells (L-5) at a given external NaCl concentration. (c) Growth of salt-tolerant cells is markedly suppressed upon replacement of NaCl by KCl, whereas the growth of salt-sensitive cells is only slightly affected. Accumulation of K+ and Cl− accompanies the inhibition of growth. Experiments carried out with sodium and potassium sulfate suggest that the toxic effect is due to the accumulated Cl−. (d) Removal of Ca2+ from the growth medium severely inhibits the growth of salt-tolerant cells in the presence of NaCl, while it has a minor effect on growth of salt-sensitive cells in the presence of NaCl. (e) Electron micrographs show that the salt-tolerant cells have very big vacuoles when exposed to salt, while the size of the vacuoles of the salt-sensitive cells does not change. Images Fig. 3 PMID:16663067

Human Rhabdomyosarcoma Cell Lines for Rhabdomyosarcoma Research: Utility and Pitfalls

PubMed Central

Hinson, Ashley R. P.; Jones, Rosanne; Crose, Lisa E. S.; Belyea, Brian C.; Barr, Frederic G.; Linardic, Corinne M.

2013-01-01

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis. PMID:23882450

Essential Oil Content of the Rhizome of Curcuma purpurascens Bl. (Temu Tis) and Its Antiproliferative Effect on Selected Human Carcinoma Cell Lines

PubMed Central

Hong, Sok-Lai; Lee, Guan-Serm; Ahmed Hamdi, Omer Abdalla; Awang, Khalijah; Aznam Nugroho, Nurfina

2014-01-01

Curcuma purpurascens Bl., belonging to the Zingiberaceae family, is known as temu tis in Yogyakarta, Indonesia. In this study, the hydrodistilled dried ground rhizome oil was investigated for its chemical content and antiproliferative activity against selected human carcinoma cell lines (MCF7, Ca Ski, A549, HT29, and HCT116) and a normal human lung fibroblast cell line (MRC5). Results from GC-MS and GC-FID analysis of the rhizome oil of temu tis showed turmerone as the major component, followed by germacrone, ar-turmerone, germacrene-B, and curlone. The rhizome oil of temu tis exhibited strong cytotoxicity against HT29 cells (IC50 value of 4.9 ± 0.4 μg/mL), weak cytotoxicity against A549, Ca Ski, and HCT116 cells (with IC50 values of 46.3 ± 0.7, 32.5 ± 1.1, and 35.0 ± 0.3 μg/mL, resp.), and no inhibitory effect against MCF7 cells. It exhibited mild cytotoxicity against a noncancerous human lung fibroblast cell line (MRC5), with an IC50 value of 25.2 ± 2.7 μg/mL. This is the first report on the chemical composition of this rhizome's oil and its selective antiproliferative effect on HT29. The obtained data provided a basis for further investigation of the mode of cell death. PMID:25177723

Lucidumol C, a new cytotoxic lanostanoid triterpene from Ganoderma lingzhi against human cancer cells.

PubMed

Amen, Yhiya M; Zhu, Qinchang; Tran, Hai-Bang; Afifi, Mohamed S; Halim, Ahmed F; Ashour, Ahmed; Mira, Amira; Shimizu, Kuniyoshi

2016-07-01

A new oxygenated lanostane-type triterpene, named lucidumol C, together with six known compounds, was isolated from the chloroform extract of the fruiting bodies of Ganoderma lingzhi. Structures were established based on extensive spectroscopic and chemical studies. Potential cytotoxic activities of the isolated compounds were evaluated against human colorectal carcinoma (HCT-116, Caco-2), human liver carcinoma (HepG2), and human cervical carcinoma (HeLa) cell lines using WST-1 reagent. Selectivity was evaluated using normal human fibroblast cells (TIG-1 and HF19). Among the compounds, lucidumol C showed potent selective cytotoxicity against HCT-116 cells with an IC50 value of 7.86 ± 4.56 µM and selectivity index (SI) >10 with remarkable cytotoxic activities against Caco-2, HepG2 and HeLa cell lines.

Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.

PubMed

Feary, Marc; Racher, Andrew J; Young, Robert J; Smales, C Mark

2017-01-01

In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for increasing mAb productivity from GS-CHOK1SV cell lines. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:17-25, 2017. © 2016 American Institute of Chemical Engineers.

Recombinant protein production from stable mammalian cell lines and pools.

PubMed

Hacker, David L; Balasubramanian, Sowmya

2016-06-01

We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cytometric analysis, genetic manipulation and antibiotic selection of the snail embryonic cell line Bge from Biomphalaria glabrata, the intermediate host of Schistosoma mansoni.

PubMed

Rinaldi, Gabriel; Yan, Hongbin; Nacif-Pimenta, Rafael; Matchimakul, Pitchaya; Bridger, Joanna; Mann, Victoria H; Smout, Michael J; Brindley, Paul J; Knight, Matty

2015-07-01

The invertebrate cell line, Bge, from embryos of the snail Biomphalaria glabrata, remains to date the only established cell line from any species of the Phylum Mollusca. Since its establishment in 1976 by Eder Hansen, few studies have focused on profiling its cytometrics, growth characteristics or sensitivity to xenobiotics. Bge cells are reputed to be challenging to propagate and maintain. Therefore, even though this cell line is a noteworthy resource, it has not been studied widely. With growing interest in functional genomics, including genetic transformation, to elucidate molecular aspects of the snail intermediate hosts responsible for transmission of schistosomiasis, and aiming to enhance the convenience of maintenance of this molluscan cell line, we deployed the xCELLigene real time approach to study Bge cells. Doubling times for three isolates of Bge, termed CB, SL and UK, were longer than for mammalian cell lines - longer than 40 h in complete Bge medium supplemented with 7% fetal bovine serum at 25°C, ranging from ∼42 h to ∼157 h when 40,000 cells were seeded. To assess the potential of the cells for genetic transformation, antibiotic selection was explored. Bge cells were sensitive to the aminonucleoside antibiotic puromycin (from Streptomyces alboniger) from 5 μg/ml to 200 ng/ml, displaying a half maximal inhibitory concentration (IC50) of ∼1.91 μg/ml. Sensitivity to puromycin, and a relatively quick kill time (<48 h in 5 μg/ml) facilitated use of this antibiotic, together with the cognate resistance gene (puromycin N-acetyl-transferase) for selection of Bge cells transformed with the PAC gene (puroR). Bge cells transfected with a plasmid encoding puroR were partially rescued when cultured in the presence of 5 μg/ml of puromycin. These findings pave the way for the development of functional genomic tools applied to the host-parasite interaction during schistosomiasis and neglected tropical trematodiases at large. Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Anti-apoptosis Effect of Decoy Receptor 3 in Cholangiocarcinoma Cell Line TFK-1

PubMed Central

Xu, Ying-Chen; Cui, Jing; Zhang, Li-Jun; Zhang, Dong-Xin; Xing, Bing-Chen; Huang, Xiong-Wei-Ye; Wu, Ji-Xiang; Liang, Chao-Jie; Li, Guang-Ming

2018-01-01

Background: Decoy receptor 3 (DcR3) is a protein with anti-apoptotic effect that belongs to the tumor necrosis factor receptor superfamily. DcR3 is highly expressed in a variety of malignant tumors including cholangiocarcinoma and its expression was found to be related to the clinical stage, the invasion, and the metastasis of the tumor. This in vitro study aimed to investigate the effect of downregulated expression of DcR3 on cell viability, cell apoptosis, and cell cycle in cholangiocarcinoma cell line TFK-1. Methods: Three different cell lines were cultured: human cholangiocarcinoma TFK-1, human biliary epithelial carcinoma HuCCT-1, and human cholangiocarcinoma RBE. The cholangiocarcinoma cell line with the highest expression of DcR3 was selected for further investigation. The expression of DcR3 was silenced/knocked down by transfection with DcR3-siRNA in the selected cell line. Various biological phenotype parameters such as cell viability, apoptosis, and cell cycle were observed. Results: The mRNA and protein levels of DcR3 were measured in the three cell lines, and TFK-1 was selected. After the treatment with DcR3-siRNA for 48 h, DcR3 mRNA and protein expression in the treatment group were 38.45% (P < 0.01) and 48.03% (P < 0.05) of that of the control, respectively. It was found that the cell viability decreased to 61.87% of the control group (P < 0.01) after the downregulation of DcR3 in cholangiocarcinoma cell line TFK-1 by transfection with DcR3-siRNA, while the percentage of apoptotic cells was 2.98 times as compared with the control group (P < 0.05). Compared with the control group the ratio of G0/G1 increased, and the ratio of G2/M decreased in the treatment group. However, the differences were not statistically significant. Conclusions: The effect of DcR3 on the growth and apoptosis of cholangiocarcinoma has been demonstrated. DcR3 is not only a predictive marker for malignant tumor but it is also likely to be a potential target for cancer gene therapy. Further studies should focus on exploring the binding ligand of DcR3, the signaling pathway involved, and the molecular mechanism for the regulation of DcR3 expression in cholangiocarcinoma. PMID:29271385

Anti-apoptosis Effect of Decoy Receptor 3 in Cholangiocarcinoma Cell Line TFK-1.

PubMed

Xu, Ying-Chen; Cui, Jing; Zhang, Li-Jun; Zhang, Dong-Xin; Xing, Bing-Chen; Huang, Xiong-Wei-Ye; Wu, Ji-Xiang; Liang, Chao-Jie; Li, Guang-Ming

2018-01-05

Decoy receptor 3 (DcR3) is a protein with anti-apoptotic effect that belongs to the tumor necrosis factor receptor superfamily. DcR3 is highly expressed in a variety of malignant tumors including cholangiocarcinoma and its expression was found to be related to the clinical stage, the invasion, and the metastasis of the tumor. This in vitro study aimed to investigate the effect of downregulated expression of DcR3 on cell viability, cell apoptosis, and cell cycle in cholangiocarcinoma cell line TFK-1. Three different cell lines were cultured: human cholangiocarcinoma TFK-1, human biliary epithelial carcinoma HuCCT-1, and human cholangiocarcinoma RBE. The cholangiocarcinoma cell line with the highest expression of DcR3 was selected for further investigation. The expression of DcR3 was silenced/knocked down by transfection with DcR3-siRNA in the selected cell line. Various biological phenotype parameters such as cell viability, apoptosis, and cell cycle were observed. The mRNA and protein levels of DcR3 were measured in the three cell lines, and TFK-1 was selected. After the treatment with DcR3-siRNA for 48 h, DcR3 mRNA and protein expression in the treatment group were 38.45% (P < 0.01) and 48.03% (P < 0.05) of that of the control, respectively. It was found that the cell viability decreased to 61.87% of the control group (P < 0.01) after the downregulation of DcR3 in cholangiocarcinoma cell line TFK-1 by transfection with DcR3-siRNA, while the percentage of apoptotic cells was 2.98 times as compared with the control group (P < 0.05). Compared with the control group the ratio of G0/G1increased, and the ratio of G2/M decreased in the treatment group. However, the differences were not statistically significant. The effect of DcR3 on the growth and apoptosis of cholangiocarcinoma has been demonstrated. DcR3 is not only a predictive marker for malignant tumor but it is also likely to be a potential target for cancer gene therapy. Further studies should focus on exploring the binding ligand of DcR3, the signaling pathway involved, and the molecular mechanism for the regulation of DcR3 expression in cholangiocarcinoma.

Properties of the tick-borne encephalitis virus population during persistent infection of ixodid ticks and tick cell lines.

PubMed

Belova, Oxana A; Litov, Alexander G; Kholodilov, Ivan S; Kozlovskaya, Liubov I; Bell-Sakyi, Lesley; Romanova, Lidiya Iu; Karganova, Galina G

2017-10-01

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE), a vector-borne zoonotic neuroinfection. For successful circulation in natural foci the virus has to survive in the vector for a long period of time. Information about the effect of long-term infection of ticks on properties of the viral population is of great importance. In recent years, changes in the eco-epidemiology of TBEV due to changes in distribution of ixodid ticks have been observed. These changes in TBEV-endemic areas could result in a shift of the main tick vector species, which in turn may lead to changes in properties of the virus. In the present study we evaluated the selective pressure on the TBEV population during persistent infection of various species of ticks and tick cell lines. TBEV effectively replicated and formed persistent infection in ticks and tick cell lines of the vector species (Ixodes spp.), potential vectors (Dermacentor spp.) and non-vector ticks (Hyalomma spp.). During TBEV persistence in Ixodes and Dermacentor ticks, properties of the viral population remained virtually unchanged. In contrast, persistent TBEV infection of tick cell lines from both vector and non-vector ticks favoured selection of viral variants with low neuroinvasiveness for laboratory mice and substitutions in the E protein that increased local positive charge of the virion. Thus, selective pressure on viral population may differ in ticks and tick cell lines during persistent infection. Nevertheless, virus variants with properties of the original strain adapted to mouse CNS were not eliminated from the viral population during long-term persistence of TBEV in ticks and tick cell lines. Copyright © 2017 Elsevier GmbH. All rights reserved.

Study of cytotoxic activity, podophyllotoxin, and deoxypodophyllotoxin content in selected Juniperus species cultivated in Poland.

PubMed

Och, Marek; Och, Anna; Cieśla, Łukasz; Kubrak, Tomasz; Pecio, Łukasz; Stochmal, Anna; Kocki, Janusz; Bogucka-Kocka, Anna

2015-06-01

The demand for podophyllotoxin and deoxypodophyllotoxin is still increasing and commercially exploitable sources are few and one of them, Podophyllum hexandrum Royle (Berberidaceae), is a "critically endangered" species. The first aim was to quantify the amount of podophyllotoxin and deoxypodophyllotoxin in 61 Juniperus (Cupressaceae) samples. Cytotoxic activity of podophyllotoxin and ethanolic leaf extracts of Juniperus scopulorum Sarg. "Blue Pacific" and Juniperus communis L. "Depressa Aurea" was examined against different leukemia cell lines. Ultra-performance liquid chromatography (UPLC) analysis was performed with the use of a Waters ACQUITY UPLC(TM) system (Waters Corp., Milford, MA). The peaks of podophyllotoxin and deoxypodophyllotoxin were assigned on the basis of their retention data and mass-to-charge ratio (m/z). Trypan blue assay was performed to obtain IC50 cytotoxicity values against selected leukemia cell lines. Juniperus scopulorum was characterized with the highest level of podophyllotoxin (486.7 mg/100 g DW) while Juniperus davurica Pall. contained the highest amount of deoxypodophyllotoxin (726.8 mg/100 g DW). Podophyllotoxin IC50 cytotoxicity values against J45.01 and CEM/C1 leukemia cell lines were 0.0040 and 0.0286 µg/mL, respectively. Juniperus scopulorum extract examined against J45.01 and HL-60/MX2 leukemia cell lines gave the respective IC50 values: 0.369-9.225 µg/mL. Juniperus communis extract was characterized with the following IC50 cytotoxity values against J45.01 and U-266B1 cell lines: 3.310-24.825 µg/mL. Juniperus sp. can be considered as an alternative source of podophyllotoxin and deoxypodophyllotoxin. Cytotoxic activity of podophyllotoxin and selected leaf extracts of Juniperus sp. against a set of leukemia cell lines was demonstrated.

Decursin enhances TRAIL-induced apoptosis through oxidative stress mediated- endoplasmic reticulum stress signalling in non-small cell lung cancers.

PubMed

Kim, Jaekwang; Yun, Miyong; Kim, Eun-Ok; Jung, Deok-Beom; Won, Gunho; Kim, Bonglee; Jung, Ji Hoon; Kim, Sung-Hoon

2016-03-01

The TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent due to its remarkable ability to selectively kill tumour cells. However, because most tumours exhibit resistance to TRAIL-induced apoptosis, the development of combination therapies to overcome resistance to TRAIL is required for effective cancer therapy. Cell viability and possible synergy between the plant pyranocoumarin decursin and TRAIL was measured by MTT assay and calcusyn software. Reactive oxygen species (ROS) and apoptosis were measured using dichlorodihydrofluorescein and annexin/propidium iodide in cell flow cytometry. Changes in protein levels were assessed with Western blotting. Combining decursin and TRAIL markedly decreased cell viability and increased apoptosis in TRAIL-resistant non-small-cell lung cancer (NSCLC) cell lines. Decursin induced expression of the death receptor 5 (DR5). Inhibition of DR5 attenuated apoptotic cell death in decursin + TRAIL treated NSCLC cell lines. Interestingly, induction of DR5 and CCAAT/enhancer-binding protein homologues protein by decursin was mediated through selective induction of the pancreatic endoplasmic reticulum kinase (PERK)/activating transcription factor 4 (ATF4) branch of the endoplasmic reticulum stress response pathway. Furthermore, enhancement of PERK/ATF4 signalling by decursin was mediated by ROS generation in NSCLC cell lines, but not in normal human lung cells. Decursin also markedly down-regulated expression of survivin and Bcl-xL in TRAIL-resistant NSCLC cells. ROS generation by decursin selectively activated the PERK/ATF4 axis of the endoplasmic reticulum stress signalling pathway, leading to enhanced TRAIL sensitivity in TRAIL-resistant NSCLC cell lines, partly via up-regulation of DR5. © 2015 The British Pharmacological Society.

Decursin enhances TRAIL‐induced apoptosis through oxidative stress mediated‐ endoplasmic reticulum stress signalling in non‐small cell lung cancers

PubMed Central

Kim, Jaekwang; Yun, Miyong; Kim, Eun‐Ok; Jung, Deok‐Beom; Won, Gunho; Kim, Bonglee; Jung, Ji Hoon

2016-01-01

Background and Purpose The TNF‐related apoptosis‐inducing ligand (TRAIL) is a promising anticancer agent due to its remarkable ability to selectively kill tumour cells. However, because most tumours exhibit resistance to TRAIL‐induced apoptosis, the development of combination therapies to overcome resistance to TRAIL is required for effective cancer therapy. Experimental Approach Cell viability and possible synergy between the plant pyranocoumarin decursin and TRAIL was measured by MTT assay and calcusyn software. Reactive oxygen species (ROS) and apoptosis were measured using dichlorodihydrofluorescein and annexin/propidium iodide in cell flow cytometry. Changes in protein levels were assessed with Western blotting. Key Results Combining decursin and TRAIL markedly decreased cell viability and increased apoptosis in TRAIL‐resistant non‐small‐cell lung cancer (NSCLC) cell lines. Decursin induced expression of the death receptor 5 (DR5). Inhibition of DR5 attenuated apoptotic cell death in decursin + TRAIL treated NSCLC cell lines. Interestingly, induction of DR5 and CCAAT/enhancer‐binding protein homologues protein by decursin was mediated through selective induction of the pancreatic endoplasmic reticulum kinase (PERK)/activating transcription factor 4 (ATF4) branch of the endoplasmic reticulum stress response pathway. Furthermore, enhancement of PERK/ATF4 signalling by decursin was mediated by ROS generation in NSCLC cell lines, but not in normal human lung cells. Decursin also markedly down‐regulated expression of survivin and Bcl‐xL in TRAIL‐resistant NSCLC cells. Conclusions and Implications ROS generation by decursin selectively activated the PERK/ATF4 axis of the endoplasmic reticulum stress signalling pathway, leading to enhanced TRAIL sensitivity in TRAIL‐resistant NSCLC cell lines, partly via up‐regulation of DR5. PMID:26661339

Anti-proliferative activities on HeLa cancer cell line of Thai medicinal plant recipes selected from MANOSROI II database.

PubMed

Manosroi, Jiradej; Boonpisuttinant, Korawinwich; Manosroi, Worapaka; Manosroi, Aranya

2012-07-13

The Thai/Lanna medicinal plant recipe database "MANOSROI II" contained the medicinal plant recipes of all regions in Thailand for the treatment of various diseases including anti-cancer medicinal plant recipes. To investigate anti-proliferative activity on HeLa cell lines of medicinal plant recipes selected from the Thai/Lanna medicinal plant recipe database "MANOSROI II". The forty aqueous extracts of Thai/Lanna medicinal plant recipes selected from the Thai/Lanna medicinal plant recipe database "MANOSROI II" were investigated for anti-proliferative activity on HeLa cell line by SRB assay. The apoptosis induction by caspase-3 activity and MMP-2 inhibition activity by zymography on HeLa cell line of the three selected aqueous extracts, which gave the highest anti-proliferative activity were determined. Phytochemicals and anti-oxidative activities including free radical scavenging activity, inhibition of lipid peroxidation and metal chelating inhibition activities were also investigated. Sixty percentages of the medicinal plant recipes selected from "MANOSROI II" database showed anti-proliferative activity on HeLa cell line. The recipes of N031(Albizia chinensis (Osbeck) Merr, Cassia fistula L., and Dargea volubilis Benth.ex Hook. etc.), N039 (Nymphoides indica L., Peltophorum pterocarpum (DC.), and Polyalthia debilis Finet et Gagnep etc.) and N040 (Nymphoides indica L. Kuntze, Sida rhombifolia L., and Xylinbaria minutiflora Pierre. etc.) gave higher anti-proliferative activity than the standard anti-cancer drug, cisplatin of 1.25, 1.29 and 30.18 times, respectively. The positive relationship between the anti-proliferative activity and the MMP-2 inhibition activity and metal chelating inhibition activity was observed, but no relationship between the anti-proliferative activity and apoptosis induction, free radical scavenging activity and lipid peroxidation inhibition activity. Phytochemicals found in these extracts were alkaloids, flavonoids, tannins and xanthones, but not anthraquinones and carotenoids. The recipe N040 exhibited the highest anti-proliferative and MMP-2 inhibition on HeLa cancer cell line at 30 and threefolds of cisplatin, respectively (p<0.05), while recipe N031 gave the highest caspase-3 activity (1.29-folds over the control) (p<0.05). This study has demonstrated that recipe N040 selected from MANOSROI II database appeared to be a good candidate with high potential for the further development as an anti-cancer agent. Crown Copyright © 2012. Published by Elsevier Ireland Ltd. All rights reserved.

Resistance of a soybean cell line to oxyfluorfen by overproduction of mitochondrial protoporphyrinogen oxidase.

PubMed

Warabi, E; Usui, K; Tanaka, Y; Matsumoto, H

2001-08-01

The diphenyl ether herbicide oxyfluorfen (2-chloro-4-trifluoromethylphenyl 3-ethoxy-4-nitrophenyl ether) inhibits protoporphyrinogen oxidase (Protox) which catalyzes the oxidation of protoporphyrinogen IX (Protogen) to protoporphyrin IX (Proto IX), the last step of the common pathway to chlorophyll and haeme biosynthesis. We have selected an oxyfluorfen-resistant soybean cell line by stepwise selection methods, and the resistance mechanism has been investigated. No growth inhibition was observed in resistant cells at a concentration of 10(-7) M oxyfluorfen, a concentration at which normal cells did not survive. While the degree of inhibition of total extractable Protox by oxyfluorfen was the same in both cell types, the enzyme activity in the mitochondrial fraction from non-treated resistant cells was about nine-fold higher than that from normal cells. Northern analysis of mitochondrial Protox revealed that the concentration of mitochondrial Protox mRNA was much higher in resistant cells than that in normal cells. There were no differences in the absorption and metabolic breakdown of oxyfluorfen. The growth of resistant cells was also insensitive to oxadiazon [5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2-(3H)- one], the other chemical class of Protox inhibitor. Therefore, the resistance of the selected soybean cell line to oxyfluorfen is probably mainly due to the overproduction of mitochondrial Protox.

Inactivation of an integrated antibiotic resistance gene in mammalian cells to re-enable antibiotic selection.

PubMed

Ni, Peiling; Zhang, Qian; Chen, Haixia; Chen, Lingyi

2014-01-01

Removing an antibiotic resistance gene allows the same antibiotic to be re-used in the next round of genetic manipulation. Here we applied the CRISPR/Cas system to disrupt the puromycin resistance gene in an engineered mouse embryonic stem cell line and then re-used puromycin selection in the resulting cells to establish stable reporter cell lines. With the CRISPR/Cas system, pre-engineered sequences, such as loxP or FRT, are not required. Thus, this technique can be used to disrupt antibiotic resistance genes that cannot be removed by the Cre-loxP and Flp-FRT systems.

A Study of the Effects of High Power Pulsed 2450 MHz Microwaves, ELF modulated Microwaves, and ELF Fields on Human Lymphocytes and Selected Cell Lines

DTIC Science & Technology

1993-01-27

Considerable effect was expended in investigating shifts in intercellular calcium of one particular cell line, Jurket, using flow cytometry methods. No...culture. The following analysis were used to characterize the immortalized cell lines: flow cytometry , electron microscopy, two-dimensional protein gel...further characterized by flow cytometry , electron microscopy, two dimensional protein electrophoresis and nuclear run-off assay. Flow cytometric analysis of

Anticancer drug sensitivity prediction in cell lines from baseline gene expression through recursive feature selection.

PubMed

Dong, Zuoli; Zhang, Naiqian; Li, Chun; Wang, Haiyun; Fang, Yun; Wang, Jun; Zheng, Xiaoqi

2015-06-30

An enduring challenge in personalized medicine is to select right drug for individual patients. Testing drugs on patients in large clinical trials is one way to assess their efficacy and toxicity, but it is impractical to test hundreds of drugs currently under development. Therefore the preclinical prediction model is highly expected as it enables prediction of drug response to hundreds of cell lines in parallel. Recently, two large-scale pharmacogenomic studies screened multiple anticancer drugs on over 1000 cell lines in an effort to elucidate the response mechanism of anticancer drugs. To this aim, we here used gene expression features and drug sensitivity data in Cancer Cell Line Encyclopedia (CCLE) to build a predictor based on Support Vector Machine (SVM) and a recursive feature selection tool. Robustness of our model was validated by cross-validation and an independent dataset, the Cancer Genome Project (CGP). Our model achieved good cross validation performance for most drugs in the Cancer Cell Line Encyclopedia (≥80% accuracy for 10 drugs, ≥75% accuracy for 19 drugs). Independent tests on eleven common drugs between CCLE and CGP achieved satisfactory performance for three of them, i.e., AZD6244, Erlotinib and PD-0325901, using expression levels of only twelve, six and seven genes, respectively. These results suggest that drug response could be effectively predicted from genomic features. Our model could be applied to predict drug response for some certain drugs and potentially play a complementary role in personalized medicine.

Structure-Activity Relationships of Nitro-Substituted Aroylhydrazone Iron Chelators with Antioxidant and Antiproliferative Activities.

PubMed

Hrušková, Kateřina; Potůčková, Eliška; Opálka, Lukáš; Hergeselová, Tereza; Hašková, Pavlína; Kovaříková, Petra; Šimůnek, Tomáš; Vávrová, Kateřina

2018-05-23

Aroylhydrazone iron chelators such as salicylaldehyde isonicotinoyl hydrazone (SIH) protect various cells against oxidative injury and display antineoplastic activities. Previous studies have shown that a nitro-substituted hydrazone, namely, NHAPI, displayed markedly improved plasma stability, selective antitumor activity, and moderate antioxidant properties. In this study, we prepared four series of novel NHAPI derivatives and explored their iron chelation activities, anti- or pro-oxidant effects, protection against model oxidative injury in the H9c2 cell line derived from rat embryonic cardiac myoblasts, cytotoxicities to the corresponding noncancerous H9c2 cells, and antiproliferative activities against the MCF-7 human breast adenocarcinoma and HL-60 human promyelocytic leukemia cell lines. Nitro substitution had both negative and positive effects on the examined properties, and we identified new structure-activity relationships. Naphthyl and biphenyl derivatives showed selective antiproliferative action, particularly in the breast adenocarcinoma MCF-7 cell line, where they exceeded the selectivity of the parent compound NHAPI. Of particular interest is a compound prepared from 2-hydroxy-5-methyl-3-nitroacetophenone and biphenyl-4-carbohydrazide, which protected cardiomyoblasts against oxidative injury at 1.8 ± 1.2 μM with 24-fold higher selectivity than SIH. These compounds will serve as leads for further structural optimization and mechanistic studies.

The Therapeutic Potential of AN-7, a Novel Histone Deacetylase Inhibitor, for Treatment of Mycosis Fungoides/Sezary Syndrome Alone or with Doxorubicin

PubMed Central

Goldfeiz, Neta; Rephaeli, Ada; Nudelman, Abraham; Weitman, Michal; Tarasenko, Nataly; Gorovitz, Batia; Maron, Leah; Yehezkel, Shiran; Amitay-Laish, Iris; Lubin, Ido; Hodak, Emmilia

2016-01-01

The 2 histone deacetylase inhibitors (HDACIs) approved for the treatment of cutaneous T-cell lymphoma (CTCL) including mycosis fungoides/sezary syndrome (MF/SS), suberoylanilide hydroxamic acid (SAHA) and romidepsin, are associated with low rates of overall response and high rates of adverse effects. Data regarding combination treatments with HDACIs is sparse. Butyroyloxymethyl diethylphosphate (AN-7) is a novel HDACI, which was found to have selective anticancer activity in several cell lines and animal models. The aim of this study was to compare the anticancer effects of AN-7 and SAHA, either alone or combined with doxorubicin, on MF/SS cell lines and peripheral blood lymphocytes (PBL) from patients with Sezary syndrome (SPBL). MyLa cells, Hut78 cells, SPBL, and PBL from healthy normal individuals (NPBL) were exposed to the test drugs, and the findings were analyzed by a viability assay, an apoptosis assay, and Western blot. AN-7 was more selectively toxic to MyLa cells, Hut78 cells, and SPBL (relative to NPBL) than SAHA and also acted more rapidly. Both drugs induced apoptosis in MF/SS cell lines, SAHA had a greater effect on MyLa cell line, while AN-7 induced greater apoptosis in SPBL; both caused an accumulation of acetylated histone H3, but AN-7 was associated with earlier kinetics; and both caused a downregulation of the HDAC1 protein in MF/SS cell lines. AN-7 acted synergistically with doxorubicin in both MF/SS cell lines and SPBL, and antagonistically with doxorubicin in NPBL. By contrast, SAHA acted antagonistically with doxorubicin on MF/SS cell lines, SPBL, and NPBL, leaving <50% viable cells. In conclusion, AN-7 holds promise as a therapeutic agent in MF/SS and has several advantages over SAHA. Our data provide a rationale for combining AN-7, but not SAHA, with doxorubicin to induce the cell death in MF/SS. PMID:26752418

The Therapeutic Potential of AN-7, a Novel Histone Deacetylase Inhibitor, for Treatment of Mycosis Fungoides/Sezary Syndrome Alone or with Doxorubicin.

PubMed

Moyal, Lilach; Feldbaum, Nataly; Goldfeiz, Neta; Rephaeli, Ada; Nudelman, Abraham; Weitman, Michal; Tarasenko, Nataly; Gorovitz, Batia; Maron, Leah; Yehezkel, Shiran; Amitay-Laish, Iris; Lubin, Ido; Hodak, Emmilia

2016-01-01

The 2 histone deacetylase inhibitors (HDACIs) approved for the treatment of cutaneous T-cell lymphoma (CTCL) including mycosis fungoides/sezary syndrome (MF/SS), suberoylanilide hydroxamic acid (SAHA) and romidepsin, are associated with low rates of overall response and high rates of adverse effects. Data regarding combination treatments with HDACIs is sparse. Butyroyloxymethyl diethylphosphate (AN-7) is a novel HDACI, which was found to have selective anticancer activity in several cell lines and animal models. The aim of this study was to compare the anticancer effects of AN-7 and SAHA, either alone or combined with doxorubicin, on MF/SS cell lines and peripheral blood lymphocytes (PBL) from patients with Sezary syndrome (SPBL). MyLa cells, Hut78 cells, SPBL, and PBL from healthy normal individuals (NPBL) were exposed to the test drugs, and the findings were analyzed by a viability assay, an apoptosis assay, and Western blot. AN-7 was more selectively toxic to MyLa cells, Hut78 cells, and SPBL (relative to NPBL) than SAHA and also acted more rapidly. Both drugs induced apoptosis in MF/SS cell lines, SAHA had a greater effect on MyLa cell line, while AN-7 induced greater apoptosis in SPBL; both caused an accumulation of acetylated histone H3, but AN-7 was associated with earlier kinetics; and both caused a downregulation of the HDAC1 protein in MF/SS cell lines. AN-7 acted synergistically with doxorubicin in both MF/SS cell lines and SPBL, and antagonistically with doxorubicin in NPBL. By contrast, SAHA acted antagonistically with doxorubicin on MF/SS cell lines, SPBL, and NPBL, leaving <50% viable cells. In conclusion, AN-7 holds promise as a therapeutic agent in MF/SS and has several advantages over SAHA. Our data provide a rationale for combining AN-7, but not SAHA, with doxorubicin to induce the cell death in MF/SS.

Effects of mycoplasmal pneumonia of swine (MPS) lung lesion-selected Landrace pigs on MPS resistance and immune competence in three-way crossbred pigs.

PubMed

Borjigin, Liushiqi; Shimazu, Tomoyuki; Katayama, Yuki; Watanabe, Kouichi; Kitazawa, Haruki; Roh, Sang-Gun; Aso, Hisashi; Katoh, Kazuo; Satoh, Masahiro; Suda, Yoshihito; Sakuma, Akiko; Nakajo, Mituru; Suzuki, Keiichi

2017-04-01

To clarify the genetic influence of mycoplasmal pneumonia of swine (MPS) lesion-selected Landrace (La) on MPS resistance and immune characteristics in three-way crossbred pigs (LaWaDa), the LaWaDa pigs were compared with the non-selected crossbred (LbWbDb) and purebred (La) pigs. The MPS lesion score in the three lines was as follows: La line < LaWaDa line < LbWbDb line, with significant differences among the lines. The proportions of myeloid cells and T cells were lower and higher, respectively, in the LaWaDa pigs compared with those in the other two lines. Messenger RNA (mRNA) expression of interleukin (IL)-6, IL-10, transforming growth factor-β, and interferon-γ in peripheral blood was significantly increased after vaccination in the La and LaWaDa lines. IL-4 mRNA expression in the LaWaDa line was intermediate to the La and LbWbDb lines. Furthermore, principal component analysis for immune traits and MPS lesions was executed to clarify the characteristics of each pig line. These findings suggest that the immune responses in the three pig lines are genetically distinct and that MPS resistance and some immunity characteristics from the La line were transmitted to the three-way crossbred pigs. © 2016 Japanese Society of Animal Science.

Biotin conjugated organic molecules and proteins for cancer therapy: A review.

PubMed

Maiti, Santanu; Paira, Priyankar

2018-02-10

The main transporter for biotin is sodium dependent multivitamin transporter (SMVT), which is overexpressed in various aggressive cancer cell lines such as ovarian (OV 2008, ID8), leukemia (L1210FR), mastocytoma (P815), colon (Colo-26), breast (4T1, JC, MMT06056), renal (RENCA, RD0995), and lung (M109) cancer cell lines. Furthermore, its overexpression was found higher to that of folate receptor. Therefore, biotin demand in the rapidly growing tumors is higher than normal tissues. Several biotin conjugated organic molecules has been reported here for selective delivery of the drug in cancer cell. Biotin conjugated molecules are showing higher fold of cytotoxicity in biotin positive cancer cell lines than the normal cell. Nanoparticles and polymer surface modified drugs and biotin mediated cancer theranostic strategy was highlighted in this review. The cytotoxicity and selectivity of the drug in cancer cells has enhanced after biotin conjugation. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Synthetic Lethal Strategy Identifies a Potent and Selective TTK and CLK2 Inhibitor for Treatment of Triple-negative Breast Cancer with a Compromised G1/S Checkpoint.

PubMed

Zhu, Dan; Xu, Shuichan; Deyanat-Yazdi, Gordafaried; Peng, Sophie X; Barnes, Leo A; Narla, Rama Krishna; Tran, Tam; Mikolon, David; Ning, Yuhong; Shi, Tao; Jiang, Ning; Raymon, Heather K; Riggs, Jennifer R; Boylan, John F

2018-06-04

Historically, phenotypic-based drug discovery has yielded a high percentage of novel drugs while uncovering new tumor biology. CC-671 was discovered using a phenotypic screen for compounds that preferentially induced apoptosis in triple negative breast cancer cell lines while sparing luminal breast cancer cell lines. Detailed in vitro kinase profiling shows CC-671 potently and selectively inhibits two kinases-TTK and CLK2. Cellular mechanism of action studies demonstrate that CC-671 potently inhibits the phosphorylation of KNL1 and SRp75, direct TTK and CLK2 substrates, respectively. Furthermore, CC-671 causes mitotic acceleration and modification of pre-mRNA splicing leading to apoptosis, consistent with cellular TTK and CLK inhibition. Correlative analysis of genomic and potency data against a large panel of breast cancer cell lines identifies breast cancer cells with a dysfunctional G1/S checkpoint as more sensitive to CC-671, suggesting synthetic lethality between G1/S checkpoint and TTK/CLK2 inhibition. Furthermore, significant in vivo CC-671 efficacy was demonstrated in two cell line-derived and one patient tumor-derived xenograft models of TNBC following weekly dosing. These findings are the first to demonstrate the unique inhibitory combination activity of a dual TTK/CLK2 inhibitor that preferably kills TNBC cells and shows synthetic lethality with a compromised G1/S checkpoint in breast cancer cell lines. Based on these data, CC-671 was moved forward for clinical development as a potent and selective TTK/CLK2 inhibitor in a subset of TNBC patients. Copyright ©2018, American Association for Cancer Research.

Quantitative proteomic analysis reveals effects of epidermal growth factor receptor (EGFR) on invasion-promoting proteins secreted by glioblastoma cells.

PubMed

Sangar, Vineet; Funk, Cory C; Kusebauch, Ulrike; Campbell, David S; Moritz, Robert L; Price, Nathan D

2014-10-01

Glioblastoma multiforme is a highly invasive and aggressive brain tumor with an invariably poor prognosis. The overexpression of epidermal growth factor receptor (EGFR) is a primary influencer of invasion and proliferation in tumor cells and the constitutively active EGFRvIII mutant, found in 30-65% of Glioblastoma multiforme, confers more aggressive invasion. To better understand how EGFR contributes to tumor aggressiveness, we investigated the effect of EGFR on the secreted levels of 65 rationally selected proteins involved in invasion. We employed selected reaction monitoring targeted mass spectrometry using stable isotope labeled internal peptide standards to quantity proteins in the secretome from five GBM (U87) isogenic cell lines in which EGFR, EGFRvIII, and/or PTEN were expressed. Our results show that cell lines with EGFR overexpression and constitutive EGFRvIII expression differ remarkably in the expression profiles for both secreted and intracellular signaling proteins, and alterations in EGFR signaling result in reproducible changes in concentrations of secreted proteins. Furthermore, the EGFRvIII-expressing mutant cell line secretes the majority of the selected invasion-promoting proteins at higher levels than other cell lines tested. Additionally, the intracellular and extracellular protein measurements indicate elevated oxidative stress in the EGFRvIII-expressing cell line. In conclusion, the results of our study demonstrate that EGFR signaling has a significant effect on the levels of secreted invasion-promoting proteins, likely contributing to the aggressiveness of Glioblastoma multiforme. Further characterization of these proteins may provide candidates for new therapeutic strategies and targets as well as biomarkers for this aggressive disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The cAMP analogs have potent anti-proliferative effects on medullary thyroid cancer cell lines.

PubMed

Dicitore, Alessandra; Grassi, Elisa Stellaria; Caraglia, Michele; Borghi, Maria Orietta; Gaudenzi, Germano; Hofland, Leo J; Persani, Luca; Vitale, Giovanni

2016-01-01

The oncogenic activation of the rearranged during transfection (RET) proto-oncogene has a main role in the pathogenesis of medullary thyroid cancer (MTC). Several lines of evidence suggest that RET function could be influenced by cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity. We evaluated the in vitro anti-tumor activity of 8-chloroadenosine-3',5'-cyclic monophosphate (8-Cl-cAMP) and PKA type I-selective cAMP analogs [equimolar combination of the 8-piperidinoadenosine-3',5'-cyclic monophosphate (8-PIP-cAMP) and 8-hexylaminoadenosine-3',5'-cyclic monophosphate (8-HA-cAMP) in MTC cell lines (TT and MZ-CRC-1)]. 8-Cl-cAMP and the PKA I-selective cAMP analogs showed a potent anti-proliferative effect in both cell lines. In detail, 8-Cl-cAMP blocked significantly the transition of TT cell population from G2/M to G0/G1 phase and from G0/G1 to S phase and of MZ-CRC-1 cells from G0/G1 to S phase. Moreover, 8-Cl-cAMP induced apoptosis in both cell lines, as demonstrated by FACS analysis for annexin V-FITC/propidium iodide, the activation of caspase-3 and PARP cleavage. On the other hand, the only effect induced by PKA I-selective cAMP analogs was a delay in G0/G1-S and S-G2/M progression in TT and MZ-CRC-1 cells, respectively. In conclusion, these data demonstrate that cAMP analogs, particularly 8-Cl-cAMP, significantly suppress in vitro MTC proliferation and provide rationale for a potential clinical use of cAMP analogs in the treatment of advanced MTC.

The T47D cell line is an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer.

PubMed

Yu, Sungryul; Kim, Taemook; Yoo, Kyung Hyun; Kang, Keunsoo

2017-05-06

Cell lines are often used as in vitro tools to mimic certain types of in vivo system; several cell lines, including MCF-7 and T47D, have been widely used in breast cancer studies without investigating the cell lines' characteristics. In this study, we compared the genome-wide binding profiles of ERα, PR, and P300, and the gene expression changes between MCF-7 and T47D cell lines that represent the luminal A subtype of breast cancer. Surprisingly, several thousand genes were differentially expressed under estrogenic condition. In addition, ERα, PR, and P300 binding to regulatory elements showed distinct genomic landscapes between MCF-7 and T47D cell lines in the same hormonal states. In particular, the T47D cell line was markedly susceptible to progesterone, whereas the MCF-7 cell line did not respond to progesterone in the presence of estrogen. Consistently, changes in the expression level of the PR-target gene, STAT5A, were only observed in the T47D cell line, not the MCF-7 cell line, when treated with progesterone. Overall, the results highlight the importance of selecting appropriate cell lines for breast cancer studies and suggest that T47D cell lines can be an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Myeloid cell leukemia-1 is an important apoptotic survival factor in triple-negative breast cancer.

PubMed

Goodwin, C M; Rossanese, O W; Olejniczak, E T; Fesik, S W

2015-12-01

Breast cancer is the second-most frequently diagnosed malignancy in US women. The triple-negative breast cancer (TNBC) subtype, which lacks expression of the estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2, afflicts 15% of patients and is refractory to current targeted therapies. Like many cancers, TNBC cells often deregulate programmed cell death by upregulating anti-apoptotic proteins of the B-cell CLL/lymphoma 2 (Bcl-2) family. One family member, myeloid cell leukemia-1 (Mcl-1), is commonly amplified in TNBC and correlates with a poor clinical prognosis. Here we show the effect of silencing Mcl-1 and Bcl-2-like protein 1 isoform 1 (Bcl-xL) expression on viability in a panel of seventeen TNBC cell lines. Cell death was observed in a subset upon Mcl-1 knockdown. In contrast, Bcl-xL knockdown only modestly reduced viability, indicating that Mcl-1 is a more important survival factor. However, dual silencing of both Mcl-1 and Bcl-xL reduced viability in most cell lines tested. These proliferation results were recapitulated by BH3 profiling experiments. Treatment with a Bcl-xL and Bcl-2 peptide had only a moderate effect on any of the TNBC cell lines, however, co-dosing an Mcl-1-selective peptide with a peptide that inhibits Bcl-xL and Bcl-2 was effective in each line tested. Similarly, the selective Bcl-xL inhibitor WEHI-539 was only weakly cytotoxic across the panel, but sensitization by Mcl-1 knockdown markedly improved its EC50. ABT-199, which selectively inhibits Bcl-2, did not synergize with Mcl-1 knockdown, indicating the relatively low importance of Bcl-2 in these lines. Mcl-1 sensitivity is not predicted by mRNA or protein levels of a single Bcl-2 family member, except for only a weak correlation for Bak and Bax protein expression. However, a more comprehensive index composed of Mcl-1, Bcl-xL, Bim, Bak and Noxa protein or mRNA expression correlates well with Mcl-1 sensitivity in TNBC and can also predict Mcl-1 dependency in non-small cell lung cancer cell lines.

Tofacitinib induces G1 cell-cycle arrest and inhibits tumor growth in Epstein-Barr virus-associated T and natural killer cell lymphoma cells.

PubMed

Ando, Shotaro; Kawada, Jun-Ichi; Watanabe, Takahiro; Suzuki, Michio; Sato, Yoshitaka; Torii, Yuka; Asai, Masato; Goshima, Fumi; Murata, Takayuki; Shimizu, Norio; Ito, Yoshinori; Kimura, Hiroshi

2016-11-22

Epstein-Barr virus (EBV) infects not only B cells, but also T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoma. These lymphoid malignancies are refractory to conventional chemotherapy. We examined the activation of the JAK3/STAT5 pathway in EBV-positive and -negative B, T and NK cell lines and in cell samples from patients with EBV-associated T cell lymphoma. We then evaluated the antitumor effects of the selective JAK3 inhibitor, tofacitinib, against these cell lines in vitro and in a murine xenograft model. We found that all EBV-positive T and NK cell lines and patient samples tested displayed activation of the JAK3/STAT5 pathway. Treatment of these cell lines with tofacitinib reduced the levels of phospho-STAT5, suppressed proliferation, induced G1 cell-cycle arrest and decreased EBV LMP1 and EBNA1 expression. An EBV-negative NK cell line was also sensitive to tofacitinib, whereas an EBV-infected NK cell line was more sensitive to tofacitinib than its parental line. Tofacitinib significantly inhibited the growth of established tumors in NOG mice. These findings suggest that tofacitinib may represent a useful therapeutic agent for patients with EBV-associated T and NK cell lymphoma.

Human CD4 T cell epitopes selective for Vaccinia versus Variola virus.

PubMed

Probst, Alicia; Besse, Aurore; Favry, Emmanuel; Imbert, Gilles; Tanchou, Valérie; Castelli, Florence Anne; Maillere, Bernard

2013-04-01

Due to the high degree of sequence identity between Orthopoxvirus species, the specific B and T cell responses raised against these viruses are largely cross-reactive and poorly selective. We therefore searched for CD4 T cell epitopes present in the conserved parts of the Vaccinia genome (VACV) but absent from Variola viruses (VARV), with a view to identifying immunogenic sequences selective for VACV. We identified three long peptide fragments from the B7R, B10R and E7R proteins by in silico comparisons of the poxvirus genomes, and evaluated the recognition of these fragments by VACV-specific T cell lines derived from healthy donors. For the 12 CD4 T cell epitopes identified, we assessed their binding to common HLA-DR allotypes and their capacity to induce peptide-specific CD4 T-cell lines. Four peptides from B7R and B10R displayed a broad binding specificity for HLA-DR molecules and induced multiple T cell lines from healthy donors. Besides their absence from VARV, the two B10R peptide sequences were mutated in the Cowpox virus and completely absent from the Monkeypox genome. This work contributes to the development of differential diagnosis of poxvirus infections. Copyright © 2012 Elsevier Ltd. All rights reserved.

A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines

PubMed Central

Sciamanna, Ilaria; Gualtieri, Alberto; Cossetti, Cristina; Osimo, Emanuele Felice; Ferracin, Manuela; Macchia, Gianfranco; Aricò, Eleonora; Prosseda, Gianni; Vitullo, Patrizia; Misteli, Tom; Spadafora, Corrado

2013-01-01

LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the ‘normal’ small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the transformed state in tumor cells. PMID:24345856

Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase.

PubMed

He, Shan; Li, Yangyang; Chen, Yang; Zhu, Yue; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

2016-08-01

Pigs are the most economically important livestock, but pig cell lines useful for physiological studies and/or vaccine development are limited. Although several pig cell lines have been generated by oncogene transformation or human telomerase reverse transcriptase (TERT) immortalization, these cell lines contain viral sequences and/or antibiotic resistance genes. In this study, we established a new method for generating pig cell lines using the Sleeping Beauty (SB) transposon-mediated ectopic expression of porcine telomerase reverse transcriptase (pTERT). The performance of the new method was confirmed by generating a pig fibroblast cell (PFC) line. After transfection of primary PFCs with the SB transposon system, one cell clone containing the pTERT expression cassette was selected by dilution cloning and passed for different generations. After passage for more than 40 generations, the cell line retained stable expression of ectopic pTERT and continuous growth potential. Further characterization showed that the cell line kept the fibroblast morphology, growth curve, population doubling time, cloning efficiency, marker gene expression pattern, cell cycle distribution and anchorage-dependent growth property of the primary cells. These data suggest that the new method established is useful for generating pig cell lines without viral sequence and antibiotic resistant gene.

Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis.

PubMed Central

Arsenakis, M; Hubenthal-Voss, J; Campadelli-Fiume, G; Pereira, L; Roizman, B

1986-01-01

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells. Images PMID:3022001

Effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine on several parameters of Epstein-Barr virus infection.

PubMed

Zhang, Z X; Liu, Y X; Chen, H C; Allaudeen, H S; De Clercq, E

1984-01-01

The selective and potent anti-herpesvirus drug, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU), has been examined for its inhibitory effects on several parameters of Epstein-Barr virus (EBV) infection in the lymphoblastoid cell lines Raji, P3HR-1, B-95-8 and P3 hybrid cells (a human embryo oropharyngeal cell line fused with a nasopharyngeal carcinoma cell line). At a dosage of 0.03 to 0.1 mM, BVdU caused a marked inhibition of (i) spontaneous viral capsid antigen (VCA) expression in B-95-8 and P3 hybrid cells, (ii) VCA expression and DNA synthesis in B-95-8 cells induced with croton oil and n-butyrate, (iii) early antigen (EA) expression and DNA synthesis in Raji cells superinfected with EBV, and (iv) VCA expression and DNA synthesis in B-95-8 cells superinfected with EBV. In its inhibitory effects on these various parameters of EBV infection, BVdU appears to be comparable to acyclovir [9-(2-hydroxyethoxymethyl)guanine], another selective anti-herpesvirus drug which has been previously recognized as an effective inhibitor of EBV replication.

Cytotoxic activity of kenaf (Hibiscus cannabinus L.) seed extract and oil against human cancer cell lines.

PubMed

Wong, Yu Hua; Tan, Wai Yan; Tan, Chin Ping; Long, Kamariah; Nyam, Kar Lin

2014-05-01

To examine the cytotoxic properties of both the kenaf (Hibiscus cannabinus L.) seed extract and kenaf seed oil on human cervical cancer, human breast cancer, human colon cancer and human lung cancer cell lines. The in vitro cytotoxic activity of the kenaf (Hibiscus cannabinus L.) seed extract and kenaf seed oil on human cancer cell lines was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and sulforhodamine B assays. Cell morphological changes were observed by using an inverted light microscope. The kenaf seed extract (KSE) exhibited a lower IC50 than kenaf seed oil (KSO) in all of the cancer cell lines. Morphological alterations in the cell lines after KSE and KSO treatment were observed. KSE and KSO possessed effective cytotoxic activities against all the cell lines been selected. KSE and KSO could be potential sources of natural anti-cancer agents. Further investigations on using kenaf seeds for anti-proliferative properties are warranted.

Cytotoxic activity of kenaf (Hibiscus cannabinus L.) seed extract and oil against human cancer cell lines

PubMed Central

Wong, Yu Hua; Tan, Wai Yan; Tan, Chin Ping; Long, Kamariah; Nyam, Kar Lin

2014-01-01

Objective To examine the cytotoxic properties of both the kenaf (Hibiscus cannabinus L.) seed extract and kenaf seed oil on human cervical cancer, human breast cancer, human colon cancer and human lung cancer cell lines. Methods The in vitro cytotoxic activity of the kenaf (Hibiscus cannabinus L.) seed extract and kenaf seed oil on human cancer cell lines was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and sulforhodamine B assays. Cell morphological changes were observed by using an inverted light microscope. Results The kenaf seed extract (KSE) exhibited a lower IC50 than kenaf seed oil (KSO) in all of the cancer cell lines. Morphological alterations in the cell lines after KSE and KSO treatment were observed. KSE and KSO possessed effective cytotoxic activities against all the cell lines been selected. Conclusions KSE and KSO could be potential sources of natural anti-cancer agents. Further investigations on using kenaf seeds for anti-proliferative properties are warranted. PMID:25183141

The insulin-like growth factor-1 receptor inhibitor PPP produces only very limited resistance in tumor cells exposed to long-term selection.

PubMed

Vasilcanu, D; Weng, W-H; Girnita, A; Lui, W-O; Vasilcanu, R; Axelson, M; Larsson, O; Larsson, C; Girnita, L

2006-05-25

The cyclolignan PPP was recently demonstrated to inhibit the activity of insulin-like growth factor-1 receptor (IGF-1R), without affecting the highly homologous insulin receptor. In addition, PPP caused complete regression of xenografts derived from various types of cancer. These data highlight the use of this compound in cancer treatment. However, a general concern with antitumor agents is development of resistance. In light of this problem, we aimed to investigate whether malignant cells may develop serious resistance to PPP. After trying to select 10 malignant cell lines, with documented IGF-1R expression and apoptotic responsiveness to PPP treatment (IC50s less than 0.1 microM), only two survived an 80-week selection but could only tolerate maximal PPP doses of 0.2 and 0.5 microM, respectively. Any further increase in the PPP dose resulted in massive cell death. These two cell lines were demonstrated not to acquire any essential alteration in responsiveness to PPP regarding IGF-1-induced IGF-1R phosphorylation. Neither did they exhibit any increase in expression of the multidrug resistance proteins MDR1 or MRP1. Consistently, they did not exhibit decreased sensitivity to conventional cytostatic drugs. Rather, the sensitivity was increased. During the first half of the selection period, both cell lines responded with a temporary and moderate increase in IGF-1R expression, which appeared to be because of an increased transcription of the IGF-1R gene. This increase in IGF-1R might be necessary to make cells competent for further selection but only up to a PPP concentration of 0.2 and 0.5 microM. In conclusion, malignant cells develop no or remarkably weak resistance to the IGF-1R inhibitor PPP.

Cell type-specific manipulation with GFP-dependent Cre recombinase.

PubMed

Tang, Jonathan C Y; Rudolph, Stephanie; Dhande, Onkar S; Abraira, Victoria E; Choi, Seungwon; Lapan, Sylvain W; Drew, Iain R; Drokhlyansky, Eugene; Huberman, Andrew D; Regehr, Wade G; Cepko, Constance L

2015-09-01

There are many transgenic GFP reporter lines that allow the visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. We developed a method that exploits GFP for gene manipulation, Cre recombinase dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, which led to effective recombination selectively in GFP-labeled cells. Furthermore, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP(+) cells, we found that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins.

The costs of using unauthenticated, over-passaged cell lines: how much more data do we need?

PubMed

Hughes, Peyton; Marshall, Damian; Reid, Yvonne; Parkes, Helen; Gelber, Cohava

2007-11-01

Increasing data demonstrate that cellular cross-contamination, misidentified cell lines, and the use of cultures at high-passage levels contribute to the generation of erroneous and misleading results as well as wasted research funds. Contamination of cell lines by other lines has been recognized and documented back to the 1950s. Based on submissions to major cell repositories in the last decade, it is estimated that between 18% and 36% of cell lines may be contaminated or misidentified. More recently, problems surrounding practices of over-subculturing cells are being identified. As a result of selective pressures and genetic drift, cell lines, when kept in culture too long, exhibit reduced or altered key functions and often no longer represent reliable models of their original source material. A review of papers showing significant experimental variances between low- and high-passage cell culture numbers, as well as contaminated lines, makes a strong case for using verified, tested cell lines at low- or defined passage numbers. In the absence of cell culture guidelines, mandates from the National Institutes of Health (NIH) and other funding agencies or journal requirements, it becomes the responsibility of the scientific community to perform due diligence to ensure the integrity of cell cultures used in research.

Production of Zebrafish Offspring from Cultured Female Germline Stem Cells

PubMed Central

Wong, Ten-Tsao; Tesfamichael, Abraham; Collodi, Paul

2013-01-01

Zebrafish female germline stem cell (FGSC) cultures were generated from a transgenic line of fish that expresses Neo and DsRed under the control of the germ cell specific promoter, ziwi [Tg(ziwi:neo);Tg(ziwi:DsRed)]. Homogeneous FGSC cultures were established by G418 selection and continued to express ziwi for more than 6 weeks along with the germ cell markers nanos3, dnd, dazl and vasa. A key component of the cell culture system was the use of a feeder cell line that was initiated from ovaries of a transgenic line of fish [Tg(gsdf:neo)] that expresses Neo controlled by the zebrafish gonadal soma derived factor (gsdf) promoter. The feeder cell line was selected in G418 and engineered to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and glial-cell-line derived neurotrophic factor (Gdnf). These factors were shown to significantly enhance FGSC growth, survival and germline competency in culture. Results from cell transplantation experiments revealed that the cultured FGSCs were able to successfully colonize the gonad of sterile recipient fish and generate functional gametes. Up to 20% of surviving recipient fish that were injected with the cultured FGSCs were fertile and generated multiple batches of normal offspring for at least 6 months. The FGSC cultures will provide an in vitro system for studies of zebrafish germ cell growth and differentiation and their high frequency of germline transmission following transplantation could form the basis of a stem cell-mediated strategy for gene transfer and manipulation of the zebrafish genome. PMID:23671620

Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

PubMed Central

Gardell, Alison M.; Qin, Qin; Rice, Robert H.; Li, Johnathan; Kültz, Dietmar

2014-01-01

Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish. PMID:24797371

The impact of intermittent versus continuous exposure to EGFR tyrosine kinase inhibitor on selection of EGFR T790M-mutant drug-resistant clones in a lung cancer cell line carrying activating EGFR mutation

PubMed Central

Lee, Youngjoo; Choi, Yu-Ra; Kim, Kyoung-Yeon; Shin, Dong Hoon

2016-01-01

Drug-resistant cell lines are essential tools for investigating the mechanisms of resistance to molecular-targeted anti-cancer drugs. However, little is known about how to establish clinically relevant drug-resistant cell lines. Our study examined the impact of a drug-free period on the establishment of a cell line with clinically relevant resistance to molecular-targeted drugs. We used PC9 cells, a lung cancer cell line carrying EGFR mutation, because this is a validated target for EGFR tyrosine kinase inhibitors (TKI). PC9 cells were intermittently or continuously exposed to increasing concentrations of gefitinib (0.01 μM to 1.0 μM) and the emergence of the most common acquired resistance mutation in EGFR, T790M, was determined. T790M was detected at a 25-fold lower drug concentration in cells continuously exposed to gefitinib (PC9/GRc) than in cells intermittently exposed to gefitinib (PC9/GRi) (0.04 μM vs 1.0 μM, respectively). The mutation frequencies at those drug concentrations were 19.8% and 8.0% in PC9/GRc and PC9/GRi cells, respectively. After drug-free culture for 8 weeks, resistance to gefitinib decreased in the PC9/GRi cells but not in the PC9/GRc cells. In the PC9/GRc cells, the frequency of the T790M mutation was consistently about 20% from 0.04 μM to 1.0 μM of gefitinib. In the PC9/GRc cells, the T790M mutation was detected in all single-cell clones, at frequencies ranging from 7.0% to 37.0%, with a median of 19.5% (95% confidence interval, 17.3%–20.9%). In conclusion, compared with intermittent drug exposure, continuous exposure might select better minor drug-resistant clones when creating cell lines resistant to molecular-targeted drugs. PMID:27270313

The impact of intermittent versus continuous exposure to EGFR tyrosine kinase inhibitor on selection of EGFR T790M-mutant drug-resistant clones in a lung cancer cell line carrying activating EGFR mutation.

PubMed

Lee, Youngjoo; Choi, Yu-Ra; Kim, Kyoung-Yeon; Shin, Dong Hoon

2016-07-12

Drug-resistant cell lines are essential tools for investigating the mechanisms of resistance to molecular-targeted anti-cancer drugs. However, little is known about how to establish clinically relevant drug-resistant cell lines. Our study examined the impact of a drug-free period on the establishment of a cell line with clinically relevant resistance to molecular-targeted drugs. We used PC9 cells, a lung cancer cell line carrying EGFR mutation, because this is a validated target for EGFR tyrosine kinase inhibitors (TKI). PC9 cells were intermittently or continuously exposed to increasing concentrations of gefitinib (0.01 μM to 1.0 μM) and the emergence of the most common acquired resistance mutation in EGFR, T790M, was determined. T790M was detected at a 25-fold lower drug concentration in cells continuously exposed to gefitinib (PC9/GRc) than in cells intermittently exposed to gefitinib (PC9/GRi) (0.04 μM vs 1.0 μM, respectively). The mutation frequencies at those drug concentrations were 19.8% and 8.0% in PC9/GRc and PC9/GRi cells, respectively. After drug-free culture for 8 weeks, resistance to gefitinib decreased in the PC9/GRi cells but not in the PC9/GRc cells. In the PC9/GRc cells, the frequency of the T790M mutation was consistently about 20% from 0.04 μM to 1.0 μM of gefitinib. In the PC9/GRc cells, the T790M mutation was detected in all single-cell clones, at frequencies ranging from 7.0% to 37.0%, with a median of 19.5% (95% confidence interval, 17.3%-20.9%). In conclusion, compared with intermittent drug exposure, continuous exposure might select better minor drug-resistant clones when creating cell lines resistant to molecular-targeted drugs.

Antitumor Agents. 272. Structure–Activity Relationships and In Vivo Selective Anti-Breast Cancer Activity of Novel Neo-tanshinlactone Analogs

PubMed Central

Dong, Yizhou; Shi, Qian; Pai, Huei-Chen; Peng, Chieh-Yu; Pan, Shiow-Lin; Teng, Che-Ming; Nakagawa-Goto, Kyoko; Yu, Donglei; Liu, Yi-Nan; Wu, Pei-Chi; Bastow, Kenneth F.; Morris-Natschke, Susan L.; Brossi, Arnold; Lang, Jing-Yu; Hsu, Jennifer L.; Hung, Mien-Chie; Lee, Eva Y.-H. P.; Lee, Kuo-Hsiung

2010-01-01

Neo-tanshinlactone (1) and its previously reported analogs, such as 2, are potent and selective in vitro anti-breast cancer agents. The synthetic pathway to 2 was optimized from seven to five steps, with a better overall yield. Structure–activity relationships studies on these compounds revealed some key molecular determinants for this family of anti-breast agents. Several derivatives (19-21 and 24) exerted potent and selective anti-breast cancer activity with IC50 values of 0.3, 0.2, 0.1 and 0.1 μg/mL, respectively, against the ZR-75-1 cell lines. Compound 24 was two- to three-fold more potent than 1 against SK-BR-3 and ZR-75-1. Importantly, 21 exhibited high selectivity; it was 23 times more active against ZR-75-1 than MCF-7. Compound 20 had an approximately 12-fold ratio of SK-BR-3/MCF-7 selectivity. In addition, analog 2 showed potent activity against a ZR-75-1 xenograft model, but not PC-3 and MDA-MB-231 xenografts, as well as high selectivity against breast cancer cell line compared with normal breast tissue-derived cell lines. Further development of lead compounds 19-21 and 24 as clinical trial candidates is warranted. PMID:20148565

Akt mediated ROS-dependent selective targeting of mutant KRAS tumors.

PubMed

Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

2014-10-01

Reactive oxygen species (ROS) play a critical role in a variety of cellular processes, ranging from cell survival and proliferation to cell death. Previously, we reported the ability of a small molecule compound, C1, to induce ROS dependent autophagy associated apoptosis in human cancer cell lines and primary tumor cells (Wong C. et al. 2010). Our ongoing investigations have unraveled a hitherto undefined novel signaling network involving hyper-phosphorylation of Akt and Akt-mediated ROS production in cancer cell lines. Interestingly, drug-induced Akt activation is selectively seen in cell lines that carry mutant KRAS; HCT116 cells that carry the V13D KRAS mutation respond favorably to C1 while HT29 cells expressing wild type KRAS are relatively resistant. Of note, not only does the compound target mutant KRAS expressing cells but also induces RAS activation as evidenced by the PAK pull down assay. Corroborating this, pharmacological inhibition as well as siRNA mediated silencing of KRAS or Akt, blocked C1-induced ROS production and rescued tumor colony forming ability in HCT116 cells. To further confirm the involvement of KRAS, we made use of mutant KRAS transformed RWPE-1 prostate epithelial cells. Notably, drug-induced ROS generation and death sensitivity was significantly higher in RWPE-1-KRAS cells than the RWPE-1-vector cells, thus confirming the results obtained with mutant KRAS colorectal carcinoma cell line. Lastly, we made use of HCT116 mutant KRAS knockout cells (KO) where the mutant KRAS allele had been deleted, thus expressing a single wild-type KRAS allele. Exposure of the KO cells to C1 failed to induce Akt activation and mitochondrial ROS production. Taken together, results show the involvement of activated Akt in ROS-mediated selective targeting of mutant KRAS expressing tumors, which could have therapeutic implications given the paucity of chemotherapeutic strategies specifically targeting KRAS mutant cancers. Copyright © 2014. Published by Elsevier Inc.

Expression of apoptosis-regulatory genes in lung tumour cell lines: relationship to p53 expression and relevance to acquired drug resistance.

PubMed Central

Reeve, J. G.; Xiong, J.; Morgan, J.; Bleehen, N. M.

1996-01-01

As a first step towards elucidating the potential role(s) of bcl-2 and bcl-2-related genes in lung tumorigenesis and therapeutic responsiveness, the expression of these genes has been examined in a panel of lung cancer cell lines derived from untreated and treated patients, and in cell lines selected in vitro for multidrug resistance. Bcl-2 was hyperexpressed in 15 of 16 small-cell lung cancer (SCLC) cell lines and two of five non-small-cell lung cancer (NSCLC) lines compared with normal lung and brain, and hyperexpression was not chemotherapy related. Bcl-x was hyperexpressed in the majority of SCLC and NSCLC cell lines as compared with normal tissues, and all lung tumour lines preferentially expressed bcl-x1-mRNA, the splice variant form that inhibits apoptosis. Bax gene transcripts were hyperexpressed in most SCLC and NSCLC cell lines examined compared with normal adult tissues. Mutant p53 gene expression was detected in the majority of the cell lines and no relationship between p53 gene expression and the expression of either bcl-2, bcl-x or bax was observed. No changes in bcl-2, bcl-x and bax gene expression were observed in multidrug-resistant cell lines compared with their drug-sensitive counterparts. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8630278

Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies

PubMed Central

Lehmann, Brian D.; Bauer, Joshua A.; Chen, Xi; Sanders, Melinda E.; Chakravarthy, A. Bapsi; Shyr, Yu; Pietenpol, Jennifer A.

2011-01-01

Triple-negative breast cancer (TNBC) is a highly diverse group of cancers, and subtyping is necessary to better identify molecular-based therapies. In this study, we analyzed gene expression (GE) profiles from 21 breast cancer data sets and identified 587 TNBC cases. Cluster analysis identified 6 TNBC subtypes displaying unique GE and ontologies, including 2 basal-like (BL1 and BL2), an immunomodulatory (IM), a mesenchymal (M), a mesenchymal stem–like (MSL), and a luminal androgen receptor (LAR) subtype. Further, GE analysis allowed us to identify TNBC cell line models representative of these subtypes. Predicted “driver” signaling pathways were pharmacologically targeted in these cell line models as proof of concept that analysis of distinct GE signatures can inform therapy selection. BL1 and BL2 subtypes had higher expression of cell cycle and DNA damage response genes, and representative cell lines preferentially responded to cisplatin. M and MSL subtypes were enriched in GE for epithelial-mesenchymal transition, and growth factor pathways and cell models responded to NVP-BEZ235 (a PI3K/mTOR inhibitor) and dasatinib (an abl/src inhibitor). The LAR subtype includes patients with decreased relapse-free survival and was characterized by androgen receptor (AR) signaling. LAR cell lines were uniquely sensitive to bicalutamide (an AR antagonist). These data may be useful in biomarker selection, drug discovery, and clinical trial design that will enable alignment of TNBC patients to appropriate targeted therapies. PMID:21633166

Characterisation of the p53 pathway in cell lines established from TH-MYCN transgenic mouse tumours.

PubMed

Chen, Lindi; Esfandiari, Arman; Reaves, William; Vu, Annette; Hogarty, Michael D; Lunec, John; Tweddle, Deborah A

2018-03-01

Cell lines established from the TH-MYCN transgenic murine model of neuroblastoma are a valuable preclinical, immunocompetent, syngeneic model of neuroblastoma, for which knowledge of their p53 pathway status is important. In this study, the Trp53 status and functional response to Nutlin-3 and ionising radiation (IR) were determined in 6 adherent TH-MYCN transgenic cell lines using Sanger sequencing, western blot analysis and flow cytometry. Sensitivity to structurally diverse MDM2 inhibitors (Nutlin-3, MI-63, RG7388 and NDD0005) was determined using XTT proliferation assays. In total, 2/6 cell lines were Trp53 homozygous mutant (NHO2A and 844MYCN+/+) and 1/6 (282MYCN+/-) was Trp53 heterozygous mutant. For 1/6 cell lines (NHO2A), DNA from the corresponding primary tumour was found to be Trp53 wt. In all cases, the presence of a mutation was consistent with aberrant p53 signalling in response to Nutlin-3 and IR. In comparison to TP53 wt human neuroblastoma cells, Trp53 wt murine control and TH-MYCN cell lines were significantly less sensitive to growth inhibition mediated by MI-63 and RG7388. These murine Trp53 wt and mutant TH-MYCN cell lines are useful syngeneic, immunocompetent neuroblastoma models, the former to test p53-dependent therapies in combination with immunotherapies, such as anti-GD2, and the latter as models of chemoresistant relapsed neuroblastoma when aberrations in the p53 pathway are more common. The spontaneous development of Trp53 mutations in 3 cell lines from TH-MYCN mice may have arisen from MYCN oncogenic driven and/or ex vivo selection. The identified species-dependent selectivity of MI-63 and RG7388 should be considered when interpreting in vivo toxicity studies of MDM2 inhibitors.

An Online Compendium of CHO RNA-Seq Data Allows Identification of CHO Cell Line-specific Transcriptomic Signatures.

PubMed

Singh, Ankita; Kildegaard, Helene F; Andersen, Mikael R

2018-05-15

Chinese hamster ovary (CHO) cell lines can fold, assemble and modify proteins post-translationally to produce human-like proteins; as a consequence, it is the single most common expression systems for industrial production of recombinant therapeutic proteins. A thorough knowledge of cultivation conditions of different CHO cell lines has been developed over the last decade, but comprehending gene or pathway-specific distinctions between CHO cell lines at transcriptome level remains a challenge. To address these challenges, we compiled a compendium of 23 RNA-Seq studies from public and in-house data on CHO cell lines, i.e. CHO-S, CHO-K1 and DG44. Significantly differentially expressed (DE) genes particularly related to subcellular structure and macromolecular categories were used to identify differences between the cell lines. A R-based web application was developed specifically for CHO cell lines to further visualize expression values across different cell lines, and make available the normalized full CHO data set graphically as a CHO research community resource. This study quantitatively categorizes CHO cell lines based on patterns at transcriptomic level and detects gene and pathway specific key distinctions among sibling cell lines. Studies such as this can be used to select desired characteristics across various CHO cell lines. Furthermore, the availability of the data as an internet-based application can be applied to broad range of CHO engineering applications. This article is protected by copyright. All rights reserved.

A phenotypic screening approach to identify anticancer compounds derived from marine fungi.

PubMed

Ellinger, Bernhard; Silber, Johanna; Prashar, Anjali; Landskron, Johannes; Weber, Jonas; Rehermann, Sarah; Müller, Franz-Josef; Smith, Stephen; Wrigley, Stephen; Taskén, Kjetil; Gribbon, Philip; Labes, Antje; Imhoff, Johannes F

2014-04-01

This study covers the isolation, testing, and identification of natural products with anticancer properties. Secondary metabolites were isolated from fungal strains originating from a variety of marine habitats. Strain culture protocols were optimized with respect to growth media composition and fermentation conditions. From these producers, isolated compounds were screened for their effect on the viability and proliferation of a subset of the NCI60 panel of cancer cell lines. Active compounds of interest were identified and selected for detailed assessments and structural elucidation using nuclear magnetic resonance. This revealed the majority of fungal-derived compounds represented known anticancer chemotypes, confirming the integrity of the process and the ability to identify suitable compounds. Examination of effects of selected compounds on cancer-associated cell signaling pathways used phospho flow cytometry in combination with 3D fluorescent cell barcoding. In parallel, the study addressed the logistical aspects of maintaining multiple cancer cell lines in culture simultaneously. A potential solution involving microbead-based cell culture was investigated (BioLevitator, Hamilton). Selected cell lines were cultured in microbead and 2D methods and cell viability tests showed comparable compound inhibition in both methods (R2=0.95). In a further technology assessment, an image-based assay system was investigated for its utility as a possible complement to ATP-based detection for quantifying cell growth and viability in a label-free manner.

CCI-007, a novel small molecule with cytotoxic activity against infant leukemia with MLL rearrangements

PubMed Central

Middlemiss, Shiloh M.C.; Wen, Victoria W.; Clifton, Molly; Kwek, Alan; Liu, Bing; Mayoh, Chelsea; Bongers, Angelika; Karsa, Mawar; Pan, Sukey; Cruikshank, Sarah; Scandlyn, Marissa; Hoang, Wendi; Imamura, Toshihiko; Kees, Ursula R.; Gudkov, Andrei V.; Chernova, Olga B.

2016-01-01

There is an urgent need for the development of less toxic, more selective and targeted therapies for infants with leukemia characterized by translocation of the mixed lineage leukemia (MLL) gene. In this study, we performed a cell-based small molecule library screen on an infant MLL-rearranged (MLL-r) cell line, PER-485, in order to identify selective inhibitors for MLL-r leukemia. After screening initial hits for a cytotoxic effect against a panel of 30 cell lines including MLL-r and MLL wild-type (MLL-wt) leukemia, solid tumours and control cells, small molecule CCI-007 was identified as a compound that selectively and significantly decreased the viability of a subset of MLL-r and related leukemia cell lines with CALM-AF10 and SET-NUP214 translocation. CCI-007 induced a rapid caspase-dependent apoptosis with mitochondrial depolarization within twenty-four hours of treatment. CCI-007 altered the characteristic MLL-r gene expression signature in sensitive cells with downregulation of the expression of HOXA9, MEIS1, CMYC and BCL2, important drivers in MLL-r leukemia, within a few hours of treatment. MLL-r leukemia cells that were resistant to the compound were characterised by significantly higher baseline gene expression levels of MEIS1 and BCL2 in comparison to CCI-007 sensitive MLL-r leukemia cells. In conclusion, we have identified CCI-007 as a novel small molecule that displays rapid toxicity towards a subset of MLL-r, CALM-AF10 and SET-NUP214 leukemia cell lines. Our findings suggest an important new avenue in the development of targeted therapies for these deadly diseases and indicate that different therapeutic strategies might be needed for different subtypes of MLL-r leukemia. PMID:27317766

Studies on mimicry of naturally occurring annonaceous acetogenins: non-THF analogues leading to remarkable selective cytotoxicity against human tumor cells.

PubMed

Zeng, Bu-Bing; Wu, Yikang; Jiang, Sheng; Yu, Qian; Yao, Zhu-Jun; Liu, Zhong-Hai; Li, Hong-Yan; Li, Yan; Chen, Xiao-Guang; Wu, Yu-Lin

2003-01-03

A class of structurally simplified analogues of the naturally occurring annonaceous acetogenins were developed, amongst which some non-THF analogues showed remarkable cytotoxicities against tumor cell lines, as well as good selectivity between human tumor cells and normal cells. The synthetic routes were significantly shortened because of the removal of the chiral centers bearing the THF rings on the natural templates. This simplification also provides access to the parallel synthesis of these mimics by a combinatorial strategy. The remaining stereogenic centers at the positions alpha to the ethereal links were introduced by the Chiron approach from the easily accessible chiral building blocks 6a and/or 6b, made in turn from L-ascorbic acid or D-mannitol, while the one in the butenolide segment was taken from L-lactate. All four diastereomeric non-THF analogues 2a-2d showed remarkable activity against the HCT-8 cell line, and better differentiation was found when testing against the HT-29 cell line. It was also discovered that both the butenolide and ethylene glycol subunits play essential roles in the cytotoxicities against tumor cell lines, while the 10-substituted hydroxy group and the absolute configuration of methyl group at the butenolide moiety are less important for their activity.

Selective loss of nucleoside carrier expression in rat hepatocarcinomas.

PubMed

Dragan, Y; Valdés, R; Gomez-Angelats, M; Felipe, A; Javier Casado, F; Pitot, H; Pastor-Anglada, M

2000-08-01

Evidence that hepatoma cell lines show differential expression of concentrative nucleoside transporters (CNT1 and CNT2) prompted us to study the transporter proteins in 2 models of hepatocarcinogenesis, the chemically induced Solt and Farber model and the albumin-SV40 large T antigen (Alb-SV40) transgenic rat. CNT1 expression was lower in tumor biopsy specimens from Alb-SV40 rat livers than in normal tissue. Immunocytochemistry revealed that the CNT1 protein was indeed absent in the tumor lesions. CNT1 was also absent in a cell line, L25, derived from the Alb-SV40 transgenic rat liver tumors, whereas another cell line, L37, derived from the normal-appearing parenchyma, retained the expression of both carrier isoforms. The protein expression correlated with the nucleoside transport properties of these cell lines. Moreover, although CNT2 expression was highly dependent on the growth characteristics of the 2 cell lines, as was CNT1 (albeit to a lower extent) in L37 cells, it was not expressed in L25 cells at any stage of cell growth. In contrast to the transgenic model of hepatocarcinogenesis, in the chemically induced tumors the expression of CNT2 was lower, although still detectable. In summary, these data indicate that hepatocarcinogenesis leads to a selective loss or diminished expression of nucleoside carrier isoforms, a feature that may be relevant to our understanding of the molecular basis of the bioavailability of those drugs that are nucleoside derivatives and may be substrates of these carriers. The transport properties and isoform-expression profile of the L25 and L37 cell lines make them suitable hepatocyte culture models with which to study nucleoside transport processes and drug sensitivity.

Cytogenetics of small cell carcinoma of the lung.

PubMed

Wurster-Hill, D H; Cannizzaro, L A; Pettengill, O S; Sorenson, G D; Cate, C C; Maurer, L H

1984-12-01

Nineteen cell lines derived from various malignant tissues of 15 patients with small cell carcinoma of the lung (SCCL) have been studied. The results showed heterogeneity in all cell lines, with no one consistent abnormality among them. Cell lines from 11 of the patients had minute and double minute chromosomes, and cell lines from 2 patients had abnormally banding regions, designated as ABRs, as distinguished from homogeneously staining regions (HSRs). The latter 2 and several of the former cell lines were derived from specimens taken before the patients were placed on therapy. All but 2 of the cell lines had a constant marker load, consisting of 24%-35% of the complement. Some markers remained stable through months and years of culture life, while other markers came and went. Chromosomes #1, #6 and #11 were most frequently involved in marker formation in the cell lines, and these were compared to similar markers in direct bone marrow preparations. Chromosome #1 markers were of variable structure, whereas #6 and #11 most often took the form of 6q- and 11p+ markers, with breakpoints most frequently at 6q23-25 and 11p11-12. A 3p- marker was found in a minority of cell lines. All of these markers were also found in direct marrow preparations from some patients with SCCL. Nonmonoclonal tumors arose from inoculation of bimodal cell lines into nude mice, but population selection by undetermined mechanism was evident. Cytogenetic parameters showed no positive correlation with hormone production by these cell lines.

5-(Furan-2-yl)-4-(3,4,5-trimethoxyphenyl)-3H-1,2-dithiol-3-one oxime (6f), a new synthetic compound, causes human fibrosarcoma HT-1080 cell apoptosis by disrupting tubulin polymerisation and inducing G2/M arrest.

PubMed

Zuo, Daiying; Pang, Lili; Shen, Jiwei; Guan, Qi; Bai, Zhaoshi; Zhang, Huijuan; Li, Yao; Lu, Guodong; Zhang, Weige; Wu, Yingliang

2017-06-01

In the current study, we synthesized a series of new compounds targeting tubulin and tested their anti-proliferative activities. Among these new synthetic com-pounds, 5-(furan-2-yl)-4-(3,4,5-trimethoxyphenyl)-3H-1,2-dithiol-3-one oxime (6f) exhibited significant anti-proliferative activity against different human cancer cell lines including human gastric adenocarcinoma SGC-7901, human non-small cell lung cancer A549, and human fibrosarcoma HT-1080. As a result, 6f was selected to further test the sensitivity to different cancer cell lines including human cervical cancer cell line HeLa, human breast cancer cell line MCF-7, non-small cell lung cancer cell line A549, human liver carcinoma cell line HepG-2, human oral squamous cell carcinoma cell lines KB, SGC-7901 and HT-1080. Among these cell lines, HT-1080 and HeLa are the most sensitive. Therefore, HT-1080 was selected to further explore the properties of anti-proliferative activity and the underlying mechanisms. Our data proved that 6f exhibited strong anti-proliferative effects against HT-1080 cells in a time- and dose-dependent manner. We showed that the growth inhibitory effect of 6f in HT-1080 cells was related with microtubule depolymerisation. Molecular docking studies revealed that 6f interacted and bound efficiently with the colchicine-binding site of tubulin. In addition, 6f treatment induced G2/M cell cycle arrest dose-dependently and subsequently induced cell apoptosis. Western blot study indicated that upregulation of cyclin B1 and p-cdc2 was related with G2/M arrest. 6f-induced cell apoptosis was associated with both mitochondrial and death receptor pathway. In conclusion, our data showed that 6f, among the newly synthetic compounds, exhibited highest anti-proliferative activity by disrupting the microtubule polymerisation, causing G2/M arrest and subsequently inducing cell apoptosis in HT-1080 cells. Hence, 6f is a promising microtubule depolymerising agent for the treatment of various cancers especially human fibrosarcoma.

[Application of dhfr gene negative Chinese hamster ovary cell line to express hepatitis B virus surface antigen].

PubMed

Yi, Y; Zhang, M; Liu, C

2001-06-01

To set up an efficient expressing system for recombinant hepatitis B virus surface antigen (HBsAg) in dhfr gene negative CHO cell line. HBsAg gene expressing plasmid pCI-dhfr-S was constructed by integrating HBsAg gene into plasmid pCI which carries dhfr gene. The HBsAg expressing cell line was set up by transfection of plasmid pCI-dhfr-S into dhfr gene negative CHO cell line in the way of lipofectin. Under the selective pressure of MTX, 18 of 28 clonized cell lines expressed HBsAg, 4 of them reached a high titer of 1:32 and protein content 1-3 micrograms/ml. In this study, the high level expression of HBsAg demonstrated that the dhfr negative mammalian cell line when recombined with plasmid harboring the corresponding deleted gene can efficiently express the foreign gene. The further steps toward building optimum conditions of the expressing system and the increase of expressed product are under study.

PI3K pathway dependencies in endometrioid endometrial cancer cell lines

PubMed Central

Weigelt, Britta; Warne, Patricia H; Lambros, Maryou B; Reis-Filho, Jorge S; Downward, Julian

2013-01-01

Purpose Endometrioid endometrial cancers (EECs) frequently harbor coexisting mutations in PI3K pathway genes, including PTEN, PIK3CA, PIK3R1, and KRAS. We sought to define the genetic determinants of PI3K pathway inhibitor response in EEC cells, and whether PTEN-mutant EEC cell lines rely on p110β signaling for survival. Experimental Design Twenty-four human EEC cell lines were characterized for their mutation profile and activation state of PI3K and MAPK signaling pathway proteins. Cells were treated with pan-class I PI3K, p110α and p110β isoform-specific, allosteric mTOR, mTOR kinase, dual PI3K/mTOR, MEK and RAF inhibitors. RNA interference (RNAi) was employed to assess effects of KRAS silencing in EEC cells. Results EEC cell lines harboring PIK3CA and PTEN mutations were selectively sensitive to the pan-class I PI3K inhibitor GDC-0941 and allosteric mTOR inhibitor Temsirolimus, respectively. Subsets of EEC cells with concurrent PIK3CA and/or PTEN and KRAS mutations were sensitive to PI3K pathway inhibition, and only 2/6 KRAS-mutant cell lines showed response to MEK inhibition. KRAS RNAi silencing did not induce apoptosis in KRAS-mutant EEC cells. PTEN-mutant EEC cell lines were resistant to the p110β inhibitors GSK2636771 and AZD6482, and only in combination with the p110α selective inhibitor A66, a decrease in cell viability was observed. Conclusions Targeted pan-PI3K and mTOR inhibition in EEC cells may be most effective in PIK3CA-mutant and PTEN-mutant tumors, respectively, even in a subset of EECs concurrently harboring KRAS mutations. Inhibition of p110β alone may not be sufficient to sensitize PTEN-mutant EEC cells and combination with other targeted agents may be required. PMID:23674493

PI3K pathway dependencies in endometrioid endometrial cancer cell lines.

PubMed

Weigelt, Britta; Warne, Patricia H; Lambros, Maryou B; Reis-Filho, Jorge S; Downward, Julian

2013-07-01

Endometrioid endometrial cancers (EEC) frequently harbor coexisting mutations in phosphoinositide 3-kinase (PI3K) pathway genes, including PTEN, PIK3CA, PIK3R1, and KRAS. We sought to define the genetic determinants of PI3K pathway inhibitor response in EEC cells, and whether PTEN-mutant EEC cell lines rely on p110β signaling for survival. Twenty-four human EEC cell lines were characterized for their mutation profile and activation state of PI3K and mitogen-activated protein kinase (MAPK) signaling pathway proteins. Cells were treated with pan-class I PI3K, p110α, and p110β isoform-specific, allosteric mTOR, mTOR kinase, dual PI3K/mTOR, mitogen-activated protein/extracellular signal-regulated kinase (MEK), and RAF inhibitors. RNA interference (RNAi) was used to assess effects of KRAS silencing in EEC cells. EEC cell lines harboring PIK3CA and PTEN mutations were selectively sensitive to the pan-class I PI3K inhibitor GDC-0941 and allosteric mTOR inhibitor temsirolimus, respectively. Subsets of EEC cells with concurrent PIK3CA and/or PTEN and KRAS mutations were sensitive to PI3K pathway inhibition, and only 2 of 6 KRAS-mutant cell lines showed response to MEK inhibition. KRAS RNAi silencing did not induce apoptosis in KRAS-mutant EEC cells. PTEN-mutant EEC cell lines were resistant to the p110β inhibitors GSK2636771 and AZD6482, and only in combination with the p110α selective inhibitor A66 was a decrease in cell viability observed. Targeted pan-PI3K and mTOR inhibition in EEC cells may be most effective in PIK3CA- and PTEN-mutant tumors, respectively, even in a subset of EECs concurrently harboring KRAS mutations. Inhibition of p110β alone may not be sufficient to sensitize PTEN-mutant EEC cells and combination with other targeted agents may be required. ©2013 AACR.

Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: Characterization of a Selective Allosteric Kinase Inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagashima, Kumiko; Shumway, Stuart D.; Sathyanarayanan, Sriram

2013-11-20

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitivemore » kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.« less

Selective effects of quercetin on the cell growth and antioxidant defense system in normal versus transformed mouse hepatic cell lines.

PubMed

Son, Young-Ok; Lee, Kyung-Yeol; Kook, Sung-Ho; Lee, Jeong-Chae; Kim, Jong-Ghee; Jeon, Young-Mi; Jang, Yong-Suk

2004-10-19

Quercetin is a dietary anticancer chemical that is capable of inducing apoptosis in tumor cells. However, little is known about its biological effect on nonmalignant cells, although the effect is one of the critical criteria to evaluate the clinical efficacy of the anticancer agent. In this study, we investigated the effects of quercetin on cell growth and apoptosis using embryonic normal hepatic cell line (BNL CL.2) and its SV40-transformed cell line (BNL SV A.8). We also evaluated the effects of quercetin on the antioxidant defense system in those cells. BNL SV A.8 cells were more sensitive to quercetin-mediated cytotoxicity than BNL CL.2 cells. In addition, the enzyme assays showed that quercetin actively stimulated the antioxidant defense systems including superoxide dismutase, catalase, glutathione, and glutathione reductase only in the BNL CL.2 cells. In particular, quercetin significantly reduced superoxide dismutase activity and increased the malonaldehyde content in BNL SV A.8 cells. These are thought to be closely related to quercetin-mediated apoptosis. Our findings suggest that quercetin is a dietary flavonoid that is capable of inducing selective growth inhibition and apoptosis in hepatic tumor cells, but not in normal cells.

Tofacitinib induces G1 cell-cycle arrest and inhibits tumor growth in Epstein-Barr virus-associated T and natural killer cell lymphoma cells

PubMed Central

Ando, Shotaro; Kawada, Jun-ichi; Watanabe, Takahiro; Suzuki, Michio; Sato, Yoshitaka; Torii, Yuka; Asai, Masato; Goshima, Fumi; Murata, Takayuki; Shimizu, Norio; Ito, Yoshinori; Kimura, Hiroshi

2016-01-01

Epstein-Barr virus (EBV) infects not only B cells, but also T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoma. These lymphoid malignancies are refractory to conventional chemotherapy. We examined the activation of the JAK3/STAT5 pathway in EBV-positive and -negative B, T and NK cell lines and in cell samples from patients with EBV-associated T cell lymphoma. We then evaluated the antitumor effects of the selective JAK3 inhibitor, tofacitinib, against these cell lines in vitro and in a murine xenograft model. We found that all EBV-positive T and NK cell lines and patient samples tested displayed activation of the JAK3/STAT5 pathway. Treatment of these cell lines with tofacitinib reduced the levels of phospho-STAT5, suppressed proliferation, induced G1 cell-cycle arrest and decreased EBV LMP1 and EBNA1 expression. An EBV-negative NK cell line was also sensitive to tofacitinib, whereas an EBV-infected NK cell line was more sensitive to tofacitinib than its parental line. Tofacitinib significantly inhibited the growth of established tumors in NOG mice. These findings suggest that tofacitinib may represent a useful therapeutic agent for patients with EBV-associated T and NK cell lymphoma. PMID:27732937

Expression of the FAP gene in non-fibroblast human cell lines. Development of cancer-associated fibroblast models.

PubMed

Tyulkina, D V; Pleshkan, V V; Alekseenko, I V; Kopantseva, M R; Sverdlov, E D

2016-09-01

The fibroblast activation protein (FAP) is selectively expressed in cancer-associated fibroblasts (CAFs) and facilitates tumor progression, which makes this protein an attractive therapeutic target. There are difficulties in obtaining CAFs for studying the function and suppression of FAP. In this work, the expression level of FAP was determined by PCR assay in 25 human cell lines and 8 surgical samples of tumor stroma. The expression of FAP was observed in all tumor stroma samples and in four cell lines: NGP-127, SJCRH30, SJSA-1, and A375. The level of FAP expression in NGP-127, SJCRH30, and SJSA-1 lines as well as in CAFs of patients was comparable, which makes these cell lines a possible model for studying FAP.

Antiproliferative activity of synthetic fatty acid amides from renewable resources.

PubMed

dos Santos, Daiane S; Piovesan, Luciana A; D'Oca, Caroline R Montes; Hack, Carolina R Lopes; Treptow, Tamara G M; Rodrigues, Marieli O; Vendramini-Costa, Débora B; Ruiz, Ana Lucia T G; de Carvalho, João Ernesto; D'Oca, Marcelo G Montes

2015-01-15

In the work, the in vitro antiproliferative activity of a series of synthetic fatty acid amides were investigated in seven cancer cell lines. The study revealed that most of the compounds showed antiproliferative activity against tested tumor cell lines, mainly on human glioma cells (U251) and human ovarian cancer cells with a multiple drug-resistant phenotype (NCI-ADR/RES). In addition, the fatty methyl benzylamide derived from ricinoleic acid (with the fatty acid obtained from castor oil, a renewable resource) showed a high selectivity with potent growth inhibition and cell death for the glioma cell line-the most aggressive CNS cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

Transduction of human IL-9 receptor cDNA into TF1 cells induces IL-9 dependency and erythroid differentiation.

PubMed

Xiao, M; Luo, Z; Mantel, C; Broxmeyer, H E; Lu, L

2000-02-01

Human growth factor-dependent cell line TF1, which lacks interleukin (IL)-9 receptors (R) and does not grow in IL-9, was transduced with a retroviral vector containing human IL-9R cDNA and a selection marker. An IL-9-dependent TF1 cell line, which could also grow in other cytokines, was established after selection in G418 and could produce mature RBC in response to cytokine stimulation. TF1 cells transduced with the same viral vector without the IL-9R insert cDNA (mock control) and then selected responded the same as nontransduced TF1 cells. They failed to grow in response to IL-9 and did not generate RBC. An increased number and size of burst-forming units-erythroid (BFU-E)-like colonies were detected from IL-9R-transduced TF1 cells, compared with mock-transduced cells, in response to erythropoietin (EPO) and IL-9. To evaluate self-renewal and differentiation capacity, colony-replating assays were performed in the presence of IL-3, GM-CSF, IL-9, and EPO. After four replatings, the cloning efficiency of IL-9R-transduced TF1 cells decreased from 98% to 38%, most likely due to terminal erythroid cell differentiation. In contrast, no change in replating efficiency was detected in mock-transduced cells. TF1 cells stably expressing IL-9R and responding to IL-9 can serve as a cell line model to study the intracellular signals mediating IL-9-induced erythroid cell proliferation and differentiation.

Human ovarian cancer stem/progenitor cells are stimulated by doxorubicin but inhibited by Mullerian inhibiting substance

PubMed Central

Meirelles, Katia; Benedict, Leo Andrew; Dombkowski, David; Pepin, David; Preffer, Frederic I.; Teixeira, Jose; Tanwar, Pradeep Singh; Young, Robert H.; MacLaughlin, David T.; Donahoe, Patricia K.; Wei, Xiaolong

2012-01-01

Women with late-stage ovarian cancer usually develop chemotherapeutic-resistant recurrence. It has been theorized that a rare cancer stem cell, which is responsible for the growth and maintenance of the tumor, is also resistant to conventional chemotherapeutics. We have isolated from multiple ovarian cancer cell lines an ovarian cancer stem cell-enriched population marked by CD44, CD24, and Epcam (3+) and by negative selection for Ecadherin (Ecad−) that comprises less than 1% of cancer cells and has increased colony formation and shorter tumor-free intervals in vivo after limiting dilution. Surprisingly, these cells are not only resistant to chemotherapeutics such as doxorubicin, but also are stimulated by it, as evidenced by the significantly increased number of colonies in treated 3+Ecad− cells. Similarly, proliferation of the 3+Ecad− cells in monolayer increased with treatment, by either doxorubicin or cisplatin, compared with the unseparated or cancer stem cell-depleted 3−Ecad+ cells. However, these cells are sensitive to Mullerian inhibiting substance (MIS), which decreased colony formation. MIS inhibits ovarian cancer cells by inducing G1 arrest of the 3+Ecad− subpopulation through the induction of cyclin-dependent kinase inhibitors. 3+Ecad− cells selectively expressed LIN28, which colocalized by immunofluorescence with the 3+ cancer stem cell markers in the human ovarian carcinoma cell line, OVCAR-5, and is also highly expressed in transgenic murine models of ovarian cancer and in other human ovarian cancer cell lines. These results suggest that chemotherapeutics may be stimulative to cancer stem cells and that selective inhibition of these cells by treating with MIS or targeting LIN28 should be considered in the development of therapeutics. PMID:22308459

Human ovarian cancer stem/progenitor cells are stimulated by doxorubicin but inhibited by Mullerian inhibiting substance.

PubMed

Meirelles, Katia; Benedict, Leo Andrew; Dombkowski, David; Pepin, David; Preffer, Frederic I; Teixeira, Jose; Tanwar, Pradeep Singh; Young, Robert H; MacLaughlin, David T; Donahoe, Patricia K; Wei, Xiaolong

2012-02-14

Women with late-stage ovarian cancer usually develop chemotherapeutic-resistant recurrence. It has been theorized that a rare cancer stem cell, which is responsible for the growth and maintenance of the tumor, is also resistant to conventional chemotherapeutics. We have isolated from multiple ovarian cancer cell lines an ovarian cancer stem cell-enriched population marked by CD44, CD24, and Epcam (3+) and by negative selection for Ecadherin (Ecad-) that comprises less than 1% of cancer cells and has increased colony formation and shorter tumor-free intervals in vivo after limiting dilution. Surprisingly, these cells are not only resistant to chemotherapeutics such as doxorubicin, but also are stimulated by it, as evidenced by the significantly increased number of colonies in treated 3+Ecad- cells. Similarly, proliferation of the 3+Ecad- cells in monolayer increased with treatment, by either doxorubicin or cisplatin, compared with the unseparated or cancer stem cell-depleted 3-Ecad+ cells. However, these cells are sensitive to Mullerian inhibiting substance (MIS), which decreased colony formation. MIS inhibits ovarian cancer cells by inducing G1 arrest of the 3+Ecad- subpopulation through the induction of cyclin-dependent kinase inhibitors. 3+Ecad- cells selectively expressed LIN28, which colocalized by immunofluorescence with the 3+ cancer stem cell markers in the human ovarian carcinoma cell line, OVCAR-5, and is also highly expressed in transgenic murine models of ovarian cancer and in other human ovarian cancer cell lines. These results suggest that chemotherapeutics may be stimulative to cancer stem cells and that selective inhibition of these cells by treating with MIS or targeting LIN28 should be considered in the development of therapeutics.

A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system.

PubMed

Aranda, Alejandro; Bezunartea, Jaione; Casales, Erkuden; Rodriguez-Madoz, Juan R; Larrea, Esther; Prieto, Jesus; Smerdou, Cristian

2014-12-01

We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.

Characterization of the Apoptotic Response Induced by the Cyanine Dye D112: A Potentially Selective Anti-Cancer Compound

PubMed Central

Yang, Ning; Gilman, Paul; Mirzayans, Razmik; Sun, Xuejun; Touret, Nicolas; Weinfeld, Michael; Goping, Ing Swie

2015-01-01

Chemotherapeutic drugs that are used in anti-cancer treatments often cause the death of both cancerous and noncancerous cells. This non-selective toxicity is the root cause of untoward side effects that limits the effectiveness of therapy. In order to improve chemotherapeutic options for cancer patients, there is a need to identify novel compounds with higher discrimination for cancer cells. In the past, methine dyes that increase the sensitivity of photographic emulsions have been investigated for anti-cancer properties. In the 1970's, Kodak Laboratories initiated a screen of approximately 7000 dye structural variants for selective toxicity. Among these, D112 was identified as a promising compound with elevated toxicity against a colon cancer cell line in comparison to a non-transformed cell line. Despite these results changing industry priorities led to a halt in further studies on D112. We decided to revive investigations on D112 and have further characterized D112-induced cellular toxicity. We identified that in response to D112 treatment, the T-cell leukemia cell line Jurkat showed caspase activation, mitochondrial depolarization, and phosphatidylserine externalization, all of which are hallmarks of apoptosis. Chemical inhibition of caspase enzymatic activity and blockade of the mitochondrial pathway through Bcl-2 expression inhibited D112-induced apoptosis. At lower concentrations, D112 induced growth arrest. To gain insight into the molecular mechanism of D112 induced mitochondrial dysfunction, we analyzed the intracellular localization of D112, and found that D112 associated with mitochondria. Interestingly, in the cell lines that we tested, D112 showed increased toxicity toward transformed versus non-transformed cells. Results from this work identify D112 as a potentially interesting molecule warranting further investigation. PMID:25927702

Effect of glucose transport inhibitors on vincristine efflux in multidrug-resistant murine erythroleukaemia cells overexpressing the multidrug resistance-associated protein (MRP) and two glucose transport proteins, GLUT1 and GLUT3.

PubMed Central

Martell, R. L.; Slapak, C. A.; Levy, S. B.

1997-01-01

The relationship between mammalian facilitative glucose transport proteins (GLUT) and multidrug resistance was examined in two vincristine (VCR)-selected murine erythroleukaemia (MEL) PC4 cell lines. GLUT proteins, GLUT1 and GLUT3, were constitutively coexpressed in the parental cell line and also in the VCR-selected cell lines. Increased expression of the GLUT1 isoform was noted both in the PC-V40 (a non-P-glycoprotein, mrp-overexpressing subline) and in the more resistant PC-V160 (overexpressing mrp and mdr3) cell lines. Overexpression of GLUT3 was detected only in the PC-V160 subline. An increased rate of facilitative glucose transport (Vmax) and level of plasma membrane GLUT protein expression paralleled increased VCR resistance, active VCR efflux and decreased VCR steady-state accumulation in these cell lines. Glucose transport inhibitors (GTIs), cytochalasin B (CB) and phloretin blocked the active efflux and decreased steady-state accumulation of VCR in the PC-V40 subline. GTIs did not significantly affect VCR accumulation in the parental or PC-V160 cells. A comparison of protein sequences among GLUT1, GLUT3 and MRP revealed a putative cytochalasin B binding site in MRP, which displayed 44% sequence similarity/12% identity with that previously identified in GLUT1 and GLUT3; these regions also exhibited a similar hydropathy plot pattern. The findings suggested that CB bound to MRP and directly or indirectly lowered VCR efflux and/or CB bound to one or both GLUT proteins, which acted to lower the VCR efflux mediated by MRP. This is the first report of a non-neuronal murine cell line that expressed GLUT3. Images Figure 3 PMID:9010020

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Toki, Yasumichi; Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp; Tanaka, Hiroki

2016-08-05

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncatedmore » peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.« less

Preservation of high glycolytic phenotype by establishing new acute lymphoblastic leukemia cell lines at physiologic oxygen concentration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheard, Michael A., E-mail: msheard@chla.usc.edu; Ghent, Matthew V., E-mail: mattghent@gmail.com; Cabral, Daniel J., E-mail: dcabral14@gmail.com

2015-05-15

Cancer cells typically exhibit increased glycolysis and decreased mitochondrial oxidative phosphorylation, and they continue to exhibit some elevation in glycolysis even under aerobic conditions. However, it is unclear whether cancer cell lines employ a high level of glycolysis comparable to that of the original cancers from which they were derived, even if their culture conditions are changed to physiologically relevant oxygen concentrations. From three childhood acute lymphoblastic leukemia (ALL) patients we established three new pairs of cell lines in both atmospheric (20%) and physiologic (bone marrow level, 5%) oxygen concentrations. Cell lines established in 20% oxygen exhibited lower proliferation, survival,more » expression of glycolysis genes, glucose consumption, and lactate production. Interestingly, the effects of oxygen concentration used during cell line initiation were only partially reversible when established cell cultures were switched from one oxygen concentration to another for eight weeks. These observations indicate that ALL cell lines established at atmospheric oxygen concentration can exhibit relatively low levels of glycolysis and these levels are semi-permanent, suggesting that physiologic oxygen concentrations may be needed from the time of cell line initiation to preserve the high level of glycolysis commonly exhibited by leukemias in vivo. - Highlights: • Establishing new ALL cell lines in 5% oxygen resulted in higher glycolytic expression and function. • Establishing new ALL cell lines in 5% oxygen resulted in higher proliferation and lower cell death. • The divergent metabolic phenotypes selected in 5% and 20% oxygen are semi-permanent.« less

Supplementation of Nucleosides During Selection can Reduce Sequence Variant Levels in CHO Cells Using GS/MSX Selection System.

PubMed

Tang, Danming; Lam, Cynthia; Louie, Salina; Hoi, Kam Hon; Shaw, David; Yim, Mandy; Snedecor, Brad; Misaghi, Shahram

2018-01-01

In the process of generating stable monoclonal antibody (mAb) producing cell lines, reagents such as methotrexate (MTX) or methionine sulfoximine (MSX) are often used. However, using such selection reagent(s) increases the possibility of having higher occurrence of sequence variants in the expressed antibody molecules due to the effects of MTX or MSX on de novo nucleotide synthesis. Since MSX inhibits glutamine synthase (GS) and results in both amino acid and nucleoside starvation, it is questioned whether supplementing nucleosides into the media could lower sequence variant levels without affecting titer. The results show that the supplementation of nucleosides to the media during MSX selection decreased genomic DNA mutagenesis rates in the selected cells, probably by reducing nucleotide mis-incorporation into the DNA. Furthermore, addition of nucleosides enhance clone recovery post selection and does not affect antibody expression. It is further observed that nucleoside supplements lowered DNA mutagenesis rates only at the initial stage of the clone selection and do not have any effect on DNA mutagenesis rates after stable cell lines are established. Therefore, the data suggests that addition of nucleosides during early stages of MSX selection can lower sequence variant levels without affecting titer or clone stability in antibody expression. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Non-p-glycoprotein-mediated multidrug resistance in detransformed rat cells selected for resistance to methylglyoxal bis(guanylhydrazone).

PubMed

Weber, J M; Sircar, S; Horvath, J; Dion, P

1989-11-01

Three independent variants (G2, G4, G5), resistant to methylglyoxal bis(guanylhydrazone), an anticancer drug, have been isolated by single step selection from an adenovirus-transformed rat brain cell line (1). These variants display selective cross-resistance to several natural product drugs of dissimilar structure and action. Multidrug resistance has recently been shown to be caused by overexpression of the membrane-associated p-glycoprotein, most often caused by amplification of the mdr gene. Several types of experiments were conducted to determine whether the observed drug resistance in our cell lines could be due to changes at the mdr locus. The following results were obtained: (a) the mdr locus was not amplified; (b) transcription of the mdr gene and p-glycoprotein synthesis were not increased; (c) multidrug resistance cell lines, which carry an amplified mdr locus, were not cross-resistant to methylglyoxal bis(guanylhydrazone); (d) verapamil did not reverse the resistance of G cells or mdr cells to methylglyoxal bis(guanylhydrazone), nor that of G cells to vincristine; and (e) methylglyoxal bis(guanylhydrazone) resistance was recessive and depended on a block to drug uptake, as opposed to mdr cells which are dominant and express increased drug efflux. The results obtained suggest that the drug resistance in the G2, G4, and G5 cells was atypical and may be due to a mechanism distinct from that mediated by the mdr locus.

The cytotoxic action of the CD56+ fraction of cytokine-induced killer cells against a K562 cell line is mainly restricted to the natural killer cell subset.

PubMed

Chieregato, Katia; Zanon, Cristina; Castegnaro, Silvia; Bernardi, Martina; Amati, Eliana; Sella, Sabrina; Rodeghiero, Francesco; Astori, Giuseppe

2017-01-01

Cytokine-induced killer cells are polyclonal T cells generated ex vivo and comprise two main subsets: the CD56- fraction, possessing an alloreactive potential caused by T cells (CD3+CD56-), and the CD56+ fraction, characterised by a strong antitumour capacity induced by natural killer-like T cells (NK-like T, CD3+CD56+) and natural killer cells (NK, CD3-CD56+ bright). We investigated the cytotoxic action of selected CD56+ cell subpopulations against a human chronic myeloid leukaemia (K562) cell line. After immunomagnetic selection of the CD56+ cell fraction, NK bright cells (CD3-CD56+ bright) and two subsets of NK-like T cells (CD3+CD56+), called NK-like T CD56 dim and NK-like T CD56 bright, could be identified. The cytotoxic effect against K562 cells was mainly exerted by the NK bright subpopulation and resulted to be inversely correlated with the percentage of NK-like T CD56 dim cells in the culture. The lytic action appeared to be independent of cell degranulation as suggested by the lack of change in the expression of CD107a. We conclude that the cytotoxic action of CD56+ cells against a K562 cell line is mainly due to the NK cells.

Multiparameter cell affinity chromatography: separation and analysis in a single microfluidic channel.

PubMed

Li, Peng; Gao, Yan; Pappas, Dimitri

2012-10-02

The ability to sort and capture more than one cell type from a complex sample will enable a wide variety of studies of cell proliferation and death and the analysis of disease states. In this work, we integrated a pneumatic actuated control layer to an affinity separation layer to create different antibody-coating regions on the same fluidic channel. The comparison of different antibody capture capabilities to the same cell line was demonstrated by flowing Ramos cells through anti-CD19- and anti-CD71-coated regions in the same channel. It was determined that the cell capture density on the anti-CD19 region was 2.44 ± 0.13 times higher than that on the anti-CD71-coated region. This approach can be used to test different affinity molecules for selectivity and capture efficiency using a single cell line in one separation. Selective capture of Ramos and HuT 78 cells from a mixture was also demonstrated using two antibody regions in the same channel. Greater than 90% purity was obtained on both capture areas in both continuous flow and stop flow separation modes. A four-region antibody-coated device was then fabricated to study the simultaneous, serial capture of three different cell lines. In this case the device showed effective capture of cells in a single separation channel, opening up the possibility of multiple cell sorting. Multiparameter sequential blood sample analysis was also demonstrated with high capture specificity (>97% for both CD19+ and CD4+ leukocytes). The chip can also be used to selectively treat cells after affinity separation.

Augmenting Trastuzumab Therapy against Breast Cancer through Selective Activation of NK Cells

DTIC Science & Technology

2014-12-01

purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Breast cancer cell lines including MCF7 (A and E...purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Chromium-labeled breast cancer cell lines including MCF7 (A...and Whiteside, T.L. 2007. A novel multiparametric flow cytometry -based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons

Changes of heterogeneous cell populations in the Ishikawa cell line during long-term culture: Proposal for an in vitro clonal evolution model of tumor cells.

PubMed

Kasai, Fumio; Hirayama, Noriko; Ozawa, Midori; Iemura, Masashi; Kohara, Arihiro

2016-06-01

Genomic changes in tumor cell lines can occur during culture, leading to differences between cell lines carrying the same name. In this study, genome profiles between low and high passages were investigated in the Ishikawa 3-H-12 cell line (JCRB1505). Cells contained between 43 and 46 chromosomes and the modal number changed from 46 to 45 during culture. Cytogenetic analysis revealed that a translocation t(9;14), observed in all metaphases, is a robust marker for this cell line. Single-nucleotide polymorphism microarrays showed a heterogeneous copy number in the early passages and distinct profiles at late passages. These results demonstrate that cell culture can lead to elimination of ancestral clones by sequential selection, resulting in extensive replacement with a novel clone. Our observations on Ishikawa cells in vitro are different from the in vivo heterogeneity in which ancestral clones are often retained during tumor evolution and suggest a model for in vitro clonal evolution. Copyright © 2016 Elsevier Inc. All rights reserved.

An SSH library responsive to azadirachtin A constructed in Spodoptera litura Fabricius cell lines.

PubMed

Yan, Chao; Zhang, Zhi-Xiang; Xu, Han-Hong

2012-05-31

The present study revealed differentially expressed genes responsive to azadirachtin A (Aza) in Spodoptera litura cell line through suppression subtractive hybridization. In the Aza-responsive SSH library, approximately 270 sequences represent 53 different identified genes encoding proteins with various predicted functions, and the percentages of the gene clusters were 26.09% (genetic information processing), 11.41% (cell growth and death), 7.07% (metabolism), 6.52% (signal transduction/transport) and 2.72% (immunity), respectively. Eleven clones homologous to identified genes were selected to be confirmed through quantitative real time polymerase chain reaction. Among the eleven clones validated, all but one transcript of lipase showed an increase in SL cell line collected from ETA, whereas the transcripts of other genes were lower in the SL cell line collected from ETA compared with that of UETA. These genes were considered to be related to the response of SL cell line to Aza. These will provide a new clue to uncover the molecular mechanisms of Aza acting on SL cell line. Copyright © 2012 Elsevier B.V. All rights reserved.

Selective Targeting of CTNNB1-, KRAS- or MYC-Driven Cell Growth by Combinations of Existing Drugs

PubMed Central

Uitdehaag, Joost C. M.; de Roos, Jeroen A. D. M.; van Doornmalen, Antoon M.; Prinsen, Martine B. W.; Spijkers-Hagelstein, Jill A. P.; de Vetter, Judith R. F.; de Man, Jos; Buijsman, Rogier C.; Zaman, Guido J. R.

2015-01-01

The aim of combination drug treatment in cancer therapy is to improve response rate and to decrease the probability of the development of drug resistance. Preferably, drug combinations are synergistic rather than additive, and, ideally, drug combinations work synergistically only in cancer cells and not in non-malignant cells. We have developed a workflow to identify such targeted synergies, and applied this approach to selectively inhibit the proliferation of cell lines with mutations in genes that are difficult to modulate with small molecules. The approach is based on curve shift analysis, which we demonstrate is a more robust method of determining synergy than combination matrix screening with Bliss-scoring. We show that the MEK inhibitor trametinib is more synergistic in combination with the BRAF inhibitor dabrafenib than with vemurafenib, another BRAF inhibitor. In addition, we show that the combination of MEK and BRAF inhibitors is synergistic in BRAF-mutant melanoma cells, and additive or antagonistic in, respectively, BRAF-wild type melanoma cells and non-malignant fibroblasts. This combination exemplifies that synergistic action of drugs can depend on cancer genotype. Next, we used curve shift analysis to identify new drug combinations that specifically inhibit cancer cell proliferation driven by difficult-to-drug cancer genes. Combination studies were performed with compounds that as single agents showed preference for inhibition of cancer cells with mutations in either the CTNNB1 gene (coding for β-catenin), KRAS, or cancer cells expressing increased copy numbers of MYC. We demonstrate that the Wnt-pathway inhibitor ICG-001 and trametinib acted synergistically in Wnt-pathway-mutant cell lines. The ERBB2 inhibitor TAK-165 was synergistic with trametinib in KRAS-mutant cell lines. The EGFR/ERBB2 inhibitor neratinib acted synergistically with the spindle poison docetaxel and with the Aurora kinase inhibitor GSK-1070916 in cell lines with MYC amplification. Our approach can therefore efficiently discover novel drug combinations that selectively target cancer genes. PMID:26018524

Artepillin C induces selective oxidative stress and inhibits migration and invasion in a comprehensive panel of human cervical cancer cell lines.

PubMed

Souza, Raquel Pantarotto; de Souza Bonfim-Mendonca, Patricia; Damke, Gabrielle Marconi Zago Ferreira; De Assis Carvalho, Analine Rosa Barquez; Ratti, Bianca Altrao; de Oliveira Dembogurski, Djaceli Sampaio; da Silva, Vania Ramos Sela; Silva, Sueli Oliveira; da Silva, Denise Brentan; Bruschi, Marcos Luciano; Maria-Engler, Silvya Stuchi; Consolaro, Marcia Edilaine Lopes

2018-06-03

Artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) is the main bioactive component of Brazilian green propolis, and possesses, among other things, anticancer properties. However, to the best of our knowledge, there are no studies of artepillin C in cervical cancer. To explore a new therapeutic candidate for cervical cancer, we have evaluated the effects of artepillin C on cellular viability in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16- and 18-positive) and C33A (HPV-negative) cells compared to a spontaneously immortalized human epithelial cell line (HaCaT). Our results demonstrated that artepillin C had a selective effect on cellular viability and could induce apoptosis possibly by intrinsic pathway, likely a result of oxidative stress, in all cancer-derived cell lines but not in HaCaT. Additionally, artepillin C was able to inhibit the migration and invasion of cancer cells. Thus, artepillin C appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV types. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Selective targeting of KRAS-Mutant cells by miR-126 through repression of multiple genes essential for the survival of KRAS-Mutant cells

PubMed Central

Hara, Toshifumi; Jones, Matthew F.; Subramanian, Murugan; Li, Xiao Ling; Ou, Oliver; Zhu, Yuelin; Yang, Yuan; Wakefield, Lalage M.; Hussain, S. Perwez; Gaedcke, Jochen; Ried, Thomas; Luo, Ji; Caplen, Natasha J.; Lal, Ashish

2014-01-01

MicroRNAs (miRNAs) regulate the expression of hundreds of genes. However, identifying the critical targets within a miRNA-regulated gene network is challenging. One approach is to identify miRNAs that exert a context-dependent effect, followed by expression profiling to determine how specific targets contribute to this selective effect. In this study, we performed miRNA mimic screens in isogenic KRAS-Wild-type (WT) and KRAS-Mutant colorectal cancer (CRC) cell lines to identify miRNAs selectively targeting KRAS-Mutant cells. One of the miRNAs we identified as a selective inhibitor of the survival of multiple KRAS-Mutant CRC lines was miR-126. In KRAS-Mutant cells, miR-126 over-expression increased the G1 compartment, inhibited clonogenicity and tumorigenicity, while exerting no effect on KRAS-WT cells. Unexpectedly, the miR-126-regulated transcriptome of KRAS-WT and KRAS-Mutant cells showed no significant differences. However, by analyzing the overlap between miR-126 targets with the synthetic lethal genes identified by RNAi in KRAS-Mutant cells, we identified and validated a subset of miR-126-regulated genes selectively required for the survival and clonogenicity of KRAS-Mutant cells. Our strategy therefore identified critical target genes within the miR-126-regulated gene network. We propose that the selective effect of miR-126 on KRAS-Mutant cells could be utilized for the development of targeted therapy for KRAS mutant tumors. PMID:25245095

Selective cytotoxicity of the antibacterial peptide ABP-dHC-Cecropin A and its analog towards leukemia cells.

PubMed

Sang, Ming; Zhang, Jiaxin; Zhuge, Qiang

2017-05-15

Some cationic antibacterial peptides, with typical amphiphilic α-helical conformations in a membrane-mimicking environment, exhibit anticancer properties as a result of a similar mechanism of action towards both bacteria and cancer cells. We previously reported the cDNA sequence of the antimicrobial peptide ABP-dHC-Cecropin A precursor cloned from drury (Hyphantria cunea) (dHC). In the present study, we synthesized and structurally characterized ABP-dHC-Cecropin A and its analog, ABP-dHC-Cecropin A-K(24). Circular dichroism spectroscopy showed that ABP-dHC-Cecropin A and its analog adopt a well-defined α-helical structure in a 50% trifluorethanol solution. The cytotoxicity and cell selectivity of these peptides were further examined in three leukemia cell lines and two non-cancerous cell lines. The MTT assay indicated both of these peptides have a concentration-dependent cytotoxic effect in leukemia cells, although the observed cytotoxicity was greater with ABP-dHC-Cecropin A-K(24) treatment, whereas they were not cytotoxic towards the non-cancerous cell lines. Moreover, ABP-dHC-Cecropin A and its analog had a lower hemolytic effect in human red blood cells. Together, these results suggest the peptides are selectively cytotoxic towards leukemia cells. Confocal laser scanning microscopy determined that the peptides were concentrated at the surface of the leukemia cells, and changes in the cell membrane were determined with a permeability assay, which suggested that the anticancer activity of ABP-dHC-Cecropin A and its analog is a result of its presence at the leukemia cell membrane. ABP-dHC-Cecropin A and its analog may represent a novel anticancer agent for leukemia therapy, considering its cancer cell selectivity and relatively low cytotoxicity in normal cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Autumn Royal and Ribier Grape Juice Extracts Reduced Viability and Metastatic Potential of Colon Cancer Cells.

PubMed

Valenzuela, Manuel; Bastias, Lorena; Montenegro, Iván; Werner, Enrique; Madrid, Alejandro; Godoy, Patricio; Párraga, Mario; Villena, Joan

2018-01-01

Antioxidants are known to be beneficial to health. This paper evaluates the potential chemopreventive and anticancer properties of phenolic compounds present in grape juice extracts (GJE) from Autumn Royal and Ribier varieties. The effects of these GJE on viability (SRB day assay) and metastatic potential (migration and invasion parameters) of colon cancer cell lines HT-29 and SW-480 were evaluated. The effects of GJE on two matrix metalloproteinase gene expressions (MMP2 and MMP9) were also evaluated via qRT-PCR. In the former, GJE reduced cell viability in both cell lines in a dose-dependent manner. GJE treatment also reduced cell migration and invasion. Moreover, MMP-2 and MMP-9 gene expression diminished depending on extract and on cell type. Conclusions . These results provide novel information concerning anticancer properties of selected GJE by revealing selective cytotoxicity and the ability to reduce invasiveness of colon cancer cells.

Autumn Royal and Ribier Grape Juice Extracts Reduced Viability and Metastatic Potential of Colon Cancer Cells

PubMed Central

Valenzuela, Manuel; Bastias, Lorena; Montenegro, Iván; Werner, Enrique; Madrid, Alejandro; Godoy, Patricio

2018-01-01

Antioxidants are known to be beneficial to health. This paper evaluates the potential chemopreventive and anticancer properties of phenolic compounds present in grape juice extracts (GJE) from Autumn Royal and Ribier varieties. The effects of these GJE on viability (SRB day assay) and metastatic potential (migration and invasion parameters) of colon cancer cell lines HT-29 and SW-480 were evaluated. The effects of GJE on two matrix metalloproteinase gene expressions (MMP2 and MMP9) were also evaluated via qRT-PCR. In the former, GJE reduced cell viability in both cell lines in a dose-dependent manner. GJE treatment also reduced cell migration and invasion. Moreover, MMP-2 and MMP-9 gene expression diminished depending on extract and on cell type. Conclusions. These results provide novel information concerning anticancer properties of selected GJE by revealing selective cytotoxicity and the ability to reduce invasiveness of colon cancer cells. PMID:29552079

SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson's disease.

PubMed

Xie, Hong-rong; Hu, Lin-sen; Li, Guo-yi

2010-04-20

To evaluate the human neuroblastoma SH-SY5Y cell line as an in vitro model of dopaminergic (DAergic) neurons for Parkinson's disease (PD) research and to determine the effect of differentiation on this cell model. The data of this review were selected from the original reports and reviews related to SH-SY5Y cells published in Chinese and foreign journals (Pubmed 1973 to 2009). After searching the literature, 60 articles were selected to address this review. The SH-SY5Y cell line has become a popular cell model for PD research because this cell line posses many characteristics of DAergic neurons. For example, these cells express tyrosine hydroxylase and dopamine-beta-hydroxylase, as well as the dopamine transporter. Moreover, this cell line can be differentiated into a functionally mature neuronal phenotype in the presence of various agents. Upon differentiation, SH-SY5Y cells stop proliferating and a constant cell number is subsequently maintained. However, different differentiating agents induce different neuronal phenotypes and biochemical changes. For example, retinoic acid induces differentiation toward a cholinergic neuronal phenotype and increases the susceptibility of SH-SY5Y cells to neurotoxins and neuroprotective agents, whereas treatment with retinoic acid followed by phorbol ester 12-O-tetradecanoylphorbol-13-acetate results in a DAergic neuronal phenotype and decreases the susceptibility of cells to neurotoxins and neuroprotective agents. Some differentiating agents also alter kinetics of 1-methyl-4-phenyl-pyridinium (MPP(+)) uptake, making SH-SY5Y cells more similar to primary mesencephalic neurons. Differentiated and undifferentiated SH-SY5Y cells have been widely used as a cell model of DAergic neurons for PD research. Some differentiating agents afford SH-SY5Y cells with more potential for studying neurotoxicity and neuroprotection and are thus more relevant to experimental PD research.

Innate responses to gene knockouts impact overlapping gene networks and vary with respect to resistance to viral infection.

PubMed

Liu, Yonghong; Liu, Yuanyuan; Wu, Jiaming; Roizman, Bernard; Zhou, Grace Guoying

2018-04-03

Analyses of the levels of mRNAs encoding IFIT1, IFI16, RIG-1, MDA5, CXCL10, LGP2, PUM1, LSD1, STING, and IFNβ in cell lines from which the gene encoding LGP2, LSD1, PML, HDAC4, IFI16, PUM1, STING, MDA5, IRF3, or HDAC 1 had been knocked out, as well as the ability of these cell lines to support the replication of HSV-1, revealed the following: ( i ) Cell lines lacking the gene encoding LGP2, PML, or HDAC4 (cluster 1) exhibited increased levels of expression of partially overlapping gene networks. Concurrently, these cell lines produced from 5 fold to 12 fold lower yields of HSV-1 than the parental cells. ( ii ) Cell lines lacking the genes encoding STING, LSD1, MDA5, IRF3, or HDAC 1 (cluster 2) exhibited decreased levels of mRNAs of partially overlapping gene networks. Concurrently, these cell lines produced virus yields that did not differ from those produced by the parental cell line. The genes up-regulated in cell lines forming cluster 1, overlapped in part with genes down-regulated in cluster 2. The key conclusions are that gene knockouts and subsequent selection for growth causes changes in expression of multiple genes, and hence the phenotype of the cell lines cannot be ascribed to a single gene; the patterns of gene expression may be shared by multiple knockouts; and the enhanced immunity to viral replication by cluster 1 knockout cell lines but not by cluster 2 cell lines suggests that in parental cells, the expression of innate resistance to infection is specifically repressed.

Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System.

PubMed

Grobarczyk, Benjamin; Franco, Bénédicte; Hanon, Kevin; Malgrange, Brigitte

2015-10-01

Genome engineering and human iPS cells are two powerful technologies, which can be combined to highlight phenotypic differences and identify pathological mechanisms of complex diseases by providing isogenic cellular material. However, very few data are available regarding precise gene correction in human iPS cells. Here, we describe an optimized stepwise protocol to deliver CRISPR/Cas9 plasmids in human iPS cells. We highlight technical issues especially those associated to human stem cell culture and to the correction of a point mutation to obtain isogenic iPS cell line, without inserting any resistance cassette. Based on a two-steps clonal isolation protocol (mechanical picking followed by enzymatic dissociation), we succeed to select and expand corrected human iPS cell line with a great efficiency (more than 2% of the sequenced colonies). This protocol can also be used to obtain knock-out cell line from healthy iPS cell line by the NHEJ pathway (with about 15% efficiency) and reproduce disease phenotype. In addition, we also provide protocols for functional validation tests after every critical step.

Withanolides from Aeroponically Grown Physalis peruviana and Their Selective Cytotoxicity to Prostate Cancer and Renal Carcinoma Cells.

PubMed

Xu, Ya-Ming; Wijeratne, E M Kithsiri; Babyak, Ashley L; Marks, Hanna R; Brooks, Alan D; Tewary, Poonam; Xuan, Li-Jiang; Wang, Wen-Qiong; Sayers, Thomas J; Gunatilaka, A A Leslie

2017-07-28

Investigation of aeroponically grown Physalis peruviana resulted in the isolation of 11 new withanolides, including perulactones I-L (1-4), 17-deoxy-23β-hydroxywithanolide E (5), 23β-hydroxywithanolide E (6), 4-deoxyphyperunolide A (7), 7β-hydroxywithanolide F (8), 7β-hydroxy-17-epi-withanolide K (9), 24,25-dihydro-23β,28-dihydroxywithanolide G (10), and 24,25-dihydrowithanolide E (11), together with 14 known withanolides (12-25). The structures of 1-11 were elucidated by the analysis of their spectroscopic data, and 12-25 were identified by comparison of their spectroscopic data with those reported. All withanolides were evaluated for their cytotoxic activity against a panel of tumor cell lines including LNCaP (androgen-sensitive human prostate adenocarcinoma), 22Rv1 (androgen-resistant human prostate adenocarcinoma), ACHN (human renal adenocarcinoma), M14 (human melanoma), SK-MEL-28 (human melanoma), and normal human foreskin fibroblast cells. Of these, the 17β-hydroxywithanolides (17-BHWs) 6, 8, 9, 11-13, 15, and 19-22 showed selective cytotoxic activity against the two prostate cancer cell lines LNCaP and 22Rv1, whereas 13 and 20 exhibited selective toxicity for the ACHN renal carcinoma cell line. These cytotoxicity data provide additional structure-activity relationship information for the 17-BHWs.

Cell Context Dependent p53 Genome-Wide Binding Patterns and Enrichment at Repeats

DOE PAGES

Botcheva, Krassimira; McCorkle, Sean R.

2014-11-21

The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We reportmore » distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). In conclusion, our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways.« less

Molecular characterization of immortalized normal and dysplastic oral cell lines.

PubMed

Dickman, Christopher T D; Towle, Rebecca; Saini, Rajan; Garnis, Cathie

2015-05-01

Cell lines have been developed for modeling cancer and cancer progression. The molecular background of these cell lines is often unknown to those using them to model disease behaviors. As molecular alterations are the ultimate drivers of cell phenotypes, having an understanding of the molecular make-up of these systems is critical for understanding the disease biology modeled. Six immortalized normal, one immortalized dysplasia, one self-immortalized dysplasia, and two primary normal cell lines derived from oral tissues were analyzed for DNA copy number changes and changes in both mRNA and miRNA expression using SMRT-v.2 genome-wide tiling comparative genomic hybridization arrays, Agilent Whole Genome 4x44k expression arrays, and Exiqon V2.M-RT-PCR microRNA Human panels. DNA copy number alterations were detected in both normal and dysplastic immortalized cell lines-as well as in the single non-immortalized dysplastic cell line. These lines were found to have changes in expression of genes related to cell cycle control as well as alterations in miRNAs that are deregulated in clinical oral squamous cell carcinoma tissues. Immortal lines-whether normal or dysplastic-had increased disruption in expression relative to primary lines. All data are available as a public resource. Molecular profiling experiments have identified DNA, mRNA, and miRNA alterations for a panel of normal and dysplastic oral tissue cell lines. These data are a valuable resource to those modeling diseases of the oral mucosa, and give insight into the selection of model cell lines and the interpretation of data from those lines. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Role of epigenetic factors in the selection of the alternative splicing isoforms of human KRAS in colorectal cancer cell lines.

PubMed

Riffo-Campos, Ángela L; Gimeno-Valiente, Francisco; Rodríguez, Fernanda M; Cervantes, Andrés; López-Rodas, Gerardo; Franco, Luis; Castillo, Josefa

2018-04-17

Mutation-driven activation of KRAS is crucial to cancer development. The human gene yields four mRNA splicing isoforms, 4A and 4B being translated to protein. Their different properties and oncogenic potential have been studied, but the mechanisms deciding the ratio 4A/4B are not known. To address this issue, the expression of the four KRAS isoforms was determined in 9 human colorectal cancer cell lines. HCT116 and SW48 were further selected because they present the highest difference in the ratio 4A/4B (twice as much in HCT116 than in SW48). Chromatin structure was analysed at the exon 4A, characteristic of isoform 4A, at its intronic borders and at the two flanking exons. The low nucleosome occupancy at exon 4A in both cell lines may result in a fast transcriptional rate, which would explain the general lower abundance of isoform 4A, also found in cells and tissues by other authors, but due to its similarity between both cell lines, chromatin structure does not influence alternative splicing. DNA methylation downstream exon 4A significantly differs in HCT116 and SW48 cells, but the CCCTC-binding factor, which affects the processivity of RNA polymerase and the alternative splicing, does not bind the differentially methylated sequences. Quantitative epigenetic analysis at mononucleosomal level revealed significant differences between both cell lines in H3K4me3, H3K27me3, H3K36me3, H3K9ac, H3K27ac and H4K20me1, and the inhibition of some histone-modifying enzymes alters the ratio 4A/4B. It can be concluded that the epigenetic modification of histones has an influence on the selection of isoforms 4A and 4B.

Cell Type-Specific Manipulation with GFP-Dependent Cre Recombinase

PubMed Central

Tang, Jonathan C Y; Rudolph, Stephanie; Dhande, Onkar S; Abraira, Victoria E; Choi, Seungwon; Lapan, Sylvain; Drew, Iain R; Drokhlyansky, Eugene; Huberman, Andrew D; Regehr, Wade G; Cepko, Constance L

2016-01-01

Summary There are many transgenic GFP reporter lines that allow visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. Here we report the creation of a method that exploits GFP for gene manipulation, Cre Recombinase Dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, leading to effective recombination selectively in GFP-labeled cells. Further, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP+ cells, we demonstrate that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins. PMID:26258682

Mechanisms of Cellular Membrane Effects of TCDD, Selected Perfluorinated Acids and Polyhalogenated Aromatic Hydrocarbons

DTIC Science & Technology

1985-02-01

toxic response of both cell lines after treatment with the perfluorinated acids ( perfluoro -n-octanoic acid, 9-H hexadecafluoro-n-nonanoic acid, and...throughout the experiments to eliminate the effect of serum on toxicity. The results for perfluoro -n-octanoic acid ( PFOA ) in both cell lines are presented in...the TK+/+ cells but not in the TK+/- cells. The results for the perfluoro -n-decanoic acid ( PFDA ) are presented in Table 3. A dose-response reldtionship

Selecting Cells for Bioartificial Liver Devices and the Importance of a 3D Culture Environment: A Functional Comparison between the HepaRG and C3A Cell Lines.

PubMed

van Wenum, Martien; Adam, Aziza A A; Hakvoort, Theodorus B M; Hendriks, Erik J; Shevchenko, Valery; van Gulik, Thomas M; Chamuleau, Robert A F M; Hoekstra, Ruurdtje

2016-01-01

Recently, the first clinical trials on Bioartificial Livers (BALs) loaded with a proliferative human hepatocyte cell source have started. There are two cell lines that are currently in an advanced state of BAL development; HepaRG and HepG2/C3A. In this study we aimed to compare both cell lines on applicability in BALs and to identify possible strategies for further improvement. We tested both cell lines in monolayer- and BAL cultures on growth characteristics, hepatic differentiation, nitrogen-, carbohydrate-, amino acid- and xenobiotic metabolism. Interestingly, both cell lines adapted the hepatocyte phenotype more closely when cultured in BALs; e.g. monolayer cultures produced lactate, while BAL cultures showed diminished lactate production (C3A) or conversion to elimination (HepaRG), and urea cycle activity increased upon BAL culturing in both cell lines. HepaRG-BALs outperformed C3A-BALs on xenobiotic metabolism, ammonia elimination and lactate elimination, while protein synthesis was comparable. In BAL cultures of both cell lines ammonia elimination correlated positively with glutamine production and glutamate consumption, suggesting ammonia elimination was mainly driven by the balance between glutaminase and glutamine synthetase activity. Both cell lines lacked significant urea cycle activity and both required multiple culture weeks before reaching optimal differentiation in BALs. In conclusion, culturing in BALs enhanced hepatic functionality of both cell lines and from these, the HepaRG cells are the most promising proliferative cell source for BAL application.

Effect of capping agents on the cytotoxicity of silver nanoparticles in human normal and cancer skin cell lines

NASA Astrophysics Data System (ADS)

Netchareonsirisuk, Ponsawan; Puthong, Songchan; Dubas, Stephan; Palaga, Tanapat; Komolpis, Kittinan

2016-11-01

Silver nanoparticles (AgNPs) are among the most widely used nanomaterials in medical and consumer products. However, safety in the uses of AgNPs is still controversial. The toxicity of AgNPs toward various cell types has been reported to depend on the surface properties of the nanoparticles. In this study, the effect of AgNPs with the average size of 5-15 nm on the viability of the CCD-986SK human normal skin fibroblast cell line and A375 human malignant melanoma cell line was evaluated. Comparative toxicity studies, based on MTT assay, were performed by using either sodium alginate or poly (4-styrenesulfonic acid-co-maleic acid) sodium salt (PSSMA) as capping agent in the nanoparticle preparation. The cytotoxicity tests revealed that AgNO3 alone was highly toxic to both cell types while both alginate and PSSMA alone were not toxic. AgNPs capped with alginate were selectively toxic to the cancer cell line but not to the normal cell line while AgNPs capped with PSSMA were toxic to both cancer and normal cell lines. Judging from the 50 % inhibition concentration (IC50), it was found that the cancer cell line was more sensitive to AgNPs than the normal cell line. Study on the mode of cell death by annexin V and propidium iodide staining revealed that AgNPs induced more apoptotic cell death (84-90 %) than necrosis (8-12 %) in the skin cancer cell line. These results suggest that the toxicity of AgNPs depended on the type of capping agent and the type of cell line.

A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality.

PubMed

Grassian, A R; Scales, T M E; Knutson, S K; Kuntz, K W; McCarthy, N J; Lowe, C E; Moore, J D; Copeland, R A; Keilhack, H; Smith, J J; Wickenden, J A; Ribich, S

2015-01-01

Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining whether a cell line is dependent on a specific gene product for proliferation or survival. In our initial studies using CRISPR-Cas9 to verify the dependence on EZH2 activity for proliferation of a SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor (MRT) cell line, we noted that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity, at least one allele of EZH2 remains functional. To verify this, we developed an assay to analyze 10s-100s of clonal cell populations for target gene disruption using restriction digest and fluorescent fragment length analyses. Our results clearly show that in cell lines in which EZH2 is essential for proliferation, at least one potentially functional allele of EZH2 is retained in the clones that survive. This assay clearly indicates whether or not a specific gene is essential for survival and/or proliferation in a given cell line. Such data can aid the development of more robust therapeutics by increasing confidence in target selection.

[Construction of BAD Lentivirus Vector and Its Effect on Proliferation in A549 Cell Lines].

PubMed

Huang, Na; He, Yan-qi; Zhu, Jing; Li, Wei-min

2015-05-01

To construct the recombinant lentivirus expressing vector BAD (Bcl-2-associated death protein) gene and to study its effect on A549 cell proliferation. The BAD gene was amplified from plasmid pAV-MCMV-BAD-GFP by PCR. The purified BAD gene fragment was inserted into a lentivirus vector (pLVX-IRES-ZsGreen 1), and the insertion was identified by PCR, restriction endonuclease analysis and DNA sequencing. A549 cells were then transfected with the packaged recombinant lentivirus, and resistant cell clones were selected with flow cytometry. The expression of BAD in A549 cell lines stably transduction with a lentivirus was examined using Western blot. The effect of BAD overexpression on proliferation of A549 cells was evaluated by using CCK-8 kit. Restriction enzyme digestion and DNA sequencing showed that the full-length BAD gene (507 bp) had been successfully subcloned into the lentiviral vector to result in the recombinant vector pLVX-IRES-ZsGreen 1. Monoclonal cell lines BAD-A549 was produced after transfection with the recombinant lentivirus and selected with flow cytometry. Stable expression of BAD protein was verified by Western blot. In vitro, the OD value in BAD group was significantly lower than that of control groups from 120-144 h (P<0. 05). A549 cell lines stably transduced with a lentivirus expressing the BAD gene had been successfully generated. In vitro, BAD overexpression significantly inhibited A549 cells proliferation.

Targeting tumor cells via EGF receptors: selective toxicity of an HBEGF-toxin fusion protein.

PubMed

Chandler, L A; Sosnowski, B A; McDonald, J R; Price, J E; Aukerman, S L; Baird, A; Pierce, G F; Houston, L L

1998-09-25

Over-expression of the epidermal growth factor receptor (EGFR) is a hallmark of numerous solid tumors, thus providing a means of selectively targeting therapeutic agents. Heparin-binding epidermal growth factor (HBEGF) binds to EGFRs with high affinity and to heparan sulfate proteoglycans, resulting in increased mitogenic potential compared to other EGF family members. We have investigated the feasibility of using HBEGF to selectively deliver a cytotoxic protein into EGFR-expressing tumor cells. Recombinant fusion proteins consisting of mature human HBEGF fused to the plant ribosome-inactivating protein saporin (SAP) were expressed in Escherichia coli. Purified HBEGF-SAP chimeras inhibited protein synthesis in a cell-free assay and competed with EGF for binding to receptors on intact cells. A construct with a 22-amino-acid flexible linker (L22) between the HBEGF and SAP moieties exhibited an affinity for the EGFR that was comparable to that of HBEGF. The sensitivity to HBEGF-L22-SAP was determined for a variety of human tumor cell lines, including the 60 cell lines comprising the National Cancer Institute Anticancer Drug Screen. HBEGF-L22-SAP was cytotoxic in vitro to a variety of EGFR-bearing cell lines and inhibited growth of EGFR-over-expressing human breast carcinoma cells in vivo. In contrast, the fusion protein had no effect on small-cell lung carcinoma cells, which are EGFR-deficient. Our results demonstrate that fusion proteins composed of HBEGF and SAP exhibit targeting specificity and cytotoxicity that may be of therapeutic value in treating a variety of EGFR-bearing malignancies.

Impact of media and antifoam selection on monoclonal antibody production and quality using a high throughput micro‐bioreactor system

PubMed Central

Velugula‐Yellela, Sai Rashmika; Williams, Abasha; Trunfio, Nicholas; Hsu, Chih‐Jung; Chavez, Brittany; Yoon, Seongkyu

2017-01-01

Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used throughout manufacturing. It is important to identify high impact components early on during the lifecycle of a biotechnology‐derived product. While cell culture media selection is of obvious importance to the health and productivity of mammalian bioreactor operations, other components such as antifoam selection can also play an important role in bioreactor cell culture. Silicone polymer‐based antifoams were known to have negative impacts on cell health, production, and downstream filtration and purification operations. High throughput screening in micro‐scale bioreactors provides an efficient strategy to identify initial operating parameters. Here, we utilized a micro‐scale parallel bioreactor system to study an IgG1 producing CHO cell line, to screen Dynamis, ProCHO5, PowerCHO2, EX‐Cell Advanced, and OptiCHO media, and 204, C, EX‐Cell, SE‐15, and Y‐30 antifoams and their impacts on IgG1 production, cell growth, aggregation, and process control. This study found ProCHO5, EX‐Cell Advanced, and PowerCHO2 media supported strong cellular growth profiles, with an IVCD of 25‐35 × 106 cells‐d/mL, while maintaining specific antibody production (Qp > 2 pg/cell‐d) for our model cell line and a monomer percentage above 94%. Antifoams C, EX‐Cell, and SE‐15 were capable of providing adequate control of foaming while antifoam 204 and Y‐30 noticeably stunted cellular growth. This work highlights the utility of high throughput micro bioreactors and the importance of identifying both positive and negative impacts of media and antifoam selection on a model IgG1 producing CHO cell line. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:262–270, 2018 PMID:29086492

Novel Biomarker Discovery for Diagnostic and Therapeutic Strategies in Prostate Cancer

DTIC Science & Technology

2014-03-01

aptamers that distinguish between prostate cancers that are likely to remain organ-confined and those with potential to metastasize, The scope of this...pilot is to generate DNA aptamers that selectively react with a prostate cancer cell line that remains confined to the prostate (LNCaP) vs. a...subpopulation of this cell line that has acquired the ability to metastasize aggressively, employing Cell-Selex and Aptamer -Facilitated Biomarker Discovery

Guidelines for the use of cell lines in biomedical research

PubMed Central

Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

2014-01-01

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise. PMID:25117809

Guidelines for the use of cell lines in biomedical research.

PubMed

Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

2014-09-09

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

Selective inhibition of esophageal cancer cells by combination of HDAC inhibitors and Azacytidine

PubMed Central

Ahrens, Theresa D; Timme, Sylvia; Hoeppner, Jens; Ostendorp, Jenny; Hembach, Sina; Follo, Marie; Hopt, Ulrich T; Werner, Martin; Busch, Hauke; Boerries, Melanie; Lassmann, Silke

2015-01-01

Esophageal cancers are highly aggressive tumors with poor prognosis despite some recent advances in surgical and radiochemotherapy treatment options. This study addressed the feasibility of drugs targeting epigenetic modifiers in esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) cells. We tested inhibition of histone deacetylases (HDACs) by SAHA, MS-275, and FK228, inhibition of DNA methyltransferases by Azacytidine (AZA) and Decitabine (DAC), and the effect of combination treatment using both types of drugs. The drug targets, HDAC1/2/3 and DNMT1, were expressed in normal esophageal epithelium and tumor cells of ESCC or EAC tissue specimens, as well as in non-neoplastic esophageal epithelial (Het-1A), ESCC (OE21, Kyse-270, Kyse-410), and EAC (OE33, SK-GT-4) cell lines. In vitro, HDAC activity, histone acetylation, and p21 expression were similarly affected in non-neoplastic, ESCC, and EAC cell lines post inhibitor treatment. Combined MS-275/AZA treatment, however, selectively targeted esophageal cancer cell lines by inducing DNA damage, cell viability loss, and apoptosis, and by decreasing cell migration. Non-neoplastic Het-1A cells were protected against HDACi (MS-275)/AZA treatment. RNA transcriptome analyses post MS-275 and/or AZA treatment identified novel regulated candidate genes (up: BCL6, Hes2; down: FAIM, MLKL), which were specifically associated with the treatment responses of esophageal cancer cells. In summary, combined HDACi/AZA treatment is efficient and selective for the targeting of esophageal cancer cells, despite similar target expression of normal and esophageal cancer epithelium, in vitro and in human esophageal carcinomas. The precise mechanisms of action of treatment responses involve novel candidate genes regulated by HDACi/AZA in esophageal cancer cells. Together, targeting of epigenetic modifiers in esophageal cancers may represent a potential future therapeutic approach. PMID:25923331

Data set of the protein expression profiles of Luminal A, Claudin-low and overexpressing HER2+ breast cancer cell lines by iTRAQ labelling and tandem mass spectrometry

PubMed Central

Calderón-González, Karla Grisel; Valero Rustarazo, Ma Luz; Labra-Barrios, Maria Luisa; Bazán-Méndez, César Isaac; Tavera-Tapia, Alejandra; Herrera-Aguirre, Marí;aEsther; Sánchez del Pino, Manuel M.; Gallegos-Pérez, José Luis; González-Márquez, Humberto; Hernández-Hernández, Jose Manuel; León-Ávila, Gloria; Rodríguez-Cuevas, Sergio; Guisa-Hohenstein, Fernando; Luna-Arias, Juan Pedro

2015-01-01

Breast cancer is the most common and the leading cause of mortality in women worldwide. There is a dire necessity of the identification of novel molecules useful in diagnosis and prognosis. In this work we determined the differentially expression profiles of four breast cancer cell lines compared to a control cell line. We identified 1020 polypeptides labelled with iTRAQ with more than 95% in confidence. We analysed the common proteins in all breast cancer cell lines through IPA software (IPA core and Biomarkers). In addition, we selected the specific overexpressed and subexpressed proteins of the different molecular classes of breast cancer cell lines, and classified them according to protein class and biological process. Data in this article is related to the research article “Determination of the protein expression profiles of breast cancer cell lines by Quantitative Proteomics using iTRAQ Labelling and Tandem Mass Spectrometry” (Calderón-González et al. [1] in press). PMID:26217805

Xenobiotic metabolism in the fish hepatic cell lines Hepa-E1 and RTH-149, and the gill cell lines RTgill-W1 and G1B: Biomarkers of CYP450 activity and oxidative stress.

PubMed

Franco, Marco E; Sutherland, Grace E; Lavado, Ramon

2018-04-01

The use of fish cell cultures has proven to be an effective tool in the study of environmental and aquatic toxicology. Valuable information can be obtained from comparisons between cell lines from different species and organs. In the present study, specific chemicals were used and biomarkers (e.g. 7-Ethoxyresorufin-O-deethylase (EROD) activity and reactive oxygen species (ROS)) were measured to assess the metabolic capabilities and cytotoxicity of the fish hepatic cell lines Hepa-E1 and RTH-149, and the fish gill cell lines RTgill-W1 and G1B. These cell lines were exposed to β-naphthoflavone (BNF) and benzo[a]pyrene (BaP), the pharmaceutical tamoxifen (TMX), and the organic peroxide tert-butylhydroperoxide (tBHP). Cytotoxicity in gill cell lines was significantly higher than in hepatic cells, with BNF and TMX being the most toxic compounds. CYP1-like associated activity, measured through EROD activity, was only detected in hepatic cells; Hepa-E1 cells showed the highest activity after exposure to both BNF and BaP. Significantly higher levels of CYP3A-like activity were also observed in Hepa-E1 cells exposed to TMX, while gill cell lines presented the lowest levels. Measurements of ROS and antioxidant enzymes indicated that peroxide levels were higher in gill cell lines in general. However, levels of superoxide were significantly higher in RTH-149 cells, where no distinctive increase of superoxide-related antioxidants was observed. The present study demonstrates the importance of selecting adequate cell lines in measuring specific metabolic parameters and provides strong evidence for the fish hepatocarcinoma Hepa-E1 cells to be an excellent alternative in assessing metabolism of xenobiotics, and in expanding the applicability of fish cell lines for in vitro studies. Copyright © 2018 Elsevier Inc. All rights reserved.

Establishment of two basal-like breast cancer cell lines with extremely low tumorigenicity from Taiwanese premenopausal women.

PubMed

Kuo, Wen-Ling; Ueng, Shir-Hwa; Wu, Chun-Hsing; Lee, Li-Yu; Lee, Yun-Shien; Yu, Ming-Chin; Chen, Shin-Cheh; Yu, Chi-Chang; Tsai, Chi-Neu

2018-04-01

The research of carcinogenetic mechanisms of breast cancer in different ethnic backgrounds is an interesting field, as clinical features of breast cancers vary among races. High premenopausal incidence is distinctive in East-Asian breast cancer. However, human cell lines derived from Asian primary breast tumor are rare. To provide alternative cell line models with a relevant genetic background, we aimed to establish breast cancer cell lines from Taiwanese patients of Han-Chinese ethnicity. Fresh tissue from mammary tumors were digested into organoids, plated and grown in basal serum-free medium of human mammary epithelial cells (HuMEC) with supplements. Cells were further enriched by positive selection with CD326 (epithelial cell adhesion molecule; EpCAM)-coated micro-magnetic beads. Two breast cancer cell lines derived from premenopausal women were successfully established by this method, and named Chang-Gung Breast Cancer 01 (CGBC 01) and 02 (CGBC 02). These two cell lines had a similar phenotype with weak expression of estrogen receptor (ER), progesterone receptor (PR), and without amplification of receptor tyrosine protein kinase erbB-2 (HER2/neu). Genome-wide Single Nucleotide Polymorphism (SNP) array showed multiple copy number alterations in both cell lines. Based on gene expression profiles, CGBC 01 and 02 were clustered into basal-like subtype with reference to the breast cancer cell line gene expression database. The tumorigenicity of both cell lines was extremely low in both anchorage-independence assay and transplantation into the mammary fat pads of nude mice. CGBC 01 and CGBC 02 are low tumorigenic breast cancer cell lines, established from Han-Chinese premenopausal breast cancer patients, which serve as in vitro models in studying the biological features of Asian breast cancer.

Apoptosis induced by tumor necrosis factor-alpha in rat hepatocyte cell lines expressing hepatitis B virus.

PubMed Central

Guilhot, S.; Miller, T.; Cornman, G.; Isom, H. C.

1996-01-01

Three well differentiated SV40-immortalized rat hepatocyte cell lines, CWSV1, CWSV2, and CWSV14, and Hepatitis B Virus (HBV)-producing cell lines derived from them were examined for sensitivity to tumor necrosis factor (TNF)-alpha. CWSV1, CWSV2, and CWSV14 cells were co-transfected with a DNA construct containing a dimer of the HBV genome and the neo gene and selected in G418 to generate stable cell lines. Characterization of these cell lines indicated that they contain integrated HBV DNA, contain low molecular weight HBV DNA compatible with the presence of HBV replication intermediates, express HBV transcripts, and produce HBV proteins. The viability of CWSV1, CWSV2, and CWSV2 cells was not significantly altered when they were treated with TNF-alpha at concentrations as high as 20,000 U/ml. The HBV-expressing CWSV1 cell line, SV1di36, and the HBV-expressing CWSV14 cell line, SV14di208, were also not killed when treated with TNF-alpha. However, the HBV-expressing CWSV2 cell line, SV2di366, was extensively killed when treated with TNF-alpha at concentrations ranging from 200 to 20,000 U/ml. Analysis of several different HBV-producing CWSV2 cell lines indicated that TNF-alpha killing depended upon the level of HBV expression. The TNF-alpha-induced cell killing in high HBV-producing CWSV2 cell lines was accompanied by the presence of an oligonucleosomal DNA ladder characteristic of apoptosis. Images Figure 2 Figure 3 Figure 4 Figure 6 Figure 9 Figure 10 Figure 11 PMID:8774135

Expression of Metallothionein and Vascular Endothelial Growth Factor Isoforms in Breast Cancer Cells.

PubMed

Wierzowiecka, Barbara; Gomulkiewicz, Agnieszka; Cwynar-Zajac, Lucja; Olbromski, Mateusz; Grzegrzolka, Jedrzej; Kobierzycki, Christopher; Podhorska-Okolow, Marzenna; Dziegiel, Piotr

2016-01-01

Metallothioneins (MTs) are low-molecular-weight and cysteine-rich proteins that bind heavy metal ions and oxygen-free radicals. MTs are commonly expressed in various tissues of mammals and are involved in regulation of cell proliferation and differentiation, and may be engaged in angiogenesis. Expression of MTs has been studied in many cancer types, especially breast cancer. The research results indicate that MTs may play important, although not yet fully known, roles in cancer angiogenesis. The aim of this study was to analyze the level of gene expression of selected MT isoforms induced with zinc ions in correlation with vascular endothelial growth factor (VEGF) isoforms in in vitro models of breast cancer. The studies were carried out in three breast cancer cell lines (MCF-7, SK-BR-3, MDA-MB-231). An epithelial cell line derived from normal breast tissue (Me16c) was used as a control. The levels of expression of selected MT isoforms and selected genes involved in angiogenesis were studied with real-time PCR. Expression of different MT isoforms was induced by zinc ions to differing degrees in individual breast cancer cell lines. An increase in the expression of some MT isoforms was associated with a slight increase in the level of expression of VEGFA. The research results may indicate certain correlation between an increased expression of selected MT isoforms and a pro-angiogenic factor VEGF in specific types of breast cancer cells. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

GDC-0941, a novel class I selective PI3K inhibitor, enhances the efficacy of docetaxel in human breast cancer models by increasing cell death in vitro and in vivo.

PubMed

Wallin, Jeffrey J; Guan, Jane; Prior, Wei Wei; Lee, Leslie B; Berry, Leanne; Belmont, Lisa D; Koeppen, Hartmut; Belvin, Marcia; Friedman, Lori S; Sampath, Deepak

2012-07-15

Docetaxel is a front-line standard-of-care chemotherapeutic drug for the treatment of breast cancer. Phosphoinositide 3-kinases (PI3K) are lipid kinases that regulate breast tumor cell growth, migration, and survival. The current study was intended to determine whether GDC-0941, an orally bioavailable class I selective PI3K inhibitor, enhances the antitumor activity of docetaxel in human breast cancer models in vitro and in vivo. A panel of 25 breast tumor cell lines representing HER2+, luminal, and basal subtypes were treated with GDC-0941, docetaxel, or the combination of both drugs and assayed for cellular viability, modulation of PI3K pathway markers, and apoptosis induction. Drug combination effects on cellular viability were also assessed in nontransformed MCF10A human mammary epithelial cells. Human xenografts of breast cancer cell lines and patient-derived tumors were used to assess efficacy of GDC-0941 and docetaxel in vivo. Combination of GDC-0941 and docetaxel decreased the cellular viability of breast tumor cell lines in vitro but to variable degrees of drug synergy. Compared with nontransformed MCF10A cells, the addition of both drugs resulted in stronger synergistic effects in a subset of tumor cell lines that were not predicted by breast cancer subtype. In xenograft models, GDC-0941 enhanced the antitumor activity of docetaxel with maximum combination efficacy observed within 1 hour of administering both drugs. GDC-0941 increased the rate of apoptosis in cells arrested in mitosis upon cotreatment with docetaxel. GDC-0941 augments the efficacy of docetaxel by increasing drug-induced apoptosis in breast cancer models.

New Inhibitors of the Peripheral Site in Acetycholinesterase that Specifically Block Organophosphorylation

DTIC Science & Technology

1999-09-01

P~ "I" C 3CH20--P-- 0 at 28 *C. S2 cells were cotransfected with pPac carrying the hygromycin CH3 0 C3&13! phosphotransferase gene for selection of...cells with hygromycin B. After selection with 0.2 mg/ml hygromycin B, monoclonal cell lines were isolated from colonies formed using a modified soft

Cell cloning-on-the-spot by using an attachable silicone cylinder.

PubMed

Park, Hong Bum; Son, Wonseok; Chae, Dong Han; Lee, Jisu; Kim, Il-Woung; Yang, Woomi; Sung, Jae Kyu; Lim, Kyu; Lee, Jun Hee; Kim, Kyung-Hee; Park, Jong-Il

2016-06-10

Cell cloning is a laboratory routine to isolate and keep particular properties of cultured cells. Transfected or other genetically modified cells can be selected by the traditional microbiological cloning. In addition, common laboratory cell lines are prone to genotypic drift during their continual culture, so that supplementary cloning steps are often required to maintain correct lineage phenotypes. Here, we designed a silicone-made attachable cloning cylinder, which facilitated an easy and bona fide cloning of interested cells. This silicone cylinder was easy to make, showed competent stickiness to laboratory plastics including culture dishes, and hence enabled secure isolation and culture for days of selected single cells, especially, on the spots of preceding cell-plating dishes under microscopic examination of visible cellular phenotypes. We tested the silicone cylinder in the monoclonal subcloning from a heterogeneous population of a breast cancer cell line, MDA-MB-231, and readily established independent MDA-MB-231 subclones showing different sublineage phenotypes. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency.

PubMed

Waddell, D; Ullman, B

1983-04-10

From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.

Anti-tumor and anti-angiogenic ergosterols from Ganoderma lucidum

NASA Astrophysics Data System (ADS)

Chen, Shaodan; Yong, Tianqiao; Zhang, Yifang; Su, Jiyan; Jiao, Chunwei; Xie, Yizhen

2017-10-01

This study was carried out to isolate chemical constituents from the lipid enriched fraction of Ganoderma lucidum extract and to evaluate their anti-proliferative effect on cancer cell lines and human umbilical vein endothelial cells. Ergosterol derivatives (1-14) were isolated from the lipid enriched fraction of G. lucidum. Their structures were established on the basis of spectroscopic analyses or by comparison of mass and NMR spectral data with those reported previously. Amongst, compound 1 was isolated and identified as a new compound. All the compounds were evaluated for their inhibitory effect on tumor cells and human umbilical vein endothelial cells in vitro. Compounds 9-13 displayed inhibitory activity against two tumor cell lines and human umbilical vein endothelial cells, which indicated that these four compounds had both anti-tumor and anti-angiogenesis activities. Compound 2 had significant selective inhibition against two tumor cell lines, while 3 exhibited selective inhibition against human umbilical vein endothelial cells. The structure–activity relationships for inhibiting human HepG2 cells were revealed by 3D-QASR. Ergosterol content in different parts of the raw material and products of G. lucidum was quantified. This study provides a basis for further development and utilization of ergosterol derivatives as natural nutraceuticals and functional food ingredients, or as source of new potential antitumor or anti-angiogenesis chemotherapy agent.

Targeting malignant B cells with an immunotoxin against ROR1

PubMed Central

Baskar, Sivasubramanian; Wiestner, Adrian; Wilson, Wyndham H.; Pastan, Ira; Rader, Christoph

2012-01-01

The selective cell surface expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) has made ROR1 a novel and promising target for therapeutic monoclonal antibodies (mAbs). Four mouse mAbs generated by hybridoma technology exhibited specific binding to human ROR1. Epitope mapping studies showed that two mAbs (2A2 and 2D11) recognized N-terminal epitopes in the extracellular region of ROR1 and the other two (1A1 and 1A7) recognized C-terminal epitopes. A ROR1- immunotoxin (BT-1) consisting of truncated Pseudomonas exotoxin A (PE38) and the VH and VL fragments of 2A2-IgG was made recombinantly. Both 2A2-IgG and BT-1 showed dose-dependent and selective binding to primary CLL and MCL cells and MCL cell lines. Kinetic analyses revealed 0.12-nM (2A2-IgG) to 65-nM (BT-1) avidity/affinity to hROR1, depicting bivalent and monovalent interactions, respectively. After binding to cell surface ROR1, 2A2-IgG and BT-1 were partially internalized by primary CLL cells and MCL cell lines, and BT-1 induced profound apoptosis of ROR1-expressing MCL cell lines in vitro (EC50 = 16 pM–16 nM), but did not affect ROR1-negative cell lines. Our data suggest that ROR1-immunotoxins such as BT-1 could serve as targeted therapeutic agents for ROR1-expressing B cell malignancies and other cancers. PMID:22531447

CRISPR/Cas9 Genetic Modification of CYP3A5 *3 in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism.

PubMed

Dorr, Casey R; Remmel, Rory P; Muthusamy, Amutha; Fisher, James; Moriarity, Branden S; Yasuda, Kazuto; Wu, Baolin; Guan, Weihua; Schuetz, Erin G; Oetting, William S; Jacobson, Pamala A; Israni, Ajay K

2017-08-01

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 engineering of the CYP3A5 *3 locus (rs776746) in human liver cell line HuH-7 ( CYP3A5 *3/*3 ) has led to three CYP3A5 *1 cell lines by deletion of the exon 3B splice junction or point mutation. Cell lines CYP3A5 *1/*3 sd (single deletion), CYP3A5 *1/*1 dd (double deletion), or CYP3A5 *1/*3 pm (point mutation) expressed the CYP3A5 *1 mRNA and had elevated CYP3A5 mRNA ( P < 0.0005 for all engineered cell lines) and protein expression compared with HuH-7. In metabolism assays, HuH-7 had less tacrolimus (all P < 0.05) or midazolam (MDZ) (all P < 0.005) disappearance than all engineered cell lines. HuH-7 had less 1-OH MDZ (all P < 0.0005) or 4-OH (all P < 0.005) production in metabolism assays than all bioengineered cell lines. We confirmed CYP3A5 metabolic activity with the CYP3A4 selective inhibitor CYP3CIDE. This is the first report of genomic CYP3A5 bioengineering in human cell lines with drug metabolism analysis. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Quantitative structure-cytotoxicity relationship of piperic acid amides.

PubMed

Shimada, Chiyako; Uesawa, Yoshihiro; Ishihara, Mariko; Kagaya, Hajime; Kanamoto, Taisei; Terakubo, Shigemi; Nakashima, Hideki; Takao, Koichi; Miyashiro, Takaki; Sugita, Yoshiaki; Sakagami, Hiroshi

2014-09-01

A total of 12 piperic acid amides, including piperine, were subjected to quantitative structure-activity relationship (QSAR) analysis, based on their cytotoxicity, tumor selectivity and anti-HIV activity, in order to find new biological activities. Cytotoxicity against four human oral squamous cell carcinoma (OSCC) cell lines and three human oral normal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Tumor selectivity was evaluated by the ratio of the mean 50% cytotoxic concentration (CC50) against normal oral cells to that against OSCC cell lines. Anti-HIV activity was evaluated by the ratio of the CC50 to 50% HIV infection-cytoprotective concentration (EC50). Physicochemical, structural, and quantum-chemical parameters were calculated based on the conformations optimized by LowModeMD method followed by density functional theory method. All compounds showed low-to-moderate tumor selectivity, but no anti-HIV activity. N-Piperoyldopamine ( 8: ) which has a catechol moiety, showed the highest tumor selectivity, possibly due to its unique molecular shape and electrostatic interaction, especially its largest partial equalization of orbital electronegativities and vsurf descriptors. The present study suggests that molecular shape and ability for electrostatic interaction are useful parameters for estimating the tumor selectivity of piperic acid amides. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Altered synthesis and processing of oligosaccharides of vesicular stomatitis virus glycoprotein in different lectin-resistant Chinese hamster ovary cell lines.

PubMed

Hunt, L A

1980-08-01

To determine the particular intracellular steps in the glycosylation of the vesicular stomatitis virus (VSV) glycoprotein that were altered in several lectin-resistant CHO cell lines, VSV-infected parental and mutant cells were pulse-labeled for 30 and 120 min with [3H]mannose and [3H]glucosamine. Cell-associated viral glycopeptides were analyzed by gel filtration combined with specific glycosidase digestions and compared with the corresponding mature virion oligosaccharides. The intracellular glycosylation of the VSV glycoprotein in a mutant cell line resistant to phytohemagglutinin was identical to that in the normal cells except for a complete block in processing at a specific step in the final trimming of the oligomannosyl core from five to three mannoses. The results demonstrated that a double-mutant cell line selected from the phytohemagglutinin-resistant cells for resistance to concanavalin A had an additional defect in one of the earliest stages of glycosylation, resulting in smaller precursor oligosaccharides linked to protein.

Altered synthesis and processing of oligosaccharides of vesicular stomatitis virus glycoprotein in different lectin-resistant Chinese hamster ovary cell lines.

PubMed Central

Hunt, L A

1980-01-01

To determine the particular intracellular steps in the glycosylation of the vesicular stomatitis virus (VSV) glycoprotein that were altered in several lectin-resistant CHO cell lines, VSV-infected parental and mutant cells were pulse-labeled for 30 and 120 min with [3H]mannose and [3H]glucosamine. Cell-associated viral glycopeptides were analyzed by gel filtration combined with specific glycosidase digestions and compared with the corresponding mature virion oligosaccharides. The intracellular glycosylation of the VSV glycoprotein in a mutant cell line resistant to phytohemagglutinin was identical to that in the normal cells except for a complete block in processing at a specific step in the final trimming of the oligomannosyl core from five to three mannoses. The results demonstrated that a double-mutant cell line selected from the phytohemagglutinin-resistant cells for resistance to concanavalin A had an additional defect in one of the earliest stages of glycosylation, resulting in smaller precursor oligosaccharides linked to protein. Images PMID:6255177

Increased Activity of [gamma]-Glutamylcysteine Synthetase in Tomato Cells Selected for Cadmium Tolerance.

PubMed

Chen, J.; Goldsbrough, P. B.

1994-09-01

Two cell lines of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) were systematically compared for their capacity to tolerate cadmium. Unselected CdS cells died in the presence of 0.3 mM CdCl2. CdR6-0 cells, which were selected from CdS, survived and grew in medium supplemented with 0.3 mM CdCl2. Growth of CdR6-0 cells under this condition was accompanied by synthesis of cadmium-binding phytochelatins and maintenance of cellular glutathione (GSH) levels. CdR6-0 cells also exhibited increased tolerance to buthionine sulfoximine, in both the presence and absence of 0.1 mM CdCl2. The specific activity of [gamma]-glutamylcysteine synthetase (EC 6.3.2.2) was approximately 2-fold higher in CdR6-0 cells than in CdS cells, whereas there was no difference between cell lines in specific activity of GSH synthetase (EC 6.3.2.3). Increased activity of the first enzyme of GSH biosynthesis in CdR6-0 cells, presumably a result of selection for increased cadmium tolerance, provides an enhanced capacity to synthesize GSH and to maintain the production of phytochelatins in response to cadmium. This adaptation may contribute to the enhanced cadmium tolerance of CdR6-0 cells.

Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line.

PubMed

Thomas, Andrew J; Hailey, Dale W; Stawicki, Tamara M; Wu, Patricia; Coffin, Allison B; Rubel, Edwin W; Raible, David W; Simon, Julian A; Ou, Henry C

2013-03-06

Cisplatin, one of the most commonly used anticancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analog of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line.

Assessing Estrogen-Induced Proliferative Response in an Endometrial Cancer Cell Line Using a Universally Applicable Methodological Guide.

PubMed

Parkes, Christina; Kamal, Areege; Valentijn, Anthony J; Alnafakh, Rafah; Gross, Stephane R; Barraclough, Roger; Moss, Diana; Kirwan, John; Hapangama, Dharani K

2018-01-01

Translational endometrial cancer (EC) research benefits from an in vitro experimental approach using EC cell lines. We demonstrated the steps that are required to examine estrogen-induced proliferative response, a simple yet important research question pertinent to EC, and devised a pragmatic methodological workflow for using EC cell lines in experimental models. Comprehensive review of all commercially available EC cell lines was carried out, and Ishikawa cell line was selected to study the estrogen responsiveness with HEC1A, RL95-2, and MFE280 cell lines as comparators where appropriate, examining relevant differential molecular (steroid receptors) and functional (phenotype, anchorage-independent growth, hormone responsiveness, migration, invasion, and chemosensitivity) characteristics in 2-dimensional and 3-dimensional cultures in vitro using immunocytochemistry, immunofluorescence, quantitative polymerase chain reaction, and Western blotting. In vivo tumor, formation, and chemosensitivity were also assessed in a chick chorioallantoic membrane model. Short tandem repeat analysis authenticated the purchased cell lines, whereas gifted cells deviated significantly from the published profile. We demonstrate the importance of prior assessment of the suitability of each cell line for the chosen in vitro experimental technique. Prior establishment of baseline, nonenriched conditions was required to induce a proliferative response to estrogen. The chorioallantoic membrane model was a suitable in vivo multicellular animal model for EC for producing rapid and reproducible data. We have developed a methodological guide for EC researchers when using endometrial cell lines to answer important translational research questions (exemplified by estrogen-responsive cell proliferation) to facilitate robust data, while saving time and resources.

Novel urea and bis-urea primaquine derivatives with hydroxyphenyl or halogenphenyl substituents: Synthesis and biological evaluation.

PubMed

Perković, I; Antunović, M; Marijanović, I; Pavić, K; Ester, K; Kralj, M; Vlainić, J; Kosalec, I; Schols, D; Hadjipavlou-Litina, D; Pontiki, E; Zorc, B

2016-11-29

A series of novel compounds 3a-j and 6a-j with primaquine and hydroxyl or halogen substituted benzene moieties bridged by urea or bis-urea functionalities were designed, synthesized and evaluated for biological activity. The title compounds were prepared using benzotriazole as the synthon, through several synthetic steps. 3-[3,5-Bis(trifluoromethyl)phenyl]-1-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}urea (3j) was the most active urea and 1-[({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)amino]-3-[3-(trifluoromethyl)phenyl]urea (6h) the most active bis-urea derivative in antiproliferative screening in vitro against eight tested cancer cell lines. Urea derivatives 3a-g with hydroxy group or one halogen atom showed moderate antiproliferative effects against all the tested cell lines, but stronger activity against breast carcinoma MCF-7 cell line, while trifluoromethyl derivatives 3h-j showed antiproliferative effects against all the tested cell lines in low micromolar range. Finally, bis-ureas with hydroxy and fluoro substituents 6a-d showed extreme selectivity and chloro or bromo derivatives 6e-g high selectivity against MCF-7 cells (IC 50 0.1-2.6 μM). p-Fluoro derivative 6d, namely 3-(4-fluorophenyl)-1-[({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)amino]urea, is the most promising compound. Further biological experiments showed that 6d affected cell cycle and induced cell death of MCF-7 cell line. Due to its high activity against MCF-7 cell line (IC 50 0.31 μM), extreme selectivity and full agreement with the Lipinski's and Gelovani's rules for prospective small molecular drugs, 6d may be considered as a lead compound in development of breast carcinoma drugs. Urea 3b and almost all bis-ureas showed high antioxidant activity in DPPH assay, but urea derivatives were more active in lipid peroxidation test. Only few compounds exhibited weak inhibition of soybean lipoxygenase. Compound 3j exhibited the strongest antimicrobial activity in susceptibility assay in vitro (MIC = 1.6-12.5 μg ml -1 ). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Heat shock protein 90-mediated peptide-selective presentation of cytosolic tumor antigen for direct recognition of tumors by CD4(+) T cells.

PubMed

Tsuji, Takemasa; Matsuzaki, Junko; Caballero, Otavia L; Jungbluth, Achim A; Ritter, Gerd; Odunsi, Kunle; Old, Lloyd J; Gnjatic, Sacha

2012-04-15

Tumor Ag-specific CD4(+) T cells play important functions in tumor immunosurveillance, and in certain cases they can directly recognize HLA class II-expressing tumor cells. However, the underlying mechanism of intracellular Ag presentation to CD4(+) T cells by tumor cells has not yet been well characterized. We analyzed two naturally occurring human CD4(+) T cell lines specific for different peptides from cytosolic tumor Ag NY-ESO-1. Whereas both lines had the same HLA restriction and a similar ability to recognize exogenous NY-ESO-1 protein, only one CD4(+) T cell line recognized NY-ESO-1(+) HLA class II-expressing melanoma cells. Modulation of Ag processing in melanoma cells using specific molecular inhibitors and small interfering RNA revealed a previously undescribed peptide-selective Ag-presentation pathway by HLA class II(+) melanoma cells. The presentation required both proteasome and endosomal protease-dependent processing mechanisms, as well as cytosolic heat shock protein 90-mediated chaperoning. Such tumor-specific pathway of endogenous HLA class II Ag presentation is expected to play an important role in immunosurveillance or immunosuppression mediated by various subsets of CD4(+) T cells at the tumor local site. Furthermore, targeted activation of tumor-recognizing CD4(+) T cells by vaccination or adoptive transfer could be a suitable strategy for enhancing the efficacy of tumor immunotherapy.

Identification of Mutation Accumulation as Resistance Mechanism Emerging in First-Line Osimertinib Treatment.

PubMed

Uchibori, Ken; Inase, Naohiko; Nishio, Makoto; Fujita, Naoya; Katayama, Ryohei

2018-04-24

The survival of patients with EGFR mutation-positive lung cancer has dramatically improved since the introduction of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Recently, osimertinib showed significantly prolonged progression-free survival than first-generation EGFR-TKI in first-line treatment, suggesting that a paradigm change that would move osimetinib to first-line treatment is indicated. We performed N-ethyl-N-nitrosourea (ENU) mutagenesis screening to uncover the resistant mechanism in first- and second-line osimertinib treatment. Ba/F3 cells harboring EGFR activating-mutation with or without secondary resistant mutation were exposed to ENU for 24 hours to introduce random mutations and selected with gefitinib, afatinib, or osimertinib. Mutations of emerging resistant cells were assessed. The resistance of T790M and C797S to gefitinib and osimertinib, respectively, was prevalent in the mutagenesis screening with the Ba/F3 cells harboring activating-mutation alone. From C797S/activating-mutation expressing Ba/F3, the additional T790M was a major resistant mechanism in gefitinib and afatinib selection and the additional T854A and L792H were minor resistance mechanisms only in afatinib selection. However, the additional T854A or L792H mediated resistance to all classes of EGFR-TKI. Surprisingly, no resistant clone due to secondary mutation emerged from activating-mutation alone in the gefitinib + osimertinib selection. We showed the resistance mechanism to EGFR-TKI focusing on first- and second-line osimertinib using ENU mutagenesis screening. Additional T854A and L792H on C797S/activating-mutation were found as afatinib resistance and not as gefitinib resistance. Thus, compared to afatinib, the first-generation EGFR-TKI might be preferable as second-line treatment to C797S/activating-mutation emerging after first-line osimertinib treatment. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Establishment of optimized MDCK cell lines for reliable efflux transport studies.

PubMed

Gartzke, Dominik; Fricker, Gert

2014-04-01

Madin-Darby canine kidney (MDCK) cells transfected with human MDR1 gene (MDCK-MDR1) encoding for P-glycoprotein (hPgp, ABCB1) are widely used for transport studies to identify drug candidates as substrates of this efflux protein. Therefore, it is necessary to rely on constant and comparable expression levels of Pgp to avoid false negative or positive results. We generated a cell line with homogenously high and stable expression of hPgp through sorting single clones from a MDCK-MDR1 cell pool using fluorescence-activated cell sorting (FACS). To obtain control cell lines for evaluation of cross-interactions with endogenous canine Pgp (cPgp) wild-type cells were sorted with a low expression pattern of cPgp in comparison with the MDCK-MDR1. Expression of other transporters was also characterized in both cell lines by quantitative real-time PCR and Western blot. Pgp function was investigated applying the Calcein-AM assay as well as bidirectional transport assays using (3) H-Digoxin, (3) H-Vinblastine, and (3) H-Quinidine as substrates. Generated MDCK-MDR1 cell lines showed high expression of hPgp. Control MDCK-WT cells were optimized in showing a comparable expression level of cPgp in comparison with MDCK-MDR1 cell lines. Generated cell lines showed higher and more selective Pgp transport compared with parental cells. Therefore, they provide a significant improvement in the performance of efflux studies yielding more reliable results. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Antiproliferative action of Xylopia aethiopica fruit extract on human cervical cancer cells.

PubMed

Adaramoye, Oluwatosin A; Sarkar, Jayanta; Singh, Neetu; Meena, Sanjeev; Changkija, Bendangla; Yadav, Prem P; Kanojiya, Sanjeev; Sinha, Sudhir

2011-10-01

The anticancer potential of Xylopia aethiopica fruit extract (XAFE), and the mechanism of cell death it elicits, was investigated in various cell lines. Treatment with XAFE led to a dose-dependent growth inhibition in most cell lines, with selective cytotoxicity towards cancer cells and particularly the human cervical cancer cell line C-33A. In this study, apoptosis was confirmed by nuclear fragmentation and sub-G(0)/G(1) phase accumulation. The cell cycle was arrested at the G(2)/M phase with a decreased G(0)/G(1) population. A semi-quantitative gene expression study revealed dose-dependent up-regulation of p53 and p21 genes, and an increase in the Bax/Bcl-2 ratio. These results indicate that XAFE could be a potential therapeutic agent against cancer since it inhibits cell proliferation, and induces apoptosis and cell cycle arrest in C-33A cells. Copyright © 2011 John Wiley & Sons, Ltd.

GAPD and tubulin are suitable internal controls for qPCR analysis of oral squamous cell carcinoma cell lines.

PubMed

Campos, M S; Rodini, C O; Pinto-Júnior, D S; Nunes, F D

2009-02-01

The selection of housekeeping genes is critical for gene expression studies. To address this issue, four candidate housekeeping genes, including several commonly used ones, were investigated in oral squamous cell carcinoma cell lines. A simple quantitative RT-PCR approach was employed by comparing relative expression of the four candidate genes within two cancerous cell lines (HN6 and HN31) and one noncancerous cell line (HaCaT) treated or not with EGF and TGF-beta1. Data were analyzed using ANOVA followed by the NormFinder software program. On this basis, stability of the candidate housekeeping genes was ranked and non statistical differences were found using ANOVA test. On the other hand, the NormFinder was able to show that GAPD and TUBB presented the less variable results, representing appropriated housekeeping genes for the samples and conditions analyzed. In conclusion, this study suggests that the GAPD and the TUBB represent adequate normalizers for gene profiling studies in OSCC cell lines, covering, respectively, high and low expression levels genes.

The phosphoinositide 3-kinase α selective inhibitor BYL719 enhances the effect of the protein kinase C inhibitor AEB071 in GNAQ/GNA11-mutant uveal melanoma cells.

PubMed

Musi, Elgilda; Ambrosini, Grazia; de Stanchina, Elisa; Schwartz, Gary K

2014-05-01

G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (sotrastaurin) and PI3K/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11-mutant cells with AEB071 versus no activity in wild-type cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of myristoylated alanine-rich C-kinase substrate, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal antiproliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11-mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ- and GNA11-mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ-mutant model. These findings suggest a new therapy treatment option for G-protein-mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.

The Phosphoinositide 3-Kinaseα Selective Inhibitor, BYL719, Enhances the Effect of the Protein Kinase C Inhibitor, AEB071, in GNAQ/GNA11 Mutant Uveal Melanoma Cells

PubMed Central

Musi, Elgilda; Ambrosini, Grazia; de Stanchina, Elisa; Schwartz, Gary K.

2014-01-01

G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-Kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (Sotrastaurin) and PI3k/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11 mutant cells with AEB071 versus no activity in WT cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of MARCKS, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal anti-proliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11 mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ and GNA11 mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ mutant model. These findings suggest a new therapy treatment option for G-protein mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy. PMID:24563540

Involvement of triacylglycerol in the metabolism of fatty acids by cultured neuroblastoma and glioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, H.W.; Clarke, J.T.; Spence, M.W.

1982-12-01

The metabolism (chain elongation, desaturation, and incorporation into complex lipids) of thirteen different radiolabeled fatty acids and acetate was examined in N1E-115 neuroblastoma and C-6 glioma cell lines in culture. During 6-hr incubations, all fatty acids were extensively (14-80%) esterified to complex lipids, mainly choline phosphoglycerides and triacylglycerol. With trienoic and tetraenoic substrates, inositol and ethanolamine phosphoglycerides also contained up to 30% of the labeled fatty acids; plasmalogen contained up to half of the label in the ethanolamine phosphoglyceride fraction of neuroblastoma cells. Chain elongation and delta 9, delta 6, and delta 5 desaturation occurred in both cell lines; deltamore » 4 desaturation was not observed. Seemingly anomalous utilization of arachidic acid and some selectivity based on the geometric configuration of double bonds was observed. These studies indicate that these cell lines are capable of modulating cellular membrane composition by a combination of selective exclusion and removal of inappropriate acyl chains and of modification of other acyl chains by desaturation and chain elongation. The time courses and patterns of modification and incorporation of exogenous substrates into phospholipids and triacylglycerol suggest that exogenous unsaturated fatty acid may be incorporated into triacylglycerol and later released for further metabolism and incorporation into phospholipids. This supports a role for triacylglycerol in the synthesis of membrane complex lipids in cell lines derived from neural tissue.« less

Establishment of a cell line producing bone morphogenetic protein from a human osteosarcoma.

PubMed

Takaoka, K; Yoshikawa, H; Masuhara, K; Sugamoto, K; Tsuda, T; Aoki, Y; Ono, K; Sakamoto, Y

1989-07-01

A human osteosarcoma cell line was established from a biopsy specimen from a 13-year-old girl. The osteosarcoma tissue was maintained in athymic nude mice (Balb C nu/nu) by serial transplantation for three years. The tumor was excised from a host mouse and digested with collagenase. The isolated cells were cultured by 98 passages in 14 months, and clones of osteosarcoma cells were obtained by limiting dilution. A clone named human osteosarcoma cell 6 (H-OS-6) that showed the osteoblastic phenotypes of productions of bone morphogenetic protein (BMP) and alkaline phosphatase and a response to human parathyroid hormone (h-PTH 1-34) was selected. The morphology of its chromosomes indicated its human origin. This human osteosarcoma cell line is unique in producing BMP under in vitro conditions.

Influence of water content on the laser-induced breakdown spectroscopy analysis of human cell pellet

NASA Astrophysics Data System (ADS)

Moon, Youngmin; Han, Jung Hyun; Lee, Jong Jin; Jeong, Sungho

2015-12-01

The effects of water content change in a biological sample on the emission signal intensity and intensity ratio during LIBS analysis were investigated. To examine the effects of water content only avoiding matrix effects, a homogeneous human cell pellet consisting of cultured human immortalized keratinocyte cell only was used as the sample. LIBS spectra of the human cell pellet sample produced with a Q-switched Nd:YAG laser (λ = 532 nm, τ = 5 ns, top-hat profile) and a six-channel CCD spectrometer (spectral range = 187-1045 nm, spectral resolution = 0.1 nm) revealed that most of the emission lines observed from a tissue sample were also observable from the human cell pellet. The intensity and intensity ratio of the emission lines varied significantly as the water content of the human cell pellet was changed. It was found that a typically selected internal standard in LIBS analysis of biological samples such as carbon could produce inconsistent results, whereas the ratio of properly selected emission lines such as Mg(II) 280.270 nm and Ca(II) 396.847 nm was nearly independent of sample water content.

Fred Hutchinson Cancer Research Center (FHCRC1): Functional Landscape of the Human Kinome in MYCN Amplified and Non-amplified Neuroblastoma | Office of Cancer Genomics

Cancer.gov

In order to identify candidate drugs targets that exhibit lethality only in the context of MYCN amplification, we carried out a set of siRNA screens focused on the kinome, targeting ~713 kinases, utilizing human neuroblastoma cells lines with or without MYCN amplification. The neuroblastoma cell lines were: SK-N-BE2 (MYCN amplified) and SK-N-AS (non-amplified).  The kinase Hits for the MYCN amplified cell line were selected using a combination of their differential activity when compared to the non-MYCN amplified cells and also ranked by P-values, based on the replicates.

Fred Hutchinson Cancer Research Center (FHCRC-1): Functional Landscape of the Human Kinome in MYCN Amplified and Non-amplified Neuroblastoma | Office of Cancer Genomics

Cancer.gov

In order to identify candidate drugs targets that exhibit lethality only in the context of MYCN amplification, we carried out a set of siRNA screens focused on the kinome, targeting ~713 kinases, utilizing human neuroblastoma cells lines with or without MYCN amplification. The neuroblastoma cell lines were: SK-N-BE2 (MYCN amplified) and SK-N-AS (non-amplified). The kinase Hits for the MYCN amplified cell line were selected using a combination of their differential activity when compared to the non-MYCN amplified cells and also ranked by P-values, based on the replicates.

Recombinase-mediated cassette exchange (RMCE) for monoclonal antibody expression in the commercially relevant CHOK1SV cell line.

PubMed

Zhang, Lin; Inniss, Mara C; Han, Shu; Moffat, Mark; Jones, Heather; Zhang, Baohong; Cox, Wendy L; Rance, James R; Young, Robert J

2015-01-01

To meet product quality and cost parameters for therapeutic monoclonal antibody (mAb) production, cell lines are required to have excellent growth, stability, and productivity characteristics. In particular, cell line generation stability is critical to the success of a program, especially where high cell line generation numbers are required for large in-market supply. However, a typical process for developing such cell lines is laborious, lengthy, and costly. In this study, we applied a FLP/FRT recombinase-mediated cassette exchange (RMCE) system to build a site-specific integration (SSI) system for mAb expression in the commercially relevant CHOK1SV cell line. Using a vector with a FRT-flanked mAb expression cassette, we generated a clonal cell line with good productivity, long-term production stability, and low mAb gene-copy number indicating the vector was located in a 'hot-spot.' A SSI host cell line was made by removing the mAb genes from the 'hot-spot' by RMCE, creating a 'landing pad' containing two recombination cassettes that allow targeting of one or two copies of recombinant genes. Cell lines made from this host exhibited excellent growth and productivity profiles, and stability for at least 100 generations in the absence of selection agents. Importantly, while clones containing two copies had higher productivity than single copy clones, both were stable over many generations. Taken together, this study suggests the use of FLP-based RMCE to develop SSI host cells for mAb production in CHOK1SV offers significant savings in both resources and overall cell line development time, leading to a shortened 'time-to-clinic' for therapeutic mAbs. © 2015 American Institute of Chemical Engineers.

Novel ligands for cancer diagnosis: selection of peptide ligands for identification and isolation of B-cell lymphomas.

PubMed

McGuire, Michael J; Samli, Kausar N; Chang, Ya-Ching; Brown, Kathlynn C

2006-04-01

Lymphoma and leukemia account for nearly 8% of cancer fatalities each year. Present treatments do not differentiate between normal and malignant cells. New reagents that distinguish malignant cells and enable the isolation of these cells from the normal background will enhance the molecular characterization of disease and specificity of treatment. Peptide ligands were selected from a phage-displayed peptide library by biopanning on the B-cell lymphoma line, A20. The isolated peptides were assessed as reagents for identification and isolation of lymphoma cells by flow cytometry and cell capture with magnetic beads. Two novel peptides and one obtained previously on cardiomyocytes were selected. A20 cells bind phage displaying these peptides 250- to 450-fold over control phage. These phage bind to other bone marrow-derived cancel lines including some macrophage and T cells but do not bind to normal splenocytes. Synthetic constructs of these peptides have binding affinities comparable to B-cell-specific antibodies. Similar to antibodies, these peptides can be used in flow cytometry and magnetic bead capture to distinguish lymphoma cells from normal splenocytes. Bone marrow-derived malignant cells express cell surface markers that can be used to distinguish them from normal cells. These results demonstrate the ability to use an unbiased screen to rapidly generate high-affinity peptide ligands for identification and isolation of lymphoma cells.

Exosomes secreted by placental stem cells selectively inhibit growth of aggressive prostate cancer cells.

PubMed

Peak, Taylor C; Praharaj, Prakash P; Panigrahi, Gati K; Doyle, Michael; Su, Yixin; Schlaepfer, Isabel R; Singh, Ravi; Vander Griend, Donald J; Alickson, Julie; Hemal, Ashok; Atala, Anthony; Deep, Gagan

2018-05-23

The current paradigm in the development of new cancer therapies is the ability to target tumor cells while avoiding harm to noncancerous cells. Furthermore, there is a need to develop novel therapeutic options against drug-resistant cancer cells. Herein, we characterized the placental-derived stem cell (PLSC) exosomes (PLSC Exo ) and evaluated their anti-cancer efficacy in prostate cancer (PCa) cell lines. Nanoparticle tracking analyses revealed the size distribution (average size 131.4 ± 0.9 nm) and concentration of exosomes (5.23 × 10 10 ±1.99 × 10 9 per ml) secreted by PLSC. PLSC Exo treatment strongly inhibited the viability of enzalutamide-sensitive and -resistant PCa cell lines (C4-2B, CWR-R1, and LNCaP cells). Interestingly, PLSC Exo treatment had no effect on the viability of a non-neoplastic human prostate cell line (PREC-1). Mass spectrometry (MS) analyses showed that PLSC Exo are loaded with 241 proteins and mainly with saturated fatty acids. Further, Ingenuity Pathway Analysis analyses of proteins loaded in PLSC Exo suggested the role of retinoic acid receptor/liver x receptor pathways in their biological effects. Together, these results suggest the novel selective anti-cancer effects of PLSC Exo against aggressive PCa cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Evaluation of Cytarabine Against Ewing Sarcoma Xenografts by the Pediatric Preclinical Testing Program

PubMed Central

Houghton, Peter J.; Morton, Christopher L.; Kang, Min; Reynolds, C. Patrick; Billups, Catherine A.; Favours, Edward; Payne-Turner, Debbie; Tucker, Chandra; Smith, Malcolm A.

2015-01-01

Treatment with the nucleoside analog cytarabine has been shown to mimic changes in gene expression associated with down-regulation of the EWS-FLI1 oncogene in Ewing sarcoma cell lines, selectively inhibit their growth in vitro, and cause tumor regression in athymic nude mice. For this report cytarabine was studied in vitro against a panel of 23 pediatric cancer cell lines and in vivo against 6 Ewing sarcoma xenografts. Acute lymphoblastic leukemia cell lines were the most sensitive to cytarabine in vitro (median IC50 9 nM), while Ewing sarcoma cell lines showed intermediate sensitivity (median IC50 232 nM). Cytarabine at a dose of 150 mg/kg administered daily 5× failed to significantly inhibit growth of five xenograft models, but reduced growth rate of the A673 xenograft by 50%. Cytarabine shows no differential in vitro activity against Ewing sarcoma cell lines and is ineffective in vivo against Ewing sarcoma xenografts at the dose and schedule studied. PMID:20979180

Analysis of proviral integration in human mammary epithelial cell lines immortalized by retroviral infection with a temperature-sensitive SV40 T-antigen construct.

PubMed

Stamps, A C; Davies, S C; Burman, J; O'Hare, M J

1994-06-15

A panel of eight conditionally immortal lines derived by infection of human breast epithelial cells with an amphotropic retrovirus transducing a ts mutant of SV40 large T-antigen was analyzed with respect to individual retroviral integration patterns. Each line contained multiple integration sites which were clonal and stable over extended passage. Similar integration patterns were observed between individual lines arising separately from the same stock of pre-immortal cells, suggesting a common progenitor. Retroviral integration analysis of pre-immortal cells at different stages of pre-crisis growth showed changes indicative of a progressive transition from polyclonality to clonality as the cells approached crisis. Each of the immortal lines contained a sub-set of the integration sites of their pre-immortal progenitors, with individual combinations and copy numbers of sites. Since all the cell lines appeared to originate from single foci in separate flasks, it is likely that each set arose from a common clone of pre-immortal cells as the result of separate genetic events. There was no evidence from this analysis to suggest that specific integration sites played any part either in the selection of pre-crisis clones or in the subsequent establishment of immortal lines.

Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells.

PubMed

Blochlinger, K; Diggelmann, H

1984-12-01

The DNA coding sequence for the hygromycin B phosphotransferase gene was placed under the control of the regulatory sequences of a cloned long terminal repeat of Moloney sarcoma virus. This construction allowed direct selection for hygromycin B resistance after transfection of eucaryotic cell lines not naturally resistant to this antibiotic, thus providing another dominant marker for DNA transfer in eucaryotic cells.

Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells.

PubMed Central

Blochlinger, K; Diggelmann, H

1984-01-01

The DNA coding sequence for the hygromycin B phosphotransferase gene was placed under the control of the regulatory sequences of a cloned long terminal repeat of Moloney sarcoma virus. This construction allowed direct selection for hygromycin B resistance after transfection of eucaryotic cell lines not naturally resistant to this antibiotic, thus providing another dominant marker for DNA transfer in eucaryotic cells. Images PMID:6098829

Development of cortical orientation selectivity in the absence of visual experience with contour

PubMed Central

Hussain, Shaista; Weliky, Michael

2011-01-01

Visual cortical neurons are selective for the orientation of lines, and the full development of this selectivity requires natural visual experience after eye opening. Here we examined whether this selectivity develops without seeing lines and contours. Juvenile ferrets were reared in a dark room and visually trained by being shown a movie of flickering, sparse spots. We found that despite the lack of contour visual experience, the cortical neurons of these ferrets developed strong orientation selectivity and exhibited simple-cell receptive fields. This finding suggests that overt contour visual experience is unnecessary for the maturation of orientation selectivity and is inconsistent with the computational models that crucially require the visual inputs of lines and contours for the development of orientation selectivity. We propose that a correlation-based model supplemented with a constraint on synaptic strength dynamics is able to account for our experimental result. PMID:21753023

Retinal ganglion cells with distinct directional preferences differ in molecular identity, structure, and central projections.

PubMed

Kay, Jeremy N; De la Huerta, Irina; Kim, In-Jung; Zhang, Yifeng; Yamagata, Masahito; Chu, Monica W; Meister, Markus; Sanes, Joshua R

2011-05-25

The retina contains ganglion cells (RGCs) that respond selectively to objects moving in particular directions. Individual members of a group of ON-OFF direction-selective RGCs (ooDSGCs) detect stimuli moving in one of four directions: ventral, dorsal, nasal, or temporal. Despite this physiological diversity, little is known about subtype-specific differences in structure, molecular identity, and projections. To seek such differences, we characterized mouse transgenic lines that selectively mark ooDSGCs preferring ventral or nasal motion as well as a line that marks both ventral- and dorsal-preferring subsets. We then used the lines to identify cell surface molecules, including Cadherin 6, CollagenXXVα1, and Matrix metalloprotease 17, that are selectively expressed by distinct subsets of ooDSGCs. We also identify a neuropeptide, CART (cocaine- and amphetamine-regulated transcript), that distinguishes all ooDSGCs from other RGCs. Together, this panel of endogenous and transgenic markers distinguishes the four ooDSGC subsets. Patterns of molecular diversification occur before eye opening and are therefore experience independent. They may help to explain how the four subsets obtain distinct inputs. We also demonstrate differences among subsets in their dendritic patterns within the retina and their axonal projections to the brain. Differences in projections indicate that information about motion in different directions is sent to different destinations.

Ethanolic and aqueous extracts derived from Australian fungi inhibit cancer cell growth in vitro.

PubMed

Beattie, Karren D; Ulrich, Rahel; Grice, I Darren; Uddin, Shaikh J; Blake, Tony B; Wood, Kyle A; Steele, Jules; Iu, Fontaine; May, Tom W; Tiralongo, Evelin

2011-01-01

Fifteen Australian macrofungi were investigated for cytotoxic activity. Ethanol, cold and hot water extracts of each species were screened for cytotoxic activity against normal mouse fibroblast cells (NIH/3T3), healthy human epithelial kidney cells (HEK-293), four cancer cell lines, gastric adenocarcinoma cells (AGS), two mammary gland adenocarcinoma cells (MDA-MB-231, MCF7) and colorectal adenocarcinoma cells (HT-29) with a validated MTT assay. Most extracts derived from Omphalotus nidiformis, Cordyceps cranstounii and Cordyceps gunnii demonstrated significant cytotoxic activity toward a variety of cancer cell lines. In contrast only some extracts from Coprinus comatus, Cordyceps hawkesii, Hypholoma fasciculare, Lepista nuda, Leratiomyces ceres and Ophiocordyceps robertsii displayed significant cytotoxic activity, which was usually selective for only one or two cancer cell lines tested. The least cytotoxic species evaluated in this study were Agaricus bitorquis, Coprinopsis atrametaria, Psathyrella asperospora, Russula clelandii, Tricholoma sp. AU2 and Xerula mundroola.

[Effect of EMP-1 gene on human esophageal cancer cell line].

PubMed

Wang, Hai-tao; Liu, Zhi-hua; Wang, Xiu-qin; Wu, Min

2002-03-01

EMP-1 was selected from a series of differential expressed genes obtained from cDNA microarray in the authors' lab. Epithelial membrane pnteiu-1 gene (EMP-1) was expressed 6 fold lower in esophageal cancer than in normal tissue. The authors further designed the experiment to study the effect of human EMP-1 gene on human esophageal cancer cell line in order to explain the function of this gene on the carcinogensis and progression esophageal cancer. EMP-1 gene was cloned into eukaryotic vector and transfected into the human esophageal cancer cell line. The transfection effect was qualified by Western blot and RT-PCR method. The cell growth curve was observed and the cell cycle was checked by FACS method. EMP-1 was transfected into EC9706 cell line and its expression was up-regulated. The cell growth is accelerated and expression of EMP-1 is linked to induction of S phase arrest. EMP-1 gene has some relationship with carcinogenesis of esophagus.

Cytotoxic 2',5'-dihydroxychalcones with unexpected antiangiogenic activity.

PubMed

Nam, Nguyen-Hai; Kim, Yong; You, Young-Jae; Hong, Dong-Ho; Kim, Hwan-Mook; Ahn, Byung-Zun

2003-02-01

A series of 2',5'-dihydroxychalcones were synthesized and evaluated for cytotoxicity against tumor cell lines and human umbilical venous endothelial cells (HUVEC). It was found that chalcones with electron-withdrawing substituents on the B ring exhibited potent cytotoxicity against a variety of tumor cell lines while compounds with electron-releasing groups were less potent in general. Those compounds with B ring replaced by extended or heteroaromatic rings exhibited significant bioactivity. Several compounds were shown to have marked cytotoxic selectivity towards HUVECs. Especially, among the synthesized compounds, 2-chloro-2',5'-dihydroxychalcone (2-3) showed the highest selectivity index up to 66 in comparison to HCT116 cells. This compound also exhibited strong inhibitory effects on the HUVEC tube formation in an in vitro model. When administered into BDF1 mice bearing Lewis lung carcinoma cells at 50 mg kg(-1) day(-1), 2-3 was found to inhibit the growth of tumor mass by 60.5%.

Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin

PubMed Central

Oulhen, Nathalie; Wessel, Gary M.

2016-01-01

Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3′UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability. PMID:27424271

Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.

PubMed

Oulhen, Nathalie; Wessel, Gary M

2016-10-01

Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3'UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparison of protein expression between human livers and the hepatic cell lines HepG2, Hep3B, and Huh7 using SWATH and MRM-HR proteomics: Focusing on drug-metabolizing enzymes.

PubMed

Shi, Jian; Wang, Xinwen; Lyu, Lingyun; Jiang, Hui; Zhu, Hao-Jie

2018-04-01

Human hepatic cell lines are widely used as an in vitro model for the study of drug metabolism and liver toxicity. However, the validity of this model is still a subject of debate because the expressions of various proteins in the cell lines, including drug-metabolizing enzymes (DMEs), can differ significantly from those in human livers. In the present study, we first conducted an untargeted proteomics analysis of the microsomes of the cell lines HepG2, Hep3B, and Huh7, and compared them to human livers using a sequential window acquisition of all theoretical mass spectra (SWATH) method. Furthermore, high-resolution multiple reaction monitoring (MRM-HR), a targeted proteomic approach, was utilized to compare the expressions of pre-selected DMEs between human livers and the cell lines. In general, the SWATH quantifications were in good agreement with the MRM-HR analysis. Over 3000 protein groups were quantified in the cells and human livers, and the proteome profiles of human livers significantly differed from the cell lines. Among the 101 DMEs quantified with MRM-HR, most were expressed at substantially lower levels in the cell lines. Thus, appropriate caution must be exercised when using these cell lines for the study of hepatic drug metabolism and toxicity. Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Generation and validation of PAX7 reporter lines from human iPS cells using CRISPR/Cas9 technology.

PubMed

Wu, Jianbo; Hunt, Samuel D; Xue, Haipeng; Liu, Ying; Darabi, Radbod

2016-03-01

Directed differentiation of iPS cells toward various tissue progenitors has been the focus of recent research. Therefore, generation of tissue-specific reporter iPS cell lines provides better understanding of developmental stages in iPS cells. This technical report describes an efficient strategy for generation and validation of knock-in reporter lines in human iPS cells using the Cas9-nickase system. Here, we have generated a knock-in human iPS cell line for the early myogenic lineage specification gene of PAX7. By introduction of site-specific double-stranded breaks (DSB) in the genomic locus of PAX7 using CRISPR/Cas9 nickase pairs, a 2A-GFP reporter with selection markers has been incorporated before the stop codon of the PAX7 gene at the last exon. After positive and negative selection, single cell-derived human iPS clones have been isolated and sequenced for in-frame positioning of the reporter construct. Finally, by using a nuclease-dead Cas9 activator (dCas9-VP160) system, the promoter region of PAX7 has been targeted for transient gene induction to validate the GFP reporter activity. This was confirmed by flow cytometry analysis and immunostaining for PAX7 and GFP. This technical report provides a practical guideline for generation and validation of knock-in reporters using CRISPR/Cas9 system. Published by Elsevier B.V.

Evaluation of in silico pharmacokinetic properties and in vitro cytotoxic activity of selected newly synthesized N-succinimide derivatives.

PubMed

Milosevic, Natasa P; Kojic, Vesna; Curcic, Jelena; Jakimov, Dimitar; Milic, Natasa; Banjac, Nebojsa; Uscumlic, Gordana; Kaliszan, Roman

2017-04-15

Design of a new drug entity is usually preceded by analysis of quantitative structure activity (properties) relationships, QSA(P)R. Six newly synthesized succinimide derivatives have been determined for (i) in silico physico-chemical descriptors, pharmacokinetic and toxicity predictors, (ii) in vitro biological activity on four different carcinoma cell lines and on normal fetal lung cells and (iii) lipophilicity on liquid chromatography. All compounds observed were predicted for good permeability and solubility, good oral absorption rate and moderate volume of distribution as well as for modest blood brain permeation, followed by acceptable observed toxicity. In silico determined lipophilicity, permeability through jejunum and aqueous solubility were correlated with experimentally obtained lipophilic constants (by use of high pressure liquid chromatography) and linear correlations were obtained. Absorption rate and volume of distribution were predicted by chromatographic lipophilicity measurements while permeation through blood bran barrier was predicted dominantly by molecular size defined with molecular weight. Five compounds have demonstrated antiproliferative activity toward cervix carcinoma HeLa cell lines; three were cytotoxic against breast carcinoma MCF-7 cells, while one inhibited proliferation of colon carcinoma HT-29 cell lines. Only one compound was cytotoxic toward normal cell lines, while other compounds were proven as safe. Antiproliferative potential against HeLa cells was described as exponential function of lipophilicity. Based on obtained results, lead compounds were selected. Copyright © 2017 Elsevier B.V. All rights reserved.

Marking Embryonic Stem Cells with a 2A Self-Cleaving Peptide: A NKX2-5 Emerald GFP BAC Reporter

PubMed Central

Hsiao, Edward C.; Yoshinaga, Yuko; Nguyen, Trieu D.; Musone, Stacy L.; Kim, Judy E.; Swinton, Paul; Espineda, Isidro; Manalac, Carlota; deJong, Pieter J.; Conklin, Bruce R.

2008-01-01

Background Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available. Methodology Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice. Conclusions Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library. PMID:18596956

Chaetocin: a promising new antimyeloma agent with in vitro and in vivo activity mediated via imposition of oxidative stress

PubMed Central

Isham, Crescent R.; Tibodeau, Jennifer D.; Jin, Wendy; Xu, Ruifang; Timm, Michael M.

2007-01-01

Chaetocin, a thiodioxopiperazine natural product previously unreported to have anticancer effects, was found to have potent antimyeloma activity in IL-6–dependent and –independent myeloma cell lines in freshly collected sorted and unsorted patient CD138+ myeloma cells and in vivo. Chaetocin largely spares matched normal CD138− patient bone marrow leukocytes, normal B cells, and neoplastic B-CLL (chronic lymphocytic leukemia) cells, indicating a high degree of selectivity even in closely lineage-related B cells. Furthermore, chaetocin displays superior ex vivo antimyeloma activity and selectivity than doxorubicin and dexamethasone, and dexamethasone- or doxorubicin-resistant myeloma cell lines are largely non–cross-resistant to chaetocin. Mechanistically, chaetocin is dramatically accumulated in cancer cells via a process inhibited by glutathione and requiring intact/unreduced disulfides for uptake. Once inside the cell, its anticancer activity appears mediated primarily through the imposition of oxidative stress and consequent apoptosis induction. Moreover, the selective antimyeloma effects of chaetocin appear not to reflect differential intracellular accumulation of chaetocin but, instead, heightened sensitivity of myeloma cells to the cytotoxic effects of imposed oxidative stress. Considered collectively, chaetocin appears to represent a promising agent for further study as a potential antimyeloma therapeutic. PMID:17090648

Synthesis of sulfadimethoxine based surfactants and their evaluation as antitumor agents.

PubMed

Khowdiary, Manal Mohmed; Mostafa, Nashwa S

2016-01-01

Synthesized CO (II) and Pt (II) of sulfadimethoxine. These compounds were tested for potential antitumor activity against two of human tumor cell lines, colon carcinoma cell line [Hct116], and breast carcinoma cell line MCF7. The structures of the resulting compounds have been investigated by elemental, FT-IR and H 1 NMR analyzes to insure the purity and confirmed the structures of them. The surface properties studies and octanol/water partition coefficients, Po/w were measured. The synthesized compounds exhibit biological activities with the lowest log Po/w and critical micelle concentration (CMC) values. In addition, in this article we provide an insight into this subject in order to increase the drug bioavailability. Inhibitory activity against colon carcinoma cells was detected for Pt and cobalt ion complex with IC50 = 4.5, 2.2 µg and against breast carcinoma cells IC50 = 18.2, 5.7 µg, respectively. The main goal of cancer therapy is to attain the maximum therapeutic damage of tumor cells in combination with a minimum concentration of the drug. This can be achieved in principle via selective antitumor preparations, the cytostatic effects of which would be restricted within tumor tissue. While 100% selectivity may be impractical, the achievement of reasonably high selectivity seems to be a feasible aim. Platinum and cobalt complex surfactants in our research affect tumor tissue at a very low concentration at values lower than their CMC values; this indicate that the sulfadimethoxine complexes merit further investigation as potential antitumor drugs.

Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line

PubMed Central

Thomas, Andrew J.; Hailey, Dale W.; Stawicki, Tamara M.; Wu, Patricia; Coffin, Allison B.; Rubel, Edwin W.; Raible, David W.; Simon, Julian A.; Ou, Henry C.

2013-01-01

Cisplatin, one of the most commonly used anti-cancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analogue of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line. PMID:23467357

Dual effect of F-actin targeted carrier combined with antimitotic drug on aggressive colorectal cancer cytoskeleton: Allying dissimilar cell cytoskeleton disrupting mechanisms.

PubMed

Taranejoo, Shahrouz; Janmaleki, Mohsen; Pachenari, Mohammad; Seyedpour, Seyed Morteza; Chandrasekaran, Ramya; Cheng, Wenlong; Hourigan, Kerry

2016-11-20

A recent approach to colon cancer therapy is to employ selective drugs with specific extra/intracellular sites of action. Alteration of cytoskeletal protein reorganization and, subsequently, to cellular biomechanical behaviour during cancer progression highly affects the cancer cell progress. Hence, cytoskeleton targeted drugs are an important class of cancer therapy agents. We have studied viscoelastic alteration of the human colon adenocarcinoma cell line, SW48, after treatment with a drug delivery system comprising chitosan as the carrier and albendazole as the microtubule-targeting agent (MTA). For the first time, we have evaluated the biomechanical characteristics of the cell line, using the micropipette aspiration (MA) method after treatment with drug delivery systems. Surprisingly, employing a chitosan-albendazole pair, in comparison with both neat materials, resulted in more significant change in the viscoelastic parameters of cells, including the elastic constants (K 1 and K 2 ) and the coefficient of viscosity (μ). This difference was more pronounced for cancer cells after 48h of the treatment. Microtubule and actin microfilament (F-actin) contents in the cell line were studied by immunofluorescent staining. Good agreement was observed between the mechanical characteristics results and microtubule/F-actin contents of the treated SW48 cell line, which declined after treatment. The results showed that chitosan affected F-actin more, while MTA was more effective for microtubules. Toxicity studies were performed against two cancer cell lines (SW48 and MCF10CA1h) and compared to normal cells, MCF10A. The results showed cancer selectiveness, safety of formulation, and enhanced anticancer efficacy of the CS/ABZ conjugate. This study suggests that employing such a suitable pair of drug-carriers with dissimilar sites of action, thus allying the different cell cytoskeleton disrupting mechanisms, may provide a more efficient cancer therapy approach. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of a Rat Primary Cell-Based Blood-Brain Barrier Model With Epithelial and Brain Endothelial Cell Lines: Gene Expression and Drug Transport.

PubMed

Veszelka, Szilvia; Tóth, András; Walter, Fruzsina R; Tóth, Andrea E; Gróf, Ilona; Mészáros, Mária; Bocsik, Alexandra; Hellinger, Éva; Vastag, Monika; Rákhely, Gábor; Deli, Mária A

2018-01-01

Cell culture-based blood-brain barrier (BBB) models are useful tools for screening of CNS drug candidates. Cell sources for BBB models include primary brain endothelial cells or immortalized brain endothelial cell lines. Despite their well-known differences, epithelial cell lines are also used as surrogate models for testing neuropharmaceuticals. The aim of the present study was to compare the expression of selected BBB related genes including tight junction proteins, solute carriers (SLC), ABC transporters, metabolic enzymes and to describe the paracellular properties of nine different culture models. To establish a primary BBB model rat brain capillary endothelial cells were co-cultured with rat pericytes and astrocytes (EPA). As other BBB and surrogate models four brain endothelial cells lines, rat GP8 and RBE4 cells, and human hCMEC/D3 cells with or without lithium treatment (D3 and D3L), and four epithelial cell lines, native human intestinal Caco-2 and high P-glycoprotein expressing vinblastine-selected VB-Caco-2 cells, native MDCK and MDR1 transfected MDCK canine kidney cells were used. To test transporter functionality, the permeability of 12 molecules, glucopyranose, valproate, baclofen, gabapentin, probenecid, salicylate, rosuvastatin, pravastatin, atorvastatin, tacrine, donepezil, was also measured in the EPA and epithelial models. Among the junctional protein genes, the expression level of occludin was high in all models except the GP8 and RBE4 cells, and each model expressed a unique claudin pattern. Major BBB efflux (P-glycoprotein or ABCB1) and influx transporters (GLUT-1, LAT-1) were present in all models at mRNA levels. The transcript of BCRP (ABCG2) was not expressed in MDCK, GP8 and RBE4 cells. The absence of gene expression of important BBB efflux and influx transporters BCRP, MRP6, -9, MCT6, -8, PHT2, OATPs in one or both types of epithelial models suggests that Caco-2 or MDCK models are not suitable to test drug candidates which are substrates of these transporters. Brain endothelial cell lines GP8, RBE4, D3 and D3L did not form a restrictive paracellular barrier necessary for screening small molecular weight pharmacons. Therefore, among the tested culture models, the primary cell-based EPA model is suitable for the functional analysis of the BBB.

In vitro models.

PubMed

Mather, Jennie Powell

2012-02-01

The current resurgence of interest in the cancer stem cell (CSC) hypothesis as possibly providing a unifying theory of cancer biology is fueled by the growing body of work on normal adult tissue stem cells and the promise that CSC may hold the key to one of the central problems of clinical oncology: tumor recurrence. Many studies suggest that the microenvironment plays a role, perhaps a seminal one, in cancer development and progression. In addition, the possibility that the stem cell-like component of tumors is capable of rapid and reversible changes of phenotype raises questions concerning studies with these populations and the application of what we learn to the clinical situation. These types of questions are extremely difficult to study using in vivo models or freshly isolated cells. Established cell lines grown in defined conditions provide important model systems for these studies. There are three types of in vitro models for CSCs: (a) selected subpopulations of existing tumor lines (derived from serum-containing medium; (b) creation of lines from tumor or normal cells by genetic manipulation; or (c) direct in vitro selection of CSC from tumors or sorted tumor cells using defined serum-free conditions. We review the problems associated with creating and maintaining in vitro cultures of CSCs and the progress to date on the establishment of these important models. Copyright © 2011 AlphaMed Press.

The 5-HT{sub 2A} serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sonier, Brigitte; Arseneault, Madeleine; Institut National de la Recherche Scientifique-Institut Armand-Frappier, Montreal, Que.

2006-05-19

Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT{sub 2A} serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTTmore » proliferation assay. We have demonstrated that the 5-HT{sub 2A} receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT{sub 2A} receptor present in this cell line is identical to the 5-HT{sub 2A} receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT{sub 2A} receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT{sub 2A} receptor subtype, which is fully expressed in this cell line.« less

Identification of small-molecule inhibitors of the colorectal cancer oncogene Krüppel-like factor 5 expression by ultrahigh-throughput screening.

PubMed

Bialkowska, Agnieszka B; Crisp, Melissa; Bannister, Thomas; He, Yuanjun; Chowdhury, Sarwat; Schürer, Stephan; Chase, Peter; Spicer, Timothy; Madoux, Franck; Tian, Chenlu; Hodder, Peter; Zaharevitz, Daniel; Yang, Vincent W

2011-11-01

The transcription factor Krüppel-like factor 5 (KLF5) is primarily expressed in the proliferative zone of the mammalian intestinal epithelium, where it regulates cell proliferation. Studies showed that inhibition of KLF5 expression reduces proliferation rates in human colorectal cancer cells and intestinal tumor formation in mice. To identify chemical probes that decrease levels of KLF5, we used cell-based ultrahigh-throughput screening (uHTS) to test compounds in the public domain of NIH, the Molecular Libraries Probe Production Centers Network library. The primary screen involved luciferase assays in the DLD-1/pGL4.18hKLF5p cell line, which stably expressed a luciferase reporter driven by the human KLF5 promoter. A cytotoxicity counterscreen was done in the rat intestinal epithelial cell line, IEC-6. We identified 97 KLF5-selective compounds with EC(50) < 10 μmol/L for KLF5 inhibition and EC(50) > 10 μmol/L for IEC-6 cytotoxicity. The two most potent compounds, CIDs (PubChem Compound IDs) 439501 and 5951923, were further characterized on the basis of computational, Western blot, and cell viability analyses. Both of these compounds, and two newly synthesized structural analogs of CID 5951923, significantly reduced endogenous KLF5 protein levels and decreased viability of several colorectal cancer cell lines without any apparent impact on IEC-6 cells. Finally, when tested in the NCI-60 panel of human cancer cell lines, compound CID 5951923 was selectively active against colon cancer cells. Our results show the feasibility of uHTS in identifying novel compounds that inhibit colorectal cancer cell proliferation by targeting KLF5.

Identification of Small-Molecule Inhibitors of the Colorectal Cancer Oncogene Krüppel-Like Factor 5 Expression by Ultrahigh-Throughput Screening

PubMed Central

Bialkowska, Agnieszka B.; Crisp, Melissa; Bannister, Thomas; He, Yuanjun; Chowdhury, Sarwat; Schürer, Stephan; Chase, Peter; Spicer, Timothy; Madoux, Franck; Tian, Chenlu; Hodder, Peter; Zaharevitz, Daniel; Yang, Vincent W.

2011-01-01

The transcription factor Krüppel-like factor 5 (KLF5) is primarily expressed in the proliferative zone of the mammalian intestinal epithelium where it regulates cell proliferation. Studies showed that inhibition of KLF5 expression reduces proliferation rates in human colorectal cancer cells and intestinal tumor formation in mice. To identify chemical probes that decrease levels of KLF5, we used cell-based ultrahigh-throughput screening (uHTS) to test compounds in the NIH’s public domain, the Molecular Libraries Probe Production Centers Network (MLPCN) library. The primary screen involved luciferase assays in the DLD-1/pGL4.18hKLF5p cell line, which stably expressed a luciferase reporter driven by the human KLF5 promoter. A cytotoxicity counterscreen was performed in the rat intestinal epithelial cell line, IEC-6. We identified 97 KLF5-selective compounds with EC50<10 µM for KLF5 inhibition and EC50>10 µM for IEC-6 cytotoxicity. The two most potent compounds, CIDs (PubChem Compound IDs) 439501 and 5951923, were further characterized based on computational, Western blot, and cell viability analyses. Both of these compounds and two newly-synthesized structural analogs of CID 5951923 significantly reduced endogenous KLF5 protein levels and decreased viability of several colorectal cancer cell lines without any apparent impact on IEC-6 cells. Finally, when tested in the NCI-60 panel of human cancer cell lines, compound CID 5951923 was selectively active against colon cancer cells. Our results demonstrate the feasibility of uHTS in identifying novel compounds that inhibit colorectal cancer cell proliferation by targeting KLF5. PMID:21885866

Effects of icotinib, a novel epidermal growth factor receptor tyrosine kinase inhibitor, in EGFR-mutated non-small cell lung cancer.

PubMed

Yang, Guangdie; Yao, Yinan; Zhou, Jianya; Zhao, Qiong

2012-06-01

Epidermal growth factor receptor (EGFR) is one of the most promising targets for non-small cell lung cancer (NSCLC). Our study demonstrated the antitumor effects of icotinib hydrochloride, a highly selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI), in two EGFR-mutated lung cancer cell lines compared to A549, a cell line without EGFR mutations. We incubated PC-9 and HCC827 human lung cancer cell lines both with (E746-A750) mutations with various concentrations of icotinib and gefitinib for 48 h. Cell proliferation and migration were determined using a real-time cell invasion and migration assay and cytotoxicity assay. Apoptosis was assessed by measuring Annexin V staining using flow cytometry. The antitumor effects of icotinib compared to gefitinib were similar and were most effective in reducing the proliferation of EGFR-mutated cells compared to non-mutated controls. Our results suggest the possibility of icotinib as a new therapeutic agent of EGFR-mutated cancer cells, which has the potential to be used in the first-line treatment of EGFR-mutated NSCLC.

Resolution of Novel Pancreatic Ductal Adenocarcinoma Subtypes by Global Phosphotyrosine Profiling*

PubMed Central

Humphrey, Emily S.; Su, Shih-Ping; Nagrial, Adnan M.; Hochgräfe, Falko; Pajic, Marina; Lehrbach, Gillian M.; Parton, Robert G.; Yap, Alpha S.; Horvath, Lisa G.; Chang, David K.; Biankin, Andrew V.; Wu, Jianmin; Daly, Roger J.

2016-01-01

Comprehensive characterization of signaling in pancreatic ductal adenocarcinoma (PDAC) promises to enhance our understanding of the molecular aberrations driving this devastating disease, and may identify novel therapeutic targets as well as biomarkers that enable stratification of patients for optimal therapy. Here, we use immunoaffinity-coupled high-resolution mass spectrometry to characterize global tyrosine phosphorylation patterns across two large panels of human PDAC cell lines: the ATCC series (19 cell lines) and TKCC series (17 cell lines). This resulted in the identification and quantification of over 1800 class 1 tyrosine phosphorylation sites and the consistent segregation of both PDAC cell line series into three subtypes with distinct tyrosine phosphorylation profiles. Subtype-selective signaling networks were characterized by identification of subtype-enriched phosphosites together with pathway and network analyses. This revealed that the three subtypes characteristic of the ATCC series were associated with perturbations in signaling networks associated with cell-cell adhesion and epithelial-mesenchyme transition, mRNA metabolism, and receptor tyrosine kinase (RTK) signaling, respectively. Specifically, the third subtype exhibited enhanced tyrosine phosphorylation of multiple RTKs including the EGFR, ERBB3 and MET. Interestingly, a similar RTK-enriched subtype was identified in the TKCC series, and 'classifier' sites for each series identified using Random Forest models were able to predict the subtypes of the alternate series with high accuracy, highlighting the conservation of the three subtypes across the two series. Finally, RTK-enriched cell lines from both series exhibited enhanced sensitivity to the small molecule EGFR inhibitor erlotinib, indicating that their phosphosignature may provide a predictive biomarker for response to this targeted therapy. These studies highlight how resolution of subtype-selective signaling networks can provide a novel taxonomy for particular cancers, and provide insights into PDAC biology that can be exploited for improved patient management. PMID:27259358

Genetic selection for resistance to mycoplasmal pneumonia of swine (MPS) in the Landrace line influences the expression of soluble factors in blood after MPS vaccine sensitization.

PubMed

Shimazu, Tomoyuki; Borjigin, Liushiqi; Katayama, Yuki; Li, Meihua; Satoh, Takumi; Watanabe, Kouichi; Kitazawa, Haruki; Roh, Sang-gun; Aso, Hisashi; Kazuo, Katoh; Suda, Yoshihito; Sakuma, Akiko; Nakajo, Mituru; Suzuki, Keiichi

2014-04-01

We recently developed a Landrace line that is resistant to mycoplasmal pneumonia of swine (MPS) infection by genetic selection for five generations, and we reported that the immunophenotype of this line is different from that of the non-selected line in terms of changes in peripheral blood leukocyte population after MPS vaccination. This study followed up previous findings demonstrating changes in soluble factors in blood, namely, hormones, Mycoplasma hyopneumoniae-specific immunoglobulin G (IgG), and cytokines. These two lines were injected with MPS vaccine on days -7 and 0 after blood sampling on those days, and blood samples were collected on days -14, -7, 0, 2, 7 and 14. We found changes in the levels of many hormones and cytokines in both lines. However, we found that only growth hormone (GH) and interferon (IFN)-γ levels were statistically different between these two lines. GH concentration was reduced (day 0) and IFN-γ concentration was increased (day 14) in the MPS-selected line compared with the non-selected line, despite unchanged IFN-γ messenger RNA expression in blood cells. Although detailed mechanisms underlying these phenotypes remain unsolved, these traits would be useful to improve MPS resistance in pig production and provide an insight into MPS infection. © 2013 Japanese Society of Animal Science.

The effects of estradiol and selective estrogen receptor modulators on gene expression and messenger RNA stability in immortalized sheep endometrial stromal cells and human endometrial adenocarcinoma cells.

PubMed

Farnell, Yuhua Z; Ing, Nancy H

2003-03-01

The purpose of this study was to identify an endometrial cell line that maintained the E2 up-regulation of estrogen receptor (ER) mRNA by enhanced message stability and to assess its dependence on ER protein. Estradiol (E2) effects on gene expression were measured in three cell lines: one immortalized from sheep endometrial stroma (ST) and two from human endometrial adenocarcinomas (Ishikawa and ECC-1). E2 up-regulated ER mRNA levels in ST and Ishikawa cells, but down-regulated ER mRNA levels in ECC-1 cells. E2 up-regulated progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and transforming growth factor-alpha (TGF-alpha) in both Ishikawa and ECC-1 cells. The selective estrogen receptor modulator ICI 182,780 antagonized the E2-induced up-regulation of ER and/or PR mRNA levels in all three cells, while another, GW 5638, antagonized the up-regulation of PR mRNA in Ishikawa and ECC-1 cells. In mechanistic studies, E2 had no effect on ER mRNA stability in ST cells and it destabilized ER mRNA in ECC-1 cells. Thus, Ishikawa cells appear to be the most physiologically relevant cell line in which to study the up-regulation of ER mRNA levels by enhanced mRNA stability. Its antagonism by ICI 182,780 reveals that ER protein is involved in this E2 response.

An oncolytic adenovirus vector combining enhanced cell-to-cell spreading, mediated by the ADP cytolytic protein, with selective replication in cancer cells with deregulated wnt signaling.

PubMed

Toth, Karoly; Djeha, Hakim; Ying, Baoling; Tollefson, Ann E; Kuppuswamy, Mohan; Doronin, Konstantin; Krajcsi, Peter; Lipinski, Kai; Wrighton, Christopher J; Wold, William S M

2004-05-15

We have constructed a novel oncolytic adenovirus (Ad) vector named VRX-009 that combines enhanced cell spread with tumor-specific replication. Enhanced spread, which could significantly increase antitumor efficacy, is mediated by overexpression of the Ad cytolytic protein named ADP (also known as E3-11.6K). Replication of VRX-009 is restricted to cells with a deregulated wnt signal transduction pathway by replacement of the wild-type Ad E4 promoter with a synthetic promoter consisting of five consensus binding sites for the T-cell factor transcription factor. Tumor-selective replication is indicated by several lines of evidence. VRX-009 expresses E4ORF3, a representative Ad E4 protein, only in colon cancer cell lines. Furthermore, VRX-009 replicates preferentially in colon cancer cell lines as evidenced by virus productivity 2 orders of magnitude higher in SW480 colon cancer cells than in A549 lung cancer cells. Replication in primary human bronchial epithelial cells and human umbilical vein endothelial cells was also significantly lower than in SW480 cells. When tested in human tumor xenografts in nude mice, VRX-009 effectively suppressed the growth of SW480 colon tumors but not of A549 lung tumors. VRX-009 may provide greater level of antitumor efficacy than standard oncolytic Ad vectors in tumors in which a defect in wnt signaling increases the level of nuclear beta-catenin.

The Broad Spectrum Receptor Tyrosine Kinase Inhibitor Dovitinib Suppresses Growth of BRAF Mutant Melanoma Cells in Combination with Other Signaling Pathway Inhibitors

PubMed Central

Langdon, Casey G.; Held, Matthew A.; Platt, James T.; Meeth, Katrina; Iyidogan, Pinar; Mamillapalli, Ramanaiah; Koo, Andrew B.; Klein, Michael; Liu, Zongzhi; Bosenberg, Marcus W.; Stern, David F.

2016-01-01

Summary BRAF inhibitors have revolutionized treatment of mutant BRAF metastatic melanomas. However, resistance develops rapidly following BRAF inhibitor treatment. We have found that BRAF-mutant melanoma cell lines are more sensitive than wild-type BRAF cells to the small molecule tyrosine kinase inhibitor dovitinib. Sensitivity is associated with inhibition of a series of known dovitinib targets. Dovitinib in combination with several agents inhibits growth more effectively than either agent alone. These combinations inhibit BRAF-mutant melanoma and colorectal carcinoma cell lines, including cell lines with intrinsic or selected BRAF inhibitor resistance. Hence, combinations of dovitinib with second agents are potentially effective therapies for BRAF-mutant melanomas, regardless of their sensitivity to BRAF inhibitors. PMID:25854919

A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines.

PubMed

Wang, Fei; Guo, Tao; Jiang, Hongmei; Li, Ruobi; Wang, Ting; Zeng, Ni; Dong, Guanghui; Zeng, Xiaowen; Li, Daochuan; Xiao, Yongmei; Hu, Qiansheng; Chen, Wen; Xing, Xiumei; Wang, Qing

2018-06-01

Gene knockdown and knockout using RNAi and CRISPR/Cas9 allow for efficient evaluation of gene function, but it is unclear how the choice of technology can influence the results. To compare the phenotypes obtained using siRNA and CRISPR/Cas9 technologies, aldehyde dehydrogenase 2 (ALDH2) was selected as an example. In this study, we constructed one HepG2 cell line with a homozygous mutation in the fifth exon of ALDH2 (ALDH2-KO1 cell) using the eukaryotic CRISPR/Cas9 expression system followed by the limited dilution method and one HepG2 cell line with different mutations in the ALDH2 gene (ALDH2-KO2 cell) using the lentivirus CRISPR/Cas9 system. Additionally, one ALDH2-knockdown (KD) HepG2 cell line was created using siRNA. The reproducibility of these methods was further verified in the HEK293FT cell line. We found that the mRNA expression level of ALDH2 was significantly decreased and the protein expression level of ALDH2 was completely abolished in the ALDH2-KO cell lines, but not in ALDH2-KD cells. Furthermore, the functional activity of ALDH2 was also markedly disrupted in the two ALDH2-KO cell lines compared with ALDH2-KD and wild-type cells. The lack of ALDH2 expression mediated by CRIPSR/Cas9 resulted in a more dramatic increase in the cellular susceptibility to chemical-induced reactive oxygen species generation, cytotoxicity, apoptosis, and inflammation, especially at low concentrations compared with ALDH2-KD and WT cells. Therefore, we consider the gene knockout cell line created by CRISPR/Cas9 to be a more useful tool for identifying the function of a gene.

RUNX3 confers sensitivity to pheophorbide a-photodynamic therapy in human oral squamous cell carcinoma cell lines.

PubMed

Moon, Sook; Bae, Jung Yoon; Son, Hwa-Kyung; Lee, Doo Young; Park, Gyeongju; You, Hyun; Ko, Hyojin; Kim, Yong-Chul; Kim, Jin

2015-02-01

Photodynamic therapy (PDT) with photosensitizer is one of the promising modalities for cancer treatment. For clinical use of PDT, screening process should be preceded to enhance sensitivity to PDT. Thus, we investigated a molecular biomarker to determine the sensitivity to pheophorbide a (Pa)-PDT in immortalized human oral keratinocytes (IHOK) and oral squamous cell carcinoma (OSCC) cell lines. Two IHOK and several OSCC cell lines were used. After Pa-PDT, cell viability was reduced by more than 50%, and reactive oxygen species were generated in IHOK and OSCC cell lines. Additionally, apoptosis occurred in PDT-treated cells. IHOK(S) and IHOK(P), the two IHOK cell lines derived from the same source, showed a difference in cytotoxicity after Pa-PDT. To explain this difference in cytotoxicity, we looked at the expression of Wnt signaling-related genes in these two cell lines, for the morphology of IHOK(S) which was spindle like and elongated and distinct from IHOK(P) and the parent cell. Among the relevant genes, runt-related transcription factor 3 (RUNX3), an apoptosis-related gene, was selected as a potential marker that confers sensitivity to PDT. We found that the cytotoxicity by Pa-PDT was proportional to RUNX3 expression in OSCC cell lines. Additionally, knockdown of RUNX3 expression reduced cytotoxicity by Pa-PDT, suggesting that RUNX3 might be a biomarker to determine sensitivity to Pa-PDT. This was the first study to find a new target molecule that enhances Pa-PDT effects in IHOK and OSCC cell lines. Hence, the development of a PDT-dependent biomarker could provide a novel approach to improve the effects of PDT on oral precancerous and cancerous lesions.

Production of medakafish chimeras from a stable embryonic stem cell line.

PubMed

Hong, Y; Winkler, C; Schartl, M

1998-03-31

Embryonic stem (ES) cell lines provide a unique tool for introducing targeted or random genetic alterations through gene replacement, insertional mutagenesis, and gene addition because they offer the possibility for in vitro selection for the desired, but extremely rare, recombinant genotypes. So far only mouse blastocyst embryos are known to have the competence to give rise to such ES cell lines. We recently have established a stable cell line (Mes1) from blastulae of the medakafish (Oryzias latipes) that shows all characteristics of mouse ES cells in vitro. Here, we demonstrate that Mes1 cells also have the competence for chimera formation; 90% of host blastulae transplanted with Mes1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. The Mes1 cells contributed to numerous organs derived from all three germ layers and differentiated into various types of functional cells, most readily observable in pigmented chimeras. These features suggest the possibility that Mes1 cells may be a fish equivalent of mouse ES cells and that medaka can be used as another system for the application of the ES cell technology.

Production of medakafish chimeras from a stable embryonic stem cell line

PubMed Central

Hong, Yunhan; Winkler, Christoph; Schartl, Manfred

1998-01-01

Embryonic stem (ES) cell lines provide a unique tool for introducing targeted or random genetic alterations through gene replacement, insertional mutagenesis, and gene addition because they offer the possibility for in vitro selection for the desired, but extremely rare, recombinant genotypes. So far only mouse blastocyst embryos are known to have the competence to give rise to such ES cell lines. We recently have established a stable cell line (Mes1) from blastulae of the medakafish (Oryzias latipes) that shows all characteristics of mouse ES cells in vitro. Here, we demonstrate that Mes1 cells also have the competence for chimera formation; 90% of host blastulae transplanted with Mes1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. The Mes1 cells contributed to numerous organs derived from all three germ layers and differentiated into various types of functional cells, most readily observable in pigmented chimeras. These features suggest the possibility that Mes1 cells may be a fish equivalent of mouse ES cells and that medaka can be used as another system for the application of the ES cell technology. PMID:9520425

Protein degradation in a LAMP-2-deficient B-lymphoblastoid cell line from a patient with Danon disease.

PubMed

Sánchez-Lanzas, Raul; Alvarez-Castelao, Beatriz; Bermejo, Teresa; Ayuso, Teresa; Tuñón, Teresa; Castaño, José G

2016-08-01

Danon disease, a condition characterized by cardiomyopathy, myopathy, and intellectual disability, is caused by mutations in the LAMP-2 gene. Lamp-2A protein, generated by alternative splicing from the Lamp-2 pre-mRNA, is reported to be the lysosomal membrane receptor essential for the chaperone-mediated autophagic pathway (CMA) aimed to selective protein targeting and translocation into the lysosomal lumen for degradation. To study the relevance of Lamp-2 in protein degradation, a lymphoblastoid cell line was obtained by EBV transformation of B-cells from a Danon patient. The derived cell line showed no significant expression of Lamp-2 protein. The steady-state mRNA and protein levels of alpha-synuclein, IΚBα, Rcan1, and glyceraldehyde-3-phosphate dehydrogenase, four proteins reported to be selective substrates of the CMA pathway, were similar in control and Lamp-2-deficient cells. Inhibition of protein synthesis showed that the half-life of alpha-synuclein, IΚBα, and Rcan1 was similar in control and Lamp-2-deficient cells, and its degradation prevented by proteasome inhibitors. Both in control and Lamp-2-deficient cells, induction of CMA and macroautophagy by serum and aminoacid starvation of cells for 8h produced a similar decrease in IΚBα and Rcan1 protein levels and was prevented by the addition of lysosome and autophagy inhibitors. In conclusion, the results presented here showed that Lamp-2 deficiency in human lymphoblastoid cells did not modify the steady-state levels or the degradation of several protein substrates reported as selective substrates of the CMA pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

Lapatinib sensitivities of two novel trastuzumab-resistant HER2 gene-amplified gastric cancer cell lines.

PubMed

Oshima, Yukiko; Tanaka, Harunari; Murakami, Hiroki; Ito, Yuichi; Furuya, Tomomi; Kondo, Eisaku; Kodera, Yasuhiro; Nakanishi, Hayao

2014-01-01

Trastuzumab (Tmab) resistance is a major clinical problem to be resolved in patients with HER2-positive gastric cancers. However, in contrast to the situation for HER2-positive breast cancer lines, the Tmab-resistant gastric cancer preclinical models that are needed to develop a new therapy to overcome this problem are not yet available. We developed three new cell lines from HER2 gene-amplified gastric cancer cell lines (GLM-1, GLM-4, NCI N-87) by a new in vivo selection method consisting of the repeated culture of small residual peritoneal metastasis but not subcutaneous tumor after Tmab treatment. We then evaluated the anti-tumor efficacy of lapatinib for these Tmab-resistant cells. We successfully isolated two Tmab-resistant cell lines (GLM1-HerR2(3), GLM4-HerR2) among the three tested cell lines. These resistant cells differed from the parental cells in their flat morphology and rapid growth in vitro, but HER2, P95HER2 expression, and Tmab binding were essentially the same for the parental and resistant cells. MUC4 expression was up- or downregulated depending on the cell line. These resistant cells were still sensitive to lapatinib, similar to the parental cells, in vitro. This growth inhibition of the Tmab-resistant cells by lapatinib was due to both G1 cell-cycle arrest and apoptosis induction via effective blockade of the PI3K/Akt and MAPK pathways. A preclinical study confirmed that the Tmab-resistant tumors are significantly susceptible to lapatinib. These results suggest that lapatinib has antitumor activity against the Tmab-resistant gastric cancer cell lines, and that these cell lines are useful for understanding the mechanism of Tmab resistance and for developing a new molecular therapy for Tmab-resistant HER2-positive gastric cancers.

Arginase treatment prevents the recovery of canine lymphoma and osteosarcoma cells resistant to the toxic effects of prolonged arginine deprivation.

PubMed

Wells, James W; Evans, Christopher H; Scott, Milcah C; Rütgen, Barbara C; O'Brien, Timothy D; Modiano, Jaime F; Cvetkovic, Goran; Tepic, Slobodan

2013-01-01

Rapidly growing tumor cells require a nutrient-rich environment in order to thrive, therefore, restricting access to certain key amino acids, such as arginine, often results in the death of malignant cells, which frequently display defective cell cycle check-point control. Healthy cells, by contrast, become quiescent and remain viable under arginine restriction, displaying full recovery upon return to arginine-rich conditions. The use of arginase therapy to restrict available arginine for selectively targeting malignant cells is currently under investigation in human clinical trials. However, the suitability of this approach for veterinary uses is unexplored. As a prelude to in vivo studies in canine malignancies, we examined the in vitro effects of arginine-deprivation on canine lymphoid and osteosarcoma cell lines. Two lymphoid and 2 osteosarcoma cell lines were unable to recover following 6 days of arginine deprivation, but all remaining cell lines displayed full recovery upon return to arginine-rich culture conditions. These remaining cell lines all proved susceptible to cell death following the addition of arginase to the cultures. The lymphoid lines were particularly sensitive to arginase, becoming unrecoverable after just 3 days of treatment. Two of the osteosarcoma lines were also susceptible over this time-frame; however the other 3 lines required 6-8 days of arginase treatment to prevent recovery. In contrast, adult progenitor cells from the bone marrow of a healthy dog were able to recover fully following 9 days of culture in arginase. Over 3 days in culture, arginase was more effective than asparaginase in inducing the death of lymphoid lines. These results strongly suggest that short-term arginase treatment warrants further investigation as a therapy for lymphoid malignancies and osteosarcomas in dogs.

Patient-Specific B-Cell Antibody Factories to Treat Metastatic Disease

DTIC Science & Technology

2013-08-01

Immortalization of these selected clones using Epstein - Barr viral transformation provides a method to maintain these antibody producing cell lines as a...2013 Please see next page. None provided. Patient-Specific B-Cell Antibody Factories to Treat Metastatic Disease Kevin Claffey University of

Discovery of potent cytotoxic ortho-aryl chalcones as new scaffold targeting tubulin and mitosis with affinity-based fluorescence.

PubMed

Zhu, Cuige; Zuo, Yinglin; Wang, Ruimin; Liang, Baoxia; Yue, Xin; Wen, Gesi; Shang, Nana; Huang, Lei; Chen, Yu; Du, Jun; Bu, Xianzhang

2014-08-14

A series of new ortho-aryl chalcones have been designed and synthesized. Many of these compounds were found to exhibit significant antiproliferation activity toward a panel of cancer cell lines. Selected compounds show potent cytotoxicity against several drug resistant cell lines including paclitaxel (Taxol) resistant human ovarian carcinoma cells, vincristine resistant human ileocecum carcinoma cells, and doxorubicin resistant human breast carcinoma cells. Further investigation revealed that active analogues could inhibit the microtubule polymerization by binding to colchicine site and thus induce multipolar mitosis, G2/M phase arrest, and apoptosis of cancer cells. Furthermore, affinity-based fluorescence enhancement was observed during the binding of active compounds with tubulin, which greatly facilitated the determination of tubulin binding site of the compounds. Finally, selected compound 26 was found to exhibit obvious in vivo antitumor activity in A549 tumor xenografts model. Our systematic studies implied a new scaffold targeting tubulin and mitosis for novel antitumor drug discovery.

A boosted negative bit-line SRAM with write-assisted cell in 45 nm CMOS technology

NASA Astrophysics Data System (ADS)

Bhatnagar, Vipul; Kumar, Pradeep; Pandey, Neeta; Pandey, Sujata

2018-02-01

A new 11 T SRAM cell with write-assist is proposed to improve operation at low supply voltage. In this technique, a negative bit-line voltage is applied to one of the write bit-lines, while a boosted voltage is applied to the other write bit-line where transmission gate access is used in proposed 11 T cell. Supply voltage to one of the inverters is interrupted to weaken the feedback. Improved write feature is attributed to strengthened write access devices and weakened feedback loop of cell at the same time. Amount of boosting required for write performance improvement is also reduced due to feedback weakening, solving the persistent problem of half-selected cells and reliability reduction of access devices with the other suggested boosted and negative bit-line techniques. The proposed design improves write time by 79%, 63% and slower by 52% with respect to LP 10 T, WRE 8 T and 6 T cells respectively. It is found that write margin for the proposed cell is improved by about 4×, 2.4× and 5.37× compared to WRE8 T, LP10 T and 6 T respectively. The proposed cell with boosted negative bit line (BNBL) provides 47%, 31%, and 68.4% improvement in write margin with respect to no write-assist, negative bit line (NBL) and boosted bit line (BBL) write-assist respectively. Also, new sensing circuit with replica bit-line is proposed to give a more precise timing of applying boosted voltages for improved results. All simulations are done on TSMC 45 nm CMOS technology.

Antitumor effect of cordycepin (3'-deoxyadenosine) on mouse melanoma and lung carcinoma cells involves adenosine A3 receptor stimulation.

PubMed

Nakamura, Kazuki; Yoshikawa, Noriko; Yamaguchi, Yu; Kagota, Satomi; Shinozuka, Kazumasa; Kunitomo, Masaru

2006-01-01

An attempt was made to elucidate the molecular targetfor the antitumor effects of cordycepin (3'-deoxyadenosine) using non-selective and selective adenosine A1, A2a, A2b and A3 receptor agonists and antagonists. Although adenosine and 2'-deoxyadenosine (up to 100 microM) had no effect, cordycepin showed remarkable inhibitory effects on the growth curves of B16-BL6 mouse melanoma (IC50= 39 microM) and mouse Lewis lung carcinoma (IC50 = 48 microM) cell lines in vitro. Among the adenosine receptor agonists and antagonists used (up to 100 microM), only 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), a selective adenosine A3 receptor agonist, notably inhibited the growth of both mouse tumor cell lines (B16-BL6; IC50 = 5 microM, LLC; 14 microM). In addition, the tumor growth inhibitory effect of cordycepin was antagonized by 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191), a selective adenosine A3 receptor antagonist. These results suggest that cordycepin exerts inhibitory effects on the growth of mouse melanoma and lung carcinoma cells by stimulating adenosine A3 receptors on tumor cells.

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC.

PubMed

Slusser-Nore, Andrea; Larson-Casey, Jennifer L; Zhang, Ruowen; Zhou, Xu Dong; Somji, Seema; Garrett, Scott H; Sens, Donald A; Dunlevy, Jane R

2016-01-01

This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As(+3)) and cadmium (Cd(+2))-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd(+2)-and As(+3)-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice. Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As(+3)-and Cd(+2)-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres. It was shown that the As(+3)-and Cd(+2)-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells. Tumor initiating cells isolated from SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC

PubMed Central

Slusser-Nore, Andrea; Larson-Casey, Jennifer L.; Zhang, Ruowen; Zhou, Xu Dong; Somji, Seema; Garrett, Scott H.; Sens, Donald A.; Dunlevy, Jane R.

2016-01-01

Background This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As+3) and cadmium (Cd+2)-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd+2-and As+3-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice. Methods Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As+3-and Cd+2-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres. Results It was shown that the As+3-and Cd+2-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As+3-and Cd+2-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells. Conclusions Tumor initiating cells isolated from SPARC-transfected As+3-and Cd+2-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA. PMID:26783756

A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies.

PubMed

Roy, Gargi; Martin, Tom; Barnes, Arnita; Wang, Jihong; Jimenez, Rod Brian; Rice, Megan; Li, Lina; Feng, Hui; Zhang, Shu; Chaerkady, Raghothama; Wu, Herren; Marelli, Marcello; Hatton, Diane; Zhu, Jie; Bowen, Michael A

2018-04-01

The conserved glycosylation site Asn 297 of a monoclonal antibody (mAb) can be decorated with a variety of sugars that can alter mAb pharmacokinetics and recruitment of effector proteins. Antibodies lacking the core fucose at Asn 297 (afucosylated mAbs) show enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and increased efficacy. Here, we describe the development of a robust platform for the manufacture of afucosylated therapeutic mAbs by engineering a Chinese hamster ovary (CHO) host cell line to co-express a mAb with GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose to a dead-end product, resulting in the production of afucosylated mAb (GlymaxX™ Technology, ProBioGen). Expression of the mAb and RMD genes was coordinated by co-transfection of separate mAb and RMD vectors or use of an internal ribosome entry site (IRES) element to link the translation of RMD with either the glutamine synthase selection marker or the mAb light chain. The GS-IRES-RMD vector format was more suitable for the rapid generation of high yielding cell lines, secreting afucosylated mAb with titers exceeding 6.0 g/L. These cell lines maintained production of afucosylated mAb over 60 generations, ensuring their suitability for use in large-scale manufacturing. The afucosylated mAbs purified from these RMD-engineered cell lines showed increased binding in a CD16 cellular assay, demonstrating enhancement of ADCC compared to fucosylated control mAb. Furthermore, the afucosylation in these mAbs could be controlled by simple addition of L-fucose in the culture medium, thereby allowing the use of a single cell line for production of the same mAb in fucosylated and afucosylated formats for multiple therapeutic indications.

Toxicity evaluation of some traditional African spices on breast cancer cells and isolated rat hepatic mitochondria.

PubMed

Choumessi, Aphrodite T; Loureiro, Rute; Silva, Ana M; Moreira, Ana C; Pieme, Anatole C; Tazoacha, Asonganyi; Oliveira, Paulo J; Penlap, Véronique B

2012-11-01

Fagara leprieuri (FL), Fagara xanthoxyloïdes (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) are used in many African countries as food spices or in traditional medicine to treat several maladies. In this work, we (a) investigate whether the crude spice extracts present selective cytotoxicity for breast cancer cell lines and (b) investigate whether the same extracts affect the bioenergetics and calcium susceptibility of isolated liver mitochondrial fractions. All extracts were cytotoxic to the cell lines studied, with the exception of MW, which was less toxic for a normal cell line. Interestingly, some of the extracts did not depolarize mitochondria in intact breast cancer MCF-7 cells, although this effect was observed in a normal breast cancer cell line (MCF-12A). All extracts increased hepatic mitochondrial state 2/4 respiration and decreased the respiratory control ratio and the transmembrane electric potential. Also, the extracts induced the mitochondrial permeability transition (MPT). Mitochondrial toxicity may be part of the mechanism by which the spices tested cause inhibition of proliferation and death in the cell lines tested. This study also warrants caution in the excessive use of these spices for human consumption. Copyright © 2012 Elsevier Ltd. All rights reserved.

Metabolic Response to NAD Depletion across Cell Lines Is Highly Variable.

PubMed

Xiao, Yang; Kwong, Mandy; Daemen, Anneleen; Belvin, Marcia; Liang, Xiaorong; Hatzivassiliou, Georgia; O'Brien, Thomas

2016-01-01

Nicotinamide adenine dinucleotide (NAD) is a cofactor involved in a wide range of cellular metabolic processes and is a key metabolite required for tumor growth. NAMPT, nicotinamide phosphoribosyltransferase, which converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), the immediate precursor of NAD, is an attractive therapeutic target as inhibition of NAMPT reduces cellular NAD levels and inhibits tumor growth in vivo. However, there is limited understanding of the metabolic response to NAD depletion across cancer cell lines and whether all cell lines respond in a uniform manner. To explore this we selected two non-small cell lung carcinoma cell lines that are sensitive to the NAMPT inhibitor GNE-617 (A549, NCI-H1334), one that shows intermediate sensitivity (NCI-H441), and one that is insensitive (LC-KJ). Even though NAD was reduced in all cell lines there was surprising heterogeneity in their metabolic response. Both sensitive cell lines reduced glycolysis and levels of di- and tri-nucleotides and modestly increased oxidative phosphorylation, but they differed in their ability to combat oxidative stress. H1334 cells activated the stress kinase AMPK, whereas A549 cells were unable to activate AMPK as they contain a mutation in LKB1, which prevents activation of AMPK. However, A549 cells increased utilization of the Pentose Phosphate pathway (PPP) and had lower reactive oxygen species (ROS) levels than H1334 cells, indicating that A549 cells are better able to modulate an increase in oxidative stress. Inherent resistance of LC-KJ cells is associated with higher baseline levels of NADPH and a delayed reduction of NAD upon NAMPT inhibition. Our data reveals that cell lines show heterogeneous response to NAD depletion and that the underlying molecular and genetic framework in cells can influence the metabolic response to NAMPT inhibition.

[Partial purification of peptides present in the Tityus macrochirus (Buthidae) scorpion venom and preliminary assessment of their cytotoxicity].

PubMed

Rincón-Cortés, Clara Andrea; Reyes-Montaño, Edgar Antonio; Vega-Castro, Nohora Angélica

2017-06-01

Scorpion venom contains peptides with neurotoxic action primarily active on ion channels in the nervous system of insects and mammals. They are also characterized as cytolytic and anticancer, biological characteristics that have not yet been reported for the Tityus macrochirus venom. To assess if the total T. macrochirus venom and the fraction of partially purified peptides decrease the viability of various tumor-derived cell lines. The scorpion venom was collected by electrical stimulation and, subsequently, subjected to chromatography, electrophoresis, and ultrafiltration with Amicon Ultra 0.5® membranes for the partial identification and purification of its peptides. The cytotoxic activity of the venom and the peptides fraction trials on tumor-derived cell lines were carried out by the MTT method. The T. macrochirus scorpion venom has peptides with molecular weights ranging between 3 and 10 kDa. They were partially purified using the ultrafiltration technique, and assessed by the RP-HPLC method. Cytotoxicity trials with the whole T. macrochirus venom showed a higher viability decrease on the PC3 cell line compared to the other cell lines assessed, while the partially purified peptides decreased the HeLa cell line viability. Peptides in the T. macrochirus scorpion venom showed cytotoxic activity on some tumorderived cell lines. We observed some degree of selectivity against other cell lines assessed.

Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

PubMed

Posner, M R; Santos, D J; Elboim, H S; Tumber, M B; Frackelton, A R

1989-04-01

Peripheral blood mononuclear cells from a patient with chronic myelogenous leukemia (CML), in remission, were depleted of CD8-positive T-cells and cultured with Epstein-Barr virus. Four of 20 cultures (20%) secreted human IgG antibodies selectively reactive with the cell surfaces of certain human leukemia cell lines. Three polyclonal, Epstein-Barr virus-transformed, B-cell lines were expanded and fused with the human-mouse myeloma analogue HMMA2.11TG/O. Antibody from secreting clones HL 1.2 (IgG1), HL 2.1 (IgG3), and HL 3.1 (IgG1) have been characterized. All three react with HL-60 (promyelocytic), RWLeu4 (CML promyelocytic), and U937 (monocytic), but not with KG-1 (myeloblastic) or K562 (CML erythroid). There is no reactivity with T-cell lines, Burkitt's cell lines, pre-B-leukemia cell lines, or an undifferentiated CML cell line, BV173. Leukemic cells from two of seven patients with acute myelogenous leukemia and one of five with acute lymphocytic leukemia react with all three antibodies. Normal lymphocytes, monocytes, polymorphonuclear cells, red blood cells, bone marrow cells, and platelets do not react. Samples from patients with other diverse hematopoietic malignancies showed no reactivity. Immunoprecipitations suggest that the reactive antigen(s) is a lactoperoxidase iodinatable series of cell surface proteins with molecular weights of 42,000-54,000 and a noniodinatable protein with a molecular weight of 82,000. Based on these data these human monoclonal antibodies appear to react with myelomonocytic leukemic cells and may detect a leukemia-specific antigen or a highly restricted differentiation antigen.

Synthesis and Evaluation of the Tumor Cell Growth Inhibitory Potential of New Putative HSP90 Inhibitors.

PubMed

Bizarro, Ana; Sousa, Diana; Lima, Raquel T; Musso, Loana; Cincinelli, Raffaella; Zuco, Vantina; De Cesare, Michelandrea; Dallavalle, Sabrina; Vasconcelos, M Helena

2018-02-13

Heat shock protein 90 (HSP90) is a well-known target for cancer therapy. In a previous work, some of us have reported a series of 3-aryl-naphtho[2,3- d ]isoxazole-4,9-diones as inhibitors of HSP90. In the present work, various compounds with new chromenopyridinone and thiochromenopyridinone scaffolds were synthesized as potential HSP90 inhibitors. Their binding affinity to HSP90 was studied in vitro. Selected compounds ( 5 and 8 ) were further studied in various tumor cell lines regarding their potential to cause cell growth inhibition, alter the cell cycle profile, inhibit proliferation, and induce apoptosis. Their effect on HSP90 client protein levels was also confirmed in two cell lines. Finally, the antitumor activity of compound 8 was studied in A431 squamous cell carcinoma xenografts in nude mice. Our results indicated that treatment with compounds 5 and 8 decreased the proliferation of tumor cell lines and compound 8 induced apoptosis. In addition, these two compounds were able to downregulate selected proteins known as "clients" of HSP90. Finally, treatment of xenografted mice with compound 5 resulted in a considerable dose-dependent inhibition of tumor growth. Our results show that two new compounds with a chromenopyridinone and thiochromenopyridinone scaffold are promising putative HSP90 inhibitors causing tumor cell growth inhibition.

Knockdown of Oncogenic KRAS in Non-Small Cell Lung Cancers Suppresses Tumor Growth and Sensitizes Tumor Cells to Targeted Therapy

PubMed Central

Sunaga, Noriaki; Shames, David S.; Girard, Luc; Peyton, Michael; Larsen, Jill E.; Imai, Hisao; Soh, Junichi; Sato, Mitsuo; Yanagitani, Noriko; Kaira, Kyoichi; Xie, Yang; Gazdar, Adi F.; Mori, Masatomo; Minna, John D.

2011-01-01

Oncogenic KRAS is found in >25% of lung adenocarcinomas, the major histologic subtype of non-small cell lung cancer (NSCLC), and is an important target for drug development. To this end, we generated four NSCLC lines with stable knockdown selective for oncogenic KRAS. As expected, stable knockdown of oncogenic KRAS led to inhibition of in vitro and in vivo tumor growth in the KRAS mutant NSCLC cells, but not in NSCLC cells that have wild-type KRAS (but mutant NRAS). Surprisingly, we did not see large-scale induction of cell death and the growth inhibitory effect was not complete. To further understand the ability of NSCLCs to grow despite selective removal of mutant KRAS expression, we performed microarray expression profiling of NSCLC cell lines with or without mutant KRAS knockdown and isogenic human bronchial epithelial cell lines (HBECs) with and without oncogenic KRAS. We found that while the MAPK pathway is significantly down-regulated after mutant KRAS knockdown, these NSCLCs showed increased levels of phospho-STAT3 and phospho-EGFR, and variable changes in phospho-Akt. In addition, mutant KRAS knockdown sensitized the NSCLCs to p38 and EGFR inhibitors. Our findings suggest that targeting oncogenic KRAS by itself will not be sufficient treatment but may offer possibilities of combining anti-KRAS strategies with other targeted drugs. PMID:21306997

Indole-based hydrazide-hydrazones and 4-thiazolidinones: synthesis and evaluation as antitubercular and anticancer agents.

PubMed

Cihan-Üstündağ, Gökçe; Şatana, Dilek; Özhan, Gül; Çapan, Gültaze

2016-01-01

A new series of indolylhydrazones (6) and indole-based 4-thiazolidinones (7, 8) have been designed, synthesized and screened for in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv. 4-Thiazolidinone derivatives 7g-7j, 8g, 8h and 8j displayed notable antituberculosis (anti-TB) activity showing 99% inhibition at MIC values ranging from 6.25 to 25.0 µg/ml. Compounds 7g, 7h, 7i, 8h and 8j demonstrated anti-TB activity at concentrations 10-fold lower than those cytotoxic for the mammalian cell lines. The indolylhydrazone derivative 6b has also been evaluated for antiproliferative activity against human cancer cell lines at the National Cancer Institute (USA). Compound 6b showed an interesting anticancer profile against different human tumor-derived cell lines at sub-micromolar concentrations with obvious selectivity toward colon cancer cell line COLO 205.

From Ugly Duckling to Swan: Unexpected Identification from Cell-SELEX of an Anti-Annexin A2 Aptamer Targeting Tumors

PubMed Central

Cibiel, Agnes; Nguyen Quang, Nam; Gombert, Karine; Thézé, Benoit; Garofalakis, Anikitos; Ducongé, Frédéric

2014-01-01

Background Cell-SELEX is now widely used for the selection of aptamers against cell surface biomarkers. However, despite negative selection steps using mock cells, this method sometimes results in aptamers against undesirable targets that are expressed both on mock and targeted cells. Studying these junk aptamers might be useful for further applications than those originally envisaged. Methodology/Principal Findings Cell-SELEX was performed to identify aptamers against CHO-K1 cells expressing human Endothelin type B receptor (ETBR). CHO-K1 cells were used for negative selection of aptamers. Several aptamers were identified but no one could discriminate between both cell lines. We decided to study one of these aptamers, named ACE4, and we identified that it binds to the Annexin A2, a protein overexpressed in many cancers. Radioactive binding assays and flow cytometry demonstrated that the aptamer was able to bind several cancer cell lines from different origins, particularly the MCF-7 cells. Fluorescence microscopy revealed it could be completely internalized in cells in 2 hours. Finally, the tumor targeting of the aptamer was evaluated in vivo in nude mice xenograft with MCF-7 cells using fluorescence diffuse optical tomography (fDOT) imaging. Three hours after intravenous injection, the aptamer demonstrated a significantly higher uptake in the tumor compared to a scramble sequence. Conclusions/Significance Although aptamers could be selected during cell-SELEX against other targets than those initially intended, they represent a potential source of ligands for basic research, diagnoses and therapy. Here, studying such aptamers, we identify one with high affinity for Annexin A2 that could be a promising tool for biomedical application. PMID:24489826

Continuous hematopoietic cell lines as model systems for leukemia-lymphoma research.

PubMed

Drexler, H G; Matsuo, A Y; MacLeod, R A

2000-11-01

Along with other improvements, the advent of continuous human leukemia-lymphoma (LL) cell lines as a rich resource of abundant, accessible and manipulable living cells has contributed significantly to a better understanding of the pathophysiology of hematopoietic tumors. The first LL cell lines, Burkitt's lymphoma-derived lines, were established in 1963. Since then, more than 1000 cell lines have been described, although not all of them in full detail. The major advantages of continuous cell lines is the unlimited supply and worldwide availability of identical cell material, and the infinite viable storability in liquid nitrogen. LL cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro under preservation of most cellular features, and specific genetic alterations. The most practical classification of LL cell lines assigns them to one of the physiologically occurring cell lineages, based on their immunophenotype, genotype and functional features. Truly malignant cell lines must be discerned from Epstein-Barr virus (EBV)-immortalized normal cells, using various distinguishing parameters. However, the picture is not quite so straightforward, as some types of LL cell lines are indeed EBV+, and some EBV+ normal cell lines carry also genetic aberrations and may mimic malignancy-associated features. Apart from EBV and human T-cell leukemia virus in some lines, the majority of wild-type LL cell lines are virus-negative. The efficiency of cell line establishment is rather low and the deliberate establishment of new LL cell lines remains by and large an unpredictable random process. Difficulties in establishing continuous cell lines may be caused by the inappropriate selection of nutrients and growth factors for these cells. Clearly, a generally suitable microenvironment for hematopoietic cells, either malignant or normal, cannot yet be created in vitro. The characterization and publication of new LL cell lines should provide important and informative core data, attesting to their scientific significance. Large percentages of LL cell lines are contaminated with mycoplasma (about 30%) or are cross-contaminated with other cell lines (about 15-20%). Solutions to these problems are sensitive detection, effective elimination and rigorous prevention of mycoplasma infection, and proper, regular authentication of cell lines. The underlying cause, however, appears to be negligent cell culture practice. The willingness of investigators to make their LL cell lines available to others is all too often limited. There is a need in the scientific community for clean and authenticated high-quality LL cell lines to which every scientist has access. These are offered by various institutionalized public cell line banks. It has been argued that LL cell lines are genetically unstable (both cytogenetically and molecular genetically). For instance, cell lines are supposed to acquire numerical and structural chromosomal alterations and various types of mutations (e.g. point mutations) in vitro. We present evidence that while nearly 100% of all LL cell lines indeed carry genetic alterations, these alterations appear to be stable rather than unstable. As an example of the practical utility of LL cell lines, the recent advances in studies of classical and molecular cytogenetics, which in large part were made possible by cell lines, are highlighted. A list of the most useful, robust and publicly available reference cell lines that may be used for a variety of experimental purposes is proposed. Clearly, by opening new avenues for investigation, studies of LL cell lines have provided seminal insights into the biology of hematopoietic neoplasia. Over a period of nearly four decades, these initially rather exotic cell cultures, known only to a few specialists, have become ubiquitous powerful research tools that are available to every investigator.

Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line

PubMed Central

Senevirathne, Mahinda; Kim, Soo-Hyun

2010-01-01

Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H2O2-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H2O2-induced cell damage in vitro. PMID:20607062

Specific inhibitors of mammalian DNA polymerase species.

PubMed

Mizushina, Yoshiyuki

2009-06-01

In screening of selective inhibitors of eukaryotic DNA polymerases (pols) for 15 years, more than 100 inhibitors have been discovered from natural and chemical sources. Some compounds selectively inhibit the activities of mammalian pols, and in particular, dehydroaltenusin and curcumin derivatives, such as monoacetyl-curcumin, were found to be specific inhibitors of pol alpha and pol lambda, respectively. Dehydroaltenusin was isolated from a fungus (Alternaria tennuis), and this compound inhibited cell proliferation of human cancer cell lines by arresting the cells at the S-phase, and was effective in suppressing the growth on nude mice of solid tumors of human cervical cancer cell line HeLa. Curcumin derivatives had anti-12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory activity with the same tendency as pol lambda inhibitory activity. These compounds might be useful not only as "molecular probes" for pol research, but also as biomedical and chemotherapeutic drugs for anti-cancer or anti-inflammation.

Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line.

PubMed

Senevirathne, Mahinda; Kim, Soo-Hyun; Jeon, You-Jin

2010-06-01

Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H(2)O(2)-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H(2)O(2)-induced cell damage in vitro.

[The influence of immobilized fibronectin on karyotypic variability of two rat kangaroo kidney cell lines].

PubMed

Polianskaia, G G; Goriachaia, T S; Pinaev, G P

2007-01-01

The numerical and structural karyotypic variability has been investigated in "markerless" Rat kangaroo kidney cell lines NBL-3-17 and NBL-3-11 when cultivating on a fibronectin-coated surface. In cell line NBL-3-17, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with modal number of chromosomes, and an increase in frequency of cells with lower chromosomal number. Many new additional structural variants of the karyotype (SVK) appear. The observed alterations seem to be due preference adhesion of cells with lower chromosome number, disturbances of mitotic apparatus and selection of SVK, which are more adopted to changes in culture conditions. Detachment of cells from the fibronectin-coated surface, followed by 5 days cultivation on a hydrophilic surface restored control distribution. In cell line NBL-3-11, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of numerical karyotypic variability did not change compared to control variants. In cell line NBL-3-17 the frequency of chromosomal aberrations under cultivation on the fibronectin-coated surface for 1, 2, 4 and 8 days did not change relative to control variants. In cell line NBL-3-11 the frequency of chromosomal aberrations under the same conditions significantly increases, mainly at the expence of chromosomal, chromatid breaks and dicentrics (telomeric association) relative to control variants. We discuss possible reasons of differences in the character of numerical and structural karyotypic variability between cell lines NBL-3-17 (hypotriploid) and NBL-3-11 (hypodiploid) under cultivation on fibronectin. The reasons of the observed interline karyotypic differences possibly consist in peculiarity of karyotypic structure of cell line NBL-3-11 and in the change of gene expression, namely in a dose of certain functioning genes in the hypotryploid cell line NBL-3-17.

[The establishment of the immortalized mouse brain microvascular pericytes model and its preliminary application in screening of cerebrovascular toxicants].

PubMed

Zhao, H P; Gao, Y F; Xia, D; Zhao, Z Q; Wu, S; Wang, X H; Liu, H X; Xiao, C; Xing, X M; He, Y

2018-05-06

Objective: To establish the immortalized mouse brain microvascular pericytes model and to apply to the cerebrovascular toxicants screening study. Methods: Brain pericytes were isolated from 3 weeks of mice by tissue digestion. Immortalized pericyte cell line was constructed by infecting with LT retrovirus. Monoclone was selected to purify the immortalized pericyte cell line. The pericyte characteristics and purity were explored by immunocytochemistry. Cell proliferation was measured by using the Pomega MTS cell Proliferation Colorimetric Assay Kit. Pericytes were treated with 0, 160, 320, 640, 1 280, 2 560 μmol/L lead acetate, 0, 5, 10, 20, 40, 80 μmol/L cadmium chloride and 0, 5, 10, 20, 40, 80 μmol/L sodium arsenite in 24 hours. Cell toxicity of each group was determined by MTS assay, median lethal dose (LD(50)) was calculated in linear regression. Results: Mouse brain pericytes were successfully isolated by tissue separation and enzyme digestion method. After immortalized by LT retroviruses, monoclone was selected and expanded to establish pericyte cell line. The brain pericytes exhibited typical long spindle morphology and positive staining for α-SMA and Vimentin. The proliferation of brain pericytes cell lines was very slowly, and the doubling time was about 48 hours. The proliferation of immortalized brain pericytes cell lines was very quickly, and the doubling time was about 24 hours. After lead acetate, cadmium chloride and sodium arsenite treatment for 24 hours respectively, gradual declines in cell viability were observed. The LD(50) of lead acetate was 2 025.0 μmol/L, the LD(50) of cadmium chloride was 36.6 μmol/L, and the LD(50) of sodium arsenite was 33.2 μmol/L. Conclusion: The immortalized mouse brain microvascular pericyte model is established successfully by infecting with LT retrovirus, and can be applied to screen cerebrovascular toxicants. The toxicity of these toxicants to immortalized mouse brain microvascular pericyte is in sequence: sodium arsenite,cadmium chloride, lead acetate.

Imidazopyridine-fused [1,3]-diazepinones part 2: Structure-activity relationships and antiproliferative activity against melanoma cells.

PubMed

Bellet, Virginie; Lichon, Laure; Arama, Dominique P; Gallud, Audrey; Lisowski, Vincent; Maillard, Ludovic T; Garcia, Marcel; Martinez, Jean; Masurier, Nicolas

2017-01-05

We recently described a pyrido-imidazodiazepinone derivative which could be a promising hit compound for the development of new drugs acting against melanoma cells. In this study, a series of 28 novel pyrido-imidazodiazepinones were synthesized and screened for their in vitro cytotoxic activities against the melanoma MDA-MB-435 cell line. Among the derivatives, seven of them showed 50% growth inhibitory activity at 1 μM concentration, and high selectivity against the melanoma cell line MDA-MB-435. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Mass spectrometry (LC-MS/MS) identified proteomic biosignatures of breast cancer in proximal fluid.

PubMed

Whelan, Stephen A; He, Jianbo; Lu, Ming; Souda, Puneet; Saxton, Romaine E; Faull, Kym F; Whitelegge, Julian P; Chang, Helena R

2012-10-05

We have begun an early phase of biomarker discovery in three clinically important types of breast cancer using a panel of human cell lines: HER2 positive, hormone receptor positive and HER2 negative, and triple negative (HER2-, ER-, PR-). We identified and characterized the most abundant secreted, sloughed, or leaked proteins released into serum free media from these breast cancer cell lines using a combination of protein fractionation methods before LC-MS/MS mass spectrometry analysis. A total of 249 proteins were detected in the proximal fluid of 7 breast cancer cell lines. The expression of a selected group of high abundance and/or breast cancer-specific potential biomarkers including thromobospondin 1, galectin-3 binding protein, cathepsin D, vimentin, zinc-α2-glycoprotein, CD44, and EGFR from the breast cancer cell lines and in their culture media were further validated by Western blot analysis. Interestingly, mass spectrometry identified a cathepsin D protein single-nucleotide polymorphism (SNP) by alanine to valine replacement from the MCF-7 breast cancer cell line. Comparison of each cell line media proteome displayed unique and consistent biosignatures regardless of the individual group classifications, demonstrating the potential for stratification of breast cancer. On the basis of the cell line media proteome, predictive Tree software was able to categorize each cell line as HER2 positive, HER2 negative, and hormone receptor positive and triple negative based on only two proteins, muscle fructose 1,6-bisphosphate aldolase and keratin 19. In addition, the predictive Tree software clearly identified MCF-7 cell line overexpresing the HER2 receptor with the SNP cathepsin D biomarker.

Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells.

PubMed

Spidel, Jared L; Vaessen, Benjamin; Chan, Yin Yin; Grasso, Luigi; Kline, J Bradford

2016-12-01

Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential. A protocol was developed to decrease the time, cost, and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol and from the stable cell line process by utilizing bicistronic plasmids to generate stable semi-clonal cell pools facilitated an increased throughput of the entire process from plasmid assembly through transient transfections and selection of stable semi-clonal cell pools. Furthermore, the time required by a single individual to clone, express, and select stable cell pools in a high-throughput format was reduced from 4 to 6months to only 4 to 6weeks. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Modulators of sensitivity and resistance to inhibition of PI3K identified in a pharmacogenomic screen of the NCI-60 human tumor cell line collection.

PubMed

Kwei, Kevin A; Baker, Joffre B; Pelham, Robert J

2012-01-01

The phosphoinositide 3-kinase (PI3K) signaling pathway is significantly altered in a wide variety of human cancers, driving cancer cell growth and survival. Consequently, a large number of PI3K inhibitors are now in clinical development. To begin to improve the selection of patients for treatment with PI3K inhibitors and to identify de novo determinants of patient response, we sought to identify and characterize candidate genomic and phosphoproteomic biomarkers predictive of response to the selective PI3K inhibitor, GDC-0941, using the NCI-60 human tumor cell line collection. In this study, sixty diverse tumor cell lines were exposed to GDC-0941 and classified by GI(50) value as sensitive or resistant. The most sensitive and resistant cell lines were analyzed for their baseline levels of gene expression and phosphorylation of key signaling nodes. Phosphorylation or activation status of both the PI3K-Akt signaling axis and PARP were correlated with in vitro response to GDC-0941. A gene expression signature associated with in vitro sensitivity to GDC-0941 was also identified. Furthermore, in vitro siRNA-mediated silencing of two genes in this signature, OGT and DDN, validated their role in modulating sensitivity to GDC-0941 in numerous cell lines and begins to provide biological insights into their role as chemosensitizers. These candidate biomarkers will offer useful tools to begin a more thorough understanding of determinants of patient response to PI3K inhibitors and merit exploration in human cancer patients treated with PI3K inhibitors.

Modulators of Sensitivity and Resistance to Inhibition of PI3K Identified in a Pharmacogenomic Screen of the NCI-60 Human Tumor Cell Line Collection

PubMed Central

Kwei, Kevin A.; Baker, Joffre B.; Pelham, Robert J.

2012-01-01

The phosphoinositide 3-kinase (PI3K) signaling pathway is significantly altered in a wide variety of human cancers, driving cancer cell growth and survival. Consequently, a large number of PI3K inhibitors are now in clinical development. To begin to improve the selection of patients for treatment with PI3K inhibitors and to identify de novo determinants of patient response, we sought to identify and characterize candidate genomic and phosphoproteomic biomarkers predictive of response to the selective PI3K inhibitor, GDC-0941, using the NCI-60 human tumor cell line collection. In this study, sixty diverse tumor cell lines were exposed to GDC-0941 and classified by GI50 value as sensitive or resistant. The most sensitive and resistant cell lines were analyzed for their baseline levels of gene expression and phosphorylation of key signaling nodes. Phosphorylation or activation status of both the PI3K-Akt signaling axis and PARP were correlated with in vitro response to GDC-0941. A gene expression signature associated with in vitro sensitivity to GDC-0941 was also identified. Furthermore, in vitro siRNA-mediated silencing of two genes in this signature, OGT and DDN, validated their role in modulating sensitivity to GDC-0941 in numerous cell lines and begins to provide biological insights into their role as chemosensitizers. These candidate biomarkers will offer useful tools to begin a more thorough understanding of determinants of patient response to PI3K inhibitors and merit exploration in human cancer patients treated with PI3K inhibitors. PMID:23029544

Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana plumbaginifolia.

PubMed

Kaye, C; Crawford, N M; Malmberg, R L

1997-04-01

We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.

Establishment and Characterization of a Highly Tumourigenic and Cancer Stem Cell Enriched Pancreatic Cancer Cell Line as a Well Defined Model System

PubMed Central

Fredebohm, Johannes; Boettcher, Michael; Eisen, Christian; Gaida, Matthias M.; Heller, Anette; Keleg, Shereen; Tost, Jörg; Greulich-Bode, Karin M.; Hotz-Wagenblatt, Agnes; Lathrop, Mark; Giese, Nathalia A.; Hoheisel, Jörg D.

2012-01-01

Standard cancer cell lines do not model the intratumoural heterogeneity situation sufficiently. Clonal selection leads to a homogeneous population of cells by genetic drift. Heterogeneity of tumour cells, however, is particularly critical for therapeutically relevant studies, since it is a prerequisite for acquiring drug resistance and reoccurrence of tumours. Here, we report the isolation of a highly tumourigenic primary pancreatic cancer cell line, called JoPaca-1 and its detailed characterization at multiple levels. Implantation of as few as 100 JoPaca-1 cells into immunodeficient mice gave rise to tumours that were histologically very similar to the primary tumour. The high heterogeneity of JoPaca-1 was reflected by diverse cell morphology and a substantial number of chromosomal aberrations. Comparative whole-genome sequencing of JoPaca-1 and BxPC-3 revealed mutations in genes frequently altered in pancreatic cancer. Exceptionally high expression of cancer stem cell markers and a high clonogenic potential in vitro and in vivo was observed. All of these attributes make this cell line an extremely valuable model to study the biology of and pharmaceutical effects on pancreatic cancer. PMID:23152778

Cell death induced by Morarah and Khaltita in hepatoma cancer cells (Huh-7).

PubMed

Baig, Saeeda; Alamgir, Mohiuddin

2009-10-01

To compare the combined and isolated growth inhibitory effects of Morarah and Khaltita (herbs) on hepatoma cell lines (Huh-7), through induction of apoptosis or necrosis. Comparative controlled in-vitro study. The Molecular Biology Laboratory, The Aga Khan University, Karachi, from June to December 2006. The growth of hepatoma cell lines (Huh-7) was checked by adding Khaltita and Morarah to the cells before culture in a 24 well plate. Six wells were selected and labeled for each of the four variables (controls, Khaltita, Morarah and mixture). After 2 days, cells were studied under an inverted phase contrast microscope and fields were recorded. Approximately four fields per slide of higher intensity were selected randomly to determine the dead cell density, and the procedure was repeated 10 or more times. Frequency and percentages were calculated for dead or alive cells in controls, Morarah, Khaltita and their mixture. Chi-square was used to compare the qualitative variables. P-values < 0.05 were considered significant. Morarah and Khaltita were found to induce statistically significant (p < 0.001) cell death in hepatoma cell lines (Huh-7). At a magnification of 40x, the controls showed 1% dead cells compared to 91% in Morarah, 83% in Khaltita and 73% in combined mixture of Khaltita and Morarah. At magnification of 20x, the controls showed 4% dead cells compared to 44% in Morarah, 47% in Khaltita and 49% in the combined mixture of Khaltita and Morarah. Morarah and Khaltita induced cell death in cultured hepatoma cells (Huh-7).

Evaluation of Antioxidative and Cytotoxic Activities of Streptomyces pluripotens MUSC 137 Isolated from Mangrove Soil in Malaysia

PubMed Central

Ser, Hooi-Leng; Ab Mutalib, Nurul-Syakima; Yin, Wai-Fong; Chan, Kok-Gan; Goh, Bey-Hing; Lee, Learn-Han

2015-01-01

Streptomyces pluripotens MUSC 137 was isolated from mangrove soil obtained from Tanjung Lumpur, Pahang, Malaysia. We investigated the phylogenetic, genomic, biochemical, and phenotypic characteristics of this strain. Uniquely adapted microorganisms from mangrove habitats have previously yielded compounds of biopharmaceutical interest. In order to examine the bioactivities possessed by the strain, fermentation extract was prepared through solvent extraction method prior to bioactivities screenings. Antioxidant activity was examined via DPPH assay while the cytotoxic effect was assessed by means of examining the activity of the extract against selected human cancer cell lines, namely colon cancer cells (HCT-116, Caco-2, SW480, and HT-29), breast cancer cell (MCF-7), lung cancer cell (A549), prostate cancer cell (DU145), and cervical cancer cell (Ca Ski). The results revealed MUSC 137 possesses significant antioxidant activity and demonstrates cytotoxic effect against several cancer cell lines tested. The results indicated MCF-7 cells were most susceptible to the extract with the lowest IC50 (61.33 ± 17.10 μg/mL), followed by HCT-116 and A549. Additionally, selective index (SI) showed that MUSC 137 extract was less toxic against normal cell lines when compared to MCF-7 and HCT-116 cells. The extract was further subjected to chemical analysis using GC–MS and revealed the presence of deferoxamine and pyrrolizidines related compounds which may account for the antioxidant and cytotoxic properties observed. PMID:26733951

SU-C-204-04: Irradiation of Human Cell Lines Using Various Ions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Y; McMahon, S; Kaminuma, T

2016-06-15

Purpose: The purpose of this study is to investigate and quantify the biological effects of ion radiation using several human cell lines. We aim to answer the question of whether carbon ion the most ideal ion species for heavy ion radiotherapy. Methods: The cells were irradiated at different positions along the pristine Bragg peak of several ions with different atomic number. The biological effectiveness was evaluated using the clonogenic cell survival assay. Irradiation of three human lung cancer cell lines and a fibroblast cell line were undertaken using the charged particle beam at the NASA Space Radiation Laboratory at Brookhavenmore » National Lab. Four mono-energetic ion beams (carbon, oxygen, helium and lithium) were used to irradiate the cells. Water or media-filled T25 flasks were lined up along the beam line so that the cell-containing surfaces of the flasks were placed at a specific depth along the pristine Bragg curve. Four depths along the curve, representing entrance point, rising peak, peak and distal fall off, were selected to determine biological effectiveness. Gaf-chromic films were placed between the flasks to monitor the irradiation as soon as it was finished. Results: For all ion radiations, the maximum cell killing effect occurs at either peak or distal fall off, depending on the cell lines. For instance, for the fibroblast cell line AGO1522, RBEs of 1.4, 1.2, 1.4 and 1.9 were observed at the Bragg peak for Helium, Lithium, Carbon and Oxygen ions. Comparing positions, RBEs of 0.9, 1.2, 1.4 and 1.8 were observed for carbon irradiation of AGO-1522 cells positions corresponding to entrance, rising peak, peak and distal fall off. Conclusion: RBE values differ with position in the Bragg peak, ion species and cell line. Ions other than carbon may prove more effective in certain irradiation conditions and may contribute to optimized heavy ion therapy.« less

Targeting activated Akt with GDC-0068, a novel selective Akt inhibitor that is efficacious in multiple tumor models.

PubMed

Lin, Jie; Sampath, Deepak; Nannini, Michelle A; Lee, Brian B; Degtyarev, Michael; Oeh, Jason; Savage, Heidi; Guan, Zhengyu; Hong, Rebecca; Kassees, Robert; Lee, Leslie B; Risom, Tyler; Gross, Stefan; Liederer, Bianca M; Koeppen, Hartmut; Skelton, Nicholas J; Wallin, Jeffrey J; Belvin, Marcia; Punnoose, Elizabeth; Friedman, Lori S; Lin, Kui

2013-04-01

We describe the preclinical pharmacology and antitumor activity of GDC-0068, a novel highly selective ATP-competitive pan-Akt inhibitor currently in clinical trials for the treatment of human cancers. The effect of GDC-0068 on Akt signaling was characterized using specific biomarkers of the Akt pathway, and response to GDC-0068 was evaluated in human cancer cell lines and xenograft models with various genetic backgrounds, either as a single agent or in combination with chemotherapeutic agents. GDC-0068 blocked Akt signaling both in cultured human cancer cell lines and in tumor xenograft models as evidenced by dose-dependent decrease in phosphorylation of downstream targets. Inhibition of Akt activity by GDC-0068 resulted in blockade of cell-cycle progression and reduced viability of cancer cell lines. Markers of Akt activation, including high-basal phospho-Akt levels, PTEN loss, and PIK3CA kinase domain mutations, correlate with sensitivity to GDC-0068. Isogenic PTEN knockout also sensitized MCF10A cells to GDC-0068. In multiple tumor xenograft models, oral administration of GDC-0068 resulted in antitumor activity ranging from tumor growth delay to regression. Consistent with the role of Akt in a survival pathway, GDC-0068 also enhanced antitumor activity of classic chemotherapeutic agents. GDC-0068 is a highly selective, orally bioavailable Akt kinase inhibitor that shows pharmacodynamic inhibition of Akt signaling and robust antitumor activity in human cancer cells in vitro and in vivo. Our preclinical data provide a strong mechanistic rationale to evaluate GDC-0068 in cancers with activated Akt signaling. ©2012 AACR.

BINDING OF SOLUBLE IMMUNE COMPLEXES TO HUMAN LYMPHOBLASTOID CELLS

PubMed Central

Theofilopoulos, Argyrios N.; Dixon, Frank J.; Bokisch, Viktor A.

1974-01-01

In the present work we studied the expression of membrane-bound Ig (MBIg) as well as receptors for IgG Fc and complement on nine human lymphoblastoid cell lines. When MBIg and receptors for IgG Fc were compared, four categories of cell lines could be distinguished: (a) cell lines having both MBIg and receptors for IgG Fc, (b) cell lines having MBIg but lacking receptors for IgG Fc, (c) cell lines lacking MBIg but having receptors for IgG Fc, and (d) cell lines lacking both MBIg and receptors for IgG Fc. Two types of receptors for complement could be detected on the cell lines studied, one for C3-C3b and one for C3d. When sensitized red cells carrying C3b or C3d were used for rosette tests, three categories of cell lines could be distinguished: (a) cell lines having receptors for C3b and C3d, (b) cell lines having receptors only for C3d and (c) cell lines lacking both receptors. However, when a more sensitive immunofluorescent method was used instead of the rosette technique, it was found that cell lines unable to form rosettes with EAC1423bhu were able to bind soluble C3 or C3b which indicated the presence of these receptors on the cell surface. Inhibition experiments showed that receptors for C3-C3b and receptors for C3d are distinct and that receptors for C3-C3b and C3d are different from receptors for IgG Fc. A cell line (Raji) without MBIg but with receptors for IgG Fc, C3-C3b, and C3d was selected for use in studying the binding mechanism of soluble immune complexes to cell surface membrane. Aggregated human gamma globulin was used in place of immune complexes. Immune complexes containing complement bind to Raji cells only via receptors for complement, namely receptors for C3-C3b and C3d. Binding of immune complexes containing complement to cells is much greater than that of complexes without complement. Immune complexes bound to cells via receptors for complement can be partially released from the cell surface by addition of normal human serum as well as isolated human C3 or C3b. We postulate that such release is due to competition of immune complex bound C3b and free C3 or C3b for the receptors on Raji cells. PMID:4139225

Alterations of mitochondrial DNA in CEM cells selected for resistance toward ddC toxicity.

PubMed

Bjerke, M; Franco, M; Johansson, M; Balzarini, J; Karlsson, A

2006-01-01

2 ',3 '-dideoxycytidine (ddC) is a nucleoside analog that has been shown to produce a delayed toxicity which may be due to the depletion of mitochondrial DNA (mtDNA). In order to gain further understanding of the events involved in mitochondrial toxicity, two different CEM cell lines were selected for resistance to the delayed ddC toxicity.

Evaluating hepatocellular carcinoma cell lines for tumour samples using within-sample relative expression orderings of genes.

PubMed

Ao, Lu; Guo, You; Song, Xuekun; Guan, Qingzhou; Zheng, Weicheng; Zhang, Jiahui; Huang, Haiyan; Zou, Yi; Guo, Zheng; Wang, Xianlong

2017-11-01

Concerns are raised about the representativeness of cell lines for tumours due to the culture environment and misidentification. Liver is a major metastatic destination of many cancers, which might further confuse the origin of hepatocellular carcinoma cell lines. Therefore, it is of crucial importance to understand how well they can represent hepatocellular carcinoma. The HCC-specific gene pairs with highly stable relative expression orderings in more than 99% of hepatocellular carcinoma but with reversed relative expression orderings in at least 99% of one of the six types of cancer, colorectal carcinoma, breast carcinoma, non-small-cell lung cancer, gastric carcinoma, pancreatic carcinoma and ovarian carcinoma, were identified. With the simple majority rule, the HCC-specific relative expression orderings from comparisons with colorectal carcinoma and breast carcinoma could exactly discriminate primary hepatocellular carcinoma samples from both primary colorectal carcinoma and breast carcinoma samples. Especially, they correctly classified more than 90% of liver metastatic samples from colorectal carcinoma and breast carcinoma to their original tumours. Finally, using these HCC-specific relative expression orderings from comparisons with six cancer types, we identified eight of 24 hepatocellular carcinoma cell lines in the Cancer Cell Line Encyclopedia (Huh-7, Huh-1, HepG2, Hep3B, JHH-5, JHH-7, C3A and Alexander cells) that are highly representative of hepatocellular carcinoma. Evaluated with a REOs-based prognostic signature for hepatocellular carcinoma, all these eight cell lines showed the same metastatic properties of the high-risk metastatic hepatocellular carcinoma tissues. Caution should be taken for using hepatocellular carcinoma cell lines. Our results should be helpful to select proper hepatocellular carcinoma cell lines for biological experiments. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Adaptive Roles of SSY1 and SIR3 During Cycles of Growth and Starvation in Saccharomyces cerevisiae Populations Enriched for Quiescent or Nonquiescent Cells.

PubMed

Wloch-Salamon, Dominika M; Tomala, Katarzyna; Aggeli, Dimitra; Dunn, Barbara

2017-06-07

Over its evolutionary history, Saccharomyces cerevisiae has evolved to be well-adapted to fluctuating nutrient availability. In the presence of sufficient nutrients, yeast cells continue to proliferate, but upon starvation haploid yeast cells enter stationary phase and differentiate into nonquiescent (NQ) and quiescent (Q) cells. Q cells survive stress better than NQ cells and show greater viability when nutrient-rich conditions are restored. To investigate the genes that may be involved in the differentiation of Q and NQ cells, we serially propagated yeast populations that were enriched for either only Q or only NQ cell types over many repeated growth-starvation cycles. After 30 cycles (equivalent to 300 generations), each enriched population produced a higher proportion of the enriched cell type compared to the starting population, suggestive of adaptive change. We also observed differences in each population's fitness suggesting possible tradeoffs: clones from NQ lines were better adapted to logarithmic growth, while clones from Q lines were better adapted to starvation. Whole-genome sequencing of clones from Q- and NQ-enriched lines revealed mutations in genes involved in the stress response and survival in limiting nutrients ( ECM21 , RSP5 , MSN1 , SIR4 , and IRA2 ) in both Q and NQ lines, but also differences between the two lines: NQ line clones had recurrent independent mutations affecting the Ssy1p-Ptr3p-Ssy5p (SPS) amino acid sensing pathway, while Q line clones had recurrent, independent mutations in SIR3 and FAS1 Our results suggest that both sets of enriched-cell type lines responded to common, as well as distinct, selective pressures. Copyright © 2017 Wloch-Salamon et al.

[Streptococcus group B--association with Aerobic vaginitis and ability to human cell lines activation].

PubMed

Romanik, Małgorzata; Kafel, Joanna; Lagergård, Teresa; Martirosian, Gayane

2007-01-01

The aim of this study was to estimate: the frequency of aerobic vaginitis, susceptibility of the GBS isolated from vagina of non-pregnant women with and without cervicitis to selected antibiotics and chemotherapeutics and the proinflammatory cytokines production by HeLa, THP-I, U - 937 cells after stimulation by vaginal GBS. Our results indicated low frequency of the aerobic vaginitis -4.5% among non-pregnant young women and ability of the vaginal GBS to release proinflammatory cytokines by human cell lines in vitro.

Tocotrienol-rich fraction of palm oil induces cell cycle arrest and apoptosis selectively in human prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Srivastava, Janmejai K.; Department of Urology, University Hospitals of Cleveland, Cleveland, OH 44106; Gupta, Sanjay

2006-07-28

One of the requisite of cancer chemopreventive agent is elimination of damaged or malignant cells through cell cycle inhibition or induction of apoptosis without affecting normal cells. In this study, employing normal human prostate epithelial cells (PrEC), virally transformed normal human prostate epithelial cells (PZ-HPV-7), and human prostate cancer cells (LNCaP, DU145, and PC-3), we evaluated the growth-inhibitory and apoptotic effects of tocotrienol-rich fraction (TRF) extracted from palm oil. TRF treatment to PrEC and PZ-HPV-7 resulted in almost identical growth-inhibitory responses of low magnitude. In sharp contrast, TRF treatment resulted in significant decreases in cell viability and colony formation inmore » all three prostate cancer cell lines. The IC{sub 5} values after 24 h TRF treatment in LNCaP, PC-3, and DU145 cells were in the order 16.5, 17.5, and 22.0 {mu}g/ml. TRF treatment resulted in significant apoptosis in all the cell lines as evident from (i) DNA fragmentation (ii) fluorescence microscopy, and (iii) cell death detection ELISA, whereas the PrEC and PZ-HPV-7 cells did not undergo apoptosis, but showed modestly decreased cell viability only at a high dose of 80 {mu}g/ml. In cell cycle analysis, TRF (10-40 {mu}g/ml) resulted in a dose-dependent G0/G1 phase arrest and sub G1 accumulation in all three cancer cell lines but not in PZ-HPV-7 cells. These results suggest that the palm oil derivative TRF is capable of selectively inhibiting cellular proliferation and accelerating apoptotic events in prostate cancer cells. TRF offers significant promise as a chemopreventive and/or therapeutic agent against prostate cancer.« less

Sensitizing basal-like breast cancer to chemotherapy using nanoparticles conjugated with interference peptide

NASA Astrophysics Data System (ADS)

Sorolla, A.; Ho, D.; Wang, E.; Evans, C. W.; Ormonde, C. F. G.; Rashwan, R.; Singh, R.; Iyer, K. Swaminathan; Blancafort, P.

2016-04-01

Basal-like breast cancers are highly aggressive malignancies associated with very poor prognosis. Although these cancers may initially respond to first-line treatment, they become highly resistant to standard chemotherapy in the metastatic setting. Chemotherapy resistance in basal-like breast cancers is associated with highly selective overexpression of the homeobox transcription factor Engrailed 1 (EN1). Herein, we propose a novel therapeutic strategy using poly(glycidyl methacrylate) nanoparticles decorated with poly(acrylic acid) that enable dual delivery of docetaxel and interference peptides designed to block or inhibit EN1 (EN1-iPep). We demonstrate that EN1-iPep is highly selective in inducing apoptotic cell death in basal-like cancer cells with negligible effects in a non-neoplastic human mammary epithelial cell line. Furthermore, we show that treatment with EN1-iPep results in a highly synergistic pharmacological interaction with docetaxel in inhibiting cancer cell growth. The incorporation of these two agents in a single nanoformulation results in greater anticancer efficacy than current nanoparticle-based treatments used in the clinical setting.Basal-like breast cancers are highly aggressive malignancies associated with very poor prognosis. Although these cancers may initially respond to first-line treatment, they become highly resistant to standard chemotherapy in the metastatic setting. Chemotherapy resistance in basal-like breast cancers is associated with highly selective overexpression of the homeobox transcription factor Engrailed 1 (EN1). Herein, we propose a novel therapeutic strategy using poly(glycidyl methacrylate) nanoparticles decorated with poly(acrylic acid) that enable dual delivery of docetaxel and interference peptides designed to block or inhibit EN1 (EN1-iPep). We demonstrate that EN1-iPep is highly selective in inducing apoptotic cell death in basal-like cancer cells with negligible effects in a non-neoplastic human mammary epithelial cell line. Furthermore, we show that treatment with EN1-iPep results in a highly synergistic pharmacological interaction with docetaxel in inhibiting cancer cell growth. The incorporation of these two agents in a single nanoformulation results in greater anticancer efficacy than current nanoparticle-based treatments used in the clinical setting. Electronic supplementary information (ESI) available. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ob00583g

Synthesis, DNA binding ability and anticancer activity of 2-heteroaryl substituted benzimidazoles linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates.

PubMed

Kamal, Ahmed; Pogula, Praveen Kumar; Khan, Mohammed Naseer Ahmed; Seshadri, Bobburi Naga; Sreekanth, Kokkonda

2013-08-01

As a continuation of our efforts to develop the benzimidazole-PBD conjugates as potential anticancer agents, a series of heteroaryl substituted benzimidazole linked PBD conjugates has been synthesized and evaluated for their anticancer potential in 60 human cancer cell lines. Most of the compounds exhibited promising anticancer activity and interestingly, compounds 4c and 4d displayed significant activity in most of the cell lines tested. Whereas, compound 4e showed selectivity in renal cancer cells with GI50 values of <10 and 70 nM against RXF 393 and UO-31 cell lines, respectively. Further, these compounds also showed significant DNA-binding affinity by thermal denaturation study using duplex form of calf thymus (CT) DNA.

Fluorescent Probes of the Apoptolidins and their Utility in Cellular Localization Studies

PubMed Central

DeGuire, Sean M.; Earl, David C.; Du, Yu; Crews, Brenda A.; Jacobs, Aaron T.; Ustione, Alessandro; Daniel, Cristina; Chong, Katherine; Marnett, Lawrence J.; Piston, David W.; Bachmann, Brian O.; Sulikowski, Gary A.

2014-01-01

Apoptolidin A has been described as among the top 0.1% most cell selective cytotoxic agents to be evaluated in the NCI 60 cell line panel. The molecular structure of apoptolidin A consists of a 20-membered macrolide with mono- and disaccharide moieties located at C9 and C27, respectively. In contrast to apoptolidin A, the aglycone (apoptolidinone) shows no cytotoxicity (>10 μM) when evaluated against several tumor cell lines. Apoptolidin H, the C27 deglycosylated analog of apoptolidin A, was produced by targeted glycosyl transferase gene deletion and displayed sub-micromolar activity against H292 lung carcinoma cells. Selective esterification of the C2′ hydroxyl group of apoptolidins A and H with 5-azidopentanoic acid afforded azido functionalized derivatives of potency equal to their parent macrolide. Azido apoptolidins readily underwent strain-promoted alkyne azido cycloaddition (SPAAC) reactions to provide access to fluorescent and biotin functionalized probes. Microscopy studies demonstrate apoptolidins A and H localize in the mitochondria of H292 human lung carcinoma cells. PMID:25430909

Development of a chemically defined platform fed-batch culture media for monoclonal antibody-producing CHO cell lines with optimized choline content.

PubMed

Kuwae, Shinobu; Miyakawa, Ichiko; Doi, Tomohiro

2018-01-11

A chemically defined platform basal medium and feed media were developed using a single Chinese hamster ovary (CHO) cell line that produces a monoclonal antibody (mAb). Cell line A, which showed a peak viable cell density of 5.9 × 10 6  cells/mL and a final mAb titer of 0.5 g/L in batch culture, was selected for the platform media development. Stoichiometrically balanced feed media were developed using glucose as an indicator of cell metabolism to determine the feed rates of all other nutrients. A fed-batch culture of cell line A using the platform fed-batch medium yielded a 6.4 g/L mAb titer, which was 12-fold higher than that of the batch culture. To examine the applicability of the platform basal medium and feed media, three other cell lines (A16, B, and C) that produce mAbs were cultured using the platform fed-batch medium, and they yielded mAb titers of 8.4, 3.3, and 6.2 g/L, respectively. The peak viable cell densities of the three cell lines ranged from 1.3 × 10 7 to 1.8 × 10 7  cells/mL. These results show that the nutritionally balanced fed-batch medium and feeds worked well for other cell lines. During the medium development, we found that choline limitation caused a lower cell viability, a lower mAb titer, a higher mAb aggregate content, and a higher mannose-5 content. The optimal choline chloride to glucose ratio for the CHO cell fed-batch culture was determined. Our platform basal medium and feed media will shorten the medium-development time for mAb-producing cell lines.

Selective Cell Adhesion and Biosensing Applications of Bio-Active Block Copolymers Prepared by CuAAC/Thiol-ene Double Click Reactions.

PubMed

Oyman Eyrilmez, Gizem; Doran, Sean; Murtezi, Eljesa; Demir, Bilal; Odaci Demirkol, Dilek; Coskunol, Hakan; Timur, Suna; Yagci, Yusuf

2015-09-01

N-Acetyl-l-cysteine (NAC)-capped poly(methyl methacrylate)-b-polycaprolactone block copolymer (PMMA-b-PCL-NAC) was prepared using the previously described one-pot photoinduced sequential CuAAC/thiol-ene double click procedure. PMMA-b-PCL-NAC had previously shown good applicability as a matrix for cell adhesion of cells from the Vero cell line (African green monkey kidney epithelial). Here, in this work, PMMA-b-PCL-NAC served as an excellent immobilization matrix for biomolecule conjugation. Covalent binding of RGD (R: arginine, G: glycine, and D: aspartic acid) peptide sequence onto the PMMA-b-PCL-NAC-coated surface was performed via EDC chemistry. RGD-modified PMMA-b-PCL-NAC (PMMA-b-PCL-NAC-RGD) as a non-toxic cell proliferation platform was used for selective "integrin αvβ3-mediated cell adhesion and biosensing studies. Both optical and electrochemical techniques were used to monitor the adhesion differences between "integrin αvβ3" receptor positive and negative cell lines on to the designed biofunctional surfaces. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Effects of Allicin, a Reactive Sulfur Species from Garlic, on a Selection of Mammalian Cell Lines

PubMed Central

Gruhlke, Martin C. H.; Nicco, Carole; Batteux, Frederic; Slusarenko, Alan J.

2016-01-01

Garlic (Allium sativum L.) has been used as a spice and medicinal plant since ancient times. Garlic produces the thiol-reactive defence substance, allicin, upon wounding. The effects of allicin on human lung epithelium carcinoma (A549), mouse fibroblast (3T3), human umbilical vein endothelial cell (HUVEC), human colon carcinoma (HT29) and human breast cancer (MCF7) cell lines were tested. To estimate toxic effects of allicin, we used a standard MTT-test (methylthiazoltetrazolium) for cell viability and 3H-thymidine incorporation for cell proliferation. The glutathione pool was measured using monobromobimane and the formation of reactive species was identified using 2′,7′-dichlorofluoresceine-diacetate. The YO-PRO-1 iodide staining procedure was used to estimate apoptosis. Allicin reduced cell viability and cell proliferation in a concentration dependent manner. In the bimane test, it was observed that cells treated with allicin showed reduced fluorescence, suggesting glutathione oxidation. The cell lines tested differed in sensitivity to allicin in regard to viability, cell proliferation and glutathione oxidation. The 3T3 and MCF-7 cells showed a higher proportion of apoptosis compared to the other cell types. These data show that mammalian cell lines differ in their sensitivity and responses to allicin. PMID:28035949

High hydrostatic pressure affects antigenic pool in tumor cells: Implication for dendritic cell-based cancer immunotherapy.

PubMed

Urbanova, Linda; Hradilova, Nada; Moserova, Irena; Vosahlikova, Sarka; Sadilkova, Lenka; Hensler, Michal; Spisek, Radek; Adkins, Irena

2017-07-01

High hydrostatic pressure (HHP) can be used to generate dendritic cell (DC)-based active immunotherapy for prostate, lung and ovarian cancer. We showed here that HHP treatment of selected human cancer cell lines leads to a degradation of tumor antigens which depends on the magnitude of HHP applied and on the cancer cell line origin. Whereas prostate or ovarian cell lines displayed little protein antigen degradation with HHP treatment up to 300MPa after 2h, tumor antigens are hardly detected in lung cancer cell line after treatment with HHP 250MPa at the same time. On the other hand, quick reduction of tumor antigen-coding mRNA was observed at HHP 200MPa immediately after treatment in all cell lines tested. To optimize the DC-based active cellular therapy protocol for HHP-sensitive cell lines the immunogenicity of HHP-treated lung cancer cells at 150, 200 and 250MPa was compared. Lung cancer cells treated with HHP 150MPa display characteristics of immunogenic cell death, however cells are not efficiently phagocytosed by DC. Despite induction of the highest number of antigen-specific CD8 + T cells, 150 MPa-treated lung cancer cells survive in high numbers. This excludes their use in DC vaccine manufacturing. HHP of 200MPa treatment of lung cancer cells ensures the optimal ratio of efficient immunogenic killing and delivery of protein antigens in DC. These results represent an important pre-clinical data for generation of immunogenic killed lung cancer cells in ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa). Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Carrier-selective interlayer materials for silicon solar cell contacts

NASA Astrophysics Data System (ADS)

Xue, Muyu; Islam, Raisul; Chen, Yusi; Chen, Junyan; Lu, Ching-Ying; Mitchell Pleus, A.; Tae, Christian; Xu, Ke; Liu, Yi; Kamins, Theodore I.; Saraswat, Krishna C.; Harris, James S.

2018-04-01

This work presents titanium oxide (TiOx) and nickel oxide (NiOx) as promising carrier-selective interlayer materials for metal-interlayer-semiconductor contacts for silicon solar cells. The electron-conducting, hole-blocking behavior of TiOx and the opposite carrier-selective behavior of NiOx are investigated using the transmission-line-method. The Fermi level depinning effect and the tunneling resistance are demonstrated to be dependent on the interlayer oxide thickness and annealing temperature. NiOx is furthermore experimentally demonstrated to be capable of improving the effective minority carrier lifetime by quasi-steady-state photoconductance method. Our study demonstrates that TiOx and NiOx can be effective carrier-selective materials for Si solar cells and provides a framework for characterizing carrier-selective contacts.

The Pathway Analysis of Micrornas Regulated Drug-Resistant Responses in HeLa Cells.

PubMed

Yang, Yubo; Dai, Cuihong; Cai, Zhipeng; Hou, Aiju; Cheng, Dayou; Wu, Guanying; Li, Jing; Cui, Jie; Xu, Dechang

2016-03-01

Chemotherapy is the main strategy in the treatment of cancer; however, the development of drug-resistance is the obstacle in long-term treatment of cervical cancer. Cisplatin is one of the most common drugs used in cancer therapy. Recently, accumulating evidence suggests that miRNAs are involved in various bioactivities in oncogenesis. It is not unexpected that miRNAs play a key role in acquiring of drug-resistance in the progression of tumor. In this study, we induced and maintained four levels of cisplatin-resistant HeLa cell lines (HeLa/CR1, HeLa/CR2, HeLa/CR3, and HeLa/CR4). According to the previous studies and existing evidence, we selected five miRNAs (miR-183, miR-182, miR-30a, miR-15b, and miR-16) and their potential target mRNAs as our research targets. The real-time RT-PCR was adopted to detect the relative expression of miRNAs and their mRNAs. The results show that miR-182 and miR-15b were up-regulated in resistant cell lines, while miR-30a was significantly down-regulated. At the same time, their targets are related to drug resistance. Compared to their parent HeLa cell line, the expression of selected miRNAs in resistant cell lines altered. The alteration suggests that HeLa cell drug resistance is associated with distinct miRNAs, which indicates that miRNAs may be one of the therapy targets in the treatment of cervical cancer by sensitizing cell to chemotherapy. We suggested a possible network diagram based on the existing theory and the preliminary results of candidate miRNAs and their targets in HeLa cells during development of drug resistance.

[Cloning goat producing human lactoferrin with genetically modified donor cells selected by single or dual markers].

PubMed

An, Liyou; Yuan, Yuguo; Yu, Baoli; Yang, Tingjia; Cheng, Yong

2012-12-01

We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.

Targeting Nrf2 with wogonin overcomes cisplatin resistance in head and neck cancer.

PubMed

Kim, Eun Hye; Jang, Hyejin; Shin, Daiha; Baek, Seung Ho; Roh, Jong-Lyel

2016-11-01

A principal limitation to the clinical use of cisplatin is the high incidence of chemoresistance to this drug. Combination treatments with other drugs may help to circumvent this problem. Wogonin, one of the major natural flavonoids, is known to reverse multidrug resistance in several types of cancers. We investigated the ability of wogonin to overcome cisplatin resistance in head and neck cancer (HNC) cells and further clarified its molecular mechanisms of action. Two cisplatin-resistant HNC cell lines (AMC-HN4R and -HN9R) and their parental and other human HNC cell lines were used. The effects of wogonin, either alone or in combination with cisplatin, were assessed in HNC cells and normal cells using cell cycle and death assays and by measuring cell viability, reactive oxygen species (ROS) production, and protein expression, and in tumor xenograft mouse models. Wogonin selectively killed HNC cells but spared normal cells. It inhibited nuclear factor erythroid 2-related factor 2 and glutathione S-transferase P in cisplatin-resistant HNC cells, resulting in increased ROS accumulation in HNC cells, an effect that could be blocked by the antioxidant N-acetyl-L-cysteine. Wogonin also induced selective cell death by targeting the antioxidant defense mechanisms enhanced in the resistant HNC cells and activating cell death pathways involving PUMA and PARP. Hence, wogonin significantly sensitized resistant HNC cells to cisplatin both in vitro and in vivo. Wogonin is a promising anticancer candidate that induces ROS accumulation and selective cytotoxicity in HNC cells and can help to overcome cisplatin-resistance in this cancer.

Ferulic acid reverses ABCB1-mediated paclitaxel resistance in MDR cell lines.

PubMed

Muthusamy, Ganesan; Balupillai, Agilan; Ramasamy, Karthikeyan; Shanmugam, Mohana; Gunaseelan, Srithar; Mary, Beaulah; Prasad, N Rajendra

2016-09-05

Multidrug resistance (MDR) remains a major obstacle in cancer chemotherapy. The use of the dietary phytochemicals as chemosensitizing agents to enhance the efficacy of conventional cytostatic drugs has recently gained the attention as a plausible approach for overcoming the drug resistance. The aim of this study was to investigate whether a naturally occurring diet-based phenolic acid, ferulic acid, could sensitize paclitaxel efficacy in ABCB1 overexpressing (P-glycoprotein) colchicine selected KB Ch(R)8-5 cell line. In vitro drug efflux assays demonstrated that ferulic acid inhibits P-glycoprotein transport function in drug resistant KB Ch(R)8-5 cell lines. However, ferulic acid significantly downregulates ABCB1 expression in a concentration dependent manner. Cytotoxicity assay reveals that ferulic acid decreased paclitaxel resistance in KBCh(R)8-5 and HEK293/ABCB1 cells, which indicates its chemosensitizing potential. Clonogenic cell survival assay and apoptotic morphological staining further confirm the chemosensitizing potential of ferulic acid in drug resistant KB Ch(R)8-5 cell lines. Ferulic acid treatment enhances paclitaxel mediated cell cycle arrest and upregulates paclitaxel-induced apoptotic signaling in KB resistant cells. Hence, it has been concluded that downregulation of ABCB1 and subsequent induction of paclitaxel-mediated cell cycle arrest and apoptotic signaling may be the cause for the chemosensitizing potential of ferulic acid in P-gp overexpressing cell lines. Copyright © 2016 Elsevier B.V. All rights reserved.

Development and characterization of a mouse floxed Bmp2 osteoblast cell line that retains osteoblast genotype and phenotype

PubMed Central

Wu, Li-an; Feng, Junsheng; Wang, Lynn; Mu, Yan-dong; Baker, Andrew; Donly, Kevin J.; Harris, Stephen E.; MacDougall, Mary; Chen, Shuo

2011-01-01

Bone morphogenetic protein 2 (Bmp2) is essential for osteoblast differentiation and osteogenesis. Generation of floxed Bmp2 osteoblast cell lines is a valuable tool for studying the effects of Bmp2 on osteoblast differentiation and its signaling pathways during skeletal metabolism. Due to relatively limited sources of primary osteoblasts, we have developed cell lines that serve as good surrogate models for the study of osteoblast cell differentiation and bone mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 osteoblast cell lines. Primary mouse floxed Bmp2 osteoblasts were transfected with pSV3-neo and clonally selected. These transfected cells were verified by PCR and immunohistochemistry. To determine the genotype and phenotype of the immortalized cells, cell morphology, proliferation, differentiation and mineralization were analyzed. Also, expression of osteoblast-related gene markers including Runx2, Osx, ATF4, Dlx3, bone sialoprotein, dentin matrix protein 1, osteonectin, osteocalcin and osteopontin were examined by quantitative RT-PCR and immunohistochemistry. These results showed that immortalized floxed Bmp2 osteoblasts had a higher proliferation rate but preserved their genotypic and phenotypic characteristics similar to the primary cells. Thus, we, for the first time, describe the development of immortalized mouse floxed Bmp2 osteoblast cell lines and present a useful model to study osteoblast biology mediated by BMP2 and its downstream signaling transduction pathways. PMID:21271257

Sleeping Beauty transposon-based system for rapid generation of HBV-replicating stable cell lines.

PubMed

Wu, Yong; Zhang, Tian-Ying; Fang, Lin-Lin; Chen, Zi-Xuan; Song, Liu-Wei; Cao, Jia-Li; Yang, Lin; Yuan, Quan; Xia, Ning-Shao

2016-08-01

The stable HBV-replicating cell lines, which carry replication-competent HBV genome stably integrated into the genome of host cell, are widely used to evaluate the effects of antiviral agents. However, current methods to generate HBV-replicating cell lines, which are mostly dependent on random integration of foreign DNA via plasmid transfection, are less-efficient and time-consuming. To address this issue, we constructed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable cell lines carrying replication-competent HBV genome of different genotype. This vector contains a Sleeping Beauty transposon containing HBV 1.3-copy genome with an expression cassette of the SV40 promoter driving red fluorescent protein (mCherry) and self-cleaving P2A peptide linked puromycin resistance gene (PuroR). In addition, a PGK promoter-driven SB100X hyperactive transposase cassette is placed in the outside of the transposon in the same plasmid.The HBV-replicating stable cells could be obtained from pTSMP-HBV transfected HepG2 cells by red fluorescence-activated cell sorting and puromycin resistant cell selection within 4-week. Using this system, we successfully constructed four cell lines carrying replication-competent HBV genome of genotypes A-D. The replication and viral protein expression profiles of these cells were systematically characterized. In conclusion, our study provides a high-efficiency strategy to generate HBV-replicating stable cell lines, which may facilitate HBV-related virological study. Copyright © 2016. Published by Elsevier B.V.

Dihydroartemisinin is an inhibitor of ovarian cancer cell growth.

PubMed

Jiao, Yang; Ge, Chun-min; Meng, Qing-hui; Cao, Jian-ping; Tong, Jian; Fan, Sai-jun

2007-07-01

To investigate the anticancer activity of dihydroartemisinin (DHA), a derivative of antimalaria drug artemisinin in a panel of human ovarian cancer cell lines. Cell growth was determined by the MTT viability assay. Apoptosis and cell cycle progression were evaluated by a DNA fragmentation gel electro-phoresis, flow cytometry assay, and TUNEL assay; protein and mRNA expression were analyzed by Western blotting and RT-PCR assay. Artemisinin and its derivatives, including artesunate, arteether, artemether, arteannuin, and DHA, exhibit anticancer growth activities in human ovarian cancer cells. Among them, DHA is the most effective in inhibiting cell growth. Ovarian cancer cell lines are more sensitive (5-10-fold) to DHA treatment compared to normal ovarian cell lines. DHA at micromolar dose levels exhibits a dose- and time-dependent cytotoxicity in ovarian cancer cell lines. Furthermore, DHA induced apoptosis and G2 cell cycle arrest, accompanied by a decrease of Bcl-xL and Bcl-2 and an increase of Bax and Bad. The promising results show for the first time that DHA inhibits the growth of human ovarian cancer cells. The selective inhibition of ovarian cancer cell growth, apoptosis induction, and G2 arrest provide in vitro evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of ovarian cancer.

Production of human monoclonal IgG antibodies against Rhesus (D) antigen.

PubMed Central

Bron, D; Feinberg, M B; Teng, N N; Kaplan, H S

1984-01-01

An Epstein-Barr virus (EBV)-transformed human B-cell line ( LB4r ) producing anti-Rhesus [Rho(D) antigen] antibody was fused with a non-immunoglobulin-producing mouse-human heteromyeloma ( SHM - D33 ) and selected in hypoxanthine/aminopterin/thymidine medium containing 0.5 microM ouabain. Surviving hybrids found to secrete specific anti-Rho(D) antibody were cloned by limiting dilution. Two clones (D4-B2 and E10-C1) producing high levels (12 and 20 micrograms/ml per 10(6) cells per 24 hr, respectively) of monospecific antibody (IgG3, lambda chain) were selected for expansion and further characterization. Compared to the parental cell line ( LB4r ), these hybridoma cell lines presented several advantages: antibody production was increased 10-fold, cloning efficiency was improved, and the EBV genome was not retained. Antibody production has been stable for greater than 8 months. These human monoclonal anti-Rho(D) antibodies have demonstrated utility in routine blood-group typing. They may also prove useful in the biochemical and genetic characterization of the Rh antigen system. Most important, they offer a source of Rh-immune globulin for the prevention of Rh immunization and alloimmune hemolytic disease of the newborn. Images PMID:6427767

Cytotoxic and antibacterial angucycline- and prodigiosin-analogues from the deep-sea derived Streptomyces sp. SCSIO 11594.

PubMed

Song, Yongxiang; Liu, Guangfu; Li, Jie; Huang, Hongbo; Zhang, Xing; Zhang, Hua; Ju, Jianhua

2015-03-16

Two new C-glycoside angucyclines, marangucycline A (1) and marangucycline B (2), along with three known compounds, dehydroxyaquayamycin (3), undecylprodigiosin (4) and metacycloprodigiosin (5), have been identified as products of the deep-sea sediment strain Streptomyces sp. SCSIO 11594. New structures were elucidated on the basis of HRESIMS, 1D and 2D NMR analyses and comparisons to previously reported datasets. Compounds 2 and 4 displayed in vitro cytotoxicity against four cancer cell lines A594, CNE2, HepG2, MCF-7 superior to those obtained with cisplatin, the positive control. Notably, compound 2 bearing a keto-sugar displayed significant cytotoxicity against cancer cell lines with IC50 values ranging from 0.24 to 0.56 μM; An IC50 value of 3.67 μM was found when using non-cancerous hepatic cell line HL7702, demonstrating the cancer cell selectivity of 2. Compounds 1-3 were proved to have weak antibacterial activities against Enterococcus faecalis ATCC29212 with an MIC value of 64.0 μg/mL. Moreover, 3 displayed selective antibacterial activity against methicillin-resistant Staphylococcus epidermidis shhs-E1 with an MIC value of 16.0 μg/mL.

Design, synthesis, antiproliferative activity and docking studies of quinazoline derivatives bearing 2,3-dihydro-indole or 1,2,3,4-tetrahydroquinoline as potential EGFR inhibitors.

PubMed

OuYang, Yiqiang; Zou, Wensheng; Peng, Liang; Yang, Zunhua; Tang, Qidong; Chen, Mengzi; Jia, Shuang; Zhang, Hong; Lan, Zhou; Zheng, Pengwu; Zhu, Wufu

2018-05-09

Eight series of quinazoline derivatives bearing 2,3-dihydro-indole or 1,2,3,4-tetrahydroquinoline were designed, synthesized and evaluated for the IC 50 values against three cancer cell lines (A549, MCF-7 and PC-3). Most of the forty nine target compounds showed excellent antiproliferative activity against one or several cancer cell lines. The compound 13a showed the best activity against A549, MCF-7 and PC-3 cancer cell lines, with the IC 50 values of 1.09 ± 0.04 μM, 1.34 ± 0.13 μM and 1.23 ± 0.09 μM, respectively. Eight selected compounds were further selected to evaluated for the inhibitory activity against EGFR kinase. Three of them showed equal activity against EGFR kinase to positive control afatinib. AnnexinV-FITC, propidium iodide (PI) double staining and acridine orange single staining results indicated that the compound 13a could induce apoptosis of human lung cancer A549 cells. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Constraint based modeling of metabolism allows finding metabolic cancer hallmarks and identifying personalized therapeutic windows.

PubMed

Bordel, Sergio

2018-04-13

In order to choose optimal personalized anticancer treatments, transcriptomic data should be analyzed within the frame of biological networks. The best known human biological network (in terms of the interactions between its different components) is metabolism. Cancer cells have been known to have specific metabolic features for a long time and currently there is a growing interest in characterizing new cancer specific metabolic hallmarks. In this article it is presented a method to find personalized therapeutic windows using RNA-seq data and Genome Scale Metabolic Models. This method is implemented in the python library, pyTARG. Our predictions showed that the most anticancer selective (affecting 27 out of 34 considered cancer cell lines and only 1 out of 6 healthy mesenchymal stem cell lines) single metabolic reactions are those involved in cholesterol biosynthesis. Excluding cholesterol biosynthesis, all the considered cell lines can be selectively affected by targeting different combinations (from 1 to 5 reactions) of only 18 metabolic reactions, which suggests that a small subset of drugs or siRNAs combined in patient specific manners could be at the core of metabolism based personalized treatments.

Functional characterisation of osteosarcoma cell lines and identification of mRNAs and miRNAs associated with aggressive cancer phenotypes

PubMed Central

Lauvrak, S U; Munthe, E; Kresse, S H; Stratford, E W; Namløs, H M; Meza-Zepeda, L A; Myklebost, O

2013-01-01

Background: Osteosarcoma is the most common primary malignant bone tumour, predominantly affecting children and adolescents. Cancer cell line models are required to understand the underlying mechanisms of tumour progression and for preclinical investigations. Methods: To identify cell lines that are well suited for studies of critical cancer-related phenotypes, such as tumour initiation, growth and metastasis, we have evaluated 22 osteosarcoma cell lines for in vivo tumorigenicity, in vitro colony-forming ability, invasive/migratory potential and proliferation capacity. Importantly, we have also identified mRNA and microRNA (miRNA) gene expression patterns associated with these phenotypes by expression profiling. Results: The cell lines exhibited a wide range of cancer-related phenotypes, from rather indolent to very aggressive. Several mRNAs were differentially expressed in highly aggressive osteosarcoma cell lines compared with non-aggressive cell lines, including RUNX2, several S100 genes, collagen genes and genes encoding proteins involved in growth factor binding, cell adhesion and extracellular matrix remodelling. Most notably, four genes—COL1A2, KYNU, ACTG2 and NPPB—were differentially expressed in high and non-aggressive cell lines for all the cancer-related phenotypes investigated, suggesting that they might have important roles in the process of osteosarcoma tumorigenesis. At the miRNA level, miR-199b-5p and mir-100-3p were downregulated in the highly aggressive cell lines, whereas miR-155-5p, miR-135b-5p and miR-146a-5p were upregulated. miR-135b-5p and miR-146a-5p were further predicted to be linked to the metastatic capacity of the disease. Interpretation: The detailed characterisation of cell line phenotypes will support the selection of models to use for specific preclinical investigations. The differentially expressed mRNAs and miRNAs identified in this study may represent good candidates for future therapeutic targets. To our knowledge, this is the first time that expression profiles are associated with functional characteristics of osteosarcoma cell lines. PMID:24064976

Antileishmanial pharmacomodulation in 8-nitroquinolin-2(1H)-one series.

PubMed

Kieffer, Charline; Cohen, Anita; Verhaeghe, Pierre; Paloque, Lucie; Hutter, Sébastien; Castera-Ducros, Caroline; Laget, Michèle; Rault, Sylvain; Valentin, Alexis; Rathelot, Pascal; Azas, Nadine; Vanelle, Patrice

2015-05-15

An antileishmanial pharmacomodulation at position 4 of 8-nitroquinolin-2(1H)-one was conducted by using the Sonogashira and Suzuki-Miyaura coupling reactions. A series of 25 derivatives was tested in vitro on the promastigote stage of Leishmania donovani along with an in vitro cytotoxicity evaluation on the human HepG2 cell line. Only the derivatives bearing a phenyl moiety at position 4 of the quinoline ring displayed interesting biologic profile, when the phenyl moiety was substituted at the para position by a Br or Cl atom, or by a CF3 group. Among them, molecules 17 and 19 were the most selective and were then tested in vitro on the intracellular amastigote stage of both L. donovani and Leishmania infantum, in parallel with complementary in vitro cytotoxicity assays on the macrophage cell lines THP-1 and J774A.1. Molecule 19 showed no activity on the amastigote stages of the parasites and some cytotoxicity on the J774A.1 cell line while molecule 17, less cytotoxic than 19, showed anti-amastigote activity in L. infantum, being 3 times less active than miltefosine but more active and selective than pentamidine. Nevertheless, hit-molecule 17 did not appear as selective as the parent compound. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hybridization of downregulated-COMT transgenic switchgrass lines with field selected switchgrass for improved biomass traits

USDA-ARS?s Scientific Manuscript database

Transgenic switchgrass (Panicum virgatum L.) has been produced for improved cell walls for biofuels. Downregulated caffeic acid 3-O-methyltransferase (COMT) switchgrass produced significantly more biomass and biofuel than the non-transgenic progenitor line. In the present study we sought to further...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Seong-Su, E-mail: seong-su-han@uiowa.edu; Han, Sangwoo; Kamberos, Natalie L.

Highlights: • PL inhibits the proliferation of B-ALL cell lines irrespective of GC-resistance. • PL selectively kills B-ALL cells by increasing ROS, but not normal counterpart. • PL does not sensitize majority of B-ALL cells to DEX. • PL represses the network of constitutively activated TFs and modulates their target genes. • PL may serve as a new therapeutic molecule for GC-resistant B-ALL. - Abstract: Piperlongumine (PL), a pepper plant alkaloid from Piper longum, has anti-inflammatory and anti-cancer properties. PL selectively kills both solid and hematologic cancer cells, but not normal counterparts. Here we evaluated the effect of PL onmore » the proliferation and survival of B-cell acute lymphoblastic leukemia (B-ALL), including glucocorticoid (GC)-resistant B-ALL. Regardless of GC-resistance, PL inhibited the proliferation of all B-ALL cell lines, but not normal B cells, in a dose- and time-dependent manner and induced apoptosis via elevation of ROS. Interestingly, PL did not sensitize most of B-ALL cell lines to dexamethasone (DEX). Only UoC-B1 exhibited a weak synergistic effect between PL and DEX. All B-ALL cell lines tested exhibited constitutive activation of multiple transcription factors (TFs), including AP-1, MYC, NF-κB, SP1, STAT1, STAT3, STAT6 and YY1. Treatment of the B-ALL cells with PL significantly downregulated these TFs and modulated their target genes. While activation of AURKB, BIRC5, E2F1, and MYB mRNA levels were significantly downregulated by PL, but SOX4 and XBP levels were increased by PL. Intriguingly, PL also increased the expression of p21 in B-ALL cells through a p53-independent mechanism. Given that these TFs and their target genes play critical roles in a variety of hematological malignancies, our findings provide a strong preclinical rationale for considering PL as a new therapeutic agent for the treatment of B-cell malignancies, including B-ALL and GC-resistant B-ALL.« less

Effects of selective inhibitors of Aurora kinases on anaplastic thyroid carcinoma cell lines.

PubMed

Baldini, Enke; Tuccilli, Chiara; Prinzi, Natalie; Sorrenti, Salvatore; Antonelli, Alessandro; Gnessi, Lucio; Morrone, Stefania; Moretti, Costanzo; Bononi, Marco; Arlot-Bonnemains, Yannick; D'Armiento, Massimino; Ulisse, Salvatore

2014-10-01

Aurora kinases are serine/threonine kinases that play an essential role in cell division. Their aberrant expression and/or function induce severe mitotic abnormalities, resulting in either cell death or aneuploidy. Overexpression of Aurora kinases is often found in several malignancies, among which is anaplastic thyroid carcinoma (ATC). We have previously demonstrated the in vitro efficacy of Aurora kinase inhibitors in restraining cell growth and survival of different ATC cell lines. In this study, we sought to establish which Aurora might represent the preferential drug target for ATC. To this end, the effects of two selective inhibitors of Aurora-A (MLN8237) and Aurora-B (AZD1152) on four human ATC cell lines (CAL-62, BHT-101, 8305C, and 8505C) were analysed. Both inhibitors reduced cell proliferation in a time- and dose-dependent manner, with IC50 ranges of 44.3-134.2 nM for MLN8237 and of 9.2-461.3 nM for AZD1152. Immunofluorescence experiments and time-lapse videomicroscopy yielded evidence that each inhibitor induced distinct mitotic phenotypes, but both of them prevented the completion of cytokinesis. As a result, poliploidy increased in all AZD1152-treated cells, and in two out of four cell lines treated with MLN8237. Apoptosis was induced in all the cells by MLN8237, and in BHT-101, 8305C, and 8505C by AZD1152, while CAL-62 exposed to AZD1152 died through necrosis after multiple rounds of endoreplication. Both inhibitors were capable of blocking anchorage-independent cell growth. In conclusion, we demonstrated that either Aurora-A or Aurora-B might represent therapeutic targets for the ATC treatment, but inhibition of Aurora-A appears more effective for suppressing ATC cell proliferation and for inducing the apoptotic pathway. © 2014 Society for Endocrinology.

A BCWD-resistant line of rainbow trout exhibits higher abundance of IgT+ B cells and heavy chain tau transcripts compared to a susceptible line following challenge with Flavobacterium psychrophilum

USDA-ARS?s Scientific Manuscript database

Bacterial Cold Water Disease (BCWD) is a common, chronic disease in rainbow trout, and is caused by the gram-negative bacterium Flavobacterium psychrophilum (Fp). Through selective breeding, the National Center for Cool and Cold Water Aquaculture has generated a genetic line that is highly resistant...

Thiazole-based nitrogen mustards: Design, synthesis, spectroscopic studies, DFT calculation, molecular docking, and antiproliferative activity against selected human cancer cell lines

NASA Astrophysics Data System (ADS)

Łączkowski, Krzysztof Z.; Świtalska, Marta; Baranowska-Łączkowska, Angelika; Plech, Tomasz; Paneth, Agata; Misiura, Konrad; Wietrzyk, Joanna; Czaplińska, Barbara; Mrozek-Wilczkiewicz, Anna; Malarz, Katarzyna; Musioł, Robert; Grela, Izabela

2016-09-01

Synthesis, characterization and investigation of antiproliferative activity of ten thiazole-based nitrogen mustard against human cancer cells lines (MV4-11, A549, MCF-7 and HCT116) and normal mouse fibroblast (BALB/3T3) is presented. The structures of novel compounds were determined using 1H and 13C NMR, FAB(+)-MS, and elemental analyses. Among the derivatives, 5b, 5c, 5e, 5f and 5i were found to exhibit high activity against human leukaemia MV4-11 cells with IC50 values of 2.17-4.26 μg/ml. The cytotoxic activity of compound 5c and 5f against BALB/3T3 cells is up to 20 times lower than against cancer cell lines. Our results also show that compounds 5e and 5i have very strong activity against MCF-7 and HCT116 with IC50 values of 3.02-4.13 μg/ml. Moreover, spectroscopic characterization and cellular localization for selected compound were performed. In order to identify potential drug targets we perform computer simulations with DNA-binding site of hTopoI and hTopoII and quantum chemical calculation of interaction and binding energies in complexes of the five most active compounds with guanine.

Immunotechnology: Preparation of Immunotherapeutic Reagents and Development of Immunopharmacologic Vaccines.

DTIC Science & Technology

1981-10-01

Cryopreservation and HLA Typing of the Panel We have assembled a group of volunteer and paid blood donors, some of whom have been selected on the basis of...reagents used as controls. The cell donor panel is typed for human histocompatibility antigens. Dr. A.H. Johnson determines the HLA -A,B,C phenotypes and Dr...2 microglobulin and anti- HLA antibodies. Screening the fusions on glutaraldehyde fixed monolayers of B lymphoblastoid lines, T cell leukaemic lines

Suvanine analogs from a Coscinoderma sp. marine sponge and their cytotoxicities against human cancer cell lines.

PubMed

Lee, Jeong-Woo; Lee, Hyi-Seung; Shin, Jongheon; Kang, Jong Soon; Yun, Jieun; Shin, Hee Jae; Lee, Jong Seok; Lee, Yeon-Ju

2015-06-01

Nine suvanine analogs including suvanine phenethylammonium salt and two new compounds were isolated from the marine sponge Coscinoderma sp., collected from Chuuk State, Federated States of Micronesia. The structures of the new compounds were elucidated by 2D NMR and HRMS analyses. Suvanine and a new analog exhibited weak but selective cytotoxicity against colon (HCT-15), lung (NCI-H23), stomach (NUGC-3), and prostate (PC-3) cancer cell lines.

The ethanol extract of Scutellaria baicalensis and the active compounds induce cell cycle arrest and apoptosis including upregulation of p53 and Bax in human lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao Jiayu; Morgan, Winston A.; Sanchez-Medina, Alberto

2011-08-01

Despite a lack of scientific authentication, Scutellaria baicalensis is clinically used in Chinese medicine as a traditional adjuvant to chemotherapy of lung cancer. In this study, cytotoxicity assays demonstrated that crude ethanolic extracts of S. baicalensis were selectively toxic to human lung cancer cell lines A549, SK-LU-1 and SK-MES-1 compared with normal human lung fibroblasts. The active compounds baicalin, baicalein and wogonin did not exhibit such selectivity. Following exposure to the crude extracts, cellular protein expression in the cancer cell lines was assessed using 2D gel electrophoresis coupled with MALDI-TOF-MS/Protein Fingerprinting. The altered protein expression indicated that cell growth arrestmore » and apoptosis were potential mechanisms of cytotoxicity. These observations were supported by PI staining cell cycle analysis using flow cytometry and Annexin-V apoptotic analysis by fluorescence microscopy of cancer cells treated with the crude extract and pure active compounds. Moreover, specific immunoblotting identification showed the decreased expression of cyclin A results in the S phase arrest of A549 whereas the G{sub 0}/G{sub 1} phase arrest in SK-MES-1 cells results from the decreased expression of cyclin D1. Following treatment, increased expression in the cancer cells of key proteins related to the enhancement of apoptosis was observed for p53 and Bax. These results provide further insight into the molecular mechanisms underlying the clinical use of this herb as an adjuvant to lung cancer therapy. - Research Highlights: > Scutellaria baicalensis is a clinical adjuvant to lung cancer chemotherapy in China. > Scutellaria ethanol extracts selectively toxic to A549, SK-LU-1 and SK-MES-1. > Baicalin, baicalein and wogonin were toxic to all lung cancer cell lines. > Proteomics identified increased p53 and BAX in response to Scutellaria extracts.« less

Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

PubMed

Hertsenberg, Andrew J; Funderburgh, James L

2016-01-01

Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

Enhanced and selective permeability of gold nanoparticles functionalized with cell penetrating peptide derived from maurocalcine animal toxin.

PubMed

Khamehchian, Sedigheh; Nikkhah, Maryam; Madani, Rasool; Hosseinkhani, Saman

2016-11-01

Functionalization of gold nanoparticles (GNPs) is suitable for many applications such as biomedical imaging, clinical diagnosis, and targeted delivery by conjugating cell-penetrating peptides (CPPs). Here, we investigated intracellular uptake of GNP conjugated to MCaUF1-9(Ala) , a CPP derived from maurocalcine (MCa) animal toxin, and compared it with TAT functionalized GNP. Peptide conjugated GNP was characterized using UV-Visible spectroscopy, dynamic light scattering, zeta potential, and transmission electron microscopy. Uptake of MCaUF1-9(Ala) and TAT functionalized GNPs was evaluated in three cell lines, HeLa, MDA-MB-231, and A431, using dark field imaging and atomic absorption spectroscopy. According to peptide sequences and type of cells different cell penetrating activity was observed. Peptide functionalized GNP had little effect on cell viability and respect to net charge difference between peptide, showed interesting selectivity against three cell types. Peptide conjugated to GNPs displayed higher uptake than bare GNPs in the all cell lines except HeLa cell with lowest internalization. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2693-2700, 2016. © 2016 Wiley Periodicals, Inc.

Cytosine-based nucleoside analogs are selectively lethal to DNA mismatch repair-deficient tumour cells by enhancing levels of intracellular oxidative stress

PubMed Central

Hewish, M; Martin, S A; Elliott, R; Cunningham, D; Lord, C J; Ashworth, A

2013-01-01

Background: DNA mismatch repair deficiency is present in a significant proportion of a number of solid tumours and is associated with distinct clinical behaviour. Methods: To identify the therapeutic agents that might show selectivity for mismatch repair-deficient tumour cells, we screened a pair of isogenic MLH1-deficient and MLH1-proficient tumour cell lines with a library of clinically used drugs. To test the generality of hits in the screen, selective agents were retested in cells deficient in the MSH2 mismatch repair gene. Results: We identified cytarabine and other related cytosine-based nucleoside analogues as being selectively toxic to MLH1 and MSH2-deficient tumour cells. The selective cytotoxicity we observed was likely caused by increased levels of cellular oxidative stress, as it could be abrogated by antioxidants. Conclusion: We propose that cytarabine-based chemotherapy regimens may represent a tumour-selective treatment strategy for mismatch repair-deficient cancers. PMID:23361057

Genetically fluorescent melanoma bone and organ metastasis models.

PubMed

Yang, M; Jiang, P; An, Z; Baranov, E; Li, L; Hasegawa, S; Al-Tuwaijri, M; Chishima, T; Shimada, H; Moossa, A R; Hoffman, R M

1999-11-01

We report here the establishment and metastatic properties of bright, highly stable, green fluorescent protein (GFP) expression transductants of the B16 mouse malignant melanoma cell line and the LOX human melanoma line. The highly fluorescent malignant melanoma cell lines allowed the visualization of skeletal and multiorgan metastases after i.v. injection of B16 cells in C57BL/6 mice and intradermal injection of LOX cells in nude mice. The melanoma cell lines were transduced with the pLEIN expression retroviral vector containing the GFP and neomycin resistance genes. Stable B16F0 and LOX clones expressing high levels of GFP were selected stepwise in vitro in levels of G418 of up to 800 microg/ml. Extensive bone and bone marrow metastases of B16F0 were visualized by GFP expression when the animals were sacrificed 3 weeks after cell implantation. Metastases for both cell lines were visualized in many organs, including the brain, lung, pleural membrane, liver, kidney, adrenal gland, lymph nodes, skeleton, muscle, and skin by GFP fluorescence. This is the first observation of experimental skeletal metastases of melanoma, which was made possible by GFP expression. These models should facilitate future studies of the mechanism and therapy of bone and multiorgan metastasis of melanoma.

Modification of meta-iodobenzylguanidine uptake in neuroblastoma cells by elevated temperature.

PubMed Central

Armour, A.; Mairs, R. J.; Gaze, M. N.; Wheldon, T. E.

1994-01-01

Successful imaging or treatment of neuroblastoma with 131I-meta-iodobenzylguanidine (131I-mIBG) depends on the selectivity of active (type 1) uptake of mIBG in neuroblastoma cells relative to passive (type 2) uptake present in most normal tissues. This study investigates the effects of moderately elevated temperature (39-41 degrees C) on the cellular uptake of 131I-mIBG in two neuroblastoma cell lines [SK-N-BE(2c) and IMR-32] and in a non-neuronal (ovarian carcinoma) cell line (A2780). In SK-N-BE(2c), a cell line with high active uptake capacity, the specific (type 1) uptake was reduced by 75% (P < 0.001) at 39 degrees C. Both IMR-32 and A2780 have a low capacity for accumulation of mIBG by active uptake. These cell lines demonstrated a statistically significant increase in accumulation at 39 degrees C, mainly as a result of increased non-specific transport. At 41 degrees C uptake of 131I-mIBG was reduced in all cell lines. Thus, the active component of mIBG uptake is more vulnerable to increased temperature than the passive component. It seems probable that moderately increased temperature will have an unfavourable effect on the therapeutic differential for targeted radiotherapy of neuroblastoma using radiolabelled mIBG. PMID:8080728

Image classification of human carcinoma cells using complex wavelet-based covariance descriptors.

PubMed

Keskin, Furkan; Suhre, Alexander; Kose, Kivanc; Ersahin, Tulin; Cetin, A Enis; Cetin-Atalay, Rengul

2013-01-01

Cancer cell lines are widely used for research purposes in laboratories all over the world. Computer-assisted classification of cancer cells can alleviate the burden of manual labeling and help cancer research. In this paper, we present a novel computerized method for cancer cell line image classification. The aim is to automatically classify 14 different classes of cell lines including 7 classes of breast and 7 classes of liver cancer cells. Microscopic images containing irregular carcinoma cell patterns are represented by subwindows which correspond to foreground pixels. For each subwindow, a covariance descriptor utilizing the dual-tree complex wavelet transform (DT-[Formula: see text]WT) coefficients and several morphological attributes are computed. Directionally selective DT-[Formula: see text]WT feature parameters are preferred primarily because of their ability to characterize edges at multiple orientations which is the characteristic feature of carcinoma cell line images. A Support Vector Machine (SVM) classifier with radial basis function (RBF) kernel is employed for final classification. Over a dataset of 840 images, we achieve an accuracy above 98%, which outperforms the classical covariance-based methods. The proposed system can be used as a reliable decision maker for laboratory studies. Our tool provides an automated, time- and cost-efficient analysis of cancer cell morphology to classify different cancer cell lines using image-processing techniques, which can be used as an alternative to the costly short tandem repeat (STR) analysis. The data set used in this manuscript is available as supplementary material through http://signal.ee.bilkent.edu.tr/cancerCellLineClassificationSampleImages.html.

Image Classification of Human Carcinoma Cells Using Complex Wavelet-Based Covariance Descriptors

PubMed Central

Keskin, Furkan; Suhre, Alexander; Kose, Kivanc; Ersahin, Tulin; Cetin, A. Enis; Cetin-Atalay, Rengul

2013-01-01

Cancer cell lines are widely used for research purposes in laboratories all over the world. Computer-assisted classification of cancer cells can alleviate the burden of manual labeling and help cancer research. In this paper, we present a novel computerized method for cancer cell line image classification. The aim is to automatically classify 14 different classes of cell lines including 7 classes of breast and 7 classes of liver cancer cells. Microscopic images containing irregular carcinoma cell patterns are represented by subwindows which correspond to foreground pixels. For each subwindow, a covariance descriptor utilizing the dual-tree complex wavelet transform (DT-WT) coefficients and several morphological attributes are computed. Directionally selective DT-WT feature parameters are preferred primarily because of their ability to characterize edges at multiple orientations which is the characteristic feature of carcinoma cell line images. A Support Vector Machine (SVM) classifier with radial basis function (RBF) kernel is employed for final classification. Over a dataset of 840 images, we achieve an accuracy above 98%, which outperforms the classical covariance-based methods. The proposed system can be used as a reliable decision maker for laboratory studies. Our tool provides an automated, time- and cost-efficient analysis of cancer cell morphology to classify different cancer cell lines using image-processing techniques, which can be used as an alternative to the costly short tandem repeat (STR) analysis. The data set used in this manuscript is available as supplementary material through http://signal.ee.bilkent.edu.tr/cancerCellLineClassificationSampleImages.html. PMID:23341908

Identification of cytotoxic mediators and their putative role in the signaling pathways during docosahexaenoic acid (DHA)-induced apoptosis of cancer cells.

PubMed

Das, Moitreyi; Das, Sumantra

2016-12-01

Docosahexaenoic acid (DHA), an important w-3 fatty acid exhibits differential behavior in cancer cells of neural origin when compared to that in normal healthy astrocytes. Treatment of C6 glioma and SH-SY5Y cell lines and primary astrocytes, representing the neoplastic cells and normal healthy cells respectively, with 100 µM DHA for 24 h showed significant loss of cell viability in the both the cancer cells as determined by MTT assay, whereas the primary astrocytes cultures were unaffected. Such loss of cell viability was due to apoptosis as confirmed by TUNEL staining and caspase-3 activation in cancer cells. Proteomic approach, employing 2-dimensional gel electrophoresis (2DE), difference gel electrophoresis (DIGE), and MALDI-TOF-TOF analysis identified six proteins which unlike in the astrocytes, were differently altered in the cancer cells upon exposure to DHA, suggesting their putative contribution in causing apoptosis in these cells. Of these, annexin A2, calumenin, pyruvate kinase M2 isoform, 14-3-3ζ were downregulated while aldo keto reductase-1B8 (AKR1B8) and glutathione-S-transferase P1 subunit (GSTP1) showed upregulation by DHA in the cancer cells. siRNA-mediated knockdown of AKR1B8 and GSTP1 inhibit DHA-induced apoptosis confirming their role in apoptotic process. Furthermore, western blot analysis identified upregulation of PPARα and the MAP kinases, JNK and p38 as well as increased ROS production selectively in the cell lines. Results suggest that DHA selectively induces apoptosis in the neural cell lines by regulating the expression of the above proteins to activate multiple apoptotic pathways which in association with excess ROS and activated MAPKs promote cell death.

Differences in growth properties of endometrial cancer in three dimensional (3D) culture and 2D cell monolayer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chitcholtan, Kenny, E-mail: kenny.chitcholtan@otago.ac.nz; Asselin, Eric, E-mail: Eric.Asselin@uqtr.ca; Parent, Sophie, E-mail: Sophie.Parent@uqtr.ca

2013-01-01

Three-dimensional (3D) in vitro models have an invaluable role in understanding the behaviour of tumour cells in a well defined microenvironment. This is because some aspects of tumour characteristics cannot be fully recapitulated in a cell monolayer (2D). In the present study, we compared growth patterns, expression of signalling molecules, and metabolism-associated proteins of endometrial cancer cell lines in 3D and 2D cell cultures. Cancer cells formed spherical structures in 3D reconstituted basement membrane (3D rBM), and the morphological appearance was cell line dependent. Cell differentiation was observed after 8 days in the 3D rBM. There was reduced proliferation, detectedmore » by less expression of PCNA in 3D rBM than in 2D cell monolayers. The addition of exogenous epidermal growth factor (EGF) to cancer cells induced phosphorylation of EGFR and Akt in both cell culture conditions. The uptake of glucose was selectively altered in the 3D rBM, but there was a lack of association with Glut-1 expression. The secretion of vascular endothelial growth factor (VEGF) and prostaglandin E{sub 2} (PGE{sub 2}) was selectively altered in 3D rBM, and it was cell line dependent. Our data demonstrated that 3D rBM as an in vitro model can influence proliferation and metabolism of endometrial cancer cell behaviour compared to 2D cell monolayer. Changes are specific to individual cell types. The use of 3D rBM is, therefore, important in the in vitro study of targeted anticancer therapies.« less

Validation of a polymerase chain reaction aided transcript titration assay (PATTY) for topoisomerase II in lung cancer samples.

PubMed

Dingemans, A M; Van Ark-Otte, J; Smit, E F; Postmus, P E; Giaccone, G

This report describes the validation of a polymerase chain reaction aided transcript titration assay (PATTY) for tumor samples. The results obtained with the PATTY were compared to those of RNase protection in a set of 7 human lung cancer cell lines and in 23 non-small cell lung cancer samples derived from resected patients. Whereas between PATTY and RNase protection assay a good correlation was observed in the cell lines (r = 0.74, p = 0.057), no correlation was observed within the tumor samples (r = 0.06, p = 0.78). This was also the case when only tumors with a high percentage of tumor cells (> 90%) were selected. Although PATTY is a valuable tool to measure mRNA expression in cell lines, our results caution the use of PATTY in human tumor samples without proper validation. The possible causes of these results are discussed.

Identification of FDA-approved oncology drugs with selective potency in high-risk childhood ependymoma.

PubMed

Donson, Andrew M; Amani, Vladimir; Warner, Elliot A; Griesinger, Andrea M; Witt, Davis A; Mulcahy Levy, Jean M; Hoffman, Lindsey M; Hankinson, Todd C; Handler, Michael H; Vibhakar, Rajeev; Dorris, Kathleen; Foreman, Nicholas K

2018-06-20

Children with ependymoma are cured in less than 50% of cases, with little improvement in outcome over the last several decades. Chemotherapy has not impacted survival in ependymoma, due in part to a lack of preclinical models that has precluded comprehensive drug testing. We recently developed two human ependymoma cell lines harboring high-risk phenotypes which provided us with an opportunity to execute translational studies. ependymoma and other pediatric brain tumor cell lines were subject to a large-scale comparative drug screen of ependymoma -approved oncology drugs for rapid clinical application. The results of this in vitro study were combined with in silico prediction of drug sensitivity to identify ependymoma -selective compounds, which were validated by dose curve and time course modelling. Mechanisms of ependymoma -selective antitumor effect were further investigated using transcriptome and proteome analyses. We identified three classes of oncology drugs that showed ependymoma -selective anti-tumor effect, namely (i) fluorinated pyrimidines (5-fluorouracil, carmofur and floxuridine), (ii) retinoids (bexarotene, tretinoin and isotretinoin), and (iii) a subset of small-molecule multi-receptor tyrosine kinase inhibitors (axitinib, imatinib and pazopanib). Axitinib's anti-tumor mechanism in ependymoma cell lines involved inhibition of PDGFR-alpha and PDGFR-beta and was associated with reduced mitosis-related gene expression and cellular senescence. The clinically available, ependymoma -selective oncology drugs identified by our study have the potential to critically inform design of upcoming clinical studies in ependymoma, in particular for those children with recurrent ependymoma who are in the greatest need of novel therapeutic approaches. Copyright ©2018, American Association for Cancer Research.

Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos.

PubMed

Behboodi, E; Bondareva, A; Begin, I; Rao, K; Neveu, N; Pierson, J T; Wylie, C; Piero, F D; Huang, Y J; Zeng, W; Tanco, V; Baldassarre, H; Karatzas, C N; Dobrinski, I

2011-03-01

Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo-derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES-like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA-1, and SSEA-4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5-6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells. Copyright © 2011 Wiley-Liss, Inc.

Selectivity of Pinus sylvestris extract and essential oil to estrogen-insensitive breast cancer cells Pinus sylvestris against cancer cells.

PubMed

Hoai, Nguyen Thi; Duc, Ho Viet; Thao, Do Thi; Orav, Anne; Raal, Ain

2015-10-01

So far, the anticancer action of pine tree extracts has mainly been shown for the species distributed widely around the Asian countries. Therefore, this study was performed to examine the potential cytotoxicity of Scots pine (Pinus sylvestris L.) native also to the European region and growing widely in Estonia. The cytotoxic activity of methanol extract and essential oil of Scots pine needles was determined by sulforhodamine B assay in different human cancer cell lines. This needle extract was found to suppress the viability of several human cancer cell lines showing some selectivity to estrogen receptor negative breast cancer cells, MDA-MB-231(half maximal inhibitory concentration [IC50] 35 μg/ml) in comparison with estrogen receptor-positive breast cancer cells, MCF-7 (IC50 86 μg/ml). It is the strongest cytotoxic effect at all measured, thus far for the needles and leaves extracts derived from various pine species, and is also the first study comparing the anticancer effects of pine tree extracts on molecularly different human breast cancer cells. The essential oil showed the stronger cytotoxic effect to both negative and positive breast cancer cell lines (both IC50 29 μg/ml) than pine extract (IC50 42 and 80 μg/ml, respectively). The data from this report indicate that Scots pine needles extract and essential oil exhibits some potential as chemopreventive or chemotherapeutic agent for mammary tumors unresponsive to endocrine treatment.

Arachidonic acid-induced Ca2+ entry and migration in a neuroendocrine cancer cell line.

PubMed

Goswamee, Priyodarshan; Pounardjian, Tamar; Giovannucci, David R

2018-01-01

Store-operated Ca 2+ entry (SOCE) has been implicated in the migration of some cancer cell lines. The canonical SOCE is defined as the Ca 2+ entry that occurs in response to near-maximal depletion of Ca 2+ within the endoplasmic reticulum. Alternatively, arachidonic acid (AA) has been shown to induce Ca 2+ entry in a store-independent manner through Orai1/Orai3 hetero-multimeric channels. However, the role of this AA-induced Ca 2+ entry pathway in cancer cell migration has not been adequately assessed. The present study investigated the involvement of AA-induced Ca 2+ entry in migration in BON cells, a model gastro-enteropancreatic neuroendocrine tumor (GEPNET) cell line using pharmacological and gene knockdown methods in combination with live cell fluorescence imaging and standard migration assays. We showed that both the store-dependent and AA-induced Ca 2+ entry modes could be selectively activated and that exogenous administration of AA resulted in Ca 2+ entry that was pharmacologically distinct from SOCE. Also, whereas homomeric Orai1-containing channels appeared to largely underlie SOCE, the AA-induced Ca 2+ entry channel required the expression of Orai3 as well as Orai1. Moreover, we showed that AA treatment enhanced the migration of BON cells and that this migration could be abrogated by selective inhibition of the AA-induced Ca 2+ entry. Taken together, these data revealed that an alternative Orai3-dependent Ca 2+ entry pathway is an important signal for GEPNET cell migration.

In Vitro Differentiation and Propagation of Urothelium from Pluripotent Stem Cell Lines.

PubMed

Osborn, Stephanie L; Kurzrock, Eric A

2018-01-01

Bioengineering of bladder tissue, particularly for those patients who have advanced bladder disease, requires a source of urothelium that is healthy, capable of significant proliferation in vitro and immunologically tolerated upon transplant. As pluripotent stem cells have the potential to fulfill such criteria, they provide a critical cell source from which urothelium might be derived in vitro and used clinically. Herein, we describe the in vitro differentiation of urothelium from the H9 human embryonic stem cell (hESC) line through the definitive endoderm (DE) phase via selective culture techniques. The protocol can be used to derive urothelium from other hESCs or human-induced pluripotent stem cells.

Intracellular distribution of Photofrin in malignant and normal endothelial cell lines.

PubMed

Saczko, J; Mazurkiewicz, M; Chwiłkowska, A; Kulbacka, J; Kramer, G; Ługowski, M; Snietura, M; Banaś, T

2007-01-01

Compared to current treatments including surgery, radiation therapy, and chemotherapy, PDT offers the advantage of an effective and selective method of destroying diseased tissues without damaging surrounding healthy tissues. One of the aspects of antitumour effectiveness of PDT is related to the distribution of photosensitizing drugs. The localization of photosensitizers in cytoplasmic organelles during PDT plays a major role in the cell destruction; therefore, intracellular localization of Ph in malignant and normal cells was investigated. The cell lines used throughout the study were: human malignant A549, MCF-7, Me45 and normal endothelial cell line HUV-EC-C. After incubation with Ph cells were examined using fluorescence and confocal microscopy to visualize the photosensitizer accumulation. For cytoplasm and mitochondria identification, cells were stained with CellTracker Green and MitoTracker Green, respectively. Distribution of Ph was different in malignant and normal cells and dependent on the incubation time. The maximal concentration of Ph in two malignant cell lines (A549 and MCF-7) was observed after 4 hours of incubation, and the most intensive signal was observed around the nuclear envelope. Intracellular distribution of Ph in the Me45 cell line showed that the fluorescence emitted by Ph overlaid that from MitoTracker. This indicates preferential accumulation of the sensitizer in mitochondria. Our results based on the mitochondrial localization support the idea that PDT can contribute to elimination of malignant cells by inducing apoptosis, which is of physiological significance.

Study of small-cell lung cancer cell-based sensor and its applications in chemotherapy effects rapid evaluation for anticancer drugs.

PubMed

Guohua, Hui; Hongyang, Lu; Zhiming, Jiang; Danhua, Zhu; Haifang, Wan

2017-11-15

Small cell lung cancer (SCLC) is a smoking-related cancer disease. Despite improvement in clinical survival, SCLC outcome remains extremely poor. Cisplatin (DDP) is the first-line chemotherapy drug for SCLC, but the choice of second-line chemotherapy drugs is not clear. In this paper, a SCLC cell-based sensor was proposed, and its applications in chemotherapy effects rapid evaluation for anticancer drugs were investigated. SCLC cell lines lung adenocarcinoma cell (LTEP-P) and DDP-resistant lung adenocarcinoma cell (LTEP-P/DDP-1.0) are cultured on carbon screen-printed electrode (CSPE) to fabricate integrated cell-based sensor. Several chemotherapy anticancer drugs, including cisplatin, ifosmamide, gemcitabine, paclitaxel, docetaxel, vinorelbine, etoposide, camptothecin, and topotecan, are selected as experimental chemicals. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests are conducted to evaluate chemotherapy drug effects on LTEP-P and LTEP-P/DDP-1.0 cell lines. Electrical cell-substrate impedance sensing (ECIS) responses to anti-tumor chemicals are measured and processed by double-layered cascaded stochastic resonance (DCSR). Cisplatin solutions in different concentrations measurement results demonstrate that LTEP-P cell-based sensor presents quantitative analysis abilities for cisplatin and topotecan. Cisplatin and its mixtures can also be discriminated. Results demonstrate that LTEP-P cell-based sensor sensitively evaluates chemotherapy drugs' apoptosis function to SCLC cells. LTEP-P/DDP-1.0 cell-based sensor responses demonstrate that gemcitabine, vinorelbine, and camptothecin are ideal second-line drugs for clinical post-cisplatin therapy than other drugs according to MTT test results. This work provides a novel way for SCLC second-line clinical chemotherapy drug screening. Copyright © 2017 Elsevier B.V. All rights reserved.

Esters of Quinoxaline 1ˏ4-Di-N-oxide with Cytotoxic Activity on Tumor Cell Lines Based on NCI-60 Panel

PubMed Central

Rivera, Gildardo; Ahmad Shah, Syed Shoaib; Arrieta-Baez, Daniel; Palos, Isidro; Mongue, Antonio; Sánchez-Torres, Luvia Enid

2017-01-01

Quinoxalines display diverse and interesting pharmacological activities as antibacterial, antiviral, antiparasitic and anticancer agents. Particularly, their 1ˏ4-di-N-oxide derivatives have proved to be cytotoxic agents that are active under hypoxic conditions as that of solid tumours. A new series of quinoxaline 1ˏ4-di-N-oxide substitutes at 7-position with esters group were synthetized and characterized by infrared (IR), proton nuclear magnetic resonance (1H-NMR), spectroscopy, and elemental analysis. Seventeen derivatives (M1-M3, E1-E8, P1-P3 and DR1-DR3) were selected and evaluated for antitumor activities using the NCI-60 human tumor cell lines screen. Results showed that E7, P3 and E6 were the most active compounds against the cell lines tested. Substitutions at 7-position with esters group not necessarily affect the biological activity, but the nature of the esters group could exert an influence on the selectivity. Additionally, substitutions at 2-position influenced the cytotoxic activity of the compounds. PMID:29201086

Esters of Quinoxaline 1`4-Di-N-oxide with Cytotoxic Activity on Tumor Cell Lines Based on NCI-60 Panel.

PubMed

Rivera, Gildardo; Ahmad Shah, Syed Shoaib; Arrieta-Baez, Daniel; Palos, Isidro; Mongue, Antonio; Sánchez-Torres, Luvia Enid

2017-01-01

Quinoxalines display diverse and interesting pharmacological activities as antibacterial, antiviral, antiparasitic and anticancer agents. Particularly, their 1`4-di- N -oxide derivatives have proved to be cytotoxic agents that are active under hypoxic conditions as that of solid tumours. A new series of quinoxaline 1`4-di- N -oxide substitutes at 7-position with esters group were synthetized and characterized by infrared (IR), proton nuclear magnetic resonance ( 1 H-NMR), spectroscopy, and elemental analysis. Seventeen derivatives (M1-M3, E1-E8, P1-P3 and DR1-DR3) were selected and evaluated for antitumor activities using the NCI-60 human tumor cell lines screen. Results showed that E7, P3 and E6 were the most active compounds against the cell lines tested. Substitutions at 7-position with esters group not necessarily affect the biological activity, but the nature of the esters group could exert an influence on the selectivity. Additionally, substitutions at 2-position influenced the cytotoxic activity of the compounds.

Targeting of phage particles towards endothelial cells by antibodies selected through a multi-parameter selection strategy.

PubMed

Mandrup, Ole A; Lykkemark, Simon; Kristensen, Peter

2017-02-10

One of the hallmarks of cancer is sustained angiogenesis. Here, normal endothelial cells are activated, and their formation of new blood vessels leads to continued tumour growth. An improved patient condition is often observed when angiogenesis is prevented or normalized through targeting of these genomically stable endothelial cells. However, intracellular targets constitute a challenge in therapy, as the agents modulating these targets have to be delivered and internalized specifically to the endothelial cells. Selection of antibodies binding specifically to certain cell types is well established. It is nonetheless a challenge to ensure that the binding of antibodies to the target cell will mediate internalization. Previously selection of such antibodies has been performed targeting cancer cell lines; most often using either monovalent display or polyvalent display. In this article, we describe selections that isolate internalizing antibodies by sequential combining monovalent and polyvalent display using two types of helper phages, one which increases display valence and one which reduces background. One of the selected antibodies was found to mediate internalization into human endothelial cells, although our results confirms that the single stranded nature of the DNA packaged into phage particles may limit applications aimed at targeting nucleic acids in mammalian cells.

Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations

PubMed Central

Kamitaki, Nolan; Mitchell, Jana; Avior, Yishai; Mello, Curtis; Kashin, Seva; Mekhoubad, Shila; Ilic, Dusko; Charlton, Maura; Saphier, Genevieve; Handsaker, Robert E.; Genovese, Giulio; Bar, Shiran; Benvenisty, Nissim; McCarroll, Steven A.; Eggan, Kevin

2017-01-01

Human pluripotent stem cells (hPSCs) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with acquisition of large copy number variants (CNVs) that provide mutant cells with a growth advantage in culture1–3. However, the nature, extent, and functional impact of other acquired genome sequence mutations in cultured hPSCs is not known. Here, we sequenced the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hESC) lines, including 26 lines prepared for potential clinical use4. We then applied computational strategies for identifying mutations present in a subset of cells5. Though such mosaic mutations were generally rare, we identified five unrelated hESC lines that carried six mutations in the TP53 gene that encodes the tumor suppressor P53. Notably, the TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We used droplet digital PCR to demonstrate that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that P53 mutation confers selective advantage. When we then mined published RNA sequencing data from 117 hPSC lines, we observed another nine TP53 mutations, all resulting in coding changes in the DNA binding domain of P53. Strikingly, in three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from loss of heterozygosity at the TP53 locus. As the acquisition and favored expansion of cancer-associated mutations in hPSCs may go unnoticed during most applications, we suggest that careful genetic characterization of hPSCs and their differentiated derivatives should be carried out prior to clinical use. PMID:28445466

Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics.

PubMed

Yang, Andrew C; du Bois, Haley; Olsson, Niclas; Gate, David; Lehallier, Benoit; Berdnik, Daniela; Brewer, Kyle D; Bertozzi, Carolyn R; Elias, Joshua E; Wyss-Coray, Tony

2018-06-13

Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyr Y43G ) and a phenylalanyl ( MmPhe T413G ) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyr Y43G and MmPhe T413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyr Y43G and MmPhe T413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.

N-ω-chloroacetyl-L-ornithine has in-vitro activity against cancer cell lines and in-vivo activity against ascitic and solid tumors.

PubMed

Vargas-Ramírez, Alba L; Medina-Enríquez, Miriam M; Cordero-Rodríguez, Neira I; Ruiz-Cuello, Tatiana; Aguilar-Faisal, Leopoldo; Trujillo-Ferrara, José G; Alcántara-Farfán, Verónica; Rodríguez-Páez, Lorena

2016-07-01

N-ω-chloroacetyl-L-ornithine (NCAO) is an ornithine decarboxylase (ODC) inhibitor that is known to exert cytotoxic and antiproliferative effects on three neoplastic human cancer cell lines (HeLa, MCF-7, and HepG2). Here, we show that NCAO has antiproliferative activity in 13 cancer cell lines, of diverse tissue origin from human and mice, and in a mouse cancer model in vivo. All cell lines were sensitive to NCAO after 72 h of treatment (the EC50 ranged from 1 to 50.6 µmol/l). The Ca Ski cell line was the most sensitive (EC50=1.18±0.07 µmol/l) and MDA-MB-231 was the least sensitive (EC50=50.6±0.3 µmol/l). This ODC inhibitor showed selectivity for cancer cells, exerting almost no cytotoxic effect on the normal Vero cell line (EC50>1000 µmol/l). NCAO induced apoptosis and inhibited tumor cell migration in vitro. Furthermore, in vivo, this compound (at 50 and 100 mg/kg, daily intraperitoneal injection for 7 days) exerted potent antitumor activity against both solid and ascitic tumors in a mouse model using the myeloma (Ag8) cell line. At these same two doses, the toxicological evaluation showed that NCAO has no obvious systemic toxicity. The current results suggest that the antitumor activity is exerted by apoptosis related not only to a local but also a systemic cytotoxic effect exerted by NCAO on tumor cells. The applications for NCAO as an antitumor agent may be extensive; however, further studies are needed to ascertain the antitumor activity on other types of tumor in vivo and to determine the precise molecular mechanism of its activity.

CD30 downregulation, MMAE resistance, and MDR1 upregulation are all associated with resistance to brentuximab vedotin

PubMed Central

Chen, Robert; Hou, Jessie; Newman, Edward; Kim, Young; Donohue, Cecile; Liu, Xueli; Thomas, Sandra H.; Forman, Stephen J.; Kane, Susan E.

2015-01-01

Brentuximab vedotin (BV) is an antibody-drug conjugate that specifically delivers the potent cytotoxic drug MMAE to CD30-positive cells. BV is FDA-approved for treatment of relapsed/refractory Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL); however, many patients do not achieve complete remission and develop BV resistant disease. We selected for BV-resistant HL (L428) and ALCL (Karpas-299) cell lines using either constant (ALCL) or pulsatile (HL) exposure to BV. We confirmed drug resistance by MTS assay, and analyzed CD30 expression in resistant cells by flow cytometry, qRT-PCR, and Western blotting. We also measured drug exporter expression, MMAE resistance, and intracellular MMAE concentrations in BV-resistant cells. Additionally, tissue biopsy samples from 10 HL and 5 ALCL patients who had relapsed or progressed after BV treatment were analyzed by immunohistocytochemistry for CD30 expression. The resistant ALCL cell line, but not the HL cell line, demonstrated downregulated CD30 expression compared to the parental cell line. In contrast, the HL cell line, but not the ALCL cell line, exhibited MMAE resistance and increased expression of the MDR1 drug exporter compared to the parental line. For both HL and ALCL, samples from patients relapsed/resistant on BV persistently expressed CD30 by immunohistocytochemistry. One HL patient sample expressed MDR1 by immunohistocytochemistry. Although loss of CD30 expression is a possible mode of BV resistance in ALCL in vitro models, this has not been confirmed in patients. MMAE resistance and MDR1 expression are possible modes of BV resistance for HL both in vitro and in patients. PMID:25840583

Infrasound sensitizes human glioblastoma cells to cisplatin-induced apoptosis.

PubMed

Rachlin, Kenneth; Moore, Dan H; Yount, Garret

2013-11-01

The development of nontoxic agents that can selectively enhance the cytotoxicity of chemotherapy is an important aim in oncology. This study evaluates the ability of infrasound exposure to sensitize glioblastoma cells to cisplatin-induced apoptosis. The infrasound was delivered using a device designed to replicate the unique infrasound emissions measured during external Qigong treatments. Human glioblastoma cell lines harboring wild-type p53 (U87) or mutant p53 (U251, SF210, and SF188) were treated in culture with cisplatin, infrasound emissions, or the combination of the 2 agents. Induction of apoptosis was quantified after 24 hours by flow cytometry following annexin V/propidium iodide staining. Infrasound emissions alone, delivered at moderate levels (~10 mPa) with dynamic frequency content (7-13 Hz), did not induce apoptosis, yet combining infrasound with cisplatin augmented the induction of apoptosis by cisplatin in all the 4 cell lines (P < .05). Increased cellular uptake of the fluorophore calcein associated with infrasound exposure was quantified by fluorescence microscopy as well as flow cytometry, demonstrating increased cell membrane permeability. The 4 cell lines differed in the degree to which infrasound exposure increased calcein uptake, and these differences were predictive of the extent to which infrasound enhanced cisplatin-induced apoptosis. When exposed to specific frequencies, membrane permeabilization also appeared to be differentially responsive for each cell line, suggesting the potential for selective targeting of tissue types using isolated infrasonic frequencies. Additionally, the pressure amplitudes used in this study were several orders of magnitude less than those used in similar studies involving ultrasound and shock waves. The results of this study provide support for using infrasound to enhance the chemotherapeutic effects of cisplatin in a clinical setting.

Mechanisms of allele-selective down-regulation of HLA class I in Burkitt's lymphoma.

PubMed

Imreh, M P; Zhang, Q J; de Campos-Lima, P O; Imreh, S; Krausa, P; Browning, M; Klein, G; Masucci, M G

1995-07-04

Burkitt lymphomas (BL) that arise in HLA-AII-positive individuals are characterized by selective loss/down-regulation of the HLA AII polypeptide. We have investigated the molecular basis of such down-regulation by comparing 5 pairs of BL lines and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) derived from the normal B cells of the same individuals. The presence of apparently intact HLA AII genes was confirmed in all 5 BL/LCL pairs by polymerase chain reaction (PCR) typing and by Southern-blot hybridization with HLA A locus-specific probes. Northern-blot analysis with locus- and allele-specific probes revealed a significantly lower expression or absence of AII-specific mRNA in all 5 BL lines compared to the corresponding LCLs. Up-regulation of AII-specific mRNA was achieved by IFN alpha treatment of 2 BL lines with low HLA AII expression (BL-28 and BL-72) while the treatment had no effect in 3 BL lines (WWI-BL, WW2-BL and BL41) that did not express the endogenous gene. HLA AII expression was restored by transfection of the gene in WWI-BL whereas transfectants of BL-41 remained AII-negative. An HLA-AII-promoter-driven chloramphenicol acetyl transferase reporter gene (pAIICAT) was active in WWI-BL but not in BL-41. HLA-AII was expressed in hybrids of BL-41 with an AII-positive LCL, while expression of the endogenous HLA AII gene could not be restored by fusion of BL-41 with an AII-negative LCL, although an adequate set of transcription factors was present in the hybrid. Our results suggest that genetic defects and lack of transcription factors may contribute to the selective down-regulation of HLA AII in BL cells.

In vitro effects of dental cements on hard and soft tissues associated with dental implants.

PubMed

Rodriguez, Lucas C; Saba, Juliana N; Chung, Kwok-Hung; Wadhwani, Chandur; Rodrigues, Danieli C

2017-07-01

Dental cements for cement-retained restorations are often chosen based on clinician preference for the product's material properties, mixing process, delivery mechanism, or viscosity. The composition of dental cement may play a significant role in the proliferation or inhibition of different bacterial strains associated with peri-implant disease, and the effect of dental cements on host cellular proliferation may provide further insight into appropriate cement material selection. The purpose of this in vitro study was to investigate the cellular host response of bone cells (osteoblasts) and soft tissue cells (gingival fibroblasts) to dental cements. Zinc oxide (eugenol and noneugenol), zinc phosphate, and acrylic resin cements were molded into pellets and directly applied to confluent preosteoblast (cell line MC3T3 E1) or gingival fibroblast cell cultures (cell line HGF) to determine cellular viability after exposure. Controls were defined as confluent cell cultures with no cement exposure. Direct contact cell culture testing was conducted following International Organization for Standardization 10993 methods, and all experiments were performed in triplicate. To compare either the MC3T3 E1 cell line, or the HGF cell line alone, a 1-way ANOVA test with multiple comparisons was used (α=.05). To compare the MC3T3 E1 cell line results and the HGF cell line results, a 2-way ANOVA test with multiple comparisons was used (α=.05). The results of this study illustrated that while both bone and soft tissue cell lines were vulnerable to the dental cement test materials, the soft tissue cell line (human gingival fibroblasts) was more susceptible to reduced cellular viability after exposure. The HGF cell line was much more sensitive to cement exposure. Here, the acrylic resin, zinc oxide (eugenol), and zinc phosphate cements significantly reduced cellular viability after exposure with respect to HGF cells only. Within the limitation of this in vitro cellular study, the results indicated that cell response to various implant cements varied significantly, with osteoblast proliferation much less affected than gingival fibroblast cells. Furthermore, the zinc oxide noneugenol dental cement appeared to affect the cell lines significantly less than the other test cements. Copyright © 2016 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.

Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines.

PubMed

Horsch, Marion; Aguilar-Pimentel, Juan Antonio; Bönisch, Clemens; Côme, Christophe; Kolster-Fog, Cathrine; Jensen, Klaus T; Lund, Anders H; Lee, Icksoo; Grossman, Lawrence I; Sinkler, Christopher; Hüttemann, Maik; Bohn, Erwin; Fuchs, Helmut; Ollert, Markus; Gailus-Durner, Valérie; de Angelis, Martin Hrabĕ; Beckers, Johannes

2015-01-01

We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype-envirotype interactions for other diseases.

Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines

PubMed Central

Bönisch, Clemens; Côme, Christophe; Kolster-Fog, Cathrine; Jensen, Klaus T.; Lund, Anders H.; Lee, Icksoo; Grossman, Lawrence I.; Sinkler, Christopher; Hüttemann, Maik; Bohn, Erwin; Fuchs, Helmut; Ollert, Markus; Gailus-Durner, Valérie; Hrabĕ de Angelis, Martin; Beckers, Johannes

2015-01-01

We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype – envirotype interactions for other diseases. PMID:26263558

NFATC3-PLA2G15 Fusion Transcript Identified by RNA Sequencing Promotes Tumor Invasion and Proliferation in Colorectal Cancer Cell Lines.

PubMed

Jang, Jee-Eun; Kim, Hwang-Phill; Han, Sae-Won; Jang, Hoon; Lee, Si-Hyun; Song, Sang-Hyun; Bang, Duhee; Kim, Tae-You

2018-06-14

This study was designed to identify novel fusion transcripts (FTs) and their functional significance in colorectal cancer lines. We performed paired-end RNA sequencing of 28 colorectal cancer (CRC) cell lines. FT candidates were identified using TopHat-fusion, ChimeraScan, and FusionMap tools and further experimental validation was conducted through reverse transcription-polymerase chain reaction and Sanger sequencing. FT was depleted in human CRC line and the effects on cell proliferation, cell migration, and cell invasion were analyzed. 1,380 FT candidates were detected through bioinformatics filtering. We selected 6 candidate FTs, including 4 inter-chromosomal and 2 intra-chromosomal FTs and each FT was found in at least 1 of the 28 cell lines. Moreover, when we tested 19 pairs of CRC tumor and adjacent normal tissue samples, NFATC3-PLA2G15 FT was found in 2. Knockdown of NFATC3-PLA2G15 using siRNA reduced mRNA expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, twist, and fibronectin and increased mesenchymal-epithelial transition markers of E-cadherin, claudin-1, and FOXC2 in colo-320 cell line harboring NFATC3-PLA2G15 FT. The NFATC3-PLA2G15 knockdown also inhibited invasion, colony formation capacity, and cell proliferation. These results suggest that that NFATC3-PLA2G15 FTs may contribute to tumor progression by enhancing invasion by EMT and proliferation.

The Tick Cell Biobank: A global resource for in vitro research on ticks, other arthropods and the pathogens they transmit.

PubMed

Bell-Sakyi, Lesley; Darby, Alistair; Baylis, Matthew; Makepeace, Benjamin L

2018-05-31

Tick cell lines are increasingly used in many fields of tick and tick-borne disease research. The Tick Cell Biobank was established in 2009 to facilitate the development and uptake of these unique and valuable resources. As well as serving as a repository for existing and new ixodid and argasid tick cell lines, the Tick Cell Biobank supplies cell lines and training in their maintenance to scientists worldwide and generates novel cultures from tick species not already represented in the collection. Now part of the Institute of Infection and Global Health at the University of Liverpool, the Tick Cell Biobank has embarked on a new phase of activity particularly targeted at research on problems caused by ticks, other arthropods and the diseases they transmit in less-developed, lower- and middle-income countries. We are carrying out genotypic and phenotypic characterisation of selected cell lines derived from tropical tick species. We continue to expand the culture collection, currently comprising 63 cell lines derived from 18 ixodid and argasid tick species and one each from the sand fly Lutzomyia longipalpis and the biting midge Culicoides sonorensis, and are actively engaging with collaborators to obtain starting material for primary cell cultures from other midge species, mites, tsetse flies and bees. Outposts of the Tick Cell Biobank will be set up in Malaysia, Kenya and Brazil to facilitate uptake and exploitation of cell lines and associated training by scientists in these and neighbouring countries. Thus the Tick Cell Biobank will continue to underpin many areas of global research into biology and control of ticks, other arthropods and vector-borne viral, bacterial and protozoan pathogens. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

Anti-leukaemic activity of the TYK2 selective inhibitor NDI-031301 in T-cell acute lymphoblastic leukaemia.

PubMed

Akahane, Koshi; Li, Zhaodong; Etchin, Julia; Berezovskaya, Alla; Gjini, Evisa; Masse, Craig E; Miao, Wenyan; Rocnik, Jennifer; Kapeller, Rosana; Greenwood, Jeremy R; Tiv, Hong; Sanda, Takaomi; Weinstock, David M; Look, A Thomas

2017-04-01

Activation of tyrosine kinase 2 (TYK2) contributes to the aberrant survival of T-cell acute lymphoblastic leukaemia (T-ALL) cells. Here we demonstrate the anti-leukaemic activity of a novel TYK2 inhibitor, NDI-031301. NDI-031301 is a potent and selective inhibitor of TYK2 that induced robust growth inhibition of human T-ALL cell lines. NDI-031301 treatment of human T-ALL cell lines resulted in induction of apoptosis that was not observed with the JAK inhibitors tofacitinib and baricitinib. Further investigation revealed that NDI-031301 treatment uniquely leads to activation of three mitogen-activated protein kinases (MAPKs), resulting in phosphorylation of ERK, SAPK/JNK and p38 MAPK coincident with PARP cleavage. Activation of p38 MAPK occurred within 1 h of NDI-031301 treatment and was responsible for NDI-031301-induced T-ALL cell death, as pharmacological inhibition of p38 MAPK partially rescued apoptosis induced by TYK2 inhibitor. Finally, daily oral administration of NDI-031301 at 100 mg/kg bid to immunodeficient mice engrafted with KOPT-K1 T-ALL cells was well tolerated, and led to decreased tumour burden and a significant survival benefit. These results support selective inhibition of TYK2 as a promising potential therapeutic strategy for T-ALL. © 2017 John Wiley & Sons Ltd.

Synthesis, characterization, X-ray crystal structures of heterocyclic Schiff base compounds and in vitro cholinesterase inhibition and anticancer activity

NASA Astrophysics Data System (ADS)

Arafath, Md. Azharul; Adam, Farook; Al-Suede, Fouad Saleih R.; Razali, Mohd R.; Ahamed, Mohamed B. Khadeer; Abdul Majid, Amin Malik Shah; Hassan, Mohd Zaheen; Osman, Hasnah; Abubakar, Saifullah

2017-12-01

Four heterocyclic embedded Schiff base derivatives (1-4) were synthesized and characterized by melting point, elemental analysis, FTIR, 1H, 13C NMR, UV-Visible spectral data. The structures of compounds 1, 2 and 4 were successfully established through single crystal X-ray diffraction analysis. In vitro cholinesterase inhibition assays showed that the cyclized derivative 1 displayed higher BuChE enzyme inhibitory activity with IC50 value of 1.45 ± 0.09 μM. The anti-proliferative efficacies of the compounds were also evaluated using human colorectal HCT 116 and breast MCF-7 adenocarcinoma cell lines. In addition, a human normal endothelial cell line (Ea.hy926) was also tested to assess the safety and selectivity of the compounds towards normal and cancer cells, respectively. Among the compounds tested, compound 2 displayed potent cytotoxic effect (IC50 = 34 μM) against HCT 116 cells with highest selectivity index of 3.1 with respect to the normal endothelial cells. Whereas, compound 4 exhibited significant anti-proliferative effect (IC50 = 21.1 μM) against MCF-7 cells with highest selectivity index of 3.3 with respect to the normal endothelial cells. The docking result of these compounds against hAChE showed potent activities with different binding modes. These compounds could be a promising pharmacological agent to treat cancer and Alzheimer's disease.

A BCWD-resistant line of rainbow trout exhibits higher abundance of IgT+ B cells and heavy chain tau transcripts compared to a susceptible line following challenge with Flavobacterium psychrophilum.

PubMed

Zwollo, Patty; Hennessey, Erin; Moore, Catherine; Marancik, David P; Wiens, Gregory D; Epp, Lidia

2017-09-01

Bacterial Cold Water Disease (BCWD) is a common, chronic disease in rainbow trout, and is caused by the gram-negative bacterium Flavobacterium psychrophilum (Fp). Through selective breeding, the National Center for Cool and Cold Water Aquaculture has generated a genetic line that is highly resistant to Fp challenge, designated ARS-Fp-R (or R-line), as well as a susceptible "control" line, ARS-Fp-S (S-line). In previous studies, resistance to Fp had been shown to correlate with naive animal spleen size, and further, naïve R-line trout had been shown to have a lower abundance of IgM + and IgM ++ cells compared to S-line fish. Here we wished to first determine whether the abundance of IgT + and/or IgT ++ cells differed between the two lines in naïve fish, and if so, how these patterns differed after in vivo challenge with Fp. Fp challenge was by intramuscular injection of live Fp and tissue collections were on days 5, 6, and/or 28 post-challenge, in two independent challenge experiments. Flow cytometric and gene expression analyses revealed that naïve R-line fish had a higher abundance of IgT + B cells in their anterior kidney, spleen, and blood, compared to S line fish. Further, that after Fp challenge, this difference was maintained between the two lines. Lastly, abundance of IgT + B cells and expression of secHCtau correlated with lower Fp pathogen loads in challenged fish. In the anterior kidney, IgM + B cell abundance correlated with increased Fp loads. Together, these results suggest that IgT + B lineage cells may have a protective function in the immune response to Fp. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation of tumor antigen-specific single-chain variable fragments using a chimeric antigen receptor bicistronic retroviral vector in a Mammalian screening protocol.

PubMed

Lipowska-Bhalla, Grazyna; Gilham, David E; Hawkins, Robert E; Rothwell, Dominic G

2013-12-01

The clinical potential of chimeric antigen receptors in adoptive cellular therapy is beginning to be realized with several recent clinical trials targeting CD19 showing promising results in advanced B cell malignancies. This increased efficacy corresponds with improved engineering of the chimeric receptors with the latest-generation receptors eliciting greater signaling and proliferation potential. However, the antigen-binding single-chain variable fragment (scFv) domain of the receptors is critical in determining the activity of the chimeric receptor-expressing T cells, as this determines specificity and affinity to the tumor antigen. In this study, we describe a mammalian T cell line screening protocol employing a 2A-based bicistronic retroviral vector to isolate functional scFvs. This approach involves expression of the scFv library in a chimeric antigen receptor, and is based on selection of clones capable of stimulating CD69 upregulation in a T cell line and has a number of advantages over previously described methods in that the use of a 2A cassette ensures the exclusion of nonexpressing scFvs and the screening using a chimeric receptor in a mammalian T cell line ensures selection in the optimum context for therapeutic use. Proof-of-principle experiments show that the protocol was capable of a 10(5)-fold enrichment of positive clones after three rounds of selection. Furthermore, an antigen-specific clone was successfully isolated from a partially enriched scFv library, confirming the strength of the protocol. This approach has the potential to identify novel scFvs of use in adoptive T cell therapy and, potentially, wider antibody-based applications.

Low incidence of DNA sequence variation in human induced pluripotent stem cells generated by non-integrating plasmid expression

PubMed Central

Cheng, Linzhao; Hansen, Nancy F.; Zhao, Ling; Du, Yutao; Zou, Chunlin; Donovan, Frank X.; Chou, Bin-Kuan; Zhou, Guangyu; Li, Shijie; Dowey, Sarah N.; Ye, Zhaohui; Chandrasekharappa, Settara C.; Yang, Huanming; Mullikin, James C.; Liu, P. Paul

2012-01-01

Summary The utility of induced pluripotent stem cells (iPSCs) as models to study diseases and as sources for cell therapy depends on the integrity of their genomes. Despite recent publications of DNA sequence variations in the iPSCs, the true scope of such changes for the entire genome is not clear. Here we report the whole-genome sequencing of three human iPSC lines derived from two cell types of an adult donor by episomal vectors. The vector sequence was undetectable in the deeply sequenced iPSC lines. We identified 1058–1808 heterozygous single nucleotide variants (SNVs), but no copy number variants, in each iPSC line. Six to twelve of these SNVs were within coding regions in each iPSC line, but ~50% of them are synonymous changes and the remaining are not selectively enriched for known genes associated with cancers. Our data thus suggest that episome-mediated reprogramming is not inherently mutagenic during integration-free iPSC induction. PMID:22385660

Oxaphosphinanes: new therapeutic perspectives for glioblastoma.

PubMed

Clarion, Ludovic; Jacquard, Carine; Sainte-Catherine, Odile; Loiseau, Séverine; Filippini, Damien; Hirlemann, Marie-Hélène; Volle, Jean-Noël; Virieux, David; Lecouvey, Marc; Pirat, Jean-Luc; Bakalara, Norbert

2012-03-08

This paper reports the design and the synthesis of a new family of compounds, the phostines, belonging to the [1,2]oxaphosphinane family. Twenty-six compounds have been screened for their antiproliferative activity against a large panel of NCI cancer cell lines. Because of its easy synthesis and low EC(50) value (500 nM against the C6 rat glioma cell line), compound 3.1a was selected for further biological study. Moreover, the specific biological effect of 3.1a on the glioblastoma phylogenetic cluster from the NCI is dependent on its stereochemistry. Within that cluster, 3.1a has a higher antiproliferative activity than Temozolomide and is more potent than paclitaxel for the SF295 and SNB75 cell lines. In constrast with paclitaxel and vincristine, 3.1a is devoid of astrocyte toxicity. The original activity spectrum of 3.1a on the NCI cancer cell line panel allows the development of this family for use in association with existing drugs, opening new therapeutic perspectives.

Fibroblast growth factor 2 restrains Ras-driven proliferation of malignant cells by triggering RhoA-mediated senescence.

PubMed

Costa, Erico T; Forti, Fábio L; Matos, Tatiana G F; Dermargos, Alexandre; Nakano, Fábio; Salotti, Jacqueline; Rocha, Kátia M; Asprino, Paula F; Yoshihara, Celina K; Koga, Marianna M; Armelin, Hugo A

2008-08-01

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant Y1 adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated beta-galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Y1 adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either Y1 or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Ras-dependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RhoA-GTP. Surprisingly, attempts to select FGF2-resistant cells from the Y1 and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Ras-dependent malignant cells could rarely overcome.

Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stark, M.; Naiman, T.; Canaani, D.

1989-08-15

In a previous work, an immortal xeroderma pigmentosum cell line belonging to complementation group C was complemented to a UV-resistant phenotype by transfection with a human cDNA clone library. We now report that the primary transformants selected for UV-resistance also acquired normal levels of DNA repair. This was assessed both by measurement of UV-induced ({sup 3}H)thymidine incorporation and by equilibrium sedimentation analysis of repair-DNA synthesis. Therefore, the transduced DNA element which confers normal UV-resistance also corrects the excision repair defect of the xeroderma pigmentosum group C cell line.

Epigenetic inactivation of VGF associated with Urothelial Cell Carcinoma and its potential as a non-invasive biomarker using urine.

PubMed

Hayashi, Masamichi; Bernert, Heike; Kagohara, Luciane Tsukamoto; Maldonado, Leonel; Brait, Mariana; Schoenberg, Mark; Bivalacqua, Trinity; Netto, George J; Koch, Wayne; Sidransky, David; Hoque, Mohammad O

2014-05-30

To identify new epigenetic markers and further characterize Urothelial Cell Carcinoma (UCC), we tested the promoter methylation (PM) status of 19 genes previously identified as cancer specific methylated genes in other solid tumors. We used bisulfite sequencing, methylation specific PCR and quantitative methylation specific PCR (QMSP) to test the PM status of 19 genes in urothelial cancer cell lines. Among the 19 genes tested, VGF was found to be completely methylated in several UCC cell lines. VGF QMSP analysis showed that methylation values of almost all the primary 19 UCC tissues were higher than the paired normal tissues (P=0.009). In another cohort, 12/35 (34.3%) of low grade UCC cases displayed VGF methylation. As a biomarker for non-invasive detection of UCC, VGF showed a significantly higher frequency of methylation in urine from UCC cases (8/20) compared to controls (1/20) (P=0.020). After treatment of cell lines with 5-Aza-2'-deoxycytidine, VGF was robustly re-expressed. Forced expression of VGF in bladder cancer cell lines inhibited cell growth. Selection of candidates from genome-wide screening approach in other solid tumors successfully identified UCC specific methylated genes.

Peptidomic analysis of human cell lines

PubMed Central

Gelman, Julia S.; Sironi, Juan; Castro, Leandro M.; Ferro, Emer S.; Fricker, Lloyd D.

2011-01-01

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEK293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the P1 position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells. PMID:21204522

In vitro antiplasmodial and cytotoxic activities of sesquiterpene lactones from Vernonia fimbrillifera Less. (Asteraceae).

PubMed

Bordignon, Annélise; Frédérich, Michel; Ledoux, Allison; Campos, Pierre-Eric; Clerc, Patricia; Hermann, Thomas; Quetin-Leclercq, Joëlle; Cieckiewicz, Ewa

2018-06-01

Due to the in vitro antiplasmodial activity of leaf extracts from Vernonia fimbrillifera Less. (Asteraceae), a bioactivity-guided fractionation was carried out. Three sesquiterpene lactones were isolated, namely 8-(4'-hydroxymethacrylate)-dehydromelitensin (1), onopordopicrin (2) and 8α-[4'-hydroxymethacryloyloxy]-4-epi-sonchucarpolide (3). Their structures were elucidated by spectroscopic methods (1D and 2D NMR and MS analyses) and by comparison with published data. The isolated compounds exhibited antiplasmodial activity with IC 50 values ≤ 5 μg/mL. Cytotoxicity of the compounds against a human cancer cell line (HeLa) and a mouse lung epithelial cell line (MLE12) was assessed to determine selectivity. Compound 3 displayed promising selective antiplasmodial activity (SI > 10).

Understanding the Role of TSC1/2 in Cerebellar Purkinje Neurons

DTIC Science & Technology

2017-09-01

patient-specific iPSC lines and rescue the disease phenotypes in patient specific neurons in vitro. We will employ CRISPR -Caspase 9 (Cas9) genome...development from human stem cells. For Aim 2. We at BCH have successfully generated CRISPR -cas9-mediated correction of TSC2- microdeletion in TSC...patient (TSC2+/-) derived hiPSC line (Figure 2). We used puromycin selection for isolation of the CRISPR -cas9 and ssODN transfected cells, and with

Production of coagulation factor VII in human cell lines Sk-Hep-1 and HKB-11.

PubMed

Corrêa de Freitas, Marcela Cristina; Bomfim, Aline de Sousa; Mizukami, Amanda; Picanço-Castro, Virgínia; Swiech, Kamilla; Covas, Dimas Tadeu

2017-09-01

Recombinant factor VII (rFVII) is the main therapeutic choice for hemophilia patients who have developed inhibitory antibodies against conventional treatments (FVIII and FIX). Because of the post-translational modifications, rFVII needs to be produced in mammalian cell lines. In this study, for the first time, we have shown efficient rFVII production in HepG2, Sk-Hep-1, and HKB-11 cell lines. Experiments in static conditions for a period of 96 h showed that HepG2-FVII produced the highest amounts of rhFVII, with an average of 1843 ng/mL. Sk-hep-1-FVII cells reached a maximum protein production of 1432 ng/mL and HKB-11-FVII cells reached 1468 ng/mL. Sk-Hep-1-rFVII and HKB-11-rFVII were selected for the first step of scale-up. Over 10 days of spinner flask culture, HKB-11 and SK-Hep-1 cells showed a cumulative production of rFVII of 152 μg and 202.6 μg in 50 mL, respectively. Thus, these human cell lines can be used for an efficient production of recombinant FVII. With more investment in basic research, human cell lines can be optimized for the commercial production of different bio therapeutic proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Methotrexate induces high level of apoptosis in canine lymphoma/leukemia cell lines.

PubMed

Pawlak, Aleksandra; Kutkowska, Justyna; Obmińska-Mrukowicz, Bożena; Rapak, Andrzej

2017-10-01

Methotrexate is an antimetabolite used in the treatment of cancer and non-malignant diseases including rheumatoid arthritis, psoriasis and graft vs. host disease. Combination therapy with methotrexate was successful in the treatment of canine lymphoma, mammary tumor and invasive urinary bladder cancer. Lymphoma, the most common hematopoietic cancer in dogs, and leukemia are sensitive to chemotherapy, which is why methotrexate may be an important treatment option for these diseases. Although methotrexate is already used in veterinary oncology its effects on canine cancer cells has not been tested. The aim of the study was to evaluate for the first time methotrexate concentration-dependent cytotoxicity and its capability of inducing apoptosis in selected canine lymphoma/leukemia cell lines: CLBL-1, GL-1 and CL-1 as a first step before the in vitro development of new therapeutic options with the use of methotrexate. Methotrexate exhibited concentration-dependent inhibitory effect on proliferation of all the examined cell lines with different degree of apoptosis induction. The most methotrexate sensitive cells belonged to CL-1 cell line derived from T cell neoplasia and previously characterized by high resistance to the majority of anticancer drugs used in the therapy of lymphoma/leukemia in dogs. Canine lymphoma and leukemia cell lines are sensitive to methotrexate, and this drug may be useful in effective treatment of canine neoplasms and especially of T-type leukemia/lymphoma. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Novel Polyphenol Conjugate Sensitizes Cisplatin-Resistant Head and Neck Cancer Cells to Cisplatin via Nrf2 Inhibition.

PubMed

Kim, Eun Hye; Jang, Hyejin; Roh, Jong-Lyel

2016-11-01

Many cancer cells show acquired resistance to chemotherapeutic agents, such as cisplatin. This is a major cause of cancer treatment failure, and novel agents to overcome resistance are thus urgently required. A novel synthetic polyphenol conjugate, (E)-3-(3,5-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (DPP-23), selectively kills tumor cells via the reactive oxygen species (ROS)-mediated unfolded protein response. We investigated the ability of DPP-23 to overcome cisplatin resistance in head and neck cancer (HNC) cells and further clarified its molecular mechanisms of action. Cisplatin-resistant HNC cell lines and their parental and other HNC cell lines were used. The effects of cisplatin and DPP-23 were assessed alone and in combination in HNC and normal cells using cell viability, cell cycle, and cell death assays, by measuring glutathione (GSH), ROS, and protein levels, and via preclinical mouse studies. DPP-23 induced selective cell death in HNC cells, including cisplatin-resistant HNC cells, but spared normal cells, via cellular GSH depletion and ROS accumulation. The effect was blocked by the antioxidant N-acetyl-L-cysteine. DPP-23 activated p53 and its related cell death pathways via a robust accumulation of cellular ROS that involved inhibition of nuclear factor erythroid 2-related factor 2 antioxidant defense mechanisms. Thus, DPP-23 significantly overcame cisplatin resistance in HNC cells in vitro and in vivo As a promising anticancer strategy, ROS generation and subsequent selective cancer cell killing by DPP-23 might help to overcome cisplatin resistance in HNC. Mol Cancer Ther; 15(11); 2620-9. ©2016 AACR. ©2016 American Association for Cancer Research.

Anti-proliferative activity of 2,6-dichloro-9- or 7-(ethoxycarbonylmethyl)-9H- or 7H-purines against several human solid tumour cell lines.

PubMed

Morales, Fátima; Ramírez, Alberto; Conejo-García, Ana; Morata, Cynthia; Marchal, Juan A; Campos, Joaquín M

2014-04-09

As leads we took several benzo-fused seven- and six-membered scaffolds linked to the pyrimidine or purine moieties with notable anti-proliferative activity against human breast, colon and melanoma cancerous cell lines. We then decided to maintain the double-ringed nitrogenous bases and change the other components to the ethyl acetate moiety. This way six purine and two 5-fluorouracil derivatives were obtained and evaluated against the MCF-7, HCT-116, A-375 and G-361 cancer cell lines. Two QSARs are obtained between the anti-proliferative IC₅₀ values for compounds 26-33 and the clog P against the melanoma cell lines A-375 and G-361. Our results show that two of the analogues [ethyl 2-(2,6-dichloro-9H- or 7H-purine-9- or 7-yl)acetates (30 and 33, respectively)] are potent cytotoxic agents against all the tumour cell lines assayed, showing single-digit micromolar IC₅₀ values. This exemplifies the potential of our previously reported purine compounds to qualify as lead structures for medicinal chemistry campaigns, affording simplified analogues easy to synthesize and with a noteworthy bioactivity. The selective activity of 30 and 33 against the melanoma cell line A-375, via apoptosis, supposes a great advantage for a future therapeutic use. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Impact of cell lines included in enterovirus isolation protocol on perception of nonpolio enterovirus species C diversity.

PubMed

Adeniji, Johnson Adekunle; Faleye, Temitope Oluwasegun Cephas

2014-10-01

There has been under-reporting of nonpolio enterovirus species Cs (NPESCs) in Nigeria despite the fact that most isolates recovered from the Nigerian vaccine derived poliovirus serotype 2 (VDPV2) outbreak were recombinants with nonstructural region of NPESC origin. It has been suggested that cell lines included in enterovirus isolation protocols might account for this phenomenon and this study examined this suggestion. Fifteen environmental samples concentrated previously and analysed using L20B and RD cell lines as part of the poliovirus environmental surveillance (ES) program in Nigeria were randomly selected and inoculated into two cell lines (MCF-7 and LLC-MK2). Isolates were identified as enteroviruses and species C members using different RT-PCR assays, culture in L20B cell line and sequencing of partial VP1. Forty-eight (48) isolates were recovered from the 15 samples, 47 (97.9%) of which were enteroviruses. Of the enteroviruses, 32 (68.1%) belonged to enterovirus species C (EC) of which 19 (40.4%) were polioviruses and 13 (27.7%) were NPESC members. All 13 NPESC isolates were recovered on MCF-7. Results of the study show that NPESCs are circulating in Nigeria and their under-reporting was due to the combination of cell lines used for enterovirus isolation in previous reports. Copyright © 2014 Elsevier B.V. All rights reserved.

CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients.

PubMed

Pardanani, A; Lasho, T; Smith, G; Burns, C J; Fantino, E; Tefferi, A

2009-08-01

Somatic mutations in Janus kinase 2 (JAK2), including JAK2V617F, result in dysregulated JAK-signal transducer and activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an ATP-competitive small molecule that potently inhibits JAK1/JAK2 kinases (IC(50)=11 and 18 nM, respectively), with significantly less activity against other kinases, including JAK3 (IC(50)=155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC(50) approximately 1500 nM) or Ba/F3-MPLW515L cells (IC(50)=200 nM), but has considerably less activity against BCR-ABL harboring K562 cells (IC=58 000 nM). Cell lines harboring mutated JAK2 alleles (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was inhibited in HEL cells with an IC(50)=400 nM. Furthermore, CYT387 selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin. Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of MPN harboring mutated JAK2 and MPL alleles.

Traditional Aboriginal Preparation Alters the Chemical Profile of Carica papaya Leaves and Impacts on Cytotoxicity towards Human Squamous Cell Carcinoma.

PubMed

Nguyen, Thao T; Parat, Marie-Odile; Shaw, Paul N; Hewavitharana, Amitha K; Hodson, Mark P

2016-01-01

Carica papaya leaf decoction, an Australian Aboriginal remedy, has been used widely for its healing capabilities against cancer, with numerous anecdotal reports. In this study we investigated its in vitro cytotoxicity on human squamous cell carcinoma cells followed by metabolomic profiling of Carica papaya leaf decoction and leaf juice/brewed leaf juice to determine the effects imparted by the long heating process typical of the Aboriginal remedy preparation. MTT assay results showed that in comparison with the decoction, the leaf juice not only exhibited a stronger cytotoxic effect on SCC25 cancer cells, but also produced a significant cancer-selective effect as shown by tests on non-cancerous human keratinocyte HaCaT cells. Furthermore, evidence from testing brewed leaf juice on these two cell lines suggested that the brewing process markedly reduced the selective effect of Carica papaya leaf on SCC25 cancer cells. To tentatively identify the compounds that contribute to the distinct selective anticancer activity of leaf juice, an untargeted metabolomic approach employing Ultra High Performance Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry followed by multivariate data analysis was applied. Some 90 and 104 peaks in positive and negative mode respectively were selected as discriminatory features from the chemical profile of leaf juice and >1500 putative compound IDs were obtained via database searching. Direct comparison of chromatographic and tandem mass spectral data to available reference compounds confirmed one feature as a match with its proposed authentic standard, namely pheophorbide A. However, despite pheophorbide A exhibiting cytotoxic activity on SCC25 cancer cells, it did not prove to be the compound contributing principally to the selective activity of leaf juice. With promising results suggesting stronger and more selective anticancer effects when compared to the Aboriginal remedy, Carica papaya leaf juice warrants further study to explore its activity on other cancer cell lines, as well as investigation to confirm the identity of compounds contributing to its selective effect, particularly those compounds altered by the long heating process applied during the traditional Aboriginal remedy preparation.

Traditional Aboriginal Preparation Alters the Chemical Profile of Carica papaya Leaves and Impacts on Cytotoxicity towards Human Squamous Cell Carcinoma

PubMed Central

Nguyen, Thao T.; Parat, Marie-Odile; Shaw, Paul N.; Hewavitharana, Amitha K.; Hodson, Mark P.

2016-01-01

Carica papaya leaf decoction, an Australian Aboriginal remedy, has been used widely for its healing capabilities against cancer, with numerous anecdotal reports. In this study we investigated its in vitro cytotoxicity on human squamous cell carcinoma cells followed by metabolomic profiling of Carica papaya leaf decoction and leaf juice/brewed leaf juice to determine the effects imparted by the long heating process typical of the Aboriginal remedy preparation. MTT assay results showed that in comparison with the decoction, the leaf juice not only exhibited a stronger cytotoxic effect on SCC25 cancer cells, but also produced a significant cancer-selective effect as shown by tests on non-cancerous human keratinocyte HaCaT cells. Furthermore, evidence from testing brewed leaf juice on these two cell lines suggested that the brewing process markedly reduced the selective effect of Carica papaya leaf on SCC25 cancer cells. To tentatively identify the compounds that contribute to the distinct selective anticancer activity of leaf juice, an untargeted metabolomic approach employing Ultra High Performance Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry followed by multivariate data analysis was applied. Some 90 and 104 peaks in positive and negative mode respectively were selected as discriminatory features from the chemical profile of leaf juice and >1500 putative compound IDs were obtained via database searching. Direct comparison of chromatographic and tandem mass spectral data to available reference compounds confirmed one feature as a match with its proposed authentic standard, namely pheophorbide A. However, despite pheophorbide A exhibiting cytotoxic activity on SCC25 cancer cells, it did not prove to be the compound contributing principally to the selective activity of leaf juice. With promising results suggesting stronger and more selective anticancer effects when compared to the Aboriginal remedy, Carica papaya leaf juice warrants further study to explore its activity on other cancer cell lines, as well as investigation to confirm the identity of compounds contributing to its selective effect, particularly those compounds altered by the long heating process applied during the traditional Aboriginal remedy preparation. PMID:26829042

Soft fibrin gels promote selection and growth of tumorigenic cells

NASA Astrophysics Data System (ADS)

Liu, Jing; Tan, Youhua; Zhang, Huafeng; Zhang, Yi; Xu, Pingwei; Chen, Junwei; Poh, Yeh-Chuin; Tang, Ke; Wang, Ning; Huang, Bo

2012-08-01

The identification of stem-cell-like cancer cells through conventional methods that depend on stem cell markers is often unreliable. We developed a mechanical method for selecting tumorigenic cells by culturing single cancer cells in fibrin matrices of ~100 Pa in stiffness. When cultured within these gels, primary human cancer cells or single cancer cells from mouse or human cancer cell lines grew within a few days into individual round colonies that resembled embryonic stem cell colonies. Subcutaneous or intravenous injection of 10 or 100 fibrin-cultured cells in syngeneic or severe combined immunodeficiency mice led to the formation of solid tumours at the site of injection or at the distant lung organ much more efficiently than control cancer cells selected using conventional surface marker methods or cultured on conventional rigid dishes or on soft gels. Remarkably, as few as ten such cells were able to survive and form tumours in the lungs of wild-type non-syngeneic mice.

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

PubMed Central

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Deletion of ribosomal protein genes is a common vulnerability in human cancer, especially in concert with TP53 mutations.

PubMed

Ajore, Ram; Raiser, David; McConkey, Marie; Jöud, Magnus; Boidol, Bernd; Mar, Brenton; Saksena, Gordon; Weinstock, David M; Armstrong, Scott; Ellis, Steven R; Ebert, Benjamin L; Nilsson, Björn

2017-04-01

Heterozygous inactivating mutations in ribosomal protein genes (RPGs) are associated with hematopoietic and developmental abnormalities, activation of p53, and altered risk of cancer in humans and model organisms. Here we performed a large-scale analysis of cancer genome data to examine the frequency and selective pressure of RPG lesions across human cancers. We found that hemizygous RPG deletions are common, occurring in about 43% of 10,744 cancer specimens and cell lines. Consistent with p53-dependent negative selection, such lesions are underrepresented in TP53 -intact tumors ( P  ≪ 10 -10 ), and shRNA-mediated knockdown of RPGs activated p53 in TP53 -wild-type cells. In contrast, we did not see negative selection of RPG deletions in TP53 -mutant tumors. RPGs are conserved with respect to homozygous deletions, and shRNA screening data from 174 cell lines demonstrate that further suppression of hemizygously deleted RPGs inhibits cell growth. Our results establish RPG haploinsufficiency as a strikingly common vulnerability of human cancers that associates with TP53 mutations and could be targetable therapeutically. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Assessment of citalopram and escitalopram on neuroblastoma cell lines: Cell toxicity and gene modulation

PubMed Central

Sakka, Laurent; Delétage, Nathalie; Chalus, Maryse; Aissouni, Youssef; Sylvain-Vidal, Valérie; Gobron, Stéphane; Coll, Guillaume

2017-01-01

Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (p<2.26 10−7), -24.1 (p<5.6 10−9) and -17.7 (p<1.2 10−7). CCNE1, AURKA, IGF2, MYCN and ERBB2 were more moderately down-regulated by both molecules. Glioma markers E2F1, DAPK1 and CCND1 were down-regulated. Citalopram displayed more powerful action with broader and distinct spectrum of action than escitalopram. PMID:28467792

Assessment of citalopram and escitalopram on neuroblastoma cell lines. Cell toxicity and gene modulation.

PubMed

Sakka, Laurent; Delétage, Nathalie; Chalus, Maryse; Aissouni, Youssef; Sylvain-Vidal, Valérie; Gobron, Stéphane; Coll, Guillaume

2017-06-27

Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (p<2.26 10-7), -24.1 (p<5.6 10-9) and -17.7 (p<1.2 10-7). CCNE1, AURKA, IGF2, MYCN and ERBB2 were more moderately down-regulated by both molecules. Glioma markers E2F1, DAPK1 and CCND1 were down-regulated. Citalopram displayed more powerful action with broader and distinct spectrum of action than escitalopram.

Tricyclic Benzo[cd]azulenes Selectively Inhibit Activities of Pim Kinases and Restrict Growth of Epstein-Barr Virus-Transformed Cells

PubMed Central

Santio, Niina M.; Arnaudova, Ralica; Eerola, Sini K.; Rainio, Eeva-Marja; Aumüller, Ingo B.; Yli-Kauhaluoma, Jari; Koskinen, Päivi J.

2013-01-01

Oncogenic Pim family kinases are often overexpressed in human hematopoietic malignancies as well as in solid tumours. These kinases contribute to tumorigenesis by promoting cell survival and by enhancing resistance against chemotherapy and radiation therapy. Furthermore, we have recently shown that they increase the metastatic potential of adherent cancer cells. Here we describe identification of tricyclic benzo[cd]azulenes and their derivatives as effective and selective inhibitors of Pim kinases. These compounds inhibit Pim autophosphorylation and abrogate the anti-apoptotic effects of Pim kinases. They also reduce cancer cell motility and suppress proliferation of lymphoblastoid cell lines infected and immortalized by the Epstein-Barr virus. Thus, these novel Pim-selective inhibitors provide promising compounds for both research and therapeutic purposes. PMID:23405147

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K., E-mail: tfrey@gsu.edu

Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drugmore » selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.« less

Study designs to investigate Nox1 acceleration of neoplastic progression in immortalized human epithelial cells by selection of differentiation resistant cells.

PubMed

Sattayakhom, Apsorn; Chunglok, Warangkana; Ittarat, Wanida; Chamulitrat, Walee

2014-01-01

To investigate the role of NADPH oxidase homolog Nox1 at an early step of cell transformation, we utilized human gingival mucosal keratinocytes immortalized by E6/E7 of human papillomavirus (HPV) type 16 (GM16) to generate progenitor cell lines either by chronic ethanol exposure or overexpression with Nox1. Among several cobblestone epithelial cell lines obtained, two distinctive spindle cell lines - FIB and NuB1 cells were more progressively transformed exhibiting tubulogenesis and anchorage-independent growth associated with increased invasiveness. These spindle cells acquired molecular markers of epithelial mesenchymal transition (EMT) including mesenchymal vimentin and simple cytokeratins (CK) 8 and 18 as well as myogenic alpha-smooth muscle actin and caldesmon. By overexpression and knockdown experiments, we showed that Nox1 on a post-translational level regulated the stability of CK18 in an ROS-, phosphorylation- and PKCepilon-dependent manner. PKCepilon may thus be used as a therapeutic target for EMT inhibition. Taken together, Nox1 accelerates neoplastic progression by regulating structural intermediate filaments leading to EMT of immortalized human gingival epithelial cells.

Increased expression of the PI3K catalytic subunit p110δ underlies elevated S6 phosphorylation and protein synthesis in an individual with autism from a multiplex family.

PubMed

Poopal, Ashwini C; Schroeder, Lindsay M; Horn, Paul S; Bassell, Gary J; Gross, Christina

2016-01-01

Dysfunctions in the PI3K/mTOR pathway have gained a lot of attention in autism research. This was initially based on the discovery of several monogenic autism spectrum disorders with mutations or defects in PI3K/mTOR signaling components. Recent genetic studies corroborate that defective PI3K/mTOR signaling might be a shared pathomechanism in autism disorders of so far unknown etiology, but functional molecular analyses in human cells are rare. The goals of this study were to perform a functional screen of cell lines from patients with idiopathic autism for defects in PI3K/mTOR signaling, to test if further functional analyses are suitable to detect underlying molecular mechanisms, and to evaluate this approach as a biomarker tool to identify therapeutic targets. We performed phospho-S6- and S6-specific ELISA experiments on 21 lymphoblastoid cell lines from the AGRE collection and on 37 lymphoblastoid cell lines from the Simons Simplex Collection and their healthy siblings. Cell lines from one individual with increased S6 phosphorylation and his multiplex family were analyzed in further detail to identify upstream defects in PI3K signaling associated with autism diagnosis. We detected significantly increased S6 phosphorylation in 3 of the 21 lymphoblastoid cell lines from AGRE compared to a healthy control and in 1 of the 37 lymphoblastoid cell lines from the Simons Simplex Collection compared to the healthy sibling. Further analysis of cells from one individual with elevated S6 phosphorylation showed increased expression of the PI3K catalytic subunit p110δ, which was also observed in lymphoblastoid cells from other autistic siblings but not unaffected members in his multiplex family. The p110δ-selective inhibitor IC87114 reduced elevated S6 phosphorylation and protein synthesis in this cell line. Our results suggest that functional analysis of PI3K/mTOR signaling is a biomarker tool to identify disease-associated molecular defects that could serve as therapeutic targets in autism. Using this approach, we discovered impaired signaling and protein synthesis through the PI3K catalytic subunit p110δ as an underlying molecular defect and potential treatment target in select autism spectrum disorders. Increased p110δ activity was recently associated with schizophrenia, and our results suggest that p110δ may also be implicated in autism.

Studies on the secondary metabolites of a Pseudoalteromonas sp. isolated from sediments collected at the northeastern coast of Brazil.

PubMed

Arthaud, Isabelle D B; Rodrigues, Felipe A R; Jimenez, Paula C; Montenegro, Raquel C; Angelim, Alysson L; Maciel, Vânia M M; Silveira, Edilberto R; Freitas, Hozana P S; Sousa, Thiciana S; Pessoa, Otília D L; Lotufo, Tito M C; Costa-Lotufo, Letícia V

2012-02-01

Continuing search for anticancer compounds from the marine environment, we have studied microorganisms that inhabit intertidal sediments of the northeastern Brazilian coast. Of the 32 strains isolated, 13 were selected for biological evaluation of their crude extracts. The acetate extract obtained from a Gram-negative bacterium was strongly active against cancer cell lines with IC(50) values that ranged from 0.04 (HL60 leukemia cells) to 0.26 μg/ml (MDA MB-435 melanoma cells). The bacterium was identified as a Pseudoalteromonas sp. based on 16S rRNA gene sequencing. A bioassay-guided fractionation of the active extract led to the isolation of prodigiosin, a well-known tripyrrole red pigment with immunosuppressive and anticancer activities. Further experiments with ErbB-2 overexpressing cell lines, including HB4a-C3.6 (moderate overexpression), HB4a-C5.2 (high overexpression), and the parental HB4a cell line, were performed. Prodigiosin was moderately active toward HB4a cells with an IC(50) of 4.6 μg/ml, while it was 115 and 18 times more active toward HB4a-C3.6 cells (IC(50) of 0.04 μg/ml) and HB4a-C5.2 (IC(50) of 0.26 μg/ml) cells, respectively. These data suggest that, in spite of its previously described apoptosis-inducing properties, prodigiosin can selectively recognize cells overexpressing ErbB-2, which could be highly appealing in human breast cancer therapy. Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.

Novel method for in vitro depletion of T cells by monoclonal antibody-targeted photosensitization.

PubMed

Berki, T; Németh, P

1998-02-01

An immunotargeting method (called photo-immunotargeting) has been developed for selective in vitro cell destruction. The procedure combines the photosensitizing (toxic) effect of light-induced dye-molecules, e.g., hematoporphyrin (HP) and the selective binding ability of monoclonal antibodies (mAb) to cell surface molecules. The photosensitizer HP molecules were covalently attached to monoclonal antibodies (a-Thy-1) recognizing an antigen on the surface of T lymphocytes, and used for T cell destruction. To increase the selectivity of the conventional targeting methods, a physical activation step (local light irradiation) as a second degree of specificity was employed. The HP in conjugated form was sufficient to induce T cell (thymocytes, EL-4 cell line) death after irradiation at 400 nm, at tenfold lower concentration compared to the photosensitizing effect of unbound HP. The selective killing of T lymphocytes (bearing the Thy-1 antigen) in a mixed cell population was demonstrated after a treatment with the phototoxic conjugate and light irradiation. This method can be useful for selective destruction of one population (target cell) in an in vitro heterogeneous cell mixture, e.g., in bone marrow transplants for T cell depletion to avoid graft vs. host reaction.

A characterization of the ZFL cell line and primary hepatocytes as in vitro liver cell models for the zebrafish (Danio rerio).

PubMed

Eide, Marta; Rusten, Marte; Male, Rune; Jensen, Knut Helge Midtbø; Goksøyr, Anders

2014-02-01

The zebrafish (Danio rerio) is a widely used model species in biomedical research. The ZFL cell line, established from zebrafish liver, and freshly isolated primary hepatocytes from zebrafish have been used in several toxicological studies. However, no previous report has compared and characterized these two systems at the level of gene expression. The aim of this study was to evaluate the ZFL cell line in comparison to primary hepatocytes as in vitro models for studying effects of environmental contaminants in zebrafish liver. Using quantitative real-time PCR, the basal level and transcriptional induction potential of key genes involved in toxic responses in the ZFL cell line, primary hepatocytes and whole liver from zebrafish were compared. The study showed that the ZFL cells have lower levels of mRNA of most selected genes compared to zebrafish liver. The induced gene transcription following exposure to ligand was much lower in ZFL cells compared to zebrafish primary hepatocytes at the doses tested. Importantly, oestrogen receptor and vitellogenin genes showed low basal transcription and no induction response in the ZFL cell line. In conclusion, it appears that primary hepatocytes are well suited for studying environmental contaminants including xenoestrogens, but may show large sex-dependent differences in gene transcription. The ZFL cell line shows potential in toxicological studies involving the aryl hydrocarbon receptor pathway. However, low potential for transcriptional induction of genes in general should be expected, especially notable when studying estrogenic responses. Copyright © 2013 Elsevier B.V. All rights reserved.

Induction of apoptosis in human colorectal cancer cell line, HCT-116 by a vanadium- Schiff base complex.

PubMed

Sinha, Abhinaba; Banerjee, Kaushik; Banerjee, Arpita; Sarkar, Avijit; Ahir, Manisha; Adhikary, Arghya; Chatterjee, Mitali; Choudhuri, Soumitra Kumar

2017-08-01

Vanadium compounds are well known for their therapeutic interventions against several diseases. Various biochemical attributes of vanadium complexes inspired us to evaluate the cancer cell killing efficacy of the vanadium complex, viz., vanadyl N-(2-hydroxyacetophenone) glycinate [VO(NG) 2 ]. Previously we showed that VO(NG) 2 is an effective anticancer agent in in vitro and in vivo cancer models and imposed miniscule side effects. Herein we report that VO(NG) 2 is significantly cytotoxic to various cancer cell lines. Furthermore, this redox active vanadyl complex altered the redox homeostatsis of many human cancer cell lines significantly. VO(NG) 2 actuates programmed cell death in human colorectal carcinoma cells(HCT-116) through mitochondrial outer membrane permeabilization but in caspase independent manner, possibly by altering cellular redox status and by inflicting DNA damage. Thus, the present work is an attempt to provide many evidences regarding the potent and selective chemotherapeutic efficacy of the novel VO(NG) 2 . Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Cytotoxicity of titanium and silicon dioxide nanoparticles

NASA Astrophysics Data System (ADS)

Wagner, Stefanie; Münzer, Simon; Behrens, Peter; Scheper, Thomas; Bahnemann, Detlef; Kasper, Cornelia

2009-05-01

Different TiO2 and SiO2 nanoparticles have been tested concerning their toxicity on selected mammalian cell lines. Various powders and suspensions, all of which consist of titanium or silicon dioxide nanoparticles have been examined. These particles differ in the crystal structure, the size and the BET-surface area. There was also a classification in fixed particles and in particles easily accessible in solution. With focus on the possible adsorption of the nanoparticles into the human organism, via skin and via respiratory tract, the effects on fibroblasts (NIH-3T3) and on a human lung adenocarcinoma epithelial cell line were examined. Additionally, the particles were tested with HEP-G2 cells, which are often used as model cell line for biocompatibility tests, and PC-12 cells, a rat adrenal pheochromocytoma cell line. The viability of the cells was examined by the MTT-test. The viability results were found to partly depend on the type of cells used. The experimental results show that the adhesion of the cells on the different powders strongly depends on the type of cell lines as well as on the type of powder. It was found that the lower viability of some cells on the powder coatings is not only caused by a cytotoxicity effect of the powders, but is also due to a lower adhesion of the cells on the particle surfaces. Furthermore, it could be shown that the physical properties of the powders cannot be easily correlated to any observed biological effect. While some powders show a significant suppression of the cell growth, others with similar physical properties indicate no toxic effect.

Cytotoxicity of South-African medicinal plants towards sensitive and multidrug-resistant cancer cells.

PubMed

Saeed, Mohamed E M; Meyer, Marion; Hussein, Ahmed; Efferth, Thomas

2016-06-20

Traditional medicine plays a major role for primary health care worldwide. Cancer belongs to the leading disease burden in industrialized and developing countries. Successful cancer therapy is hampered by the development of resistance towards established anticancer drugs. In the present study, we investigated the cytotoxicity of 29 extracts from 26 medicinal plants of South-Africa against leukemia cell lines, most of which are used traditionally to treat cancer and related symptoms. We have investigated the plant extracts for their cytotoxic activity towards drug-sensitive parental CCRF-CEM leukemia cells and their multidrug-resistant P-glycoprotein-overexpressing subline, CEM/ADR5000 by means of the resazurin assay. A panel of 60 NCI tumor cell lines have been investigated for correlations between selected phytochemicals from medicinal plants and the expression of resistance-conferring genes (ABC-transporters, oncogenes, tumor suppressor genes). Seven extracts inhibited both cell lines (Acokanthera oppositifolia, Hypoestes aristata, Laurus nobilis, Leonotis leonurus, Plectranthus barbatus, Plectranthus ciliates, Salvia apiana). CEM/ADR5000 cells exhibited a low degree of cross-resistance (3.35-fold) towards the L. leonurus extract, while no cross-resistance was observed to other plant extracts, although CEM/ADR5000 cells were highly resistant to clinically established drugs. The log10IC50 values for two out of 14 selected phytochemicals from these plants (acovenoside A and ouabain) of 60 tumor cell lines were correlated to the expression of ABC-transporters (ABCB1, ABCB5, ABCC1, ABCG2), oncogenes (EGFR, RAS) and tumor suppressors (TP53). Sensitivity or resistance of the cell lines were not statistically associated with the expression of these genes, indicating that multidrug-resistant, refractory tumors expressing these genes may still respond to acovenoside A and ouabain. The bioactivity of South African medicinal plants may represent a basis for the development of strategies to treat multidrug-resistant tumors either by phytotherapeutic approaches with whole plant preparations or by classical drug development with isolated compounds such as acovenoside A or ouabain. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

MTH1 deficiency selectively increases non-cytotoxic oxidative DNA damage in lung cancer cells: more bad news than good?

PubMed

Abbas, Hussein H K; Alhamoudi, Kheloud M H; Evans, Mark D; Jones, George D D; Foster, Steven S

2018-04-16

Targeted therapies are based on exploiting cancer-cell-specific genetic features or phenotypic traits to selectively kill cancer cells while leaving normal cells unaffected. Oxidative stress is a cancer hallmark phenotype. Given that free nucleotide pools are particularly vulnerable to oxidation, the nucleotide pool sanitising enzyme, MTH1, is potentially conditionally essential in cancer cells. However, findings from previous MTH1 studies have been contradictory, meaning the relevance of MTH1 in cancer is still to be determined. Here we ascertained the role of MTH1 specifically in lung cancer cell maintenance, and the potential of MTH1 inhibition as a targeted therapy strategy to improve lung cancer treatments. Using siRNA-mediated knockdown or small-molecule inhibition, we tested the genotoxic and cytotoxic effects of MTH1 deficiency on H23 (p53-mutated), H522 (p53-mutated) and A549 (wildtype p53) non-small cell lung cancer cell lines relative to normal MRC-5 lung fibroblasts. We also assessed if MTH1 inhibition augments current therapies. MTH1 knockdown increased levels of oxidatively damaged DNA and DNA damage signaling alterations in all lung cancer cell lines but not normal fibroblasts, despite no detectable differences in reactive oxygen species levels between any cell lines. Furthermore, MTH1 knockdown reduced H23 cell proliferation. However, unexpectedly, it did not induce apoptosis in any cell line or enhance the effects of gemcitabine, cisplatin or radiation in combination treatments. Contrastingly, TH287 and TH588 MTH1 inhibitors induced apoptosis in H23 and H522 cells, but only increased oxidative DNA damage levels in H23, indicating that they kill cells independently of DNA oxidation and seemingly via MTH1-distinct mechanisms. MTH1 has a NSCLC-specific p53-independent role for suppressing DNA oxidation and genomic instability, though surprisingly the basis of this may not be reactive-oxygen-species-associated oxidative stress. Despite this, overall our cell viability data indicates that targeting MTH1 will likely not be an across-the-board effective NSCLC therapeutic strategy; rather it induces non-cytotoxic DNA damage that could promote cancer heterogeneity and evolution.

Simple Monitoring of Gene Targeting Efficiency in Human Somatic Cell Lines Using the PIGA Gene

PubMed Central

Karnan, Sivasundaram; Konishi, Yuko; Ota, Akinobu; Takahashi, Miyuki; Damdindorj, Lkhagvasuren; Hosokawa, Yoshitaka; Konishi, Hiroyuki

2012-01-01

Gene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvement of this technology and its application to a broad range of cell lines. In this study, we utilized phosphatidylinositol glycan anchor biosynthesis class A (PIGA), a gene essential for the synthesis of glycosylphosphatidyl inositol (GPI) anchors, as a reporter of gene targeting events in human somatic cell lines. Targeted disruption of PIGA was quantitatively detected with FLAER, a reagent that specifically binds to GPI anchors. Using this PIGA-based reporter system, we successfully detected adeno-associated virus (AAV)-mediated gene targeting events both with and without promoter-trap enrichment of gene-targeted cell population. The PIGA-based reporter system was also capable of reproducing previous findings that an AAV-mediated gene targeting achieves a remarkably higher ratio of homologous versus random integration (H/R ratio) of targeting vectors than a plasmid-mediated gene targeting. The PIGA-based system also detected an approximately 2-fold increase in the H/R ratio achieved by a small negative selection cassette introduced at the end of the AAV-based targeting vector with a promoter-trap system. Thus, our PIGA-based system is useful for monitoring AAV-mediated gene targeting and will assist in improving gene targeting technology in human somatic cell lines. PMID:23056640

[Development of a mouse cell line containing stably integrated copies of pMCLacI/Neo plasmid: a model for studying mutations in vitro].

PubMed

Lu, Y; Li, H; Fu, J

2000-04-01

To establish a suitable model for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells. The NIH3T3 cells were transfected with the linearized pMCLacI/Neo DNAs by liposome-mediated transfection, and grew in the presence of G418. One drug resistant cell clone was selected to proliferate and to be analyzed with Southern blot and RT-PCR analyses on its genomic DNAs. (1) Multiple copies of pMCLacI/Neo plasmid DNA were intactly integrated in the genomic DNAs of the cell clone. (2) One of lac I target genes in the integrated plasmid could be transcribed in the NIH3T3 cells while the other could not. (3) The pMCLacI/Neo plasmid DNA could be efficiently rescued from the genomic DNAs of the cell clone with the average rescue efficiency of 410 cfu/microg DNA. The NIH3T3 cell line containing copies of a stably integrated pMCLacI/Neo has been established. The two lacI target genes in the cell line could imitate the functional states of expressed and non-expressed genes in mammalian cells respectively. The cell line will be a useful model for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells.

Genetic and small molecule inhibition of arylamine N-acetyltransferase 1 reduces anchorage-independent growth in human breast cancer cell line MDA-MB-231.

PubMed

Stepp, Marcus W; Doll, Mark A; Carlisle, Samantha M; States, J Christopher; Hein, David W

2018-04-01

Arylamine N-acetyltransferase 1 (NAT1) expression is reported to affect proliferation, invasiveness, and growth of cancer cells. MDA-MB-231 breast cancer cells were engineered such that NAT1 expression was elevated or suppressed, or treated with a small molecule inhibitor of NAT1. The MDA-MB-231 human breast cancer cell lines were engineered with a scrambled shRNA, a NAT1 specific shRNA or a NAT1 overexpression cassette stably integrated into a single flippase recognition target (FRT) site facilitating incorporation of these different genetic elements into the same genomic location. NAT1-specific shRNA reduced NAT1 activity in vitro by 39%, increased endogenous acetyl coenzyme A levels by 35%, and reduced anchorage-independent growth (sevenfold) without significant effects on cell morphology, growth rates, anchorage-dependent colony formation, or invasiveness compared to the scrambled shRNA cell line. Despite 12-fold overexpression of NAT1 activity in the NAT1 overexpression cassette transfected MDA-MB-231 cell line, doubling time, anchorage-dependent cell growth, anchorage-independent cell growth, and relative invasiveness were not changed significantly when compared to the scrambled shRNA cell line. A small molecule (5E)-[5-(4-hydroxy-3,5-diiodobenzylidene)-2-thioxo-1,3-thiazolidin-4-one (5-HDST) was 25-fold more selective towards the inhibition of recombinant human NAT1 than N-acetyltransferase 2. Incubation of MDA-MB-231 cell line with 5-HDST resulted in 60% reduction in NAT1 activity and significant decreases in cell growth, anchorage-dependent growth, and anchorage-independent growth. In summary, inhibition of NAT1 activity by either shRNA or 5-HDST reduced anchorage-independent growth in the MDA-MB-231 human breast cancer cell line. These findings suggest that human NAT1 could serve as a target for the prevention and/or treatment of breast cancer. © 2018 Wiley Periodicals, Inc.

Phase II Trial of Atezolizumab As First-Line or Subsequent Therapy for Patients With Programmed Death-Ligand 1-Selected Advanced Non-Small-Cell Lung Cancer (BIRCH).

PubMed

Peters, Solange; Gettinger, Scott; Johnson, Melissa L; Jänne, Pasi A; Garassino, Marina C; Christoph, Daniel; Toh, Chee Keong; Rizvi, Naiyer A; Chaft, Jamie E; Carcereny Costa, Enric; Patel, Jyoti D; Chow, Laura Q M; Koczywas, Marianna; Ho, Cheryl; Früh, Martin; van den Heuvel, Michel; Rothenstein, Jeffrey; Reck, Martin; Paz-Ares, Luis; Shepherd, Frances A; Kurata, Takayasu; Li, Zhengrong; Qiu, Jiaheng; Kowanetz, Marcin; Mocci, Simonetta; Shankar, Geetha; Sandler, Alan; Felip, Enriqueta

2017-08-20

Purpose BIRCH was designed to examine the efficacy of atezolizumab, a humanized anti-programmed death-ligand 1 (PD-L1) monoclonal antibody, in advanced non-small-cell lung cancer (NSCLC) across lines of therapy. Patients were selected on the basis of PD-L1 expression on tumor cells (TC) or tumor-infiltrating immune cells (IC). Patients and Methods Eligible patients had advanced-stage NSCLC, no CNS metastases, and zero to two or more lines of prior chemotherapy. Patients whose tumors expressed PD-L1 using the SP142 immunohistochemistry assay on ≥ 5% of TC or IC (TC2/3 or IC2/3 [TC or IC ≥ 5% PD-L1-expressing cells, respectively]) were enrolled. Atezolizumab 1,200 mg was administered intravenously every 3 weeks. Efficacy-evaluable patients (N = 659) comprised three cohorts: first line (cohort 1; n = 139); second line (cohort 2; n = 268); and third line or higher (cohort 3; n = 252). The primary end point was independent review facility-assessed objective response rate (ORR; Response Evaluation Criteria in Solid Tumors [RECIST] version 1.1). Secondary end points included median duration of response, progression-free survival, and overall survival (OS). Results BIRCH met its primary objective of demonstrating a significant ORR versus historical controls. With a minimum of 12 months of follow-up, the independent review facility-assessed ORR was 18% to 22% for the three cohorts, and 26% to 31% for the TC3 or IC3 subgroup; most responses are ongoing. Responses occurred regardless of EGFR or KRAS mutation status. The median OS from an updated survival analysis (minimum of 20 month follow up) for cohort 1 was 23.5 months (26.9 months for TC3 or IC3 patients); the median OS in cohorts 2 and 3 was 15.5 and 13.2 months, respectively. The safety profile was similar across cohorts and consistent with previous atezolizumab monotherapy trials. Conclusion BIRCH demonstrated responses with atezolizumab monotherapy in patients with PD-L1-selected advanced NSCLC, with good tolerability. PD-L1 status may serve as a predictive biomarker for identifying patients most likely to benefit from atezolizumab.

Stem/progenitor cell-like properties of desmoglein 3dim cells in primary and immortalized keratinocyte lines.

PubMed

Wan, Hong; Yuan, Ming; Simpson, Cathy; Allen, Kirsty; Gavins, Felicity N E; Ikram, Mohammed S; Basu, Subham; Baksh, Nuzhat; O'Toole, Edel A; Hart, Ian R

2007-05-01

We showed previously that primary keratinocytes selected for low desmoglein 3 (Dsg3) expression levels exhibited increased colony-forming efficiency and heightened proliferative potential relative to cells with higher Dsg3 expression levels, characteristics consistent with a more "stem/progenitor cell-like" phenotype. Here, we have confirmed that Dsg3(dim) cells derived from cultured primary human adult keratinocytes have comparability with alpha(6)(bri)/CD71(dim) stem cells in terms of colony-forming efficiency. Moreover, these Dsg3(dim) cells exhibit increased reconstituting ability in in vitro organotypic culture on de-epidermalized dermis (DED); they are small, actively cycling cells, and they express elevated levels of various p63 isoforms. In parallel, using the two immortalized keratinocyte cell lines HaCaT and NTERT, we obtained essentially similar though occasionally different findings. Thus, reduced colony-forming efficiency by Dsg3(bri) cells consistently was observed in both cell lines even though the cell cycle profile and levels of p63 isoforms in the bri and dim populations differed between these two cell lines. Dsg3(dim) cells from both immortalized lines produced thicker and better ordered hierarchical structural organization of reconstituted epidermis relative to Dsg3(bri) and sorted control cells. Dsg3(dim) HaCaT cells also show sebocyte-like differentiation in the basal compartment of skin reconstituted after a 4-week organotypic culture. No differences in percentages of side population cells (also a putative marker of stem cells) were detected between Dsg3(dim) and Dsg3(bri) populations. Taken together our data indicate that Dsg3(dim) populations from primary human adult keratinocytes and long-term established keratinocyte lines possess certain stem/progenitor cell-like properties, although the side population characteristic is not one of these features. Disclosure of potential conflicts of interest is found at the end of this article.

Small Molecules that Suppress IGF-Activated Prostate Cancers

DTIC Science & Technology

2006-04-01

selectively impair the growth of IGF2-overexpressing hepatocellular carcinoma cells. For cell viability assays, IGF2-expressing cells were plated at a...produced at high levels in liver tumors (27). We identified three chemically analogous compounds that killed IGF2-overexpressing hepatocellular carcinoma cells... hepatocellular carcinoma cell lines that we recently characterized2 indicated that one of the three chemi- cals, 94G6, exhibited the highest cytotoxicity

Genomic instability and tumorigenic induction in immortalized human bronchial epithelial cells by heavy ions

NASA Astrophysics Data System (ADS)

Hei, T. K.; Piao, C. Q.; Wu, L. J.; Willey, J. C.; Hall, E. J.

1998-11-01

Carcinogenesis is postulated to be a progressive multistage process characterized by an increase in genomic instability and clonal selection with each mutational event endowing a selective growth advantage. Genomic instability as manifested by the amplification of specific gene fragments is common among tumor and transformed cells. In the present study, immortalized human bronchial (BEP2D) cells were irradiated with graded doses of either 1GeV/nucleon 56Fe ions or 150 keV/μm alpha particles. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Tumorigenic cells showed neither ras mutations nor deletion in the p16 tumor suppressor gene. In contrast, they harbored mutations in the p53 gene and over-expressed cyclin D1. Genomic instability among transformed cells at various stage of the carcinogenic process was examined based on frequencies of PALA resistance. Incidence of genomic instability was highest among established tumor cell lines relative to transformed, non-tumorigenic and control cell lines. Treatment of BEP2D cells with a 4 mM dose of the aminothiol WR-1065 significantly reduced their neoplastic transforming response to 56Fe particles. This model provides an opportunity to study the cellular and molecular mechanisms involved in malignant transformation of human epithelial cells by heavy ions.

The selective Aurora B kinase inhibitor AZD1152 is a potential new treatment for multiple myeloma.

PubMed

Evans, Robert P; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Maxwell, Christopher A; Keats, Jonathan J; Belch, Andrew R; Pilarski, Linda M; Lai, Raymond; Reiman, Tony

2008-02-01

Aurora kinases are potential targets for cancer therapy. Previous studies have validated Aurora kinase A as a therapeutic target in multiple myeloma (MM), and have demonstrated in vitro anti-myeloma effects of small molecule Aurora kinase inhibitors that inhibit both Aurora A and B. This study demonstrated that Aurora B kinase was strongly expressed in myeloma cell lines and primary plasma cells. The selective Aurora B inhibitor AZD1152-induced apoptotic death in myeloma cell lines at nanomolar concentrations, with a cell cycle phenotype consistent with that reported previously for Aurora B inhibition. In some cases, AZD1152 in combination with dexamethasone showed increased anti-myeloma activity compared with the use of either agent alone. AZD1152 was active against sorted CD138(+) BM plasma cells from myeloma patients but also, as expected, was toxic to CD138(-) marrow cells from the same patients. In a murine myeloma xenograft model, AZD1152-inhibited tumour growth at well-tolerated doses and induced cell death in established tumours, with associated mild, transient leucopenia. AZD1152 shows promise in these preclinical studies as a novel treatment for MM.

Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library.

PubMed

Yu, Bing; Ni, Ming; Li, Wen-Han; Lei, Ping; Xing, Wei; Xiao, Dai-Wen; Huang, Yu; Tang, Zhen-Jie; Zhu, Hui-Fen; Shen, Guan-Xin

2005-07-14

To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in E.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M(r) value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.

Functional significance of CD105-positive cells in papillary renal cell carcinoma.

PubMed

Matak, Damian; Brodaczewska, Klaudia K; Szczylik, Cezary; Koch, Irena; Myszczyszyn, Adam; Lipiec, Monika; Lewicki, Slawomir; Szymanski, Lukasz; Zdanowski, Robert; Czarnecka, Anna M

2017-01-05

CD105 was postulated as a renal cell carcinoma (RCC) stem cell marker, and CD133 as a putative RCC progenitor. Hypoxia, a natural microenvironment that prevails in tumors, was also incorporated into the study, especially in terms of the promotion of hypothetical stem-like cell properties. Within this study, we verify the existence of CD105+ and CD133+ populations in selected papillary subtype RCC (pRCC) cell lines. Both populations were analyzed for correlation with stem-like cell properties, such as stemness gene expression, and sphere and colony formation. For the preliminary analysis, several RCC cell lines were chosen (786-O, SMKT-R2, Caki-2, 796-P, ACHN, RCC6) and the control was human kidney cancer stem cells (HKCSC) and renal cells of embryonic origin (ASE-5063). Four cell lines were chosen for further investigation: Caki-2 (one of the highest numbers of CD105+ cells; primary origin), ACHN (a low number of CD105+ cells; metastatic origin), HKCSC (putative positive control), and ASE-5063 (additional control). In 769-P and RCC6, we could not detect a CD105+ population. Hypoxia variously affects pRCC cell growth, and mainly diminishes the stem-like properties of cells. Furthermore, we could not observe the correlation of CD105 and/or CD133 expression with the enhancement of stem-like properties. Based on this analysis, CD105/CD133 cannot be validated as cancer stem cell markers of pRCC cell lines.

New cytotoxic diarylheptanoids from the rhizomes of Alpinia officinarum Hance.

PubMed

Liu, Dan; Liu, Yan-Wen; Guan, Fu-Qin; Liang, Jing-Yu

2014-07-01

Two new dimeric diarylheptanoids, named Alpinin C (1) and D (2), a new natural product of diarylheptanoid (3) along with three known diarylheptanoids (4-6) were isolated from the rhizomes of Alpinia officinarum Hance. Their structures were elucidated based on extensive spectroscopic analyses (1D and 2D NMR, HRTOFMS, IR). The isolated compounds were evaluated for their cytotoxicity against human tumor cell lines HepG2, MCF-7, T98G and B16-F10. Compound 1 showed selective cytotoxicity against cell lines of MCF-7 and T98G, while compound 6 showed significant cytotoxicity to the all tested tumor cell lines with IC50 in the range from 8.46 to 22.68 μmol/L. Copyright © 2014 Elsevier B.V. All rights reserved.

FLAX OIL FROM TRANSGENIC LINUM USITATISSIMUM SELECTIVELY INHIBITS IN VITRO PROLIFERATION OF HUMAN CANCER CELL LINES.

PubMed

Gebarowski, Tomasz; Gebczak, Katarzyna; Wiatrak, Benita; Kulma, Anna; Pelc, Katarzyna; Czuj, Tadeusz; Szopa, Jan; Gasiorowski, Kazimierz

2017-03-01

Emulsions made of oils from transgenic flaxseeds significantly decreased in vitro proliferation of six tested human cancer cell lines in 48-h cultures, as assessed with the standard sulforhodamine assay. However, the emulsions also increased proliferation rate of normal human dermal fibroblasts and, to a lower extend, of keratinocytes. Both inhibition of in vitro proliferation of human cancer cell lines and stimulation of proliferation of normal dermal fibroblasts and keratinocytes were especially strong with the emulsion type B and with emulsion type M. Oils from seeds of transgenic flax type B and M should be considered as valuable adjunct to standard cytostatic therapy of human cancers and also could be applied to improve the treatment of skin lesions in wound healing.

The HDAC6/8/10 inhibitor TH34 induces DNA damage-mediated cell death in human high-grade neuroblastoma cell lines.

PubMed

Kolbinger, Fiona R; Koeneke, Emily; Ridinger, Johannes; Heimburg, Tino; Müller, Michael; Bayer, Theresa; Sippl, Wolfgang; Jung, Manfred; Gunkel, Nikolas; Miller, Aubry K; Westermann, Frank; Witt, Olaf; Oehme, Ina

2018-06-09

High histone deacetylase (HDAC) 8 and HDAC10 expression levels have been identified as predictors of exceptionally poor outcomes in neuroblastoma, the most common extracranial solid tumor in childhood. HDAC8 inhibition synergizes with retinoic acid treatment to induce neuroblast maturation in vitro and to inhibit neuroblastoma xenograft growth in vivo. HDAC10 inhibition increases intracellular accumulation of chemotherapeutics through interference with lysosomal homeostasis, ultimately leading to cell death in cultured neuroblastoma cells. So far, no HDAC inhibitor covering HDAC8 and HDAC10 at micromolar concentrations without inhibiting HDACs 1, 2 and 3 has been described. Here, we introduce TH34 (3-(N-benzylamino)-4-methylbenzhydroxamic acid), a novel HDAC6/8/10 inhibitor for neuroblastoma therapy. TH34 is well-tolerated by non-transformed human skin fibroblasts at concentrations up to 25 µM and modestly impairs colony growth in medulloblastoma cell lines, but specifically induces caspase-dependent programmed cell death in a concentration-dependent manner in several human neuroblastoma cell lines. In addition to the induction of DNA double-strand breaks, HDAC6/8/10 inhibition also leads to mitotic aberrations and cell-cycle arrest. Neuroblastoma cells display elevated levels of neuronal differentiation markers, mirrored by formation of neurite-like outgrowths under maintained TH34 treatment. Eventually, after long-term treatment, all neuroblastoma cells undergo cell death. The combination of TH34 with plasma-achievable concentrations of retinoic acid, a drug applied in neuroblastoma therapy, synergistically inhibits colony growth (combination index (CI) < 0.1 for 10 µM of each). In summary, our study supports using selective HDAC inhibitors as targeted antineoplastic agents and underlines the therapeutic potential of selective HDAC6/8/10 inhibition in high-grade neuroblastoma.

Electrophysiological characteristics of embryonic stem cell-derived cardiomyocytes are cell line-dependent.

PubMed

Hannes, Tobias; Wolff, Marie; Doss, Michael Xavier; Pfannkuche, Kurt; Haustein, Moritz; Müller-Ehmsen, Jochen; Sachinidis, Agapios; Hescheler, Jürgen; Khalil, Markus; Halbach, Marcel

2015-01-01

Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs) requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs) of different murine ESC lines. Two wild-type (D3 and R1) and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7) were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP) and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC) promoter. Action potentials (APs) were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Spontaneous AP frequency and AP duration (APD) as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully. © 2015 S. Karger AG, Basel.

A novel mechanism of EML4-ALK rearrangement mediated by chromothripsis in a patient-derived cell line.

PubMed

Kodama, Tatsushi; Motoi, Noriko; Ninomiya, Hironori; Sakamoto, Hiroshi; Kitada, Kunio; Tsukaguchi, Toshiyuki; Satoh, Yasuko; Nomura, Kimie; Nagano, Hiroko; Ishii, Nobuya; Terui, Yasuhito; Hatake, Kiyohiko; Ishikawa, Yuichi

2014-11-01

EML4-ALK is a driver oncogene in non-small-cell lung cancer (NSCLC) and has been developed into a promising molecular target for antitumor agents. Although EML4-ALK is reported to be formed by inversion of chromosome 2, other mechanisms of this gene fusion remain unknown. This study aimed to examine the mechanism of EML4-ALK rearrangement using a novel cell line with the EML4-ALK fusion gene. An EML4-ALK-positive cell line, termed JFCR-LC649, was established from pleomorphic carcinoma, a rare subtype of NSCLC. We investigated the chromosomal aberrations using fluorescence in situ hybridization and comparative genomic hybridization (CGH). Alectinib/CH5424802, a selective ALK inhibitor, was evaluated in the antitumor activity against JFCR-LC649 in vitro and in vivo xenograft model. We established an EML4-ALK-positive cell line, termed JFCR-LC649, derived from a patient with NSCLC and revealed that the JFCR-LC649 cells harbor variant 3 of the EML4-ALK fusion with twofold copy number gain. Interestingly, comparative genomic hybridization and metaphase-fluorescence in situ hybridization analysis showed that in addition to two normal chromosome 2, JFCR-LC649 cells contained two aberrant chromosome 2 that were fragmented and scattered. These observations provided the first evidence that EML4-ALK fusion in JFCR-LC649 cells was formed in chromosome 2 by a distinct mechanism of genomic rearrangement, termed chromothripsis. Furthermore, a selective ALK inhibitor alectinib/CH5424802 suppressed tumor growth of the JFCR-LC649 cells through inhibition of phospho-ALK in vitro and in vivo in a xenograft model. Our results suggested that chromothripsis may be a mechanism of oncogenic rearrangement of EML4-ALK. In addition, alectinib was effective against EML4-ALK-positive tumors with ALK copy number gain mediated by chromothripsis.

Selective Activation of N,N'-Diacyl Rhodamine Pro-fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE).

PubMed

Abney, Kristopher K; Ramos-Hunter, Susan J; Romaine, Ian M; Godwin, J Shawn; Sulikowski, Gary A; Weaver, Charles David

2018-04-21

This study reports the synthesis and testing of a family of rhodamine pro-fluorophores and an enzyme capable of converting pro-fluorophores to Rhodamine 110. We prepared a library of simple N,N'-diacyl rhodamines and investigated Porcine Liver Esterase (PLE) as an enzyme to activate rhodamine-based pro-fluorophores. A PLE-expressing cell line generated an increase in fluorescence rapidly upon pro-fluorophore addition demonstrating the rhodamine pro-fluorophores are readily taken up and fluorescent upon PLE-mediated release. Rhodamine pro-fluorophore amides trifluoroacetamide (TFAm) and proponamide (PAm) appeared to be the best substrates using a cell-based assay using PLE expressing HEK293. Our pro-fluorophore series showed diffusion into live cells and resisted endogenous hydrolysis. The use of our engineered cell line containing the exogenous enzyme PLE demonstrated the rigorousness of amide masking when compared to cells not containing PLE. This simple and selective pro-fluorophore rhodamine pair with PLE offers the potential to be used in vitro and in vivo fluorescence based assays. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis of mono Mannich bases of 2-(4-hydroxybenzylidene)-2,3-dihydroinden-1-one and evaluation of their cytotoxicities.

PubMed

Tugrak, Mehtap; Yamali, Cem; Sakagami, Hiroshi; Gul, Halise Inci

2016-10-01

Chalcones and Mannich bases are a group of compounds known for their cytotoxicities. In this study restricted chalcone analogue, compound 2-(4-hydroxybenzylidene)-2,3-dihydroinden-1-one MT1, was used as a starting compound to synthesize new mono Mannich bases since Mannich bases may induce more cytotoxicity than chalcone analogue that they are derived from by producing additional alkylating center for cellular thiols. In this study, cyclic and acyclic amines were used to synthesize Mannich bases. All compounds were tested against Ca9-22 (gingival carcinoma), HSC-2, HSC-3 and HSC-4 (oral squamous cell carcinoma) as tumour cell lines and HGF (gingival fibroblasts), HPC (pulp cells) and HPLF (periodontal ligament fibroblasts) human normal oral cells as non tumour cell lines. Cytotoxicity, selectivity index (SI) values and potency selectivity expression (PSE) values expressed as a percentage were determined for the compounds. According to data obtained, the compound MT8 with the highest PSE value bearing N-methylpiperazine moiety seems to be a good candidate to develop new cytotoxic compounds and is suited for further investigation.

[Using of cell biocomposite material in tissue engineering of the urinary bladder].

PubMed

Glybochko, P V; Olefir, Yu V; Alyaev, Yu G; Butnaru, D V; Bezrukov, E A; Chaplenko, A A; Zharikova, T M

2017-06-01

In a systematic review, to present an overview of the current situation in the field of tissue engineering of urinary bladder related to the use of cell lines pre-cultured on matrices. The selection of eligible publications was conducted according to the method described in the article Glybochko P.V. et al. "Tissue engineering of urinary bladder using acellular matrix." At the final stage, studies investigating the application of matrices with human and animal cell lines were analyzed. Contemporary approaches to using cell-based tissue engineering of the bladder were analyzed, including the formation of 3D structures from several types of cells, cell layers and genetic modification of injected cells. The most commonly used cell lines are urothelial cells, mesenchymal stem cells and fibroblasts. The safety and efficacy of any types of composite cell structures used in the cell-based bladder tissue engineering has not been proven sufficiently to warrant clinical studies of their usefulness. The results of cystoplasty of rat bladder are almost impossible to extrapolate to humans; besides, it is difficult to predict possible side effects. For the transition to clinical trials, additional studies on relevant animal models are needed.

Proteasome inhibition blocks NF-κB and ERK1/2 pathways, restores antigen expression and sensitizes resistant human melanoma to TCR-engineered CTLs

PubMed Central

Jazirehi, Ali R.; Economou, James S.

2012-01-01

Adoptive cell transfer (ACT) of ex vivo engineered autologous lymphocytes encoding high-affinity MART-1/HLA-A*0201-specific T-cell receptor (TCR) α/β chains (F5 CTL), densely infiltrate into sites of metastatic disease, mediating dramatic but partial clinical responses in melanoma patients. We hypothesized that MART-1 down-modulation in addition to aberrant apoptotic/survival signaling could confer resistance to death signals delivered by transgenic CTLs. To explore this hypothesis, we established an in vitro model of resistant (R) lines from MART-1+/HLA-A*0201+ F5 CTL-sensitive parental (P) lines under serial F5 CTL-selective pressure. We have recently reported that several melanoma R lines, while retaining MART-1 expression, exhibited constitutive NF-κB activation and over-expression of NF-κB-dependent resistance factors. Another established melanoma cell line M244, otherwise sensitive to F5 CTL, yielded R lines after serial F5 CTL selective pressure which had both reduced MART-1 expression levels, thus, could not be recognized, and were resistant to CTL-delivered apoptotic death signals. The proteasome inhibitor bortezomib blocked NF-κB activity, decreased phopspho-ERK1/2, increased phospho-JNK levels, reduced expression of resistance-factors, restored MART-1 expression to sufficient levels, which in combination allowed M244R lines be sensitized to F5 CTL-killing. These findings suggest that proteasome inhibition in immune resistant tumors can restore proapoptotic signaling and improve tumor antigen expression. PMID:22532603

Avian germplasm preservation: embryonic stem cells or primordial germ cells?

PubMed

Petitte, J N

2006-02-01

Presently, avian genetic resources are best maintained as living collections of birds. Unfortunately, these stocks have been under constant pressure to be destroyed because of the decline in the number of Poultry Science Departments and pressures to cut costs at land grant institutions. Cryopreservation of semen is often suggested as a means to bank avian germplasm. However, this is only applicable for single-gene traits and does not allow for full reconstitution of the genetics of the original line. Over the last 15 yr, advances in the manipulation of the early chick embryo, manipulation of primordial germ cells (PGC), and the culture of embryonic stem cells (ESC) suggests that cryopreservation of blastodermal cells, ESC, or PGC might offer a means to preserve the entire genome of highly selected, specialized stocks of poultry. Freezing each of these cell types is possible with varying degrees of efficiency. Similarly, the effectiveness of generating germ line chimeras using blastodermal cells, ESC, or PGC also varies greatly. Other factors that must be considered include the choice of the recipient lines to develop the germ line chimeras and the number of individuals needed to reconstitute the line. Finally, the low efficiency rate of reconstitution and the high cost associated with current technologies makes these approaches prohibitive. Significant challenges remain to be overcome before the entire genome of poultry stocks can be routinely cryoperserved and reconstituted.

High efficacy of the BCL-2 inhibitor ABT199 (venetoclax) in BCL-2 high-expressing neuroblastoma cell lines and xenografts and rational for combination with MCL-1 inhibition

PubMed Central

Bate-Eya, Laurel T.; den Hartog, Ilona J.M.; van der Ploeg, Ida; Schild, Linda; Koster, Jan; Santo, Evan E.; Westerhout, Ellen M.; Versteeg, Rogier; Caron, Huib N.; Molenaar, Jan J.; Dolman, M. Emmy M.

2016-01-01

The anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in neuroblastoma and plays an important role in oncogenesis. In this study, the selective BCL-2 inhibitor ABT199 was tested in a panel of neuroblastoma cell lines with diverse expression levels of BCL-2 and other BCL-2 family proteins. ABT199 caused apoptosis more potently in neuroblastoma cell lines expressing high BCL-2 and BIM/BCL-2 complex levels than low expressing cell lines. Effects on cell viability correlated with effects on BIM displacement from BCL-2 and cytochrome c release from the mitochondria. ABT199 treatment of mice with neuroblastoma tumors expressing high BCL-2 levels only resulted in growth inhibition, despite maximum BIM displacement from BCL-2 and the induction of a strong apoptotic response. We showed that neuroblastoma cells might survive ABT199 treatment due to its acute upregulation of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) and BIM sequestration by MCL-1. In vitro inhibition of MCL-1 sensitized neuroblastoma cell lines to ABT199, confirming the pivotal role of MCL-1 in ABT199 resistance. Our findings suggest that neuroblastoma patients with high BCL-2 and BIM/BCL-2 complex levels might benefit from combination treatment with ABT199 and compounds that inhibit MCL-1 expression. PMID:27056887

Antileukemic Activity of Tillandsia recurvata and Some of its Cycloartanes

PubMed Central

LOWE, HENRY I.C.; TOYANG, NGEH J.; WATSON, CHARAH T.; AYEAH, KENNETH N.N.; BRYANT, JOSEPH

2015-01-01

Background Approximately 250,000 deaths were caused by leukemia globally in 2012 and about 40%-50% of all leukemia diagnoses end-up in death. Medicinal plants are a rich source for the discovery of new drugs against leukemia and other types of cancers. To this end, we subjected the Jamaican ball moss (Tillandsia recurvata) and its cycloartanes, as well as some analogs, to in vitro screening against a number of leukemia cell lines. The WST-1 anti-proliferation assay was used to determine the anticancer activity of ball moss and two cycloartanes isolated from ball moss and four of their analogs against four leukemia cell lines (HL-60, K562, MOLM-14, monoMac6). Ball moss crude methanolic extract showed activity with a 50% inhibition concentration (IC50) value of 3.028 μg/ml against the Molm-14 cell line but was ineffective against HL-60 cells. The six cycloartanes tested demonstrated varying activity against the four leukemia cancer cell lines with IC50 values ranging from 1.83 μM to 18.3 μM. Five out of the six cycloartanes demonstrated activity, while one was inactive against all four cell lines. The preliminary activity demonstrated by the Jamaican ball moss and its cycloartanes against selected leukemia cell lines continues to throw light on the broad anticancer activity of ball moss. Further studies to evaluate the efficacy of these molecules in other leukemia cell lines are required in order to validate the activity of these molecules, as well as to determine their mechanisms of action and ascertain the activity in vivo in order to establish efficacy and safety profiles. PMID:24982361

Antileukemic activity of Tillandsia recurvata and some of its cycloartanes.

PubMed

Lowe, Henry I C; Toyang, Ngeh J; Watson, Charah T; Ayeah, Kenneth N N; Bryant, Joseph

2014-07-01

Approximately 250,000 deaths were caused by leukemia globally in 2012 and about 40%-50% of all leukemia diagnoses end-up in death. Medicinal plants are a rich source for the discovery of new drugs against leukemia and other types of cancers. To this end, we subjected the Jamaican ball moss (Tillandsia recurvata) and its cycloartanes, as well as some analogs, to in vitro screening against a number of leukemia cell lines. The WST-1 anti-proliferation assay was used to determine the anticancer activity of ball moss and two cycloartanes isolated from ball moss and four of their analogs against four leukemia cell lines (HL-60, K562, MOLM-14, monoMac6). Ball moss crude methanolic extract showed activity with a 50% inhibition concentration (IC50) value of 3.028 μg/ml against the Molm-14 cell line but was ineffective against HL-60 cells. The six cycloartanes tested demonstrated varying activity against the four leukemia cancer cell lines with IC50 values ranging from 1.83 μM to 18.3 μM. Five out of the six cycloartanes demonstrated activity, while one was inactive against all four cell lines. The preliminary activity demonstrated by the Jamaican ball moss and its cycloartanes against selected leukemia cell lines continues to throw light on the broad anticancer activity of ball moss. Further studies to evaluate the efficacy of these molecules in other leukemia cell lines are required in order to validate the activity of these molecules, as well as to determine their mechanisms of action and ascertain the activity in vivo in order to establish efficacy and safety profiles. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signal-regulated kinases 1 and 2.

PubMed

Dussault, Nathalie; Ducas, Eric; Racine, Claudia; Jacques, Annie; Paré, Isabelle; Côté, Serge; Néron, Sonia

2008-11-01

In the treatment of autoimmune diseases, intravenous Igs (IVIg) are assumed to modulate immune cells through the binding of surface receptors. IVIg act upon definite human B cell populations to modulate Ig repertoire, and such modulation might proceed through intracellular signaling. However, the heterogeneity of human B cell populations complicates investigations of the intracellular pathways involved in IVIg-induced B cell modulation. The aim of this study was to establish a model allowing the screening of IVIg signal transduction in human B cell lines and to attempt transposing observations made in cell lines to normal human B lymphocytes. Nine human B cell lines were treated with IVIg with the goal of selecting the most suitable model for human B lymphocytes. The IgG(+) DB cell line, whose response was similar to that of human B lymphocytes, showed reduced IVIg modulation following addition of PD98059, an inhibitor of extracellular signal-regulated protein kinase 1/2 (ERK1/2). The IVIg-induced ERK1/2 phosphorylation was indeed proportional to the dosage of monomeric IVIg used when tested on DB cells as well as Pfeiffer cells, another IgG(+) cell line. In addition, two other intermediates, Grb2-associated binder 1 (Gab1) and Akt, showed increased phosphorylation in IVIg-treated DB cells. IVIg induction of ERK1/2 phosphorylation was finally observed in peripheral human B lymphocytes, specifically within the IgG(+) B cell population. In conclusion, IVIg immunomodulation of human B cells can thus be linked to intracellular transduction pathways involving the phosphorylation of ERK1/2, which in combination with Gab1 and Akt, may be related to B cell antigen receptor signaling.

Cytotoxic and Antibacterial Angucycline- and Prodigiosin- Analogues from the Deep-Sea Derived Streptomyces sp. SCSIO 11594

PubMed Central

Song, Yongxiang; Liu, Guangfu; Li, Jie; Huang, Hongbo; Zhang, Xing; Zhang, Hua; Ju, Jianhua

2015-01-01

Two new C-glycoside angucyclines, marangucycline A (1) and marangucycline B (2), along with three known compounds, dehydroxyaquayamycin (3), undecylprodigiosin (4) and metacycloprodigiosin (5), have been identified as products of the deep-sea sediment strain Streptomyces sp. SCSIO 11594. New structures were elucidated on the basis of HRESIMS, 1D and 2D NMR analyses and comparisons to previously reported datasets. Compounds 2 and 4 displayed in vitro cytotoxicity against four cancer cell lines A594, CNE2, HepG2, MCF-7 superior to those obtained with cisplatin, the positive control. Notably, compound 2 bearing a keto-sugar displayed significant cytotoxicity against cancer cell lines with IC50 values ranging from 0.24 to 0.56 μM; An IC50 value of 3.67 μM was found when using non-cancerous hepatic cell line HL7702, demonstrating the cancer cell selectivity of 2. Compounds 1–3 were proved to have weak antibacterial activities against Enterococcus faecalis ATCC29212 with an MIC value of 64.0 μg/mL. Moreover, 3 displayed selective antibacterial activity against methicillin-resistant Staphylococcus epidermidis shhs-E1 with an MIC value of 16.0 μg/mL. PMID:25786061

Histone deacetylase inhibitors selectively suppress expression of HDAC7.

PubMed

Dokmanovic, Milos; Perez, Gisela; Xu, Weisheng; Ngo, Lang; Clarke, Cathy; Parmigiani, Raphael B; Marks, Paul A

2007-09-01

There are 18 histone deacetylases (HDAC) generally divided into four classes based on homology to yeast HDACs. HDACs have many protein substrates in addition to histones that are involved in regulation of gene expression, cell proliferation, and cell death. Inhibition of HDACs can cause accumulation of acetylated forms of these proteins, thus altering their function. HDAC inhibitors (HDACi), such as the hydroxamic acid-based vorinostat (suberoylanilide hydroxamic acid), inhibit the zinc-containing classes I, II, and IV, but not the NAD(+)-dependent class III, enzymes. HDACis are a group of novel anticancer agents. Vorinostat is the first HDACi approved for clinical use in the treatment of the cancer cutaneous T-cell lymphoma. Factors affecting expression of HDACs are not well understood. This study focuses on the effect of the HDACi vorinostat on the expression of class I and class II HDACs. We found that vorinostat selectively down-regulates HDAC7 with little or no effect on the expression of other class I or class II HDACs. Fourteen cell lines were examined, including normal, immortalized, genetically transformed, and human cancer-derived cell lines. Down-regulation of HDAC7 by vorinostat is more pronounced in transformed cells sensitive to inhibitor-induced cell death than in normal cells or cancer cells resistant to induced cell death. Modulation of HDAC7 levels by small interfering RNA-mediated knockdown or by HDAC7 overexpression is associated with growth arrest but without detectable changes in acetylation of histones or p21 gene expression. Selective down-regulation of HDAC7 protein may serve as a marker of response of tumors to HDACi.

Anti-proliferative and apoptosis induction activities of extracts from Thai medicinal plant recipes selected from MANOSROI II database.

PubMed

Manosroi, Jiradej; Sainakham, Mathukorn; Manosroi, Worapaka; Manosroi, Aranya

2012-05-07

ETHONOPHARMACOLOGICAL RELEVANCES: Traditional medicines have long been used by the Thai people. Several medicinal recipes prepared from a mixture of plants are often used by traditional medicinal practitioners for the treatment of many diseases including cancer. The recipes collected from the Thai medicinal text books were recorded in MANOSROI II database. Anticancer recipes were searched and selected by a computer program using the recipe indication keywords including Ma-reng and San which means cancer in Thai, from the database for anticancer activity investigation. To investigate anti-cancer activities of the Thai medicinal plant recipes selected from the "MANOSROI II" database. Anti-proliferative and apoptotic activities of extracts from 121 recipes selected from 56,137 recipes in the Thai medicinal plant recipe "MANOSROI II" database were investigated in two cancer cell lines including human mouth epidermal carcinoma (KB) and human colon adenocarcinoma (HT-29) cell lines using sulforhodamine B (SRB) assay and acridine orange (AO) and ethidium bromide (EB) staining technique, respectively. In the SRB assay, recipes NE028 and, S003 gave the highest anti-proliferation activity on KB and HT29 with the IC(50) values of 2.48±0.24 and 6.92±0.49μg/ml, respectively. In the AO/EB staining assay, recipes S016 and NE028 exhibited the highest apoptotic induction in KB and HT-29 cell lines, respectively. This study has demonstrated that the three Thai medicinal plant recipes selected from "MANOSROI II" database (NE028, S003 and S016) gave active anti-cancer activities according to the NCI classification which can be further developed for anti-cancer treatment. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Prospects for the use of plant cell cultures in food biotechnology.

PubMed

Davies, Kevin M; Deroles, Simon C

2014-04-01

Plant cell cultures can offer continuous production systems for high-value food and health ingredients, independent of geographical or environmental variations and constraints. Yet despite many improvements in culture technologies, cell line selection, and bioreactor design, there are few commercial successes. This is principally due to the culture yield and market price of food products not being sufficient to cover the plant cell culture production costs. A better understanding of the underpinning biological mechanisms that control the target metabolite biosynthetic pathways may allow the metabolic engineering of cell lines to provide for economically competitive product yields. However, uncertainty around the regulatory and public acceptance of products derived from engineered cell cultures presents a barrier to the uptake of the technology by food product companies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Selectivity of Pinus sylvestris extract and essential oil to estrogen-insensitive breast cancer cells Pinus sylvestris against cancer cells

PubMed Central

Hoai, Nguyen Thi; Duc, Ho Viet; Thao, Do Thi; Orav, Anne; Raal, Ain

2015-01-01

Background: So far, the anticancer action of pine tree extracts has mainly been shown for the species distributed widely around the Asian countries. Objective: Therefore, this study was performed to examine the potential cytotoxicity of Scots pine (Pinus sylvestris L.) native also to the European region and growing widely in Estonia. Materials and Methods: The cytotoxic activity of methanol extract and essential oil of Scots pine needles was determined by sulforhodamine B assay in different human cancer cell lines. Results: This needle extract was found to suppress the viability of several human cancer cell lines showing some selectivity to estrogen receptor negative breast cancer cells, MDA-MB-231(half maximal inhibitory concentration [IC50] 35 μg/ml) in comparison with estrogen receptor-positive breast cancer cells, MCF-7 (IC50 86 μg/ml). It is the strongest cytotoxic effect at all measured, thus far for the needles and leaves extracts derived from various pine species, and is also the first study comparing the anticancer effects of pine tree extracts on molecularly different human breast cancer cells. The essential oil showed the stronger cytotoxic effect to both negative and positive breast cancer cell lines (both IC50 29 μg/ml) than pine extract (IC50 42 and 80 μg/ml, respectively). Conclusion: The data from this report indicate that Scots pine needles extract and essential oil exhibits some potential as chemopreventive or chemotherapeutic agent for mammary tumors unresponsive to endocrine treatment. PMID:26664017

Cancer-specific SNPs originate from low-level heteroplasmic variants in human mitochondrial genomes of a matched cell line pair.

PubMed

Hedberg, Annica; Knutsen, Erik; Løvhaugen, Anne Silje; Jørgensen, Tor Erik; Perander, Maria; Johansen, Steinar D

2018-04-19

Low-level mitochondrial heteroplasmy is a common phenomenon in both normal and cancer cells. Here, we investigate the link between low-level heteroplasmy and mitogenome mutations in a human breast cancer matched cell line by high-throughput sequencing. We identified 23 heteroplasmic sites, of which 15 were common between normal cells (Hs578Bst) and cancer cells (Hs578T). Most sites were clustered within the highly conserved Complex IV and ribosomal RNA genes. Two heteroplasmic variants in normal cells were found as fixed mutations in cancer cells. This indicates a positive selection of these variants in cancer cells. RNA-Seq analysis identified upregulated L-strand specific transcripts in cancer cells, which include three mitochondrial long non-coding RNA molecules. We hypothesize that this is due to two cancer cell-specific mutations in the control region.

Withaferin-A induces apoptosis in osteosarcoma U2OS cell line via generation of ROS and disruption of mitochondrial membrane potential.

PubMed

Li, A-X; Sun, M; Li, X

2017-03-01

Withaferin-A (WF-A) is a well-known dietary compound isolated from Withania somnifera. It has marked pharmacological potential and has been shown to exhibit antiproliferative activity against several types of cancerous cells. Currently, the main focus of anti-cancer therapeutic development is to identify apoptosis-inducing drug-like molecules. Osteosarcoma is a rare type of bone cancer affecting humans. The objective of the present study was therefore to evaluate the antitumor potential of WF-A against several osteosarcoma cell lines. MTT assay was used to evaluate WF-A against osteosarcoma cell lines and to calculate the IC50. DAPI staining was used to confirm the apoptosis-inducing potential of WF-A. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, and Western blotting were used to confirm the basis of apoptosis. The results of the present study revealed that WF-A exhibited strong antiproliferative activity against all the cells lines, with IC50 ranging from 0.32 to 7.6 µM. The lowest IC50 (0.32 µM) was observed against U2OS cell line and, therefore, it was selected for further analysis. DAPI staining indicated that WF-A exhibited antiproliferative activity via induction of apoptosis. Moreover, WF-A induced a ROS-mediated reduction in mitochondrial membrane potential in a dose-dependent manner and activation of caspase-3 in osteosarcoma cells. We suggest that WF-A may prove a potent therapeutic agent for inducing apoptosis in osteosarcoma cell lines via generation of ROS and disruption of mitochondrial membrane potential.

Rho kinase inhibitor Y-27632 and Accutase dramatically increase mouse embryonic stem cell derivation.

PubMed

Zhang, Peng; Wu, Xinglong; Hu, Chunchao; Wang, Pengbo; Li, Xiangyun

2012-01-01

Although it has been 30 yr since the development of derivation methods for mouse embryonic stem (ES) cells, the biology of derivation of ES cells is poorly understood and the efficiency varies dramatically between cell lines. Recently, the Rho kinase inhibitor Y-27632 and the cell dissociation reagent Accutase were reported to significantly inhibit apoptosis of human ES cells during passaging. Therefore, in the current study, C57BL/6×129/Sv mouse blastocysts were used to evaluate the effect of the combination of the two reagents instead of using the conventional 129 line in mouse ES cell derivation. The data presented in this study suggests that the combination of Y-27632 and Accutase significantly increases the efficiency of mouse ES cell derivation; furthermore, no negative side effects were observed with Y-27632 and Accutase treatment. The newly established ES cell lines retain stable karyotype, surface markers expression, formed teratomas, and contributed to viable chimeras and germline transmission by tetraploid complementation assay. In addition, Y-27632 improved embryoid body formation of ES cells. During ES cell microinjection, Y-27632 prevented the formation of dissociation-induced cell blebs and facilitates the selection and the capture of intact cells. The methods presented in this study clearly demonstrate that inhibition of Rho kinase with Y-27632 and Accutase dissociation improve the derivation efficiently and reproducibility of mouse ES cell generation which is essential for reducing variability in the results obtained from different cell lines.

[Preparation and application of monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit].

PubMed

Yan, Xiaofei; Wu, Litao; DU, Xiaojuan; Li, Jing; Zhang, Fujun; Han, Yan; Lyu, Shemin; Li, Dongmin

2016-12-01

Objective To prepare monoclonal antibodies against DR region (897DVEDSYGQQWTYEQR911) of Na + -K + -ATPase α1 subunit and identify their properties. Methods BALB/c mice were immunized with DR-keyholelimpet hemocyanin (KLH). Splenocytes from the immunized mice were collected and subsequently fused with SP2/0 mouse myeloma cells. Positive hybridoma clones were obtained after cell fusion and selection. ELISA was used to detect DR antibody titer in the cell supernatants. DR region-specific monoclonal antibodies were analyzed by dot blotting, Western blotting and immunofluorescence assay. Na + -K + -ATPase activity was detected by SensoLyte R FDP Protein Phosphatase Assay Kit and the protective effect of the monoclonal antibody against high glucose-induced cell injury was assessed in H9c2 cells. Results Three hybridoma cell lines which secreted stable DR monoclonal antibody were obtained. The strongest positive cell line, named DRm217, was selected to prepare ascites. Dot blotting, Western blotting and immunofluorescence assay showed that DRm217 recognized specially DR region of Na + -K + -ATPase and bound on H9c2 cell membranes. DRm217 stimulated Na + -K + -ATPase activity and alleviated high glucose-induced H9c2 cells injury. Conclusion The monoclonal antibodies against DR region of Na + -K + -ATPase α1 subunit is prepared.

Multiple protocadherins are expressed in brain microvascular endothelial cells and might play a role in tight junction protein regulation.

PubMed

Dilling, Christina; Roewer, Norbert; Förster, Carola Y; Burek, Malgorzata

2017-10-01

Protocadherins (Pcdhs) are a large family of cadherin-related molecules. They play a role in cell adhesion, cellular interactions, and development of the central nervous system. However, their expression and role in endothelial cells has not yet been characterized. Here, we examined the expression of selected clustered Pcdhs in endothelial cells from several vascular beds. We analyzed human and mouse brain microvascular endothelial cell (BMEC) lines and primary cells, mouse myocardial microvascular endothelial cell line, and human umbilical vein endothelial cells. We examined the mRNA and protein expression of selected Pcdhs using RT-PCR, Western blot, and immunostaining. A strong mRNA expression of Pcdhs was observed in all endothelial cells tested. At the protein level, Pcdhs-gamma were detected using an antibody against the conserved C-terminal domain of Pcdhs-gamma or an antibody against PcdhgC3. Deletion of highly expressed PcdhgC3 led to differences in the tight junction protein expression and mRNA expression of Wnt/mTOR (mechanistic target of rapamycin) pathway genes as well as lower transendothelial electrical resistance. Staining of PcdhgC3 showed diffused cytoplasmic localization in mouse BMEC. Our results suggest that Pcdhs may play a critical role in the barrier-stabilizing pathways at the blood-brain barrier.

Fabrication and evaluation of novel zeolite membranes to control the neoplastic activity and anti-tumoral drug treatments in human breast cancer cells. Part 1: Synthesis and characterization of Pure Zeolite Membranes and Mixed Matrix Membranes for adhesion and growth of cancer cells.

PubMed

Tavolaro, Palmira; Martino, Guglielmo; Andò, Sebastiano; Tavolaro, Adalgisa

2016-12-01

Novel pure and hybrid zeolite membranes were prepared with appropriate different physicochemical characteristics such as frameworks, hydrophilicity, crystal size, chemical composition, acid-base properties (Point of Zero Charge, PZC) and surface morphology and used in inorganic cell/scaffold constructs. Because the control of cell interactions, as the adhesion, proliferation, remodelling and mobility, is important for differentiation and progression of tumors, this work focused on response of cancer cells adhered and grown on synthesized zeolite surfaces in order to study the influence of these scaffolds in controlled conditions. We have selected the MCF-7 and MDA-MB-231 human breast cancer cell line as model tumor cell lines. This study showed that all the zeolite membranes synthesized are excellent scaffolds because they are very selective materials to support the adhesion and growth of neoplastic cells. All zeolite scaffolds were characterized by FESEM, FTIR ATR, XRD, AFM, PZC and contact angle analyses. Cell adhesion, viability and morphology were measured by count, MTT assay and FESEM microphotography analysis, at various incubation times. Copyright © 2016. Published by Elsevier B.V.

Mechanism of arctigenin-mediated specific cytotoxicity against human lung adenocarcinoma cell lines.

PubMed

Susanti, Siti; Iwasaki, Hironori; Inafuku, Masashi; Taira, Naoyuki; Oku, Hirosuke

2013-12-15

The lignan arctigenin (ARG) from the herb Arctium lappa L. possesses anti-cancer activity, however the mechanism of action of ARG has been found to vary among tissues and types of cancer cells. The current study aims to gain insight into the ARG mediated mechanism of action involved in inhibiting proliferation and inducing apoptosis in lung adenocarcinoma cells. This study also delineates the cancer cell specificity of ARG by comparison with its effects on various normal cell lines. ARG selectively arrested the proliferation of cancer cells at the G0/G1 phase through the down-regulation of NPAT protein expression. This down-regulation occurred via the suppression of either cyclin E/CDK2 or cyclin H/CDK7, while apoptosis was induced through the modulation of the Akt-1-related signaling pathway. Furthermore, a GSH synthase inhibitor specifically enhanced the cytotoxicity of ARG against cancer cells, suggesting that the intracellular GSH content was another factor influencing the susceptibility of cancer cells to ARG. These findings suggest that specific cytotoxicity of ARG against lung cancer cells was explained by its selective modulation of the expression of NPAT, which is involved in histone biosynthesis. The cytotoxicity of ARG appeared to be dependent on the intracellular GSH level. Copyright © 2013 Elsevier GmbH. All rights reserved.

Solamargine, a bioactive steroidal alkaloid isolated from Solanum aculeastrum induces non-selective cytotoxicity and P-glycoprotein inhibition.

PubMed

Burger, Trevor; Mokoka, Tsholofelo; Fouché, Gerda; Steenkamp, Paul; Steenkamp, Vanessa; Cordier, Werner

2018-05-02

Solanum aculeastrum fruits are used by some cancer sufferers as a form of alternative treatment. Scientific literature is scarce concerning its anticancer activity, and thus the aim of the study was to assess the in vitro anticancer and P-glycoprotein inhibitory potential of extracts of S. aculeastrum fruits. Furthermore, assessment of the combinational effect with doxorubicin was also done. The crude extract was prepared by ultrasonic maceration. Liquid-liquid extraction yielded one aqueous and two organic fractions. Bioactive constituents were isolated from the aqueous fraction by means of column chromatography, solid phase extraction and preparative thin-layer chromatography. Confirmation of bioactive constituent identity was done by nuclear magnetic resonance and ultra-performance liquid chromatography mass spectrometry. The crude extract and fractions were assessed for cytotoxicity and P-glycoprotein inhibition in both cancerous and non-cancerous cell lines using the sulforhodamine B and rhodamine-123 assays, respectively. Both the crude extract and aqueous fraction was cytotoxic to all cell lines, with the SH-SY5Y neuroblastoma cell line being most susceptible to exposure (IC 50  = 10.72 μg/mL [crude], 17.21 μg/mL [aqueous]). Dose-dependent P-glycoprotein inhibition was observed for the crude extract (5.9 to 18.9-fold at 100 μg/mL) and aqueous fraction (2.9 to 21.2 at 100 μg/mL). The steroidal alkaloids solamargine and solanine were identified. While solanine was not bioactive, solamargine displayed an IC 50 of 15.62 μg/mL, and 9.1-fold P-glycoprotein inhibition at 100 μg/mL against the SH-SY5Y cell line. Additive effects were noted for combinations of doxorubicin against the SH-SY5Y cell line. The crude extract and aqueous fraction displayed potent non-selective cytotoxicity and noteworthy P-glycoprotein inhibition. These effects were attributed to solamargine. P-glycoprotein inhibitory activity was only present at concentrations higher than those inducing cytotoxicity, and thus does not appear to be the likely mechanism for the enhancement of doxorubicin's cytotoxicity. Preliminary results suggest that non-selective cytotoxicity may hinder drug development, however, further assessment of the mode of cell death is necessary to determine the route forward.

Undecylprodigiosin selectively induces apoptosis in human breast carcinoma cells independent of p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ho, T.-F.; Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan; Department of Medical Technology, Central Taiwan University of Science and Technology, Taichung 40605, Taiwan

2007-12-15

Undecylprodigiosin (UP) is a bacterial bioactive metabolite produced by Streptomyces and Serratia. In this study, we explored the anticancer effect of UP. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47D and one nonmalignant human breast epithelial cell line, MCF-10A, were tested in this study. We found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines in a dose- and time-dependent manner. In contrast, UP showed limited toxicity to MCF-10A cells, indicating UP's cytotoxic effect is selective for malignant cells. UP's cytotoxic effect was due to apoptosis, as confirmed by positive TUNEL signals, annexin V-binding, caspasemore » 9 activation and PARP cleavage. Notably, UP-induced apoptosis was blocked by the pan-caspase inhibitor z-VAD.fmk, further indicating the involvement of caspase activity. Moreover, UP caused a marked decrease of the levels of antiapoptotic BCL-X{sub L}, Survivin and XIAP while enhancing the levels of proapoptotic BIK, BIM, MCL-1S and NOXA, consequently favoring induction of apoptosis. Additionally, we found that cells with functional p53 (MCF-7, T47D) or mutant p53 (BT-20, MDA-MB-231) were both susceptible to UP's cytotoxicity. Importantly, UP was able to induce apoptosis in MCF-7 cells with p53 knockdown by RNA interference, confirming the dispensability of p53 in UP-induced apoptosis. Overall, our results establish that UP induces p53-independent apoptosis in breast carcinoma cells with no marked toxicity to nonmalignant cells, raising the possibility of its use as a new chemotherapeutic drug for breast cancer irrespective of p53 status.« less

Multidrug resistance-selective antiproliferative activity of Piper amide alkaloids and synthetic analogues.

PubMed

Wang, Yue-Hu; Goto, Masuo; Wang, Li-Ting; Hsieh, Kan-Yen; Morris-Natschke, Susan L; Tang, Gui-Hua; Long, Chun-Lin; Lee, Kuo-Hsiung

2014-10-15

Twenty-five amide alkaloids (1-25) from Piper boehmeriifolium and 10 synthetic amide alkaloid derivatives (39-48) were evaluated for antiproliferative activity against eight human tumor cell lines, including chemosensitive and multidrug-resistant (MDR) cell lines. The results suggested tumor type-selectivity. 1-[7-(3,4,5-Trimethoxyphenyl)heptanoyl]piperidine (46) exhibited the best inhibitory activity (IC50=4.94 μM) against the P-glycoprotein (P-gp)-overexpressing KBvin MDR sub-line, while it and all other tested compounds, except 9, were inactive (IC50 >40 μM) against MDA-MB-231 and SK-BR-3. Structure-activity relationships (SARs) indicated that (i) 3,4,5-trimethoxy phenyl substitution is critical for selectivity against KBvin, (ii) the 4-methoxy group in this pattern is crucial for antiproliferative activity, (iii) double bonds in the side chain are not needed for activity, and (iv), in arylalkenylacyl amide alkaloids, replacement of an isobutylamino group with pyrrolidin-1-yl or piperidin-1-yl significantly improved activity. Further study on Piper amides is warranted, particularly whether side chain length affects the ability to overcome the MDR cancer phenotype. Copyright © 2014 Elsevier Ltd. All rights reserved.

Single-cell-based breeding: Rational strategy for the establishment of cell lines from a single cell with the most favorable properties.

PubMed

Yoshimoto, Nobuo; Kuroda, Shun'ichi

2014-04-01

For efficient biomolecule production (e.g., antibodies, recombinant proteins), mammalian cells with high expression rates should be selected from cell libraries, propagated while maintaining a homogenous expression rate, and subsequently stabilized at their high expression rate. Clusters of isogenic cells (i.e., colonies) have been used for these processes. However, cellular heterogeneity makes it difficult to obtain cell lines with the highest expression rates by using single-colony-based breeding. Furthermore, even among the single cells in an isogenic cell population, the desired cell properties fluctuate stochastically during long-term culture. Therefore, although the molecular mechanisms underlying stochastic fluctuation are poorly understood, it is necessary to establish excellent cell lines in order to breed single cells to have higher expression, higher stability, and higher homogeneity while suppressing stochastic fluctuation (i.e., single-cell-based breeding). In this review, we describe various methods for manipulating single cells and facilitating single-cell analysis in order to better understand stochastic fluctuation. We demonstrated that single-cell-based breeding is practical and promising by using a high-throughput automated system to analyze and manipulate single cells. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Cytogenetic characterization of three cell lines derived from primary cervical tumors of different histologic grade

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hann, E.; Beauregard, L.; Mikumo, R.

Braum et al.(1993) established three cell lines from keratinizing and nonkeratinizing cervical carcinomas. These cell lines were subsequently analyzed for growth properties and the physical state of the human papillomavirus type 16 genome. TC140, derived from a keratinizing cervical tumor, contains human papillomavirus type 16 in the episomal state. TC-146A and TC-146B, derived from a nonkeratinizing large-cell cervical carcinoma, contain human papillomavirus type 16 in the integrated state. The goal of the present study was to cytogenetically characterize these cell lines, developed from cervical carcinoma with a defined histopathology, in order to shed additional light on the biological basis ofmore » the histological and clinical heterogeneity of cervical cancers. Information on solid tumors has been limited because they are often difficult to culture and the karyotypes on the available metaphases are often complex with unidentifiable markers. The chromosomes of these three cell lines were characterized in the present study using GTG-banding. For cell line 140, the most striking chromosomal abnormalities noted were the presence of an i(5p) or i(12p) marker, an isochromosome 8q marker and multiple copies of chromosome 9. For cell line 146A, the most notable chromosomal abnormalities noted were the presence of a marker chromosome 7 with additional materials present on the long arms, an isochomosome of the long arms of chromosome 8 and a question of chromosome 19 markers. For cell line 146B, the most notable chromosomal abnormalities were found to be a deleted X chromosome, a marker chromosome 7 with additional material on the long arm, an isochromosome 8q marker, and isochromosome 16q marker and one or more copies of an isochromosome 17q marker. Fluorescent in situ hybridization experiments performed using select probes further corroborate the results of the above-mentioned conventional cytogenetic studies.« less

Predicting selective drug targets in cancer through metabolic networks

PubMed Central

Folger, Ori; Jerby, Livnat; Frezza, Christian; Gottlieb, Eyal; Ruppin, Eytan; Shlomi, Tomer

2011-01-01

The interest in studying metabolic alterations in cancer and their potential role as novel targets for therapy has been rejuvenated in recent years. Here, we report the development of the first genome-scale network model of cancer metabolism, validated by correctly identifying genes essential for cellular proliferation in cancer cell lines. The model predicts 52 cytostatic drug targets, of which 40% are targeted by known, approved or experimental anticancer drugs, and the rest are new. It further predicts combinations of synthetic lethal drug targets, whose synergy is validated using available drug efficacy and gene expression measurements across the NCI-60 cancer cell line collection. Finally, potential selective treatments for specific cancers that depend on cancer type-specific downregulation of gene expression and somatic mutations are compiled. PMID:21694718

Anti-tumour potential of a gallic acid-containing phenolic fraction from Oenothera biennis.

PubMed

Pellegrina, Chiara Dalla; Padovani, Giorgia; Mainente, Federica; Zoccatelli, Gianni; Bissoli, Gaetano; Mosconi, Silvia; Veneri, Gianluca; Peruffo, Angelo; Andrighetto, Giancarlo; Rizzi, Corrado; Chignola, Roberto

2005-08-08

A phenolic fraction purified form defatted seeds of Oenothera biennis promoted selective apoptosis of human and mouse bone marrow-derived cell lines following first-order kinetics through a caspase-dependent pathway. In non-leukemia tumour cell lines, such as human colon carcinoma CaCo(2) cells and mouse fibrosarcoma WEHI164 cells, this fraction inhibited (3)H-thymidine incorporation but not cell death or cell cycle arrest. Human peripheral blood mononuclear cells showed low sensitivity to treatment. Single bolus injection of the phenolic fraction could delay the growth of established myeloma tumours in syngeneic animals. HPLC and mass spectrometry analysis revealed that the fraction contains gallic acid. However, the biological activity of the fraction differs from the activity of this phenol and hence it should be attributed to other co-purified molecules which remain still unidentified.

Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model.

PubMed

Swift, Brenna E; Williams, Brent A; Kosaka, Yoko; Wang, Xing-Hua; Medin, Jeffrey A; Viswanathan, Sowmya; Martinez-Lopez, Joaquin; Keating, Armand

2012-07-01

Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89-99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma tumor burden in a xenograft mouse model was reduced by intravenous NK-92 cell therapy. Since multiple myeloma colony frequency correlates with survival, our observations have important clinical implications and suggest that clinical studies of NK cell lines to treat MM are warranted.

Selective synthesis and characterization of chlorins as sensitizers for photodynamic therapy

NASA Astrophysics Data System (ADS)

Montforts, Franz-Peter; Kusch, Dirk; Hoper, Frank; Braun, Stefan; Gerlach, Benjamin; Brauer, Hans-Dieter; Schermann, Guido; Moser, Joerg G.

1996-04-01

Chlorin type sensitizers have ideal photophysical properties for an application in PDT. The basic chlorin framework of these sensitizers has to be modified by attachment of lipophilic and hydrophilic residues to achieve a good cell uptake and tumor enrichment. In the present study we describe the selective synthesis of amphiphilic chlorins starting from the readily accessible red blood pigment heme. The photophysical properties of the well defined synthetic chlorins are characterized by photophysical investigations. The kinetic of cell uptake, the localization in the cell and the photodynamic behavior of the amphiphilic sensitizers are demonstrated by incubation of A 375 cancer cell lines with structurally different chlorins.

Automatic microscopy for mitotic cell location.

NASA Technical Reports Server (NTRS)

Herron, J.; Ranshaw, R.; Castle, J.; Wald, N.

1972-01-01

Advances are reported in the development of an automatic microscope with which to locate hematologic or other cells in mitosis for subsequent chromosome analysis. The system under development is designed to perform the functions of: slide scanning to locate metaphase cells; conversion of images of selected cells into binary form; and on-line computer analysis of the digitized image for significant cytogenetic data. Cell detection criteria are evaluated using a test sample of 100 mitotic cells and 100 artifacts.

Concise Review: Fluorescent Reporters in Human Pluripotent Stem Cells: Contributions to Cardiac Differentiation and Their Applications in Cardiac Disease and Toxicity.

PubMed

Den Hartogh, Sabine C; Passier, Robert

2016-01-01

In the last decade, since the first report of induced pluripotent stem cells, the stem cell field has made remarkable progress in the differentiation to specialized cell-types of various tissues and organs, including the heart. Cardiac lineage- and tissue-specific human pluripotent stem cell (hPSC) reporter lines have been valuable for the identification, selection, and expansion of cardiac progenitor cells and their derivatives, and for our current understanding of the underlying molecular mechanisms. In order to further advance the use of hPSCs in the fields of regenerative medicine, disease modeling, and preclinical drug development in cardiovascular research, it is crucial to identify functionally distinct cardiac subtypes and to study their biological signaling events and functional aspects in healthy and diseased conditions. In this review, we discuss the various strategies that have been followed to generate and study fluorescent reporter lines in hPSCs and provide insights how these reporter lines contribute to a better understanding and improvement of cell-based therapies and preclinical drug and toxicity screenings in the cardiac field. © AlphaMed Press.

Complexing Methylene Blue with Phosphorus Dendrimers to Increase Photodynamic Activity.

PubMed

Dabrzalska, Monika; Janaszewska, Anna; Zablocka, Maria; Mignani, Serge; Majoral, Jean Pierre; Klajnert-Maculewicz, Barbara

2017-02-23

The efficiency of photodynamic therapy is limited mainly due to low selectivity, unfavorable biodistribution of photosensitizers, and long-lasting skin sensitivity to light. However, drug delivery systems based on nanoparticles may overcome the limitations mentioned above. Among others, dendrimers are particularly attractive as carriers, because of their globular architecture and high loading capacity. The goal of the study was to check whether an anionic phosphorus dendrimer is suitable as a carrier of a photosensitizer-methylene blue (MB). As a biological model, basal cell carcinoma cell lines were used. We checked the influence of the MB complexation on its singlet oxygen production ability using a commercial fluorescence probe. Next, cellular uptake, phototoxicity, reactive oxygen species (ROS) generation, and cell death were investigated. The MB-anionic dendrimer complex (MB-1an) was found to generate less singlet oxygen; however, the complex showed higher cellular uptake and phototoxicity against basal cell carcinoma cell lines, which was accompanied with enhanced ROS production. Owing to the obtained results, we conclude that the photodynamic activity of MB complexed with an anionic dendrimer is higher than free MB against basal cell carcinoma cell lines.

Herpes Simplex Virus Selectively Induces Expression of the CC Chemokine RANTES/CCL5 in Macrophages through a Mechanism Dependent on PKR and ICP0

PubMed Central

Melchjorsen, Jesper; Pedersen, Finn S.; Mogensen, Søren C.; Paludan, Søren R.

2002-01-01

Recruitment of leukocytes is essential for eventual control of virus infections. Macrophages represent a leukocyte population involved in the first line of defense against many infections, including herpes simplex virus (HSV) infection. Through presentation of antigens to T cells and production of cytokines and chemokines, macrophages also constitute an important link between the innate and adaptive immune systems. Here, we have investigated the chemokine expression profile of macrophages after HSV infection and the virus-cell interactions involved. By reverse transcription-PCR and cDNA arrays, we found that HSV type 1 (HSV-1) and HSV-2 induced expression of the CC chemokine RANTES/CCL5 in murine macrophage cell lines and peritoneal cells. The CXC chemokine BCA-1/CXCL13 was also induced in peritoneal cells. Twenty-six other chemokines tested were not affected. Accumulation of RANTES mRNA was detectable after 5 h of infection, was sensitive to UV irradiation of the virus, and was preceded by accumulation of viral immediate-early mRNA and proteins. The viral components responsible for initiation of RANTES expression were examined with virus mutants and RAW 264.7 macrophage-like cells expressing a dominant negative mutant of the double-stranded-RNA-activated protein kinase (PKR). The PKR mutant cell line displayed reduced constitutive and HSV-inducible RANTES expression compared to the control cell line. HSV-1 mutants deficient in genes encoding the immediate-early proteins ICP4, ICP22, and ICP27 remained fully capable of inducing RANTES expression in macrophages. By contrast, the ability of an ICP0-deficient HSV-1 mutant to induce RANTES expression was compromised. Thus, HSV selectively induces expression of RANTES in macrophages through a mechanism dependent on cellular PKR and viral ICP0. PMID:11861845

Quantitative Structure-Cytotoxicity Relationship of Oleoylamides.

PubMed

Sakagami, Hiroshi; Uesawa, Yoshihiro; Ishihara, Mariko; Kagaya, Hajime; Kanamoto, Taisei; Terakubo, Shigemi; Nakashima, Hideki; Takao, Koichi; Sugita, Yoshiaki

2015-10-01

Eighteen oleoylamides were subjected to quantitative structure-activity relationship analysis based on their cytotoxicity, tumor selectivity and anti-HIV activity, in order to assess their biological activities. Cytotoxicity against four human oral squamous cell carcinoma (OSCC) cell lines and five human oral normal cells (gingival fibroblast, periodontal ligament fibroblast, pulp cell, oral keratinocyte, primary gingival epithelial cells) was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Tumor-selectivity (TS) was evaluated by the ratio of the mean 50% cytotoxic concentration (CC50) against normal human oral cells to that against OSCC cell lines. Potency-selectivity expression (PSE) was determined by the ratio of TS to CC50 against OSCC. Anti-HIV activity was evaluated by the ratio of CC50 to the concentration leading to 50% cytoprotection from HIV infection (EC50). Physicochemical, structural and quantum-chemical parameters were calculated based on the conformations optimized by the LowModeMD method. Among 18 derivatives, compounds 8: with a catechol group) and 18: with a (2-pyridyl)amino group) had the highest TS. On the other hand, doxorubicin and 5-fluorouracil (5-FU) were more highly cytotoxic to normal epithelial cells, displaying unexpectedly lower TS and PSE values. None of the compounds had anti-HIV activity. Among 330 chemical descriptors, 75, 73 and 19 descriptors significantly correlated to the cytotoxicity to normal and tumor cells, and TS, respectively. Multivariate statistics with chemical descriptors for molecular polarization and hydrophobicity may be useful for the evaluation of cytotoxicity and TS of oleoylamides. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Human hybrid hybridoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tiebout, R.F.; van Boxtel-Oosterhof, F.; Stricker, E.A.M.

1987-11-15

Hybrid hybridomas are obtained by fusion of two cells, each producing its own antibody. Several authors have reported the construction of murine hybrid hybridomas with the aim to obtain bispecific monoclonal antibodies. The authors have investigated, in a model system, the feasibility of constructing a human hybrid hybridoma. They fused two monoclonal cell lines: an ouabain-sensitive and azaserine/hypoxanthine-resistant Epstein-Barr virus-transformed human cell line that produces an IgG1kappa antibody directed against tetanus toxiod and an azaserine/hypoxanthine-sensitive and ouabain-resistant human-mouse xenohybrid cell line that produces a human IgG1lambda antibody directed against hepatitis-B surface antigen. Hybrid hybridoma cells were selected in culture mediummore » containing azaserine/hypoxanthine and ouabain. The hybrid nature of the secreted antibodies was analyzed by means of two antigen-specific immunoassay. The results show that it is possible, with the combined use of transformation and xenohybridization techniques, to construct human hybrid hybridomas that produce bispecific antibodies. Bispecific antibodies activity was measured by means of two radioimmunoassays.« less

Label retention identifies a multipotent mesenchymal stem cell-like population in the postnatal thymus.

PubMed

Osada, Masako; Singh, Varan J; Wu, Kenmin; Sant'Angelo, Derek B; Pezzano, Mark

2013-01-01

Thymic microenvironments are essential for the proper development and selection of T cells critical for a functional and self-tolerant adaptive immune response. While significant turnover occurs, it is unclear whether populations of adult stem cells contribute to the maintenance of postnatal thymic epithelial microenvironments. Here, the slow cycling characteristic of stem cells and their property of label-retention were used to identify a K5-expressing thymic stromal cell population capable of generating clonal cell lines that retain the capacity to differentiate into a number of mesenchymal lineages including adipocytes, chondrocytes and osteoblasts suggesting a mesenchymal stem cell-like phenotype. Using cell surface analysis both culture expanded LRCs and clonal thymic mesenchymal cell lines were found to express Sca1, PDGFRα, PDGFRβ,CD29, CD44, CD49F, and CD90 similar to MSCs. Sorted GFP-expressing stroma, that give rise to TMSC lines, contribute to thymic architecture when reaggregated with fetal stroma and transplanted under the kidney capsule of nude mice. Together these results show that the postnatal thymus contains a population of mesenchymal stem cells that can be maintained in culture and suggests they may contribute to the maintenance of functional thymic microenvironments.

Maximizing the Therapeutic Efficacy of Imatinib Mesylate-Loaded Niosomes on Human Colon Adenocarcinoma Using Box-Behnken Design.

PubMed

Kassem, Mohammed A; El-Sawy, Hossam S; Abd-Allah, Fathy I; Abdelghany, Tamer M; El-Say, Khalid M

2017-01-01

This research purposed to formulate an optimized imatinib mesylate (IM)-loaded niosomes to improve its chemotherapeutic efficacy. The influence of 3 formulation factors on niosomal vesicular size (Y 1 ), zeta potential (Y 2 ), entrapment capacity percentage (Y 3 ), the percentage of initial drug release after 2 h (Y 4 ), and the percentage of cumulative drug release after 24 h (Y 5 ) were studied and optimized using Box-Behnken design. Optimum desirability was specified and the optimized formula was prepared, stability tested, morphologically examined, checked for vesicular bilayer formation and evaluated for its in vitro cytotoxicity on 3 different cancer cell lines namely MCF-7, HCT-116, and HepG-2 in addition to 1 normal cell line to ensure its selectivity against cancer cells. The actual responses of the optimized IM formulation were 425.36 nm, -62.4 mV, 82.96%, 18.93%, and 89.45% for Y 1 , Y 2 , Y 3 , Y 4 , and Y 5 , respectively. The optimized IM-loaded niosomes confirmed the spherical vesicular shape imaged by both light and electron microscopes and further proven by differential scanning calorimetry. Moreover, the optimized formula exhibited improved stability on storage at 4 ± 2°C and superior efficacy on MCF7, HCT-116, and HepG2 as IC 50 values were 6.7, 16.4, and 7.3 folds less than those of free drug, respectively. Interestingly, IC 50 of the optimized formula against normal cell line was ranged from 3 to 11 folds higher than in different cancer cells indicating a higher selectivity of the optimized formula to cancer cells. In conclusion, the incorporation of IM in niosomes enhanced its efficacy and selectivity toward cancer cells, presenting a promising tool to fight cancer using this approach. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Effects of Escherichia coli challenge and transport stress on hematology and serum chemistry values of three genetic lines of turkeys.

PubMed

Huff, G R; Huff, W E; Rath, N C; Anthony, N B; Nestor, K E

2008-11-01

Three lines of turkeys were compared for response to an Escherichia coli challenge followed by transport stress (transport). The turkey lines were a slow-growing line selected for increased egg production (egg line), a fast-growing line selected for increased 16-wk BW (F line), and a commercial line (Comm line). Birds were challenged at 14 wk of age with an air sac injection of 5,000 to 10,000 cfu of E. coli. At 8 d postchallenge, birds were subjected to a transport stress procedure that included 12 h of holding time in a transport vehicle. The following morning all birds (n = 10 to 19 birds/line) were bled. Whole blood was analyzed using the Cell-Dyn 3500 blood analysis system (Abbott Diagnostics), and serum chemistry was measured using the Express Plus analyzer (Ciba-Corning Diagnostics Corp.). Transport significantly decreased the levels of hematocrit, hemoglobin, mean cell volume, mean corpuscular hemoglobin, glucose, triglycerides, cholesterol, phosphorus, iron, albumin, and alkaline phosphatase (AP) and increased the levels of uric acid, blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, and creatine kinase. Line differences were variable, but the levels of both iron and AP were least in the fastest-growing Comm line birds and greatest in the slowest-growing egg-line birds with intermediate values in the F line. Iron and AP were also the only parameters influenced by sex, with males having greater levels of both compared with females. The creatine kinase levels were more than 6-fold greater in transported Comm line birds, and iron levels of transported Comm males were 3-fold less than controls. Previously, the growth rate of these lines was positively correlated with increased heterophil to lymphocyte ratios and susceptibility to colibacillosis. The differences seen in the Comm line for these commonly measured blood parameters suggest that they may be useful for profiling flocks to determine their response to transport stress and feed withdrawal.

Epigenetic inactivation of VGF associated with Urothelial Cell Carcinoma and its potential as a non-invasive biomarker using urine

PubMed Central

Kagohara, Luciane Tsukamoto; Maldonado, Leonel; Brait, Mariana; Schoenberg, Mark; Bivalacqua, Trinity; Netto, George J; Koch, Wayne; Sidransky, David; Hoque, Mohammad O.

2014-01-01

Background: To identify new epigenetic markers and further characterize Urothelial Cell Carcinoma (UCC), we tested the promoter methylation (PM) status of 19 genes previously identified as cancer specific methylated genes in other solid tumors. Methods: We used bisulfite sequencing, methylation specific PCR and quantitative methylation specific PCR (QMSP) to test the PM status of 19 genes in urothelial cancer cell lines. Results: Among the 19 genes tested, VGF was found to be completely methylated in several UCC cell lines. VGF QMSP analysis showed that methylation values of almost all the primary 19 UCC tissues were higher than the paired normal tissues (P=0.009). In another cohort, 12/35 (34.3%) of low grade UCC cases displayed VGF methylation. As a biomarker for non-invasive detection of UCC, VGF showed a significantly higher frequency of methylation in urine from UCC cases (8/20) compared to controls (1/20) (P=0.020). After treatment of cell lines with 5-Aza-2'-deoxycytidine, VGF was robustly re-expressed. Forced expression of VGF in bladder cancer cell lines inhibited cell growth. Conclusion: Selection of candidates from genome-wide screening approach in other solid tumors successfully identified UCC specific methylated genes. PMID:24830820

RB1 status in triple negative breast cancer cells dictates response to radiation treatment and selective therapeutic drugs.

PubMed

Robinson, Tyler J W; Liu, Jeff C; Vizeacoumar, Frederick; Sun, Thomas; Maclean, Neil; Egan, Sean E; Schimmer, Aaron D; Datti, Alessandro; Zacksenhaus, Eldad

2013-01-01

Triple negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which only chemotherapy and radiation therapy are currently available. The retinoblastoma (RB1) tumor suppressor is frequently lost in human TNBC. Knockdown of RB1 in luminal BC cells was shown to affect response to endocrine, radiation and several antineoplastic drugs. However, the effect of RB1 status on radiation and chemo-sensitivity in TNBC cells and whether RB1 status affects response to divergent or specific treatment are unknown. Using multiple basal-like and claudin-low cell lines, we hereby demonstrate that RB-negative TNBC cell lines are highly sensitive to gamma-irradiation, and moderately more sensitive to doxorubicin and methotrexate compared to RB-positive TNBC cell lines. In contrast, RB1 status did not affect sensitivity of TNBC cells to multiple other drugs including cisplatin (CDDP), 5-fluorouracil, idarubicin, epirubicin, PRIMA-1(met), fludarabine and PD-0332991, some of which are used to treat TNBC patients. Moreover, a non-biased screen of ∼3400 compounds, including FDA-approved drugs, revealed similar sensitivity of RB-proficient and -deficient TNBC cells. Finally, ESA(+)/CD24(-/low)/CD44(+) cancer stem cells from RB-negative TNBC lines were consistently more sensitive to gamma-irradiation than RB-positive lines, whereas the effect of chemotherapy on the cancer stem cell fraction varied irrespective of RB1 expression. Our results suggest that patients carrying RB-deficient TNBCs would benefit from gamma-irradiation as well as doxorubicin and methotrexate therapy, but not necessarily from many other anti-neoplastic drugs.

A Synthetic Interaction Screen Identifies Factors Selectively Required for Proliferation and TERT Transcription in p53-Deficient Human Cancer Cells

PubMed Central

Park, Sung Mi; Zhu, Lihua J.; Debily, Marie-anne; Kittler, Ellen L. W.; Zapp, Maria L.; Lapointe, David; Gobeil, Stephane; Virbasius, Ching-Man; Green, Michael R.

2012-01-01

Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi)–based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53−) human cancer cells. We find that compared to p53-competent (p53+) human cancer cell lines, diverse p53− human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53− cells, RNAi–mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53− but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53− cancer cells. PMID:23284306

Gene expression analysis of rheumatoid arthritis synovial lining regions by cDNA microarray combined with laser microdissection: up-regulation of inflammation-associated STAT1, IRF1, CXCL9, CXCL10, and CCL5

PubMed Central

Yoshida, S; Arakawa, F; Higuchi, F; Ishibashi, Y; Goto, M; Sugita, Y; Nomura, Y; Niino, D; Shimizu, K; Aoki, R; Hashikawa, K; Kimura, Y; Yasuda, K; Tashiro, K; Kuhara, S; Nagata, K; Ohshima, K

2012-01-01

Objectives The main histological change in rheumatoid arthritis (RA) is the villous proliferation of synovial lining cells, an important source of cytokines and chemokines, which are associated with inflammation. The aim of this study was to evaluate gene expression in the microdissected synovial lining cells of RA patients, using those of osteoarthritis (OA) patients as the control. Methods Samples were obtained during total joint replacement from 11 RA and five OA patients. Total RNA from the synovial lining cells was derived from selected specimens by laser microdissection (LMD) for subsequent cDNA microarray analysis. In addition, the expression of significant genes was confirmed immunohistochemically. Results The 14 519 genes detected by cDNA microarray were used to compare gene expression levels in synovial lining cells from RA with those from OA patients. Cluster analysis indicated that RA cells, including low- and high-expression subgroups, and OA cells were stored in two main clusters. The molecular activity of RA was statistically consistent with its clinical and histological activity. Expression levels of signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor 1 (IRF1), and the chemokines CXCL9, CXCL10, and CCL5 were statistically significantly higher in the synovium of RA than in that of OA. Immunohistochemically, the lining synovium of RA, but not that of OA, clearly expressed STAT1, IRF1, and chemokines, as was seen in microarray analysis combined with LMD. Conclusions Our findings indicate an important role for lining synovial cells in the inflammatory and proliferative processes of RA. Further understanding of the local signalling in structural components is important in rheumatology. PMID:22401175

Development of recombinant adeno-associated virus vectors carrying small interfering RNA (shHec1)-mediated depletion of kinetochore Hec1 protein in tumor cells.

PubMed

Li, L; Yang, L; Scudiero, D A; Miller, S A; Yu, Z-X; Stukenberg, P T; Shoemaker, R H; Kotin, R M

2007-05-01

Transcript depletion using small interfering RNA (siRNA) technology represents a potentially valuable technique for the treatment of cancer. However, delivering therapeutic quantities of siRNA into solid tumors by chemical transfection is not feasible, whereas viral vectors efficiently transduce many human tumor cell lines. Yet producing sufficient quantities of viral vectors that elicit acute and selective cytotoxicity remains a major obstacle for preclinical and clinical trials. Using the invertebrate Spodoptera frugiperda (Sf9) cell line, we were able to produce high titer stocks of cytotoxic recombinant adeno-associated virus (rAAV) that express short hairpin RNA (shRNA) and that efficiently deplete Hec1 (highly expressed in cancer 1), or Kntc2 (kinetochore-associated protein 2), a kinetochore protein directly involved in kinetochore microtubule interactions, chromosome congression and spindle checkpoint signaling. Depletion of Hec1 protein results in persistent spindle checkpoint activation followed by cell death. Because Hec1 expression and activity are only present in mitotic cells, non-dividing cells were not affected by rAAV treatment. On the basis of the results of screening 56 human tumor cell lines with three different serotype vectors, we used a tumor xenograft model to test the effects in vivo. The effects of the shHec1 vector were evident in sectioned and stained tumors. The experiments with rAAV-shRNA vectors demonstrate the utility of producing vectors in invertebrate cells to obtain sufficient concentrations and quantities for solid tumor therapy. This addresses an important requirement for cancer gene therapy, to produce cytotoxic vectors in sufficient quantities and concentrations to enable quantitative transduction and selective killing of solid tumor cells.

Increased expression of annexin I and thioredoxin detected by two-dimensional gel electrophoresis of drug resistant human stomach cancer cells.

PubMed

Sinha, P; Hütter, G; Köttgen, E; Dietel, M; Schadendorf, D; Lage, H

1998-11-18

The therapy of advanced cancer using chemotherapy alone or in combination with radiation or hyperthermia yields an overall response rate of about 20-50%. This success is often marred by the development of resistance to cytostatic drugs. Our aim was to study the global analysis of protein expression in the development of chemoresistance in vitro. We therefore used a cell culture model derived from the gastric carcinoma cell line EPG 85-257P. A classical multidrug-resistant subline EPG85-257RDB selected to daunorubicin and an atypical multidrug-resistant cell variant EPG85-257RNOV selected to mitoxantrone, were analysed using two-dimensional electrophoresis in immobilized pH-gradients (pH 4.0-8.0) in the first dimension and linear polyacrylamide gels (12%) in the second dimension. After staining with coomassie brilliant blue, image analysis was performed using the PDQuest system. Spots of interest were isolated using preparative two-dimensional electrophoresis and subjected to microsequencing. A total of 241 spots from the EPG85-257RDB-standard and 289 spots from the EPG85-257RNOV-standard could be matched to the EPG85-257P-standard. Microsequencing after enzymatic hydrolysis in gel, mass spectrometric data and sequencing of the peptides after their fractionation using microbore HPLC identified that two proteins annexin I and thioredoxin were overexpressed in chemoresistant cell lines. Annexin I was present in both the classical and the atypical multidrug-resistant cells. Thioredoxin was found to be overexpressed only in the atypical multidrug-resistant cell line.

Oxidative Stress Triggered by Apigenin Induces Apoptosis in a Comprehensive Panel of Human Cervical Cancer-Derived Cell Lines.

PubMed

Souza, Raquel P; Bonfim-Mendonça, Patrícia de S; Gimenes, Fabrícia; Ratti, Bianca A; Kaplum, Vanessa; Bruschi, Marcos L; Nakamura, Celso V; Silva, Sueli O; Maria-Engler, Silvya S; Consolaro, Marcia E L

2017-01-01

Recently, the cytotoxic effects of apigenin (4',5,7-trihydroxyflavone), particularly its marked inhibition of cancer cell viability both in vitro and in vivo, have attracted the attention of the anticancer drug discovery field. Despite this, there are few studies of apigenin in cervical cancer, and these studies have mostly been conducted using HeLa cells. To evaluate the possibility of apigenin as a new therapeutic candidate for cervical cancer, we evaluated its cytotoxic effects in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16 and HPV 18-positive), and C33A (HPV-negative) cells in comparison to a nontumorigenic spontaneously immortalized human epithelial cell line (HaCaT). Our results demonstrated that apigenin had a selective cytotoxic effect and could induce apoptosis in all cervical cancer cell lines which were positively marked with Annexin V, but not in HaCaT (control cells). Additionally, apigenin was able to induce mitochondrial redox impairment, once it increased ROS levels and H 2 O 2 , decreased the Δ ψm , and increased LPO. Still, apigenin was able to inhibit migration and invasion of cancer cells. Thus, apigenin appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV genotypes.

Oxidative Stress Triggered by Apigenin Induces Apoptosis in a Comprehensive Panel of Human Cervical Cancer-Derived Cell Lines

PubMed Central

Souza, Raquel P.; Gimenes, Fabrícia; Ratti, Bianca A.; Kaplum, Vanessa; Bruschi, Marcos L.; Nakamura, Celso V.; Maria-Engler, Silvya S.

2017-01-01

Recently, the cytotoxic effects of apigenin (4′,5,7-trihydroxyflavone), particularly its marked inhibition of cancer cell viability both in vitro and in vivo, have attracted the attention of the anticancer drug discovery field. Despite this, there are few studies of apigenin in cervical cancer, and these studies have mostly been conducted using HeLa cells. To evaluate the possibility of apigenin as a new therapeutic candidate for cervical cancer, we evaluated its cytotoxic effects in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16 and HPV 18-positive), and C33A (HPV-negative) cells in comparison to a nontumorigenic spontaneously immortalized human epithelial cell line (HaCaT). Our results demonstrated that apigenin had a selective cytotoxic effect and could induce apoptosis in all cervical cancer cell lines which were positively marked with Annexin V, but not in HaCaT (control cells). Additionally, apigenin was able to induce mitochondrial redox impairment, once it increased ROS levels and H2O2, decreased the Δψm, and increased LPO. Still, apigenin was able to inhibit migration and invasion of cancer cells. Thus, apigenin appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV genotypes. PMID:28191273

Withaferin-A Induces Apoptosis in Osteosarcoma U2OS Cell Line via Generation of ROS and Disruption of Mitochondrial Membrane Potential.

PubMed

Zhang, Hui-Liang; Zhang, Hong

2017-01-01

Withaferin-A (WF-A) is a well-known dietary compound isolated from Withania sominifera . It has tremendous pharmacological potential and has been shown to exhibit antiproliferative activity against several types of cancerous cells. Currently, the main focus of anti-cancer therapeutic development is to identify apoptosis inducing drug-like molecules. Osteosarcoma is a rare type of osteocancer, affecting human. The present study therefore focused on the evaluation of antitumor potential of WF-A against several osteosarcoma cell lines. MTT assay was used to evaluate WF-A against osteosarcoma cell lines and to calculate the IC 50 . DAPI staining was used to confirm the apoptosis inducing potential of WF-A. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, and Western blotting were used to confirm the basis of apoptosis. The results revealed that that WF-A exhibited strong antiproliferative activity against all the cells lines, with IC 50 ranging from 0.32 to 7.6 μM. The lowest IC 50 (0.32 μM) was observed against U2OS cell line and therefore it was selected for further analysis. DAPI staining indicated that WF-A exhibited antiproliferative activity via induction of apoptosis. Moreover, WF-A induced ROS-mediated reduction in mitochondrial membrane potential ΔΨm) in a dose-dependent manner and activation of caspase-3 in osteosarcoma cells. We propose that WF-A may prove a potent therapeutic agent for inducing apoptosis in osteosarcoma cell lines via generation of ROS and disruption of mitochondrial membrane potential. WF-A exhibits strong anticancer activity against osteosarcoma cell linesAntiproliferative activity of WF-A is via induction of apoptosisWF-A induced ROS-mediated reduction in mitochondrial membrane potentialWF-A induced expression of caspase-3 in osteosarcoma cells. Abbreviations used: WA: Withaferin A; ROS: Reactive oxygen species; OS: Osteosarcoma; MMP: Mitochondrial membrane potential.

A Cre Mouse Line for Probing Irradiance- and Direction-Encoding Retinal Networks

PubMed Central

Sabbah, Shai

2017-01-01

Abstract Cell type-specific Cre driver lines have revolutionized the analysis of retinal cell types and circuits. We show that the transgenic mouse Rbp4-Cre selectively labels several retinal neuronal types relevant to the encoding of absolute light intensity (irradiance) and visual motion. In the ganglion cell layer (GCL), most marked cells are wide-field spiking polyaxonal amacrine cells (ACs) with sustained irradiance-encoding ON responses that persist during chemical synaptic blockade. Their arbors spread about 1 mm across the retina and are restricted to the inner half of the ON sublamina of the inner plexiform layer (IPL). There, they costratify with dendrites of M2 intrinsically photosensitive retinal ganglion cells (ipRGCs), to which they are tracer coupled. We propose that synaptically driven and intrinsic photocurrents of M2 cells pass through gap junctions to drive AC light responses. Also marked in this mouse are two types of RGCs. R-cells have a bistratified dendritic arbor, weak directional tuning, and irradiance-encoding ON responses. However, they also receive excitatory OFF input, revealed during ON-channel blockade. Serial blockface electron microscopic (SBEM) reconstruction confirms OFF bipolar input, and reveals that some OFF input derives from a novel type of OFF bipolar cell (BC). R-cells innervate specific layers of the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC). The other marked RGC type (RDS) is bistratified, transient, and ON-OFF direction selective (DS). It apparently innervates the nucleus of the optic tract (NOT). The Rbp4-Cre mouse will be valuable for targeting these cell types for further study and for selectively manipulating them for circuit analysis. PMID:28466070

Synthesis and in vitro antiproliferative activities of (5-aryl-1,2,4-oxadiazole-3-yl) methyl d-ribofuranosides.

PubMed

Avanzo, Romina E; Padrón, José M; D'Accorso, Norma B; Fascio, Mirta L

2017-08-15

The emergence of multidrug resistance cell lines is one of the major obstacles in the success of cancer chemotherapeutic treatment. Therefore, it remains a big challenge the development of new and effective drugs to defeat cancer. The presence of nitrogen heterocycles in the architectural design of drugs has led to the discovery of new leading compounds. Herein, we report the synthesis, characterization and in vitro antiproliferative activity against six cancer cell lines of d-ribofuranoside derivatives bearing a 1,2,4-oxadiazolic ring, with the aim of developing new active compounds. Most of these derivatives exhibit significant antiproliferative activities in the micromolar range. Noteworthy, the most potent compound of the series showed better selectivity towards the more resistant colon cancer cell line WiDr. Copyright © 2017 Elsevier Ltd. All rights reserved.

Gene mutations and increased levels of p53 protein in human squamous cell carcinomas and their cell lines.

PubMed Central

Burns, J. E.; Baird, M. C.; Clark, L. J.; Burns, P. A.; Edington, K.; Chapman, C.; Mitchell, R.; Robertson, G.; Soutar, D.; Parkinson, E. K.

1993-01-01

Using immunocytochemical and Western blotting techniques we have demonstrated the presence of abnormally high levels of p53 protein in 8/24 (33%) of human squamous cell carcinomas (SCC) and 9/18 (50%) of SCC cell lines. There was a correlation between the immunocytochemical results obtained with eight SCC samples and their corresponding cell lines. Direct sequencing of PCR-amplified, reverse transcribed, p53 mRNA confirmed the expression of point mutations in six of the positive cell lines and detected in-frame deletions in two others. We also detected two stop mutations and three out-of-frame deletions in five lines which did not express elevated levels of p53 protein. Several of the mutations found in SCC of the tongue (3/7) were in a region (codons 144-166) previously identified as being a p53 mutational hot spot in non-small cell lung tumours (Mitsudomi et al., 1992). In 11/13 cases only the mutant alleles were expressed suggesting loss or reduced expression of the wild type alleles in these cases. Six of the mutations were also detected in the SCCs from which the lines were derived, strongly suggesting that the mutations occurred, and were selected, in vivo. The 12th mutation GTG-->GGG (valine-->glycine) at codon 216 was expressed in line SCC-12 clone B along with an apparently normal p53 allele and is to our knowledge a novel mutation. Line BICR-19 also expressed a normal p53 allele in addition to one where exon 10 was deleted. Additionally 15 of the SCC lines (including all of those which did not show elevated p53 protein levels) were screened for the presence of human papillomavirus types 16 and 18 and were found to be negative. These results are discussed in relation to the pathogenesis of SCC and the immortalisation of human keratinocytes in vitro. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8390283

Differentiation and Characterization of Myeloid Cells

PubMed Central

Gupta, Dipti; Shah, Hetavi Parag; Malu, Krishnakumar; Berliner, Nancy; Gaines, Peter

2015-01-01

Recent molecular studies of myeloid differentiation have utilized several in vitro models of myelopoiesis, generated from either ex vivo differentiated bone marrow progenitors or induced immortalized myeloid cell lines. Ex vivo differentiation begins with an enriched population of bone marrow-derived hematopoietic stem cells generated by lineage depletion and/or positive selection for CD34+ antigen (human) or Sca-1+ (mouse) cells, which are then expanded and subsequently induced in vitro in a process that recapitulates normal myeloid development. Myeloid cell lines include two human leukemic cell lines, NB-4 and HL-60, which have been demonstrated to undergo retinoic acid–induced myeloid development, however, both cell lines exhibit defects in the upregulation of late-expressed neutrophil-specific genes. Multiple murine factor–dependent cell models of myelopoiesis are also available that express the full range of neutrophil maturation markers, including: 32Dcl3 cells, which undergo G-CSF-induced myeloid maturation, EML/EPRO cells, which develop into mature neutrophils in response to cytokines and retinoic acid, and ER-Hoxb8 cells, which undergo myeloid maturation upon removal of estradial in the maintenance medium. In this unit, the induction of myeloid maturation in each of these model systems is described, including their differentiation to either neutrophils or macrophages, if applicable. Commonly used techniques to test for myeloid characteristics of developing cells are also described, including flow cytometry and real time RT-PCR. Together, these assays provide a solid foundation for in vitro investigations of myeloid development with either human or mouse models. PMID:24510620

Improved selectivity and cytotoxic effects of irinotecan via liposomal delivery: A comparative study on Hs68 and HeLa cells.

PubMed

Casadó, Ana; Mora, Margarita; Sagristá, Maria Lluïsa; Rello-Varona, Santi; Acedo, Pilar; Stockert, Juan Carlos; Cañete, Magdalena; Villanueva, Angeles

2017-11-15

Irinotecan (CPT-11) is an effective chemotherapeutic agent widely used to treat different cancers. Otherwise, the liposomal delivery of anti-tumor agents has been shown to be a promising strategy. The aim of this study has been to analyze the effect of liposomal CPT-11 (CPT-11lip) on two human cell lines (Hs68 and HeLa) to establish the suitability of this CPT-11 nanocarrier. We have demonstrated the highest uptake of CPT-11lip in comparison with that of CPT-11sol, in lactate buffer, and that CPT-11lip was internalized in the cells through an endocytic process whereas CPT-11sol does so by passive diffusion. CPT-11lip was not cytotoxic to normal fibroblast Hs68 cells, but induced a massive apoptosis accompanied by cell senescence in HeLa cells. CPT-11lip treatment modified the morphology of HeLa cells, induced different cell cycle alterations and accumulated into lysosomes in both cell lines. In particular, CPT-11lip treatment showed that surviving HeLa cells remained in a state of senescence whereas only a temporal growth arrest was induced in Hs68 cells. Results of RT-PCR indicated that the different responses in Hs68 (survival) and HeLa cells (apoptotic death), seemed to be induced by a p53- and p53- independent mechanism, respectively. An analysis of DNA damage also determined that released CPT-11 from liposomes was able to reach the nucleus and exert a genotoxic effect in both cell lines, which was repaired in Hs68 but not in HeLa cells. All results indicate that phospholipid-cholesterol liposomes possess optimum properties for CPT-11 delivery, being biocompatible and selectively cytotoxic against HeLa tumorigenic cells. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

PIM1 kinase regulates cell death, tumor growth and chemotherapy response in triple-negative breast cancer.

PubMed

Brasó-Maristany, Fara; Filosto, Simone; Catchpole, Steven; Marlow, Rebecca; Quist, Jelmar; Francesch-Domenech, Erika; Plumb, Darren A; Zakka, Leila; Gazinska, Patrycja; Liccardi, Gianmaria; Meier, Pascal; Gris-Oliver, Albert; Cheang, Maggie Chon U; Perdrix-Rosell, Anna; Shafat, Manar; Noël, Elodie; Patel, Nirmesh; McEachern, Kristen; Scaltriti, Maurizio; Castel, Pau; Noor, Farzana; Buus, Richard; Mathew, Sumi; Watkins, Johnathan; Serra, Violeta; Marra, Pierfrancesco; Grigoriadis, Anita; Tutt, Andrew N

2016-11-01

Triple-negative breast cancers (TNBCs) have poor prognosis and lack targeted therapies. Here we identified increased copy number and expression of the PIM1 proto-oncogene in genomic data sets of patients with TNBC. TNBC cells, but not nonmalignant mammary epithelial cells, were dependent on PIM1 for proliferation and protection from apoptosis. PIM1 knockdown reduced expression of the anti-apoptotic factor BCL2, and dynamic BH3 profiling of apoptotic priming revealed that PIM1 prevents mitochondrial-mediated apoptosis in TNBC cell lines. In TNBC tumors and their cellular models, PIM1 expression was associated with several transcriptional signatures involving the transcription factor MYC, and PIM1 depletion in TNBC cell lines decreased, in a MYC-dependent manner, cell population growth and expression of the MYC target gene MCL1. Treatment with the pan-PIM kinase inhibitor AZD1208 impaired the growth of both cell line and patient-derived xenografts and sensitized them to standard-of-care chemotherapy. This work identifies PIM1 as a malignant-cell-selective target in TNBC and the potential use of PIM1 inhibitors for sensitizing TNBC to chemotherapy-induced apoptotic cell death.

A multi-landing pad DNA integration platform for mammalian cell engineering

PubMed Central

Gaidukov, Leonid; Wroblewska, Liliana; Teague, Brian; Nelson, Tom; Zhang, Xin; Liu, Yan; Jagtap, Kalpana; Mamo, Selamawit; Tseng, Wen Allen; Lowe, Alexis; Das, Jishnu; Bandara, Kalpanie; Baijuraj, Swetha; Summers, Nevin M; Zhang, Lin; Weiss, Ron

2018-01-01

Abstract Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three ‘landing pad’ recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection. We used this method to controllably integrate up to nine copies of a monoclonal antibody, representing about 100 kb of heterologous DNA in 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload resulted in a linear increase in antibody expression. Overall, this multi-copy site-specific integration platform allows for controllable and reproducible insertion of large amounts of DNA into stable genomic sites, which has broad applications for mammalian synthetic biology, recombinant protein production and biomanufacturing. PMID:29617873

Cytotoxic activity of some medicinal plants from hamedan district of iran.

PubMed

Behzad, Sahar; Pirani, Atefeh; Mosaddegh, Mahmoud

2014-01-01

Medicinal plants have been investigated for possible anti-cancer effects. The aim of the present study was to examine the cytotoxic activity of several medicinal plants on different tumor cell lines. 11 selected plant species which have been used in folkloric prescriptions were collected from different sites of Hamedan district of Iran. The methanolic extracts of the plants were prepared and their cytotoxic effects on four human cancer cell lines (A549, human lung adenocarcinoma; MCF7, human breast adenocarcinoma; HepG2, hepatocellular carcinoma and HT-29, human colon carcinoma) and one normal cell line (MDBK, bovine kidney) were examined using the MTT assay. Three of these were exhibited antiproliferative activity against one or more of the cell lines. The extract from Primula auriculata demonstrated the highest cytotoxicity with IC50 of 25.79, 35.79 and 43.34 μg.mL-1 against MCF7, HepG2 and HT- 29 cells, respectively. For some of the plants, their traditional use was correlated with the cytotoxic results, whereas for others the results may support the non-cytotoxicity of species used traditionally as natural remedies. The cytotoxic species could be considered as potential of anticancer compounds.

Disrupting CCT-β : β-tubulin selectively kills CCT-β overexpressed cancer cells through MAPKs activation

PubMed Central

Liu, Yan-Jin; Kumar, Vathan; Lin, Yuan-Feng; Liang, Po-Huang

2017-01-01

We have previously demonstrated the ability of I-Trp to disrupt the protein–protein interaction of β-tubulin with chaperonin-containing TCP-1β (CCT-β). This caused more severe apoptosis in multidrug-resistant MES-SA/Dx5, compared to MES-SA, due to its higher CCT-β overexpression. In this study, we screened a panel of cancer cell lines, finding CCT-β overexpression in the triple-negative breast cancer cell line MDA-MB-231, colorectal cancer cell lines Colo205 and HCT116, and a gastric cancer cell line MKN-45. Thus, I-Trp killed these cancers with sub- to low-μM EC50, whereas it was non-toxic to MCF-10A. We then synthesized analogs of I-Trp and evaluated their cytotoxicity. Furthermore, apoptotic mechanism investigations revealed the activation of both protein ubiquitination/degradation and ER-associated protein degradation pathways. These pathways proceeded through activation of MAPKs at the onset of CCT-β : β-tubulin complex disruption. We thus establish an effective strategy to treat CCT-β overexpressed cancers by disrupting the CCT-β : β-tubulin complex. PMID:28906489

In Vitro Growth Inhibitory Activities of Natural Products from Irciniid Sponges against Cancer Cells: A Comparative Study

PubMed Central

BenRedjem Romdhane, Yosr; Elbour, Monia; Carbone, Marianna; Ciavatta, Maria Letizia; Gavagnin, Margherita; Mathieu, Véronique; Lefranc, Florence; Ktari, Leila; Ben Mustapha, Karim; Boudabous, Abdellatif; Kiss, Robert

2016-01-01

Marine sponges of the Irciniidae family contain both bioactive furanosesterterpene tetronic acids (FTAs) and prenylated hydroquinones (PHQs). Both classes of compounds are known for their anti-inflammatory, antioxidant, and antimicrobial properties and known to display growth inhibitory effects against various human tumor cell lines. However, the different experimental conditions of the reported in vitro bioassays, carried out on different cancer cell lines within separate studies, prevent realistic actual discrimination between the two classes of compounds from being carried out in terms of growth inhibitory effects. In the present work, a chemical investigation of irciniid sponges from Tunisian coasts led to the purification of three known FTAs and three known PHQs. The in vitro growth inhibitory properties of the six purified compounds have been evaluated in the same experiment in a panel of five human and one murine cancer cell lines displaying various levels of sensitivity to proapoptotic stimuli. Surprisingly, FTAs and PHQs elicited distinct profiles of growth inhibitory-responses, differing by one to two orders of magnitude in favor of the PHQs in all cell lines. The obtained comparative results are discussed in the light of a better selection of drug candidates from natural sources. PMID:27597966

From Never Born Proteins to Minimal Living Cells: two projects in synthetic biology.

PubMed

Luisi, Pier Luigi; Chiarabelli, Cristiano; Stano, Pasquale

2006-12-01

The Never Born Proteins (NBPs) and the Minimal Cell projects are two currently developed research lines belonging to the field of synthetic biology. The first deals with the investigation of structural and functional properties of de novo proteins with random sequences, selected and isolated using phage display methods. The minimal cell is the simplest cellular construct which displays living properties, such as self-maintenance, self-reproduction and evolvability. The semi-synthetic approach to minimal cells involves the use of extant genes and proteins in order to build a supramolecular construct based on lipid vesicles. Results and outlooks on these two research lines are shortly discussed, mainly focusing on their relevance to the origin of life studies.

Hybridization of downregulated-COMT transgenic switchgrass lines with field-selected switchgrass for improved biomass traits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baxter, Holly L.; Alexander, Lisa W.; Mazarei, Mitra

Transgenic switchgrass (Panicum virgatum L.) has been produced for improved cell walls for biofuels. For instance, downregulated caffeic acid 3-O-methyltransferase (COMT) switchgrass produced significantly more biomass and biofuel than the non-transgenic progenitor line. In this present study we sought to further improve biomass characteristics by crossing the downregulated COMT T 1 lines with high-yielding switchgrass accessions in two genetic backgrounds ('Alamo' and 'Kanlow'). Crosses and T 2 progeny analyses were made under greenhouse conditions to assess maternal effects, plant morphology and yield, and cell wall traits. Female parent type influenced morphology, but had no effect on cell wall traits. Tmore » 2 hybrids produced with T 1 COMT-downregulated switchgrass as the female parent were taller, produced more tillers, and produced 63% more biomass compared with those produced using the field selected accession as the female parent. Transgene status (presence or absence of transgene) influenced both growth and cell wall traits. T 2 transgenic hybrids were 7% shorter 80 days after sowing and produced 43% less biomass than non-transgenic null-segregant hybrids. Cell wall-related differences included lower lignin content, reduced syringyl-to-guaiacyl (S/G) lignin monomer ratio, and a 12% increase in total sugar release in the T 2 transgenic hybrids compared to non-transgenic null segregants. This is the first study to evaluate the feasibility of transferring the low-recalcitrance traits associated with a transgenic switchgrass line into high-yielding field varieties in an attempt to improve growth-related traits. Lastly, our results provide insights into the possible improvement of switchgrass productivity via biotechnology paired with plant breeding.« less

Hybridization of downregulated-COMT transgenic switchgrass lines with field-selected switchgrass for improved biomass traits

DOE PAGES

Baxter, Holly L.; Alexander, Lisa W.; Mazarei, Mitra; ...

2016-01-21

Transgenic switchgrass (Panicum virgatum L.) has been produced for improved cell walls for biofuels. For instance, downregulated caffeic acid 3-O-methyltransferase (COMT) switchgrass produced significantly more biomass and biofuel than the non-transgenic progenitor line. In this present study we sought to further improve biomass characteristics by crossing the downregulated COMT T 1 lines with high-yielding switchgrass accessions in two genetic backgrounds ('Alamo' and 'Kanlow'). Crosses and T 2 progeny analyses were made under greenhouse conditions to assess maternal effects, plant morphology and yield, and cell wall traits. Female parent type influenced morphology, but had no effect on cell wall traits. Tmore » 2 hybrids produced with T 1 COMT-downregulated switchgrass as the female parent were taller, produced more tillers, and produced 63% more biomass compared with those produced using the field selected accession as the female parent. Transgene status (presence or absence of transgene) influenced both growth and cell wall traits. T 2 transgenic hybrids were 7% shorter 80 days after sowing and produced 43% less biomass than non-transgenic null-segregant hybrids. Cell wall-related differences included lower lignin content, reduced syringyl-to-guaiacyl (S/G) lignin monomer ratio, and a 12% increase in total sugar release in the T 2 transgenic hybrids compared to non-transgenic null segregants. This is the first study to evaluate the feasibility of transferring the low-recalcitrance traits associated with a transgenic switchgrass line into high-yielding field varieties in an attempt to improve growth-related traits. Lastly, our results provide insights into the possible improvement of switchgrass productivity via biotechnology paired with plant breeding.« less

Synthesis, spectroscopic, physicochemical and structural characterization of tetrandrine-based macrocycles functionalized with acridine and anthracene groups: DNA binding and anti-proliferative activity.

PubMed

Calvillo-Páez, Viviana; Sotelo-Mundo, Rogerio R; Leyva-Peralta, Mario; Gálvez-Ruiz, Juan Carlos; Corona-Martínez, David; Moreno-Corral, Ramón; Escobar-Picos, Raymundo; Höpfl, Herbert; Juárez-Sánchez, Octavio; Lara, Karen Ochoa

2018-04-25

In this work, we report on the synthesis of two new mono-alkylated tetrandrine derivatives with acridine and anthracene units, MAcT and MAnT. The compounds were fully characterized by physicochemical techniques and single-crystal X-ray diffraction analysis. In addition, both derivatives were studied as nucleotide receptors and double-stranded DNA binders in aqueous phosphate buffer at pH = 7.2 using UV-vis and fluorescence spectroscopy. According to the molecular recognition studies, MAcT and MAnT exhibit high affinity (K ∼ 10 5  M -1 ) and selectivity for ds-DNA, presumably in an intercalation mode. Finally, the anti-proliferative effects of the tetrandrine derivatives on different cancer cell lines were explored, revealing promising activities. Particularly, the mono-anthracene tetrandrine derivative MAnT showed an IC 50 of 2.74 μg/mL on the HeLa cervical cancer cell line, representing a value 3.3 times smaller than that obtained for unsubstituted tetrandrine. Examination of the cytotoxic effects on the HeLa cell line by inverted microscopy suggests that the cell death mechanism consists basically in apoptosis. The molecular modelling of three ds-DNA-MAcT complexes, suggested that the macrocycles may use an intercalation binding mode towards DNA. MAcT is predicted to bind into the major groove of the ds-DNA providing non-covalent interactions such as electrostatic, van der Waals and hydrophobic interactions that lead to selectivity. Overall experimental data supports the mode of action of MAnT and MAcT as cytotoxic compounds against cancer cell lines via a DNA interaction mechanism. Copyright © 2018 Elsevier B.V. All rights reserved.

Plumbagin induces cell cycle arrest and autophagy and suppresses epithelial to mesenchymal transition involving PI3K/Akt/mTOR-mediated pathway in human pancreatic cancer cells

PubMed Central

Wang, Feng; Wang, Qi; Zhou, Zhi-Wei; Yu, Song-Ning; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Wang, Dong; Yang, Yin-Xue; Yang, Tianxing; Sun, Tao; Li, Min; Qiu, Jia-Xuan; Zhou, Shu-Feng

2015-01-01

Plumbagin (PLB), an active naphthoquinone compound, has shown potent anticancer effects in preclinical studies; however, the effect and underlying mechanism of PLB for the treatment of pancreatic cancer is unclear. This study aimed to examine the pancreatic cancer cell killing effect of PLB and investigate the underlying mechanism in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that PLB exhibited potent inducing effects on cell cycle arrest in PANC-1 and BxPC-3 cells via the modulation of cell cycle regulators including CDK1/CDC2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. PLB treatment concentration- and time-dependently increased the percentage of autophagic cells and significantly increased the expression level of phosphatase and tensin homolog, beclin 1, and the ratio of LC3-II over LC3-I in both PANC-1 and BxPC-3 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin and p38 mitogen-activated protein kinase (p38 MAPK) pathways and activation of 5′-AMP-dependent kinase as indicated by their altered phosphorylation, contributing to the proautophagic activities of PLB in both cell lines. Furthermore, SB202190, a selective inhibitor of p38 MAPK, and wortmannin, a potent, irreversible, and selective PI3K inhibitor, remarkably enhanced PLB-induced autophagy in PANC-1 and BxPC-3 cells, indicating the roles of PI3K and p38 MAPK mediated signaling pathways in PLB-induced autophagic cell death in both cell lines. In addition, PLB significantly inhibited epithelial to mesenchymal transition phenotype in both cell lines with an increase in the expression level of E-cadherin and a decrease in N-cadherin. Moreover, PLB treatment significantly suppressed the expression of Sirt1 in both cell lines. These findings show that PLB promotes cell cycle arrest and autophagy but inhibits epithelial to mesenchymal transition phenotype in pancreatic cancer cells with the involvement of PI3K/protein kinase B/mammalian target of rapamycin and p38 MAPK mediated pathways. PMID:25632222

Whole Blood Transcriptomics Is Relevant to Identify Molecular Changes in Response to Genetic Selection for Feed Efficiency and Nutritional Status in the Pig.

PubMed

Jégou, Maëva; Gondret, Florence; Vincent, Annie; Tréfeu, Christine; Gilbert, Hélène; Louveau, Isabelle

2016-01-01

The molecular mechanisms underlying feed efficiency need to be better understood to improve animal efficiency, a research priority to support a competitive and sustainable livestock production. This study was undertaken to determine whether pig blood transcriptome was affected by differences in feed efficiency and by ingested nutrients. Growing pigs from two lines divergently selected for residual feed intake (RFI) and fed isoproteic and isocaloric diets contrasted in energy source and nutrients were considered. Between 74 and 132 days of age, pigs (n = 12 by diet and by line) received a regular diet rich in cereals and low in fat (LF) or a diet where cereals where partially substituted by lipids and fibers (HF). At the end of the feeding trial, the total number of white blood cells was not affected by the line or by the diet, whereas the red blood cell number was higher (P<0.001) in low RFI than in high RFI pigs. Analysis of the whole blood transcriptome using a porcine microarray reveals a higher number of probes differentially expressed (DE) between RFI lines than between diets (2,154 versus 92 probes DE, P<0.01). This corresponds to 528 overexpressed genes and 477 underexpressed genes in low RFI pigs compared with high RFI pigs, respectively. Overexpressed genes were predominantly associated with translational elongation. Underexpressed genes were mainly involved in the immune response, regulation of inflammatory response, anti-apoptosis process, and cell organization. These findings suggest that selection for RFI has affected the immune status and defense mechanisms of pigs. Genes DE between diets were mainly related to the immune system and lipid metabolism. Altogether, this study demonstrates the usefulness of the blood transcriptome to identify the main biological processes affected by genetic selection and feeding strategies.

Whole Blood Transcriptomics Is Relevant to Identify Molecular Changes in Response to Genetic Selection for Feed Efficiency and Nutritional Status in the Pig

PubMed Central

Jégou, Maëva; Gondret, Florence; Vincent, Annie; Tréfeu, Christine; Gilbert, Hélène; Louveau, Isabelle

2016-01-01

The molecular mechanisms underlying feed efficiency need to be better understood to improve animal efficiency, a research priority to support a competitive and sustainable livestock production. This study was undertaken to determine whether pig blood transcriptome was affected by differences in feed efficiency and by ingested nutrients. Growing pigs from two lines divergently selected for residual feed intake (RFI) and fed isoproteic and isocaloric diets contrasted in energy source and nutrients were considered. Between 74 and 132 days of age, pigs (n = 12 by diet and by line) received a regular diet rich in cereals and low in fat (LF) or a diet where cereals where partially substituted by lipids and fibers (HF). At the end of the feeding trial, the total number of white blood cells was not affected by the line or by the diet, whereas the red blood cell number was higher (P<0.001) in low RFI than in high RFI pigs. Analysis of the whole blood transcriptome using a porcine microarray reveals a higher number of probes differentially expressed (DE) between RFI lines than between diets (2,154 versus 92 probes DE, P<0.01). This corresponds to 528 overexpressed genes and 477 underexpressed genes in low RFI pigs compared with high RFI pigs, respectively. Overexpressed genes were predominantly associated with translational elongation. Underexpressed genes were mainly involved in the immune response, regulation of inflammatory response, anti-apoptosis process, and cell organization. These findings suggest that selection for RFI has affected the immune status and defense mechanisms of pigs. Genes DE between diets were mainly related to the immune system and lipid metabolism. Altogether, this study demonstrates the usefulness of the blood transcriptome to identify the main biological processes affected by genetic selection and feeding strategies. PMID:26752050

Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts.

PubMed

Milovancev, Milan; Hilgart-Martiszus, Ian; McNamara, Michael J; Goodall, Cheri P; Seguin, Bernard; Bracha, Shay; Wickramasekara, Samanthi I

2013-06-13

Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis.

Anticancer activity of the new photosensitizers: dose and cell type dependence

NASA Astrophysics Data System (ADS)

Gyulkhandanyan, Grigor V.; Ghambaryan, Sona S.; Amelyan, Gayane V.; Ghazaryan, Robert K.; Haroutiunian, Samvel G.; Gyulkhandanyan, Aram G.; Gasparyan, Gennadi H.

2005-04-01

The necessity of researches of antitumor efficiency of new photosensitizers (PS) is explained by the opportunity of their application in photodynamic therapy of tumors. PS, selectively accumulated in cancer cells and activated by the light, generate the active oxygen species that cause apoptosis. Earlier, it was shown that PS chlorin e6 (0.3-0.5 μg/ml) induces rat embryo fibroblast-like cell apoptosis. In present work antitumor activity of the new photosensitizers, water-soluble cationic porphyrins and their metal complexes, is investigated. The dose-dependent destruction of cancer cells was shown on PC-12 (pheochromocytoma, rat adrenal gland) and Jurkat (human lymphoma) cell lines. Meso-tetra-[4-N-(2 `- oxyethyl) pyridyl] porphyrin (TOEPyP) and chlorin e6 possessed the same toxicity at LD50 dose on PC-12 cell line, whereas phototoxicity of TOEPyP was 3 times less compared to chlorin e6(LD50=0.2 and 0.075 μg/ml accordingly). The results have shown weak photosensitizing effect of Zn-and Ag-derivatives of TOEPyP on PC-12 cell line. TOEPyP and Zn-TOEPyP (0.1 - 50 μg/ml) were non-toxic for Jurkat cell line, whereas Ag-TOEPyP was toxic at 10 μg/ml (LD90). TOEPyP and chlorin e6 have shown phototoxic effect in the same dose range (LD50=0.5 and 0.3 μg/ml accordingly). The investigation of toxic and phototoxic effects of the new porphyrins revealed significantly different sensitivity of various cell lines to PSs.

Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines.

PubMed

Chen, Di; Xin, Xiao-Xuan; Qian, Hao-Cheng; Yu, Zhang-Yin; Shen, Li-Rong

2016-06-01

Royal jelly (RJ) is a well-known bioactive substance. It contains large amounts of major royal jelly proteins (MRJPs), which express growth-factor-like activity in several animal and human cell lines. However, the question on whether MRJPs possess growth-factor-like activity on all types of cell cultures remains. In order to determine whether MRJPs can be used as an alternative to fetal bovine serum (FBS) in different types of human cell culture, the proliferation of the complex serum with different ratios of MRJPs/FBS (M/F) was evaluated on five cell lines: 293T, HFL-I, 231, HCT116, and Changliver using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The proliferation activity of the combination of the complex M/F serum with cytokines on the test cell lines was also measured. The results demonstrated that the complex serum with M/F 6/4 possessed the highest proliferation activity similar to or in excess of FBS. However, no activity of complex medium with M/F 6/4 was observed in 231 cells, indicating a selectivity of MRJPs on cell types. Compared with the complex medium with M/F 6/4, the complex medium with M/F 6/4 together with two cytokines, epidermal growth factor (EGF) and insulin-transferrin-selenium (ITS), promoted proliferations of Changliver, 293T, HCT116, and HFL-I by 18.73%‒56.19% (P<0.01). Our findings demonstrate that MRJPs could partially replace FBS in culturing many human cell lines.

Plant genetic transformation efficiency of selected Malaysian rice based on selectable marker gene (hptII).

PubMed

Htwe, Nwe Nwe; Ling, Ho Chai; Zaman, Faridah Qamaruz; Maziah, Mahmood

2014-04-01

Rice is one of the most important cereal crops with great potential for biotechnology progress. In transformation method, antibiotic resistance genes are routinely used as powerful markers for selecting transformed cells from surrounding non-transformed cells. In this study, the toxicity level of hygromycin was optimized for two selected mutant rice lines, MR219 line 4 and line 9. The mature embryos were isolated and cultured on an MS medium with different hygromycin concentrations (0, 20, 40, 60, 80 and 100 mg L(-1)). Evidently, above 60 mg L(-1) was effective for callus formation and observed completely dead. Further there were tested for specific concentration (0-60). Although, 21.28% calli survived on the medium containing 45 mg L(-1) hygromycin, it seemed suitable for the identification of putative transformants. These findings indicated that a system for rice transformation in a relatively high frequency and the transgenes are stably expressed in the transgenic plants. Green shoots were regenerated from the explant under hygromycin stress. RT-PCR using hptII and gus sequence specific primer and Southern blot analysis were used to confirm the presence of the transgene and to determine the transformation efficiency for their stable integration in regenerated plants. This study demonstrated that the hygromycin resistance can be used as an effective marker for rice transformation.

New Radiation Therapy Systems: Applications to Human Cancer Treatment and Novel Drug Discovery

DTIC Science & Technology

2010-07-01

that basal kinase activity of PKG (likely involving selectively the PKG-Iα isoform) plays a critically important role in pro- moting the DNA synthesis...pro- moting DNA synthesis in human ovarian cancer cells may be independent of p53 mutational status and whether the cells are resistant to cisplatin...pro- moting cell proliferation in both cell lines. Thus, the data suggest that PKG-Iα kinase activity in human ovarian cancer cells plays an

Rewiring carbohydrate catabolism differentially affects survival of pancreatic cancer cell lines with diverse metabolic profiles

PubMed Central

Tataranni, Tiziana; Agriesti, Francesca; Ruggieri, Vitalba; Mazzoccoli, Carmela; Simeon, Vittorio; Laurenzana, Ilaria; Scrima, Rosella; Pazienza, Valerio; Capitanio, Nazzareno; Piccoli, Claudia

2017-01-01

An increasing body of evidence suggests that targeting cellular metabolism represents a promising effective approach to treat pancreatic cancer, overcome chemoresistance and ameliorate patient's prognosis and survival. In this study, following whole-genome expression analysis, we selected two pancreatic cancer cell lines, PANC-1 and BXPC-3, hallmarked by distinct metabolic profiles with specific concern to carbohydrate metabolism. Functional comparative analysis showed that BXPC-3 displayed a marked deficit of the mitochondrial respiratory and oxidative phosphorylation activity and a higher production of reactive oxygen species and a reduced NAD+/NADH ratio, indicating their bioenergetic reliance on glycolysis and a different redox homeostasis as compared to PANC-1. Both cell lines were challenged to rewire their metabolism by substituting glucose with galactose as carbon source, a condition inhibiting the glycolytic flux and fostering full oxidation of the sugar carbons. The obtained data strikingly show that the mitochondrial respiration-impaired-BXPC-3 cell line was unable to sustain the metabolic adaptation required by glucose deprivation/substitution, thereby resulting in a G2\\M cell cycle shift, unbalance of the redox homeostasis, apoptosis induction. Conversely, the mitochondrial respiration-competent-PANC-1 cell line did not show clear evidence of cell sufferance. Our findings provide a strong rationale to candidate metabolism as a promising target for cancer therapy. Defining the metabolic features at time of pancreatic cancer diagnosis and likely of other tumors, appears to be crucial to predict the responsiveness to therapeutic approaches or coadjuvant interventions affecting metabolism. PMID:28476035

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds.

PubMed

Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Naïra; Rau, Frédérique; Jollet, Arnaud; Edom-Vovard, Frédérique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois; Mouly, Vincent; Klein, Arnaud F; Furling, Denis

2017-04-01

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations. © 2017. Published by The Company of Biologists Ltd.

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds

PubMed Central

Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Naïra; Rau, Frédérique; Jollet, Arnaud; Edom-Vovard, Frédérique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois; Mouly, Vincent; Klein, Arnaud F.

2017-01-01

ABSTRACT Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations. PMID:28188264

Functional importance of GLP-1 receptor species and expression levels in cell lines.

PubMed

Knudsen, Lotte Bjerre; Hastrup, Sven; Underwood, Christina Rye; Wulff, Birgitte Schjellerup; Fleckner, Jan

2012-04-10

Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands. Copyright © 2011 Elsevier B.V. All rights reserved.

Novel menadione hybrids: Synthesis, anticancer activity, and cell-based studies.

PubMed

Prasad, Chakka Vara; Nayak, Vadithe Lakshma; Ramakrishna, Sistla; Mallavadhani, Uppuluri Venkata

2018-01-01

A series of novel menadione-based triazole hybrids were designed and synthesized by employing copper-catalyzed azide-alkyne cycloaddition (CuAAC). All the synthesized hybrids were characterized by their spectral data ( 1 H NMR, 13 C NMR, IR, and HRMS). The synthesized compounds were evaluated for their anticancer activity against five selected cancer cell lines including lung (A549), prostate (DU-145), cervical (Hela), breast (MCF-7), and mouse melanoma (B-16) using MTT assay. The screening results showed that majority of the synthesized compounds displayed significant anticancer activity. Among the tested compounds, the triazoles 5 and 6 exhibited potent activity against all cell lines. In particular, compound 6 showed higher potency than the standard tamoxifen and parent menadione against MCF-7 cell line. Flow cytometric analysis revealed that compound 6 arrested cell cycle at G0/G1 phase and induced apoptotic cell death which was further confirmed by Hoechst staining, measurement of mitochondrial membrane potential (ΔΨm) and Annexin-V-FITC assay. Thus, compound 6 can be considered as lead molecule for further development as potent anticancer therapeutic agent. © 2017 John Wiley & Sons A/S.

Identification of novel indole based heterocycles as selective estrogen receptor modulator.

PubMed

Singla, Ramit; Prakash, Kunal; Bihari Gupta, Kunj; Upadhyay, Shishir; Dhiman, Monisha; Jaitak, Vikas

2018-04-24

In the present study, we have designed and synthesized indole derivatives by coalescing the indole nucleus with chromene carbonitrile and dihydropyridine nucleus. Two compounds 5c and 6d were selected from series I and II after sequential combinatorial library generation, docking, absorption, distribution, metabolism and excretion (ADME) filtering, anti-proliferative activity, cytotoxicity, and ER-α competitor assay kit by utilizing estrogen receptor-α (ER-α) dominant T47D BC cells line and PBMCs (Peripheral Blood Mononuclear Cells). Cell imaging experiment suggested that both the compounds successfully cross cellular biomembrane and accumulate in nuclear, cytoplasmic and plasma membrane region. Semiquantitative RT-PCR and Western blotting experiments further supported that both compounds reduced the expression of mRNA and receptor protein of ER-α, thereby preventing downstream transactivation and signaling pathway in T47D cells line. Current findings imply that 5c and 6d represent novel ER-α antagonists and may be used in the development of chemotherapy for the management of BC. Copyright © 2018 Elsevier Inc. All rights reserved.

Activated MET acts as a salvage signal after treatment with alectinib, a selective ALK inhibitor, in ALK-positive non-small cell lung cancer.

PubMed

Kogita, Akihiro; Togashi, Yosuke; Hayashi, Hidetoshi; Banno, Eri; Terashima, Masato; De Velasco, Marco A; Sakai, Kazuko; Fujita, Yoshihiko; Tomida, Shuta; Takeyama, Yoshifumi; Okuno, Kiyotaka; Nakagawa, Kazuhiko; Nishio, Kazuto

2015-03-01

Non-small cell lung cancer (NSCLC) carrying echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearrangements is hypersensitive to ALK inhibitors, including crizotinib and alectinib. Crizotinib was initially designed as a MET inhibitor, whereas alectinib is a selective ALK inhibitor. The MET signal, which is inhibited by crizotinib but not by alectinib, is dysregulated in many human cancers. However, the role of the MET signal in ALK-positive NSCLC remains unclear. In this study, we found that hepatocyte growth factor (HGF), ligand of MET, mediated the resistance to alectinib, but not to crizotinib, via the MET signal in ALK-positive NSCLC cell lines (H3122 and H2228 cell lines). In addition, alectinib activated the MET signal even in the absence of HGF and the inhibition of the MET signal enhanced the efficacy of alectinib. These findings suggest that activated MET acts as a salvage signal in ALK-positive NSCLC. This novel role of the MET signal in ALK-positive NSCLC may pave the way for further clinical trials examining MET inhibitors.

Tumor specific cytotoxicity of arctigenin isolated from herbal plant Arctium lappa L.

PubMed

Susanti, Siti; Iwasaki, Hironori; Itokazu, Yukiyoshi; Nago, Mariko; Taira, Naoyuki; Saitoh, Seikoh; Oku, Hirosuke

2012-10-01

The effectiveness of cancer chemotherapy is often limited by the toxicity to other tissues in the body. Therefore, the identification of non-toxic chemotherapeutics from herbal medicines remains to be an attractive goal to advance cancer treatments. This study evaluated the cytotoxicity profiles of 364 herbal plant extracts, using various cancer and normal cell lines. The screening found occurrence of A549 (human lung adenocarcinoma) specific cytotoxicity in nine species of herbal plants, especially in the extract of Arctium lappa L. Moreover, purification of the selective cytotoxicity in the extract of Arctium lappa L. resulted in the identification of arctigenin as tumor specific agent that showed cytotoxicity to lung cancer (A549), liver cancer (HepG2) and stomach cancer (KATO III) cells, while no cytotoxicity to several normal cell lines. Arctigenin specifically inhibited the proliferation of cancer cells, which might consequently lead to the induction of apoptosis. In conclusion, this study found that arctigenin was one of cancer specific phytochemicals, and in part responsible for the tumor selective cytotoxicity of the herbal medicine.

Potent anti-proliferative effects against oral and cervical cancers of Thai medicinal plants selected from the Thai/Lanna medicinal plant recipe database "MANOSROI III".

PubMed

Manosroi, Aranya; Akazawa, Hiroyuki; Pattamapun, Kassara; Kitdamrongtham, Worapong; Akihisa, Toshihiro; Manosroi, Worapaka; Manosroi, Jiradej

2015-07-01

Thai/Lanna medicinal plant recipes have been used for the treatment of several diseases including oral and cervical cancers. To investigate anti-proliferative activity on human cervical (HeLa) and oral (KB) cancer cell lines of medicinal plants selected from Thai/Lanna medicinal plant recipe database "MANOSROI III". Twenty-three methanolic plant crude extracts were tested for phytochemicals and anti-proliferative activity on HeLa and KB cell lines for 24 h by the sulforhodamine B (SRB) assay at the doses of 1 × 10(1)-1 × 10(-6 )mg/ml. The nine extracts with the concentrations giving 50% growth inhibition (GI50) lower than 100 µg/ml were further semi-purified by liquid/liquid partition in order to evaluate and enhance the anti-proliferative potency. All extracts contained steroids/triterpenoids, but not xanthones. The methanolic extracts of Gloriosa superba L. (Colchinaceae) root and Albizia chinensis (Osbeck) Merr. (Leguminosae-Mimosoideae) wood gave the highest anti-proliferative activity on HeLa and KB cell lines with the GI50 values of 0.91 (6.0- and 0.31-fold of cisplatin and doxorubicin) and 0.16 µg/ml (28.78- and 82.29-fold of cisplatin and doxorubicin), respectively. Hexane and methanol-water fractions of G. superba exhibited the highest anti-proliferative activity on HeLa and KB cell lines with the GI50 values of 0.15 (37- and 1.9-fold of cisplatin and doxorubicin) and 0.058 µg/ml (77.45- and 221.46-fold of cisplatin and doxorubicin), respectively. This study has demonstrated the potential of plants selected from MANOSROI III database especially G. superba and A. chinensis for further development as anti-oral and cervical cancer agents.

Antiproliferative activity of king cobra (Ophiophagus hannah) venom L-amino acid oxidase.

PubMed

Li Lee, Mui; Chung, Ivy; Yee Fung, Shin; Kanthimathi, M S; Hong Tan, Nget

2014-04-01

King cobra (Ophiophagus hannah) venom L-amino acid oxidase (LAAO), a heat-stable enzyme, is an extremely potent antiproliferative agent against cancer cells when compared with LAAO isolated from other snake venoms. King cobra venom LAAO was shown to exhibit very strong antiproliferative activities against MCF-7 (human breast adenocarcinoma) and A549 (human lung adenocarcinoma) cells, with an IC50 value of 0.04±0.00 and 0.05±0.00 μg/mL, respectively, after 72-hr treatment. In comparison, its cytotoxicity was about 3-4 times lower when tested against human non-tumourigenic breast (184B5) and lung (NL 20) cells, suggesting selective antitumour activity. Furthermore, its potency in MCF-7 and A549 cell lines was greater than the effects of doxorubicin, a clinically established cancer chemotherapeutic agent, which showed an IC50 value of 0.18±0.03 and 0.63±0.21 μg/mL, respectively, against the two cell lines. The selective cytotoxic action of the LAAO was confirmed by phycoerythrin (PE) annexin V/7-amino-actinomycin (AAD) apoptotic assay, in which a significant increase in apoptotic cells was observed in LAAO-treated tumour cells than in their non-tumourigenic counterparts. The ability of LAAO to induce apoptosis in tumour cells was further demonstrated using caspase-3/7 and DNA fragmentation assays. We also determined that this enzyme may target oxidative stress in its killing of tumour cells, as its cytotoxicity was significantly reduced in the presence of catalase (a H2O2 scavenger). In view of its heat stability and selective and potent cytotoxic action on cancer cells, king cobra venom LAAO can be potentially developed for treating solid tumours. © 2013 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).